WO2003085121A2 - Nucleic acid and corresponding protein entitled 213p1f11 useful in treatment and detection of cancer - Google Patents

Nucleic acid and corresponding protein entitled 213p1f11 useful in treatment and detection of cancer Download PDF

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Publication number
WO2003085121A2
WO2003085121A2 PCT/US2002/010220 US0210220W WO03085121A2 WO 2003085121 A2 WO2003085121 A2 WO 2003085121A2 US 0210220 W US0210220 W US 0210220W WO 03085121 A2 WO03085121 A2 WO 03085121A2
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protein
amino acid
cancer
cell
cells
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PCT/US2002/010220
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French (fr)
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WO2003085121A9 (en
Inventor
Pia M. Challita-Eid
Arthur B. Raitano
Mary Faris
Rene S. Hubert
Robert Kendall Morrison
Wangmao Ge
Aya Jakobovits
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Agensys, Inc.
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Priority to PCT/US2002/010220 priority Critical patent/WO2003085121A2/en
Priority to EP02728647A priority patent/EP1578980A4/en
Priority to AU2002258689A priority patent/AU2002258689C1/en
Priority to IL16432602A priority patent/IL164326A0/en
Priority to CA002480811A priority patent/CA2480811A1/en
Publication of WO2003085121A2 publication Critical patent/WO2003085121A2/en
Priority to IL164326A priority patent/IL164326A/en
Publication of WO2003085121A9 publication Critical patent/WO2003085121A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3038—Kidney, bladder
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in Figure 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
  • T thymidine
  • U uracil
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative.temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • an HLA or cellular immune response "vaccine” is a composition that contains or encodes one or more peptides of the invention.
  • vaccines such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide.
  • Embodiments of a 213P1F11 polynucleotide include: a 213P1F11 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 213P1F11 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U.
  • embodiments of 213P IF 11 nucleotides comprise, without limitation:
  • V a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D , from nucleotide residue number 1 through nucleotide residue number 966, including the stop codon, wherein T can also be U;
  • VIII a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2G , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
  • HLA Peptide Tables (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc.
  • HLA Peptide Tables the particular variant
  • the length of the peptide in an HLA Peptide Table the Search Peptides in Table XXIX.
  • a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant.
  • the position of each Search Peptide relative to its respective parent molecule is listed in Table XXIX.
  • a Search Peptide begins at position "X"
  • a particular Search Peptide begins at position 150 of is parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
  • nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 213P1F11.
  • antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner.
  • PNAs peptide nucleic acids
  • non-nucleic acid molecules such as phosphorothioate derivatives
  • the 213P1F11 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 213P1F1 1 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 213P1F11 mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
  • the present invention includes the use of any probe as described herein to identify and isolate a 213P1F11 or 213PlFl l related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence er se, which would comprise all or most of the sequences found in the probe used. ⁇ .A.4.) Isolation of 213PlFll-Encoding Nucleic Acid Molecules
  • a 213P1F11 coding sequence is subcloned into the retroviral vector pSRccMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 213P1F11 expressing cell lines.
  • various mammalian cell lines such as NIH 3T3, TsuPrl, 293 and rat-1
  • Various other expression systems well known in the art can also be employed.
  • Expression constructs encoding a leader peptide joined in frame to a 213P1F11 coding sequence can be used for the generation of a secreted form of recombinant 213P1F11 protein.
  • Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
  • isoleucine I
  • V valine
  • L leucine
  • Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
  • 213P1F11-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 213P1F11-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 213P1F11 protein (or variants, homologs or analogs thereof).
  • Polypeptides comprising one or more of the 213P1F11 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 213P1F11 motifs discussed above are associated with growth deregulation and because 213P1F11 is overexpressed in certain cancers (See, e.g., Table I).
  • Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C are enzymes known to be associated with the development of the malignant phenotype (see e.g.
  • candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
  • Various strategies can be utilized to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al, Mol. Immunol 32:603, 1995; Celis, E. et al, Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al, J. Immunol. 158:1796, 1997; Kawashima, I. et al, Human Immunol. 59:1, 1998).
  • This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or ⁇ Cr-release assay involving peptide sensitized target cells.
  • PBL peripheral blood lymphocytes
  • Transgenic animals that include a copy of a transgene encoding 213P1F11 can be used to examine the effect of increased expression of DNA that encodes 213P1F11. Such animals' can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • Methods for the detection of particular mRNAs in cells include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 213P1F11 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 213P1F11, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • an assay for identifying a cell that expresses a 213P1F11 gene comprises detecting the presence of 213P1F11-related protein • in the cell or secreted by the cell.
  • Various methods for the detection of proteins are well known in the art and are employed for the detection of 213P1F11-related proteins and cells that express 213P1F11-related proteins.
  • the status of 213P1F11 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 213P1F11 gene), Northern analysis and/or PCR analysis of 213P1F11 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 213P1F11 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 213P1F11 proteins and/or associations of 213P1F11 proteins with polypeptide binding partners).
  • genomic Southern analysis to examine, for example perturbations in a 213P1F11 gene
  • Northern analysis and/or PCR analysis of 213P1F11 mRNA to examine, for example alterations in the polynucleotide sequences or expression levels of 213P1F11 mRNAs
  • the invention provides methods for monitoring 213P1F11 gene products by determining the status of 213P1F11 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 213P1F11 gene products in a corresponding normal sample.
  • the presence of aberrant 213P1F11 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
  • semi-quantitative RT-PCR is used to detect and quantify 213P1F11 mRNA expression.
  • Any number of primers capable of arr ⁇ lifying 213P1F11 can be used for this purpose, including but not limited to the various primer sets specifically described herein.
  • polyclonal or monoclonal antibodies specifically reactive with the wild-type 213P1F11 protein can be used in an immunohistochemical assay of biopsied tissue.
  • An embodiment of this invention comprises a method of screening for a molecule that interacts with a 213PlFl l amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 213P1F11 amino acid sequence, allowing the population of molecules and the 213P1F11 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 213P1F11 amino acid sequence, and then separating molecules that do not interact with the 213P1F11 amino acid sequence from molecules that do.
  • the method further comprises purifying, characterizing and identifying a molecule that interacts with the 213P1F11 amino acid sequence.
  • the identified molecule can be used to modulate a function performed by 213P1F11.
  • the 213P1F11 amino acid sequence is contacted with a library of peptides.
  • Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
  • HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
  • an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-213PlFl 1 mAb preparation represents an acceptable dosing regimen.
  • the initial loading dose is administered as a 90 minute or longer infusion.
  • the periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated.
  • various factors can influence the ideal dose regimen in a particular case.
  • a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein.
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes.
  • the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays.
  • the transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection.
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity.
  • a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • the DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 213P1F11.
  • a helper T cell (HTL) peptide such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response.
  • HTL helper T cell
  • a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 213P1F11.
  • human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect.
  • a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like.
  • human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect.
  • a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like.
  • a dose may be about 10 4 cells to about 10 6 cells, about 10 6 cells to about 10 8 cells, about 10 8 to about 10" cells, or about 10 8 to about 5 x 10 10 cells.
  • a dose may also about 10 6 cells/m 2 to about 10 10 cells/m 2 , or about 10 ⁇ cells/m 2 to about 10 8 cells/m 2 .
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concenfration of 25%-75%.
  • the 213P1F11 polynucleotides and polypeptides exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I.
  • Diagnostic assays that measure the presence of 213P1F11 gene products in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA.
  • these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 213P1F11 polynucleotides and polypeptides (as well as the 213P1F11 polynucleotide probes and anti-213PlFl 1 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
  • a method of inhibiting the transcription of the 213P1F11 gene comprises contacting the 213P1F11 gene with a 213P1F11 antisense polynucleotide.
  • a method of inhibiting 213P1F11 mRNA translation comprises contacting a 213P1F11 mRNA with an antisense polynucleotide.
  • a 213P1F11 specific ribozyme is used to cleave a 213P1F11 message, thereby inhibiting translation.
  • Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 213P1F11 gene, such as 213P1F11 promoter and/or enhancer elements.
  • Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site.
  • Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, infratumor, intradermal, intraorgan, orthotopic, and the like.
  • a preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP.
  • Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the SSH DNA sequence of 166 bp did not show homology to any known gene.
  • the full- length cDNA 213P1F11 was cloned from bladder cancer cDNA. Variants of 213P1F11 were identified and these are listed in Figures 2 and 3. 213PlFl l v.l reveals 100% identity to caspase-14 precursor apoptosis- related cysteine protease protein ( Figure 4).
  • PCR-based Validation Welhnann S, et al, Specific reverse franscription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, H.P., et al, Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8.
  • PCR-based and 5' RACE Validation Brigle, K.E., et al, Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353(2): 191-8).
  • Tables XXIV through XXVII are set forth herein on a variant-by-variant basis.
  • Table XXIV shows the nucleotide sequences of transcript variant 2 through variant 4.
  • Table XXV shows the alignment of franscript variant 2 through variant 4, each with the nucleic acid sequence of 213P1F11 variant 1.
  • Table XXVI lays out amino acid translation of franscript variant 2 through variant 4 for the identified reading frame orientation.
  • Table XXVII displays alignments of the amino acid sequences encoded by splice variant 2 through variant 4, each with that of 213P1F11 variant 1.
  • Table XXVIII displays clustal alignments of 213P1F11 protein variant 1 through variant 6.
  • 213P1F11 and 213P1F11 variants are cloned into any one of a variety of expression vectors known in the art.
  • One or more of the following regions of 213P1F11 or 213P1F11 variants are expressed in these constructs, amino acids 1 to 242 of 213P1F11 variant 1, amino acids 1-230 of variant 2, amino acids 1-146 of variant 3, amino acids 1-321 of variant 4; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 213P1F11, variants, or analogs thereof.
  • pCRII In vitro transcription and translation constructs: pCRII: To generate 213P1F11 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 213P IF 11 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 213P1F11 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze, the cell and tissue expression of 213P1F11 at the RNA level.
  • the GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies.
  • the 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, e.g., of the open reading frame (ORF).
  • a proteolytic cleavage site such as the PreScissionTM recognition site in pGEX-6P-l, may be employed such that it permits cleavage of the GST tag from 213P1F11-related protein.
  • the ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in £. coli.
  • the MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies.
  • the 6X His epitope tag is generated by adding 6 histidine codons to the 3' cloning primer.
  • a Factor Xa recognition site permits cleavage of the pMAL tag from 213P1F11.
  • the pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
  • pET Constructs To express 213P1F11 in bacterial cells, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly confrolled expression of recombinant 213P1F11 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag TM that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 213P1F11 protein are expressed as arnino-terminal fusions to NusA.
  • the resulting recombinant 213P1F11 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 213P1F11 proteins.
  • Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification.
  • the Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.
  • ⁇ tag5 A 213P1F11 ORF, or portions thereof, is cloned into pTag-5.
  • the 213P1F11 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.
  • GST glutathione-S-transferase
  • HIS HIS tagged fusion proteins
  • Reactivity and specificity of serum to full length 213P1F11 variant protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells fransfected with an expression vector encoding the 213P1F11 variant 1 cDNA (see e.g., the Example entitled "Production of Recombinant 213P1F11 in Eukaryotic Systems").
  • Other recombinant 213P1F11 variant 1-expressing cells or cells endogenously expressing 213P1F11 variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later.
  • mice The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Hariow and Lane, 1988). Supematants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 213P1F11 specific antibody-producing clones.
  • HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions.
  • An analysis of the 213P1F11 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
  • recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50IU/ml (Tsai et al, Critical Reviews in Immunology 18(l-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51 Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFN ⁇ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.
  • Cultures are expanded in the absence of anti-CD3 + as follows. Those, cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5x10 CD8 cells are added to a T25 flask containing the following: l lO 6 autologous PBMC per ml which have been peptide-pulsed with 10 ⁇ g/ml peptide for two hours at 37°C and irradiated (4,200 rad); 2x10 5 frradiated (8,000 rad) EBV-fransformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 25mM 2-ME, L-glutamine and gentamicin.
  • each engineered analog is initially tested for binding to the prototype A2 supertype allele A*Q201, then, if A*0201 binding capacity is maintained, for A2- supertype cross-reactivity.
  • Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization.
  • Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 213P1F11-expressing tumors.
  • Example 16 Identification and confirmation of 213P1F11-derived sequences with HLA-DR binding motifs
  • Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al, J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR- supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al, ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
  • the 213P1F11-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 ⁇ l, DR2w2 ⁇ 2, DR6wl9, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4wl5, DR5wl 1, and DR8w2 molecules in tertiary assays.
  • This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
  • mice which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide. ,
  • Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K b chimeric gene (e.g., Vitiello et al, J. Exp. Med. 173:1007, 1991)
  • Target cells 1.0 to 1.5xl0 6
  • Peptide is added where required at a concentration of 1 ⁇ g/ml.
  • 10 4 51 Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 ⁇ l) in U-bottom 96-well plates. After a six hour incubation period at 37°C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter.
  • % 51 Cr release data is expressed as lytic units/10 6 cells.
  • One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 5I Cr release assay.
  • the lytic units/10 6 obtained in the absence of peptide is subtracted from the lytic units/10 6 obtained in the presence of peptide.
  • Example 21 Selection of CTL and HTL epitopes for inclusion in a 213PlFll-specific vaccine.
  • the peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
  • the following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
  • a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concenfration of epitopes.
  • Epitopes may be nested or overlapping (i.e., frame shifted relative to one another).
  • two 9- mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide.
  • Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide.
  • a multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source.
  • an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
  • Such a vaccine composition is administered for therapeutic or prophylactic purposes.
  • a vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 213P1F11.
  • This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid.
  • Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
  • the heavy chain is modified by deletion of the fransmembrane-cytosolic tail and COOH-terminal addition ofa sequence containing a BirA enzymatic biotinylation site.
  • the heavy chain, ⁇ 2-microglobulin, and peptide are refolded by dilution.
  • the 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium.
  • Sfreptavidin- phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
  • the resulting product is referred to as teframer-phycoerythrin.
  • the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50 x 10 6 DC per patient are typically administered, larger number of DC, such as 10 7 or 10 8 can also be provided. Such cell populations typically contain between 50- 90% DC.
  • Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
  • Naturally occurring or recombinant 213P1F11 is substantially purified by immunoafffriity chromatography using antibodies specific for 213P1F11.
  • An immunoaffinity column is constructed by covalently coupling anti-213PlFl 1 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • Media containing 213P1F11 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 213P1F11 (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/213PlFll binding (e.g., a buffer of pH 2 to pH 3, or a high concentration ofa chaofrope, such as urea or thiocyanate ion), and GCR.P is collected.
  • Example 36 Identification of Molecules Which Interact with 213P1F11
  • the effect of the 213P1F11 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 213P1F11.
  • SCID mice are injected subcutaneously on each flank with 1 x 106 of either prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) or NIH-3T3 cells containing tkNeo empty vector or 213P1F11.
  • mice can be implanted with 1 x 105 of the same cells orthotopically to determine if 213P1F11 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.
  • the antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. It is confirmed that anti-213PlFl 1 mAbs inhibit formation of 213P1F11-expressing tumors. Anti-213P1F11 mAbs inhibit formation of the androgen-independent LAPC-9- Al tumor xenografts, as well as PC3- 213P1F11, MCF7-213P1F11 and UM-UC3-213P1F11 tumors. Anti-213P1F11 mAbs also retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice.
  • the LAPC-9 xenograft which expresses a wild-type androgen receptor and produces prostate- specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra).
  • Prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) expressing 213P1F11 are generated by retroviral gene transfer as described in Hubert, R.S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci U S A, 1999.
  • Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. Circulating levels of anti-213PlFl 1 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078)
  • the effect of anti-213PlFl 1 mAbs on tumor formation is tested by using the prostate and bladder orthotopic models.
  • the orthotopic model which requires surgical attachment of tumor tissue directly on the prostate or bladder, results in a local tumor growth, development of metastasis in distal sites, and subsequent death (Fu, X., et al, Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al, Anticancer Res., 1997. 17: p. 3239-3242).
  • This feature make the orthotopic model more representative of human disease progression and allows one to follow the therapeutic effect of mAbs, as well as other therapeutic modalities, on clinically relevant end points.
  • 213P1F11-expressing tumor cells are implanted orthotopically, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-2000 ⁇ g, usually 200-500 ⁇ g, of anti- 213P1F11 Ab, or b) PBS, three times per week for two to five weeks. Mice are monitored weekly for indications of tumor growth.
  • a major advantage of the orthotopic prostate and bladder cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by histological analysis of tissue sections, including lung and lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49:938-939; Chang, S., et al, Anticancer Res., 1997. 17:3239-3242). Additionally, IHC analysis using anti-213PlFl 1 antibodies can be performed on the tissue sections.
  • mice bearing established orthotopic 213P1F11-expressing tumors are administered lOOO ⁇ g injections of either anti-213PlFl 1 mAb or PBS over a 4-week period.
  • Mice in both groups are allowed to establish a high tumor burden (1-2 weeks growth), to ensure a high frequency of metastasis formation in mouse lungs and lymph nodes. Mice are then sacrificed and their local bladder tumor and lung and lymph node tissue are analyzed for the presence of tumor cells by histology and IHC analysis.
  • anti-213P1F11 antibodies inhibit tumor formation and retard the growth of already established tumors and prolong the survival of treated mice.
  • anti- 213P1F11 mAbs demonsfrate a dramatic inhibitory effect on the spread of local prostate, bladder and breast tumors to distal sites, even in the presence ofa large tumor burden.
  • anti-213PlFl 1 mAbs are efficacious on major clinically relevant end points including lessened tumor growth, lessened metastasis, and prolongation of survival.
  • Example 39 Therapeutic and Diagnostic use of Anti-213P1F11 Antibodies in Humans.
  • Anti-213P1F11 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans.
  • Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-213PlFl 1 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues.
  • Detection of 213P1F11 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator.
  • Anti- 213P1F11 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
  • anti-213PlFl 1 mAb specifically binds to carcinoma cells.
  • anti-213PlFl 1 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 213P 1 F 11. Shedding or release of an exfracellular domain of 213P 1 F 11 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 213P IF 11 by anti-213PlFl 1 antibodies in serum and/or urine samples from suspect patients.
  • unconjugated and conjugated anti-213PlFl 1 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled "213P1F11 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo ").
  • Conjugated and unconjugated anti-213PlFl 1 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other freatments as described in following Examples.
  • Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-213P1F11 Antibodies In vivo
  • Adjunctive therapy In adjunctive therapy, patients are treated with anti-213PlFl 1 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy.
  • Primary cancer targets such as those listed in Table I, are treated under standard protocols by the addition anti- 213P1F11 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent.
  • Anti-213P1F11 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
  • Imaging Agent Through binding a radionuclide (e.g., iodine or yttrium (I 131 , Y 90 ) to anti- 213P1F11 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 213P1F11. In connection with the use of the anti-213PlFl 1 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a postoperative follow-up to determine what tumor remains and/or returns.
  • a radionuclide e.g., iodine or yttrium (I 131 , Y 90 .
  • anti-213PlFl 1 antibodies that are fully human antibodies have slower clearance; accordingly, dosing in patients with such fully human anti-213PlFl l antibodies can be lower, perhaps in the range of 50 to 300 mg/m 2 , and still remain efficacious.
  • Dosing in mg/m 2 is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.
  • safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 213P1F11. Standard tests and follow-up are utilized to monitor each of these safety concerns.
  • Anti-213P1F11 antibodies are found to be safe upon human administration.
  • the trial design includes delivery of six single doses of an anti-213PlFll antibody with dosage of antibody escalating from approximately about 25 mg/m 2 to about 275 mg/m 2 over the course of the treatment in accordance with the following schedule: Day O Day 7 Day 14 Day 21 Day 28 Day 35
  • Anti-213P1F11 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti- 213P1F11 antibodies.
  • the 213P1F11-V.3 variant protein show 100% identity to the human caspase 14 precursor (gi 6912286) over 134 amino acids, while differing from that protein by 12 aa at its C-terminus.
  • 213P1F1 l-v.4 shows 97% identity with the human caspase 14 precursor over 235 amino acids, while differing from the human caspase 14 precursor (gi 6912286) by 86 aa at its N- terminus ( Figure 4G).
  • 213P 1 F 11 -v.1 consists of 242 amino acids, with calculated molecular weight of 28.0 kDa, and pi of 5.4.
  • 213P1F11-V.1 is an intercellular protein, located in the cytosol with potential localization to the nucleus (Table XXII). Similar localization patterns are observed for 213P1F11 protein variants 1, 3, and 4 (Table XXII). Bioinformatic analysis indicates that 213P1F1-V.2 may also localize to the mitochondria (Table XXII).
  • Caspase 14 has been shown to be processed by caspase 8 and caspase 10 as well as granzyme B, resulting in two catalytic subunits, ⁇ 20 and plO (Ahmad M et al, Cancer Res. 1998, 58:5201). These 2 cleavage products are detected in human • epidermis and in vitro during keratinocyte differentiation (Eckhart L et al, J. Invest. Drmatol. 2000, 115:1148). Overexpression of caspase 14 in breast carcinoma cells MCF7 resulted in the apoptosis of these cells, suggesting that caspase 14 participates in the process of programmed cell death (Hu S et al, J. Biol. Chem. 1998, 273:29648).
  • proteins are identified that associate with 213P1F11 and mediate signaling events.
  • 213P1F11 Several pathways known to play a role in cancer biology can be regulated by 213P1F11, including phospholipid pathways such as PI3K, survival pathways such as AKT, NFkB, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).
  • luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.
  • 213P1F11 Signaling pathways activated by 213P IF 11 are mapped and used for the identification and validation of therapeutic targets.
  • 213P1F11 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and therapeutic purposes.
  • the effect of sfress on apoptosis is evaluated in control parental cells and cells expressing 213P1F11, including normal and tumor bladder cells.
  • Engineered and parental cells are treated with various chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and protein synthesis inhibitors, such as cycloheximide.
  • Cells are stained with annexin V-FITC and cell death is measured by FACS analysis.
  • the modulation of cell death by 213P1F11 can play a critical role in regulating tumor progression and tumor load.
  • the localization of 213P1F11 to the cytoplasm with potential nuclear localization (Table XXII), support the present invention use of 213P1F11 based on its role in the franscriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 213P 1F11. For this purpose, two types of experiments are performed.
  • RNA from parental and 213PlFll-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al., Thyroid. 2001. 11:41.).
  • 213P1F11 When 213P1F11 is localized to specific cell compartments, it is used as a target for diagnostic, preventative and therapeutic purposes.

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Abstract

A novel gene (designated 213P1F11) and its encoded protein, and variants thereof, are described wherein 213P1F11 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 213P1F11 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 213P1F11 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be sued to elicit a humoral or cellular immune response; antibodies or T cells reactive with 213P1F11 can be used in active or passive immunization.

Description

NUCLEIC ACID AND CORRESPONDING PROTEIN ENTITLED 213P1F11 USEFUL IN TREATMENT AND DETECTION OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FD3LD OF THE INVENTION
The invention described herein relates to a gene and its encoded protein, termed 213P1F11, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 213P1F11.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al, 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al, 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al, Clin Cancer Res 1996 Sep 2 (9): 1445-51), STEAP (Hubert, et al, Proc Natl Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al, 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3 : 1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients. An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (-2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (-1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.
Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment. Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992- 1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all inrra- abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about -0.9% per year) v e rates have increased slightly among women.
Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer. SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 213P1F11, that has now been found to be over- expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 213P1F11 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (Figure 2) and amino acid (Figure 2, and Figure 3) sequences of 213P1F11 are provided. The tissue-related profile of 213P IF 11 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 213P1F11 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all or part of the 213P1F11 genes, RNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 213PlFl l-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 213P1F11 -related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 213P1F11 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 213P IF 11 genes, mRNAs, or to 213P1F11 -encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 213P1F11. Recombinant DNA molecules containing 213P1F11 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 213P1F11 gene products are also provided. The invention further provides antibodies that bind to 213P1F11 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the entire nucleic acid sequence of Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of Figure 2 is encoded and/or the entire airώio acid sequence of Figure 2 is prepared, either of which are in respective human unit dose forms.
The invention further provides methods for detecting the presence and status of 213P1F11 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 213P1F11. A typical embodiment of this invention provides methods for monitoring 213P1F11 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 213P1F11 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 213P1F11 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 213P1F11 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 213P1F11. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 213P1F11 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 213P1F11 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 213P1F11 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 213P1F11. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 213P1F11 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 213P1F11 production) or a ribozyme effective to lyse 213P1F11 mRNA.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. The 213P1F11 SSH sequence of 166 nucleotides.
Figure 2. The cDNA and amino acid sequence of213PlFl l variant 1 clone CASP14-BrCl (also called "213P1F11 v.l" or "213PlFll variant 1" or "213P1F11") is shown in Figure 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA and amino acid sequence of 213P1F11 variant 2 (also called "213P1F11 v.2") is shown in Figure 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 409- 1096 including the stop codon. The cDNA and amino acid sequence of 213P1F11 variant 3 (also called "213P1F11 v.3") is shown in Figure 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-844 including the stop codon. The cDNA and amino acid sequence of 213P1F11 variant 4 (also called "213P1F11 v.4") is shown in Figure 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1-966 including the stop codon. The cDNA (SEQ ID. NO. and amino acid sequence of 213P1F11 variant 5 (also called "213P1F11 v.5") is shown in Figure 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA and amino acid sequence of213PlFl l variant 6 (also called "213P1F11 variant v.6") is shown in Figure 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA and amino acid sequence of 213P1F11 variant 7 (also called "213P1F11 v.7") is shown in Figure 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA and amino acid sequence of 213P1F11 variant 8 (also called "213P1F11 v.8") is shown in Figure 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. As used herein, a reference to 213P1F11 includes all variants thereof, including those shown in Figure 10. Figure 3. Amino acid sequence of 213P1F11 v.l is shown in Figure 3A; it has 242 amino acids. The amino acid sequence of 213P IF 11 v.2 is shown in Figure 3B; it has 230 amino acids. The amino acid sequence of 213P1F11 v.3 is shown in Figure 3C; it has 146 amino acids. The amino acid sequence of 213P1F11 v.4 is shown in Figure 3D; it has 321 amino acids. The amino acid sequence of 213P1F11 v.5 is shown in Figure 3E; it has 242 amino acids. The amino acid sequence of 213P1F11 v.6 is shown in Figure 3F; it has 242 amino acids. As used herein, a reference to 213P1F11 includes all variants thereof, including those shown in Figure 11.
Figure 4. The nucleic acid sequence alignment of 213P1F11 v.l with human Caspase-14 (gi 6912286) precursor mRNA is shown in Figure 4A. The amino acid sequence alignment of 213P1F11 v.l with human Caspase-14 (gi 6912286) mRNA is shown in Figure 4B. The amino acid sequence alignment of 213P1F11 v.1 with mouse Caspase-14 (gi 6753280) mRNA is shown in Figure 4C. The amino acid sequence alignment of 213P1F11 v.2 with human Caspase-14 (gi 6912286) mRNA is shown in Figure 4D. The amino acid sequence alignment of 213P1F11 v.3 with human Caspase-14 (gi 6912286) mRNA is shown in Figure 4E. The amino acid sequence alignment of 213P1F11 v.2 with mouse caspase 14 (gi 6753280) mRNA is shown in Figure 4F. The amino acid sequence alignment of 213P1F11 v.4 with human Caspase-14 (gi 6912286) mRNA is shown in Figure 4G.
Figure 5. Hydrophilicity amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828) accessed on the Protscale website located at the World Wide Web (.expasy.ch cgi-bin protscale.pl) through the ExPasy molecular biology server.
Figure 6. Hydropathicity amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 7. Percent accessible residues amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located at the World Wide Web (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 8. Average flexibility amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. I. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located at the World Wide Web (.expasy.ch/cgi- bin protscale.pl) through the ExPasy molecular biology server.
Figure 9. Beta-turn amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located at the World Wide Web (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. Figure 10. Schematic display of nucleotide variants of 213P1F11. Variants 213P1F11 v.2 and v.3 are splice variants. Variant 213P1F11 v.4 is an alternative transcript. Others are Single Nucleotide Polymorphism (also called "SNP") variants, which could also occur in any of the transcript variants that contains the base pairs. Numbers in "( )" underneath the box correspond to those of 213P1F11 v.l. The black boxes show the same sequence as 213P1F11 v. l. SNPs are indicated above the box.
Figure 11. Schematic display of protein variants of 213P1F11. Nucleotide variants 213P1F11 v.l though v.6 in Figure 10 code for protein variants 213P IF 11 v.l through 213P IF 11 v.6, respectively. Variants 213P1F11 v.7 through v.10 code the same protein as variant 213P1F11 v.l. Protein variants 213P1F11 v.5 and v.6 are variants with single amino acid variations, which may exist in transcript variants 213P1F11 v.2 through 4. The black boxes show the same sequence as 213P1F11 v.l. The numbers in "()" underneath the box correspond to those of 213P1F11 v.l. Single amino acid differences are indicated above the box.
Figure 12. Secondary structure prediction for 213P1F11 variants 1 through 4. The secondary structures of 213P1F11 variant 1 (SEQ ID NO: 3) (A), variant 2 (SEQ ID NO: 5) (B), variant 3 (SEQ ID NO:7) (C), and variant 4 (SEQ ID NO: 9) (D) were predicted using the HNN - Hierarchical Neural Network method (Guermeur, 1997, htφ://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located at the Wo?ld Wide Web (.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed for each variant.
Figure 13. Exon compositions of transcript variants of 213P1F11. Variant 213P1F11 v.2 and v.3 are splice variants. Variant 213P1F11 v.4 is an alternative transcript. Compared with 213P1F11 v.l, 213P1F11 v.2 has a longer (+74 bp at 5' end) exon 6 and variant 213P1F11 v.3 has a longer (+68 bp at 5' end) exon 5. Variant 213P1F11 v.4 has three different exons. Relative locations of exons from all variants on the chromosome are shown at the bottom. Numbers in "( )" underneath the box correspond to those of 213P1F11 v.l. Black boxes show the same sequence as 213PlFll v.l. Intron lengths are not proportional.
Figure 14. Expression of 213P1F11 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show strong expression of 213P1F11 in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool.
Figure 15. Expression of 213P1F11 v.l compared to 213P1F11 v.2 in patient cancer samples by RT-PCR. To determine the relative expression of 213P1F11 v.l compared to 213P1F11 v.2 in human cancers, primers were designed flanking the insertion in 213P1F11 v.2. Using these primers, amplification of 213P1F11 v.l will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2 will generate a PCR fragment of 249 bp as depicted in the figure. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), and 213P1F11 v.l plasmid control. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was performed at 35 cycles of amplification. Results show strong expression of 213P IF 11 v.l in bladder cancer pool, breast cancer pool, LAPC xenograft pool, and the plasmid positive control. A lower expression of the 249 bp 213P1F11 v.2 product was detected in breast cancer pool, LAPC xenograft pool, and to lower extent in bladder cancer pool.
Figure 16. Expression of 213P1F11 in normal tissues. Three multiple tissue northern blots (A and B, Clontech; C, OriGene) with 2 ug of mRNA/lane were probed with the 213P1F11 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show strong expression of 213P1F11 only in skin tissue. A weak transcript is detected in normal thymus but not in the other tissues tested.
Figure 17. Expression of 213P1F11 in bladder cancer patient tissues. RNA was extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3 and SCaBER), bladder cancer patient tumors (T) and normal tissue adjacent to bladder cancer (NAτ). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the side. Results show strong expression of 213P1F11 in the bladder tumor tissues but not in normal bladder nor in the bladder cancer cell lines.
Figure 18. Expression of 213P1F11 in prostate cancer xenografts. RNA was extracted from normal prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI prostate cancer xenografts. Northern blot with 10 μg of total RNA/lane was probed with 213P1F11 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 213P1F11 in the LAPC-9AI xenograft, but not in the other xenografts nor in normal prostate.
Figure 19. Expression of 213P1F11 in breast cancer patient tissues. RNA was extracted from normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1), breast cancer patient tumors (T) and breast cancer metastasis to lymph node (Met). Northern blots with 10 ug of total RNA were probed with the 213P IF 11 SSH fragment. Size standards in kilobases are indicated on the side. Results show strong expression of 213P1F11 in the breast tumor tissues as well as in the cancer metastasis specimen. Weak expression was also detected in the CAMA-1 cell line, but not in the other 2 breast cancer cell lines tested.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
H.) 213P1F11 Polynucleotides
H.A.) Uses of 213P1F11 Polynucleotides π.A.l.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
H.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of 213P1F11-Encoding Nucleic Acid Molecules π.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems m.) 213PlFll-related Proteins πi.A.) Motif-bearing Protein Embodiments
III.B.) Expression of 213PlFll-related Proteins
III.C.) Modifications of 213P1F11-related Proteins
III.D.) Uses of 213PlFll-related Proteins IV.) 213P1F11 Antibodies V.) 213P1F11 Cellular Immune Responses
VI.) 213P1F11 Transgenic Animals
VII.) Methods for the Detection of 213P1F11
VIII.) Methods for Monitoring the Status of 213P1F11-related Genes and Their Products
IX.) Identification of Molecules That Interact With 213P1F11
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines X.B.) 213P1F11 as a Target for Antibody-Based Therapy X.C.) 213P1F11 as a Target for Cellular Immune Responses X.C.1. Minigene Vaccines
X.C.2. Combinations of CTL Peptides with Helper Peptides
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
X.D.) Adoptive Immunotherapy X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 213P1F11. XII.) Inhibition of 213PlFll Protein Function
XII.A.) Inhibition of 213P1F11 With Intracellular Antibodies
XII.B.) Inhibition of 213P1F11 with Recombinant Proteins
XII.C.) Inhibition of 213P1F11 Transcription or Translation XII.D.) General Considerations for Therapeutic Strategies
Figure imgf000012_0001
L) Definitions:
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage Cl - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 213P1F11 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 213P1F11. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
The term "analog" refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 213P1F11-related protein). For example an analog of a 213P1F11 protein can be specifically bound by an antibody or T cell that specifically binds to 213P1F11.
The term "antibody" is used in the broadest sense. Therefore an "antibody" can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-213P1F11 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An "antibody fragment" is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-213PlFl 1 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-213PlFl 1 antibody compositions withpolyepitopic specificity.
The term "codon optimized sequences" refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences."
The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxql, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
The term "homolog" refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar>at corresponding positions.
"Human Leukocyte Antigen" or "HLA" is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al, IMMUNOLOGY, 8™ ED., Lange Publishing, Los Altos, CA (1994).
The terms "hybridize", "hybridizing", "hybridizes" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in O.lXSSC/0.1% SDS are above 55 degrees C.
The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 213P1F11 genes or that encode polypeptides other than 213P1F11 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 213P1F11 polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical or chemical methods are employed to remove the 213P1F11 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 213P IF 11 protein. Alternatively, an isolated protein can be prepared by chemical means.
The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
The terms "metastatic prostate cancer" and "metastatic disease" mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. A "motif, as in biological motif of a 213P1F11-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein- protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, "motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
A "pharmaceutical excipient" comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
"Pharmaceutically acceptable" refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and or RNA. In the art, this term if often used interchangeably with "oligonucleotide". A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in Figure 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".
An HLA "primary anchor residue" is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table TV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif. A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that bind or interact with 213P1F11, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 213P1F11 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 213P1F11 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative.temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll 0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dexfran sulfate at 42 °C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55 °C, followed by a high- stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C. "Moderately stringent conditions" are described by, but not limited to, those in Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dexfran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature,, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
An HLA "supermotif is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. As used herein "to treat" or "therapeutic" and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.
A "transgenic animal" (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A "transgene" is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.
As used herein, an HLA or cellular immune response "vaccine" is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The "one or more peptides" can include any whole unit integer from 1- 242 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, or 242 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
The term "variant" refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 213P1F11 protein shown in Figure 2 or Figure 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
The "213P1F11-related proteins" of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 213P1F11 proteins or fragments thereof, as well as fusion proteins of a 213PlFl l protein and a heterologous polypeptide are also included. Such 213PlFll proteins are collectively referred to as the 213P1F11-related proteins, the proteins of the invention, or 213P1F11. The term "213P1F11- related protein" refers to a polypeptide fragment or a 213P1F11 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, or 242 or more amino acids.
H.) 213P1F11 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 213P1F11 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 213P IF 11-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 213PlFl l gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 213P1F11 gene, mRNA, or to a 213P1F11 encoding polynucleotide (collectively, "213P1F11 polynucleotides"). In all instances when referred to in this section, T can also be U in Figure 2.
Embodiments of a 213P1F11 polynucleotide include: a 213P1F11 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 213P1F11 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 213P IF 11 nucleotides comprise, without limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in Figure 2 , wherein T can also be U;
(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2 , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B , from nucleotide residue number 404 through nucleotide residue number 1096, including the stop codon, wherein T can also be U;
(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2C , from nucleotide residue number 404 through nucleotide residue number 844, including the a stop codon, wherein T can also be U;
(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D , from nucleotide residue number 1 through nucleotide residue number 966, including the stop codon, wherein T can also be U;
(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2E , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
(VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2F , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2G , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
(LX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2H , from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U; (X) • a polynucleotide that encodes a 213P 1 F 11 -related protein that is at least 90% homologous to an entire amino acid sequence shown in Figure 2A-H ;
(XI) a polynucleotide that encodes a 213P1F11-related protein that is at least 90% identical to an entire amino acid sequence shown in Figure 2A-H ;
(XII) a polynucleotide that encodes at least one peptide set forth in Tables V-XIX;
(XIII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5 A; or of Figure 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5B; or of Figure 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5C; or of Figure 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5D;
(XIV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A in any whole number increment up to 242 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6A; or of Figure 3B in any whole number increment up to 230 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6B; or of Figure 3C in any whole number increment up to 146 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6C; or of Figure 3D in any whole number increment up to 321 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6D;
(XV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7A; or of Figure 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7B; or of Figure 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7C; or of Figure 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7D;
(XVI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8A; or of Figure 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8B; or of Figure 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure8C; or of Figure 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8D;
(XVII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of Figure 3 A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9A; or of Figure 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Beta- turn profile of Figure 9B; or of Figure 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure9C; or of Figure 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9D;
(XVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XVII).
(XIX) a peptide that is encoded by any of (Ϊ)-(XVΪIT); and
(XXI) a polynucleotide of any of (I)-(XVTfl) or peptide of (XIX) together with a pharmaceutical excipient and/or in a human unit dose form.
As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 213P1F11 polynucleotides that encode specific portions of 213P1F11 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 230, 235, 240, or 242 or more contiguous amino acids of 213P1F11.
(b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 230 contiguous amino acids of variant 2;
(c) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 146 contiguous amino acids of variant 3; or
(d) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 146 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, or 321 contiguous amino acids of variant 4.
For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 213P 1 F 11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 213P1F11 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 213P1F11 protein shown in Figure 2 or Figure 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in Figure 2 or Figure 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100 through the carboxyl terminal amino acid of the 213P1F11 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions ofa 213PlFl l protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 213P1F11 protein "or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 213P1F11 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein include 213P1F11 polynucleotide fragments encoding one or more of the biological motifs contained within a 213PlFl l protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 213P1F11 protein "or variant" set forth in Tables V-XIX.
Note that to determine the starting position of any peptide set forth in Tables V-XIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table XXIX. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table XXIX. Accordingly if a Search Peptide begins at position "X", one must add the value "X - 1" to each position in Tables V-XIX to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of is parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables V- XIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables V-XVIII and at least once in table XIX, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics: i) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0J, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 5; ii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of Figure 8; or v) a peptide region of at least 5 amino acids of a particular peptide of Figure 3', in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9.
In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 213P1F11 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 213P1F11 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
H.A.) Uses of 213P1F11 Polynucleotides
H.A.I.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 213P1F11 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 213P1F11." For example, because the 213P1F11 gene maps to this chromosome, polynucleotides that encode different regions of the 213P1F11 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al, Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al, Blood 86(10): 3905-3914 (1995) and Finger et al, P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 213P1F11 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 213P1F11 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al, Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
Furthermore, as 213P1F11 was shown to be highly expressed in bladder and other cancers, 213P1F11 polynucleotides are used in methods assessing the status of 213P1F11 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 213P1F11 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 213P1F11 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al, J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
II.A.2.) Antisense Embodiments
Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 213P1F11. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 213P1F11 polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 213P1F11. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 213P1F11 antisense oligonucleotides of the present invention include derivatives such as S- oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-l,2-benzodithiol-3-one- 1,1 -dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al, J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 213P1F11 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al, 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
The 213P1F11 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 213P1F1 1 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 213P1F11 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 213P1F11 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 213P1F11 mRNA. Optionally, 213P1F11 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 213P1F11. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 213P1F11 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996). II.A.3.) Primers and Primer Pairs
Further specific embodiments of this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 213P1F11 polynucleotide in a sample and as a means for detecting a cell expressing a 213P1F11 protein.
Examples of such probes include polypeptides comprising all or part of the human 213P1F11 cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 213P1F11 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 213PlFll mRNA.
The 213P1F11 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 213P1F11 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 213P1F11 polypeptides; as tools for modulating or inhibiting the expression of the 213P1F11 gene(s) and/or translation of the 213P1F11 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 213P1F11 or 213PlFl l related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence er se, which would comprise all or most of the sequences found in the probe used. π.A.4.) Isolation of 213PlFll-Encoding Nucleic Acid Molecules
The 213P1F11 cDNA sequences described herein enable the isolation of other polynucleotides encoding 213P1F11 gene produces), as well as the isolation of polynucleotides encoding 213P1F11 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 213P1F11 gene product as well as polynucleotides that encode analogs of 213P IF 11-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 213P1F11 gene are well known (see, for example, Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al, Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 213P1F11 gene cDNAs can be identified by probing with a labeled 213P1F11 cDNA or a fragment thereof. For example, in one embodiment, a 213P1F11 cDNA (e.g., Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 213P1F11 gene. A 213P1F11 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries ( YACs), and the like, with 213P1F11 DNA probes or primers.
II.A.5.) Recombinant Nucleic Acid Molecules and Host- Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 213P1F11 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).
The invention further provides a host- vector system comprising a recombinant DNA molecule containing a 213PlFl l polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 213P1F11 or a fragment, analog or homolog thereof can be used to generate 213P1F11 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of 213P1F11 proteins or fragments thereof are available, see for example, Sambrook et al, 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His- tag (Invitrogen) and the retroviral vector pSRcctkneo (Muller et al, 1991, MCB 11 : 1785). Using these expression vectors, 213P1F11 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production ofa 213PlFl l protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 213P1F11 and 213P1F11 mutations or analogs.
Recombinant human 213P1F11 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 213P1F11-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 213P1F11 or fragment, analog or homolog thereof, a 213P1F11-related protein is expressed in the 293T cells, and the recombinant 213P1F11 protein is isolated using standard purification methods (e.g., affinity purification using anti- 213P1F11 antibodies). In another embodiment, a 213P1F11 coding sequence is subcloned into the retroviral vector pSRccMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 213P1F11 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 213P1F11 coding sequence can be used for the generation of a secreted form of recombinant 213P1F11 protein.
As discussed herein, redundancy in the genetic code permits variation in 213P1F11 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL located at the World Wide Web .dna.affrc.go.jp/~nakamura/codon.html.
Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol Cell Biol, 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
IH.) 213PlFll-related Proteins
Another aspect of the present invention provides 213P1F11-related proteins. Specific embodiments of 213P1F11 proteins comprise a polypeptide having all or part of the amino acid sequence of human • 213P1F11 as shown in Figure 2 or Figure 3. Alternatively, embodiments of 213P1F11 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 213P1F11 shown in Figure 2 or Figure 3.
In general, naturally occurring allelic variants of human 213P1F11 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 213P1F11 protein contain conservative amino acid substitutions within the 213P1F11 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 213P1F11. One class of 213P1F11 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 213P1F11 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either'the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments (see, e.g. Table III herein; pages 13-15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al, PNAS 1992 Vol 89 10915-10919; Lei et al, J Biol Chem 1995 May 19; 270(20): 11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 213P1F11 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 213P1F11 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al, Nucl. Acids Res., /5.4331 (1986); Zoller et al, Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al, Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al, Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 213P1F11 variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
As defined herein, 213P1F11 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 213P1F11 protein having an amino acid sequence of Figure 3. As used in this sentence, "cross reactive" means that an antibody or T cell that specifically binds to a 213P1F11 variant also specifically binds to a 213P1F11 protein having an amino acid sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 213P1F11 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al, J. Immunol 2000 165(12): 6949-6955; Hebbes et al, Mol Immunol (1989) 26(9):865-73; Schwartz et al, J Immunol (1985) 135(4):2598-608.
Other classes of 213P1F11-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of Figure 3, or a fragment thereof. Another specific class of 213P1F11 protein variants or analogs comprise one or more of the 213P IF 11 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 213P1F11 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of Figure 2 or Figure 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 213P1F11 protein shown in Figure 2 or Figure 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 213P1F11 protein shown in Figure 2 or Figure 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 ofa 213PlFl l protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 213PlFl l protein shown in Figure 2 or Figure 3 , polypeptides consisting of about amino acid 70 to about amino acid 80 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 213P1F11 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 213PlFH protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 213P1F11 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 213P1F11 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
213P1F11-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 213P1F11-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 213P1F11 protein (or variants, homologs or analogs thereof).
HI.A.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 213P1F11 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 213P1F11 polypeptide sequence set forth in Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses located at the World Wide Web: pfam.wustl.edu/; http.V/searcHavmcher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; .cbs.dtu.dk/; .ebi.ac.uk/interpro/scan.html; .expasy.ch/tools/scnpsitl.html; Epimatrix™ and Epimer™, Brown University, .brown.edu/Research/TB-HIN J ab/epirmtrix/epi^ and BIMAS, bimas.dcrt.nih.gov/.).
Motif bearing subsequences of all 213P1F11 variant proteins are set forth and identified in Tables V- XIX.
Table XX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu ). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 213P1F11 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 213P1F11 motifs discussed above are associated with growth deregulation and because 213P1F11 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al, Lab Invest., 78(2): 165-174 (1998); Gaiddon et al, Endocrinology 136(10): 4331-4338 (1995); Hall et al, Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al, Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al, Biochem. Biophys. Acta 1473(l):21-34 (1999); Raju et al, Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al, J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 213P1F11 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL located at the World Wide Web .brown.edu/Research/TB-HIV_Lab/epimatr^ and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table TV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table TV.
A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 9733602 to Chesnut et al; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al, J. Immunol. 2001. 166(2): 1389-1397; Sidney et al, Hum. Immunol. 1997 58(1): 12-20; Kondo et al, Immunogenetics 1997 45(4): 249-258; Sidney et al, J. Immunol. 1996 157(8): 3480-90; and Falk et al, Nature 351: 290-6 (1991); Hunt et al, Science 255:1261-3 (1992); Parker et al, J. Immunol. 149:3580-7 (1992); Parker et al, J. Immunol. 152:163-75 (1994)); Kast et al, 1994 152(8): 3904-12; Borras-Cuesta et al, Hum Immunol. 2000 61(3): 266-278; Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al, PMID: 7895164, UI: 95202582; O'Sullivan et al, J. Immunol. 1991 147(8): 2663-2669; Alexander et al, Immunity 1994 1(9): 751-761 and Alexander et al, Immunol. Res. 1998 18(2): 79-92. Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table XXI, and or, one or more of the predicted CTL epitopes of Tables V-XIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
213P1F11-related proteins are embodied in many forms, preferably in isolated form. A purified 213P1F11 protein molecule will be substantially free of other proteins or molecules that impair the binding of 213P1F11 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 213P1F11-related proteins include purified 213P1F11-related proteins and functional, soluble 213P1F11-related proteins. In one embodiment, a functional, soluble 213P1F11 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 213P1F11 proteins comprising biologically active fragments of a 213P1F11 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 213P1F11 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 213P1F11 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
213P1F11-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou- Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson- Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-213PlFl 1 antibodies, or T cells or in identifying cellular factors that bind to 213P1F11. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157: 105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R, Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.
• CTL epitopes can be determined using specific algorithms to identify peptides within a 213P 1 F 11 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, located at the World Wide Web (.brown.edu/Research TB- HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 213P1F11 that are presented-in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, Al l, A24, B7 and B35 were predicted (Tables V-XIX). Specifically, the complete amino acid sequence of the 213P1F11 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking redisues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL sy eithi.bmi-heidelberg.com/.
The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al, Nature 351: 290-6 (1991); Hunt et al, Science 255:1261-3 (1992); Parker et al, J. Immunol. 149:3580-7 (1992); Parker et al, J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer (also refered to as "nonamer"), and 10-mer (also refered to as "decamer") peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C- terminus (see, e.g., Parker et al, J. Immunol. 149:3580-7 (1992)). Selected results of 213P1F11 predicted binding peptides are shown in Tables V-XIX herein. In Tables V-XIX, the selected candidates, 9-mers and 10-mers, and 15-mers for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al, Prostate 30:73-8 (1997) and Peshwa et al, Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
It is to be appreciated that every epitope predicted by thέ BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be "applied" to. a 213P1F11 protein in accordance with the invention. As used in this context "applied" means that a 213P1F11 protein is evaluated, e.g., visually or by computer- based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 213P1F11 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 213PlFll-related Proteins
In an embodiment described in the examples that follow, 213P1F11 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV- driven expression vector encoding 213P1F11 with a C-terminal 6XHis and MYC tag (pcDNA3.1 /mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 213P1F11 protein in transfected cells. The secreted HIS-tagged 213P1F11 in the culture media can be purified, e.g., using a nickel column using standard techniques.
II C.) Modifications of 213P1F11-related Proteins
Modifications of 213P1F11-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 213P1F11 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 213PlFl l protein. Another type of covalent modification of a 213P1F11 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 213P1F11 comprises linking a 213P1F11 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 213P1F11-related proteins of the present invention can also be modified to form a chimeric molecule comprising 213P1F11 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments ofa 213PlFl l sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of213PlFl l. A chimeric molecule can comprise a fusion of a 213P1F11-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a213PlFl 1 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 213P IF 11-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 213PlFl l polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27, 1995.
DI.D.) Uses of 213PlFll-related Proteins
The proteins of the invention have a number of different specific uses. As 213P1F11 is highly expressed in prostate and other cancers, 213P1F11-related proteins are used in methods that assess the status of 213P1F11 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions ofa 213PlFl l protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 213P1F11-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 213P1F11 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 213P1F11-related proteins that contain the amino acid residues of one or more of the biological motifs in a 213P1F11 protein are used to screen for factors that interact with that region of 213P1F11.
213P1F11 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 213P1F1 1 protein), for identifying agents or cellular factors that bind to 213P1F11 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to -diagnostic assays, cancer vaccines and methods of preparing such vaccines.
Proteins encoded by the 213P1F11 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 213P1F11 gene product. Antibodies raised against a 213P1F11 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 213P1F11 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 213P1F11-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 213P1F11 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 213PlFll-expressing cells (e.g., in radioscintigraphic imaging methods). 213P1F11 proteins are also particularly useful in generating cancer vaccines, as further described herein.
TV.) 213P1F11 Antibodies
Another aspect of the invention provides antibodies that bind to 213P1F11-related proteins. Preferred antibodies specifically bind to a 213P1F11-related protein and do not bind (or bind weakly) to peptides or proteins that are not 213P1F11-related proteins. For example, antibodies that bind 213P1F11 can bind 213P1F11-related proteins such as the homologs or analogs thereof.
213P1F11 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 213P1F11 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 213P1F11 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 213P1F11 and mutant 213P1F11-related proteins. Such assays can comprise one or more 213P1F11 antibodies capable of recognizing and binding a 213P1F11-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like. Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 213P1F11 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 213P1F11 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 213P1F11 expressing cancers such as prostate cancer.
213P1F11 antibodies are also used in methods for purifying a 213P1F11-related protein and for isolating 213P1F11 homologues and related molecules. For example, a method of purifying a 213P1F11-related protein comprises incubating a 213P1F11 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 213P1F11-related protein under conditions that permit the 213P1F11 antibody to bind to the 213P1F11-related protein; washing the solid matrix to eliminate impurities; and eluting the 213P1F11-related protein from the coupled antibody. Other uses of 213P1F11 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 213P1F11 protein.
Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 213P1F11-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Hariow, and Lane (1988); Hariow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 213P1F11 can also be used, such as a 213PlFl l GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 213P1F11-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with or without purified 213P1F11-related protein or 213P1F11 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al, 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a 213P1F11 protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 213P1F11 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 213P1F11 amino acid sequence are used to identify hydrophilic regions in the 213P1F11 structure. Regions of a 213P1F11 protem that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 213P1F11 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 213P1F11 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
213P1F11 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 213P1F11-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 213P1F11 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 213P1F11 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies,«by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al, 1986, Nature 321 : 522-525; Riechmann et al, 1988, Nature 332: 323-327; Verhoeyen et al, 1988, Science 239: 1534-1536). See also, Carter et at, 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al, 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al, 1998, Nature Biotechnology 16: 535-539). Fully human 213P1F11 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 213P1F11 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits et al, published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114598 issued 5 September 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 213P1F11 antibodies with a 213P1F11-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 213P1F11-related proteins, 213P1F11-expressing cells or extracts thereof. A 213P1F11 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 213P1F11 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al, Cancer Res. 53: 2560-2565).
V 213P1F11 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world- wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA- restricted T cells (Buus, S. et al, Cell 47: 1071, 1986; Babbitt, B. P. et al, Nature 317:359, 1985; Townsend, A. and Bodmer, H, Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al, J. ' Immunol. 160:3363, 1998; Rammensee, et al, Immunogenetics 41:178, 1995; Rammensee et al, SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol 6:52, 1994; Ruppert et al, Cell 74:929-937, 1993; Kondo et al, J. Immunol 155:4307-4312, 1995; Sidney et al, J. Immunol 157:3480- 3490, 1996; Sidney et al, Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov; 50(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al, Immunity 4:203, 1996; Fremont et al, Immunity 8:305, 1998; Stern et al, Structure 2:245, 1994; Jones, E.Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al, Nature 364:33, 1993; Guo, H. C. et al, Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al, N: re 360:364, 1992; Silver, M. L. et al, Nature 360:367, 1992; Matsumura, M. et al, Science 257:927, 1992; Madden et al, Cell 70:1035, 1992; Fremont, D. H. et al, Science 257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C, J. Mol. Biol. 219:277, 1991.)
Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s). ,
Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al, Mol. Immunol 32:603, 1995; Celis, E. et al, Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al, J. Immunol. 158:1796, 1997; Kawashima, I. et al, Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or ^Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al, J. Immunol. 26:97, 1996; Wentworth, P. A. et al, Int. Immunol. 8:651, 1996; Alexander, J. et al, J. Immunol 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a
^^Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al, Immunity 7:97, 1997; Bertoni, R. et al, J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al, J. Immunol. 159:1648, 1997; Diepolder, H. M. et al, J. Virol 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive"
T cells. At the end of the culture period, T cell activity is detected using assays including " τ release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 213P1F11 Transgenic Animals
Nucleic acids that encode a 213P1F11-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 213P1F11 can be used to clone genomic DNA that encodes 213P1F11. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 213P1F11. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 213P1F11 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 213P1F11 can be used to examine the effect of increased expression of DNA that encodes 213P1F11. Such animals' can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of 213P1F11 can be used to construct a 213P1F11 "knock out" animal that has a defective or altered gene encoding 213PlFl l as a result of homologous recombination between the endogenous gene encoding 213P1F11 and altered genomic DNA encoding 213P1F11 introduced into an embryonic cell of the animal. For example, cDNA that encodes 213P1F11 can be used to clone genomic DNA encoding 213P1F11 in accordance with established techniques. A portion of the genomic DNA encoding 213P1F11 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell. 5J.:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al, Cell. 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 213P1F11 polypeptide.
Vπ.) Methods for the Detection of 213P1F11
Another aspect of the present invention relates to methods for detecting 213P1F11 polynucleotides and 213P IF 11-related proteins, as well as methods for identifying a cell that expresses 213P1F11. The expression profile of 213P1F11 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 213P1F11 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 213P1F11 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
More particularly, the invention provides assays for the detection of 213P1F11 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 213P1F.11 polynucleotides include, for example, a 213P1F11 gene or fragment thereof, 213P1F11 mRNA, alternative splice variant 213P1F11 mRNAs, and recombinant DNA or RNA molecules that contain a 213P1F11 polynucleotide. A number of methods for arnplifying and or detecting the presence of 213P1F11 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
In one embodiment, a method for detecting a 213P1F11 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 213P1F11 polynucleotides as sense' and antisense primers to amplify 213P1F11 cDNAs therein; and detecting the presence of the amplified 213P1F11 cDNA. Optionally, the sequence of the amplified 213P1F11 cDNA can be determined.
In another embodiment, a method of detecting a 213P1F11 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 213P1F11 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 213P1F11 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 213P1F11 nucleotide sequence (see, e.g., Figure 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 213P1F11 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 213P 1F11-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 213P1F11-related protein in a biological sample comprises first contacting the sample with a 213P1F11 antibody, a 213P1F11-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 213P1F11 antibody; and then detecting the binding of 213P IF 11-related protein in the sample.
Methods for identifying a cell that expresses 213P1F11 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 213P1F11 gene comprises detecting the presence of 213P1F11 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 213P1F11 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 213P1F11, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 213P1F11 gene comprises detecting the presence of 213P1F11-related protein • in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 213P1F11-related proteins and cells that express 213P1F11-related proteins.
213P1F11 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 213P1F11 gene expression. For example, 213P1F11 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 213P1F11 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 213P1F11 expression by RT-PCR, nucleic acid hybridization or antibody binding.
Vffl.) Methods for Monitoring the Status of 213PlFll-reIated Genes and Their Products
Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al, Lab Invest. 77(5): 437-438 (1997) and Isaacs et al, Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 213P1F11 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 213P1F11 in a biological sample of interest can be compared, for example, to the status of 213P1F11 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 213P1F11 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al, J. Comp. Neural. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare 213P1F11 status in a sample.
The term "status" in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 213P1F11 expressing cells) as well as the level, and biological activity of expressed gene products (such as 213P1F11 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 213P1F11 comprises a change in the location of 213P1F11 and/or 213P1F11 expressing cells and/or an increase in 213P1F11 mRNA and/or protein expression.
213P1F11 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro- dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 213P1F11 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 213P1F11 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 213P1F11 gene), Northern analysis and/or PCR analysis of 213P1F11 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 213P1F11 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 213P1F11 proteins and/or associations of 213P1F11 proteins with polypeptide binding partners). Detectable 213P1F11 polynucleotides include, for example, a 213P1F11 gene or fragment thereof, 213P1F11 mRNA, alternative splice variants, 213P1F11 mRNAs, and recombinant DNA or RNA molecules containing a 213P1F11 polynucleotide.
The expression profile of 213P1F11 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 213P1F11 provides information useful for predicting susceptibility to particular disease stages, progression, and or tumor aggressiveness. The invention provides methods and assays for deterrnining 213P1F11 status and diagnosing cancers that express 213P1F11, such as cancers of the tissues listed in Table I. For example, because 213P1F11 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 213P1F11 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 213P1F11 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 213P1F11 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 213P1F11 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
As described above, the status of 213P1F11 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 213P1F11 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 213P1F11 expressing cells (e.g. those that express 213P1F11 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 213P IF 11-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 213P1F11 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al, Prostate 42(4): 315-317 (2000);Su et al, Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al, J Urol 1995 Aug 154(2 Pt l):474-8).
In one aspect, the invention provides methods for monitoring 213P1F11 gene products by determining the status of 213P1F11 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 213P1F11 gene products in a corresponding normal sample. The presence of aberrant 213P1F11 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 213P1F11 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 213P1F11 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 213P1F11 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 213P1F11 mRNA or express it at lower levels.
In a related errώodiment, 213P1F11 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 213P1F11 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 213P1F11 expressed in a corresponding normal sample. In one embodiment, the presence of 213P1F11 protein is evaluated, for example, using immunohistochemical methods. 213P1F11 antibodies or binding partners capable of detecting 213P1F11 protein expression are used in a variety of assay formats well known in the art for this purpose.
In a further embodiment, one can evaluate the status of 213P1F11 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al, 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 213P 1 F 11 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 213P1F11 indicates a potential loss of function or increase in tumor growth.
A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 213P1F11 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Patent Nos. 5,382,510 issued 7 September 1999, and 5,952,170 issued 17 January 1995).
Additionally, one can examine the methylation status of a 213P1F11 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al, Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7:531- 536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al, Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of 213P1F11. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 213P1F11 expression. The presence of RT-PCR amplifiable 213P1F11 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT- PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et α/., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al, 1995, Clin. Chem. 41 :1687-1688).
A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 213P1F11 mRNA or 213P1F11 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 213P1F11 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 213P1F11 in prostate or other tissue is examined, with the presence of 213P1F11 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 213P IF 11 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 213P1F11 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 213P1F11 mRNA or 213P1F11 protein expressed by tumor cells, comparing the level so determined to the level of 213P1F11 mRNA or 213P1F11 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 213P1F11 mRNA or 213P1F11 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 213P1F11 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 213P1F11 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 213P1F11 mRNA or 213P1F11 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 213P1F11 mRNA or 213P1F11 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 213P1F11 mRNA or 213P1F11 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 213P1F11 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 213P1F11 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al, 1998, Mod. Pathol. 11(6):543-51; Baisden et αt., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and another factor associated with malignancy entails detecting the overexpression of 213P1F11 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 213P1F11 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 213P1F11 and PSA mRNA in prostate tissue is examined, where the coincidence of 213P1F11 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of 213P1F11 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 213P1F11 mRNA include in situ hybridization using labeled 213P1F11 riboprobes, Northern blot and related techniques using 213P1F11 polynucleotide probes, RT- PCR analysis using primers" specific for 213P1F11, and other amplification type detection methods, such as, for • example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 213P1F11 mRNA expression. Any number of primers capable of arrφlifying 213P1F11 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 213P1F11 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules That Interact With 213P1F11
The 213P1F11 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 213P1F11, as well as pathways activated by 213P1F11 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the "two-hybrid assay"). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Patent Nos. 5,955,280 issued 21 September 1999, 5,925,523 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al, Nature 402: 4 November 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 213P1F11 protein sequences. In such methods, peptides that bind to 213P1F11 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 213PlFl l protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 213P1F11 protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.
Alternatively, cell lines that express 213P1F11 are used to identify protein-protein interactions mediated by 213P1F11. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al Biochem. Biophys. Res. Commun. 1999, 261:646-51). 213P1F11 protein can be immunoprecipitated from 213P1F11-expressing celllines using anti-213PlFl l antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of213PlFl l and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 213P1F11 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 213PlFl l's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 213P1F11-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 213P1F11 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc, Sunderland, MA, 1992). Moreover, ligands that regulate 213P1F11 function can be identified based on their ability to bind 213P1F11 and activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 213P1F11 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators which activate or inhibit 213P1F11.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 213PlFl l amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 213P1F11 amino acid sequence, allowing the population of molecules and the 213P1F11 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 213P1F11 amino acid sequence, and then separating molecules that do not interact with the 213P1F11 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 213P1F11 amino acid sequence. The identified molecule can be used to modulate a function performed by 213P1F11. In a preferred embodiment, the 213P1F11 amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 213P1F11 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 213P1F11 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
Accordingly, therapeutic approaches that inhibit the activity of a 213P1F11 protein are useful for patients suffering from a cancer that expresses 213P1F11. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 213P1F11 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 213P1F11 gene or translation of 213P1F11 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 213P1F11-related protein or 213P1F11-related nucleic acid. In view of the expression of 213P1F11, cancer vaccines prevent and/or treat 213P1F11-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al, 1995, Int. J. Cancer 63:231-237; Fong tα/., 1997, J. Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 213P1F11-related protein, or a 213PlFl l- encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 213P1F11 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al, Ann Med 1999 Feb 31(l):66-78; Maruyama et al, Cancer Immunol Immunother 2000 Jun 49(3): 123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 213P1F11 protein shown in Figure 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 213P1F11 immunogen contains a biological motif, see e.g., Tables V-XIX, or a peptide of a size range from 213P1F11 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.
The entire 213P1F11 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g.Nitiello, A. et al, J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres {see, e.g., Eldridge, et al, Molec. Immunol. 28:287-294, 1991 : Alonso et al, Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin Exp Immunol 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al, Nature 320:535, 1986; Hu, S. L. et al, Nature 320:537, 1986; Kieny, M.-P. et al, AIDS Bio/Technology 4:790, 1986; Top, F. H. et al, J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al, Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al, J. Immunol Methods. 192:25, 1996; Eldridge, J. H. et al, Sem. Hematol 30:16, 1993; Falo, L. D., Jr. et al, Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R, and Chedid, L. A. Annu. Rev. Immunol 4:369, 1986; Gupta, R. K. et al, Vaccine 11:293, 1993), liposomes (Reddy, R. et al, J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al, Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11 :957, 1993; Shiver, J. W. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al, Sem. Hematol 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
In patients with 213P IF 11 -associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the, like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides within 213P1F11 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (located at the World Wide Web .brown.edu/Research/ITi-HIV_Lab/epimatrix/epiimtrix.htrnl); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syφeithi.bmi-heidelberg.com/). In a preferred embodiment, a 213P1F11 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XIX, or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
Antibody-based Vaccines
A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 213P1F11 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 213PlFl l in a host, by contacting the host with a sufficient amount of at least one 213P1F11 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 213P1F11 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 213P IF 11-related protein or a man-made multiepitopic peptide comprising: administering 213P1F11 immunogen (e.g. a 213P1F11 protein or a peptide fragment thereof, a 213P1F11 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Patent No. 6, 146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, CA; see, e.g., Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al, Immunity 1994 1(9): 751-761 and Alexander et al, Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 213PlFl l immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 213PlFl l immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Patent No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 213P1F11, in order to generate a response to the target antigen.
Nucleic Acid Vaccines:
Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode ρrotein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 213P1F11. Constructs comprising DNA encoding a 213P1F11-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 213P1F11 protein/immunogen. Alternatively, a vaccine comprises a 213P1F11-related protein. Expression of the 213P1F11-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 213P1F11 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address located at the World Wide Web .genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al, Science 247:1465 (1990) as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno- associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 213P1F11-related protein into the patient (e.g., intramuscularly or inrradermally) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al, Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
Thus, gene delivery systems are used to deliver a 213P1F11-related nucleic acid molecule. In one embodiment, the full-length human 213P1F11 cDNA is employed. In another embodiment, 213P1F11 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
Ex Vivo Vaccines
Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 213P1F11 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al, 1996, Prostate 28:65-69; Murphy et al, 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 213P1F11 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 213P1F11 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 213P1F11 protein. Yet another embodiment involves engineering the overexpression of a 213P1F11 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al, 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al, 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al, 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al, 1997, J. Exp. Med. 186: 1177-1182). Cells that express 213P1F11 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 213P1 FI 1 as a Target for Antibody-based Therapy 213P1F11 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 213P IF 11 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic adrninistration of 213P IF 11 -immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, nonspecific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 213P1F11 are useful to treat 213P1F11- expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
213P1F11 antibodies can be introduced into a patient such that the antibody binds to 213P1F11 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 213P1F11, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region ofa 213PlFl l sequence shown in Figure 2 or Figure 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 213P1F11), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-213PlFl 1 antibody) that binds to a marker (e.g. 213P1F11) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 213P1F11, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 213PlFl l epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
Cancer immunotherapy using anti-213PlFll antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al, 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al, 1997, Blood 90:3179-3186, Tsunenari et al, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al, 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al, 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al, 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al, 1994, Cancer Res. 54:6160-6166; Velders et al, 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard t α/., 1991, J. Clin. Immunol. 11: 117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co- administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 213P1F11 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth- Ayerst, Madison, NJ, a recombinant humanized IgG kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see e.g., US Patent 5,416,064).
Although 213P IF 11 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable .the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the • chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.
Although 213P1F11 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of213PlFll expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 213P1F11 imaging, or other techniques that reliably indicate the presence and degree of 213P1F11 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
Anti-213P1F11 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-213PlFl 1 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody- dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-213PlFl 1 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 213P1F11. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-213PlFl 1 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement- mediated cell lysis, and so forth, as is generally known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 213P1F11 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-213PlFl 1 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-213PlFl 1 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-213PlFl 1 mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-213P1F11 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-213PlFl 1 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, .2, .3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-213PlFl 1 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 213P1F11 expression in the patient, the extent of circulating shed 213P1F11 antigen, the desired steady-state antibody concenfration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
Optionally, patients should be evaluated for the levels of 213P1F11 in a given sample (e.g. the levels of circulating 213P1F11 antigen and or 213P1F11 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
Anti-idiotypic anti-213PlFl 1 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 213P1F11-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate antiidiotypic anti-213PlFl 1 antibodies that mimic an epitope on a 213P1F11-related protein (see, for example, Wagner et al, 1997, Hybridoma 16: 33-40; Foon et al, 1995, J. Clin. Invest. 96:334-342; Herlyn et al, 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
X.C.) 213P1F11 as a Target for Cellular Immune Responses
Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosme-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold, (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))
Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, inttathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 213P1F11 antigen, or derives at least some therapeutic benefit when the antigen was tumor- associated.
In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, CA) molecule (described e.g., in U.S. Patent Number 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are fransfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al, Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an ICjo of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties. 6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a "dominant epitope." A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
X.C.1. Minigene Vaccines
A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
The use of multi-epitope minigenes is described below and in, Ishioka et al, J. Immunol 162:3915- 3925, 1999; An, L. and Whitton, J. L., J. Virol 71:2292, 1997; Thomson, S. A. et al, J. Immunol 157:822, 1996; Whitton, J. L. et al, J. Virol 67:348, 1993; Hanke, R. et al, Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and or motif-bearing epitopes derived 213P1F11, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 213P IF 11 (see e.g., Tables V-XIX), and an endoplasmic reticulum-franslocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA- encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells fransfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes,' antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co- expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases. Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA," is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et al, Proc. NaflAcad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmiiDNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene- encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene- encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles. Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 38), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 39), and Streptococcus 18kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 40). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, CA) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO: 41), where "X" is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all "L" natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε-and α- amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α- amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S- glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al, Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 213P1F11. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 213P1F11.
X.D. Adoptive Immunotherapy
Antigenic 213P1F11 -related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.
X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 213P1F11. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 213P1F11. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of213PlFl l- associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 213P1F11, a vaccine comprising 213P IF 11 -specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of aclrninistration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, infrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral adminisfration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers maybe used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to adminisfration.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to , approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concenfration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of aα^mnistration selected.
A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of5-107 to 5xl09 pfu. For antibodies, a freatment generally involves repeated administration of the anti-213PlFl 1 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 213P1F11 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 213P1F11 expression in the patient, the extent of circulating shed 213P1F11 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500μg - lmg, lmg - 50mg, 50mg - lOOmg, lOOmg - 200mg, 200mg - 300mg, 400mg - 500mg, 500mg - 600mg, 600mg - 700mg, 700mg - 800mg, 800mg - 900mg, 900mg - lg, or lmg - 700mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, lOmg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5 - lOmg/kg body -weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400mg m2 of body area weekly; l-600mg m2 of body area weekly; 225-400mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared- to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 10" cells, or about 108 to about 5 x 1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 10δ cells/m2 to about 108 cells/m2 .
Proteins(s) of the invention, and or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. 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 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concenfration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 213P1F11. As disclosed herein, 213P1F11 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled "Expression Analysis of 213P1F11 in Normal Tissues and Patient Specimens").
213P1F11 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al, J. Urol. 163(2): 503-5120 (2000); Polascik et al, J. Urol. Aug; 162(2):293-306 (1999) and , Fortier et al, J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al, Int J Mol Med 1999 Jul 4(1):99- 102 and Minimoto et al, Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 213P1F11 polynucleotides and polypeptides (as well as 213P1F11 polynucleotide probes and anti-213PlFl 1 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
Typical embodiments of diagnostic methods which utilize the 213P1F11 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al, Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al, J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 213P1F11 polynucleotides described herein can be utilized in the same way to detect 213P1F11 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al, Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3):233-7 (1996)), the • 213P1F11 polypeptides described herein can be utilized to generate antibodies for use in detecting 213P1F11 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 213P1F11 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 213P1F11-expressing cells (lymph node) is found to contain 213P1F11- expressing cells such as the 213P IF 11 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 213P1F11 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 213P1F11 or express 213P1F11 at a different level are found to express 213P1F11 or have an increased expression of 213P IF 11 (see, e.g., the 213P1F11 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 213P1F11) such as PSA, PSCA etc. (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)).
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods- of monitoring PSA, 213P1F11 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al, Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled "Expression Analysis of 213PlFl l in Normal Tissues and Patient Specimens," where a 213PlFl l polynucleotide fragment is used as a probe to show the expression of 213P1F11 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al, Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 213P1F11 polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.
Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 213P1F11 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 213P1F11 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 213P1F11 polypeptide shown in Figure 3).
As shown herein, the 213P1F11 polynucleotides and polypeptides (as well as the 213P IF 11 polynucleotide probes and anti-213PlFl 1 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 213P1F11 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 213P1F11 polynucleotides and polypeptides (as well as the 213P1F11 polynucleotide probes and anti-213PlFl 1 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the 213P1F11 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 213P1F11 gene maps (see the Example entitled "Chromosomal Mapping of 213P1F11" below). Moreover, in addition to their use in diagnostic assays, the 213P1F11-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(l-2): 63-9).
Additionally, 213P1F11-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of213PlFl l. For example, the amino acid or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to a 213P1F11 antigen. Antibodies or other molecules that react with 213P1F11 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XH.) Inhibition of 213P1F11 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 213P1F11 to its binding partner or its association with other protein(s) as well as methods for inhibiting 213P1F11 function.
Xπ.A.) Inhibition of 213P1F11 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 213P1F11 are introduced into 213P1F11 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-213PlFl 1 antibody is expressed intracellularly, binds to 213P1F11 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as "infrabodies", are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TJJBTECH vol. 13). Inrrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al, 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al, 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al, 1994, Gene Ther. 1: 332-337).
Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, infrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Infrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to infrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Infrabodies can also be targeted to exert function in the cytosol. For example, cytosolic infrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
In one embodiment, infrabodies are used to capture 213P1F11 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 213P1F11 infrabodies in order to achieve the desired targeting. Such 213P1F11 infrabodies are designed to bind specifically to a particular 213P1F11 domain. In another embodiment, cytosolic infrabodies that specifically bind to a 213PlFl l protein are used to prevent 213P1F11 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 213P1F11 from forming transcription complexes with other factors).
In order to specifically direct the expression of such infrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Patent No. 5,919,652 issued 6 July 1999).
XH.B.) Inhibition of 213P1F11 with Recombinant Proteins
In another approach, recombinant molecules bind to 213P1F11 and thereby inhibit 213P1F11 function. For example, these recombinant molecules prevent or inhibit 213P1F11 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 213P1F11 specific antibody molecule. In aparticular embodiment, the 213P1F11 binding domain ofa 213PlFll binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 213P1F11 ligand binding domains linked to the Fc portion of a human IgG, such as human IgGl. Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 213P1F11, whereby the dimeric fusion protein specifically binds to 213P1F11 and blocks 213P1F11 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
Xπ.C.) Inhibition of 213P1F11 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 213P1F11 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 213P1F11 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 213P1F11 gene comprises contacting the 213P1F11 gene with a 213P1F11 antisense polynucleotide. In another approach, a method of inhibiting 213P1F11 mRNA translation comprises contacting a 213P1F11 mRNA with an antisense polynucleotide. In another approach, a 213P1F11 specific ribozyme is used to cleave a 213P1F11 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 213P1F11 gene, such as 213P1F11 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 213P1F11 gene transcription factor are used to inhibit 213P1F11 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
Other factors that inhibit the transcription of 213P1F11 by interfering with 213P1F11 transcriptional activation are also useful to treat cancers expressing 213P1F11. Similarly, factors that interfere with 213P1F11 processing are useful to treat cancers that express 213P1F11. Cancer treatment methods utilizing such factors are also within the scope of the invention.
XH.D.) General Considerations for Therapeutic Strategies
Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 213P1F11 (i.e., antisense, ribozyme, polynucleotides encoding infrabodies and other 213P1F11 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 213P1F11 antisense polynucleotides, ribozymes, factors capable of interfering with 213P1F11 ■ transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for- all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 213P1F11 to a binding partner, etc.
In vivo, the effect of a 213P1F11 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3 : 402-408). For example, PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft- bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).
Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, infratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
xm.) Kits
For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 213P1F11-related protein or a 213PlFl l gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
EXAMPLES:
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention. Example 1: SSH-Generated Isolation of a cDNA Fragment of the 213P1F11 Gene
To isolate genes that are over-expressed in bladder cancer, Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues was performed, including invasive transitional cell carcinoma. The 213P1F11 SSH cDNA sequence was derived from a bladder cancer pool minus cDNAs derived from 9 normal tissues. The 213P1F11 cDNA was identified as highly expressed in the bladder cancer tissue pool, with no expression detected in normal tissues.
The SSH DNA sequence of 166 bp (Figure 1) did not show homology to any known gene. 213PlFl lv.l of 3336 bp was identified and the open reading frame cloned from bladder cancer cDNA, revealing an ORF of 242 amino acids (Figure 2 and Figure 3). Other variants of 213P1F11, were also identified and these are listed in Figures 2 and 3. 213P1F11 v.l reveals 100% identity to caspase-14 precursor apoptosis-related cysteine protease protein (Figure 4).
Materials and Methods
Human Tissues:
The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, PA). mRNA for some normal tissues were purchased from Clontech, Palo Alto, CA.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/ g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by specfrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.
DPNCDN (cDNA synthesis primer): 5'TTTTGATCAAGCTT303' (SEQ ID NO: 42)
Adaptor 1:
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 43)
3OGCCCGTCCTAG5' (SEQ ID NO: 44)
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 45)
3'CGGCTCCTAG5' (SEQ ID NO: 46)
PCR primer 1 :
5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: 47)
Nested primer (NP)1 : 5 CGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 48)
Nested primer (NP)2:
5ΑGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 49)
Suppression Subfractive Hybridization:
Suppression Subfractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.
The gene 213P1F11 sequence was derived from a bladder cancer pool minus normal tissue cDNA subtraction. The SSH DNA sequence (Figure 1) was identified.
The cDNA derived from of pool of normal tissues was used as the source of the "driver" cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the "tester" cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)+ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH 's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37°C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.
Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16°C overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72°C for 5 min.
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68°C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 70°C for 7 min. and stored at -20°C.
PCR Amplification. Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10 x reaction buffer (CLONTECH) and 0.5 μl 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75°C for 5 min., 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, and 72°C for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel elecfrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First sfrand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplifϊcation system. The manufacturer's protocol was used which included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First sfrand cDNAs from 16 different normal human tissues can be obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 50) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 51) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, lXPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and IX Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94°C for 15 sec, followed by a 18, 20, and 22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec. A final extension at 72°C was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
To determine expression levels of the 213P IF 11 gene, 5 μl of normalized first sfrand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 213P1F11 SSH sequence and are listed below:
213P1F11.1
5'- GGATACCAGGGAACGCTTGGAG - 3' (SEQ EDNO: 52) 213P1F11.2
5'- TTTGACCTTTCCTGCTCAAGTAACC - 3' (SEQ ID NO: 53)
A typical RT-PCR expression analysis is shown in Figure 14. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi- quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show sfrong expression of213PlFl l in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool, but not in the vital pools.
Example 2: Full Length Cloning of 213P1F11
The 213P1F11 SSH cDNA sequence was derived from a bladder cancer pool minus normal tissues cDNA subtraction. The SSH cDNA sequence (Figure 1) was designated 213P1F11.
The SSH DNA sequence of 166 bp (Figure 1) did not show homology to any known gene. The full- length cDNA 213P1F11 was cloned from bladder cancer cDNA. Variants of 213P1F11 were identified and these are listed in Figures 2 and 3. 213PlFl l v.l reveals 100% identity to caspase-14 precursor apoptosis- related cysteine protease protein (Figure 4).
Example 3: Chromosomal Mapping of 213P1F11
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).
213P1F11 maps to chromosome 19pl3.1 using 213P1F11 sequence and the NCBI BLAST tool: located at the World Wide Web (.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4: Expression Analysis of 213P1F11 in Normal Tissues and Patient Specimens
Expression analysis by RT-PCR demonstrated that 213P1F11 is strongly expressed in bladder cancer patient specimens (Figure 14). First sfrand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC-xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show strong expression of 213P1F11 in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool, but not in the vital pools.
To determine the relative expression of 213P IF 11 v.l compared to 213P1F11 v.2 in human cancers, primers were designed flanking the insertion in 213P1F11 v.2 (Figure 15). Using these primers, amplification of 213P1F11 v.l will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2 will generate a PCR fragment of 249 bp as depicted in Figure 15. The PCR product of 165bp will also correspond to the variants 213P1F11 v.3, v.4, v.5, v.6 and v.7. First sfrand cDNA was prepared from vital pool 1 (liver, lung and kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), and 213P1F11 v.1 plasmid control. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was performed at 35 cycles of amplification. Results show strong expression of213PlFll v.l in bladder cancer pool, breast cancer pool, LAPC xenograft pool, and the plasmid positive control. A lower expression of the 249 bp 213P1F11 v.2 product was detected in breast cancer pool, LAPC xenograft pool, and to lower extent in bladder cancer pool. Altogether these data show that expression of 213P1F11 v.l is more abundant than 213P IF 11 v.2 in human patient cancer samples.
Extensive northern blot analysis of213PlFll in multiple human normal tissues is shown in Figure 16. Strong expression was only detected in skin tissue. A weak transcript is detected in normal thymus but not in the other tissues tested.
RNA was extracted from normal prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI prostate cancer xenografts. Northern blot with 10 μg of total RNA lane was probed with 213P1F11 SSH sequence (Figure 18). Results show expression of 213P IF 11 in the LAPC-9AI xenograft, but not in the other xenografts nor in normal prostate.
Expression of 213P1F11 in patient bladder cancer specimens is shown in Figure 17. RNA was extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3 and SCaBER), bladder cancer patient tumors (T) and normal tissue adjacent to bladder cancer (NAτ)- Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the side. Results show sfrong expression of 213P 1 F 11 in the bladder tumor tissues but not in normal bladder, nor in the bladder cancer cell lines.
Figure 19 shows that 213P1F11 was expressed in breast cancer patient tissues. RNA was extracted from normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1), breast cancer patient tumors (T) and breast cancer metastasis to lymph node (Met). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Results show strong expression of 213P1F11 in the breast tumor tissues as well as in the cancer metastasis specimen. Weak expression was also detected in the CAMA-1 cell line, but not in the other 2 breast cancer cell lines tested.
The restricted expression of213PlFl l in normal tissues and the expression detected in bladder cancer, breast cancer, prostate cancer xenograft, and cancer metastases suggest that 213P1F11 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5: Transcript Variants of 213P1F11
Transcript variants are variants of matured mRNA from the same gene by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each franscript variant has a unique exon makeup, and can have different coding and/or non-coding (5' or 3' end) portions, from the original franscript. Transcript variants can code for similar or different proteins with the same or a similar function or may encode proteins with different functions, and may be expressed in the same tissue at the same time, or at different tissue, or at different times, proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, i.e., be secreted.
Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified in a full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene (see, e.g., located at the World Wide Web (.doubletwist.com/products/cl l_agentsOverview.jhtml). Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based franscript variant identification programs include FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila genomic DNA," Genome Research. 2000 April; 10(4):516-22); Grail (http://compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (http://genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C, A genomic perspective on human proteases, FEBS Lett. 2001 Jun 8; 498(2-3):214-8; de Souza, S.J., et al, Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci U S A. 2000 Nov 7; 97(23): 12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S.O., et al, Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug 17;1433(l-2):321-6; Ferranti P, et al, Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(sl)-casein, Eur J Biochem. 1997 Oct 1;249(1): 1-7. For PCR-based Validation: Welhnann S, et al, Specific reverse franscription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, H.P., et al, Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation: Brigle, K.E., et al, Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the genomic region, to which a gene maps, is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 213P1F11 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 213P1F11 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens. • The exon composition of the original transcript, designated as 213P1F11 v.l, is shown in Table XXIIIA. Using the full-length gene and EST sequences, two splice variants were identified, designated as 213P1F11 v.2 and 213PlFl l v.3. Compared with 213P1F11 v.l, splice variant 213P1F11 v.2 had a longer exon 6 while 213P1F11 had a longer exon 5. Using the computer program GenScan, one alternative transcript was identified, designated as 213P1F11 v.4. This alternative transcript had three different leading exons in place of the first two exons of 213P1F11 v.l. The exon composition of the alternative franscript 213P1F11 v.4 is shown in Table XXIIIB. Since 213P1F11 v.4 shares the same exons 5 and 6 as 213P1F11 v.l, splice variants of this alternative franscript with a longer exon 5 or a longer exon 6, or both, may exist in human tissues. In fact, each different combination of exons in spatial order, e.g., exons 1, 2, 3, 4 and 7, is a potential splice variant. Figure 13 shows the schematic alignment of exons of the two transcripts (in addition to variants 2 and 3).
Tables XXIV through XXVII are set forth herein on a variant-by-variant basis. Table XXIV shows the nucleotide sequences of transcript variant 2 through variant 4. Table XXV shows the alignment of franscript variant 2 through variant 4, each with the nucleic acid sequence of 213P1F11 variant 1. Table XXVI lays out amino acid translation of franscript variant 2 through variant 4 for the identified reading frame orientation. Table XXVII displays alignments of the amino acid sequences encoded by splice variant 2 through variant 4, each with that of 213P1F11 variant 1. Table XXVIII displays clustal alignments of 213P1F11 protein variant 1 through variant 6.
Example 6: Single Nucleotide Polymorphisms of 213P1F11
Single Nucleotide Polymorphism (SNP) is a single base pair variation in nucleotide sequences. At a specific point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the base pair make-up of one or more spots in the genome of an individual, while haplotype refers to base pair make-up of more than one varied spots on the same DNA molecule (chromosome in higher organism). SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases and some others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for disearses and discovery of genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, " SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct; 11(5):637-641 ; M. Pirmohamed and B. K. Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, " The use of single nucleotide polymorphisms in the isolation of common disease genes," Pharmacogenomics. 2000 Feb; l(l):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The predictive power of haplotypes in clinical response," Pharmacogenomics. 2000 feb; l(l):15-26).
SNPs are identified by a variety of art-accepted methods (P. Bean, "The promising voyage of SNP target discovery," Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res. 1998 Jul; 8(7):691-697; M. M. She, "Enabling large-scale pharmacogenetic studies by high- throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2): 164- 172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, "Methods for genotyping single nucleotide polymorphisms," Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol. Diagn. 2000 Dec; 5(4):329-340).
Using the methods described above, six SNPs were identified in the original franscript, 213P1F11 v.l, at positions 473 (T/C), 737 (C/A), 2027 (C/T), 2037 (T/C), 2268 (A/G) and 3196 (A/T). The transcripts or proteins with alternative alleles were designated as variants 213P1F11 v.5, v.6, v.7, v.8, v.9, and v.10. Figure 10 shows the schematic alignment of the nucleotide variants. Figure 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in Figure 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants that contains the sequence context of the SNPs , e.g., 213P1F11 v.2, 213P1F11 v.3 or 213Plfl l v.4.
Example 7: Production of Recombinant 213P1F11 in Prokaryotic Systems
To express recombinant 213P1F11 and 213P1F11 variants in prokaryotic cells, the full or partial length 213P1F11 and 213P1F11 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 213P1F11 or 213P1F11 variants are expressed in these constructs, amino acids 1 to 242 of 213P1F11 variant 1, amino acids 1-230 of variant 2, amino acids 1-146 of variant 3, amino acids 1-321 of variant 4; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 213P1F11, variants, or analogs thereof.
A. In vitro transcription and translation constructs: pCRII: To generate 213P1F11 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 213P IF 11 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 213P1F11 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze, the cell and tissue expression of 213P1F11 at the RNA level. Transcribed 213P1F11 RNA representing the cDNA amino acid coding region of the 213P1F11 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate Sytem (Promega, Corp., Madison, WI) to synthesize 213P1F11 protein.
B. Bacterial Constructs: pGEX Constructs: To generate recombinant 213P1F11 proteins in bacteria that are fused to the Glutathione S-fransferase (GST) protein, all or parts of the T- fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 213P1F11 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl- terminus. The GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-l, may be employed such that it permits cleavage of the GST tag from 213P1F11-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in £. coli. pMAL Constructs: To generate, in bacteria, recombinant 213P1F11 proteins that are fused to maltose-binding protein (MBP), all or parts of the 213P1F11 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant 213P1F11 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl-terminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6X His epitope tag is generated by adding 6 histidine codons to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 213P1F11. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds. pET Constructs: To express 213P1F11 in bacterial cells, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly confrolled expression of recombinant 213P1F11 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag ™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 213P1F11 protein are expressed as arnino-terminal fusions to NusA.
C. Yeast Constructs: pESC Constructs: To express 213P1F11 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRPl, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 213P1F11. In addition, expression in yeast yields similar post-franslational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells. pESP Constructs: To express 213P1F11 in the yeast species Saccharomyces pombe, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 213PlFl l protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti- Flag™ antibody.
Example 8: Production of Recombinant 213P1F11 in Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 213P1F11 in eukaryotic cells, the full or partial length 213P1F11 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 213P1F11 are expressed in these constructs, amino acids 1 to 242, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 213P1F11, variants, or analogs thereof. In certain embodiments a region of a specific variant of 213P1F11 is expressed that encodes an amino acid at a specific position which differs from the amino acid of any other variant found at that position. In other embodiments, a region ofa variant of 213P1F11 is expressed that lies partly or entirely within a sequence that is unique to that variant.
The constructs can be transfected into any one ofa wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-213P1F11 polyclonal serum, described herein. pcDNA4/HisMax Constructs: To express 213P1F11 in mammalian cells, a 213P1F11 ORF, or portions thereof, of 213P1F11 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6X His) epitopes fused to the anήno-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli. pcDNA3.1/MycHis Constructs: To express 213P1F11 in mammalian cells, a 213P1F11 ORF, or portions thereof, of 213P1F11 with a consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColEl origin permits selection and maintenance of the plasmid in ii. coli. pcDNA3.1/CT-GFP-TOPO Construct: To express 213P1F11 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 213PlFl l ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-teπninus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length ofa 213PlFl l protein.
PAPtag: A 213P1F11 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-teπninus ofa 213P1F11 protein while fusing the IgG signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGi signal sequence is fused to the amino- terminus of a 213P1F11 protein. The resulting recombinant 213P1F11 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 213P1F11 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli. ρtag5: A 213P1F11 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 213P1F11 protein with an amino-terminal IgGκ signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 213P1F11 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 213P IF 11 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.
PsecFc: A 213P1F11 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin GI (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgGl Fc fusion at the carboxyl-terminus of the 213P1F11 proteins, while fusing the IgGK signal sequence to N-terminus. 213P1F11 fusions utilizing the murine IgGl Fc region are also used. The resulting recombinant 213P1F11 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 213P1F11 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. pSRα Constructs: To generate mammalian cell lines that express 213P1F11 constitutively, 213P1F11 ORF, or portions thereof, of 213P1F11 are cloned into pSRα constructs. Amphotropic and ecofropic refroviruses are generated by transfection of pSRα constracts into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 213P1F11, into the host cell- lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEl origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 213P1F11 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5' gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 54) is added to cloning primer at the 3' end of the ORF. Additional pSRα constracts are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full-length 213P1F11 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 213P1F11. High virus titer leading to high level expression of 213P1F11 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 213P1F11 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 213P1F11 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To confrol expression of 213P1F11 in mammalian cells, coding sequences of 213P IF 11, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of • recombinant 213P1F11. These vectors are thereafter used to control expression of 213P1F11 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 213P1F11 proteins in a baculovirus expression system, 213P1F11 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-213PlFl 1 is co-fransfected with helper plasmid pBac-N- Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant 213P1F11 protein is then generated by infection of HighFive insect cells (Invifrogen) with purified baculovirus. Recombinant 213P1F11 protein can be detected using anti-213PlFl 1 or anti-His- tag antibody. 213P1F11 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 213P1F11. Example 9: Antigenicitv Profiles and Secondary Structure
Figure 5A-D, Figure 6A-D, Figure 7A-D, Figure 8A-D, and Figure 9A-D depict graphically five amino acid profiles of the 213P1F11 variants 1 through 4 respectively, each assessment available by accessing the ProtScale website located at the World Wide Web (.expasy.ch/cgi-bin protscale.pl) on the ExPasy molecular biology server.
These profiles: Figure 5, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); Figure 6, Hydropathicity, (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132); Figure 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); Figure 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 213P1F11 protein. Each of the above amino acid profiles of 213P1F11 were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (Figure 5), Hydropathicity (Figure 6) and Percentage Accessible Residues (Figure 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
Average Flexibility (Figure 8) and Beta-turn (Figure 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
Antigenic sequences of the 213P1F11 protein and of the variant proteins indicated, e.g., by the profiles set forth in Figure 5A-D, Figure 6A-D, Figure 7A-D, Figure 8A-D, and/or Figure 9A-D are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-213PlFll antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 213P 1 F 11 protein variants listed in Figures 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of Figure 5A-D; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figures 6 A-D; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of Figure 7 A-D; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on Figure 8 A-D; and, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figures 9 A-D. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.
The secondary structures of 213P1F11 variants 1 through 4, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the primary amino acid sequence using the HNN - Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgi- bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located at the World Wide Web (.expasy.ch/tools/). The analysis indicates that 213P1F11 variant 1 is composed 47.93% alpha helix, 11.57% extended strand, and 40.50% random coil (Figure 12A), variant 2 is composed of 38.70% alpha helix, 9.57% extended strand, and 51.74% random coil (Figure 12B), variant 3 is composed of 50.68% alpha helix, 6.85% extended sfrand, and 42.47% random coil (Figure 12C), and variant 4 is composed of 39.25% alpha helix, 12.15% extended strand, and 48.60% random coil (Figure 12D).
Analysis for the potential presence of transmembrane domains in 213P1F11 variant 1 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located at the World Wide Web (.expasy.ch/tools/) (Table XXII). The programs do not predict the presence of transmembrane domains in any of the 213P IF 11 variants, suggesting that each is a soluble protein.
Example 10: Generation of 213P1F11 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 213P1F11 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles and Secondary Structure"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., Figure 5A-D, Figure 6 A-D, Figure 7 A-D, Figure 8 A-D, or Figure 9 A-D for amino acid profiles that indicate such regions of 213P1F11 and variants).
For example, 213P1F11 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 213P1F11 variant proteins are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 1-17, amino acids 25-80, amino acids 88-108, amino acids 131-147, and 207-242 of 213P1F11 variant 1. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids l-17 of213PlFl l variant 1 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 213P1F11 variant proteins, analogs or fusion proteins thereof. For example, the 213P1F11 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
In one embodiment, a GST-fusion protein encoding amino acids 1-147, encompassing several predicted antigenic regions, is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 213P1F11 in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Umes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled "Production of Recombinant 213P1F11 in Eukaryotic Systems"), and retain post- translational modifications such as glycosylations found in native protein. In one embodiment, the full length sequence of variant 1, amino acids 1-242, is cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supematants of 293T cells stably expressing the recombinant vector. The purified Tag5 213P1F11 protein is then used as immunogen.
During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic ttehalose dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IF A). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserumby ELISA.
To test reactivity and specificity of immune serum, such as the rabbit serum derived from ' immunization with a KLH-conjugated peptide encoding amino acids 1-17 of variant 1, the full-length 213P1F11 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled "Production of Recombinant 213P1F11 in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-213PlFl 1 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 213P1F11 protein using the Western blot technique. The immune serum is then tested by the Western blot technique against 293T- 213P1F11 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 213P IF 11-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 213P1F11 are also carried out to test reactivity and specificity. Anti-serum from rabbits immunized with 213P IF 11 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-213P1F11 fusion protein encoding amino acids 1-147 is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif). The antiserum is then affinity purified by passage over a column composed ofa MBP-fusion protein also encoding amino acids 1-147 covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11: Generation of 213P1F11 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 213P1F11 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 213P1F11 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 213P1F11 protein variant sequence, regions of the 213P1F11 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., Figure 5 A-D, Figure 6 A-D, Figure 7 A-D, Figure 8 A-D, or Figure 9 A-D, and the Example entitled "Antigenicity Profiles and Secondary Structure"). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 213P1F11 variant, such as 293T-213P1F11 variant 1 or 300.19-213P1F11 variant lmurine Pre-B cells, are used to immunize mice.
To generate mAbs to a 213PlFl l variant, mice are first immunized intraperitoneally (D?) with, typically, 10-50 μg of protein immunogen or 107213P1F11-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 213P1F11 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, the full length variant 1 sequence, encoding amino acids 1-242, is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the same amino acids are cloned into an Fc- fusion secretion vector in which the 213P1F11 variant 1 sequence is fused at the ammo-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant yector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 213PlFl lvariant.
During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytomettic analyses, fusion and hybridoma generation is then caπied out with established procedures well known in the art (see, e.g., Hariow and Lane, 1988).
In one embodiment for generating 213P1F11 monoclonal antibodies, a Tag5-213P1F11 variant 1 antigen encoding amino acids 1-242, is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5-213P1F11 variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 213P1F11 variant protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells fransfected with an expression vector encoding the 213P1F11 variant 1 cDNA (see e.g., the Example entitled "Production of Recombinant 213P1F11 in Eukaryotic Systems"). Other recombinant 213P1F11 variant 1-expressing cells or cells endogenously expressing 213P1F11 variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Hariow and Lane, 1988). Supematants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 213P1F11 specific antibody-producing clones.
Monoclonal antibodies are also derived that react only with specific 213P1F11 variants. To this end, immunogens are designed to encode amino acid regions specific to the respective variant. For example, a Tag5 immunogen encoding amino acids 175-230 of variant 2 is produced, purified, and used to immunize mice to generate hybridomas. In another example, a KLH-coupled peptide encoding amino acids 135-146 of variant 3 is produced and used as immunogen. In another example amino acids 1-86 of variant 4 is fused to GST and used as immunogen. Monoclonal antibodies raised to these immunogens are then screened for reactivity to cells expressing the respective variants but not to other 213P1F11 variants. These strategies for • raising 213P1F11 variant specific monoclonal antibodies are also applied to polyclonal reagents described in the Example entitled "Generation of 213P1F11 Polyclonal Antibodies."
The binding affinity ofa 213P1F11 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 213P1F11 monoclonal antibodies prefeπed for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Example 12: HLA Class I and Class II Binding Assays
HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al, Current Protocols in Immunology 18.3.1 (1998); Sidney, et al, J. Immunol 154:247 (1995); Sette, et al, Mol. Immunol 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concenfration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
Since under these conditions [label]<[HLA] and IC50≥[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive confrol for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with differenHots of purified MHC. '
Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif- bearing peptides.
Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
Computer searches and algorithms for identification of supermotif and/or motif-bearing epitopes
The searches performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles and Secondary Structure" and Tables V-XIX employ the protein sequence data from the gene product of 213P1F11 set forth in Figures 2 and 3.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 213P1F11 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:
"ΔG" = aw x a2i x a3i x a„,- where a,-,- is a coefficient which represents the effect of the presence ofa given amino acid (/') at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount J to the free energy of binding of the peptide iπespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described in Gulukota et al, J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al, Human Immunol. 45:79-93, 1996; and Southwood et al, J. Immunol 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying./ is calculated relative to the remainder of the group, and used as the estimate of/,. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score ofa given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 supertvpe cross-reactive peptides
Protein sequences from 213P1F11 are scanned utilizing motif identification software, to identify 8-, 9- 10- and 11-mer sequences containing the HLA- A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides coπesponding to the positive- scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2 -supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.
Selection of HLA- A3 supermotif-bearing epitopes
The 213P1F11 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides coπesponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of <500 nM, often < 200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA- A3 -supertype molecules tested.
Selection of HLA-B7 supermotif bearing- epitopes
The 213P1F11 protein(s) scanned above is also analyzed for the presence of 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif. Coπesponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC50 of <500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.
Selection of Al and A24 motif-bearing epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 213P1F11 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14: Confirmation of Immunogenicity
Cross-reactive candidate CTL A2 -supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening:
The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1 -restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or fransfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10 x 106 PBMC/well in a 6-well plate. After 2 hours at 37°C, the non-adherent cells are removed by gently shaking the plates and aspirating the supematants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250x106 PBMC are processed to obtain 24xlOδ CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20xl06cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140μl beads/20xlOδ cells) and incubated for 1 hour at 4°C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at lOOxlO6 cells/ml (based on the original cell number) in PBS/AB serum containing lOOμl ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB DNAse to collect the CD 8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40μg/ml of peptide at a cell concenfration of l-2xl06/ml in the presence of 3 μg/ml β microglobulin for 4 hours at 20°C. The DC are then iπadiated (4,200 rads), washed 1 time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at lxlO5 cells/ml) are co-cultured with 0.25ml of CD8+ T-cells (at 2xl06 cell/ml) in each well ofa 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.
Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5x10δ cells/ml and irradiated at ~4200 rads. The PBMCs are plated at 2xlOδ in 0.5 ml complete medium per well and incubated for 2 hours at 37°C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with lOμg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37°C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then fransfeπed to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50IU/ml (Tsai et al, Critical Reviews in Immunology 18(l-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.
Measurement of CTL lytic activity by 51Cr release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 5ICr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with lOμg/ml peptide overnight at 37°C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200μCi of 51Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37°C. Labeled target cells are resuspended at 10δ per ml and diluted 1:10 with K562 cells at a concenfration of 3.3x106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (lOOμl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37°C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:
[(cpm of the test sample- cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 5lCr release sample- cpm of the spontaneous 51Cr release sample)]'x 100.
Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample- background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.
In situ Measurement of Human IFNγ Production as an Indicator of Peptide-specific and
Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHC03, pH8.2) overnight at 4°C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/ 10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of lxlO6 cells/ml. The plates are incubated for 48 hours at 37°C with 5% C02.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200pg/100 microliter/well and the plate incubated for two hours at 37°C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1 :4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6x with wash buffer, 100 microliter/well developing solution (TMB 1 : 1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well IM H3P04 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5xl04 CD8+ cells are added to a T25 flask containing the following: lxlO6 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2xl05 iπadiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (anti-CD3) at 30ng per ml in RPMI-1640 ' containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concenfration of 200IU/ml and every three days thereafter with fresh media at 50IU/ml. The cells are split if the cell concentration exceeds lxl06/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at lxl0δ/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those, cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5x10 CD8 cells are added to a T25 flask containing the following: l lO6 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37°C and irradiated (4,200 rad); 2x105 frradiated (8,000 rad) EBV-fransformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 25mM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 supermotif-bearing peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 213P1F11. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
Evaluation of A*03/A11 immunogenicity
HLA- A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.
Evaluation of B7 immunogenicity
Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2-ahd A3-supermotif-bearing peptides.
Peptides bearing other supermotifs/motifs, g.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology
Example 15: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.
Analoging at Primary Anchor Residues
Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.
To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*Q201, then, if A*0201 binding capacity is maintained, for A2- supertype cross-reactivity.
. Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of 5000nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al, J. Immunol. 157:2539, 1996; and Pogue et al, Proc. Natl Acad. Sci. USA 92:8166, 1995).
In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.
Analoging of HLA- A3 and B7-supermotif-bearing peptides
Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3 -supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*l 1 (prototype A3 supertype alleles). Those peptides that demonstrate < 500 nM binding capacity are then confirmed as having A3- supertype cross-reactivity.
Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a prefeπed residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al (J. Immunol. 157:3480-3490, 1996).
Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonsfrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.
Analoging at Secondary Anchor Residues
Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity ofa B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 213P1F11-expressing tumors.
Other analoging strategies Another form of peptide analoging, unrelated to anchor positions, involves the substitution ofa cysteine with -amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α- amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al, In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.
Example 16: Identification and confirmation of 213P1F11-derived sequences with HLA-DR binding motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-supermotif-bearing epitopes.
To identify 213P IF 11-derived, HLA class II HTL epitopes, a 213P1F11 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).
Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al, J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR- supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al, ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
The 213P1F11-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 βl, DR2w2 β2, DR6wl9, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4wl5, DR5wl 1, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 213P1F11-derived peptides found to bind common HLA-DR alleles are of particular interest.
Selection of DR3 motif peptides
Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.
To efficiently identify peptides that bind DR3, target 213P1F11 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol 152:5742-5748, 1994). The coπesponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.
Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.
Example 17: Immunogenicity of 213P1F11-derived HTL epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 213P1F11-expressing tumors.
Example 18: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine breadth of population coverage
This example illustrates the assessment of the breadth of population coverage ofa vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=l-(SQRT(l-af)) (see, e.g., Sidney et al, Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=l-(l-Cgf)2].
Where frequency data is not available at the level of DNA typing, coπespondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(l-A)). Confirmed members of the A3-like supertype are A3, Al 1, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the Al and A24 motifs. On average, Al is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when Al and A24 are combined with the coverage of the A2-, A3- and B7- supertype alleles is >95%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
Immunogenicity studies in humans (e.g., Bertoni et al, J. Clin. Invest. 100:503, 1997; Doolan et al, Immunity 7:97, 1997; and Threlkeld et al, J. Immunol 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M.J. and Rubinstein, A. "A course in game theory" MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.
Example 19: CTL Recognition Of Endogenously Processed Antigens After Priming
This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 5ICr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 213P1F11 expression vectors.
The results demonsfrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 213P1F11 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human Al l, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
Example 20: Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by use ofa 213PlFl l- derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 213P1F11-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.
Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al, J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide. ,
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al, J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30xlOδ cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (lOxlO6 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
Assay for cytotoxic activity: Target cells (1.0 to 1.5xl06) are incubated at 37°C in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37°C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release = 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 5ICr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5xl05 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5xl04 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: t(l/50,000)-(l/500,000)] x 106 = 18 LU. The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled "Confirmation of Immunogenicity." Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon adminisfration of such compositions.
Example 21: Selection of CTL and HTL epitopes for inclusion in a 213PlFll-specific vaccine.
This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form ofa nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
Epitopes are selected which, upon adminisfration, mimic immune responses that are correlated with 213P1F11 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 213P1F11. For example, if it has been observed that patients who spontaneously clear 213P1F11- expressing cells generate an immune response to at least three (3) from 213P1F11 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an IC50 of 500 nM or less for an HLA class I molecule, or for class II, an IC50 of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.
In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient aπay of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.
When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concenfration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9- mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 213P1F11, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 213P1F11.
Example 22: Construction of "Minigene" Multi-Epitope DNA Plasmids
This example discusses the construction ofa minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif- bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 213P1F11, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 213P1F11 to provide broad population coverage, i.e. both HLA DR- 1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct: The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction ofa minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95°C for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (lx= 10 mM KCL, 10 mM (NH4)2S04, 20 mM Tris-chloride, pH 8.75, 2 mM MgS04, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
Example 23: The Plasmid Construct and the Degree to Which It Induces Immunogenicity.
The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al, J. Immunol. 156:683- 692, 1996; Demotz et al, Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).
Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al, Immunity 1:751-761, 1994.
For example, to confirm the capacity ofa DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.
It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA- B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.
To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab- resfricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of confrol animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al, Aids Res. and Human Refroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al, Vaccine 16:439-445, 1998; Sedegah et al, Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al, Nature Med. 5:526-34, 1999).
For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated- in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse ofa recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled "Induction of CTL Responses Using a Prime Boost Protocol." Example 24: Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 213P1F11 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 213PlFll-associated tumor.
For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 213P1F11 -associated disease.
Alternatively, a composition typically comprising transfecting agents is used for the adminisfration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.
Example 25: Polyepitopic Vaccine Compositions Derived from Native 213P1F11 Sequences
A native 213P1F11 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify "relatively short" regions of the polyprotein that comprise multiple epitopes. The "relatively short" regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, "nested" epitopes is selected; it can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 213P1F11 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 213P1F11, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.
Example 26: Polyepitopic Vaccine Compositions From Multiple Antigens
The 213P1F11 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or freatment of cancer that expresses 213P1F11 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 213P1F11 as well as tumor- associated antigens that are often expressed with a target cancer associated with 213P1F11 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27: Use of peptides to evaluate an immune response
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 213P1F11. Such an analysis can be performed in a manner described by Ogg et al, Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes ("teframers") are used for a cross-sectional analysis of, for example, 213P1F11 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 213P1F11 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al, N. Engl J. Med. 337: 1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the fransmembrane-cytosolic tail and COOH-terminal addition ofa sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Sfreptavidin- phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as teframer-phycoerythrin.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the teframer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with teframer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the teframers include both A*0201- negative individuals and A*0201 -positive non-diseased donors. The percentage of cells stained with the teframer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 213P1F11 epitope, and thus the status of exposure to 213P1F11, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28: Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 213P1F11-associated disease or who have been vaccinated with a 213P1F11 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 213P1F11 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross- reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI- 1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (lOmM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4 x 105 PBMC are stimulated with peptide in 8 replicate cultures in 96- well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are fransfeπed into a 96- well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 5lCr release, based on comparison with non-diseased confrol subjects as previously described (Rehermann, et al, Nature Med. 2:1104,1108, 1996; Rehermann et al, J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al J. Clin. Invest. 98:1432-1440, 1996).
Target cell lines are autologous and allogeneic EBV-fransformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, MA) or established from the pool of patients as described (Guilhot, et al. J. Virol. 6"6:2670-2678, 1992).
Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-fransformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.
Cytolytic activity is determined in a standard 4-h, split well 5ICr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20- 50: 1 on day 14. Percent cytotoxicity is determined from the formula: 100 x [(experimental release- spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, MO). Spontaneous release is <25% of maximum release for all experiments.
The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 213P1F11 or a 213P1F11 vaccine.
Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5xl05 cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 213P1F11 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing lOU/ml IL-2. Two days later, 1 μCi 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
Example 29: Induction Of Specific CTL Response In Humans
A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:
A total of about 27 individuals are enrolled and divided into 3 groups:
Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll- Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
The vaccine is found to be both safe and efficacious. Example 30: Phase II Trials In Patients Expressing 213P1F11
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 213P1F11. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 213P1F11, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.
There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 213P1F11.
Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the freatment of 213P1F11-associated disease.
Example 31: Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to confirm the efficacy ofa DNA vaccine in transgenic mice, such as described above in the Example entitled "The Plasmid Construct and the Degree to Which It Induces Immunogenicity," can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids" in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5xl09 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following adminisfration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 213P1F11 is generated. Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC)
Vaccines comprising peptide epitopes of the invention can be admimstered using APCs, or "professional" APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 213P1F11 protein from which the epitopes in the vaccine are derived.
For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfiision into patients, the DC are washed to remove unbound peptides.
As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50 x 106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50- 90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after freatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are admimstered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood ofa given patient, and that patient is to receive 5 x 10δ DC, then the patient will be injected with a total of 2.5 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex vivo activation of CTL/HTL responses
Alternatively, ex vivo CTL or HTL responses to 213P1F11 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33: An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 213P1F11. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al, J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif- bearing, this is an alternative modality for obtaining the motif-bearing peptides coπelated with the particular HLA molecule expressed on the cell.
Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 213P1F11 to isolate peptides coπesponding to 213P1F11 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
Example 34: Complementary Polynucleotides
Sequences complementary to the 213P1F11 -encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 213P1F11. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 213P1F11. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 213P1F11-encoding franscript.
Example 35: Purification of Naturally-occurring or Recombinant 213P1F11 Using 213P1F11- Specific Antibodies
Naturally occurring or recombinant 213P1F11 is substantially purified by immunoafffriity chromatography using antibodies specific for 213P1F11. An immunoaffinity column is constructed by covalently coupling anti-213PlFl 1 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing 213P1F11 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 213P1F11 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/213PlFll binding (e.g., a buffer of pH 2 to pH 3, or a high concentration ofa chaofrope, such as urea or thiocyanate ion), and GCR.P is collected. Example 36: Identification of Molecules Which Interact with 213P1F11
213P1F11, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously aπayed in the wells of a multi-well plate are incubated with the labeled 213P1F11, washed, and any wells with labeled 213P1F11 complex are assayed. Data obtained using different concentrations of 213P1F11 are used to calculate values for the number, affinity, and association of 213P1F11 with the candidate molecules.
Example 37: In Vivo Assay for 213P1F11 Tumor Growth Promotion
The effect of the 213P1F11 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 213P1F11. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of either prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) or NIH-3T3 cells containing tkNeo empty vector or 213P1F11. At least two strategies may be used: (1) Constitutive 213P1F11 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a refrovirus, hepatitis-B vires and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under confrol of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 213P IF 11-expressing cells grow at a faster rate and whether tumors produced by 213P IF 11-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with 1 x 105 of the same cells orthotopically to determine if 213P1F11 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.
The assay is also useful to determine the 213P1F11 inhibitory effect of candidate therapeutic compositions, such as for example, 213P1F11 infrabodies, 213P1F11 antisense molecules and ribozymes.
Example 38: 213P1F11 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo
The significant expression of 213P1F11 in cancer tissues, together with its restricted expression in normal tissues, makes 213P1F11 an excellent target for antibody therapy. In cases where the monoclonal antibody target is a cell surface protein, antibodies have been shown to be efficacious at inhibiting tumor growth (See, e.g., (Saffian, D., et al, PNAS 10:1073-1078 or the World Wide Web at .pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target is not on the cell surface, such as for 213P1F11 and including PSA and PAP in prostate cancer, antibodies have still been shown to recognize and inhibit growth of cells expressing those proteins (Saffian, D.C., et al, Cancer and Metastasis Reviews, 1999. 18: p. 437-449). As with any cellular protein with a restricted expression profile, 213P1F11 is a target for T cell-based immunotherapy. Accordingly, the therapeutic efficacy of anti-213PlFl 1 mAbs in human prostate, bladder and breast cancer mouse models is investigated using in 213P1F11-expressing prostate and bladder cancer xenografts as well as prostate, bladder and breast cancer cell lines, such as those described in the Example entitled "In Vivo Assay for 213P1F11 Tumor Growth Promotion," that have been engineered to express 213P1F11.
Antibody efficacy on tumor growth and metastasis formation is confirmed, e.g., in a mouse orthotopic prostate or bladder cancer xenograft models, as well as SCID mice injected with prostate, bladder and breast cancer cell lines, such as those described in the Example entitled "In Vivo Assay for 213P1F11 Tumor Growth Promotion," designed to express or lack 213P1F11. Therapeutic efficacy of anti-213PlFl 1 mAbs in prostate cancer is also evaluated in human prostate xenograft mouse models sucha s the LAPC-9 xenografts (Craft, N., et al.,. Cancer Res, 1999. 59(19): p. 5030-6). The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. It is confirmed that anti-213PlFl 1 mAbs inhibit formation of 213P1F11-expressing tumors. Anti-213P1F11 mAbs inhibit formation of the androgen-independent LAPC-9- Al tumor xenografts, as well as PC3- 213P1F11, MCF7-213P1F11 and UM-UC3-213P1F11 tumors. Anti-213P1F11 mAbs also retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice. These results indicate the utility of anti-213PlFl 1 mAbs in the freatment of local and advanced stages of prostate, bladder or breast cancer. (See, e.g., Saffran, D., et al., PNAS 10: 1073-1078 or the World Wide Web at .pnas.org/cgi/doi/ 10.1073/pnas.051624698)
Administration of anti-213PlFl 1 mAbs retard established orthotopic tumor growth and inhibit metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 213P1F11 is an attractive target for immunotherapy and demonsfrate the therapeutic potential of anti-213P1F11 mAbs for the freatment of local and metastatic bladder cancer.
This example demonstrates that unconjugated 213P1F11 monoclonal antibodies effectively to inhibit the growth of human prostate, bladder and breast tumors grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
Tumor inhibition using multiple unconjugated 213P1F11 mAbs Materials and Methods
213P1F11 Monoclonal Antibodies:
Monoclonal antibodies are raised against 213P1F11 as described in the Example entitled "Generation of 213P1F11 Monoclonal Antibodies (mAbs)." The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation , in accordance with techniques known in the art, for their capacity to bind 213P1F11. Epitope mapping data for the anti-213PlFl 1 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 213P IF 11 protein. Immunohistochemical analysis of bladder cancer tissues and cells with these antibodies is performed.
The monoclonal antibodies are purified from ascites or hybridoma tissue culture supematants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of bladder tumor xenografts. Cancer Xenograft and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate- specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra). Prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) expressing 213P1F11 are generated by retroviral gene transfer as described in Hubert, R.S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci U S A, 1999. 96(25): 14523-8. Anti-213P1F11 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
In Vivo Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 6213P1F11-expressing cancer cells mixed at a 1 : 1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an iπelevaηt antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. Circulating levels of anti-213PlFl 1 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078)
Orthotopic injections are performed, for example, in two alternative embodiments, under anesthesia by, for example, use of ketamine/xylazine. In a first embodiment, an infravesicular injection of bladder cancer cells is administered directly through the urethra and into the bladder (Peralta, E. A., et al, J. Urol., 1999. 162:1806-1811). In a second embodiment, an incision is made through the abdominal wall, the bladder is exposed, and bladder tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are surgically glued onto the exterior wall of the bladder, termed "onplantation" (Fu, X., et al, Int. J. Cancer, 1991. 49: 938-939; Chang, S., et al, Anticancer Res., 1997. 17: p. 3239-3242). For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to the exposed the dorsal prostate. Antibodies can be administered to groups of mice at the time of tumor injection or onplantation, or after 1-2 weeks to allow tumor establishment.
Anti-213P1F1 1 ™Abς Tnhibit Growth of 213P1F11-Expressing Cancer Tumors
In one embodiment, the effect of anti-213PlFl 1 mAbs on tumor formation is tested by using the prostate and bladder orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires surgical attachment of tumor tissue directly on the prostate or bladder, results in a local tumor growth, development of metastasis in distal sites, and subsequent death (Fu, X., et al, Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al, Anticancer Res., 1997. 17: p. 3239-3242). This feature make the orthotopic model more representative of human disease progression and allows one to follow the therapeutic effect of mAbs, as well as other therapeutic modalities, on clinically relevant end points.
Accordingly, 213P1F11-expressing tumor cells are implanted orthotopically, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-2000μg, usually 200-500μg, of anti- 213P1F11 Ab, or b) PBS, three times per week for two to five weeks. Mice are monitored weekly for indications of tumor growth.
As noted, a major advantage of the orthotopic prostate and bladder cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by histological analysis of tissue sections, including lung and lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49:938-939; Chang, S., et al, Anticancer Res., 1997. 17:3239-3242). Additionally, IHC analysis using anti-213PlFl 1 antibodies can be performed on the tissue sections.
Mice bearing established orthotopic 213P1F11-expressing tumors are administered lOOOμg injections of either anti-213PlFl 1 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (1-2 weeks growth), to ensure a high frequency of metastasis formation in mouse lungs and lymph nodes. Mice are then sacrificed and their local bladder tumor and lung and lymph node tissue are analyzed for the presence of tumor cells by histology and IHC analysis.
These studies demonstrate a broad anti-tumor efficacy of antiT213PlFl 1 antibodies on initiation and progression of bladder cancer in mouse models. Anti-213P1F11 antibodies inhibit tumor formation and retard the growth of already established tumors and prolong the survival of treated mice. Moreover, anti- 213P1F11 mAbs demonsfrate a dramatic inhibitory effect on the spread of local prostate, bladder and breast tumors to distal sites, even in the presence ofa large tumor burden. Thus, anti-213PlFl 1 mAbs are efficacious on major clinically relevant end points including lessened tumor growth, lessened metastasis, and prolongation of survival.
Example 39: Therapeutic and Diagnostic use of Anti-213P1F11 Antibodies in Humans.
Anti-213P1F11 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-213PlFl 1 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 213P1F11 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti- 213P1F11 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-213PlFl 1 mAb specifically binds to carcinoma cells. Thus, anti-213PlFl 1 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 213P 1 F 11. Shedding or release of an exfracellular domain of 213P 1 F 11 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 213P IF 11 by anti-213PlFl 1 antibodies in serum and/or urine samples from suspect patients.
Anti-213P1F11 antibodies that specifically bind 213P1F11 are used in therapeutic applications for the treatment of cancers that express 213P1F11. Anti-213P1F11 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-213PlFl 1 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled "213P1F11 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo "). Conjugated and unconjugated anti-213PlFl 1 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other freatments as described in following Examples.
Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-213P1F11 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 213P1F11, and are used in the freatment of certain tumors such as those listedin Table I. Based upon a number of factors, including 213P1F11 expression levels, tumors such as those listed in Table I are presently prefeπed indications. In connection with each of these indications, three clinical approaches are successfully pursued.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-213PlFl 1 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti- 213P1F11 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-213P1F11 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-213P1F11 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti- 213P1F11 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 213P1F11. In connection with the use of the anti-213PlFl 1 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a postoperative follow-up to determine what tumor remains and/or returns. In one embodiment, a (1 U In)-213P1F11 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 213P1F11 (by analogy see, e.g., Divgi et al J. Natl Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified
Dose and Route of Adminisfration As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-213PlFl 1 antibodies can be administered with doses in the range of 5 to 400 mg/m 2 , with the lower doses used, e.g., in connection with safety studies. The affinity of anti-213PlFl 1 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-213PlFl 1 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-213PlFl l antibodies can be lower, perhaps in the range of 50 to 300 mg/m2 , and still remain efficacious. Dosing in mg/m2 , as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.
Three distinct delivery approaches are useful for delivery of anti-213PlFl 1 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site ofa tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-213PlFl 1 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-213PlFl 1 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 213P1F11 expression levels in their tumors as determined by biopsy.
As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 213P1F11. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-213P1F11 antibodies are found to be safe upon human administration.
Example 41: Human Clinical Trial Adjunctive Therapy with Human Anti-213P1F11 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-213PlFl 1 antibody in connection with the treatment ofa solid tumor, e.g., a cancer ofa tissue listed in Table I. In the study, the safety of single doses of anti-213PlFl 1 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorabicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-213PlFll antibody with dosage of antibody escalating from approximately about 25 mg/m 2 to about 275 mg/m 2 over the course of the treatment in accordance with the following schedule: Day O Day 7 Day 14 Day 21 Day 28 Day 35
mAb Dose 25 75 125 175 225 275 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 Chemotherapy + + + + + +
(standard dose)
Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 213P1F11. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.
The anti-213PlFl 1 antibodies are demonstrated to be safe and efficacious, Phase.II trials confirm the efficacy and refine optimum dosing.
Example 42: Human Clinical Trial: Monotherapy with Human Anti-213P1F11 Antibody
Anti-213P1F11 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti- 213P1F11 antibodies.
Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-213P1F11 Antibody
Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-213PlFl 1 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al J. Natl Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44: Homology Comparison of 213P1F11 to Known Sequences
The 213P1F11 gene is homologous to a previously cloned gene, namely the human caspase 14 precursor (gi 6912286) (Hu S et al, J. Biol. Chem. 1998, 273:29648), also known as mini-ICE (MICE). The 213P1F11 gene resulted in several protein variants, which share several characteristics (Table XXII), including homology to ICE family of cysteine proteases. Several variants of 213P1F11, namely 213P1F11- v.2, -v.3 and -v.4, are novel proteins that maintain some homology to the published caspase 14 precursor (gi 6912286). For example, 213P1F1 l-v.2 shows 100% identity to the human caspase 14 precursor (gi 6912286) over the first 174 aa of the protein (Figure 4D), while differing from the published caspase 14 precurspr protein by 56 amino acids at its C-terminus, thus resulting in 76% overall identity to caspase 14 precursor. 213P1F1 l-v.2 also maintains homology to the mouse caspase 14, and shows 83% homology and 72%> identity to that protein (gi 6753280) (Figure 4F). The 213P1F11-V.3 variant protein show 100% identity to the human caspase 14 precursor (gi 6912286) over 134 amino acids, while differing from that protein by 12 aa at its C-terminus. Similarly, 213P1F1 l-v.4 shows 97% identity with the human caspase 14 precursor over 235 amino acids, while differing from the human caspase 14 precursor (gi 6912286) by 86 aa at its N- terminus (Figure 4G). 213P 1 F 11 -v.1 consists of 242 amino acids, with calculated molecular weight of 28.0 kDa, and pi of 5.4. 213P1F11-V.1 is an intercellular protein, located in the cytosol with potential localization to the nucleus (Table XXII). Similar localization patterns are observed for 213P1F11 protein variants 1, 3, and 4 (Table XXII). Bioinformatic analysis indicates that 213P1F1-V.2 may also localize to the mitochondria (Table XXII).
Caspases are a family of cyteine proteases that function as effectors of apoptosis or programmed cell death (Salvesen GS, Dixit V, Cell. 1997, 91:443; Thornberry N, Lazebnik Y, Science. 281: 1312). These proteases cleave different cellular substrates in an aspartate-specific manner. Cleavage may result in activation or inactivation of the cleaved cellular proteins, but not in protein degradation (Nunez et al, Oncogene. 1998, 17:3237; Stennicke HR, Salvesen GS, Cell Death Differ 1999, 6:1054). Caspases traditionally exist as precursor proteins also known as single polypeptide zymogens consisting ofa pro- domain, and 2 catalytic subunits, p20 and p 10 and contain a conserved QACXG active site (Stennicke HR, Salvesen GS, Cell Death Differ 1999, 6:1054; Cohen M. Biochem J 1997, 326:1). Similar to other members of the caspase family, 213P1F11 contains two catalytic subumts, p20 and plO, in addition to the conserved penta-peptide active site. In 213PlFl l v.l, p20 spans aa 16-139 and plO spans aa 155-241, while the active site is located at aa 129. Similarly, 213P1F11 v.4 carries both p20 and plO subunits, while 213P1F11 v.2 and v.3 contain the p20 subunit only, indicating that all 4 variants of 213P1F11 can function in a similar manner. Caspases are activated by proteolytic cleavage of their internal aspartate by an upstream enzyme, often another caspase. However, unlike other caspases with short pro-domains, caspase 14 is not reported to associate with known caspases (Hu S et al, J. Biol. Chem. 1998, 273:29648). Caspase 14 has been shown to be processed by caspase 8 and caspase 10 as well as granzyme B, resulting in two catalytic subunits, ρ20 and plO (Ahmad M et al, Cancer Res. 1998, 58:5201). These 2 cleavage products are detected in human • epidermis and in vitro during keratinocyte differentiation (Eckhart L et al, J. Invest. Drmatol. 2000, 115:1148). Overexpression of caspase 14 in breast carcinoma cells MCF7 resulted in the apoptosis of these cells, suggesting that caspase 14 participates in the process of programmed cell death (Hu S et al, J. Biol. Chem. 1998, 273:29648).
Our findings that 213P1F11 is highly expressed in several cancers while showing a restricted expression pattern in normal tissues suggests that the 213P1F11 gene plays an important role in various cancers, including cancers of the prostate, bladder and breast. Based on its similarity to caspase 14 213P1F11 has the ability to control tumor growth, apoptosis, survival, differentiation and progression. Accordingly, when 213P1F11 functions as a regulator of cell growth and apoptosis, or expression, 213P1F11 is used for therapeutic, diagnostic, prognostic or preventative purposes. Our findings that 213P1F11 is highly expressed in several cancers while showing a restricted expression pattern in normal tissues suggests that the 213P1F11 gene plays an important role in various cancers, including cancers of the prostate, bladder and breast. Based on its similarity to caspase 14 213P1F11 has the ability to control tumor growth, apoptosis, survival, differentiation and progression. Accordingly, when 213P1F11 functions as a regulator of cell growth and apoptosis, or expression, 213P1F11 is used for therapeutic, diagnostic, prognostic or preventative purposes.
Example 45: Identification and Confirmation of Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J Neurochem. 2001; 76:217-223). Caspases participate in signal transduction processes by getting recruited to signaling complexes and cleaving specific cellular substrates including other caspases and structural proteins, ultimately resulting in morphologic changes that represent the hallmark of apoptosis (Cohen GM. Biochem J. 1997, 326: 1). Recent studies have demonstrated that caspases also cleave signaling molecules, such as the guanine nucleotide exchange factor TIAMl, leading to the inactivation of TIAMl and thereby the Rac cascade (Qi H et al, Cell Growth Differ. 2001, 12:603). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 213P1F11 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 213P1F11, including phospholipid pathways such as PI3K, survival pathways such as AKT, NFkB, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). Bioinformatic analysis revealed that 213P1F11 can become phosphorylated by serine/threonine as well as tyrosine kinases. Thus, the phosphorylation of 213P1F11 is provided by the present invention to lead to activation of the above listed pathways.
Using, e.g., Western blotting techniques the ability of 213P1F11 to regulate these pathways is confirmed. Cells expressing or lacking 213P1F11 are either left untreated or stimulated with cytokines, hormones and anti-integrin antibodies. Cell lysates are analyzed using anti-phospho-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, ρ38, AKT, PI3K, PLC and other signaling molecules. When 213P1F11 plays a role in the regulation of signaling pathways, whether individually or communally, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
To confirm that 213P1F11 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.
NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress SRE-luc, SRF/TCF/ELKl; MAPK/SAPK; gτowth/diffirentiation AP-l-luc, FOS/JUN; MAPK/SAPK PKC; growth/apoptosis/stress ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis p53-luc, p53; SAPK; growth/differentiatiori/apoptosis CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
Signaling pathways activated by 213P IF 11 are mapped and used for the identification and validation of therapeutic targets. When 213P1F11 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 46: Involvement in Tumor Progression
Some apoptosis intermediates, such as DcRl, FLICE and TRAIL-R3, function as cellular inhibitors of apoptosis by acting as decoys and interfering with normal function of the apoptotic machinery (Sheikh MS et al, Oncogene. 1999, 18:4153; Ashkenazi A, Dixit VM. Cuπ Opin Cell Biol. 1999, 11:255). When 213P1F11 functions as a decoy, it can contribute to the growth of cancer cells. The role of 213P1F11 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, bladder and breast cell lines as well as NIH 3T3 cells engineered to stably express 213P1F11. Parental cells lacking 213P1F11 and cells expressing 213P1F11 are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, Grimes JA, Djamgoz MB. Prostate. 2000;44:61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7:288).
To confirm the role of 213P1F11 in the transformation process, its effect in colony forming assays is investigated. Parental NIH3T3 cells lacking 213P1F11 are compared to NHI-3T3 cells expressing 213P1F11, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000, 60:6730).
To confirm the role of213PlFl l in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999, 59:6010). Control cells, including prostate, colon, bladder and kidney cell lines lacking 213P1F11 are compared to cells expressing 213P1F11. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
213P1F11 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 213P 1 F 11 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel- Malek ZA. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the GI, S, and G2M phases of the cell cycle. Alternatively, the effect of sfress on apoptosis is evaluated in control parental cells and cells expressing 213P1F11, including normal and tumor bladder cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 213P1F11 can play a critical role in regulating tumor progression and tumor load.
When 213P1F11 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 47: Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays, endothelial cell tube formation, and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the effect of 213P1F11 on angiogenesis is confirmed. For example, endothelial cells engineered to express 213P1F11 are evaluated using tube formation and proliferation assays. The effect of 213P1F11 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 213P1F11 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5- 15 days later using immunohistochemistry techniques. When 213P1F11 affects angiogenesis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes
Example 48: Regulation of Transcription
The localization of 213P1F11 to the cytoplasm with potential nuclear localization (Table XXII), support the present invention use of 213P1F11 based on its role in the franscriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 213P 1F11. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 213PlFll-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al., Thyroid. 2001. 11:41.).
In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELKl-luc, ARE-luc, p53-luc, and CRE-luc. These franscriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.
When 213P1F11 plays a role in gene regulation, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 49: Subcellular Localization of 213P1F11
The cellular location of 213P1F11 can be assessed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). A variety of cell lines, including prostate, bladder and breast cell lines as well as cell lines engineered to express 213P1F11 are separated into nuclear, cytosolic and membrane fractions. Gene expression and location in nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble protein fractions are tested using Western blotting techniques.
Alternatively, 293T cells can be transfected with an expression vector encoding individual genes, HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen) and the subcellular localization of these genes is determined as described above. In short, the transfected cells can be harvested and subjected to a differential subcellular fractionation protocol (Pemberton, P.A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.). Location of the HIS-tagged genes is followed by Western blotting.
Using 213P1F11 antibodies, it is possible to demonstrate cellular localization by immuno fluorescence and immunohistochemistry. For example, cells expressing or lacking 213P1F11 are adhered to a microscope slide and stained with anti-213PlFl 1 specific Ab. Cells are incubated with an FITC- coupled secondary anti-species Ab, and analyzed by fluorescent microscopy.
When 213P1F11 is localized to specific cell compartments, it is used as a target for diagnostic, preventative and therapeutic purposes.
Example 50: 213P1F11 Proteolytic Activity
The similarity of 213P1F11 to casapase cysteine proteases supports the use of 213P1F11 as a- protease. Protease activity can be confirmed using on in vitro protease assay coupled to detection of protein fragments by western blotting (Hu S et al, above; Slee E et al, J Biol Chem 2001, 276:7320). In one embodiment, recombinant 213P1F11 protein is incubated with apoptotic subsfrates, including other caspases known to associate with caspase 14, namely caspase 2 and caspase 4, as well as recombinant RIP and poly(ADP-ribose) polymerase (i.e. PARP) (Slee E et al, J Biol Chem 2001, 276:7320; Hayakawa et al, Apoptosis. 2002, 7: 107). Proteins are separated by SDS-Page and analyzed by western blotting with substrate specific antibodies. In another embodiment, 213P1F11 activity is compared in confrol cells lacking 213P1F11 and cells expressing 213P1F11. Cell lysates from control and 213P1F11 expressing cells are incubated in the presence of the recombinant substrates listed above. Whole proteins are analyzed by western blotting with antibodies directed to the apoptotic substrates.
When 213P1F11 functions as a protease, it is used as a target for diagnostic, preventative and therapeutic purposes
Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.)
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
TABLE I: Tissues that Express 213P1F11 When Malignant
Bladder Prostate Breast
TABLE II: Amino Acid Abbreviations
Figure imgf000122_0001
TABLE III: Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See URL at the World Wide Web (.ikp.unibe.ch/manual/blosum62.html )
A C D E F G H I K L M N P Q R S T V W Y .
4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A
9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C
6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D
5 -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E
6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 •3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G
8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H
4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I
5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K
4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L
5 -2 -2 0 -1 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N
7 -1 -2 -1 -1 -2 -4 -3 P
5 1 0 -1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S
5 0 -2 -2 T 4 -3 -1 V 11 2 W 7 Y
TABLE IV HLA Class I/II Motifs/Supermotifs
TABLE IV (A): HLA Class I Supermotifs/Motifs
Figure imgf000124_0001
Bolded residues are preferred, italicized residues are less prefeπed: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.
TABLE IV (B): HLA Class H Supermotif
Figure imgf000124_0002
TABLE TV (C): HLA Class II Motifs
MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 1 8 9
DR4 prefeπed FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE
DR1 prefeπed MFLIVWY PAMQ VMAΎSPLIC M AVM deleterious C CH FD CWD GDE D
DR7 prefeπed MFLIVWY M W A TVMSACTPL M IV deleterious C G GRD N G
CO c DR3 MOTIFS 1° anchor 1 1° anchor 4 1° anchor 6 D motif a LIVMFY D O prefeπed motif b LFVMFAY DNQEST KRH prefeπed
DR MFLIVWY VMSTACPLI o
Supermotif
Italicized residues indicate less prefeπed or "tolerated" residues
TABLE IV (D): HLA Class I Supermotifs
POSITION: 1 C-terminus
SUPER- MOTIFS
Al 1° Anchor 1° Anchor TIL VMS FWY
A2 1° Anchor 1° Anchor LTVMATQ LIVMAT
A3 prefeπed 1° Anchor YFW YFW YFW P 1° Anchor O VSMATLI (4/5) (3/5) (4/5) (4/5) RK D deleterious DE (3/5); DE O P (5/5) (4/5)
' A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM
B7 preferred FWY (5/5) 1° Anchor FWY FWY 1 "Anchor o LIVM (3/5) (4/5) (3/5) VΪLFMWYA deleterious DE (3/5); DE G QN DE
P(5/5); (3/5) (4/5) (4/5) (4/5)
G(4/5);
A(3/5);
QN(3/5)
B27 1° Anchor 1 "Anchor RHK FYLWMIVA
B44 1° Anchor 1° Anchor ED FWYLIMVA
B58 1° Anchor 1° Anchor ATS FΨYLIVMA
B62 1° Anchor 1° Anchor QUVMP F YMIVLA
TABLE IV (E): HLA Class I Motifs
POSITION: 1 2 3 4 5 6 7 8 9 C-terminus or C-terminus
Al preferred GFY l°Anchor DEA YFW P DEQN YFW 1 "Anchor
9-mer W STM Y O deleterious DE RHKLIVMP A G A D O Al preferred GRHK ASTCLIVM 1 "Anchor GSTC ASTC LIVM DE 1 "Anchor
9-mer DBAS Y deleterious A RHKDEPY DE PQN RHK PG GP
FW o Al prefeπed YFW 1 "Anchor DEAQN A YFWQN PASTC GDE P 1 "Anchor
10-mer STM Y deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A
Ul Al prefeπed YFW STCLΓVM 1 "Anchor A YFW PG G YFW 1 "Anchor
10-mer DEAS Y deleterious RHK RHKDEPY P G PRHK QN
FW
A2.1 prefeπed YFW 1 "Anchor YFW STC YFW A P 1 "Anchor
9-mer LMIVQAT VLIMAT deleterious DEP DERKH RKH DERKH
TABLE IV (E): HLA Class I Motifs, continued
POSITION: 1 2 3 4 5 6 7 8 9 C-Terminus
A2.1 prefeπed AYFW 1 "Anchor LVEVI G G FYWL 1 "Anchor
10-mer IMIVQA VIM VLIMAT T deleterious DEP DE RKHA P RKH DERKH RKH
A3 prefeπed RHK 1 "Anchor YFW PRHKYFW A YFW P 1 "Anchor LMVISA K.Y HFA O TFCGD D deleterious DEP DE O Al l preferred A 1 "Anchor YFW YFW A YFW YFW P 1 "Anchor
VTLMIS YLRYH
AGNCD deleterious DEP A G O A24 prefeπed YFWRHK 1 "Anchor STC YFW YFW 1 "Anchor
9-mer YFWAt FLIW deleterious DEG DE G QNP DERH G AQN 6 K
A24 prefeπed 1 "Anchor P YFWP P 1 "Anchor
10-mer YFW FLIW deleterious GDE QN RHK DE A QN DEA
A3101 prefeπed RHK 1 "Anchor YFW P YFW YFW AP TAnchor MVTALIS RK deleterious DEP DE ADE DE DE DE
A3301 prefeπed 1 "Anchor YFW AYFW TAnchor
MVALF/ RK
ST deleterious GP DE
TABLE IV (E): HLA Class I Motifs, continued
POSITION C-Terminus
A6801 prefeπed YFWSTC 1° Anchor YFWLIV YFW 1 "Anchor
AVTMSLI M RK deleterious GP DEG RHK
B0702 prefeπed RHKFW 1° Anchor RHK RHK RHK RHK PA 1 "Anchor
CO c Y LMFWYAIV
CD deleterious DEQNP DEP DE DE GDE QN DE CO
B3501 prefeπed FWYLIV 1 "Anchor FWY FWY 1 "Anchor M P LMFWY/F m A co
I deleterious AGP G m m
B51 prefeπed LIVMFW 1 "Anchor FWY STC FWY G FWY 1 "Anchor
Y P UVFWYAM c deleterious AGPDER DE G DEQN GDE m HKSTC r
B5301 preferred LIVMFW 1 "Anchor FWY STC FWY LIVMFWY FWY 1 "Anchor
Y P IMFWYΛZ, V deleterious AGPQN RHKQN DE
B5401 preferred FWY 1 "Anchor FWYL LIVM ALIVM FWYAP 1 "Anchor
P IVM ATTVLMF
WY deleterious GPQNDE GDES RHKDE DE QNDGE DE TC
Italicized residues indicate less prefeπed or "tolerated" residues. The information in this Table is specific for 9-mers unless otherwise specified.
Table V v. l-Al-9mers: 213P1F11
Pos 123456789 Score
97 GEDGEMVKL 0.050 Portion of SEQ ID
86 LMAHGREGF 0.050 NO: 3;
142 TVGGDEIVM 0.050 each start
37 DALEHMFRQ 0.050 position is
80 SCAFWLMA 0.050 specified,
119 ALRAKPKVY 0.050 the length
11 KYD SGAR 0.050 of each peptide is 9
81 CAFW MAH 0.050 amino
108 LFEALNNKN 0.045 acids, the
133 RGEQRDPGE 0.045 end
74 SREDPVSCA 0.045 position for
LEEEKYDMS 0.045 each peptide is
28 AREGSEEDL 0.045 the start
169 YSTVEGYIA 0.030 position
117 CQA RAKPK 0.030 plus eight
79 VSCAFW M 0.030
14 MSGAR A I 0.030
36 LDALEHMFR 0.025
46 RFESTMKR 0.025
141 ETVGGDΞIV 0.025
212 RMAEAELVQ 0.025
40 EHMFRQLRF 0.025
13 DMSGARLA 0.025
153 KDSPQTIPT 0.025
161 TYTDA HVY 0.025
EEEKYDMSG 0.022
45 QLRFESTMK 0.020
121 RAKPKVYII 0.020
49 EST KRDPT 0.015
129 IQACRGEQR 0.015
184 GSCFIQT V 0.015
199 KGHILEL T 0.013
233 STLRKR YL 0.013
30 EGSEEDLDA 0.013
77 DPVSCAFW 0.013
106 EN FEALNN 0.013
160 PTYTDALHV 0.013
128 IIQACRGEQ 0.010
20 ALILCVTKA 0.010
158 TIPTYTDAL 0.010
18 RLALILCVT 0.010
164 DALHVYSTV 0.010
111 AL NKNCQA 0.010
193 DVFTKRKGH 0.010
85 VLMABGREG 0.010
205 LLTEVTRRM 0.010
203 LE LTEVTR 0.010
147 EIVMVIKDS 0.010
110 EALNNK CQ 0.010
Figure imgf000130_0001
23 LCVTKAREG 0.010
Figure imgf000131_0001
Figure imgf000131_0002
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000133_0002
Figure imgf000134_0001
Figure imgf000134_0002
Table VI v.l-Al-lOmers: 213P1F11
Pos 12345S7890 Score
118 QALRAKPKVY 0.050 Portion of
80 SCAFWLMAH 0.050 SEQ ID
106 ENLFEAL K 0.050 NO: 3; each start
95 KGEDGE VK 0.050 position is
58 AEQFQEELEK 0.050 specified,
45 QLRFEST KR 0.050 the length
108 LFEAL NKNC 0.045 of each
62 QEELEKFQQA 0.045 peptide is
RSLEEEKYDM 10 amino
0.030 acids, the
49 ESTMKRDPTA 0.030 end
179 RHDQKGSCFI 0.025 position for
11 KYD SGARLA 0.025 each
39 LEHMFRQLRF 0.025 peptide is
166 LHVYSTVEGY 0.025 the start position
33 EED DALEHM 0.025 plus nine
227 TNPEIQSTLR 0.025
139 PGETVGGDEI 0.022
145 GDEIV VIKD 0.022
142 TVGGDEIVMV 0.020
165 A HVYSTVEG 0.020
185 SCFIQTLVDV 0.020
81 CAFW MAHG 0.020
187 FIQTLVDVFT 0.020
158 TIPTYTDALH 0.020
64 E EKFQQAID 0.018
59 EQFQEE EKF 0.015
MSNPRSLEEE 0.015
154 DSPQTIPTYT 0.015
159 IPTYTDALHV 0.013
NPRS EEEKY 0.013
143 VGGDEIV VI 0.013
111 A NNKNCQAL 0.010
19 LALI CVTKA 0.010
70 QAIDSREDPV 0.010
167 HVYSTVEGYI 0.010
125 KVYIIQACRG 0.010
114 NKNCQALRA 0.010
22 ILCVTKAREG 0.010
149 V VIKDSPQT 0.010
204 EL TEVTRRM 0.010
110 EA NNKNCQA 0.010
13 D SGARLALI 0.010
23 LCVTKAREGS 0.010
175 YIAYRHDQKG 0.010
148 IVMVIKDSPQ 0.010
84 WLMAHGREG 0.010
230 EIQST RKRL 0.010
214 AEAELVQEGK 0.010
201 HILEL TEVT 0.010
Figure imgf000135_0001
193 DVFTKRKGHI 0.010
Figure imgf000136_0001
Figure imgf000136_0002
Figure imgf000137_0001
Table VI v.4-A 1 - 1 Omers: 213P1F11
Pos 1234567890 Score
29 GQTFRLKEEQ 0.002 Portion of
41 AFRGSSVHQK 0.001 SEQ ID
20 GLRDENGECG 0.001 NO: 9; each start
73 DLSISFRNSE 0.001 position is
51 LVNDPRETQE 0.001 specified,
46 SVHQKLVWDP 0.001 the length
47 VHQK VNDPR 0.001 of each
31 TFR KEEQGR 0.001 peptide is
NDPRETQEVF 10 amino
53 0.001 acids, the
49 QKL DPRET 0,001 en
GKCQEYDKSL 0.001 position for
56 RETQEVFGGG 0.001 each
EYDKSLSVQP 0.001 peptide is
54 DPRETQEVFG 0.001 the start position
39 GRAFRGSSVH 0.001 plus nine
42 FRGSSVHQKL 0.001
61 VFGGGVGD1V 0:001
QΞYDKSLSVQ 0.001
72 RDLSISFRNS 0.001
17 KRTGLRDENG 0.001
36 EEQGRAFRGS 0.001
79 RNSETSASΞE 0.000 GKCQEYDKS 0.000
38 QGRAFRGSSV 0.000
19 TGLRDENGEC 0.000
64 GGVGDIVGRD 0.000
28 CGQTFR KEE 0.000
37 EQGRAFRGSS 0.000
78 FRNSETSASE 0.000
59 QEVFGGGVGD 0.000
77 SFRNSETSAS 0.000
16 EKRTGLRDEN 0.000
32 FRLKEEQGRA 0.000
48 HQKLVNDPRE 0.000
YDKS SVQPE 0.000
15 PEKRTGLRDE 0.000
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000139_0002
Figure imgf000140_0001
Figure imgf000140_0002
Figure imgf000141_0001
Figure imgf000141_0002
Figure imgf000142_0001
[Table VII v.4-A2-9mers: 213P1F1 1
Pos 123456789 Score
63 GGGVGDIVG 0.000 Portion of
45 SSVHQKLVN 0.000 SEQ ID
24 ENGECGQTF 0.000 NO: 9; each start
81 SETSASEEE 0.000 position is
57 ETQEVFGGG 0.000 specified,
59 QEVFGGGVG 0.000 the length
49 QKLVNDPRE 0.000 of each
GKCQEYDKS 0.000 peptide is 9 amino
52 VNDPRΞTQE 0.000 acids, the
67 GDI GRDLS 0.000 end'
78 FRNSETSAS 0.000 position for
47 VHQKLVNDP 0.000 each
32 FRLKEEQGR 0.000 peptide is
25 NGECGQTFR 0.000 the start position
42 FRGSSVHQK 0.000 plus eight
38 QGRAFRGSS 0.000
17 KRTGLRDEN 0.000
21 LRDENGECG 0.000
71 GRDLSISFR 0.000
34 LKEEQGRAF 0.000
82 ETSASEEEK 0.000
80 NSETSASEE 0.000
MGKCQEYDK 0.000
DKSLSVQPE 0.000
YDKSLSVQP 0.000
27 ECGQTFRLK 0.000
54 DPRETQEVF 0.000
31 TFR KEEQG 0.000
22 RDENGECGQ 0.000
48 HQK VNDPR 0.000
14 QPEKRTG R 0.000
41 AFRGSSVHQ 0.000
15 PEKRTGLRD 0.000
55 PRETQEVFG 0.000
EYDKS SVQ 0.000
16 EKRTGLRDE 0.000
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000144_0002
Figure imgf000145_0001
Figure imgf000145_0002
Figure imgf000146_0001
Figure imgf000146_0002
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000148_0002
Figure imgf000149_0001
Figure imgf000149_0002
Figure imgf000150_0001
Figure imgf000150_0002
Figure imgf000151_0001
Figure imgf000151_0002
Figure imgf000152_0001
Table IX v.4-A3-9mers: 213P1F11
Pos 123456789 Score
34 LKEEQGRAF 0.000 Portion of
45 SSVHQKLVN 0.000 SEQ ID
53 NDPRETQEV 0.000 NO: 9; each start
77 SFRNSETSA 0.000 position is
67 GDIVGRD S 0.000 specified,
86 SEEEKYDMS 0.000 the length
84 SASEEEKYD 0.000 of each
72 RD SISFRN 0.000 peptide is 9 amino
GKCQEYDKS 0.000 acids, the
63 GGGVGDIVG 0,000 end
28 CGQTFRLKE 0.000 position for
37 EQGRAFRGS 0.000 each
80 NSETSASEE 0.000 peptide is
66 VGDIVGRDL 0.000 the start position
17 KRTGLRDEN 0.000 plus eight
36 EEQGRAFRG 0.000
52 VNDPRETQE 0.000
79 R SETSASE 0.000
47 VHQKLVNDP 0.000
81 SETSASEEE 0.000
23 DENGECGQT 0.000
78 FR SETSAS 0.000
38 QGRAFRGSS 0.000
21 LRDENGECG 0.000
19 TG RDENGE 0.000
49 QK VNDPRE 0.000
41 AFRGSSVHQ 0.000
59 QΞVFGGGVG 0.000
31 TFRLKEEQG 0.000
YDKSLSVQP 0.000
22 RDENGECGQ 0.000
DKSLSVQPE 0.000
15 PΞKRTGLRD 0.000
EYDKSLSVQ 0.000
55 PRETQEVFG 0.000
16 EKRTGLRDE 0.000
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000154_0002
Figure imgf000155_0001
Figure imgf000155_0002
Figure imgf000156_0001
Figure imgf000156_0002
Figure imgf000157_0001
Table X v.4-A3-10mers: 213P1F11
Pos 1234567890 Score
14 QPEKRTGLRD 0.000 Portion of
27 ECGQTFRLKE 0.000 SEQ ID
37 EQGRAFRGSS 0.000 NO: 9; each start
85 ASEEEKYDMS 0.000 position is
71 GRDLSISFRN 0.000 specified,
45 SSVHQKLVND 0.000 the length
38 QGRAFRGSSV 0.000 of each
64 GGVGDI GRD 0.000 peptide is
58 TQEVFGGGVG 10 amino
0.000 acids, the
80 NSETSASEEE 0.000 end
43 RGSSVHQKLV 0.000 position for
17 KRTG RDENG 0.000 each
32 FRLKEEQGRA 0.000 peptide is
19 TG RDENGEC the start
0.000 position
54 DPRETQEVFG 0.000 plus nine
59 QEVFGGGVGD 0.000
56 RETQEVFGGG 0.000
79 RNSETSASEE 0.000
62 FGGGVGDIVG 0.000
YDKSLSVQPE 0.000
83 TSASEEEKYD 0.000
77 SFRNSETSAS 0.000
66 VGDIVGRDLS 0.000 GKCQEYDKS 0.000
21 RDENGECGQ 0.000
22 RDENGECGQT 0.000
78 FRNSETSASE 0.000
55 PRETQEVFGG 0.000
72 RDLSISFRNS 0.000
36 EEQGRAFRGS 0.000
28 CGQTFRLKEE 0.000
11 LSVQPEKRTG 0.000
49 QKLVNDPRET 0.000
EYDKSLSVQP 0.000
16 EKRTGLRDEN 0.000
15 PEKRTGLRDE 0.000
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000159_0002
Figure imgf000160_0001
Figure imgf000160_0002
Figure imgf000161_0002
Figure imgf000161_0001
Figure imgf000162_0001
Table XI v.4-Al l-9mers: 213P1F11
Pos 123456789 Score
79 RNSETSASE 0.000 Portion of
63 GGGVGDIVG 0.000 SEQ ID
EYDKS SVQ 0.000 NO: 9; each start
24 ENGECGQTF 0.000 position is
84 SASEEEKYD 0.000 specified,
67 GDIVGRD S 0.000 the length
59 QEVFGGGVG 0.000 of each
17 KRTGLRDEN 0.000 peptide is 9 amino
45 SSVHQK V 0.000 acids, the
22 RDENGECGQ 0.000 end
GKCQEYDKS 0.000 position for
86 SEEEKYDMS 0.000 each
81 SETSASEEE 0.000 peptide is
36 EEQGRAFRG 0.000 the start position
52 VNDPRETQE 0.000 plus eight
28 CGQTFRLKE 0.000
76 ISFRNSETS 0.000
74 LSISFR SE 0.000
49 QK VNDPRE 0.000
19 TG RDENGE 0.000
47 VHQK VNDP 0.000
66 VGDIVGRDL 0.000
38 QGRAFRGSS 0.000
34 LKEEQGRAF 0.000
80 NSETSASEE 0.000
YDKS SVQP 0.000
78 FRNSETSAS 0.000
21 RDENGΞCG 0.000
37 EQGRAFRGS 0.000
23 DENGECGQT 0.000
11 SVQPEKRT 0.000
15 PEKRTGLRD 0.000
73 D SISFRNS 0.000
DKSLSVQPE 0.000
55 PRETQEVFG 0.000
16 EKRTGLRDE 0.000
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000164_0002
Figure imgf000165_0001
Figure imgf000165_0002
Figure imgf000166_0001
Figure imgf000166_0002
Figure imgf000167_0001
Figure imgf000167_0002
Figure imgf000168_0001
Table XII v.4-All-10mers: 213P1F11
Pos 1234567890 Score
56 RETQEVFGGG 0.000 Portion of
68 DIVGRDLSIS 0.000 SEQ ID
23 DENGECGQTF 0.000 NO: 9; each start
37 EQGRAFRGSS 0.000 position is
79 RNSETSASEE 0.000 specified,
44 GSSVHQK 0.000 the length
EYDKSLSVQP 0.000 of each
73 D SISFR SE 0.000 peptide is
QEYDKSLSVQ 10 amino
0.000 acids, the
86 SEEEKYDMSG 0.000 end
27 ECGQTFR KE 0.000 position for
64 GGVGDIVGRD 0.000 each
59 QEVFGGGVGD 0.000 peptide is
17 KRTG RDENG 0.000 the start position
22 RDENGECGQT 0.000 plus nine
54 DPRETQEVFG 0.000
62 FGGGVGDIVG 0.000
45 SSVHQKLV D 0.000
19 TGLRDENGEC 0.000
74 LSISFRNSET 0.000
80 NSETSASEEE 0.000
MGKCQEYDKS 0.000
21 RDENGECGQ 0.000
85 ASEEEKYD S 0.000
66 VGDIVGRDLS 0.000
YDKSLSVQPE 0.000
78 FRNSETSASE 0.000
83 TSASEEEKYD 0.000
28 CGQTFRLKEE 0.000
72 RDLSISFRNS 0.000
55 PRETQEVFGG 0.000
16 EKRTGLRDEN 0.000
11 SVQPEKRTG 0.000
49 QKLVNDPRET 0.000
36 EEQGRAFRGS 0.000
15 PEKRTGLRDE 0.000
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000170_0002
Figure imgf000171_0001
Figure imgf000171_0002
Table XIII v.2-A24-9mers: 213P1F11
Pos 123456789 Score
13 QDPLY PSE 0.002 Portion of
36 PTDMIRKAH 0.001 SEQ ID
VEGPTPFQD 0.001 NO: 5; each start
40 IRKAHA SR 0.001 position is
49 PWW CSRRG 0.001 specified,
15 PLYLPSEAP 0.001 the length of each peptide is 9 amino acids, the end position for each peptide is the start position plus eight
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000174_0002
Figure imgf000175_0001
Figure imgf000175_0002
Figure imgf000176_0001
Figure imgf000176_0002
Figure imgf000177_0001
Figure imgf000177_0002
Figure imgf000178_0001
Table XIV V.4-A24-1Omers: 213P1F11
Pos 1234567890 Score
11 SVQPEKRTG 0.015 Portion of
14 QPEKRTG RD 0.015 SEQ ID
32 FRLKEEQGRA 0.015 NO: 9; each start
45 SSVHQKLVND 0.015 position is
46 SVHQKLVNDP 0.014 specified,
71 GRDLSISFRN 0.014 the length
16 EKRTGLRDEN 0.013 of each
29 GQTFR KEEQ 0.013 peptide is RDLSISFR 10 amino
70 VG 0.012 acids, the
73 DLSISFRNSE 0.012 end
20 G RDENGECG 0.012 position for
54 DPRETQEVFG 0.012 each
24 ENGECGQTFR 0.012 peptide is
27 ECGQTFRLKE 0.011 the start position
30 QTFR KEEQG 0.010 plus nine
83 TSASEEEKYD 0.010
63 GGGVGDIVGR 0.010
48 HQKLVNDPRE 0.010
62 FGGGVGDI G 0.010
56 RETQEVFGGG 0.003
35 KEEQGRAFRG 0.003
47 VHQKLVNDPR 0.002
17 KRTG RDENG 0.002
34 LKEEQGRAFR 0.002
86 SEEΞKYDMSG 0.002
DKS SVQPΞK 0.002
59 QEVFGGGVGD 0.002
78 FR SETSASE 0.002
26 GECGQTFR K 0.001
YDKS SVQPE 0.001
21 LRDENGΞCGQ 0.001
QEYDKSLSVQ 0.001
SETSASEEEK 0.001
39 GRAFRGSSVH 0.001
55 PRETQEVFGG 0.000
15 PEKRTGLRDE 0.000
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000181_0002
Figure imgf000182_0001
Figure imgf000183_0001
Table XV v.4-B7-9mers: 213P1F1 1
Pos 123456789 Score
64 GGVGDIVGR- 0.010 Portion of
18 RTGLRDENG 0.010 SEQ ID
16 EKRTG RDE 0.010 NO: 9; each start
48 HQKLVNDPR 0.010 position is
29 GQTFRLKEE 0.010 specified,
79 R SETSASE 0.010 the length
19 TG RDENGE 0.010 of each
31 TFRLKEEQG 0.010 peptide is 9 amino
CQEYDKSLS 0.006 acids, the
67 GDIVGRD S 0.003 end
17 KRTGLRDEN 0.003 position for
52 VNDPRETQE 0.003 each
25 NGECGQTFR 0.003 peptide is
80 NSETSASEE 0.003 the start position
GKCQEYDKS 0.002 plus eight
72 RDLSISFR 0.002
78 FR SETSAS 0.002
59 QEVFGGGVG 0.001
47 VHQKLVNDP 0.001
42 FRGSSVHQK 0.001
81 SETSASEEE 0.001
49 QKLVNDPRE 0.001
36 EEQGRAFRG 0.001
DKSLSVQPE 0.001
32 FRLKEEQGR 0.001
YDKSLSVQP 0.001
56 RETQEVFGG 0.001
34 LKEEQGRAF 0.001
86 SEEEKYDMS 0.001
35 KEEQGRAFR 0.000
EYDKSLSVQ 0.000
21 LRDENGECG 0.000
71 GRDLSISFR 0.000
22 RDENGECGQ 0.000
15 PEKRTGLRD 0.000
55 PRETQEVFG 0.000
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000185_0002
Figure imgf000186_0001
Figure imgf000186_0002
Table XVI- V.2-B7-1 Omers: 213P1F11
Pos 1234567890 Score
VYSTVEGPTP 0.001 Portion of
16 P YLPSEAPP 0.001 SEQ ID
28 PLWNSQDTSP 0.001 NO: 5; each start
LHVYSTVEGP 0.001 position is
12 PFQDPLYLPS 0.000 specified,
50 PWWMCSRRGK 0.000 the length of each peptide is
10 amino acids, the end position for each peptide is the start position plus nine
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000189_0002
Figure imgf000190_0001
Figure imgf000190_0002
Figure imgf000191_0001
Figure imgf000191_0002
Figure imgf000192_0001
Figure imgf000192_0002
Figure imgf000193_0001
Table XVII v.4-B35-9mers
Pos 123456789 Score
64 GGVGDIVGR 0.015 Portion of
78 FRNSΞTSAS 0.015 SEQ ID
GKCQEYDKS 0.015 NO: 9; each start
46 SVHQK VND 0.010 position is
28 CGQTFR KE 0.010 specified,
29 GQTFRLKEE 0.010 the length
30 QTFRLKEEQ 0.010 of each
60 EVFGGGVGD 0.010 peptide is 9
82 amino
ETSASEEEK 0.010 acids, the
10 SLSVQPEKR 0.010 end
27 ECGQTFR K 0.010 position for
63 GGGVGDIVG 0.010 each
67 GDIVGRDLS 0.010 peptide is
86 SEEEKYDMS the start
0.006 position
16 EKRTGLRDE 0.003 plus eight
YDKSLSVQP 0.003
56 RETQEVFGG 0.003
41 AFRGSSVHQ 0.003
31 TFR KEEQG 0.003
52 VNDPRETQE 0.003
25 NGECGQTFR 0.003
32 FRLKEEQGR 0.002
36 EEQGRAFRG 0.001
81 SETSASEEE 0.001
DKSLSVQPE 0.001
49 QKLVNDPRE 0.001
47 VHQKLVNDP 0.001
42 FRGSSVHQK 0.001
59 QEVFGGGVG 0.001
22 RDENGECGQ 0.001
21 LRDENGECG 0.001
35 KEEQGRAFR 0.001
15 PEKRTGLRD 0.000
71 GRDLSISFR 0.000
EYDKSLSVQ 0.000
55 PRETQEVFG 0.000
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000195_0002
Figure imgf000196_0001
Figure imgf000196_0002
Figure imgf000197_0001
Figure imgf000197_0002
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000199_0002
Figure imgf000200_0001
Figure imgf000200_0002
TABLE XIXA: MHC Class I Analysis of 213P1F1 1 (9-mers). Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book "MHC Ligands and Peptide Motifs" by H.G.Rammensee, J.Bachmann and S.Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes.
Figure imgf000201_0001
Figure imgf000201_0002
Figure imgf000202_0001
Figure imgf000202_0002
Figure imgf000203_0001
213P1F11 v.l : HLA-Al nonamers
Pos 1 2 3 4 5 6 7 8 9 score
170 s T V E G Y I A Y 29 Portion of
232 Q s T R K R L Y 21 SEQ ID
154 D s P Q T I P T Y 19 NO: 3; each
D G Ξ M V K Ii E N start
99 18 position is
119 A L R A K P K V Y 18 specified,
162 Y T D A L H V Y S 18 the length
206 Ii T Ξ V T R R M A 18 of each
4 P R S L E E E K Y 17 peptide is 9
136 Q R D P G E T V G 17 amino
33 E E D L D A L Ξ H 16 acids, the
E D P V S C A end position
75 R F 16 for each
161 T Y T D A Ii H V Y 16 peptide is
167 H V Y S T V Ξ G Y 16 the start
38 A L Ξ H M F R Q L 15 position
233 S T Ii R K R L Y 15 plus eight
1 M S N P R S L E E 14
31 G S Ξ E D L D A L 14
32 S E Ξ D L D A L> Ξ 14
53 K R D P T A E Q F 14
74 S R Ξ D P V S C A 14
104 K Ii Ξ N L F E A L 14
219 V Q Ξ G K A R K T 14
228 N P Ξ I Q S T L R 14
6 S Ξ E E K Y D M 13
89 - H G R E G- F L K G 13
96 K G Ξ D G E M V K 13
97 G E D G E M V K L 13
108 L F Ξ A L, N N K N 13
Figure imgf000203_0002
Figure imgf000204_0001
Figure imgf000204_0002
Figure imgf000205_0001
213P1F11 v.l : HLA-A26 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
35 D L D A L E H M F 27 Portion of
170 • S T V E G Y I A Y 27 SEQ ID
167 H V Y S T V E G Y 26 NO: 3; each start
187 F I Q T L V D V F 25 position is
60 Q F Q E E L E K F 23 specified,
78 P V S C A F V V L 23 the length
154 D S P Q T I P T Y 23 of each
104 K L E N L F E A L 22 peptide is 9
208 E V T R R M A E A 22 amino
230 E I Q S T L R K R 22 acids, the end position
38 A L E H M F R Q L 21 for each
34 E D L D A L E H M 20 peptide is
56 P T A E Q F Q E E 20 the start
94 F L K G E D G E M 20 position
142 T V G G D E I V M 20 plus eight
147 E I V M V I K D S 20
158 T I P T Y T D A L 20
193 D V F T K R K G H 20
195 F T K R K G H I L 20
119 A L R A K P K V Y 19
204 E L L T E V T R R 19
205 L L T E V T R R M 19
233 S T L R K R L Y L 19
6 S L E E Ξ K Y D M 18
101 E M V K L E N L F 18
141 E T V G G D E I V 18
157 Q T I P T Y T D A 18
201 H I L E L L T E V 18
226 K T N P Ξ I Q S T 18
13 D M S G A R L A L 17
40 E H M F R Q L R F 17
64 E L E K F Q Q A I 17
97 G E D G E M V K L 17
107 N L F E A L N N K 17
171 T V E G Y I A Y R 17
197 K R K G H I L E L 16
217 E L V Q E G K A R 16
20 A L I L C V T K A 15
31 G S E E D L D A L 15
63 E E L E K F Q Q A 15
150 M V I K D S P Q T 15
161 T Y T D A L H V Y 15
53 K R D P T A E Q F 14
75 R E D P V S C A F 14
Figure imgf000205_0002
203
(?
Figure imgf000206_0001
Figure imgf000206_0002
Figure imgf000207_0001
213P1F11 v.l : HLA-A3 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
119 A L R A K P K V Y 30 Portion of
45 Q Ii R F E S T M K 27 SEQ ID
218 L V Q E G K A R K 26 NO: 3; each
125 K V Y I I Q A C R 25 start position is
175 Y I A Y R H D Q K 24 specified,
19 L A L I L C V T K 21 the length
96 K G E D G E M V K 21 of each'
107 N L F E A L N N K 21 peptide is 9
18 R L A L I L C V T 20 amino
167 H V Y S T V E G Y 20 acids, the
191 L V D V F T K R K 20 end position for each
88 A H G R E G F L 19 peptide is
171 T V E G Y I A Y R 19 the start
20 A II I L C V T K A 18 position
78 P V S C A F V V L 18 plus eight
83 F V V L M A H G R 18
111 A L N N K N C Q A 18
165 A Ii H V Y S T V Ξ 18
189 Q T L V D V F T K 18
208 E V T R R M A E A 18
217 E Ii V Q E G K A R 18
35 D L D A L E H M F 17
117 C Q A L R A K P K 17
190 T L V D V F T K R 17
202 I L E L L T E V T 17
204 E L L T Ξ V T R R 17
21 L I L C. V T K A R 16
38 A L E H M F R Q L 16
44 R Q L R F E S T M 16
53 K R D P T A E Q F 16
142 T V G G D E I V M 16
145 G D E I V M V I K 16
Figure imgf000207_0002
Figure imgf000208_0001
Figure imgf000208_0002
213P1F11 v.l : HLA-B* Ϊ702 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
13 D M S G A R L A L 18 Portion of
77 D P V S C A F V V 17 SEQ ID
123 K P K V Y I I Q A 17 NO: 3; each
155 S P Q T I P T Y T 17 start position is
78 P V S C A F V V L 16 specified,
97 G Ξ D G E M V K L 15 the length
197 K R K G H I L E L 15 of each
11 K Y D M S G A R L 14 peptide is 9
28 A R Ξ G s E E D L 14 amino
231 I Q S T L R K R L 14 acids, the
15 s G A R L A L I L 13 end position for each
38 A I> E H M F R Q L 13 peptide is
87 M A H G R E G F L 13 the start
104 K L E N L F E A L 13 position
120 L R A K P K V Y I 13 plus eight
135 E Q R D P G E T V 13
183 K G S C F I Q T L 13
210 T R R M A E A E L 13
233 S T L R K R L Y Ii 13
3 N P R S L E E E K 12
112 L N N K N C Q A L 12
138 D P G E T V G G D 12
153 K D S P Q T I P T 12
158 T I P T Y T D A L 12
159 I P T Y T D A L H 12
198 R K G H I L E L L 12
17 A R L A L I L C V
30 E G S E E D L D A
31 G S E E D L D A L
55 D P T A E Q F Q Ξ
100 G Ξ M V K L E N L
195 F τ K R K G H I L
223 K A R K T N P E I
228 N P Ξ I Q S T L R
20 A L I L C V T K A 10
40 E H M F R Q L R F 10
Figure imgf000209_0001
57 T A E Q F Q E E It 10
71 A I D S R E D P V 10
74 S R E D P V S C A 10
75 R Ξ D P V S c A F 10
79 V S C A F V V L M 10
80 S C A F V V L M A 10
142 T V G G D E I V M 10
188 I Q T L V D V F T 10
227 T N P Ξ I Q S T L 10
14 M S G A R L A L I 9
18 R L A L I L C V T 9
49 E S T M K R D P T 9
50 S T M K R D P T A 9
53 K R D P T A E Q F 9
64 E L E K F Q Q A I 9
76 E D P V S c A F V 9
121 R A K P K V Y I I 9
141 E T V G G D E I V 9
Figure imgf000210_0001
Figure imgf000210_0002
Figure imgf000211_0002
213P1F11 v.l : HLA-B*I 38 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
121 R A K P K V Y I I 31 Portion of
195 F T K R K G H I L 31 SEQ ID
119 A L R A K P K V Y 23 NO: 3; each start
87 M A H G R E G F L 22 position is
100 G E M V K L E N L 22 specified,
197 K R K G H I L E L 22 the length
233 S T L R K R L Y L 22 of each
151 V I K D S P Q T I 21 peptide is 9
221 E G K A R K T N P 20 amino
234 T L R K R L Y L Q 20 acids, the
25 V T K A R E G S Ξ 19 end position
F L K G Ξ for each
94 D G E M 18 peptide is
123 K P K V Y I I Q A 18 the start
223 K A R K T N P E I 18 position
104 K L Ξ N L F E A L 17 plus eight
111 A L N N K N C Q A 17
210 T R R M A E A E L 17
38 A L Ξ H M F R Q L 16
40 E H M F R Q L R F 16
179 R H D Q K G S C F 16
57 T A Ξ Q F Q E E L 15
64 E L Ξ K F Q Q A I 15
158 T I P T Y T D A L 15
194 V F T K R K G H I 15
14 M S G A R L A L I 14
31 G s Ξ E D L D A L 14
63 E E L E K F Q Q A 14
3 N P R S Ii E E E K 13
9 E E K Y D M S G A 13
27 K A R E G S E E D 13
92 E G F L K G E D G 13
175 Y I A Y R H D Q K 13
7 L E Ξ E K Y D M s 12
15 S G A R L A L I L 12
16 G A R L A L I L C 12
35 D L D A Ii E H M F 12
45 Q L R F Ξ S T M K 12
49 E s T M K R D P T 12
71 A I D S R E D P V 12
97 G E D G Ξ M V K L 12
102 M V K L Ξ N L F Ξ 12
187 F I Q T L V D V F 12
227 T N P E I Q S T II 12
231 I Q s T L R K R L 12
13 D M s G A R L A L 11
23 L C V T K A R E G 11
Figure imgf000211_0001
51 T M K R D P T A Ξ 11
Figure imgf000212_0001
Figure imgf000212_0002
213P1F11 v.l : HLA-B* 1510 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
40 E H M F R Q L R F 19 Portion of
179 R H D Q K G S C F 17 SEQ ID
231 I Q S T L R K R L 16 NO: 3; each
78 P V S C A F V V L 15 start position is
97 G Ξ D σ E M V K L 15 specified,
13 D M S G A R L A L 14 the length
31 G S E E D L D A L 14 of each
57 T A E Q F Q E E L 14 peptide is 9
38 A L E H M F R Q L 13 amino
112 L N N K N C Q A L 13 acids, the
166 L H V Y S T V E G 13 end position for each
183 K G S C F I Q T L 13 peptide is
197 K R K G H I L E L 13 the start
227 T N P E I Q S T L 13 position
11 K Y D M S G A R L 12 plus eight
28 A R E G S E E D L 12
100 G E M V K L E N L 12
104 K L E N L F E A L 12
158 T I P T Y T D A Ii 12
200 G H I L E L L T Ξ 12
210 T R R M A E A E L 12
15 S G A R L A L I L 11
87 M A H G R E G F L 11
205 L L T Ξ V T R R M 11
233 S T L R K R L Y L 11
88 A H G R E G F L K 10
142 T V G G D E I V M 10
195 F T K R K G H I L 10
198 R K G H I L E L L 10
6 S L E E E K Y D M 9
75 R E D P V S C A F 9
79 V S C A F V V L M 9
86 L M A H G R E G F 9
187 F I Q T L V D V F 9
34 E D L D A L E H M 8
53 K R D P T A Ξ Q F 8
94 F L K G E D G E M 8
101 E M V K L E N L F 8
44 R Q L R F E S T H 7
60 Q F Q E E L E K F 7
22 I L C V T K A R E 6
35 D L D A L E H M F 6
72 I D S R E D P V S 6
120 L R A K P K V Y I 6
96 K G E D G E M V K 5
136 Q R D P G E T V G 5
144 G G D E I V M V I 5
145 G D E I V M V I K 5
202 I L E L L T E V T 5
206 L T E V T R R M A 5
Figure imgf000213_0001
218 L V Q E G K A R K 5
12 Y D M S G A R L A 4
37 D A L E H M F R Q 4
47 R F E S T M K R D 4
Figure imgf000214_0001
Figure imgf000214_0002
213P1F11 v.l : HLA-B*2705 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
197 K R K G H I L E L 29 Portion of
46 L R F E S T M K R 28 SEQ ID
53 K R D P T A E Q F 25 NO: 3; each
28 A R E G s E E D L 24 start position is
4 P R S L E E E K Y 22 specified,
210 T R R M A E A E L 22 the length
120 L R A K P K V Y I 21 of each
97 G Ξ D G E M V K L 19 peptide is 9
17 A R L A L I L C V 18 amino
59 E Q F Q E E L E K 18 acids, the
67 K F end position
Q Q A I D S R 18 for each
107 N L F E A L N N K 18 peptide is
125 K V Y I I Q A C R 18 the start
179 R H D Q K G S C F 18 position
204 E L L T E V T R R 18 plus eight
229 P Ξ I Q S T L R K 18
75 - R Ξ D P V S C A F 17
100 G Ξ M V K L E N L 17
218 L V Q E G K A R K 17
11 K Y D M S G A R L 16
44 R Q L R F E S T 16
90 G R E G F L K G Ξ 16
96 K G E D G Ξ M V K 16
136 Q R D P G E T V G 16
171 T V E G Y I A Y R 16
183 K G S C F I Q T L 16
190 T L V D V F T K R 16
198 R K G H I L E L L 16
211 R R M A E A E L V 16
227 T N P Ξ I Q S T L 16
19 L A L I L C V T K 15
31 G S E E D L D A L 15
40 E H M F R Q L R F 15
57 T A E Q F Q Ξ E L 15
60 Q F Q E E L E K F 15
101 E M V K L Ξ N L F 15
115 K N c Q A L R A K 15
145 G D E I V M V I K 15
154 D S P Q T I P T Y 15
203 L Ξ L L T E V T R 15
45 Q L R F E S T M K 14
81 C A F V V L M A H 14
121 R A K P K V Y I I 14
144 G G D E I V M V I 14
189 Q T L V D V F T K 14
215 E A Ξ L V Q E G K 14
217 E L V Q E G K A R 14
231 I Q S T L R K R L 14
233 S T L R K R L Y L 14
Figure imgf000215_0001
10 E K Y D M S G A R 13
13 D M S G A R L A L 13
15 S G A R L A L I L 13
21 L I L C V T K A R 13
36 L D A L E H M F R 13
Figure imgf000216_0001
Figure imgf000216_0002
Figure imgf000217_0002
213P1F11 v.l : HLA-B* 2709 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
53 K R D P T A Ξ Q F 23 Portion of
197 K R K G H I L E L 23 SEQ ID
211 R R M A E A E L V 23 NO- 3, each
17 A R L A L I L C V 22 start position is
28 A R E G S E E D L 21 specified,
210 T R R M A E A E L 20 the length
120 L R A K P K V Y I 19 of each
121 R A K P K V Y I I 15 peptide is 9
198 R K G H I L E L L 15 amino
31 G S E E D L D A L 14 acids, the
44 R Q L R F E S T M 14 end position for each
97 G E D G E M V K L 14 peptide is
100 G E M V K L E N L 14 the start
11 K Y D M S G A R L 13 position
75 R E D P V S C A F 13 plus eight
90 G R E G F L K G E 13
144 G G D E I V M V I 13
160 P T Y T D A L H V 13
233 S T L R K R L Y L 13
15 S G A R L A L I L 12
38 A L E H M F R Q L 12
46 L R F E S T M K R 12
104 K L E N L F E A L 12
140 G E T V G G D Ξ I 12
179 R H D Q K G S C F 12
183 K G S C F I Q T L 12
184 G S C F I Q T L V 12
231 I Q S T L R K R L 12
13 D M S G A R L A L 11
74 S R E D P V S C A 11
77 D P V S C A F V V 11
78 P V S C A F V V L 11
118 Q A L R A K P K V 11
Figure imgf000217_0001
136 Q R D P G E T V G 11
Figure imgf000218_0001
Figure imgf000218_0002
Figure imgf000219_0002
213P1F11 v.l : HLA-B*' 4402 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
75 R Ξ D P V S C A F 26 Portion of
97 G Ξ D G E M V K L 24 SEQ ID
100 G Ξ M V K L E N L 22 NO: 3; each
140 σ Ξ T V G G D E I 18 start position is
53 K R D P T A E Q F 17 specified,
119 A L R A K P K V Y 17 the length
13 D M S G A R L A L 16 of each
33 E E D L D A L E H 16 peptide is 9
38 A L E H M F R Q L 16 ammo
146 D Ξ I V M V I K D 16 acids, the
183 K G s C F I Q T L 16 end position for each
197 K R K G H I L E L 16 peptide is
40 E H M F R Q L R F 15 the start
63 E Ξ L E K F Q Q A 15 position
104 K L E N L F E A L 15 plus eight
154 D S P Q T I P T Y 15
158 T I P T Y T D A L 15
207 T Ξ V T R R M A Ξ 15
216 A Ξ L V Q E G K A 15
229 P Ξ I Q S T L R K 15
231 I Q s T L R K R L 15
233 s T L R K R L Y L 15
15 s G A R L A L I L 14
28 A R E G S E E D L 14
58 A Ξ Q F Q E E L Ξ 14
78 P V s C A F V V L 14
101 E M V K L E N L F 14
109 F Ξ A L N N K N C 14
161 T Y T D A L H V Y 14
170 S T V E G Y I A Y 14
203 L Ξ L L T E V T R 14
214 A Ξ A E L V Q E G 14
220 Q Ξ G K A R K T N 14
232 Q S T L R K R L Y 14
4 P R s L E E E K Y 13
31 G S E E D L D A L 13
32 S Ξ E D L D A L Ξ 13
48 F Ξ S T M K R D P 13
112 L N N K N C Q A L 13
187 F I Q T L V D V F 13
Figure imgf000219_0001
198 R K G H I L E L L 13
Figure imgf000220_0001
Figure imgf000220_0002
Figure imgf000221_0002
213P1F11 v.l: HLA-B*; 5101 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
164 D A L H V Y S T V 28 Portion of
77 D P V S C A F V V 26 SEQ ID
121 R A K P K V Y I I 23 NO: 3; each
G G D E I V M V I start
144 23 position is
223 K A R K T N P E I 23 specified,
118 Q A L R A K P K V 22 the length
37 D A L E H M F R Q 19 of each
138 D P G E T V G G D 19 peptide is 9
87 M A H G R E G F L 18 ammo
143 V G G D E I V M V 18 acids, the
176 I A Y R H D Q K G 18 end position for each
19 L A L I L C V T K 17 peptide is
57 T A E Q F Q E Ξ L 17 the start
151 V I K D S P Q T I 16 position
160 P T Y T D A L H V 16 plus eight
55 D P T A E Q F Q E 15
183 K G S C F I Q T L 15
15 S G A R L A L I L 14
81 C A F V V L M A H 14
120 L R A K P K V Y I 14
168 V Y S T V E G Y I 14
201 H I L E L L T E V 14
213 M A E A E L V Q Ξ 14
14 M S G A R L A L I 13
17 A R L A L I L C V 13
Figure imgf000221_0001
99 D G E M V K L E N 13
Figure imgf000222_0001
Figure imgf000222_0002
Figure imgf000223_0002
Figure imgf000223_0001
Figure imgf000223_0003
213P1F11 v.2: HLA-Al nonamers
Pos 1 2 3 4 5 6 7 8 9 score
9 P T P F Q D P L Y 25 Portion of
12 F Q D P L Y L P S 21 SEQ ID
36 P T D M I R K A H 17 NO: 5; each start
19 P S E A P P N P P 15 position is
5 T V Ξ G P T P F Q 12 specified,
31 S Q D T s P T D M 12 the length
4 S T V E G P T P F 10 of each
33 D T S P T D M I R 10 peptide is 9
20 S Ξ A P P N P P L 8 amino
22 A P P N P P L W N 8 acids, the
34 T S P T D M I R K 8 end position
L S R P C S for each
46 W W M 8 peptide is
3 Y S T V E G P T P 7 the start
54 S R R G K D I S 7 position
10 T P F Q D P L Y L 6 plus eight
21 E A P P N P P L W 6
40 I R K A H A L S R 6
53 C S R R G K D I S 6
6 V Ξ G P T P F Q D 5
23 P P N P P L W N S 5
7 E G P T P F Q D P 4
8 G P T P F Q D P L 4
16 L Y L P S E A P P 4
24 P N P P L N S Q 4
30 N S Q D T s P T D 4
35 S P T D M I R K A 4
45 A L S R P M C 4
2 V Y s T V E G P T 3
11 P F Q D P L Y L P 3
17 Y L P S E A P P N 3
51 W M C S R R G K D 3
15 P L Y L P S E A P 2
28 L N S Q D T S P 2
32 Q D T S P T D M I 2
39 M I R K A H A L S 2
43 A H A L S R P w 2
47 S R P W W M C S R 2
Figure imgf000224_0001
1 H V Y s T V E G P
13 Q D P L Y L P S Ξ
25 N P P L w N S Q D
27 P L N s Q D T S
50 W M C s R R G K
52 M C s R R G K D I
Figure imgf000225_0001
213P1F11 v.2: HLA-B*I )8 nonamers
213P1F11 v.2: HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score
Pos 1 2 3 4 5 6 7 8 9 score 38 D M I R K A H A L 21 Portion of
8 G P T P F Q D P L 22 Portion of 54 S R R G K D I S W 18 SEQ ID
10 T P F Q D P L Y L 22 SEQ ID 8 G P T P F Q D P L 17 NO: 5; each
20 s E A P P N P P L 17 NO: 5; each 10 T P F start
Q D P L Y L 16
N position is
22 A P P P P L N 17 start 56 R G K D I S W N F 16 position is specified,
14 D P L Y L P S E A 16 specified, 52 M C S R R G K D I 15 the length
26 P P L W N S Q D T 16 the length 20 S E A P P N P P L 13 of each
35 S P T D M I R K A 16 of each 44 H A L S R P w W 13 peptide is 9
23 P P N P P L W N S 14 peptide is 9 40 I R K A H A L S R 11 amino
18 L P S E A P P N P 13 amino 4 s T V E G P T P F 10 acids, the
38 D M I R K A H A L 11 acids, the 39 M I R K A H A L s 10 end position
P W W M c S R R for each
48 R 11 end position 35 for each s P T D M I R K A 9 peptide is
25 N P P L W N S Q D 10 D peptide is 14 P L Y L P S E A 8 the start
29 W N S Q D T S P T 10 the start 15 P L Y L P s E A P 8 position
32 Q D T S P T D M I 10 position 18 L P S E A P P N P 8 plus eight
2 V Y S T V E G P T 9 plus eight 25 N P P L W N S Q D 8
52 M C S R R G K D I 9 37 T D I R K A H A 8
4 S T V E G P T P F 8 51 W M C s R R G K D 8
5 T V E G P T P F Q 8 17 Y L P s Ξ A P P N 6
31 s Q D T S P T D M 8 21 E A P P N P P L 6
37 T D M I R K A H A 8 22 A P P N P P L W N .6
45 A L S R P W W M C 7 23 P P N P P L N S 6
56 R G K D I S w N F 7 26 P P L W N S Q D T 6
44 H A L S R P w W M 6 27 P L W N S Q D T s 6
7 Ξ G P T P F Q D P 5 32 Q D T S P T D M I 6
12 F Q D P L Y L P S 5 45 A L S R P w W M c 6
19 P S E A P P N P P 5 46 L S R P w M C s 6
43 A H A L S R P w W 5 48 R P W W c S R R 6
15 P L Y L P S E A P 4 53 C S R R G K D I S 6
39 M I R K A H A L S 4 42 K A H A L s R P 4
40 I R K A H A L S R 4 1 H V Y S T V E G P 2
54 s R R G K D I s W 4 7 E G P T P F Q D P 2
11 P F Q D P L Y L P 3 31 S Q D T S P T D M 2
16 L Y L P S Ξ A P P 3 36 P T D M I R K A H 2
21 E A P P N P P L w 3 47 S R P W M C S R 2
33 D T S P T D M I R 3 2 V Y S T V E G P T
36 P T D M I R K A H 3 3 Y S T V Ξ G P T P
42 K A H A L S R P W 3 6 V Ξ G P T P F Q D
46 L S R P W W M c S 3 12 F Q D P L Y L P S
55 R R G K D I S N 3 13 Q D P L Y L P s Ξ
3 Y S T V E G P T P 2 43 A H A L S R P
6 V Ξ G P T P F Q D 2 55 R R G K D I S w N
17 Y L P S E A P P N 2
41 R K A H A L S R P 2
50 W M C S R R G K 2
53 C S R R G K D I S 2
1 H V Y S T V E G P
9 P T P F Q D P L Y
13 Q D P L Y L P S E
24 P N P P L W N S Q
28 L N S Q D T s P
30 N S Q D T S P T D
51 C S R R G K D 213P1F11 v.2: HLA-B* [510 nonamers
Pos 1 2 3 4 5 6 7 8 9 score 213P1F11 v.2: HLA-B* 2705 nonamers
20 S E A P P N P P L 15 Portion of Pos 1 2 3 4 5 6 7 8 9 score
8 G P T P F Q D P L 13 SEQ ID 40 I R K A H A L S R 24 Portion of
10 T P F Q D P L Y L 13 NO: 5; each 47 s R P W W M C S R 21 SEQ ID
43 A H A L S R P W W 13 start 55 R R G K D I S position is w N 20 NO: 5; each
38 D I R K A H A L 11 56 R G K D I S W N F 19 start specified, position is
4 S T V E G P T P F 9 the length 48 R P W W M c S R R 17 specified,
31 S Q D T S P T D M 7 of each 4 s T V E G P T P F 16 the length
44 H A L S R P W M 7 peptide is 9 8 G P T P F Q D P L 16 of each
56 R G K D I S N F 7 ammo 10 T P F Q D P L Y L 16 peptide is 9
21 E A P P N P P L 6 acids, the 54 s R R G K D I S 16 amino
5 T V E G P T P F Q 5 end position 20 s E A P P N P P L 14 acids, the
Y S T V E G P T P for each
3 4 peptide is 38 D M I R K A H A L 14 end position
L P S E A for each
18 P P N P 4 the start 33 D T S P T D M I R 13 peptide is
34 T S P T D M I R K 4 position 34 T S P T D M I R K 13 the start
35 S P T D M I R K A 4 plus eight 31 s Q D T S P T D M 11 position
49 P W M C s R R G 4 44 H A L S R P w W M 11 plus eight
6 V E G P T P F Q D 3 50 W W M c S R R G K 11
19 P S E A P P N P P 3 9 P T P F Q D P L Y 10
23 P P N P P L W N S 3 36 P T D M I R K A H 9
30 N S Q D T S P T D 3 41 R K A H A L S R P 8
33 D T S P T D M I R 3 32 Q D T S P T D M I 7
41 R K A H A L S R P 3 23 P P N P P L W N s 6
50 W M C S R R G K 3 52 M c s R R G K D I 6
7 E G P T P F Q D P 2 14 D P L Y L P S E A 5
11 P F Q D P L Y L P 2 15 P L Y L P S E A P 5
12 F Q D P L Y L P S 2 16 L Y L P S E A P P 5
14 D P L Y L P S E A 2 35 S P T D M I R K A 5
15 P Y L P S E A P 2 5 T V E G P T P F Q 4
16 L Y L P S E A P P 2 13 Q D P L Y L P S Ξ 4
17 Y L P S E A P P N 2 18 L P S E A P P N P 4
22 A P P N P P L W N 2 24 P N P P L W N S Q 4
24 P N P P L W N S Q 2 25 N P P L W N S Q D 4
29 W N S Q D T S P T 2 27 P L W N S Q D T S 4
36 P T D M I R K A H 2 28 L W N S Q D T s P 4
37 T D M I R K A H A 2 42 K A H A L S R P W 4
40 I R K A H A L S R 2 43 A H A L S R P w W 4
42 K A H A L S R P 2 1 H V Y S T V E G P 3
45 A L S R P W W M C 2 3 Y S T V E G P T P 3
46 L S R P W W M C S 2 11 P F Q D P L Y L P 3
48 R P W W M C S R R 2 12 F Q D P L Y L P s 3
53 C S R R G K D I S 2 17 Y L P S E A P P N 3
55 R R G K D I S W N 2 22 A P P N P P L W N 3
1 H V Y S T V E G P 26 P P L W N S Q D T 3
2 V Y S T V E G P T 29 w N S Q D T S P T 3
9 P T P F Q D P L Y 30 N S Q D T S P T D 3
13 Q D P L Y L P S Ξ 39 M I R K A H A L S 3
26 P P L W N S Q D T 45 A L S R P W M C 3
27 P L W N S Q D T S 49 P w M C S R R G 3
32 Q D T S P T D M I 53 C S R R G K D I S 3
39 M I R K A H A L s 6 V E G P T P F Q D 2
47 S R P W W M C S R 19 P S E A P P N P P 2
51 W C S R R G K D 21 E A P P N P P L w 2
52 M C S R R G K D I 37 T D M I R K A H A 2
54 S R R G K D I S w 46 L S R P W w M C S 2 213P1F11 V.2: HLA-B*4402 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
20 S E A P P N P P L 24 Portion of
Figure imgf000228_0001
21 E A P P N P P L W 19. SEQ ID
38 D ' M I R K A H A L 18 NO: 5; each
213P1F11 v.2: HLA-B*" 2709 nonamers 6 V E G P T P F Q D 15 start position is
Pos 1 2 3 4 5 6 7 8 9 score 43 A H A L S R P W W 15 specified,
8 G P T P F Q D P L 15 Portion of 10 T P F Q D P L Y L 14 the length
55 R R G K D I S W N 15 SEQ ID 8 G P T P F Q D P L 13 of each
10 T P F Q D P L Y L 14 NO: 5; each 52 M C S R R G K D I 13 peptide is 9
56 R G K D I s W N F 14 start 54 S R R G K D I S W 13 amino position is
40 I R K A H A L S R 13 specified, 4 S T V E G P T P F 12 acids, the
20 s E A P P N P P L 12 the length 9 P T P F Q D P L Y 12 end position for each
38 D M I R K A H A L 12 of each 42 K A H A L S R P W 12 peptide is
44 H A L S R P W W M 11 peptide is 9 56 R G K D I S N F 12 the start
32 Q D T S P T D M I 10 amino 32 Q D T S P T D M I 9 position
47 S R P W W M C S R 10 acids, the 22 A P P N P P L W N 7 plus eight
54 S R R G K D I S W 10 end position 35 S P T D M I R K A 7 for each
4 S T V E G P T P F 9 36 P T D M I R K A H 7 peptide is
31 s Q D T S P T D M 8 the start 7 E G P T P F Q D P 5
52 M C S R R G K D I 8 position 12 F Q D P L Y L P S 5
41 R K A H A L s R P 5 plus eight 24 P N P P L W N S Q 5
48 R P W M C s R R 5 45 A L S R P W M C 5
1 H V Y S T V E G P 4 16 L Y L P S E A P P 4
16 L Y L P S E A P P 3 23 P P N P P L W N S 4
17 Y L P s E A P P N 3 25 N P P L W N S Q D 4
3 Y S T V E G P T P 2 31 S Q D T S P T D M 4
6 V E G P T P F Q D 2 33 D T S P T D M I R 4
12 F Q D P L Y L P S 2 11 P F Q D P L Y L P 3
14 D P L Y L P s E A 2 13 Q D P L Y L P S Ξ 3
15 P L Y L P s E A P 2 15 P Y L P S E A P 3
18 L P S E A P P N P 2 30 N S Q D T S P T D 3
21 E A P P N P P L W 2 34 T S P T D M I R K 3
22 A P P N P P L W N 2 44 H A L S R P W W M 3
23 P P N P P L W N S 2 46 L S R P W M C S 3 '
26 P P L W N s Q D T 2 50 W M C S R R G K 3
34 T S P T D M I R K 2 51 W M C S R R G K D 3
42 K A H A L S R P W 2 55 R R G K D I S W N 3
43 A H A L S R P W W 2 2 V Y S T V E G P T 2
5 T V E G P T P F Q 5 T V E G P T P F Q 2
9 P T P F Q D P L Y 14 D P L Y L P S Ξ A 2
11 P F Q D P L Y L P 17 Y L P S E A P P N 2
13 Q D P L Y L P S E 18 L P S E A P P N P 2
24 P N P P L W N S Q 19 P S -E A P P N P P 2
25 N P P L w N S Q D 26 P P W N S Q D T 2
28 L W N S Q D T S P 27 P L W N S Q D T S 2
29 W N S Q D T s P T 29 W N S Q D T S P T 2
30 N S Q D T S P T D 37 T D M I R K A H A 2
33 D T S P T D M I R 40 I R K A H A L S R 2
35 S P T D M I R K A 47 S R P W W M C S R 2
36 P T D M I R K A H 1 H V Y S T V E G P
45 A L S R P W W M C 3 Y S T V E G P T P
46 L S R P w w M C S 39 M I R K A H A L S
49 P W W M c S R R G 41 R K A H A L S R P
51 W M C S R R G K D 49 P W M C S R R G
Figure imgf000229_0002
Figure imgf000229_0001
213P1F11 v.3: HLA-A26 nonamers 213P1F11 v.3: HLA-B* D702 nonamers
Pos 1 2 3 4 5 6 7 8 9 score Pos 1 2 3 4 5 6 7 8 9 score
9 A T L P S P F P Y 23 Portion of 10 T L P S P F P Y L 15 Portion of
10 T L P S P F P Y L 23 SEQ ID 12 P S P F P Y L S L 15 SEQ ID
1 Y I I Q A C R G A 14 NO: 7; each 3 I Q A C R G A T L 14 NO: 7; each start
12 P S P F P Y L S L 14 start
11 L P S P F P Y L S 13 position is position is
7 R G A T L P S P F 13 specified, 2 I I Q A C R G A T 10 specified,
2 I I Q A C R G A T 11 the length 5 A C R G A T L P s 10 the length
3 I Q A C R G A T L 10 of each 7 R G A T L P S P F 9 of each
6 c R G A T L P S P 6 peptide is 9 1 Y I I Q A C R G A 6 peptide is 9
5 A C R G A T L P S 2 amino 8 G A T L P S P F P 5 amino acids, the 9 A T L P S P F P Y 4 acids, the end position 6 C R G A T L P S P 3 end position for each for each peptide is 4 Q A C R G A T L P 1 peptide is the start the start position position plus eight plus eight
11 L P S P F P Y L S 2
213P1F11 v.3: HLA-B*08 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
3 I Q A C R G A T L 19 Portion of
10 T L P S P F P Y L 16 SEQ ID
12 . P S P F P Y L S L 10 NO: 7; each
G A T L P S P F P 1 2 I I Q A C R G A T start
8 6 position is
5 A C R G A T L P S 6 specified,
213P1F11 v3: HLA-A3 nonamers 7 R G A T L P S P F 6 the length
Pos 1 2 3 4 5 6 7 8 9 score 8 G A T L P S P F P 6 of each
2 I I Q A C R G A T 15 Portion of 11 L P S P F P Y L S 6 peptide is 9
3 I Q A C R G A T L 15 SEQ ID 1 Y I I Q A C R G A 4 amino
9 A T L P S P F P Y 15 NO: 7; each 4 Q A C R G A T L P 4 acids, the start end position
7 R G A T L P S P F 13 position is for each
1 Y I I Q A C R G A 11 specified, peptide is
4 Q A C R G A T L P 11 the length the start
5 A C R G A T L P S 11 of each position.
Y L 10 peptide is 9 plus eight
10 T L P S P F P
12 P S P F P Y L s L 5 amino
6 C R G A T L P s P 4 acids, the end position for each peptide is the start
1 1 L P S P F P Y L S 4 position
8 G A T L P S P F P 1 plus eight
Figure imgf000230_0001
213P1F11 v.3: HLA-B* 4402 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
Figure imgf000231_0001
9 A T L P S P F P Y 18 Portion of
3 I Q A C R G A T L 12 SEQ ID
213P1F11 v.3: HLA-B* 2705 nonamers 7 R G A T L P S P F 12 NO: 7; each start
Pos 1 2 3 4 5 6 7 8 9 score 10 T L P S P F P Y L 12
7 R G A T L P F 16 Portion of 12 P S P F P Y L s position is P S L 12 specified,
3 I Q A C R G A T L 13 SEQ ID 5 A C R G A T L P S 6 the length
6 c R G A T L P S P 13 NO: 7; each 11 L P S P F P Y L S 6 of each
T L P S P F P Y start
9 A 13 1 Y I I Q A C R G A ' 4 peptide is 9 position is
12 P S P F P Y L S L 13 2 I I Q A C R G A T specified, 3 amino
10 T L P S P F P Y L 12 the length 4 Q A C R G A T L P 3 acids, the T L P S P F P of each 6 C R G A T L P S P 1 end position
8 G A 7 for each
4 Q A C R G A T L P 5 peptide is 9 8 G A , T L P S P F P 1 peptide is
1 Y I I Q A C R G A 4 amino the start
5 A C R G A T L P S 4 acids, the position
11 L P S P F P Y L S 2 end position plus eight for each
2 I I Q A C R G A T 1 peptide is the start position plus eight
213P1F11 v.3: HLA-B* 2709 nonamers •
Pos 1 2 3 4 5 6 7 8 9 score
7 R G A T L P S P F 12 Portion of
10 T L P S P F P Y L 12 SEQ ID
3 I Q A C R G A T L 11 NO: 7; each T L start
6 c R G A P S P 11 position is
12 P S P F P Y L S L 11 specified,
8 G A T L P S P F P 5 the length
9 A T L P S P F P Y 4 of each
5 A C R G A T L P S 2 peptide is 9
11 L P S P F P Y L S 2 amino
1 Y I I Q A C R G A 1 acids, the end position for each
Figure imgf000231_0002
peptide is the start
2 I I Q A C R G A T 1 position
4 Q A C R G A T L P 1 plus eight
Figure imgf000231_0003
Figure imgf000232_0002
213P1F11 v.4: HLA-Al nonamers
Pos 1 2 3 4 5 6 7 8 9 score
83 T S A S E E E K Y 21 Portion of
45 s S V H Q K L V N 15 SEQ ID
52 V N D P R E T Q Ξ 15 NO: 9; each start
85 A S Ξ E E K Y D M 15 position is
66 V G D I V G R D L 14 specified,
80 N S E T S A S E Ξ 14 the length
34 L K E E Q G R A F 13 of each
35 K Ξ Ξ Q G R A F R 13 peptide is 9
86 S Ξ Ξ E K Y D M S 13 ammo
4 C Q E Y D K S L S 12 acids, the
14 Q P E K R T G L R 12 end position for each
22 R D E N G Ξ C G Q 12 peptide is
25 N G E C G Q T F R 12 the start
55 P R E T Q E V F G 12 position
6 E Y D K S L S V Q 11 plus eight
21 L R D E N G E C G 11
57 E T Q E V F G G G 11
58 T Q E V F G G G V 11
71 G R D L S I S F R 10
28 C G Q T F R L K Ξ 9
10 S L S V Q P E K R 7
15 P E K R T G L R D 7
63 G G G V G D I V G 7
68 D I V G R D L S I 7
5 Q E Y D K S L S V 6
9 K S L S V Q P E K 6
12 S V Q P E K R T G 6
18 R T G L R D E N G 6
30 Q T F R L K E E Q 6
44 G S S V H Q K L V 6
82 E T S A S E E E K 6
11 L S V Q P E K R T 5
27 E C G Q T F R L K 5
61 V F G G G V G D I 5
62 F G G G V G D I V 5
Figure imgf000232_0001
67 G D I V G R D L s 5 213P1F11 v.4: HLA-A26 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
57 E T Q E V F G G G 23 Portion of
60 E V F G G G V G D 22 SEQ ID
73 D L S I S F R N S 20 NO: 9; each
24 E N G E C G start
Q T F 19 position is
68 D I V G R D L S I 19 specified,
46 S V H Q K L V N D 18 the length
54 D P R E T Q E V F 18 of each
82 E T S A S E E E K 18 peptide is 9
65 G V G D I V G R D 16 amino
69 I V G R D L S I S 15 acids, the
6 E Y D K S L S V end position
Q 14 for each
30 Q T F R L K E E Q 14 peptide is
34 L K E E Q G R A F 14 the start
13 V Q P E K R T G L 13 position
70 V G R D L S I S F 13 plus eight
83 T S A S E E E K Y 13
8 D K S L S V Q P E 12
12 S V Q P E K R T G 12
26 G E C G Q T F R L 12
33 R L K E E Q G R A 12
61 V F G G G V G D I 12
75 S I S F R N S E T 12
3 K C Q E Y D K S L 11
18 R T G L R D E N G 11
27 E C G Q T F R L K 11
37 E Q σ R A F R G S 11
41 A F R G S S V H Q 11
43 R G S S V H Q K L 11
51 L V N D P R E T Q 11
10 S L S V Q P E K R 10
16 E K R T G L R D E 10
20 G L R D E N G E C 10
85 A S E E E K Y D M 10
23 D E N G E C G Q T 9
36 Ξ E Q G R A F R G 9
50 K L V N D P R E T 9
66 V G D I V G R D L 9
31 T F R L K E Ξ Q G 8
64 G G V G D I V G R 8
77 S F R N S E T S A 8
86 S E E E K Y D M S 8
Figure imgf000233_0001
42 F R G S S V H Q K 7
71 G R D L S I S F R 7
2 G K C Q E Y D K S 6
47 V H Q K L V N D P 6
56 R E T Q E V F G G 6
7 Y D K S L s V Q P 5
29 G Q T F R L K E E 5
17 K R T G L R D E N 4
52 V N D P R Ξ T Q E 4
74 L S I S F R N S E 4
79 R N S E T S A s E 4
5 Q Ξ Y D K S L s V 3
9 K S L S V Q P E K 3
Figure imgf000234_0001
213P1F11 v.4 : HLA-A3 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
60 E V F G G G V G D 21 Portion of
40 R A F R G S S V H 20 SEQ ID
12 S V Q P E K R T G 18 NO: 9; each
69 I V G R D L S I S 18 start position is
33 R L K E E Q G R A 17 specified,
68 D I V G' R D L S I 17 the length
9 K s L S V Q P Ξ K 16 of each
10 S L S V Q P E K R 16 peptide is 9
46 S V H Q K L V N D 16 ammo
51 L V N D P R E T Q 16 acids, the
20 G L R D E N G E C 15 end position
T for each
50 K L V N D P R E 15 peptide is
75 S I S F R N S E T 14 the start
5 Q Ξ Y D K S L S V 13 position
35 K Ξ E Q G R A F R 13 plus eight
42 F R G S S V H Q K 13
24 E N G E C G Q T F 12
41 A F R G s S V H Q 12
54 D P R E T Q E V F 12
59 Q Ξ V F G G G V G 12
65 G V G D I V G R D 12
82 E T S A S E E E K 12
1 M G K C Q E Y D K 11
Figure imgf000234_0002
Figure imgf000235_0001
213P1F11 v.4: HLA-B*I )702 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
54 D P R E T Q E V F 19 Portion of
26 G E C G Q T F R L 13 SEQ ID
43 R G S S V H Q K L 13 NO: 9; each
L start
13 V Q P E K R T G 12 position is
14 Q P E K R T G L R 12 specified,
66 V G D I V G R D L 12 the length
3 K C Q E Y D K S L 11 of each
61 V F G G G V G D I 10 peptide is 9
62 F G G G V G D I V 10 amino
68 D I V G R D L s I 10 acids, the
11 L S V Q P E K R T end position
9 for each
24 E N G E C G Q T F 9 peptide is
77 S F R N s E T S A 9 the start
5 Q E Y D K S L S V 8 position
23 D E N G E C G Q T 8 plus eight
34 L K E E Q G R A F 8
39 G R A F R G S S V 8
41 A F R G S S V H Q 8
44 G S S V H Q K L V 8
70 V G R D L S I S F 8
75 S I S F R N s E T 8
85 A S Ξ Ξ E K Y D M 8
33 R L K E E Q G R A 7
Figure imgf000235_0002
50 K L V N D P R E T 7
53 N D P R E T Q E V 7 213P1F11 v.4: HLA-B* )8 nonamers
58 T Q E V F G G G V 7 Pos 1 2 3 4 5 6 7 8 9 score
60 E V F G G G V G D 6 13 V Q P E K R T G L 23 Portion of
6 E Y D K S L s V Q 4 54 D P R E T Q E V F 20 SEQ ID
8 D K S L S V Q P Ξ 4 33 R L K E E Q G R A 18 NO: 9; each
R T G L R D E N D I V G R D L S I start
17 K 4 68 18 position is
35 K E E Q G R A F R 4 31 T F R L K E E Q G 17 specified,
38 Q G R A F R G S S 4 75 S I S F R N S E T 16 the length
51 L V N D P R E T Q 4 20 G L R D E N G Ξ c 15 of each
64 G G V G D I V G R 4 14 Q P E K R T G L R 14 peptide is 9
79 R N S Ξ T S A S Ξ 4 86 S Ξ Ξ E K Y D M S 14 amino
82 Ξ T S A S E E E K 4 3 K C Q E Y D K S L 13 acids, the
R T G L R D end position
15 P E K 3 46 S V H Q K L V N D 13 for each
16 E K R T G L R D Ξ 3 48 H Q L V N D P R 13 peptide is
27 E C G Q T F R L K 3 70 V G R D L S I S F 13 the start
28 C G Q T F R L K E 3 7 Y D K S L S V Q P 11 position
36 E E Q G R A F R G 3 26 G E C G Q T F R L 11 plus eight
37 Ξ Q G R A F R G S 3 29 G Q T F R L K E Ξ 11
45 S S V H Q K L V N 3 66 V G D I V G R D L 11
52 V N D P R E T Q E 3 1 M G K c Q E Y D K 10
55 P R E T Q E V F G 3 5 Q E Y D K S L S V 10
73 D L S I S F R N S 3 15 P E K R T G L R D 10
Figure imgf000236_0002
213P1F11 v.4: HLA-B*1510 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
26 G E C G Q T F R L 14 Portion of
13 V Q E K R T G L 12 SEQ ID
47 V H Q K L V N D P 12 NO: 9; each
66 V G D I V G R D L 12 start position is
3 K C Q E Y D K S L 11 specified,
34 L K E E Q G R A F 11 the length
43 R G S S V H Q K L 11 of each
24 E N G E C G Q T F 9 peptide is 9
54 D P R E T Q E V F 9 amino
85 A S E E E K Y D M 9 acids, the
70 V G R D L S I S F 7 end position for each
12 S V Q P E K R T G 6 peptide is
49 Q K L V N D P R E 5 the start
60 E V F G G G V G D 5 position
63 G G -σ V G D I V G 5 plus eight
64 G G V G D I V G R 5
65 G V G D I V G R D 5
6 E Y D K S L S V Q 4
11 L S V Q P E K R T 4
16 E K R T G L R D E 4
27 E C G Q T F R . L K 4
35 K E E Q G R A F R 4
36 E E Q G R A F R G 4
50 K L V N D P R Ξ T 4
51 L V N D P R E T Q 4
67 G D I V G. R D L S 4
76 I S F R N S E T S 4
7 Y D K S L S V Q P 3
10 S L S V Q P E K R 3
17 K R T G L R D E N 3
20 G L R D E N G E C 3
33 R L K E E Q G R A 3
37 E Q G R A F R G S 3
40 R A F R G S S V H 3
41 A F R G S S V H Q 3
44 G S S V H Q K L V 3
45 S S V H Q K L V N 3
46 S V H Q K L V N D 3
55 P R E T Q E V F G 3
58 T Q E V F G G G V 3
59 Q E V F G G G V G 3
61 V F G G G V G D I 3
73 D L S I S F R N S
82 E T S A S E E E K 3
2 G K C Q E Y D K S 2
4 C Q Ξ Y D K S L S 2
8 D K S L S V Q P E 2
9 K S L S V Q P E K 2
14 Q P E K R T G L R 2
15 P E K R T G L R D
Figure imgf000236_0001
2
Figure imgf000237_0001
213P1F11 v.' : HLA-B* 2705 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
71 G R D S I S F R 29 Portion of
32 F R L K E E Q G R 25 SEQ ID
42 F R G S S V H Q K 23 NO: 9; each
A F R G S S V H 20 start
40 R position is
64 G G V G D I V G R 20 specified, '
9 K S L S V Q P E K 19 the length
26 G Ξ C G Q T F R L 17 of each
35 K E Ξ Q G R A F R 17 peptide is 9
43 R G S S V H Q K L 17 amino
17 K R T G L R D E N 16 acids, the
25 N G E C G Q T F R 16 end position for each
3 K C Q E Y D K S L 15 s peptide is
39 G R A F R G s V 15 the start
13 V Q P E K R T G L 14 position
54 D P R E T Q E V F 14 plus eight
70 V G R D L S I s F 14
48 H Q K L V N D P R 13
85 A S E E E K Y D 13
10 S L S V Q P E K R 12
Figure imgf000237_0002
Figure imgf000238_0001
213P1F11 v.4: HLA-B*2709 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
39 G R A F R G S S V 21 Portion of
43 R G S S V H Q K L 15 SEQ ID
26 G E C G Q T F R L 14 NO: 9; each
K R T G L R D E N 13 start
17 position is
42 F R G S S V H Q K 13 specified,
71 G R D L S I S F R 13 the length
3 K C Q E Y D K S L 12 of each
32 F R L K E E Q G R 12 peptide is 9
5 Q E Y D K S L S V 11 amino
44 G S S V H Q K L V 11 acids, the
55 " P R E T Q E V F G 11 end position for each
78 F R N S E T S A S 11 peptide is
13 V Q P E K R T G L 10 the start
21 L R D E N G E C G 10 position
66 V G D I V G R D L 10 plus eight
68 ' D I V G R D L S I 10
Figure imgf000238_0002
85 A S E E E K Y D M 10
61 V F G G G V G D I 9 213P1F11 v.4 : HLA-B*4402 nonamers
72 R D L S I S F R N 9 Pos 1 2 3 4 5 6 7 8 9 score
24 E N G E C G Q T F 8 26 G E C G Q T F R L 22 Portion of
34 L K E E Q G R A F 8 36 E Ξ Q G R A F R G 15 SEQ ID
53 N D P R E T Q E V 8 3 K C Q E Y D K S L 13 NO: 9; each
D P R E T T F start
54 Q E V F 8 24 E N G E C G Q 13 position is
58 T Q E V F G G G V 8 34 L K E E Q G R A F 13 specified,
62 F G G G V G D I V 8 5 Q E Y D K S L S V 12 the length
70 V G R D L S I s F 8 13 V Q P E K R T G L 12 of each
9 K S L S V Q P E K 6 15 P Ξ K R T G L R D 12 peptide is 9
40 R A F R G S S V H 6 23 D E N G E C G Q T 12 amino
56 R E T Q E V F G G 6 35 K E Ξ Q G R A F R 12 acids, the
G V G D I V G R 6 end position
64 G 43 R G S S V H Q K L 12 for each
65 G V G D I V G R D 6 66 V G D I V G R D L 12 peptide is
18 R T G L R D E N G 5 70 V G R D L S I S F 12 the start
33 R L K E E Q G R A 5 54 D P R E T Q E V F 11 position
22 R D E N G E C G Q 4 56 R Ξ T Q E V F G G 11 plus eight
29 G Q T F R L K E E 4 59 Q E V F G G G V G 11
49 Q K L V N D P R E 4 68 D I V G R D L S I 11
50 K L V N D P R Ξ T 4 81 S E T S A S E E Ξ 11
67 G D I V G R D L S 4 83 T S A S Ξ E E K Y 11
79 R N S E T S A S E 4 86 S Ξ Ξ E K Y D M S 11
2 G K C Q E Y D K S 3 61 V F G G G V G D I 10
Figure imgf000239_0002
213P1F11 v.4: HLA-B*5101 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
54 D P R E T Q Ξ V F 20 Portion of
62 F G G G V G D I V 18 SEQ ID
43 R G S S V H Q K L 16 NO: 9; each
66 V G D I V G R D L 16 start position is
68 D I V G R D L S I 16 specified,
5 Q E Y D K S L S V 15 the length
40 R A F R G S S V H 14 of each
61 V F G G G V G D I 14 peptide is 9
84 S A S E Iϋ £ K Y D 13 amino
13 V Q P E K R T G L 12 acids, the
14 Q P E K R T G L R 11 end position for each
3 K C Q E Y D K S L 10 peptide is
19 T G L R D E N G Ξ 10 the start
28 C G Q T F R L K E 10 position
53 N D P R E T Q E V 10 plus eight
63 G G G V G D I V G 10
44 G S S V H Q K L V 9
58 T Q E V F G G G V 9
64 G G V G D I V G R 9
70 V G R D L S I S F 9
25 N G E C G Q T F R 8
26 G E C G Q T F R L 8
39 G R A F R G S S V 8
1 M G K C Q E Y D K 7
8 D K S L S V Q P Ξ 7
38 Q G R A F R G S S 7
73 D L S I S' F R N S 7
6 E Y D K S L S V Q 6
23 D Ξ N G E C G Q T 5
34 L K E E Q G R A F 5
47 V H Q K L V N D P 5
51 L V N D P R E T Q 5
57 E T Q E V F G G G 5
76 I S F R N S E T S 5
9 K S L S V Q P E K 4
11 L S V Q P E K R T 4
21 L R D E N G E C G 4
24 Ξ N G E C G Q T F 4
32 F R L K E E Q G R 4
41 A F R G S S V H Q 4
42 F R G S S V H Q K 4
45 S S V H Q K L V N 4
Figure imgf000239_0001
65 G V G D I V G R D 4 213P1F11 v.4: RT1.A1 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
76 I S F R N S E T S 18 Portion of
83 T S A S E E E K Y 18 SEQ ID
60 E V F G G G V G D 17 NO: 9; each
85 A S E E E K Y D M start
17 position is
40 R A F R G S S V H 16 specified,
9 K S L S V Q P E K 14 the length
30 Q T F R L K E Ξ Q 13 of each
24 E N G E C G Q T F 12 peptide is 9
54 D P R E T Q E V F 12 amino
12 S V Q P E K R T G 11 acids, the
34 L K E E G R A F 11 end position
Q for each
5 Q E Y D K S L S V 10 peptide is
70 V G R D L S I S F 10 the start
51 L V N D P R E T Q 9 position
84 S A S E E E K Y D 9 plus eight
3 K C Q E Y D K S L 8
13 V Q P E K R T G L 8
32 F R L K E E Q G R 8
45 S S V H Q K L V N 8
46 S V H Q K L V N D 8
74 L S I S F R N S Ξ 8
11 L S V Q P E K R T 7
19 T G L R D E N G Ξ 7
26 G E C G Q T F R L 7
66 V G D I V G R D L 7
69 I V G R D L S I S 7
80 N S E T S A S E E 7
43 R G S S V H Q K L 6
44 G S S V H Q K L V 6
49 Q K L V N D P R Ξ 6
65 G V G D I V G R D 6
72 R D L S I S F R N 6
17 K R T G L R D Ξ N 5
2 G K C Q E Y D K S 3
35 K Ξ Ξ Q G R A F R 3
50 K L V N D P R E T 3
Figure imgf000240_0001
81 S E T S A S E E Ξ 3
86 S Ξ Ξ E K Y D M S 3
4 C Q E Y D K S L S 2
10 S L S V Q P E K R 2
18 R T G L R D E N G 2
20 G L R D E N G E C 2
21 L R D E N G E C G 2
23 D Ξ N G E C G Q T 2
28 C G Q T F R L K E 2
31 T F R L K E E Q G 2
33 R L K E E Q G R A 2
38 Q G R A F R G S S 2
41 A F R G S S V H Q 2
47 V H Q K L V N D P 2
48 H Q K L V N D P R 2
53 N D P R E T Q E V 2
56 R Ξ T Q E V F G G 2
61 V F G G G V G D I 2
Figure imgf000241_0001
TABLE XIXA, part 5: MHC Class I nonamer analysis of 213P1F11 v.5 (aa 1-242)
Figure imgf000241_0004
Figure imgf000241_0002
Figure imgf000241_0005
Figure imgf000241_0003
Figure imgf000241_0006
Figure imgf000242_0001
Figure imgf000242_0002
E13P1F11 v.5: HLA-B*4402 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
A L I L R V T K A 10 Portion of
A R L A L I L RV SEQ ID
L I L R V T K A R NO: 11;
R L A L I L R each start position is specified, the length of each peptide is 9 amino acids, the end position for each peptide is the start position
Figure imgf000242_0003
plus eight
Figure imgf000242_0004
Figure imgf000242_0005
Figure imgf000243_0002
Figure imgf000243_0001
213PTF11 v.6: HLA- A26 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
1 K L E N L F E A M 22 Portion of
4 N L F E A M N N K 17 SEQ ID
9 M N N K N C Q A L 11 NO: 13; each start
3 E N L F Ξ A N N 8 position is
5 L F E A M N N K N 7 specified,
7 E A M N N K N C Q 6 the length
2 L E N L F E A M N 2 of each
6 F E A M N N K N C 2 peptide is 9
8 A M N N K N C Q A 2 amino acids, the end position for each peptide is the start position
Figure imgf000243_0003
plus eight
213P1F11 v.6: HLA- A3 nonamers
Pos 1 2 3 4 5 6 7 8 9 score
4 N L F E A M N N K 21 Portion of
1 K L E N L F E A M 15 SEQ ID
8 A M N N K N C Q A 8 NO: 13; each start
3 E N L F E A M N N 6 position is
2 L E N L F E A M N 5 specified,
5 L F E A M N N K N 2 the length
6 F E A M N N K N C 1 of each
7 E A M N N K N C Q 1 peptide is 9
9 M N N K N C Q A L 1 amino acids, the end position for each peptide is the start position
Figure imgf000243_0004
plus eight
Figure imgf000244_0002
Figure imgf000244_0001
213P1F11 v .1: HLA-A*0201 decamers 213P1F11 v.l: HLA-A*0201 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score Pos 1 2 3 4 5 6 7 8 9 0 score
149 V M V I K D S P Q T 14 179 R H D Q K G S C F I 8
163 T D A L H V Y S T V 14 189 Q T L V D V F T K R 8
170 S T V E G Y I A Y R 14 191 L D V F T K R K G 8
218 L V Q E G K A R K T 14 199 K G H I L E L L T E 8
222 G K A R K T N P E I 14 203 L E L L T E V T R R 8
230 E I Q S T L R K R L 14 207 T E V T R R M A E A 8
6 S L E E E K Y D M S 13 43 F R Q L R F E S T M 7
85 V L M A H G R E G F 13 76 E D P V S C A F V V 7
102 M V K L E N L F E A 13 81 C A F V V L M A H G 7
134 G Ξ Q R D P G E T V 13 110 E A L N N K N C Q A 7
167 H V Y S T V E G Y I 13 137 R D P G E T V G G D 7
175 Y I A Y R H D Q K G 13 141 E T V G G D E I V M 7
204 E L L T E V T R R M 13 147 E I V M V I K D S P 7
233 S T L R K R L Y L Q 13 148 I V M V I K D S P Q 7
17 A R L A L I L C V T 12 160 P T Y T D A L H V Y 7
99 D G E M V K L E N L 12 176 I A Y R H D Q K G S 7
104 K L E N L F E A L N 12 188 I Q T L V D V F T K 7
193 D V F T K R K G H I 12 214 A E A E L V Q E G K 7
10 E K Y D M S G A R L 25 V T K A R E G S E E 6
14 M S G A R L A L I L 42 M F R Q L R F E S T 6
38 A L E H M F R Q L R 72 I D S R E D P V S C 6
41 H M F R Q L R F E S 87 M A H G R E G F L K 6
45 Q L R F E S T M K R 89 H G R E G F L K G E 6
51 T M K R D P T A E Q 97 G E D G E M V K L E 6
71 A I D S R E D P V S 101 E M V K L E N L F E 6
77 D P V S C A F V V L 114 N K N C Q A L R A K 6
78 P V S C A F V V L M 125 K V Y I I Q A C R G 6
159 I P T Y T D A L H V 139 P G E T V G G D E I 6
183 K G S C F I Q T L V 145 G D E I V M V I K D 6
194 V F T K R K G H I L 152 I K D S P Q T I P T 6
63 E Ξ L E K F Q Q A I 10 166 L H V Y S T V E G Y 6
79 V S C A F V V L M A 10 168 V Y S T V Ξ G Y 1 A 6
122 A K P K V Y I I Q A 10 171 T V E G Y I A Y R H 6
128 I I Q A C R G E Q R 10 186 C F I Q T L V D V F 6
140 G E T V G G D E I V 10 206 L T E V T R R M A E 6
153 K D S P Q T I P T Y 10 215 E A E L V Q E G K A 6
158 T I P T Y T D A L H 10 7 L E E E K Y D M S G 5
190 T L V D V F T K R K 10 11 K Y D M S G A R L A 5
210 T R R M A E A E L V 10 24 C V T K A R E G S E 5
232 Q S T L R K R L Y L 10 29 R E G S E E D L D A 5
1 M S N P R S L E E E 9 32 S E E D L D A L E H 5
5 R S L E E E K Y D M 9 33 E E D L D A L Ξ H M 5
35 D L D A L E H M F R 9 48 F E S T M. K R D P T 5
73 D S R E D P V S C A 9 83 F V V L M A H G R E 5
84 V V L M A H G R E G 9 88 A H G R E G F L K G 5
93 G F L K G E D G E M 9 112 L N N K N C Q A L R 5
151 V I K D S P Q T I P 9 115 K N C Q A L R A K P 5
213 M A E A E L V Q E G 9 118 Q A L R A K P K V Y 5
217 E L V Q E G K A R K 9 121 R A K P K V Y I I Q 5
225 R K T N P E I Q S T 9 123 K P K V Y I I Q A C 5
15 S G A R L A L I L C 8 133 R G E Q R D P G E T 5
50 S T M K R D P T A E 8 136 Q R D P G E T V G G 5
64 E L E K F Q Q A I D 8 144 G G D E I V M V I K 5
80 S C A F V V L M A H 8 155 S P Q T I P T Y T D 5
Figure imgf000246_0001
213P1F11 V .1: HLA-A*0203 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
216 A E L V Q E G K A R 9-1
10 E K Y D M S G A R L 8
13 D M Ξ G A R L A L I 8
21 L ' l L C V T K A R E 8
31 G S Ξ E D L D A L E 8
51 T M K R D P T A E Q 8
64 E L Ξ K F Q Q A 1 D 8
75 R E D P V S C A F V 8
81 C A F V V L M A H G 8
104 K L Ξ N L F E A L N 8
112 L N N K N C Q A L R 8
115 K N C Q A L R A K P 8
124 P K V Y I I Q A C R 8
158 T I P T Y T D A L H 8
170 S T V E G Y I A Y R 8
209 V T R R M A E A E L 8
217 E L V Q E G K A R K, 8
Figure imgf000247_0002
Figure imgf000247_0001
213P1F11 v.l: HLA-Al decamers 213P1F11 v.l: HLA-Al decamers
Pos 1 2 3 4 5 6 7 8 9 0 score Pos 1 2 3 4 5 6 7 8 9 0 score
191 L V D V F T K R K G 12 49 E S T M K R D P T A 4
215 E A E L V Q E G K A 12 65 L E K F Q Q A I D S 4
35 D L D A L E H M F R 77 D P V S C A F V V L 4
61 F Q E E L E K F Q Q 87 M A H G R E G F L K 4
64 E L E K F Q Q A I D 103 V K L E N L F E A L 4
90 G R E G F L K G E D 107 N L F E A L N N K N 4
108 L F E A L N N K N C 154 D S P Q T I P T Y T 4
139 P G E T V G G D E I 175 Y I A Y R H D Q K G 4
171 T V E G Y I A Y R H 13 D M S G A R L A L I 3
184 G S C F I Q T L V D 19 L A L I L C V T K A 3
189 Q T L V D V F T K R 37 D A E H M F R Q L 3
202 I L Ξ L L T E V T R 42 M F R Q L R F Ξ S T 3
213 M A E A E L V Q E G 45 Q L R F E S T M K R 3
232 Q S T L R K R L Y L 55 D P T A E Q F Q E E 3
7 L E E E K Y D M S G 10 78 P V S C A F V V L M 3
8 E E E K Y D M S G A 10 85 V L M A H G R E G F 3
14 M S G A R L A L I L 10 115 K N C Q A L R A K P 3
33 E E D L D A L E H M 10 127 Y I I Q A C R G E Q 3
47 R F E S T M K R D P 10 131 A C R G E Q R D P G 3
99 D G E M V K L E N L 10 143 V G G D E I V M V I 3
133 R G E Q R D P G E T 10 178 Y R H D Q K G S C F 3
144 G G D E I V M V I K 10 197 K R K G H I L Ξ L L 3
157 Q T I P T Y T D A L 10 205 L L T E V T R R M A 3
179 R H D Q K G S C F I 10 2 S N P R S L E E E K 2
226 K T N P E I Q S T L 10 17 A R L A L I L C V T 2
233 S T L R K R L Y L Q 10 20 A L I L C V T K A R 2
12 Y D M S G A R L A L 9 48 F E S T M K R D P T 2
1 S N P R S L E E E 8 59 E Q F Q E E L E K F 2
15 S G A R L A L I L C 8 80 S C A F V V L M A H 2
25 V T K A R E G S E E 8 84 V V L M A H G R E G 2
50 S M K R D P T A E 8 100 G E V K L E N L F 2
121 R A K P K V Y I I Q 8 111 A L N N K N C Q A L 2
170 S T V E G Y I A Y R 8 112 L N N K N C Q A L R 2
181 D Q K G S C F I Q T 8 117 C Q A L R A K P K V 2
198 R K G H I L E L L T 8 119 A L R A K P K V Y I 2
58 A E Q F Q E E L E K 7 137 R D P G E T V G G D 2
101 E M V K L E N L F E 7 142 T V G G D E I V M V 2
209 V T R R M A E A E L 7 155 S P Q T I P T Y T D 2
211 R R M A E A E L V Q 7 158 I P T Y T D A L H 2
16 G A L A L I L C V 6 165 A L H V Y S T V E G 2
29 R E G S E E D L D A 6 168 V Y S T V E G Y I A 2
39 L E H M F R Q L R F 6 183 K G S C F I Q T L V 2
56 P T A E Q F Q E E L 6 185 S C F I Q T L V D V 2
98 E D G E M V K L E N 6 186 C F I Q T L V D V F 2
105 L Ξ N L F E A L N N 6 192 V D V F T K R K G H 2
113 N N K N C Q A L R A 6 194 V F T K R K G H I L 2
159 I P T Y T D A L H V 6 210 T R R M A E A E L V 2
196 T K R K G H I L E L 6 216 A E L V Q E G K A R 2
199 K G H I L E L L T E 6 218 L V Q E G K A R K T 2
73 D S R E D P V S C A 5 227 T N P E I Q S T L R 2
94 F L K G E D G E M V 5 229 P E I Q S T L R K R 2
122 A K P K V Y I I Q A 5 10 E K Y D M S G A R L 1
224 A R K T N P E I Q S 5 18 R L A L I L C V T K 1
5 R S L E E E K Y D M 4 22 I L C V T K A R E G 1
Figure imgf000249_0002
Figure imgf000249_0001
213P1F11 v .1: HLA-A26 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
191 L V D V F T K R K G 12
201 H I L E L L T E V T 12
206 L T E V T R R M A E 12
20 A L I L C V T K A R
39 L E H M F R Q L R F
83 F V V L M A H G R E
84 V V L M A H G R E G
106 E N L F E A L N N K
125 K V Y I I Q A C R G
128 I I Q A C R G E Q R
148 I V M V I K D S P Q
167 H V Y S T V E G Y I
187 F I Q T L V D V F T
195 F T K R K G H I L E
205 L L T E V T R R M A
5 R S L E E E K Y D M 10
12 Y D M S G A R L A L 10
18 R L A L I L C V T K 10
27 K A R E G S E E D L 10
40 E H M F R Q L R F Ξ 10
45 Q L R F E S T M K R 10
63 E E L E K F Q Q A I 10
100 G E M V K L E N L F 10
118 Q A L R A K P K V Y 10
165 A L H V Y S T V E G 10
190 T L V D V F T K R K 10
231 I Q S T L R K R L Y 10
22 I L C V T K A R E G 9
38 A L E H M F R Q L R 9
43 F R Q L R F E S T M 9
46 L R F E S T M K R D 9
67 K F Q Q A I D S R E 9
86 L M A H G R E G F L 9
92 E G F L K G E D G E 9
98 E D G E M V K L E N 9
104 K L E N L F E A L N 9
119 A L R A K P K V Y I 9
202 I L E L L T E V T R 9
212 R M A E A E L V Q E 9
9 E E K Y D M S G A R 8
14 M S G A R L A L I L 8
60 Q F Q E E L E K F Q 8
81 C A F V V L M A H G 8
137 R D P G Ξ T V G G D 8
138 D P G E T V G G D E 8
144 G G D E I V M V I K 8
154 D S P Q T I P T Y T 8
229 P E I Q S T L R K R 8
232 Q S T L R K R L Y L 8
1 M S N P R S L E E E 7
47 R F E S T M K R D P 7
49 E S T M K R D P T A 7
76 E D P V S C A F V V 7
80 S C A F V V L M A H 7
Figure imgf000250_0001
Figure imgf000251_0002
Figure imgf000251_0001
213P1F11 v.l: HLA-A3 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
54 R D P T A E Q F Q E 7
73 D S R E D P V S C A 7
74 S R E D P V S C A F 7
76 E D P V S C A F V V 7
91 R E G F L K G E D G 7
105 L E N L F E A L N N 7
112 L N N K N C Q A L R 7
115 K N C Q A L R A K P 7
124 P K V Y I I Q A C R 7
137 R D P G E T V G G D 7
164 D A L H V Y S T V E 7
169 Y S T V E G Y I A Y 7
184 G S C F I Q T L V D 7
198 R K G H I L E L L T 7
219 V Q E G K A R K T N 7
223 K A R K T N P E I Q 7
224 A R K T N P Ξ I Q S 7
225 R K T N P E I Q S T 7
229 P E I Q S T L R K R 7
232 Q S T L R K R L Y L 7
11 K Y D M S G A R L, A 6
37 D L E H M F R Q L 6
53 K R D P T A E Q F Q 6
62 Q E E L E K F Q Q A 6
66 E K F Q Q A I D S R 6
67 K F Q Q A I D S R E 6
70 Q A I D S R E D P V 6
79 V S C A F V V L M A 6
110 E A L N N K N C Q A 6
122 A K P K V Y I I Q A 6
133 R G E Q R D P G E T 6
141 E T V G G D E I V M 6
143 V G G D E I V M V I 6
166 L H V Y S T V E G Y 6
173 E G Y I A Y R H D Q 6
181 D Q K G S C F I Q T 6
182 Q K G S C F I Q T L 6
185 S C F I Q T L V D V 6
196 T K R K G H I L E L 6
197 K R K G H I L E L L 6
210 T R R M A E A E L V 6
227 T N P E I Q S T L R 6
233 S T L R K R L Y L Q 6
12 Y D M S G A R L A L 5
13 D M S G A R L A L I 5
15 S G A R L A L I L C 5
28 A R E G S E E D L D 5
42 M F R Q L R F E S T 5
69 Q Q A I D S R E D P 5
123 K P K V Y I I Q A C 5
130 Q A C R G E Q R D P 5
138 D P G E T V G G D E 5
155 S P Q T I P' T Y T D 5
Figure imgf000252_0001
161 T Y T D A L H V Y S 5 213P1F11 v.l: HLA-A3 decamers
Pos 1 2 3 4 5 6 . 7 8 9 0 score
213 E A E L V Q Ξ G 2
215 E A E L V Q E G K A 2
4 P R S L E E E K Y D 1
23 L C V T K A R E G S 1
33 E E D L D A L E H M 1
46 L R F E S T M K R D 1
48 F E S T M K R D P T 1
92 E G F L K G E D G E 1
120 L R A K P K V Y I I 1
132 C R G E Q R D P G E 1
145 G D E I V M V I K D 1
213P1F11 v .1: HLA-B*0702 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
77 D P V S C A F V V L 24 Portion of
159 I P T Y T D A L H V 19 SEQ ID
12 Y D M S G A R L A L 15 NO: 3; each
T K R K G H I L E L start
196 15 position is
14 M S G A R L A L I L 14 specified,
30 E G S E E D L D A L 14 the length
27 K A R E G S E E D L 13 of each
96 K G E D G E M v -κ L 13 peptide is
111 A L N N K N C Q A L 13 10 amino
119 A L R A K P K V Y I 13 acids, the
157 Q T I P T Y T D A L 13 end position
K R for each
197 K G H I L E L L 13 peptide is
209 V T R R M A E A E L 13 the start
228 N P E I Q S T L R K 13 position
232 Q S T L R K R L Y L 13 plus nine
3 N P R S L E E E K Y 12
10 E K Y D M S G A R L 12
55 D P T A E Q F Q E E 12
86 L M A H G R E G F L 12
103 V K L E N L F Ξ A L 12 '
123 K P K V Y I I Q A C 12
226 K T N P E I Q S T L 12
230 E I Q S T L R K R L 12
13 D M S G A R L A L I 11
37 D A L E H M F R Q L 11
56 P T A E Q F Q Ξ Ξ L 11
75 R Ξ D P V S C A F V 11
78 P V S C A F V V L M 11
138 D P G E T V G G D E 11
142 T V G G D E I V M V 11
155 S P Q T I P T Y T D 11
182 Q K G S C F I Q T L 11
194 V F T K R K G H I L 11
16 G A R L A L I L C V 10
17 A R L A L I L C V T 10
29 R Ξ G S E E D L D A 10
48 F Ξ S T M K R D P T 10
79 V S C A F V V L M A 10
99 D G E M V K L E N L 10
Figure imgf000253_0001
141 E T V G G D E I V M 10 213P1F11 v .1: HLA-B*0702 decamers 213P1F11V .1: HLA-B*0702 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score Pos 1 2 3 4 5 6 7 8 9 0 score
152 I K D S P Q T I P T 10 88 A H G R Ξ G F L K G 6
183 K G S C F I Q T L V 10 102 M V K L E N L F E A 6
198 R K G H I L E L L T 10 134 G E Q R D P G E T V 6
42 M F R Q L R F E S T 9 139 P G E T V G G D E I 6
73 D S R E D P V S C A 9 140 G E T V G G D E I V 6
85 V L M A H G R E G F 9 167 H V Y S T V E G Y I 6
120 L R A K P K V Y I I 9 178 Y R H D Q K G S C F 6
122 A K P K V Y I I Q A 9 193 D V F T K R K G H I 6
143 V G G D E I V M V I 9 211 R R M A E A E L V Q 6
162 Y T D A L H V Y S T 9 218 L V Q E G K A R K T 6
179 R H D Q K G S C F I 9 71 A I D S R E D P V S 5
181 D Q K G S C F I Q T 9 72 I D S R E D P V S C 5
187 F I Q T L V D V F T 9 101 E M V K L E N L F E 5
8 E E E K Y D M S G A 8 135 E Q R D P G E T V G 5
11 K Y D M S G A R L A 8 136 Q R D P G E T V G G 5
19 . L A L I L C V T K A 8 153 K D S P Q T I P T Y 5
33 E E D L D A L E H M 8 165 A L H V Y S T V E G 5
39 L E H M F R Q L R F 8 18 R L A L I L C V T K 4
49 E S T M K R D P T A 8 20 A L I L C V T K A R 4
52 M K R D P T A E Q F 8 28 A R E G S E E D L D 4
63 E E L E K F Q Q A I 8 50 S T M K R D P T A E 4
76 E D P V S C A F V V 8 53 K R D P T A E Q F Q 4
94 F L K G E D G E M V 8 58 A E Q F Q E E L E K 4
113 N N K N C Q A L R A 8 97 G Ξ D G E M V K L E 4
117 C Q A L R A K P K V 8 98 E D G E M V K L E N 4
131 A C R G E Q R D P G 8 137 R D P G E T V G G D 4
168 V Y S T V E G Y I A 8 177 A Y R H D Q K G S C 4
185 S C F I Q T L V D V 8 184 G S C F I Q T L V D 4
186 C F I Q T L V D V F 8 212 R M A E A E L V Q E 4
201 H I L E L L T E V T 8 221 Ξ G K A R K T N P E 4
204 E L L T E V T R R M 8 223 K A R K T N P E I Q 4
210 T R R M A E A E L V 8 35 D L D A L E H M F R 3
222 G K A R K T N P E I 8 38 A L E H M F R Q L R 3
5 R S L E E E K Y D M 7 40 Ξ H M F R Q L R F E 3
34 E D L D A L E H M F 7 45 Q L R F E S T M K R 3
59 E Q F Q E E L Ξ K F 7 64 E L E K F Q Q A I D 3
62 Q E E L E K F Q Q A 7 115 K N C Q A L R A K P 3
70 Q A I D S R E D P V 7 144 G G D Ξ I V M V I K 3
74 S R E D P V S C A F 7 148 I V M V I K D S P Q 3
93 G F L K G E D G E M 7 199 K G H I L E L L T E 3
100 G E M V K L E N L F 7 202 I L E L L T E V T R 3
110 E A L N N K N C Q A 7 214 A E A E L V Q E G K 3
133 R G E Q R D P G E T 7 216 A E L V Q E G K A R 3
149 V M V I K D S P Q T 7 219 V Q E G K A R K T N 3
150 M V I K D S P Q T I 7 220 Q E G K A R K T N P 3
154 D S P Q T I P T Y T 7 231 I Q S T L R K R L Y 3
156 P Q T I P T Y T D A 7 4 P R S L E E E K Y D 2
163 T D A L H V Y S T V 7 9 E Ξ K Y D M S G A R 2
200 G H I L .Ξ L L T E V 7 32 S Ξ E D L D A L E H 2
205 L L T E V T R R M A 7 44 R Q L R F E S T M K 2
207 T E V T R R M A E A 7 51 T M K R D P T A E Q 2
215 E A E L V Q E G K A 7 69 Q Q A I D S R E D P 2
225 R K T N P E I Q S T 7 80 S C A F V V L M A H 2
43 F R Q L R F E S T M 6 82 A F V V L M A H G R 2 213P1F11 V.1: HLA-B *0702 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
151 V I K D S P Q T I P
158 T I P T Y T D A L H
160 P T Y T D A L H V Y
166 L H V Y S T V E G Y
169 Y S T V E G Y I A Y
170 S T V E G Y I A Y R
172 v E σ Y I A Y R H D
174 G Y I A Y R H D Q K
175 Y I A Y R H D Q K G
176 I A Y R H D Q K G S
180 H D Q K G S C F I Q
190 T L V D V F T K R K
191 L D V F T K R K G
192 V D V F T K R K G H
227 T N P E I Q S T L R
213P1F11 v .1: HLA-B*4402 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
63 E E L E K F Q Q A I 23 Portion of
100 G Ξ M V K L Ξ N L F 23 SEQ ID
39 L E H M F R Q L R F 21 NO: 3; each
S P Y start
153 K D Q T I P T 19 position is
157 Q T I P T Y T D A L 19 specified,
146 D Ξ I V M V I K D S 18 the length
216 A Ξ L V Q Ξ G K A R 18 of each
30 E G S E E D L D A L 16 peptide is
59 E Q F Q E E L E K F 16 10 amino
97 G Ξ D G E M V K L E 16 acids, the
111 A L N N K N C Q A L 16 end position for each
118 Q A L R A K P K V Y 16 peptide is
229 P Ξ I Q S T L R K R 16 the start
12 Y D M S G A R L A L 15 position
33 E Ξ D L D A L E H M 15 plus nine
34 E D L D A L E H M F 15
186 C F I Q T L V D V F 15
226 K T N P Ξ I Q S T L 15
230 E I Q S T L R K R L 15
9 E E K Y D M S G A R 14
32 S E E D L D A L Ξ H 14
37 D A L E H M F R Q L 14
58 A Ξ Q F Q E E L Ξ K 14
74 S R E D P V S C A F 14
75 R Ξ D P V S C A F V 14
96 K G E D G Ξ M V K L 14
103 V K L E N L F Ξ A L 14
160 P T Y T D A L H V Y 14
182 Q K G S C F I Q T L 14
196 T K R K G H I L Ξ L 14
197 K R K G H I L E L L 14
231 I Q S T L R K R L Y 14
10 E K Y D M S G A R L 13
48 F Ξ S T M K R D P T 13
52 M K R D P T A E Q F 13
Figure imgf000255_0001
62 Q Ξ E L E K F Q Q A 13 213P1F11 v.l: HLA-B*4402 decamers 213P1F11 v.l: HLA-B*4402 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score Pos 1 2 3 4 5 6 7 8 9 0 score
77 D P V S C A F V V L 13 50 S T M K R D P T A E 5
105 L E N L F E A L N N 13 53 K R D P T A E Q F Q 5
150 M V I K D S P Q T I 13 71 A I D S R E D P V S 5
169 Y S T V E G Y I A Y 13 76 E D P V S C A F V V 5
203 L E L L T E V T R R 13 82 A F V V L M A H G R 5
214 A E A E L V Q E G K 13 92 E G F L K G E D G E 5
232 Q S T L R K R L Y- L 13 106 E N L F E A L N N K 5
3 N P R S L E E E K Y 12 107 N L F E A L N N K N 5
8 E E E K Y D M S G A 12 110 E A L N N K N C Q A 5
13 D M S G A R L A L I 12 114 N K N C Q A L R A K 5
14 M S G A R L A L I L 12 131 A C R G E Q R D P G 5
20 A L I L C V T K A R 12 141 E T V G G D E I V M 5
65 L E K F Q Q A I D S 12 142 T V G G D E I V M V 5
85 V L M A H G R E G F 12 165 A L H V Y S T V E G 5
109 F E A L N N K N C Q 12 185 S C F I Q T L V D V 5
119 A L R A K P K V Y I 12 200 G H I L E L L T E V 5
134 G E Q R D P G E T V 12 219 V Q E G K A R K T N 5
27 K A R E G S E E D L 233 S T L R K R L Y L Q 5
29 R E G S E E D L D A 1 M S P R S L E E E 4
99 D G E M V K L Ξ N L 4 P R S L E E E K Y D 4
143 V G G D E I V M V I • 41 H M F R Q L R F E S 4
166 L H V Y S T V E G Y 46 L R F E S T M K R D 4
172 V E G Y I A Y R H D 70 Q A I D S R E D P V 4
178 Y R H D Q K G S C F 72 I D S R E D P V S C 4
193 D V F T K R K G H I 78 P V S C A F V V L M 4
194 V F T K R K G H I L 80 S C A F V V L M A H 4
207 T E V T R R M A E A 113 N N K N C Q A L R A 4
209 V T R R M A E A E L 116 N C Q A L R A K P K 4
7 L E E E K Y D M S G 10 121 R A K P K V Y I I Q 4
56 P T A E Q F Q E E L 10 126 V Y I I Q A C R G E 4
86 L M A H G R E G F L 10 127 Y I I Q A C R G E Q 4
91 R E G F L K G E D G 10 135 E Q R D P G E T V G 4
140 G E T V G G D E I V 10 155 S P Q T I P T Y T D 4
220 Q E G K A R K T N P 10 170 S T V E G Y I A Y R 4
120 L R A K P K V Y 1 I 9 173 E G Y I A Y R H D Q 4
122 A K P K V Y I I Q A 9 174 G Y I A Y R H D Q K 4
139 P G E T V G G D E I 9 181 D Q K G S C F I Q T 4
179 R H D Q K G S C F I 9 190 T L V D V F T K R K 4
222 G K A R K T N P E I 9 199 K G H I L E L L T E 4
167 H V Y S T V E G Y I 8 215 E A E L V Q E G K A 4
66 E K F Q Q A I D S R 7 221 E G K A R K T N P E 4
88 A H G R E G F L K G 7 225 R K T N P E I Q S T 4
17 A R L A L I L C V T 6 2 S N P R S L E E E K 3
123 K P K V Y I I Q A C 6 18 R L A L I L C V T K 3
136 Q R D P G E T V G G 6 19 L A L I L C V T K A 3
204 E L T E V T R R M 6 49 E S M K R D P T A 3
208 E V T R R -M A E A E 6 54 R D P T A E Q F Q E 3
224 A R K T N P E I Q S 6 89 H G R E G F L K G E 3
11 K Y D M S G A R L A 5 98 E D G E M V K L E N 3
15 S G A R L A L I L C 5 101 E M V K L E N L F E 3
16 G A R L A L I L C V 5 104 K L E N L F E A L N 3
28 A R E G S E E D L D 5 115 K N C Q A L R A K P 3
38 A L E H M F R Q L R 5 137 R D P G E T V G G D 3
40 E H M F R Q L R F E 5 145 G D E I V M V I K D 3
Figure imgf000257_0002
Figure imgf000257_0001
Figure imgf000258_0003
Figure imgf000258_0002
Figure imgf000258_0001
Figure imgf000259_0002
Figure imgf000259_0003
Figure imgf000259_0001
Figure imgf000260_0002
213P1F11 v .2: HLA-B*0702 decamers
Figure imgf000260_0001
Pos 1 2 3 4 5 6 7 8 9 0 score
26 N P P L W N S Q D T 16 Portion of 213P1F11 v .2: HLA-B*4402 decamers
19 L P s E A P P N P P 13 SEQ ID Pos 1 2 3 4 5 6 7 8 9 0 score
20 P s E A P P N P P L 13 NO: 5; each 21 s Ξ A P P N P P L W 26 Portion of
E G P T P F start
8 Q D P L 12 38 T D M I R K A H A L 15 SEQ ID position is
9 G P T P F Q D P L Y 12 specified, 7 V Ξ G P T P F Q D P 14 NO: 5; each
10 P T P F Q D P L Y L 12 the length 8 E G P T P F Q D P L start
14 position is
23 A P P N P P L W N S 12 of each 9 G P T P F Q D P L Y 14 specified,
36 S P T D M I R K A H 12 peptide is 10 P T P F Q D P L Y L 13 the length
38 T D M I R K A H A L 12 10 amino 32 S Q D T S P T D M I 12 of each
15 D P L Y L P S E A P 1 1 acids, the 43 K A H A L S R P W 12 peptide is
24 P P N P P L w N S Q 1 1 end position 54 for each c S R R G K D I S W 12 10 amino
49 R P W w M c S R R G 1 1 peptide is 4 Y S T V E G P T P F 11 acids, the
11 T P F Q D P L Y L P 10 the start 42 R K A H A L S R P W 11 end position w for each
27 P P L N s Q D T S 10 position 20 P s E A P P N P P L 10 peptide is
44 A H A L S R P W W M 9 plus nine 52 W M C S R R G K D I 10 the start
56 R R G K D I s W N F 9 56 R' R G K D I S W N F 10 position
4 Y S T V E G P T P F 8 22 E A P P N P P L N 8 plus nine
29 L W N S Q D T S P T 8 35 T S P T D M I R K A 7
32 S Q D T S P T D M I 8 36 S P T D M I R K A H 7
37 P T D M I R K A H A 8 23 A P P N P P W N S 6
2 H V Y S T V E G P T 7 13 F Q D P L Y L P S E 5
31 N s Q D T S P T D M 7 17 ' L Y L P S E A P P N 5
14 Q D P L Y L P S Ξ A 6 25 P N P P L W N S Q D 5
35 T S P T D M I R K A 6 34 D T S P D M I R K 5
52 W M c S R R G K D I 6 39 D M I R K A H A L S 5
40 M I R K A H A L S R 5 44 A H A L S R P W M 5
22 E A P P N P P L W N 4 46 A L S R P W W M C S 5
43 K A H A L S R P W W 4 55 S R R G K D I S w N 5
46 A L S R P w W M C S 4 11 T P F Q D P L Y L P 4
55 S R R G K D I S N 4 24 P P N P P L W N S Q 4
3 V Y S T V E G P T P 3 3 V Y S T V E G P T P 3
5 S T V E G P T P F Q 3 6 T V E G P T P F Q D 3
7 V Ξ G P T P F Q D P 3 14 Q D P L Y L P S Ξ A 3
12 P F Q D P L Y L P s 3 15 D P L Y L P S E A P 3
13 F Q D P L Y L P S E 3 19 L P S E A P P N P P 3
17 L Y L P S E A P P N 3 26 N P P L W N S Q D T 3
21 S Ξ A P P N P P L W 3 31 N S Q D T S P T D M 3
30 W N S Q D T S P T D 3 47 L S R P W W M C S R 3
34 D T S P T D M I R K 3 51 W w M C S R R G K D 3
42 R K A H A L S R P W 3 5 S T V E G P T P F Q 2
47 L S R P W W M c S R 3 12 P F Q D P L L P S 2
6 T V E G P T P F Q D 2 18 Y L P S E A P P N P 2
16 P L Y L P S E A P P 2 27 P P L W N S Q D T s 2
Figure imgf000261_0002
Figure imgf000261_0004
Figure imgf000261_0003
Figure imgf000261_0001
213P1F11 v.3: HLA-A3 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
3 I I Q A C R G A T L 22 Portion of
2 Y I I Q A C R G A T 14 SEQ ID
11 T L P S P F P Y L S 11 NO: 7; each C R G A T L P S P start
6 A 10 position is
7 C R G A T L P S P F 10 specified,
5 Q A C R G A T L P S 8 the length
9 G A T L P S P F P Y 8 of each
10 A T L P S P F P Y L 8 peptide is
12 L S P F P Y L S L 8 10 amino
4 I Q C R G A T L P 7 acids, the
8 R G A T L P S P F P 5 end position for each
1 V Y I I Q A C R G A 4 peptide is the start position plus nine
Figure imgf000262_0001
TABLE XIXB, part 4: MHC Class I decamer analysis of 213P1F11 v.4 (aa 1-321).
213P1F11 v.4: HLA-A*0201 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
12 S V Q P E K R T G L 19 Portion of
60 E V F G σ G V G D I 16 SEQ ID
65 G V G D I V G R D L 16 NO: 9; each-
10 S L S V Q P E K R T start
15 position is
52 V N D P R E T Q E V 15 specified,
57 E T Q E V F G G G V 14 the length
67 G D I V G R D L S I 14 of each
42 F R G S S V H Q K L 13 peptide is
50 K L V N D P R E T Q 13 10 amino
61 V F G G G V G D I V 13 acids, fhe
68 D I V G R D L S I S 13 end position for each
84 S A S E E E K Y D M 13 peptide is
2 G K C Q E Y D K S L 12 the start
20 G L R D E N G E C G 12 position
33 R L K E E Q G R A F 12 plus nine
38 Q G R A F R G S S V 12
Figure imgf000262_0002
Figure imgf000263_0002
Figure imgf000263_0003
Figure imgf000263_0004
Figure imgf000263_0001
Figure imgf000264_0002
Figure imgf000264_0003
Figure imgf000264_0001
Figure imgf000265_0002
Figure imgf000265_0001
Figure imgf000266_0002
Figure imgf000266_0001
E13P1F11 v.5: HLA-A*0203 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
L A L I L R V T K A 10 Portion of
A L I L R V T K R SEQ ID
L I L R V T K A R E NO: 11; each start position is specified, the length
Figure imgf000267_0001
of each peptide is
TABLE XIXB, part 5: MHC Class I decamer 10 amino analysis of 213P1F11 v.5 (aa 1 -242). acids, the
Ω13P1F11 v.5: HLA-A*0201 decamers end position
Pos for each
1 2 3 4 5 6 7 8 9 0 score peptide is
G A R L A L I L R V 20 Portion of the start
L A L I L R V T K A 18 SEQ ID position
R L A L I L R V T K 16 NO: 11; plus nine
A L I L R V T K R 14 each start position is
L I L R V T K A R E 14 specified,
I L R V T K A R E G 14 the length
A R L A L I L R V T 12 of each
S G A R L A L I R peptide is
10 R V T KA R E G S E 10 amino
L R V T K A R E G S acids, the end position for each peptide is the start position plus nine
213P1F11 v.5: HLA-A*0202 decamers
Pos 1 2 3 4 5 6 7 8 9 0 score
S G A R L A L I L R Portion of
R L A L I L R V T K SEQ ID
G A R L A L I L R V NO: 11;
Figure imgf000267_0002
L A L I L R V T K A each start position is
A R L A L I L R T specified,
A L I L R V T K A R the length of each peptide is
10 amino acids, the end position for each peptide is the start position plus nine
Figure imgf000267_0003
213P1F11 v.5: HLA-A3 decamers ! | TABLE XIXB, part 6: MHC Class I decamer
Pos 1 2 3 4 5 6 7 8 9 0 score anal sis of 213P1F11 v.6 (aa 1 -242.
4 R L A L I L R V T K 35 Portion of
6 A L I L R V T K A R 22 SEQ ID
8 I L R V T K A R E G 18 NO: 11;
R V T K A R E G S E each start
10 17 position is
7 L I L R V T K A R E 16 specified,
3 A R L A L I L R V T 11 the length
1 S G A R L A L I L R 9 of each
2 G A R L A L I L R V 7 peptide is
5 L A L I L R V T K A 4 10 amino
9 L R V T K A R E G S 1 acids, the end position for each peptide is the start position plus nine
213P1F11 v.5: HLA-B *0702 decamers
Figure imgf000268_0002
Pos 1 2 3 4 5 6 7 8 9 0 score
2 G A R L A L I L R V 10 Portion of 213P1F11 v.6: HLA-A*0202 decamers
3 A R L A L I L R V T 10 SEQ.ID Pos 1 2 3 4 5 6 7 8 9 0 score
5 L A L I L R V T K A 8 NO: 11; F E A M N N K N C Q Portion of
R L A L I L R V T K each start
4 5 E A M N N K N C.Q A SEQ ID position is
6 A L I L R V T K A R 4 specified, A M N N K N C Q A Li NO: 13;
L R V T K A R Ξ G each start
8 I 3 the length position is
10 R V T K A R E G S E 2 of each specified,
1 S G A R L A L I L R 1 peptide is the length
7 L I L R V T K A R E 1 10 amino of each
9 L R V T K A R E G S 1 acids, the peptide is end position 10 amino for each acids, the peptide is end position the start for each position peptide is plus nine the start position plus nine
Figure imgf000268_0001
Figure imgf000268_0003
Figure imgf000269_0002
Figure imgf000269_0003
Figure imgf000269_0004
Figure imgf000269_0001
TABLE XIXC: MHC Class II Analysis of 213P1F11. Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book "MHC Ligands and Peptide Motifs" by H.G.Rammensee, J.Bachmann and S.Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes.
Table XIXC, part 1: MHC Class II 15-mer analysis of 213P1F11 v.l (aa 1-242).
213PlFll .1: HLA-DRBl*010115-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
166 L H V Y S T V E G Y I A Y R H 33 Portion of
9 E E K Y D M S G A R L A L I L 29 SEQ ID
80 S C A F V V L M A H G R E G F 29 NO: 3;
192 V D V F T K R K G H I L E L L each start
29 position is
92 E G F L K G E D G E M V K L E 25 specified,
123 K P K V Y I I Q A C R G E Q R 25 the length
63 E E L E K F Q Q A I D S R E D 24 of each
145 G D E I V M V I K D S P Q T I 24 peptide is
148 I V M V I K D S P Q T I P T Y 24 15 amino
203 L E L L T E V T R R M A E A E 24 acids, the
137 R D P G E T V G G D E I V M V 23 end position for
202 I L E L L T E V T R R M A E A 23 each
8 E E E K Y D M S G A R L A L I 22 peptide is
39 L E H M F R Q L R F E S T M K 22 the start
124 P K V Y I I Q A C R G E Q R D 22 position
176 I A Y R H D Q K G S C F I Q T 22 plus
188 I Q T L V D V F T K R K G H I 22 fourteen
199 K G H I L E L L T E V T R R M 22
17 A R L A L I L C V T K A R E G 21
91 R E G F L K G E D G E M V K L 21
40 E H M F R Q L R F E S T M K R 20
99 D G E M V K L E N L F E A L N 20
208 E V T R R M A E A E L V Q E G 20
45 Q L R F E S T M K R D P T A E 19
83 F V V L M A H G R E G F L K G 19
106 E N L F E A L N N K N C Q A L 19
159 I P T Y T D A L H V Y S T V E 19
43 F R Q L R F Ξ S T M K R D P T 18
50 S T M K R D P T A E Q F Q Ξ E 18
82 A F V V L M A H G R E G F L K 18
100 G E M V K L E N L F E A L N N 18
117 C Q A L R A K P K V Y I I Q A 18
147 E I V M V I K D S P Q T I P T 18
149 V M V I K D S P Q T I P T Y T 18
184 G S C F I Q T L V D V F T K R 18
216 A E L V Q E G K A R K T N P E 18
20 A L I L C V T K A R E G S E E 17
33 E E D L D A L E H M F R Q L R 17
69 Q Q A I D S R E D P V S C A F 17
89 H G R E G F L K G E D G E M V 17
109 F E A L N N K N C Q A L R A K 17
111 A L N N K N C Q A L R A K P K 17
114 N K N C Q A L R A K P K V Y I 17
140 G E T V G G D E I V M V I K D 17
157 Q T I P T Y T D A L H V Y S T 17
183 K G S C F I Q T L V D V F T K 17
200 G H I L E L L T E V T R R M A 17
206 L T E V T R R M A E A E L V Q 17
228 N P E I Q S T L R K R L Y L Q 17
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
213P1F11 v .1: HLA-DRB1*0301 (DR17) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
228 N P E I Q S T L R K R L Y L Q 26 Portion of
83 F V V L M A H G R E G F L K G 25 SEQ ID
84 V V L M A H G R E G F L K G E 24 NO 3,
49 E S T M K R D P T A E F each start
Q Q E 23 position is
148 I V M V I K D S P Q T I P T Y 23 specified,
140 G E T V G G D E I V M V I K D 22 the length
105 L E N L F E A L N N K N c Q A 20 of each
109 F E A L N N K N C Q A L R A K 20 peptide is
19 L A L I L C V T K A R E G S E 19 15 ammo
99 D G E M V K L E N L F E A L N 19 acids, the
117 C Q A L R A K P K V Y I I Q A 19 end
E A E L V Q E G K A R K T N P position for
215 19 each
1 M S N P R S L E E E K Y D M s 18 peptide is
36 L D A L E H M F R Q L R F E S 18 the start
93 G F L K G E D G E M V K L E N 18 position
102 M V K L E N L F E A L N N K N 18 plus
145 G D E I V M V I K D S P Q T I 18 fourteen
149 V M V I K D S P Q T I P T Y T 18
158 T I P T Y T D A L H V Y s T V 18
184 G S C F I Q T L V D V F T K R 18
192 V D V F T K R K G H I L E L L 18
200 G H I L E L L T E V T R R M A 18
203 L E L L T E V T R R M A E A E 18
10 E K Y D M S G A R L A L I L C 17
22 I L C V T K A R E G S E E D L 17
25 V T K A R E G S E E D L D A L 17
32 S E Ξ D L D A L E H M F R Q L 17
39 L E H M F R Q L R F E S T M K 17
62 Q E E L E K F Q Q A I D S R E 17
67 K F Q Q A I D S R E D P V S C 17
98 E D G E M V K L E N L F E A L 17
125 K V Y I I Q A C R G E Q R D P 17
165 A L H V Y S T V E G Y I A Y R 17
175 Y I A Y R H D Q K G S c F I Q 17
189 Q T L V D V F T K R K G H I L 17
37 D A L E H M F R Q L R F E s T 16
58 A E Q F Q E E L E K F Q Q A I 16
191 L V D V F T K R K G H I L E L 16
207 T E V T R R M A Ξ A E L V Q E 16
45 Q L R F E S T M K R D P T A E 15
54 R D P T A E Q F Q E E L E K F 15
106 E N L F E A L N N K N C Q A L 15
224 A R K T N P E I Q S T L R K R 15
29 R E G S E E D L D A L E H M F 14
199 K G H I L E L L T E V T R R M 14
16 G A R L A L I L C V T K A R E 13
18 R L A L I L C V T K A R E G S 13
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
213P1F11 v .1: HLA-DRB1*0401 (DR4Dw4) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
159 I P T Y T D A L H V Y S T V E 28 Portion of
49 E S T M K R D P T A E Q F Q E 26 SEQ ID
105 L E N L F E A L N N K N C Q A 26 NO: 3;
125 K V Y I I Q A C R G E Q R D P 26 each start position is
188 I Q T L V D V F T K R K G H I 26 specified,
199 K G H I L E L L T E V T R R M 26 the length
202 I L E L L T E V T R R M A E A 26 of each
203 L Ξ L L T E V T R R M A E A E 26 peptide is
40 E H M F R Q L R F E S T M K R 22 15 amino
58 A Ξ Q F Q E E L E K F Q Q A I 22 acids, the
106 E N L F E A L N N K N C Q A L 22 end position for
166 L H V Y S T V E G Y I A Y R H 22 each
172 V E G Y I A Y R H D Q K G S C 22 peptide is
4 P R S L E E E K Y D M S G A R 20 the start
11 K Y D M S G A R L A L I L C V 20 position
16 G A R L A L I L C V T K A R E 20 plus
33 E E D L D A L E H M F R Q L R 20 fourteen
39 L E H M F R Q L R F E S T M K 20
43 F R Q L R F E S T M K R D P T 20
62 Q E E L E K F Q Q A I D S R E 20
76 E D P V S C A F V V L M A H G 20
92 E G F L K G E D G E M V K L E 20
99 D G E M V K L E N L F E A L N 20
100 G E M V K L E N L F E A L N N 20
102 M V K L E N L F E A L N N K N 20
140 G E T V G G D E I V M V I K D 20
146 D E I V M V I K D S P Q T I P 20
148 I V M V I K D S P Q T I P T Y 20
185 S C F I Q T L V D V F T K R K 20
215 E A E L V Q E G K A R K T N P 20
228 N P E I Q S T L R K R L Y L Q 20
42 M F R Q L R F E S T M K R D P 18
171 T V E G Y I A Y R H D Q K G S 18
181 D Q K G S C F I Q T L V D V F 18
212 R M A E A E L V Q E G K A R K 18
224 A R K T N P E I Q S T L R K R 18
9 E E K Y D M S G A R L A L I L 16
65 L E K F Q Q A I D S R E D P V 16
80 S C A F V V L M A H G R E G F 16
91 R E G F L K G E D G E M V K L 16
175 Y I A Y R H D Q K G S C F I Q 16
147 E I V M V I K D S P Q T I P T 15
18 R L A L I L C V T K A R E G S 14
19 L A L I L C V T K A R E G S E 14
22 I L C V T K A R E G S E E D L 14
36 L D A L E H M F R Q L R F E S 14
81 C A F V V L M A H G R E G F L 14
82 A F V V L M A H G R E G F L K 14
83 F V V L M A H G R E G F L K G 14
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
213P1F11 v .1: HLA-DRB1*1101 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
203 L E L L T E V T R R M A E A E 29 Portion of
145 G D E I V M V I K D S P Q T I 26 SEQ ID
159 I P T Y T D A L H V Y S T V E 26 NO: 3; each start
45 Q L R F E S T M K R D P T A E 25 position is
166 L H V Y S T V E G Y I A Y R H 23 specified,
189 Q T L V D V F T K R K G H I L 23 the length
19 L A L I L C V T K A R E G S E 22 of each
81 C A F V V L M A H G R E G F L 22 peptide is
36 L D A L E H M F R Q L R F E S 21 15 amino
39 L E H M F R Q L R F E S T M K 21 acids, the
33 E E D L D A L E H M F R R 20 end
Q L position for
123 K P K V Y I I Q A C R G E Q R 20 each
9 E E K Y D M S G A R L A L I L 18 peptide is
172 V E G Y I A Y R H D Q K G s c 18 the start
175 Y I A Y R H D Q K G S C F I Q 18 position
80 S C A F V V L M A H G R E G F 17 plus
40 E H M F R Q L R F E S T M K R 16 fourteen
96 K G E D G E M V K L E N L F E 16
106 E N L F E A L N N K N C Q A L 16
215 E A E L V Q E G K A R K T N P 16
228 N P E I Q S T L R K R L Y L Q 16
113 N N K N C Q A L R A K P K V Y 15
144 G G D E I V M V I K D S P Q T 15
191 L V D V F T K R K G H I L E L 15
20 A L I L C V T K A R E G S E E 14
21 L I L C V T K A R E G S E E D 14
46 L R F E S T M K R D P T A E Q 14
59 E Q F Q E E L E K F Q Q A I D 14
67 K F Q Q A I D S R E D P V s C 14
79 V S C A F V V L M A H G R E G 14
83 F V V L M A H G R E G F L K G 14
117 C Q A L R A K P K V Y I I Q A 14
125 K V Y I I Q A C R G E Q R D P 14
129 I Q A C R G E Q R D P G E T V 14
185 S C F I Q T L V D V F T K R K 14
188 I Q T L V D V F T K R K G H I 14
218 L V Q E G K A R K T N P E I Q 14
17 A R L A L I L C V T K A R E G 13
69 Q Q A I D S R E D P V S C A F 13
82 A F V V L M A H G R E G F L K 13
102 M V K L E N L F E A L N N K N 13
146 D E I V M V I K D S P Q T I P 13
149 V M V I K D S P Q T I P T Y T 13
163 T D A L H V Y S T V E G Y I A 13
173 E G Y I A Y R H D Q K G S c F 13
192 V D V F T K R K G H I L E L L 13
199 K G H I L E L L T E V T R R M 13
213 M A E A E L V Q E G K A R K T 13
4 P R S L E E E K Y D M S G A R 12
16 G A R L A L I L C V T K A R E 12
65 L E K F Q Q A I D S R E D P V 12
89 H G R E G F L K G E D G E M V 12
99 D G E M V K L E N L F E A L N 12
100 G E M V K L E N L F E A L N N 12
120 L R A K P K V Y I I Q A C R G 12
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
TABLE XIXC, part 2: MHC Class II 15-mer analysis « jf213PlFllv.2 (aa 1-230).
213P1F11 .2: HLA-DRBl*010115-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
6 L H V Y S T V E G P T P F Q D 33 Portion of
55 P W M C S R R G K D I S N 25 SEQ ID
32 P P L N S Q D T S P T D M I 23 NO: 5;
18 F Q D P L Y L P S E A P P N P 22 each start position is
21 P L Y L P S E A P P N P P L w 22 specified,
13 E G P T P F Q D P L Y L P S E 21 the length
43 T D M I R K A H A L S R P W 20 of each
20 D P L Y L P S E A P P N P P L 19 peptide is
41 S P T D M I R K A H A L S R P 19 15 amino
15 P T P F Q D P L Y L P S E A P 18 acids, the
7 H V Y S T V E G P T P F Q D P 17 end position for
19 Q D P L Y L P S E A P P N P P 17 each
31 N P P L N S Q D T S P T D M 17 peptide is
42 P T D M I R K A H A L S R P 16 the start
49 A H A L S R P W M C S R R G 16 position
17 P F Q D P L Y L P S E A P P N 15 plus
34 L W N S Q D T S P T D M I R K 15 fourteen
40 T S P T D M I R K A H A L S R 15
47 R K A H A L S R P W M C S R 15
3 T D A L H V Y S T V E G P T P 14
9 Y S T V E G P T P F Q D P L Y 14
22 L Y L P S E A P P N P P L W N 14
35 • N S Q D T S P T D M I R K A 14
53 S R P W W M C S R R G K D I S 14
2 Y T D A L H V Y S T V E G P T 13
52 L S R P M C S R R G K D I 13
4 D A L H V Y S T V Ξ G P T P F 11
54 R P W M C S R R G K D I S W 11
38 Q D T S P T D M I R K A H A L 10
1 T Y T D A L H V Y S T V E G P 9
44 D M I R K A H A L S R P W M 9
56 W W M C S R R G K D I S N F 9
5 A L H V Y S T V E G P T P F Q 8
10 S T V E G P T P F Q D P L Y L 8
11 T V E G P T P F Q D P L Y L P 8
14 G P T P F Q D P L Y L P S E A 8
16 T P F Q D P L Y L P S E A P P 8
23 Y L P S E A P P N P P L W N S 8
26 S E A P P N P P L N S Q D T 8
28 A P P N P P L W N S Q D T S P 8
Figure imgf000286_0001
213P1F11 v .2: HLA-DRB1*0301 (DR17) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
19 Q D P L Y L P S E A P P N P P 19 Portion of
5 A L H V Y S T V E G P T P F Q 17 SEQ ID
23 Y L P S E A P P N P P L N s 15 - NO: 5;
11 T V E G P T P F Q D P L Y L P 14 each start position is
42 P T D M I R K A H A L S R P w 13 specified,
3 T D A L H V Y S T V E G P T P 12 the length
14 G P T P F Q D P L Y L P S E A 12 of each
21 P L Y L P S E A P P N P P L 12 peptide is
43 T D M I R K A H A L S R P W 12 15 amino
12 V Ξ G P T P F Q D P L Y L P S 11 acids, the
38 Q D T S P T D M I R K A H A L 11 end position for
55 P W M C S R R G K D I S W N 11 each
7 H V Y S T V E G P T P F Q D P 10 peptide is
9 Y S T V E G P T P F Q D P L Y 10 the start
15 P T P F Q D P L Y L P S E A P 10 position
31 N P P L W N S Q D T S P T D M 10 plus
33 P L W N S Q D T S P T D M I R 10 fourteen
41 S P T D M I R K A H A L S R P 10
49 A H A L S R P W M C S R R G 10
13 E G P T P F Q D P L Y L P S E 9
30 P N P P L W N S Q D T S P T D 9
39 D T S P T D M I R K A H A L S 8
40 T S P T D M I R K A H A L S R 8
53 S R P M C S R R G K D I S 8
28 A P P N P P L W N S Q D T s P 7
46 I R K A H A L S R P M c s 7
54 R P W M C S R R G K D I s w 7
56 W M C S R R G K D I S N F 7
18 F Q D P L Y L P S E A P P N P 4
20 D P L Y L P S E A P P N P P L 4
48 K A H A L S R P W W M C s R R 4
8 V Y S T V E G P T P F Q D P L 3
10 ' S T V E G P T P F Q D P L Y L 3
22 L Y L P S E A P P N P P L w N 3
25 P S E A P P N P P L N S Q D 3
27 E A P P N P P L W N S Q D T S 3
2 Y T D A L H V Y S T V E G P T 2
17 P F Q D P L Y L P S E A P P N 2
24 L P S E A P P N P P L N s Q 2
Figure imgf000287_0001
213P1F11 .2: HLA-DRB1*0401 (DR4Dw4) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
32 P P L W N S Q D T S P T D M I 22 Portion of
53 S R P W W M C S R R G K D I s 22 SEQ ID
6 L H V Y S T V E G P T P F Q D 16 NO: 5;
P T P F Q D P L Y L P S E A P each start
15 16 position is
20 D P L Y L P S E A P P N P P L 16 specified,
54 R P W W M C S R R G K D I S W 16 the length
42 P T D M I R K A H A L S R P w 15 of each
3 T D A L H V Y S T V E G P T P 14 peptide is
5 A L H V Y S T V E G P T P F Q 14 15 amino
21 P L Y L P S E A P P N P P L w 14 acids, the
43 T D M I R K A H A L S R P W w 14 end R P W W M C S R R G position for
49 A H A L S 14 each
2 Y T D A L H V Y S T V E G P T 12 peptide is
8 V Y S T V E G P T P F Q D P L 12 the start
11 T V Ξ G P T P F Q D P L Y L P 12 position
18 P Q D P L Y L P S E A P P N P 12 plus
23 Y L P S E A P P N P P L W N s 12 fourteen
28 A P P N P P L W N S Q D T S P 12
29 P P N P P L W N S Q D T S P T 12
30 P N P P L W N S Q D T S P T D 12
33 P L W N S Q D T S P T D M I R 12
35 W N S Q D T S P T D M I K A 12
38 Q D T S P T D M I R K A H A L 12
40 T S P T D M I R K A H A L S R 12
45 M I R K A H A L S R P W W M C 12
46 I R K A H A L S R P W W M C S 12
55 P W W M C S R R G K D I S W N 9
9 Y S T V E G P T P F Q D P L Y 8
19 Q D P L Y L P S E A P P N P P 8
31 N P P L W N S Q D T S P T D M 8
1 T Y T D A L H V Y S T V E G P 6
7 H V Y S T V E G P T P F Q D P 6
12 V E G P T P F Q D P L Y L P S 6
13 Ξ G P T P F Q D P L Y L P S E 6
14 G P T P F Q D P L Y L P S E A 6
16 T P F Q D P L Y L P S E A P P 6
17 P F Q D P L Y L P S E A P P N 6
Figure imgf000288_0002
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000289_0002
Figure imgf000290_0001
Figure imgf000290_0002
TABLE XIXC, part 4: MHC Class II 15-mer analysis Df213PlFll v.4 (aa 1-321).
213P1F11 .4: HLA-DRB1*010115-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
63 G G G V G D I V G R D L S I S 31 Portion of
75 S I S F R N S E T S A S E E E 27 SEQ ID
4 C Q E Y D K S L S V Q P E K R 26 NO: 9;
31 T F R L K E E Q G R A F R G S 26 each start position is
59 Q E V F G G G V G D I V G R D 26 specified,
55 P R E T Q E V F G G G V G D I 24 the length
73 D L S I S F R N S E T S A S E 23 of each
67 G D I V G R D L S I S F R N S 22 peptide is
10 S L S V Q P E K R T G L R D Ξ 19 15 amino
18 R T G L R D E N G E C G Q T F 18 acids, the
36 E E Q G R A F R G S S V H Q K 18 end G V G D I V G R D L S I S F position for
64 G 18 each
21 L R D E N G E C G Q T F R L K 17 peptide is
58 T Q E V F G G G V G D I V G R 17 the start
66 V G D I V G R D L S I S F R N 17 position
29 G Q T F R L K E E Q G R A F R 16 plus
44 G S S V H Q K L V N D P R E T 16 fourteen
8 D K S L S V Q P E K R T G L R 15 .
41 A F R G S S V H Q K L V N D P 15
50 K L V N D P R E T Q E V F G G 15
70 V G R D L S I S F R N S E T S 15
2 G K C Q E Y D K S L S V Q P E 14
23 D E N G E C G Q T F R L K E E 14
7 Y D K S L S V Q P E K R T G L 13
57 E T Q E V F G G G V G D I V G 13
39 G R A F R G S S V H Q K L V N 12
85 A S E E E K Y D M S G A R L A 12
30 Q T F R L K E E Q G R A F R G 11
34 L K E E Q G R A F R G S S V H 11
71 G R D L S I S F R N S E T S A 11
12 S V Q P E K R T G L R D E N G 10
45 S S V H Q K L V N D P R E T Q 10
49 Q K L V N D P R E T Q E V F G 10
68 D I V G R D L S I S F R N S E 10
5 Q E Y D K S L S V Q P E K R T 9
15 P E K R T G L R D E N G E C G 9
40 R A F R G S S V H Q K L V N D 9
48 H Q K L V N D P R E T Q E V F 9
53 N D P R E T Q E V F G G G V G 9
56 R E T Q E V F G G G V G D I V 9
74 L S I S F R N S E T S A S E E 9
82 E T S A S E E E K Y D M S G A 9
1 M G K C Q E Y D K S L S V Q P 8
28 C G Q T F R L K E E Q G R A F 8
33 R L K E E Q G R A F R G S S V 8
35 K E Ξ Q G R A F R G S S V H Q 8
37 E Q G R A F R G S S V H Q K L 8
46- S V H Q K L V N D P R E T Q Ξ 8
51 L V N D P R E T Q E V F G G G 8
65 G V G D I V G R D L S I S F R 8
77 S F R N S E T S A S E E E K Y 8
83 T S A S E E E K Y D M S G A R 8
24 E N G E C G Q F R L K E E Q 7
32 F R L K E E Q G R A F R G S S 7
Figure imgf000292_0001
213P1F11 v .4: HLA-DRB1*0301 (DR17) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
67 G D I V G R D L S I s F R N S 29 Portion of
48 H Q K L V N D P E T Q E V F 28 SEQ ID
10 S L S V Q P Ξ K R T G L R D E 26 NO: 9;
31 T F R L K Ξ E Q G R A F R G S 21 each start position is
17 K R T G L R D E N G E C G Q T 19 specified,
29 G Q T F R L K E E Q G R A F R 18 the length
63 G G G V G D I V G R D L S I S 18 of each
8 D K S L S V Q P E K R T G L R 17 peptide is
71 G R D L S I S F R N S E T S A 17 15 amino
49 Q K L V N D P R E T Q E V F G 16 acids, the
80 N S E T S A S E E E K Y D M S 16 end position for
51 L V N D P R Ξ T Q E V F G G G 14 each
2 G K C Q E Y D K S L S V Q P E 12 peptide is
44 G S S V H Q K L V N D P R E T 12 the start
86 S E E E K Y D M S G A R L A L 12 position
18 R T G L R D E N G E C G. Q T F 11 plus
40 R A F R G S S V H Q K L V N D 11 fourteen
58 T Q E V F G G G V G D I V G R 11
62 F G G G V G D I V G R D L S I 11
66 V G D I V G R D L S I S F R N 11
75 S I S F R N S E T S A S E E E 11
39 G R A F R G S S V H Q K L V N 10
73 D L S I S F R N S E T S A S E 10
21 L R D E N G Ξ C G Q T F R L K 9
Figure imgf000293_0001
Figure imgf000294_0001
213P1F11 .4: HLA-DRBl*0401(DR4Dw4)15-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
10 S L S V Q P E K R T G L R D E 26 Portion of
48 H Q K L V N D P R E T Q E V F 26 SEQ ID
75 S I S F R N S E T S A S E E E 22 NO: 9;
G G G V G D I V G R D L S I S 20 each start
63 position is
67 G D I V G R D L S I S F R N S 20 specified,
2 G K C Q E Y D K S L S V Q P E 18 the length
30 Q T F R L K E E Q G R A F R G 18 of each
35 K E E Q G R A F R G S S V H Q 18 peptide is
41 A F R G S S V H Q K L V N D P 18 15 amino
64 G G V G D I V G R D L S I S F 18 acids, the
72 R D L S I S F R N S E T S A S 18 end position for
4 C Q E Y D K S L S V Q P E K R 16 each
39 G R A F R G S S V H Q K L V N 16 peptide is
59 Q E V F G G G V G D I V G R D 16 the start
73 D L S I S F R N S E T S A S E 15 position
8 D K S L S V Q P E K R T G L R 14 plus
18 R T G L R D E N G E C G Q T F 14 fourteen
31 T F R L K E E Q G R A F R G S 14
58 T Q E V F G G G V G D I V G R 14
71 G R D L S I S F R N S E T S A 14
6 E Y D K S L S V Q P E K R T G 12
14 Q P E K R T G L R D E N G E C 12
17 K R T G L R D E N G E C G Q T 12
23 D E N G E C G Q T F R L'K E E 12
36 E E Q G R A F R G S S V H Q K 12
38 Q G R A F R G S S V H Q K L V 12
40 R A F R G S S V H Q K L V N D 12
45 S S V H Q K L V N D P R E T Q 12
51 L V N D P R E T Q E V F G G G 12
55 P R E T Q E V F G G G V G D I 12
69 I V G R D L S I S F R N S E T 12
70 V G R D L S I S F R N S E T S 12
76 I S F R N S E T S A S E E E K 12
79 R N S E T S A S E E E K Y D M 12
82 E T S A S E E E K Y D M S G A 12
83 T S A S E E E K Y D M S G A R 12
86 S E E E K Y D M S G A R L A L 12
66 V G D I V G R D L S I S F R N 9
49 Q K L V N D P R E T Q E V F G 8
50 K L V N D P R E T Q E V F G G 7
1 M G K C Q Ξ Y D K S L S V Q P 6
5 Q E Y D K S L S V Q P E K R T 6
7 Y D K S L S V Q P E K R T G L 6
15 P E K R T G L R D E N G E C G 6
19 T G L R D E N G E C G Q T F R 6
20 G L R D E N G E C G Q T F R L 6
21 L R D E N G E C G Q T F R L K 6
22 R D E N G E C G Q T F R L K E 6
24 E N G E C G Q T F R L K E E Q 6
25 N G E C G Q T F R L K E E Q G 6
Figure imgf000295_0001
213P1F11 v .4: HLA-DRB1*1101 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
64 G G V G D I V G R D L S I S F 21 Portion of
4 C Q E Y D K S L S V Q P E K R 16 SEQ ID
75 S I S F R N S E T S A s E E E 16 NO: 9;
M G K C Q E Y D K S L s V each start
1 Q P 15 position is
9 K S L S V Q P E K R T G L R D 15 specified,
63 G G G V G D I V G R D L S I S 15 the length
10 S L S V Q P E K R T G L R D E 14 of each
15 P E K R T G L R D E N G E C G 14 peptide is
35 K E E Q G R A F R G S s V H Q 14 15 amino
48 H Q K L V N D P R E T Q E V F 14 acids, the
71 G R D L S I S F R N S E T S A 14 end position for
28 C G Q T F R L K E E Q G R A F 13 each
59 Q E V F G G G V G D I V G R D 13 peptide is
60 E V F G G G V G D I V G R D L 13 the start
66 V G D I V G R D L s I S F R N 13 position
70 V G R D L S I S F R N S E T S 13 plus
18 R T G L R D E N G E C G Q T F 12 fourteen
44 G S S V H Q K L V N D P R E T 12
73 D L S I S F R N S E T S A S E 12
40 R A F R G S S V H Q K L V N D 11
27 E C G Q T F R L K E E Q G R A 10
29 G Q T F R L K Ξ E Q G R A F R 10
39 G R A F R G S S V H Q K L V N 10
42 F R G S S V H Q K L V N D P R 10
54 D P R E T Q E V F G G G V G D 10
8 D K S L S V Q P E K R T G L R 9
Figure imgf000296_0001
Figure imgf000296_0002
213P1F11 v .5: HLA-DRB1*0301 (DR17) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
7 G A R L A L I L R V T K A R E 19 Portion of
10 L A L I L R V T K A R E G S E 19 SEQ ID
1 E K Y D M S G A R L A L I L R 17 NO: 11; L R V T K A R E G S E E D L each start
13 I 17 position is
9 R L A L I L R V T K A R E G S 13 specified,
2 K Y D M S G A R L A L I L R V 12 the length
11 A L I L R V T K A R E G S E E 12 of each
3 Y D M S G A R L A L I L R V T 10 peptide is
12 L I L R V T K A R Ξ G S E E D 7 15 amino
5 M S G A R L A L I L R V T K A 4 acids, the R V T K A R E G 3 end
8 A R L A L I L position for
6 S G A R L A L I L R V T K A R 2 each
14 L R V T K A R E G S E E D L D 2 peptide is
4 D M S G A R L A L I L R V T K 1 the start
15 R V T K A R E G S E E D L D A 1 position plus fourteen
213P1F11 .5: HLA-DRB1*0401 (DR4Dw4) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
7 G A R L A L I L R V T K A R E 26 Portion of
2 K Y D M S G A R L A L I L R V 20 SEQ ID
10 L A L I L R V T K A R E G S E 14 NO: 11;
I L R V T K A R E G S E E D L each start
13 14 position is
1 E K Y D M S G A R L A L I L R 12 specified,
5 M S G A R L A L I L R V T K A 12 the length
6 S G A R L A L I L R V T K A R 12 of each
8 A R L A L I L R V T K A R E G 12 peptide is
15 R V T K A R E G S E E D L D A 12 15 amino
9 R L A L I L R V T K A R E G S 9 acids, the
A L I A R E G S E Ξ end
11 L R V T K 8 position for
12 L I L R V T K A R E G S E E D 7 each
4 D M S G A R L A L I L R V T K 6 peptide is
3 Y D M S G A R L A L I L R V T 1 the start
14 L R V T K A R E G S E E D L D -5 position plus fourteen
213P1F11 v .5: HLA-DRB1*110115 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
10 L A L I L R V T K A R E G S E 22 Portion of
7 G A R L A L I L R V T K A R E 20 SEQ ID
11 A L I L R V T K A R E G S E E 14 NO: 11;
12 L I L R V T K A R E G S E E D 14 each start position is
8 A R L A L I L R V T K A R E G 13 specified,
1 E K Y D M S G A R L A L I L R 8 the length
6 S G A R L A L I L R V T K A R 8 of each
9 R L A L I L R V T K A R E G S 8 peptide is
13 I L R V T K A R E G S E Ξ D L 8 15 amino
2 K Y D M S G A R L A L I L R V 7 acids, the end position for each peptide is the start position plus
4 D M S G A R L A L I L R V T K 7 fourteen
TABLE XIXC, part 6: MHC Class II 15-mer analysis < 3f213PlFll v.6 (aa 1-242).
213P1F11 v .6: HLA-DRBl*010115-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
2 D G E M V K L E N L F E A M N 20 Portion of
3 G E M V K L E N L F E A M N N 18 SEQ ID
9 E N L F E A M N N K N C Q A L 17 NO: 13;
12 F E A M N N K N C Q A L R A K 17 each start position is
14 A M N N K N C Q A L R A K P K 17 specified,
5 M V K L E N L F E A M N N K N 16 the length
6 V K L E N L F E A M N N K N C 11 .of each
8 L E N L F E A M N N K N C Q A 9 peptide is
. 1 E D G E M V K L E N L F E A M 8 15 amino
4 E M V K L E N L F E A M N N K 8 acids, the
11 L F E A M N N K N C Q A L R A 8 end position for
15 M N N K N C Q A L R A K P K V 8 each
13 E A M N N K N C Q A L R A K P 3 peptide is
7 K L E N L F E A M N N K N C Q 1 the start
10 N L F E A M N N K N C Q A L R 1 position plus fourteen
213P1F11 v .6: HLA-DRB1*0301 (DR17) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
12 F Ξ A M N N K N C Q A L R A K 20 Portion of
2 D G E M V K L E N L F E A M N 19 SEQ ID
8 L E N L F E A M N N K N C Q A 19 NO: 13;
1 E D G E M V K L E N L F E A M 17 each start position is
5 M V K L Ξ N L F E A M N N K N 17 specified,
9 E N L F Ξ A M N N K N C Q A L 15 the length
3 G E M V K L E N L F E A M N N 12 of each
10 N L F E A M N N K N C Q A L R 8 peptide is
13 Ξ A M N N K N C Q A L R A K P 8 15 amino
11 L F E A M N N K N C Q A L R A 7 acids, the
4 Ξ M V K L E N L F E A M N N K 5 end N C position for
7 K L E N L F E A M N N K Q 4 each
6 V K L E N L F E A M N N K N C 3 peptide is
14 A M N N K N C Q A L R A K P K 2 the start position plus fourteen
213P1F11 .6: HLA-DRB1*0401 (DR4D 4) 15 - mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
8 L E N L F E A M N N K N C Q A 26 Portion of
2 D G E M V K L E N L F E A M N 20 SEQ ID
3 G E M V K L E N L F E A M N N 20 NO: 13;
M V K L E N L F E A M N N K N 20 each start
5 position is
9 E N L F E A M N N K N C Q A L 16 specified,
6 V K L E N L F E A M N N K N C 12 the length
7 K L E N L F E A M N N K N C Q 12 of each
11 L F E A M N N K N C Q A L R A 12 peptide is
12 F E A M N N K N C Q A L R A K 9 15 amino
4 E M V K L E N L F E A M N N K 6 acids, the
10 N L F E A M N N K N C Q A L R 6 end position for
13 E A M N N K N C Q A L R A K P 6 each
14 A M N N K N C Q A L R A K P K 6 peptide is
15 M N N K N C Q A L R A K P K V 6 the start
1 E D G E M V K L E N L F E A M -5 position plus fourteen
213P1F11 v .6: HLA-DRB1*110115-mers
Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score
9 E N L F E A M N N K N C Q A L 16 Portion of
2 D G Ξ M V K L E N L F E A M N 12 SEQ ID
3 G E M V K L E N L F E A M N N 12 NO: 13; each start
5 M V K L E N L F E A M N N K N 12 position is
8 L E N L F E A M N N K N C Q A 8 specified,
10 N L F E A M N N K N C Q A L R 8 the length
14 A M N N K N C Q A L R A K P K 8 of each
11 L F E A M N N K N C Q A L R A 6 peptide is
12 F E A M N N K N C Q A L R A K 6 15 amino
1 E D G E M V K L E N L F E A M 2 acids, the
13 E A M N N K N C Q A L R A K P 1 end position for each peptide is the start position plus fourteen
Figure imgf000301_0001
Figure imgf000302_0001
Table XXI: Motifs and Post-translational Modifications of 213P1F11
A) Post-translational Modifications of 213P1F11- v.l
Tyrosine sulfation site.
5-19 rsleeekYdmsgarl
Protein kinase C phosphorylation site
51-53 TmK
196-198 TkR
210-212 TrR
234-236 T1R
Casein kinase II phosphorylation site
6-9 SleE
32-35 SeeD
74-77 SreD
161 - 164 TytD
170 - 173 StvE
190-193 TlvD
227 - 230 TnpE
Tyrosine kinase phosphorylation site 5 - 12 RsleEek.Y
N-myristoylation site 16-21 GArlAL
Bipartite nuclear targeting sequence 211-227 RRmaeaelvqegkarkt
B) Post-translational Modifications of 213P1F11- v.2
Tyrosine sulfation site . 5-19 rsleeekYdmsgarl
Protein kinase C phosphorylation site. 51-53 TmK 220-222 SrR
Casein kinase II phosphorylation site .
6-9 SleE
32-35 SeeD
74 - 77 SreD
161 - 164 TytD
170 - 173 StvE
201 - 204 SptD
Tyrosine kinase phosphorylation site 5-12 RsleEe Y
N-myristoylation site 16-21 GArlAL NOT FURNISHED AT THE TIME OF PUBLICATION
TABLE XXII: Protein Properties of 213P1F11
Figure imgf000305_0001
TABLE XXII, continued: Protein Pro erties of 213P1F11
Figure imgf000306_0001
Figure imgf000306_0002
Table XXIIIA. Exon compositions of 213P1F11 v.l
Figure imgf000307_0001
Figure imgf000307_0002
Table XXIVA. Nucleotide sequence of transcript variant 213P1F11 v.2 (SEQ. ID. NO.55). ctgactcatt tagactctct gcctaggcca cctttgccag agggagtccc ctcagccttg 60 cgatcactca tcccattggc gttggctcca tttccacacc acagctgtgt gccaagggtg 120 tgtcatgagg tttcttgagt gacagaaaac tcaccgacaa taaagggcca ggtgattgtg 180 ccacccgatt catagaccag gcttctcagg agaaaccccg ggagattcca cactgtcagc 240 cccttctcca agatcagtac gtgggcctga ctcctcctcg gtgcccagct cagtattggc 300 aactaggaga gtagtgagat tgaacttggc cttgaggaac agctgcctct agagttggat 360 cagacaaggg tgctgagagc cgggactcac aaccaaagga gaaatgagca atccgcggtc 420 tttggaagag gagaaatatg atatgtcagg tgcccgcctg gccctaatac tgtgtgtcac 480 caaagcccgg gaaggttccg aagaagacct ggatgctctg gaacacatgt ttcggcagct 540 gagattcgaa agcaccatga aaagagaccc cactgccgag caattccagg aagagctgga 600 aaaattccag caggccatcg attcccggga agatcccgtc agttgtgcct tcgtggtact 660 catggctcac gggagggaag gcttcctcaa gggagaagat ggggagatgg tcaagctgga 720 gaatctcttc gaggccctga acaacaagaa ctgccaggcc ctgcgagcta agcccaaggt 780 gtacatcata caggcctgtc gaggagaaca aagggacccc ggtgaaacag taggtggaga 840 tgagattgtg atggtcatca aagacagccc acaaaccatc ccaacataca cagatgcctt 900 gcacgtttat tccacggtag agggacccac gcccttccag gatcccctct acctaccctc 960 tgaagctccc ccgaacccac ctctctggaa ttcccaggat acatcgccta ccgacatgat 1020 cagaaaggct catgctttat ccagaccctg gtggatgtgt tcacgaagag gaaaggacat 1080 atcttggaac ttctgacaga ggtgacccgg cggatggcag aagcagagct ggttcaagaa 1140 ggaaaagcaa ggaaaacgaa ccctgaaatc caaagcaccc tccggaaacg' gctgtatctg 1200 cagtagaagt agaaagacca ggaggagctt tccttccagc attctttctg tctcacagaa 1260 atttagaggc agctcttacc tctccccaag atcttcfegtt cccaaggcca aatggcaccc 1320 agtttctttt ccatcacacc cttcatgcag gtcctcctgt ccttattaga gcaagccagc 1380 caaaacttag cacaaggcat ggtggcaaca ttaacatcac ctccctcagg ctggactttc 1440 tatctttatt aatgcaaccg aagagaccta agagtgcatt cacttatccc actttctgtt 1500 cctgtggtct tctttctccc atgaagcaga aactggataa agctcaagat tttccataga 1560 caaaccaaag cccactcatc ccctcctacc ccaatccaac ctctgctggc tcctgcatct 1620 cacttggagg tcaaacctcc tcctgaggcc aatgcattcc caacttccag ttctttcctt 1680 taccctggag agttagtaag gtaagaacca ttctttctct ccaaaaccac tcctccttgg 1740 ctggcaagtt ggtgtcctaa ctccgttctc ttcctagctc atggcctctc tagataataa 1800 agttgtctcc tcctttctgg atctcttcct cctaacaccc ctcccctgaa accctggact 1860 ctgccctctc tccaagaaaa tccatctatt caactattct tgcattcaat tactctaaat 1920 gagagcgtgt tggagctatg gcaaattccc tgttgtcacc ttgctatttt gcagacaaca 1980 taatatttaa cctctcataa ccagagaggt taaataattt gtcaaatgca atacagtaag 2040 acagaggcaa ggacaaggtt tgacttccag cccagcctct tttccacaac ctgctaaatc 2100 ctgatccatc tgaaaacttt tctaattagt gaagatgact aataaaaatt ttccctatct 2160 ccaaggtagg agctttctgg aagtttctag aaattttcaa taaccaccag ccaaggttac 2220 ctccaggtaa ccttgcagca ccaggctgga agtcagatcg gcttcactat cttccaactc 2280 tacagcctgt atctctccat ccccagcttt gacctttcct gctcaagtaa cctacgggca 2340 catccagcgt cactaaaaac tcagggcttt tcttcccggt tactcctcca agcgttccct 2400 ggtatcctca acctcagatc ccaggttcag atttctgcag tcaatctatg acccctctct 2460 tcttgcatcc ttcatatgcc accagacacc atgcccagtc cagcctgatt ttgaaacaac 2520 tttcatgccg gtcttctctt ccctgacatg ttactgtcca ggctcaagtc ctcagcttct 2580 catatctgca tctttgcaac caacttcctc ccttgcctct ctgcttttcc atcccacttt 2640 tcatgtgtcc tccataccat ctataacagt gatctccctg gaacactcaa gaagacacaa 2700 cataccatat tatttaaaga ccagggtact ggacagtggc tcacacctgt attcccgact 2760 ttgagagtct gaagcgggag gatcacttga ggccaggagt taagagacca gcctgggcaa 2820 cacagcaaga ccctgtctct aaaaaaaaaa attaattaac tgggtatggt ggcacatgcc 2880 tgtagtccca gctactcagg aggctgaggt gggaggatga cttgagccca ggagtttgag 2940 gctgcaagga gctatgatca tgccagtgca tcccagctct aggtgagaca gtgagatccg 3000 gtctccaaaa taaatcaatc aatcaaataa agaccaaagt caaaccgcac atcaggatct 3060 ctcacaccct tccaattttg ccatctacca gcacttagct aaacccatct cccatctctt 3120 ccaccatgaa ttcactcttt σaaaaaggct aatgtcttct tactcaccct tgcctctaag 3180 cctttgetat caccatttcc cccaagctgg agggccctcc ctctcccttt acccctcttc 3240 cactacctcc cacccctact ttttccagaa agccatttcc tctctttttt ctgattgatc 3300 cttccctctc acccaggatt agatgctgga aatgaccact tctggagggc agggaacaag 3360 cccttaatct gcataatgag tgttcaataa acagttgtca aactttgaaa 3410 Table XXIVB. Nucleotide sequence of transcript variant 213P1F11 v.3 (SEQ. ID. NO.56). ctgactcatt tagactctct gcctaggcca cctttgccag agggagtccc ctcagccttg 60 cgatcactca tcccattggc gttggctcca tttccacacc acagctgtgt gccaagggtg 120 tgtcatgagg tttcttgagt gacagaaaac tcaccgacaa taaagggcca ggtgattgtg 180 ccacccgatt catagaccag gcttctcagg agaaaccccg ggagattcca cactgtcagc 240 cccttctcca agatcagtac gtgggcctga ctcctcctcg gtgcccagct cagtattggc 300 aactaggaga gtagtgagat tgaacttggc cttgaggaac agctgcctct agagttggat 360 cagacaaggg tgctgagagc cgggactcac aaccaaagga gaaatgagca atccgcggtc 420 tttggaagag gagaaatatg atatgtcagg tgcccgcctg gccctaatac tgtgtgtcac 480 caaagcccgg gaaggttccg aagaagacct ggatgctctg gaacacatgt ttcggcagct 540 gagattcgaa agcaccatga aaagagaccc cactgccgag caattccagg aagagctgga 600 aaaattccag caggccatcg attcccggga agatcccgtc agttgtgcct tcgtggtact 660 catggctcac gggagggaag gcttcctcaa gggagaagat ggggagatgg tcaagctgga 720 gaatctcttc gaggccctga acaacaagaa ctgccaggcc ctgcgagcta agcccaaggt 780 gtacatcata caggcctgtc gaggagccac cctgcccagc ccctttcctt acctttctct 840 ctgactttgc ctcctcctct tcttgttgtt tcagaacaaa gggaccccgg tgaaacagta 900 ggtggagatg agattgtgat ggtcatcaaa gacagcccac aaaccatccc aacatacaca 960 gatgccttgc acgtttattc cacggtagag ggatacatcg cctaccgaca tgatcagaaa 1020 ggctcatgct ttatccagac cctggtggat gtgttcacga agaggaaagg acatatcttg 1080 gaacttctga cagaggtgac ccggcggatg gcagaagcag agctggttca agaaggaaaa 1140 gcaaggaaaa cgaaccctga aatccaaagc accctccgga aacggctgta tctgcagtag 1200 aagtagaaag accaggagga gctttccttc cagcattctt tctgtctcac agaaatttag 1260 aggcagctct tacctctccc caagatcttc tgttcccaag gccaaatggc acccagtttc 1320 ttttccatca cacccttcat gcaggtcctc ctgtccttat tagagcaagc cagccaaaac 1380 ttagcacaag gcatggtggc aacattaaca tcacctccct caggctggac tttctatctt 1440 tattaatgca accgaagaga cctaagagtg cattcactta tcccactttc tgttcctgtg 1500 gtcttctttc tcccatgaag cagaaactgg ataaagctca agattttcca tagacaaacc 1560 aaagcccact catcccctcc taccccaatc caacctctgc tggctcctgc atctcacttg 1620 gaggtcaaac ctcctcctga ggccaatgca ttcccaactt ccagttcttt cctttaccct 1680 ggagagttag taaggtaaga accattcttt ctctccaaaa ccactcctcc ttggctggca 1740 agttggtgtc ctaactccgt tctcttccta gctcatggcc tctctagata ataaagttgt 1800 ctcctccttt ctggatctct tcctcctaac acccctcccc tgaaaccctg gactctgccc 1860 tctctccaag aaaatccatc tattcaacta ttcttgcatt caattactct aaatgagagc 1920 gtgttggagc tatggcaaat tccctgttgt caccttgcta ttttgcagac aacataatat 1980 ttaacctctc ataaccagag aggttaaata atttgtcaaa tgcaatacag taagacagag 2040 gcaaggacaa ggtttgactt ccagcccagc ctcttttcca caacctgcta aatcctgatc 2100 catctgaaaa cttttctaat tagtgaagat gactaataaa aattttccct atctccaagg 2160 taggagcttt ctggaagttt ctagaaattt tcaataacca ccagccaagg ttacctccag 2220 gtaaccttgc agcaccaggc tggaagtcag atcggcttca ctatcttcca actctacagc 2280 ctgtatctct ccatccccag ctttgacctt tcctgctcaa gtaacctacg ggcacatcca 2340 gcgtcactaa aaactcaggg cttttcttcc cggttactcc tccaagcgtt ccctggtatc 2400 ctcaacctca gatcccaggt tcagatttct gcagtcaatc tatgacccct ctcttcttgc 2460 atccttcata tgccaccaga caccatgccc agtccagcct gattttgaaa caactttcat 2520 gccggtcttc tcttccctga catgttactg tccaggctca agtcctcagc ttctcatatc 2580 tgcatctttg caaccaactt cctcccttgc ctctctgctt ttccatccca cttttcatgt 2640 gtcctccata ccatctataa cagtgatctc cctggaacac tcaagaagac acaacatacc 2700 atattattta aagaccaggg tactggacag tggctcacac ctgtattccc gactttgaga 2760 gtctgaagcg ggaggatcac ttgaggccag gagttaagag accagcctgg gcaacacagc 2820 aagaccctgt ctctaaaaaa aaaaattaat taactgggta tggtggcaca tgcctgtagt 2880 cccagctact caggaggctg aggtgggagg atgacttgag cccaggagtt tgaggctgca 2940 aggagctatg atcatgccag tgcatcccag ctctaggtga gacagtgaga tccggtctcc 3000 aaaataaatc aatcaatcaa ataaagacca aagtcaaacc gcacatcagg atctctcaca 3060 cccttccaat tttgccatct accagcactt agctaaaccc atctcccatc tcttccacca 3120 tgaattcact ctttcaaaaa ggctaatgtc ttcttactca cccttgcctc taagcctttg 3180 ctatcaccat ttcccccaag ctggagggcc ctccctctcc ctttacccct cttccactac 3240 ctcccacccc tactttttcc agaaagccat ttcctctctt ttttctgatt gatccttccc 3300 tctcacccag gattagatgc tggaaatgac cacttctgga gggcagggaa caa'gccctta 3360 atctgcataa tgagtgttca ataaacagtt gtcaaacttt gaaa 3404 Table XXIVC. Nucleotide sequence of transcript variant 213P1F11 v.4 (SEQ. ID. NO.57). atggggaaat gccaagagta tgacaaaagt ctgtctgtgc agccagagaa gagaacagga 60 ctcagagatg agaatggaga atgtggacag acattcagac tcaaggaaga gcaagggagg 120 gctttcaggg gaagttcagt ccaccagaag ctggtgaatg acccacggga gacacaggaa 180 gtttttgggg gcggagtggg ggacattgtg ggacgggatc tcagtattag cttcagaaac 240 tctgagacct ctgcaagtga ggaggagaaa tatgatatgt caggtgcccg cctggcccta 300 atactgtgtg tcaccaaagc ccgggaaggt tccgaagaag acctggatgc tctggaacac 360 atgtttcggc agctgagatt cgaaagcacc atgaaaagag accccactgc cgagcaattc 420 caggaagagc tggaaaaatt ccagcaggcc atcgattccc gggaagatcc cgtcagttgt 480 gccttcgtgg tactcatggc tcacgggagg gaaggcttcc tcaagggaga agatggggag 540 atggtcaagc tggagaatct cttcgaggcc ctgaacaaca agaactgcca ggccctgcga 600 gctaagccca aggtgtacat catacaggcc tgtcgaggag aacaaaggga ccccggtgaa 660 acagtaggtg gagatgagat tgtgatggtc atcaaagaca gcccacaaac catcccaaca 720 tacacagatg ccttgcacgt ttattccacg gtagagggat acatcgccta ccgacatgat 780 cagaaaggct catgctttat ccagaccctg gtggatgtgt tcacgaagag gaaaggacat 840 atcttggaac ttctgacaga ggtgacccgg cggatggcag aagcagagct ggttcaagaa 900 ggaaaagcaa ggaaaacgaa ccctgaaatc caaagcaccc tccggaaacg gctgtatctg 960 cagtag 966
Table XXV A. Nucleotide sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 58) and 213P1F11 v.2 (SEQ. ID. NO. 59).
213P1F11V.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11V.2 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 ************************************************************
213P1F11V.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 213P1F11V.2 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 ************************************************************
213P1F11V.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 213P1F11V.2 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 ************************************************************
213P1F11V.1 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 213P1F11V.2 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 ************************************************************
213P1F11V.1 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 213P1F11V.2 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 ************************************************************
213P1F11V.1 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 213P1F11V.2 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 ************************************************************
213P1F11V.1 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 213P1F11V.2 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 ************************************************************
213P1F11V.1 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 213P1F11V.2 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 ************************************************************
213P1F11V.1 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 213P1F11V.2 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 ************************************************************
213P1F11V.1 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 213P1F11V.2 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 ************************************************************
213P1F11V.1 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 213P1F11V.2 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 ************************************************************
213P1F11V.1 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 213P1F11V.2 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 ************************************************************
213P1F11V.1 GAATCτCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 213P1F11V.2 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 ************************************************************
213P1F11V.1 GTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGGAGA 840 213P1F11V.2 GTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGGAGA 840 ************************************************************
213P1F11V.1 TGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGCCTT 900 213P1F11V.2 TGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGCCTT 900 ************************************************************
213PlFllv .1 GCACGTTTATTCCACGGTAGAGGGA 925
213P1F11V.2 GCACGTTTATTCCACGGTAGAGGGACCCACGCCCTTCCAGGATCCCCTCTACCTACCCTC 960 ************************* 213 P1F11V . 1 - - - TACATCGCCTACCGACATGAT 946 213 P1F11V . 2 TGAAGCTCCCCCGAACCCACCTCTCTGGAATTCCCAGGATACATCGCCTACCGACATGAT 1020
* * * * * *** * ******** * * * *
213P1F11V.1 CAGAAAGGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACAT 1006 213P1F11V.2 CAGAAAGGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACAT 1080 ************************************************************
213P1F11V.1 ATCTTGGAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAA 1066 213P1F11V.2 ATCTTGGAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAA 1140 ************************************************************
213P1F11V.1 GGAAAAGCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTG 1126 213P1F11V.2 GGAAAAGCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTG 1200 ************************************************************
213P1F11V.1 CAGTAGAAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAA 1186 213P1F11V.2 CAGTAGAAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAA 1260 ************************************************************
213P1F11V.1 ATTTAGAGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCC 1246 213P1F11V.2 ATTTAGAGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCC 1320 ************************************************************
213P1F11V.1 AGTTTCTTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGC 1306 213P1F11V.2 AGTTTCTTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGC 1380 ************************************************************
213P1F11V.1 CAAAACTTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTC 1366 213P1F11V.2 CAAAACTTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTC 1440 ************************************************************
213P1F11V.1 TATCTTTATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTT 1426 213P1F11V.2 TATCTTTATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTT 1500 ************************************************************
213P1F11V.1 CCTGTGGTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGA 1486 213P1F11V.2 CCTGTGGTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGA 1560 ************************************************************
213P1F11V.1 CAAACCAAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCT 1546 213P1F11V.2 CAAACCAAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCT 1620 ************************************************************
213P1F11V.1 CACTTGGAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTT 1606 213P1F11V.2 CACTTGGAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTT 1680 ************************************************************
213P1F11V.1 TACCCTGGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGG 1666 213P1F11V.2 TACCCTGGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGG 1740 ************************************************************
213P1F11V.1 CTGGCAAGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAA 1726 213PlFll .2 CTGGCAAGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAA 1800 ************************************************************
213P1F11V.1 AGTTGTCTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACT 1786 213P1F11V.2 AGTTGTCTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACT 1860 ************************************************************
213PlFll .1 CTGCCCTCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAAT 1846 213PlFllv.2 CTGCCCTCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAAT 1920 ************************************************************
213P1F11V.1 GAGAGCGTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACA 1906 213P1F11V.2 GAGAGCGTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACA 1980 ************************************************************ 213P1F11V.1 TAATATTTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAG 1966 213P1F11V .2 TAATATTTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAG 2040 ************************************************************
213P1F11V.1 ACAGAGGCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATC 2026 213P1F11V.2 ACAGAGGCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATC 2100 ************************************************************
213P1F11V.1 CTGATCCATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCT 2086 213P1F11V.2 CTGATCCATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCT 2160 ************************************************************
213P1F11V.1 CCAAGGTAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTAC 2146 213P1F11V.2 CCAAGGTAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTAC 2220 ************************************************************
213P1F11V.1 CTCCAGGTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTC 2206 213P1F11V.2 CTCCAGGTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTC 2280 ************************************************************
213P1F11V.1 TACAGCCTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCA 2266 213P1F11V.2 TACAGCCTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCA 2340 ************************************************************
213P1F11V.1 CATCCAGCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCT 2326 213P1F11V.2 CATCCAGCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCT 2400 ************************************************************
213P1F11V.1 GGTATCCTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCT 2386 213P1F11V.2 GGTATCCTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCT 2 60 ************************************************************
213P1F11V.1 TCTTGCATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAAC 2446 213P1F11V.2 TCTTGCATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAAC 2520 ************************************************************
213P1F11V.1 TTTCATGCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCT 2506 213P1F11V.2 TTTCATGCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCT 580 ************************************************************
213P1F11V.1 CATATCTGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTT 2566 213P1F11V.2 CATATCTGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTT 2640 ************************************************************
213P1F11V.1 TCATGTGTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAA 2626 213P1F11V.2 TCATGTGTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAA 2700 ************************************************************
213P1F11V.1 CATACCATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACT 2686 213P1F11V.2 CATACCATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACT 2760 ************************************************************
213P1F11V.1 TTGAGAGTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAA 2746 213P1F11V.2 TTGAGAGTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAA 2820 ************************************************************
213P1F11V.1 CACAGCAAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCC 2806 213P1F11V.2 CACAGCAAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCC 2880 ************************************************************
213P1F11V.1 TGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAG 2866 213P1F11V.2 TGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAG 2940 ************************************************************
213P1F11V.1 GCTGCAAGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCG 2926 213P1F11V.2 GCTGCAAGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCG 3000 ************************************************************
213P1F11V.1 GTCTCCAAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCT 2986 213PlFllv.2 GTCTCCAAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCT 3060 ************************************************************
213P1F11V.1 CTCACACCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTT 3046 213P1F11V.2 CTCACACCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTT 3120 ************************************************************
213P1F11V.1 CCACCATGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAG 3106 213P1F11V.2 CCACCATGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAG 3180 ************************************************************
213P1F11V.1 CCTTTGCTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTC 3166 213P1F11V.2 CCTTTGCTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTC 3240 ************************************************************
213P1F11V.1 CACTACCTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATC 3226 213P1F11V.2 CACTACCTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATC 3300 ************************************************************
213P1F11V.1 CTTCCCTCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAG 3286 213P1F11V.2 CTTCCCTCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAG 3360 ************************************************************
213P1F11V.1 CCCTTAATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213P1F11V.2 CCCTTAATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3410 **************************************************
Table XXVB. Nucleotide sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 60) and 213P1F11 v.3 (SEQ. ID. NO. 61).
213P1F11V.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11V.3 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 ************************************************************
213P1F11V.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 20 213P1F11V.3 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 ************************************************************
213P1F11V.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 213P1F11V.3 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 ************************************************************
213P1F11V.1 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 213P1F11V.3 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 ************************************************************
213P1F11V.1 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 213P1F11V.3 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 ************************************************************
213P1F11V.1 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 213P1F11V.3 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 ************************************************************
213P1F11V.1 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 213P1F11V.3 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 ************************************************************
213P1F11V.1 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 213P1F11V.3 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 ************************************************************
213P1F11V.1 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 213P1F11V.3 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 ************************************************************
213P1F11V.1 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 213P1F11V.3 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 ************************************************************ 13PlFllv.1 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 213P1F11V.3 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 ************************************************************
213P1F11V.1 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 213P1F11V.3 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 ************************************************************
213P1F11V.1 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 213P1F11V.3 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 ************************************************************
213P1F11V.1 GTACATCATACAGGCCTGTCGAGGAG 806 213P1F11V.3 GTACATCATACAGGCCTGTCGAGGAGCCACCCTGCCCAGCCCCTTTCCTTACCTTTCTCT 840 **************************
213P1F11V.1 AACAAAGGGACCCCGGTGAAACAGTA 832 213P1F11V.3 CTGACTTTGCCTCCTCCTCTTCTTGTTGTTTCAGAACAAAGGGACCCCGGTGAAACAGTA 900
**************************
213P1F11V.1 GGTGGAGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACA 892 213P1F11V.3 GGTGGAGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACA 960 ************************************************************ 213PIFl1v .1 GATGCCTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAA 952 213P1F11V.3 GATGCCTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAA 1020 ************************************************************
213P1F11V.1 GGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTG 1012 213PlFllv.3 GGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTG 1080 ************************************************************
213PlFllv.1 GAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAA 1072 213P1F11V.3 GAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAA 1140 ************************************************************
213P1F11V.1 GCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG 1132 213P1F11V.3 GCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG 1200 ************************************************************
213P1F11V.1 AAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAG 1192 213P1F11V.3 AAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAG 1260 ************************************************************
213P1F11V.1 AGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTC 1252 213P1F11V.3 AGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTC 1320 ************************************************************
213PlFllv.1 TTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAAC 1312 213P1F11V.3 TTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAAC 1380 ************************************************************
213P1F11V.1 TTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTT 1372 213P1F11V.3 TTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTT 1440 ************************************************************
213P1F11V.1 TATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTG 1432 213P1F11V.3 TATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTG 1500 ************************************************************
213P1F11V.1 GTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACC 1492 213P1F11V.3 GTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACC 1560 ************************************************************
213P1F11V.1 AAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTG 1552 213PlFllv.3 AAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTG 1620 ************************************************************
213P1F11V.1 GAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCT 1612 213P1F11V.3 GAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCT 1680 ************************************************************
213P1F11V.1 GGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCA 1672 213P1F11V.3 GGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCA 1740 ************************************************************
213P1F11V.1 AGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGT 1732 213PlFllv.3 AGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGT 1800 ************************************************************
213P1F11V.1 CTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCC 1792 213P1F11V .3 CTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCC 1860
213P1F11V.1 TCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGC 1852 213P1F11V.3 TCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGC 1920 *************************************************************
213P1F11V.1 GTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATAT 1912 213P1F11V.3 GTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATAT 1980 ************************************************************ 213P1F11 .1 TTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAG 1972
213P1F11V.3 TTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAG 2040 ************************************************************
213P1F11V.1 GCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATC 2032 213P1F11 .3 GCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATC 2100 ************************************************************
213P1F11 .1 CATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGG 2092 213PlFllv.3 CATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGG 2160 ************************************************************
213P1F11V.1 TAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAG 2152 213P1F11V.3 TAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAG 22 0 ************************************************************
213P1F11V.1 GTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGC 2212 213P1F11V.3 GTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGC 2280 ************************************************************
213P1F11V.1 CTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCA 2272 213P1F11V.3 CTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCA 2340 ************************************************************
213P1F11V.1 GCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATC 2332 213P1F11V.3 GCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATC 2 00 ************************************************************
213P1F11V.1 CTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGC 2392 213P1F11V.3 CTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGC 2460 ************************************************************
213P1F11V.1 ATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCAT 2452 213P1F11V.3 ATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCAT 2520 ************************************************************
213P1F11V.1 GCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATC 2512 213P1F11V.3 GCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATC 2580 ************************************************************
213P1F11V.1 TGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGT 2572 213PlFllv.3 TGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGT 2640 ************************************************************
213P1F11V.1 GTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACC 2632 213P1F11V.3 GTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACC 2700 ************************************************************
213P1F11V.1 ATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGAGA 2692 213P1F11V.3 ATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGAGA 2760 ************************************************************
213P1F11V.1 GTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGC 2752 213P1F11V.3 GTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGC 2820 ************************************************************
213PlFllv.1 AAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGT 2812 213 lFllv.3 AAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGT 2880 ************************************************************
213P1F11V.1 CCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCA 2872 213P1F11V.3 CCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCA 2940 ************************************************************
213P1F11V .1 AGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCC 2932 213PlFll .3 AGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCC 3000 ************************************************************
213 PlFllv . 1 AAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACA 2992 213P1F11V.3 AAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACA 3060 ************************************************************
213P1F11V .1 CCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCA 3052 213P1F11V .3 CCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCA 3120 ************************************************************
213P1F11V.1 TGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTG 3112 213P1F11V.3 TGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTG 3180 ************************************************************
213P1F11V.1 CTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTAC 3172 213P1F11V.3 CTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTAC 32 0 ************************************************************
213P1F11V.1 CTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCC 3232 213P1F11V.3 CTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCC 3300 ************************************************************
213P1F11V.1 TCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTA 3292 213P1F11V.3 TCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTA 3360 ************************************************************
213P1F11V.1 ATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213P1F11V.3 ATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAS 3404 ********************************************
Table XXVC. Nucleotide sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 62) and 213P1F11 v.4 (SEQ. ID. NO. 63).
213P1F11V.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11V.4 - -
213P1F11V.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 213P1F11V.4
213P1F11V.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGG-TGATTGT 179
213PlFllv. ATGGGGAAAT-GCCAAGAGTATGACAAAAGTCTGTCTGT 38
* * ** * ** * * ** ** ** ***
213P1F11V.1 GCCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAG 239 213P1F11V. GCAGCCAGAGA-AGAGAACAGGACTCAGAGATGAGAAT---GGAGAATGTGGACAGACA- 93 ** ** ** * *** **** ** ** ** * ***** * ** * **
213P1F11V.1 CCCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGG 299
213PlFllv. TTCAGACTCAAGGAAGAGCAAGGGAG GGCTTTCAGGGGAAGTTCAGTCCACCAGAAG 150
* *** * ** ** * * * * * ** * * *** ** * *
213P1F11V .1 CAACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGA 359 213 PlFllv . CTGGTGAATGACCCACGGGA GACAC AGGAA GTTTTTGGGGGCGGA 195
* * ** * * * ** ***** * * * * * ***
213P1F11V.1 TCAGACAAGGGTGCTGAGAGCCGGGA-CTCACAACCAAAGG-AGAAATGAGCAATCCGCG 17 213P1F11V.4 GTGGG---GGACATTGTGGGACGGGATCTCAGTATTAGCTTCAGAAACTCTGAGACCTCT 252 * ** ** * * ***** **** * * ***** * ** *
213P1F11V.1 GTCTTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGT 477 213P1F11V.4 GCAAGTG-AGGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGT 311
* ** * **************************************************
213P1F11V.1 CACCAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCA 537 213P1F11V .4 CACCAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCA 371 ************************************************************
213P1F11V.1 GCTGAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCT 597 213P1F11V.4 GCTGAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCT 431 ************************************************************
213P1F11V.1 GGAAAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGT 657 213P1F11V.4 GGAAAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGT 491 ************************************************************
213P1F11V.1 ACTCATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCT 717 213P1F11V .4 ACTCATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCT 551
************************************************************
213P1F11V.1 GGAGAATCTCTTCGAGGCCCTGAACAACAAGAACTGGCAGGCCCTGCGAGCTAAGCCCAA 777 213PlFllv.4 GGAGAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAA 611 ************************************************************
213P1F11V .1 GGTGTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGG 837 213PIFll .4 GGTGTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGG 671 ************************************************************
213P1F11V.1 AGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGC 897 213P1F11V .4 AGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGC 731 ************************************************************
213P1F11V.1 CTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAAGGCTC 957 213P1F11V .4 CTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAAGGCTC 791 ************************************************************ 213P1F11V.1 ATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTGGAACT 1017 213P1F11V.4 ATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTGGAACT 851 ************************************************************
213P1F11V.1 TCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAAGCAAG 1077 213P1F11V.4 TCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAAGCAAG 911 ************************************************************
213P1F11V.1 GAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAGAAGTA 1137 213P1F11V.4 GAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG 966
*******************************************************
213PlFllv.1 GAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAGAGGCA 1197 213P1F11V.4
213PlFllv.1 GCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTCTTTTC 1257 213P1F11V.4
213P1F11V.1 CATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAACTTAGC 1317 213P1F11V.4
213P1F11V.1 ACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTTTATTA 1377 213P1F11V.4
213P1F11V.1 ATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTGGTCTT 1437 213P1F11V.4
213P1F11V.1 CTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACCAAAGC 1497 213P1F11V.4
213P1F11V.1 CCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTGGAGGT 1557 213PlFllv.4
213P1F11V.1 CAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCTGGAGA 161 213P1F11V.4
213P1F11V.1 GTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCAAGTTG 1677 213PlFllv.4
213P1F11V.1 GTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGTCTCCT 1737 213P1F11V.4
213PlFllv.1 CCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCCTCTCT 1797 213PlFllv.4
213PlFllv.1 CCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGCGTGTT 1857 213P1F11V.4
213P1F11V.1 GGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATATTTAAC 1917 213PlFllv.4
213P1F11V.1 CTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAGGCAAG 1977 213PlFllv.4 213P1F11V.1 GACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATCCATCT 2037 213P1F11V.4
213P1F11V.1 GAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGGTAGGA 2097 213PlFllv.4
213P1F11V.1 GCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAGGTAAC 157 213P1F11V.4
213P1F11V.1 CTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGCCTGTA 2217 213P1F11V.4
213P1F11V.1 TCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCAGCGTC 2277 213P1F11V.4
213P1F11V.1 ACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATCCTCAA 2337 213P1F11V.4
213P1F11V.1 CCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGCATCCT 2397 213P1F11V.4
213P1F11V.1 TCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCATGCCGG 2457 213PlFllv.4
213PlFllv.1 TCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATCTGCAT 2517 213PlFllv.4
213P1F11V.1 CTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGTGTCCT 2577 213P1F11V.4
213P1F11V.1 CCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACCATATT 2637 213P1F11V.4
213P1F11V.1 ATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGAGAGTCTG 2697 213P1F11V.4
213 lFllv.1 AAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGCAAGAC '2757 213P1F11V.4
213P1F11V.1 CCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGTCCCAG 2817 213P1F11V.4
213P1F11V.1 CTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCAAGGAG 2877 213P1F11V.4
213PlFllv.1 CTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCCAAAAT 2937 213P1F11V.4
213P1F11V.1 AAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACACCCTT 2997 213P1F11V.4
213P1F11V.1 CCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCATGAAT 3057 213P1F11V.4
213P1F11V.1 TCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTGCTATC 3117 213PlFllv.4
213P1F11V.1 ACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTACCTCCC 3177 213PlFllv.4
213P1F11V.1 ACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCCTCTCA 3237 213P1F11V.4
213P1F11V.1 CCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTAATCTG 3297 213P1F11V.4
213P1F11V.1 CATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213PlFllv.4
Table XXVIA. Peptide sequences of protein coded by 213P1F11 v.2 (SEQ. ID. NO. 64).
MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ 60
FQEELEKFQQ AIDSREDPVS CAFWLMAHG REGFLKGEDG EMVK ENLFE ALNNKNCQAL 120
RAKPKVYIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP TYTDALHVYS TVEGPTPFQD 180
PLY PSEAPP NPPL NSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF 230
Table XXVIB. Peptide sequences of protein coded by 213P1F11 v.3 (SEQ. ID. NO. 65).
MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ 60
FQEELEKFQQ AIDSREDPVS CAFWLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL 120
RAKPKVYIIQ ACRGATLPSP FPYLSL 146
Table XXVIC. Peptide sequences of protein coded by 213P1F11 v.4 (SEQ. ID. NO. 66)
MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE 60
VFGGGVGDIV GRDLSISFRN SETSASEEEK YDMSGARLAL ILCVTKAREG SEEDLDALEH 120
MFRQLRFEST MKRDPTAEQF QEELEKFQQA IDSREDPVSC AFWLMAHGR EGFLKGEDGE 180
MVKLENLFEA LNNKNCQALR AKPKVYIIQA CRGEQRDPGE TVGGDEIVMV IKDSPQTIPT 240
YTDALHVYST VEGYIAYRHD QKGSCFIQTL VDVFTKRKGH ILELLTEVTR RMAEAELVQE 300
GKARKTNPEI QSTLRKRLYL Q 321
Table XXVIIA. Amino acid sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 67) and 213P1F11 v.2 (SEQ. ID. NO. 68)
213P1F11V.1 MSNPRSLEEEKYDMSGA LALILCVTKAREGSEEDLDALEHMFRQ RFESTMKRDPTAEQ 60 213P1F11V .2 MSNPRSLEEEKYDMSGARLALI CVTKAREGSEEDI.DA EHMFRQLRFESTMKRDPTAEQ 60 ************************************************************
213P1F11V.1 FQEELEKFQQAIDSREDPVSCAFWLMAHGREGF KGEDGE VKLΞNLFEA N KNCQAI 120 213P1F11V.2 FQEELEKFQQAIDSREDPVSCAFWLMAHGREGF KGEDGEMVKLEN FEALN KNCQAL 120 ************************************************************
213P1F1lv .1 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDALHVYSTVEGYIAYRH 180 213P1F11 .2 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDA HVYSTVEGPTPFQD 180 ****************************************************** ..
213P1F11V.1 DQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAE VQEGKARKTNPEIQSTLRKR Y 2 0 213P1F11V.2 PLYLPSEAPPNPP SQDTSPTDMIRKAHA SRPW MCSRRGKDIS 227
213P1F11V.1 LQ- 242 213P1F11V.2 NF 230
Table XXVHB. Amino acid sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 69) and 213P1F11 v3 (SEQ. ID. NO. 70)
213PlFllv.1 MSNPRSLEEEKYDMSGARLAIil CVTKAREGSEEDLDA EHMFRQIiRFESTMKRDPTAEQ 60 213P1F11V.3 MSNPRSLEEEKYDMSGARLALI CVTKAREGSEEDLJDALEHMFRQLRFESTMKRDPTAEQ 60 ************************************************************
213P1F11V.1 FQEELEKFQQAIDSREDPVSCAFΛΠ- AHGREGFIKGEDGEMVK ENLFEAIJNNKNCQAI 120 213P1F11V.3 FQEELEKFQQAIDSREDPVSCAFVVL AHGREGFLKGEDGEMVKLENLFEALNNKNCQAIi 120 ************************************************************
213P1F11V.1 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDALHVYSTVEGYIAYRH 180 213P1F11V.3 RAKPKVYIIQACRG ATLPSPFPYLSL 146
************** *;*: * :
213P1F11V.1 DQKGSCFIQTLVDVFTKRKGHI ELLTEV RRMAEAELVQEGKARKTNPEIQSTLRKR Y 240 213P1F11V.3
213P1F11V.1 LQ 242 213P1F11V.3
Table XXVIIC. Amino acid sequence alignment of 213P1F11 v.l (SEQ. ID. NO. 71) and 213P1F11 v.4 (SEQ. ID. NO. 72).
213PlFllv.1 SNPR 5 213PlFllv.3 MGKCQEYDKSLSVQPEKRTGLRDENGECGQTFRLKEEQGRAFRGSSVHQKLVNDPRETQE 60
213P1F11V.1 SLEEEKYD SGARLALILCVTKAREGSEEDLDALEH 41 213PlFllv.3 VFGGGVGDIVGRDLSISFRNSETSASEEEKYDMSGARLALILCV KAREGSEEDLDALEH 120
. **********************************
213P1F11V.1 MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE 101 213PIFI1 .3 MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE 180 ************************************************************
213P1F11V.1 VKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT 161 13 PlFllv.3 WKLENLFEAIjNNKNCQALRAKPKWIIQACRGEQRDPGEWGGDEIVMVIKDSPQTIP^ 240 ************************************************************
213P1F11V.1 YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE 221 213PlFllv.3 YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRR AEAELVQE 300 ************************************************************
213 PlFllv.1 GKARKTNPEIQSTLRKRLYLQ 242 213P1F11V.3 GKARKTNPEIQSTLRKRLYLQ 321 *********************
Table XXVIII. Clustai Alignment of 213P1F11 protein variants. (SEQ. ID. NOs: ) v.ι_ v.2_ v.3_ .4_ MGKCQEYDKSLSVQPEKRTGLRDENGECGQTFRLKEEQGRAFRGSSVHQKLVNDPRETQE v.5_ v.6
.1_ MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.2_ MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.3_ MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.4_ VFGGGVGDIVGRDLSISFRNSETSASEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.5_ MSNPRSLEEEKYDMSGARLALILRVTKAREGSEEDLDALEH v.6_ MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH
*:> : **************** ***************** .1_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE .2_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE .3_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE .4_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE .5_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE v.6_ MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFWLMAHGREGFLKGEDGE ************************************************************ .1_ MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT v.2_ MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT .3_ MVKLENLFEALNNKNCQALRAKPKVYIIQACRGATLPSPFPYLSL v.4_ MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT .5_ MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT .6_ MVKLENLFEAMNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT A*********.********************** v.1_ YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE .2_ YTDALHVYSTVEGPTPFQDPLYLPSEAPPNPPLWNSQDTSPTDMIRKAH-ALSRP MCS v.3_ . _ YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE .5_ YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE .6 YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE
.1_ GKARKTNPEIQSTLRKRLYLQ v.2_ RRGKDISWNF v.3_ . _ GKARKTNPEIQSTLRKRLYLQ .5_ GKARKTNPEIQSTLRKRLYLQ v.6_ GKARKTNPEIQSTLRKRLYLQ
Table XXIX. Search Peptides
213P1F11 Variant 1: Nonamers, Decamers, 15-mers (aa.1-242) (SEQ. ID. NO.73)
1 MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ
61 FQEELEKFQQ AIDSREDPVS CAFWLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL
121 RAKPKVYIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP TYTDALHVYS TVEGYIAYRH
181 DQKGSCFIQT LVDVFTKRKG HILELLTEVT RRMAEAELVQ EGKARKTNPE IQSTLRKRLY
241 LQ
213P1F11 Variant 2:
Nonamers (aa. 167-230) (SEQ. ID. NO. 74)
HVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRP MCS RRGKDISWNF
Decamers (aa. 166-230) (SEQ. ID. NO. 75)
LHVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF
15-mers (aa. 161-230) (SEQ. ID. NO. 76)
TYTDALHVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF
213P1F11 Variant 3:
Nonamers (aa. 127-146) (SEQ. ID. NO. 77) YIIQ ACRGATLPSP FPYLSL
Decamers (aa.126-146) (SEQ. ID. NO. 78) VYIIQ ACRGATLPSP FPYLSL
15mers (aa. 121-146) (SEQ. ID. NO. 79) RAKPKVYIIQ ACRGATLPSP FPYLSL
213P1F11 Variant 4:
Nonamers (aa. 1-94) (SEQ. ID. NO. 80)
MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV
GRDLSISFRN SETSASEEEK YDMS
Decamers (aa. 1-95) (SEQ. ID. NO. 81)
MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV
GRDLSISFRN SETSASEEEK YDMSG
15-mers (aa. 1-100) (SEQ. ID. NO. 82)
MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV
GRDLSISFRN SETSASEEEK YDMSGARLAL
213P1F11 Variant 5:
Nonamers (aa. 16-32) (SEQ. ID. NO. 83] GARLA LILRVTKARE GS
Decamers (aa. 15-33) (SEQ. ID. NO. 84) SGARLA LILRVTKARE GSE
15-mers (aa. 10-38) (SEQ. ID. NO. 85) E KYDMSGARLA LILRVTKARE GSEEDLDA
213P1F11 Variant 6:
Nonamers (aa. 104-120) (SEQ. ID. NO. 86) KLENLFE AMNNKNCQAL
Decamers (aa. 103-121) (SEQ. ID. NO. 87) VKLENLFE AMNNKNCQAL R
15-mers (aa. 98-126) (SEQ. ID. NO. 88) EDG EMVKLENLFE AMNNKNCQAL RAKPKV

Claims

CLAIMS:
1. A composition comprising: a substance that a) modulates the status of 213P1F11, or b) a molecule that is modulated by 213P1F11 whereby the status ofa cell that expresses 213P1F11 is modulated.
2. A composition of claim 1 , further comprising a physiologically acceptable carrier.
3. A pharmaceutical composition that comprises the composition of claim 1 in a human unit dose form.
4. A composition of claim 1 wherein the substance comprises an antibody or fragment thereof that specifically binds to a 213P IF 11-related protein.
5. An antibody or fragment thereof of claim 4, which is monoclonal.
6. An antibody of claim 4, which is a human antibody, a humanized antibody or a chimeric antibody.
7. A non-human transgenic animal that produces an antibody of claim 4.
8. A hybridoma that produces an antibody of claim 5.
9. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses 213P 1 F 11 , said method comprising: providing the cytotoxic agent or the diagnostic agent conjugated to an antibody or fragment thereof of claim 4; and, exposing the cell to the antibody-agent or fragment-agent conjugate.
10. A composition of claim 1 wherein the substance comprises a polynucleotide that encodes an antibody or fragment thereof either of which immunospecifically bind to a 213P1F11-related protein.
11. A composition of claim 1 wherein the substance comprises a 213P1F11-related protein.
12. A protein of claim 11 that is at least 90% homologous to an entire amino acid sequence shown in Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
13. A composition of claim 1 wherein the substance comprises a peptide of eight, nine, ten, or eleven contiguous amino acids of Figure 2 or Tables V to XIX, or an analog thereof (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
14. A composition of claim 1 wherein the substance comprises a CTL polypeptide of the amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
15. A composition of claim 14 further limited by a. proviso that the epitope is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
16. A composition of claim 14 wherein the substance comprises a CTL polypeptide set forth in Tables V to XIX (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
17. A composition of claim 16 further limited by a proviso that the polypeptide is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
18. A composition of claim 1 wherein the substance comprises an antibody polypeptide epitope of the amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
19. A composition of claim 18 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
20. A composition of claim 18 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17) in any whole number increment up to 242 that includes an amino acid position selected from: an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5, an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 8; or an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9.
21. A composition of claim 20 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
22. A polynucleotide that encodes a protein of claim 11.
23. A polynucleotide of claim 22 that comprises a nucleic acid molecule set forth in Figure 2 (SEQ. ID. NOs. 2, 4, 6, 8, 10, 12, 14, 16).
24. A polynucleotide of claim 22 further limited by a proviso that the encoded protein is not an entire amino acid sequence of Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
25. A polynucleotide of claim 22 wherein T is substituted with U.
26. A composition of claim 1 wherein the substance comprises a polynucleotide comprising a coding sequence of a nucleic acid sequence of Figure 2 (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16).
27. A polynucleotide of claim 24 further comprising a polynucleotide that encodes a 213P1F11- related protein that is at least 90% homologous to an entire amino acid sequence shown in Figure 2 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17).
28. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 22.
29. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 25.
30. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 27.
31. A composition of claim 1 wherein the substance comprises a) a ribozyme that cleaves a polynucleotide having 213P1F11 coding sequence, or b) a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier.
32. A composition comprising the composition of claim 1 wherein the substance comprises human T cells, wherein said T cells specifically recognize a 213P1F11 peptide sequence in the context of a particular HLA molecule.
33. A method of inhibiting growth of cancer cells that expresses 213P1F11, the method comprising: administering to the cells the composition of claim 1.
34. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of: administering to said cells an antibody or fragment thereof, either of which specifically bind to a 213P1F11 -related protein.
35. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of: administering to said cells a 213P1F11-related protem.
36. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of: administering to said cells a polynucleotide comprising a 213P IF 11-related protein coding sequence or a polynucleotide complementary to a polynucleotide having a 213P1F11 coding sequence.
37. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of:
' administering to said cells a ribozyme that cleaves a polynucleotide having 213P1F11 coding sequence.
38. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11 and a particular HLA molecule, the method comprising steps of: aα'ministermg to said cells human T cells, wherein said T cells specifically recognize a 213P1F11 peptide subsequence in the context of the particular HLA molecule.
39. A method of claim 33, the method comprising steps of: administering a vector that delivers a single chain monoclonal antibody coding sequence, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express 213P1F11.
40. A method of generating a mammalian immune response directed to 213P1F11, the method comprising: exposing cells of the mammal's immune system to a portion of a) a 213P1F11-related protein and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to 213P1F11.
41. A method of generating an immune response of claim 40, said method comprising: providing a 213P1F11-related protein that comprises at least one T cell or at least one B cell epitope; and, contacting the epitope with a mammalian immune system T cell or B cell respectively, whereby the T cell or B cell is induced.
42. A method of claim 41 wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the 213P IF 11-related protein.
43. A method of claim 41 wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 213P1F11-related protein.
44. A method of claim 41 wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of cytotoxic T cell (CTL) or the antibody-producing activity of a B cell.
45. A method for detecting the presence ofa 213PlFl l -related protein or polynucleotide in a sample comprising steps of: contacting the sample with a substance of claim 1 that specifically binds to the 213P1F11-related protein or polynucleotide, respectively; and, determining that there is a complex of the substance and 213P1F11-related protein or the substance and 213P IF 11-related polynucleotide, respectively.
46. A method of claim 45 for detecting the presence ofa 213P1F11-related protein in a sample comprising steps of: contacting the sample with an antibody or fragment thereof either of which specifically bind to the 213P1F11-related protein; and, deteπnining that there is a complex of the antibody or fragment thereof and 213P1F11-related protein.
47. A method of claim 45 further comprising a step of: taking the sample from a patient who has or who is suspected of having cancer.
48. A method of claim 45 for detecting the presence of 213P1F11 mRNA in a sample comprising: producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using 213P1F11 polynucleotides as sense and antisense primers, wherein the 213P1F11 polynucleotides used as the sense and antisense primers serve to amplify 213P1F11 cDNA; and, detecting the presence of the amplified 213P1F11 cDNA.
49. A method of claim 45 for monitoring 213P1F11 gene products in a biological sample from a patient who has or who is suspected of having cancer, the method comprising: deteπnining the status of 213P1F11 gene products expressed by cells in a tissue sample from an individual; comparing the status so determined to the status of 213P1F11 gene products in a corresponding normal sample; and, identifying the presence of aberrant 213P1F11 gene products in the sample relative to the normal sample.
50. A method of monitoring the presence of cancer in an individual comprising: performing the method of claim 49 whereby the presence of elevated gene products 213P1F11 mRNA or 213P1F11 protein in the test sample relative to the normal tissue sample indicates the presence or status ofa cancer.
51. A method of claim 50 wherein the cancer occurs in a tissue set forth in Table I.
PCT/US2002/010220 2002-04-01 2002-04-01 Nucleic acid and corresponding protein entitled 213p1f11 useful in treatment and detection of cancer WO2003085121A2 (en)

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EP02728647A EP1578980A4 (en) 2002-04-01 2002-04-01 Nucleic acid and corresponding protein entitled 213p1f11 useful in treatment and detection of cancer
AU2002258689A AU2002258689C1 (en) 2002-04-01 2002-04-01 Nucleic acid and corresponding protein entitled 213P1F11 useful in treatment and detection of cancer
IL16432602A IL164326A0 (en) 2002-04-01 2002-04-01 Nucleic acid and corresponding protein entitled 238p1b2 and pharmaceutical compositions containing the same
CA002480811A CA2480811A1 (en) 2002-04-01 2002-04-01 Nucleic acid and corresponding protein entitled 213p1f11 useful in treatment and detection of cancer
IL164326A IL164326A (en) 2002-04-01 2004-09-28 Cancer-associated products and antibodies binding the same

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IL164326A0 (en) 2005-12-18
AU2002258689A1 (en) 2003-10-20
IL164326A (en) 2011-03-31
CA2480811A1 (en) 2003-10-16
AU2002258689B8 (en) 2003-10-20
EP1578980A2 (en) 2005-09-28

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