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  • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
  • A61K39/001194—Prostate specific antigen [PSA]
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  • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
  • A61K39/001195—Prostate specific membrane antigen [PSMA]
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  • 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
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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Definitions

• TITLE Enhanced Immune Response to a Vaccine
• the present invention relates to methods and compositions for enhancing an immune response to an antigen in an animal.
• a number of bacterial toxins have demonstrated immunopotentiating characteristics. These include Staphylococcal toxins (Koppler, J. et al., Science 224:811-817 (1989); White, J. et al., Cell 56:27-35 (1989); WO 98/26747; EP 839536; US Patent No. 5182109), Escherichia coli toxins (Dickinson B.L. and Clements, J.D., Infect. Immun. 63:1617-1623 (1995); Douce, G. et al., Proc. Natl. Acad. Sci. 92:1644-1648 (1995); US Patent No. 5182109) and Streptococcal, Mycoplasma arthritidal, and/or Yersinia enterocolitical toxins (WO 98/26747).
• carrier proteins e.g. tetanus toxoid (TT), diphtheria toxoid (DT)
• TT tetanus toxoid
• DT diphtheria toxoid
• haptens or weak immunogens enhance the immunogenicity of the antigens coupled to these proteins
• tetanus toxoid absorbed with aluminum salts and with preservatives such as Thimerosal (Trademark) given alone or in combination with other bacterial antigens has been used not only as a vaccine to prevent neonatal or adult tetanus (e.g. Plotkin, S.A. and Orenstein, W.A., supra, Chpt. 18, pp.
• the present inventors have determined that the immune response to an antigen can be greatly improved or enhanced if the animal is first primed with a foreign protein or inducing agent and then receives the antigen in admixture with the inducing agent.
• the immune response generated using such a protocol is enhanced several fold over when the antigen alone, without the inducer, is used.
• the method is advantageous as it provides the enhancement or augmentation of the immune response to an antigen and/or improves a vaccination protocol by allowing one to use less antigen.
• the present invention provides a method of enhancing an immune response to an antigen in an animal comprising (a) administering an inducing agent to the animal followed by (b) administering the inducing agent and the antigen to the animal.
• the antigen and/or inducing agent may be administered directly or the nucleic acid encoding the antigen and/or inducing agent may be employed.
• the nucleic acid coding for the antigen and/or inducing agent may be in a vector, plasmid, bacterial DNA or may be naked/free DNA or
• the antigen and inducing agent may additionally be administered in conjunction with at least one member selected from the group consisting of cytokines, lymphokines, co- stimulatory molecules and nucleic acids coding therefor, and adjuvants.
• the invention also includes vaccine compositions comprising an antigen and an inducing agent in admixture with a pharmaceutically acceptable diluent or carrier.
• Figure 1 shows the nucleic acid sequence of modified gp100.
• Figure 2 shows the amino acid sequence of modified gp100.
• Figure 3 shows the nucleic acid and amino acid sequence of a modified CEA.
• Figure 4 is a bar graph demonstrating the effect of tetanus toxoid priming on the immunogenicity of recombinant ALVAC (2) vectors expressing a modified gp100 gene in A2Kb transgenic mice.
• Figure 5 is a bar graph demonstrating the effect of tetanus toxoid priming on the immunogenicity of recombinant ALVAC (1) vectors expressing a modified gp100 gene in A2Kb transgenic mice.
• Figure 6 is a bar graph demonstrating the effect of tetanus toxoid priming on the immunogenicity of recombinant ALVAC vectors expressing CEA in A2Kb transgenic mice.
• Figure 7 is a bar graph demonstrating the effect of tetanus toxoid and diphtheria toxoid priming on the immunogenicity of recombinanty ALVAC vectors expressing a modified gp100 gene in A2Kb transgenic mice.
• animal as used herein includes all members of the animal kingdom including mammals, preferably humans.
• inducing agent means any agent that when used in the method of the invention can enhance, augment or improve an immune response to an antigen.
• the inducing agent enhances an immune response as the immune response to the antigen is greater when the inducing agent is administered in both steps (a) and (b) of the method of the invention than when the antigen alone is administered.
• the method of the invention may also be used to improve an immune response as in the presence of an inducing agent one can generally administer a lower concentration of the antigen than when the inducing agent is not used and still generate a comparable or perhaps enhanced immune response.
• Diphtheria toxoid CRM197, Tetanus toxoid, Pertussis toxoid, Pseudomonas aeruginosa recombinant exoprotein A and Clostridium perfringens exotoxins).
• Other proteins derived from bacteria may also be employed.
• the bacterial source may be, for example, Haemophilus influenzae, Meningococci, Pneumococci, ⁇ -hemolytic streptococci, E. coli, Vibrio, Salmonella, Staphylococci, Helicobacter and Campylobacter.
• Viral sources include influenza HA, NA or RSV capsid proteins.
• Antigens are usually proteins, but may belong to other classes of macromolecules, such as carbohydrates and the like. Protein antigens include both self antigens, such as tumor antigens and autoimmune antigens as well as non self antigens such as antigens derived from pathogenic organisms including viruses, bacteria, fungi, parasites, protozoans and yeast. Antigens may be obtained from natural sources or from host cells genetically engineered to produce the antigens.
• the term "administering" is defined as any conventional route for administering an antigen to an animal for use in the vaccine field as is known to one skilled in the art. This may include, for example, administration via the parenteral (i.e. subcutaneous, intradermal, intramuscular, etc.) or mucosal surface route.
• the antigen and inducing agent may also be administered directly to a lymphatic site for example directly into a lymph node.
• the initial step of the method of the invention i.e. step (a) administering the inducing agent to the animal, may be generally referred to as "pre-priming".
• the pre- priming of an animal can be achieved in a single dose or repeated at intervals.
• the dose of the inducing agent may vary according to factors such as the health, age, weight and sex of the animal.
• the dosage regime may be adjusted to provide the optimum induction of the immune response.
• the dosage regime can be determined and/or optimized without undue experimentation.
• the inducing agent and the antigen may be administered in various forms and combinations.
• the inducing agent and/or the antigen when either the inducing agent and/or the antigen is a protein they may be administered in the form of the protein or as a nucleic acid encoding the protein. Therefore, when either the inducing agent and/or the antigen is a protein the term "administering an inducing agent" or “administering an antigen” includes both the administration of the protein and the administration of the nucleic acid encoding the protein.
• the inducing agent may be administered as either a protein or a nucleic acid in step (a) and as a nucleic acid in step (b) and the antigen can be administered as a nucleic acid.
• the inducing agent and the antigen may be prepared as a chimeric nucleic acid sequence comprising a first nucleic acid sequence encoding an inducing agent linked to a second nucleic acid sequence encoding the antigen.
• the inducing agent and the antigen upon administration of the chimeric nucleic acid sequence to the animal, the inducing agent and the antigen will be expressed in vivo as a recombinant fusion protein.
• the inducing agent may be administered as either a protein or a nucleic acid in step (a) and as a protein in step (b) and the antigen may be administered as a protein.
• the inducing agent and antigen may be covalently linked for example they may be prepared as a recombinant fusion protein in vitro or they may be linked by other means including chemical crosslinking as described e.g., in U.S. Patent No. 5,153,312.
• crosslinkers There are several hundred crosslinkers available that can conjugate two proteins. (See for example "Chemistry of Protein Conjugation and Crosslinking". 1991 , Shans Wong, CRC Press, Ann Arbor).
• the crosslinker is generally chosen based on the reactive functional groups available or inserted on the ligand. In addition, if there are no reactive groups a photoactivatible crosslinker can be used. In certain instances, it may be desirable to include a spacer between the ligand and the oil-body protein.
• Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctional agents: m-Maleimidobenzoyl- ⁇ /-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl- ⁇ /-Hydroxysuccinimide.
• tetanus toxoid is used as an inducing agent.
• diphtheria toxoid is used as an inducing agent.
• the tetanus toxoid or diphtheria toxoid may be prepared by methodologies well known to those skilled in the art and are commercially available from Aventis Pasteur, Smithkline Beecham, Lederle, Statens Inst. etc.
• the production of the toxoid can be divided into 5 stages, namely maintenance of the working seed, mass growth from the working seed, harvest of the toxin, detoxification of the toxin, and purification of the toxoid (for example, as set out in US Patent No.
• tetanus toxoid or diphtheria toxoid is used as such, or can be further adsorbed with aluminum salts and/or admixed with preservatives such as Thimerosal (Trademark), or formulated in additional ways as will be known to those skilled in the art.
• the antigen may be synthesized in vitro using techniques well known to the person skilled in the art.
• polypeptide or “protein” is meant any chain of amino acid, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
• polypeptide or “protein” as used herein are also intended to include analogs of antigens containing one or more amino acid substitutions, insertions and/or deletions. Amino acid substitutions may be of a conserved or non-conserved nature.
• a tumor associated antigen means an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development.
• a tumor specific antigen is an antigen that is unique to tumor cells and is not expressed on normal cells.
• the term tumor antigen includes TAAs or TSAs that have been already identified and those that have yet to be identified and includes fragments, epitopes and any and all modifications to the tumor antigens.
• the tumor associated antigen can be any tumor associated antigen including, but not limited to, gp100 (Kawakami et al., J. Immunol.
• RAGE-1 Gaugler et at., Immunogenetics, 44:323-330 (1996)
• N-acetylglucosaminyltransferase-V Guilloux et at., J. Exp. Med., 183:1173-1183 (1996)
• p15 Robots et al., J. Immunol.154:5944-5950 (1995)
• tumor specific mutated antigens mutated ⁇ -catenin (Robbins et al., J. Exp.
• PSA prostate specific antigens
• PSMA prostate specific membrane antigen
• PCTA-1 Proc. Natl. Acad. Sci. USA, 93:7252-7257 (1996)
• idiotypic epitopes or antigens for example, immunoglobulin idiotypes or T cell receptor idiotypes, (Chen et al., J. Immunol., 153:4775-4787 (1994); Syrengelas et al., Nat. Med., 2:1038- 1040 (1996)); KSA (US Patent # 5348887); NY-ESO-1 (WO 98/14464).
• the nucleic acid sequence may preferably have the sequence shown in Figure 1 or SEQ. ID. NO.: 1.
• the antigen is administered as a nucleic acid sequence encoding a native CEA antigen or a modified CEA antigen having the amino acid sequence shown in Figure 3 or SEQ. ID. NO.: 4.
• the nucleic acid sequence may preferably have the sequence shown in Figure 3 or SEQ. ID. NO.: 3.
• the nucleic acid may be administered as free or naked DNA or RNA.
• the nucleic acid sequence is contained in a vector or plasmid.
• the vectors of the invention may be viral such as poxvirus, adenovirus or alphavirus.
• the viral vector is incapable of integration in recipient animal cells.
• the elements for expression from said vector may include a promoter suitable for expression in recipient animal cells.
• Poxvirus vectors that can be used include, for example, vaccinia and canary pox virus (as described in U.S. Patent Nos. 5364773, 4603112, 5762938, 5378457, 5494807, 5505941 , 5756103, 5833975 and 5990091-all of which are herein incorporated by reference).
• Fowlpox virus is the prototypic virus of the Avipox genus of the Poxvirus family. Replication of the avipox viruses is limited to avian species (Matthews, R.E.F., Intervirology, 17:42-44 (1982)) and there are no reports in the literature of avipox virus causing a productive infection in any non-avian species including man.
• This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipox virus based vectors in veterinary and human applications an attractive proposition.
• FPV has been used advantageously as a vector expressing immunogens from poultry pathogens.
• the NYVAC vector for example, is derived by deletion of specific virulence and host-range genes from the Copenhagen strain of vaccinia (Tartaglia, J. et al. (1992), supra; U.S. Patent Nos. 5364773 and 5494807-incorporated herein by reference) and has proven useful as a recombinant vector in eliciting a protective immune response against an expressed foreign antigen.
• Recombinant poxviruses can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus (described in U.S.
• Bacterial DNA useful in embodiments of the invention have been disclosed in the art. These include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette Guerin (BCG), and Streptococcus.
• Non-toxicogenic Vibrio cholerae mutant strains that are also useful as bacterial vectors in embodiments of this invention are described, for example, in US Patent No. 4,882,278 (disclosing a strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSI DNA sequences).
• An effective immunogen dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention can contain, for example, about 1x10 5 to about 1x10 9 , preferably about 1x10 6 to about 1x10 8 viable bacteria in an appropriate volume for the selected route of administration.
• Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
• Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral immunogens are described, for example, in WO 92/11361.
• Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
• bacterial strains useful as vectors in embodiments of this invention include Shigella flexneri, Streptococcus gordonii, and Bacille Calmette Guerin (as described in WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376; all of which are incorporated herein by reference).
• a polynucleotide of the invention may be inserted into the bacterial genome, can remain in a free state, or be carried on a plasmid.
• non-tissue specific promoters examples include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter.
• CMV Cytomegalovirus
• the desmin promoter is tissue-specific and drives expression in muscle cells. More generally, useful vectors have been described (i.e., WO 94/21797).
• nucleic acids coding for antigen can encode a precursor or mature form of the antigen.
• the precursor form can be homologous or heterologous.
• a eucaryotic leader sequence can be used, such as the leader sequence of the tissue-type plasminogen factor (tPA).
• a nucleic acid of the invention can be formulated according to various methods known to those who are skilled in the art. First, a nucleic acid can be used in a naked/free form, free of any delivery vehicles (such as anionic liposomes, cationic lipids, microparticles, (e.g., gold microparticles), precipitating agents (e.g., calcium phosphate)) or any other transfection-facilitating agent.
• delivery vehicles such as anionic liposomes, cationic lipids, microparticles, (e.g., gold microparticles), precipitating agents (e.g., calcium phosphate)
• precipitating agents e.g., calcium phosphate
• the amount of plasmid, naked/free DNA or RNA coding for an antigen to be administered to an animal generally depends on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the animal intended for administration (i.e. the weight, age, and general health of the animal), the mode of administration, and the type of formulation.
• a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
• the administration can be achieved in a single dose, repeated at intervals, or incorporated into prime-boost protocols (as described below).
• alum aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.
• the efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established. Notwithstanding, it does have limitations.
• alum is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response with other immunogens.
• the antibodies elicited by alum-adjuvanted antigens are mainly of the lgG1 isotype in the mouse, which may not be optimal for protection by some vaccinal agents.
• a wide range of extrinsic adjuvants can provoke potent immune responses to antigens.
• This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged with NaCI, preferably physiological saline (NaCL 9 g/l) all at once in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH.
• NaCI physiological saline
• This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form.
• the polymer concentration in the final vaccine composition will be 0.01% to 2% w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v. Persons skilled in the art can also refer to U.S. Patent No.
• Carbopol for example, 974P, 934P and 971 P.
• EMA Monsanto; which are copolymers of maleic anhydride and ethylene, linear or cross-linked, (for example cross-linked with divinyl ether)
• EMA Monsanto; which are copolymers of maleic anhydride and ethylene, linear or cross-linked, (for example cross-linked with divinyl ether)
• J. Fields et al. (Nature, 1960, 186: 778-780) for a further description of these chemicals (incorporated (herein by reference).
• compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions with which animals can be immunized, such that an effective quantity of the antigen and inducing agent (or nucleic acid coding therefor) is combined in a mixture with a pharmaceutically acceptable vehicle (for example, diluent and/or carrier).
• a pharmaceutically acceptable vehicle for example, diluent and/or carrier.
• Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences (1985), Mack Publishing Company, Easton, Pa., USA).
• mice unprimed with TT, and boosted with ALVAC recombinants in the presence or absence of TT were also examined for their capability to generate gp100-specific CTL responses.
• the analysis of the specificity of ALVAC recombinant-induced CTLs was focused on HLA-A0201 -restricted human CTL epitopes gplOO (209-217) (i.e. amino acid sequence ITDQVPFSV, SEQ.ID.NO.:5) and gplOO (280-288) (i.e. amino acid sequence YLEPGPVTA, SEQ.ID.NO.:6) of the native gplOO molecule.
• Solid phase peptide syntheses were conducted on an ABI 430A automated peptide synthesizer according to the manufacturer's standard protocols.
• the peptides were cleaved from the solid support by treatment with liquid hydrogen fluoride in a presence of thiocresole, anisole, and methyl sulfide.
• the crude products were extracted with trifluoroacetic acid (TFA) and precipitated with diethyl ether. All peptides were stored in lyophilized form at -20°C.
• Cultures were kept in a 37°C, humidified CO 2 incubator for 7 days before being tested for effector function in a standard 5 hr in vitro 51 Cr-release CTL assay as follows.
• the responders were harvested from the day 7 bulk cultures and washed twice with RPMI- 1640 medium (without bovine serum).
• the positive target was created by incubating 3-5 x 10 6 P815-A2Kb transfectant cells with 100.0 ⁇ of the specified peptide overnight in a 37°C CO 2 incubator.
• the target cells were then labeled with 51 Cr at 250.0 ⁇ Ci per 1 x 10 6 cells for 1 hr in the presence of 15.0 ⁇ of the same test peptides and 15.0 ⁇ of human ⁇ 2-microglobulin.
• In vitro re-stimulation of the in vivo generated T cells was performed by culturing in a 25 cm 2 tissue culture flask 1 x 10 8 responder cells (i.e., splenocytes) with peptide (100.0 ⁇ g per 10 8 cells). Cultures were kept in a 37°C, humidified CO 2 incubator for 7 days before being tested for effector function in a standard IFN gamma ELISPOT assay as follows. The responders were harvested from the day 7 bulk cultures and washed twice with AIM-V medium (without bovine serum).

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• 2001
  • 2001-01-05 EP EP01901075A patent/EP1246646A2/en not_active Withdrawn
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