EP1030927A1 - Expressionsplasmide für multiepitopische dna-impfstoffe - Google Patents

Expressionsplasmide für multiepitopische dna-impfstoffe

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Publication number
EP1030927A1
EP1030927A1 EP98957671A EP98957671A EP1030927A1 EP 1030927 A1 EP1030927 A1 EP 1030927A1 EP 98957671 A EP98957671 A EP 98957671A EP 98957671 A EP98957671 A EP 98957671A EP 1030927 A1 EP1030927 A1 EP 1030927A1
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EP
European Patent Office
Prior art keywords
epitopes
plasmid
nucleic acid
expression
sequence
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Withdrawn
Application number
EP98957671A
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English (en)
French (fr)
Inventor
Harry C. Ledebur, Jr.
Jeffrey L. Nordstrom
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Urigen Pharmaceuticals Inc
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Urigen Pharmaceuticals Inc
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Publication of EP1030927A1 publication Critical patent/EP1030927A1/de
Withdrawn legal-status Critical Current

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • the invention relates generally to gene therapy, in particular, the invention relates in part to improved plasmids and methods for nucleic acid based vaccines.
  • Plasmids are an essential element in genetic engineering and gene therapy. Plasmids are circular DNA molecules that can be introduced into bacterial cells by transformation which replicate autonomously in the cell. Plasmids allow for the amplification of cloned DNA. Some plasmids are present in 20 to 50 copies during cell growth, and after the arrest of protein synthesis, as many as 1000 copies per cell of a plasmid can be generated. (Suzuki et al., Genetic Analysis, p. 404, (1989).)
  • Plasmid design and construction utilizes several key factors.
  • plasmid replication origins determine plasmid copy number, which affects production yields. Plasmids that replicate to higher copy number can increase plasmid yield from a given volume of culture, but excessive copy number can be deleterious to the bacteria and lead to undesirable effects (Fitzwater, et al . , EMBO J. 7:3289-3297
  • genes that code for antibiotic resistance phenotype are included on the plasmid and antibiotics are added to kill or inhibit plasmid-free cells.
  • Most general purpose cloning vectors contain ampicillin resistance ( ⁇ -lactamase, or bla) genes. Use of ampicillin can be problematic because of the potential for residual antibiotic in the purified DNA, which can cause an allergic reaction in a treated patient.
  • ⁇ -lactam antibiotics are clinically important for disease treatment. When plasmids containing antibiotic resistance genes are used, the potential exists for the transfer of antibiotic resistance genes to a potential pathogen.
  • neo gene which is derived from the bacterial transposon Tn5.
  • the neo gene encodes resistance to kanamycin and neomycin (Smith, Vaccine 12:1515-1519 (1994)).
  • This gene has been used in a number of gene therapy studies, including several human clinical trials (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6:535-548). Due to the mechanism by which resistance is imparted, residual antibiotic and transmission of the gene to potential pathogens may be less of a problem than for ⁇ -lactams.
  • plasmid vectors have also been shown to affect transfection and expression in eukaryotic cells. Certain plasmid sequences have been shown to reduce expression of eukaryotic genes in eukaryotic cells when carried in cis (Peterson, et al . , Mol . Cell . Biol . 7:1563-1567 (1987); Yoder and Ganesan, Mol . Cell . Biol . 3:956-959 (1983); Lusky and Botchan, Nature 293:79-81 (1981); and Leite, et al . , Gene 82:351-356 (1989) ) .
  • Plasmid sequences also have been shown to fortuitously contain binding sites for transcriptional control proteins (Ghersa, et al . , Gene 151:331-332 (1994); Tully and Cidlowski, Biochem. Biophys . Res . Comm . 144:1-10 (1987); and Kushner, et al . , Mol . Endocrinol . 8:405-407 (1994)). This can cause inappropriate levels of gene expression in treated patients.
  • nucleic acid vaccines or the use of plasmid encoding antigens, has become an area of intensive research and development in the last half decade. Comprehensive reviews on nucleic acid vaccines have recently been published [M.A.
  • the use of epitopes small immunologically relevant protein sequences that are capable of inducing both cellular and humoral responses that result in a protective or therapeutic immune response against large and complex pathogens, is an attractive and amenable strategy provided by the present invention for incorporation into nucleic acid-based vaccines. If multiple epitopes are expressed in the context of a synthetic gene construct, immunity against many antigenic targets, multiple strain variants or multiple pathogens is possible. This disclosure describes the structures and characteristics of gene expression systems that are capable of expressing multiple epitopes.
  • the invention provides a method of genetic immunization comprising the step of presenting multiple epitopes to an organism in need of said immunization.
  • the multiple epitopes are presented with one or more augmenting cytokines and/or are presented with a delivery vehicle selected from the group consisting of cationic lipids, delivery peptides, and polymer based deliver systems .
  • the invention features a plasmid for expression of multiple epitopes comprising a nucleic acid sequence encoding multiple epitopes, wherein each of said epitopes is capable of creating an immune response.
  • the plasmid includes a promoter, a 5' UTR sequence, and a 3' UTR sequence, a nucleic acid sequence encoding polyubiquitin, there are spacers between the nucleic acid regions encoding each of said epitopes, there are proteolytic cleavage sites between each of said epitopes, there are ER targeting signals between each of said epitopes, there are lysosomal and/or endosomal targeting sequences between each of said epitopes.
  • the invention provides a multivalent expression system as shown in Figure 8 and selected from the group consisting of two plasmids, two genes, IRES, and alternative splicing and a method of making a plasmid producing the appropriate nucleic acid sequence.
  • Figure 1 shows a plasmid for multiple epitopes using a beads on a string approach.
  • Figure 2 shows a plasmid for multiple epitopes using beads on a string fused to polyubiquitin.
  • Figure 3 shows a plasmid for multiple epitopes using beads on a string with spacers between epitopes .
  • Figure 4 shows a plasmid for multiple epitopes using beads on a string with proteolytic cleavage sites between epitopes.
  • Figure 5 shows a plasmid for beads on a string epitopes with ER targeting sequences.
  • Figure 6 shows a plasmid for multiple epitopes with ER targeting sequences.
  • Figure 7 shows a plasmid for multiple epitopes with lysosomal/endosomal targeting sequences.
  • Figure 8 shows types of multivalent expression systems.
  • Figure 9 shows a DNA vaccine expression plasmid with two genes .
  • Figure 10 shows a design of a drug-controlled DNA vaccine expression plasmid.
  • the multiple epitopes are directly linked to each other. No spacer sequences between the epitopes are included.
  • the epitope sequences themselves are sufficient for the formation of a functional "pseudo" protein that can be processed into individual peptide epitopes via proteosome cleavage. This concept, i.e. beads-on-a-string, is supported by data that shows full CTL responses to numerous epitopes when they are placed into novel locations within different proteins
  • the present invention provides an exemplary expression system for beads-on-a-string as shown below (See Figure 1) :
  • Promoter CMV tissue-specific (e.g. APC-specific) , or synthetic promoter
  • Intron Synthetic intron that has optimized 5' ss, 3' ss and branch point sequences. Current optimal sequence is IVS 8.
  • Initiation codon AUG is placed in the context of the Kozak sequence to ensure optimal initiation of translation.
  • epitope String of epitopes, each having a length of 9-10 amino acid residues in length for class I presentation, or >10 amino acid residues for class II presentation. It appears that at least 15 epitopes may be strung together.
  • Stop codon For termination of translation. To ensure efficient termination, it is desirable to string two stop codons in tandem. 3' To ensure efficient processing of the
  • UTR/poly(A) mRNA an efficient poly (A) signal, such signal as from human growth hormone, is required .
  • Ub chains which target the substrates to the 26S proteasome, an abundant cellular protease.
  • Ub chain that is fused to the N-terminus of the multiple epitopes that are arranged as beads-on-a-string.
  • Expression plasmids with Ub fused to the antigen have been used to achieve class I presentation (Gueguen and Long, Proc . Natl . Acad. Sci . USA 93:14692-97 (1996) ) .
  • flanking sequence can profoundly influence the generation of epitopes (Yellenshaw et al . , J. Immunol . , 158:1727-33 (1997); Shastri et al., J. Immunol . , 155:4339-46 (1995); Del-Val et al . , Cell , 66:1145-93 (1991); Eggers et al . , J " . Exp. Med. 182:1865-70 (1995); Niedermann et al .
  • Spacers may facilitate the formation of epitopes that induce immunity.
  • the spacer sequence should be one that does not conform at all to the rules for class I or II epitope.
  • it may be desirable to include a hydrophobic, basic or acidic residue at the C-terminus of the spacer to facilitate cleavage between the spacer and the adjacent epitope.
  • the length of the spacer that would be optimal is not known and would have to be determined empirically.
  • viruses e.g. retroviruses, flaviviruses
  • Cleavage which occurs at specific sites, is catalyzed by host proteinases or by virally encoded proteinases.
  • the polyprotein from hepatitis C virus is structured as follows: H2N-C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH.
  • Host cell signal peptidases cleave the junctions in the region between C and NS2.
  • the viral proteinase (NS2-3 proteinase) cleaves the junction between NS2 and NS3.
  • Another viral proteinase cleaves the junctions between NS3 and NS5B .
  • One approach is to insert host cell cleavage sites between the epitope sequences. This may be achieved by insertion of the sequences that are located at the junctions between the C-El-E2-p7-NS2 proteins of the hepatitis polyprotein. Other possibilities are to utilize the recognition site for specific cellular proteases.
  • a second approach is to insert cleavage sites for the viral proteinase between the epitope sequences. Thus, the site for the sequence recognized by the NS3 proteinase, Asp/Glu-X-X-X-X-Cys/ThrlSer/Ala (Koch and Bartenschlager, Virology, 237:78-88 (1997)), may be inserted.
  • the NS3 proteinase must be also encoded by the expression plasmid.
  • two transcription units are required, one for the multiepitopes, one for the viral proteinase. See Section V. Multivalent expression plasmids for nucleic acid-based vaccines.
  • the classical pathway for antigen presentation in the context of class I involves the partial degradation of antigenic proteins into peptides by the proteasome.
  • the peptides are then transported into the endoplasmic reticulum by peptide transporters (TAP-1 and TAP-2) . It is within the lumen of the ER or cis-Golgi that the peptides are loaded into the binding pocket of the MHC class I molecules.
  • TAP-1 and TAP-2 peptide transporters
  • Ciernik et al . , J. Immunol . 2369-75 (1996) have demonstrated that the immunogenicity of an epitope may be enhanced if the epitope sequence is fused in frame with the adenovirus E3 leader sequence and expressed from a plasmid delivered by particle bombardment.
  • ER targeting signals have also been used by Overwijk et al . , Identification of a Kb-restricted Ctl Epitope of Beta- galactosidase : Potential Use in Development of Immunization Protocols for "Self” Antigens, Methods 12: 117-23 (1997) .
  • the signal sequence allows the epitope to be targeted to the ER by the standard protein translocation process.
  • the N- terminal leader sequence targets the peptide to the ER by first binding, as a nascent sequence, to the 54 kDa subunit of the SRP particle.
  • the leader sequence subsequently binds to the b-subunit of the membrane-bound transporter protein, Sec61p. Following entry into the lumen of the ER through a putative channel, a peptidase cleaves the peptide to remove the leader sequence.
  • This mechanism is independent of the TAP transporter system. This alternative mechanism may be advantageous if epitope formation by proteasome cleavage, or epitope transport by the TAP system, are limiting steps in antigen presentation.
  • a leader sequence preferably needs to be attached to the N-terminus of each epitope. Adding a leader sequence to the multiple epitopes that are arranged as beads-on-a string concept is unlikely to work, since the leader will be attached only to the first epitope sequence. Placing an individual targeting sequence on each of the epitopes that is arranged in a bead-on-a-string assembly is a possibility. However, this strategy will depend on accurate proteolytic cleavage at the N-terminus of the leader sequence and at the C-terminus of the adjoined epitope sequence. A gene expression system that utilizes alternative splicing will yield individual epitopes with their own leader sequences. The peptide epitopes produced by this strategy will not depend on random degradation of a protein precursor.
  • the only processing that is required is N-terminal processing that is associated with protein translocation.
  • the C-terminal ends of the epitopes are defined by the stop codons that are designed into the system.
  • the preprotein products may be incompletely synthesized until protein translocation through the pore into the ER has occurred.
  • the prepeptides may be synthesized in their entirety prior to ER translocation. This may expose the prepeptide to the proteasome and transport of proteins that transport epitopes to the ER by the standard pathway.
  • Table II describes the genetic elements used in the alternative splicing strategy.
  • Element Description ER Signal The N-terminal leader sequence from sequence 5' ss adenovirus E3 or preprolactin Strong 5' splice site, one that exactly matches the consensus sequence . Such a sequence is found in the synthetic intron, IVS8.
  • This sequence is derived from the intron synthetic intron, IVS8. It extends from sequence the 3' end of the 5' splice site to the 5' end of the polypyrimidine tract of the 3 'splice site.
  • 3' splice sites sites will be designed to be used equally. Thus, their relative strengths need to be mathed. This will be accomplished by introducing purines within the polypyrimidine regions of the splice site sequences.
  • Balanced splicing will be achieved by controlling the purine content of the pyrimidine-rich sequences of the 3' splice sites. In general, the greater the purine content, the weaker the splice site.
  • one way to design an appropriate alternatively spliced system for epitopes is to model the 3' splice sites of adenoviral late transcripts.
  • leader sequence after splicing, the leader sequence must be fused in frame with the peptide sequence of each epitope. Also, by altering the strengths of the 3' splice sites, the relative amounts of the epitopes may be varied. This may important if certain epitopes are more dominant than others . Table III below shows an example of a balanced set at 3' splice sites derived from the adenoviral late transcript.
  • TTGTATTCCCCTTAG ⁇ T Ad2 (14149) GTTGTATGTATCCAG ⁇ C Ad2 (16515) GTAACTATTTTGTAG ⁇ A Ad2 (17999) CCATGTCGCCGCCAG ⁇ A Ad2 (18801) ATGTTTTGTTTGAAG ⁇ T Ad2 (21649) TTCCTTCTCCTATAG ⁇ G Ad2 (24094)
  • ADRCG ADRCG . Numbers in parentheses indicate the nucleotide position of each 3' splice site. Note the locations of the purines (A or G) that interrupt the polypyrimidine (C or T) region.
  • FIG. 7 Promoter / 5' UTR / AUG / ER signal sequence / 5'ss / internal intron sequence / 3'ss-l / epitope-1 / LAMP-1 transmembrane-cytoplasmic tail / stop codon-1 / 3'ss-2 / epitope-2 / LAMP-1 transmbrane-cytoplasmic tail / stop codon-2 / 3' ss-3 / epitope-3 / LAMP-1 transmbrane- cytoplasmic tail / stop codon-3 / 3' UTR/poly(A) signal
  • each epitope is preceded by an N-terminal leader sequence (e.g.
  • adenovirus E3 adenovirus E3 and followed by the C-terminal endosomal/lysosomal targeting sequence (e.g. the transmembrane and cytoplasmic tail region of LAMP-1) .
  • endosomal targeting sequence e.g. the transmembrane and cytoplasmic tail region of LAMP-1 .
  • Another sequence that may be employed for endosomal targeting is the cytoplasmic tail of membrane immunoglobulin (Weiser et al . , Science 276:407-9 (1997); Achatz et al., Science 276:409-11 (1997)).
  • nucleic acid-based vaccines it may be important to have the capability of expressing multiple gene products.
  • expression of multiple intact antigens or multiepitope gene product may enhance the potency of the these vaccines.
  • Co-expression of costimulatory proteins, such as IL-2, IL-6, IL-12, GM-CSF, B7.1 or B7.2 have been demonstrated to enhance the immune response to an encoded antigen (Geissler et al . , J. Immunol . 158:1231-1237 (1997), Irvine et al . , (1996); Kim et al., Vaccine 15:879-83 (1997); Okada et al., J. Immunol .
  • Co-expression of proteins that facilitate peptide epitope formation such as proteolytic enzymes (e.g. the NS3 protease from hepatitis C (Koch and Bartenshlager, 1997)) or chaperone proteins (e.g. heat shock protein Hsp65 (Wells et al . , Scand. J. Immunol . , 45:605-12 1997)), may also enhance the response.
  • proteolytic enzymes e.g. the NS3 protease from hepatitis C (Koch and Bartenshlager, 1997)
  • chaperone proteins e.g. heat shock protein Hsp65 (Wells et al . , Scand. J. Immunol . , 45:605-12 1997)
  • Hsp65 heat shock protein
  • nucleic acid-based vaccine expression plasmid that has two genes is shown in Figure 9.
  • the GeneSwitch is a chimeric protein that consists of human progesterone receptor with a modified ligand binding domain, a DNA binding domain from yeast GAL4, and an activator domain from Herpes Simplex VP16.
  • a synthetic steroid mifepristone
  • the GeneSwitch protein becomes activated (binds the synthetic steroid, presumably dimerizes, and translocates to the nucleus) .
  • the activated GeneSwitch then binds to a target sequence (multiple GAL4 binding sites linked to a minimal promoter) and thereby stimulates the transcription of the desired transgene (Wang et al., Proc . Na tl . Acad . Sci . USA 91:8180- 84 (1994); Wang et al . , Nature Biotechnology 15:239-243 (1997a); Wang et al . , Gene Therapy 4:432-41 (1997b)).
  • the GeneSwitch may be used to regulate the expression of a plasmid for nucleic acid-based vaccines. It is possible that the timing of expression may influence the immune response. Thus, with a GeneSwitch regulated system, the genes that encode the multiepitopes may be turned on at a defined time after DNA delivery by the administration of the ligand (mifepristone) to the animal. If the expression plasmid persists in vivo for a long enough time, the GeneSwitch system also can be used to provide pulsatile expression of the multiepitope gene products. An example of the design of a system that is regulated by the GeneSwitch is shown in Figure 10.

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EP98957671A 1997-11-12 1998-11-09 Expressionsplasmide für multiepitopische dna-impfstoffe Withdrawn EP1030927A1 (de)

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US6513497P 1997-11-12 1997-11-12
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US20030138808A1 (en) * 1998-02-19 2003-07-24 Simard John J.L. Expression vectors encoding epitopes of target-associated antigens
US6861234B1 (en) 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
CA2416289C (en) * 2000-07-21 2012-12-04 Essentia Biosystems, Inc. Multi-component biological transport systems
DK1453471T3 (da) * 2001-11-07 2011-03-28 Mannkind Corp Ekspressionsvektorer, der koder for epitoper af antigener, og fremgangsmåde til deres konstruktion
CA3031270A1 (en) 2004-03-03 2005-12-22 Revance Therapeutics, Inc. Compositions and methods for topical diagnostic and therapeutic transport
US9211248B2 (en) 2004-03-03 2015-12-15 Revance Therapeutics, Inc. Compositions and methods for topical application and transdermal delivery of botulinum toxins
CN1961071A (zh) * 2004-03-15 2007-05-09 比奥根艾迪克Ma公司 使用可选剪接在真核细胞中表达多肽多聚体的方法和构建物
EP1861112A4 (de) 2005-03-03 2009-07-22 Revance Therapeutics Inc Zusammensetzungen und verfahren zur topischen anwendung und transdermalen abgabe von botulinumtoxinen
DE102006004612A1 (de) * 2006-02-01 2007-08-16 Ralf Dr. med. Jochem Therapeutische Zusammensetzung zur Vorbeugung und Bekämpfung von Knochenmetastasen
EP2834259A4 (de) 2012-04-02 2016-08-24 Moderna Therapeutics Inc Modifizierte polynukleotide

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EP0487587A1 (de) * 1989-08-18 1992-06-03 Chiron Corporation Rekombinante retroviren um vektorkonstruktionen an zielzellen zu liefern
EP0706319A4 (de) * 1993-01-20 1998-04-22 Biotransplant Inc Retrovirale vektoren mit der fähigkeit zur expression multimerer proteine von mehreren translationsinitiations-orten
CA2195642A1 (en) * 1994-07-27 1996-02-08 Andreas Suhrbier Polyepitope vaccines
DE19514310A1 (de) * 1995-04-18 1996-10-24 Univ Ludwigs Albert Vektoren zur Transfektion von eukaryotischen Zellen, deren Verwendung und damit transfizierte Zielzellen
US6946133B1 (en) * 1996-03-20 2005-09-20 The United States Of America As Represented By The Department Of Health And Human Services Prostate specific antigen oligo-epitope peptide

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