AU2006201797A9 - Enhancing the immune response to an antigen by presensitizing with an inducing agent prior to immunizing with the inducing agent and the antigen - Google Patents

Description

S&FRef: 599680D1 00

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Sanofi Pasteur Limited, of 1755 Steeles Avenue West, Toronto, Ontario, M2R 3T4, Canada Peter Emtage Brian H. Barber Suryprakash Sambhara Charles Dwo Yuan Sia Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Enhancing the immune response to an antigen by presensitizing with an inducing agent prior to immunizing with the inducing agent and the antigen The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c ENHANCING THE IMMUNE RESPONSE TO AN ANTIGEN BY PRESENSITIZINO WITH AN INDUCING AGENT PRIOR TO IMMUNIZING WITH THE INDUCING 0 TITLE: AGENT AND THE ANTIGEN C FIELD OF INVENTION The present invention relates to methods and compositions for enhancing an immune response to an antigen in an animal.

5 BACKGROUND TO THE INVENTION O Vaccines have been used with a high rate of efficiency to prevent INO infectious diseases caused by agents as diverse as bacteria, viruses and Sparasites (Plotkin, S.A. and Orenstein, W.A. Vaccine, 3 d ed., W.B.

Saunders, Philadelphia, U.S.A. (1991)). Furthermore, a diverse array of immunopotentiating and/or adjuvant-like materials have also been coadministered with said vaccines to augment the immune response (Gupta, R.K. and Siber, Vaccine 13:1263-1276 (1995); Cox, J.R. and Coulter, Vaccine 15:248-256 (1997); Plotkin, S.A. and Orenstein, WA., supra, pp. 36-37).

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), Escherichis coli toxins (Dickinson B.L. and Clements, 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).

Additionally, the modification of an antigen in controlled manner has also been demonstrated to enhance immunogenicity for that antigen. For example, carrier proteins tetanus toxoid diphtheria toxoid when coupled to T-independent antigens, haptens or weak immunogens enhance the immunogenicity of the antigens coupled to these proteins (Herrington, D.A. et al., Nature 328: 257-259 (1987); Nash, H. et al., Fertil.

Steril. 34: 328-335 (1980); Robbins, J.B. and Schneerson, J. Infect. Dis.

161:821-832 (1990); Powell M.F. and Newman, M.J. Vaccine Designs The Subunit and Adjuvant Approach, Plenum Publishing Corp., New York, U.S.A. (1995)).

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0 c- -2- Specifically, tetanus toxoid absorbed with aluminum salts and with O 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 Plotkin. S.A. and Orenstein, supra, Chpt.

18, pp. 441-474), but also as an agent to induce enhanced humoral immune responses against bacterial toxins/subunits or viral antigens when coupled as c- a carrier molecule thereto and/or when co-administered with the vaccine/

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0immunogen to which an immune response is desired (for example, Herrington, D.A. et al., Nature 328:257-259 (1987); Nash, H. et al., Fertil.

Steril. 34:328-335 (1980); Robbins, J.B. and Schneerson, J. Infect. Dis.

161:821-832 (1990); Kaistha, J. et al.. Indian J. Pathol. Microbiol. 39: 287-292 (1996); Mukerjee, R. and Chaturvedi, Clin. Exp. Immunol. 102:496- 500 (1995); US Patent Nos. 4673574,4751064, 5877298).

In the context of Haemophilus influenzae related conjugate vaccines utilizing tetanus toxoid or diphtheria toxoid as carrier, it has been observed that the humoral immune response to the conjugated immunogen is augmented after immune priming to the carrier (Granoff, D.M. et al., J.

Pediatr. 121: 187-194 (1992): Granoff, D.M. et al., Pediatr. Res. 85: 694-697 (1993). In contrast. Ferro and Stimson (Drug Design and Discovery 14:179- 195 (1996)) have demonstrated that animals presensitized with tetanus toxoid exhibit a significantly lower antibody response to a tetanus toxoid conjugated immunogen (gonadotrophin releasing hormone (GnRH) tetanus toxoid) by comparison to immunization with conjugated immunogen in the absence of tetanus toxoid presensitization.

In view of the foregoing, there is a need in the art to develop improved Svaccination protocols and compositions that enhance the immune response to an antigen in the vaccine.

SUMMARY OF THE INVENTION 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

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c-3such a protocol is enhanced several fold over when the antigen alone, without 0 0 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.

Accordingly, the present invention provides a method of enhancing an 0 immune response to an antigen in an animal comprising administering an inducing agent to the animal followed by administering the inducing ageni Sand the antigen to the animal.

c- In one embodiment of the invention, the inducing agent is a bacterial toxoid such as tetanus toxoid or diphtheria toxoid.

The antigen can be any antigen. In one embodiment, the antigen is selected from the group consisting of tumour antigens, pathogenic organisrr antigens, autoimmune antigens, and immunogenic fragments thereof.

The antigen and/or inducing agent may be administered directly or the nucleic acid encoding the antigen and/or inducing agent may be employed. Ir the latter case, the nucleic acid coding for the antigen and/or inducing agen' may be in a vector, plasmid, bacterial DNA or may be naked/free DNA oi

RNA.

In yet additional aspects of the invention, the antigen and inducinc agent may additionally be administered in conjunction with at least on( 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 ai antigen and an inducing agent in admixture with a pharmaceuticall! acceptable diluent or carrier.

Other features and advantages of the present invention will becomi apparent from the following detailed description. It should be understood however, that the detailed description and the specific examples whill indicating preferred embodiments of the invention are given by way c illustration only, since various changes and modifications within the spirit an scope of the invention will become apparent to those skilled in the art fror this detailed description.

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-4- BRIEF DESCRIPTION OF THE DRAWINGS 00 The invention will now be described in relation to the drawings i which: Figure 1 (and SEQ.ID.NO.:1) shows the nucleic acid sequence c modified gp100.

Figure 2 (and SEQ.ID.NO.:2) shows the amino acid sequence c 3 modified gp100.

SFigure 3 (and SEQ.ID.NO.:3 and 4) shows the nucleic acid and amine

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acid sequence of a modified CEA.

Figure 4 is a bar graph demonstrating the effect of tetanus toxoii priming on the immunogenicity of recombinant ALVAC vectors expressini a modified gp100 gene in A2Kb transgenic mice.

Figure 5 is a bar graph demonstrating the effect of tetanus toxoil priming on the immunogenicity of recombinant ALVAC vectors expressin! a modified gp100 gene in A2Kb transgenic mice.

Figure 6 is a bar graph demonstrating the effect of tetanus toxoi 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 an< diphtheria toxoid priming on the immunogenicity of recombinanty ALVAC vectors expressing a modified gp100 gene in A2Kb transgenic mice.

Figure 8 is a bar graph demonstrating the effect of tetanus toxoit priming on the immunogenicity of recombinant ALVAC vectors expressinj native or modified gp100 in A2Kb transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION As hereinbefore mentioned, the present inventors have developed ar improved vaccination protocol wherein the immune response to an antigen ic enhanced if the animal is first primed with an inducing agent and thel subsequently receives the antigen in admixture with the inducing agent.

Accordingly, the present invention provides a method of enhancing at immune response to antigen in an animal comprising administering ar effective amount of an inducing agent to the animal (sometimes referred to a, step hereinafter) followed by administering an effective amount of the 00 inducing agent and the antigen (sometimes referred to as step hereinafter) to the animal.

The term "animal" as used herein includes all members of the anima kingdom including mammals, preferably humans.

The term "enhancing an immune response" is defined as enhancing, improving or augmenting any response of the immune system, for example, ol Seither a humoral or cell-mediated nature. The enhancement of an immune CI response can be assessed using assays known to those skilled in the arl including, but not limited to, antibody assays (for example ELISA assays), antigen specific cytotoxicity assays and the production of cytokines (fol example ELISPOT assays). Preferably, the method of the present inventior enhances a cellular immune response, more preferably a cytotoxic T cel response.

The term "effective amount" of the inducing agent or the inducing agen' and the antigen means an amount effective, at dosages and for periods o time necessary to enhance an immune response.

The term "inducing agent" as used herein means any agent that wher used in the method of the invention can enhance, augment or improve ar immune response to an antigen. For example, the inducing agent enhances an immune response as the immune response to the antigen is greater wher the inducing agent is administered in both steps and of the method o 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 lowe concentration of the antigen than when the inducing agent is not used and stil generate a comparable or perhaps enhanced immune response.

The inducing agent can either be an agent to which the recipien animal is naive or to which the recipient animal has been previously exposed The inducing agent is preferably a foreign or non-self protein. Suitable proteins include, but are not limited to, natural peptides and proteins, (such abovine serum albumin) including proteins derived from bacterial, viral

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-6parasitic, fungal, mycosal and mammalian sources. In one embodiment, the 00 protein inducing agent is a bacterial toxoid derived from a bacterial toxin by their synthetic, chemical, physiochemical or genetic modification (e.g.

t- Diphtheria toxoid, CRM197, Tetanus toxoid, Pertussis toxoid, Pseudomonas

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5 aeruginosa recombinant exoprotein A and Clostridium perfringens exotoxins).

Other proteins derived from bacteria may also be employed. The bacterial c source may be, for example, Haemophilus influenzae, Meningococci, SPneumococci, P-hemolytic streptococci, E. coli, Vibrio, Salmonella,

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Staphylococci, Helicobacter and Campylobacter. Viral sources include influenza HA, NA or RSV capsid proteins.

The term "antigen" as used herein means any agent to which one wishes to generate an immune response.

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 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 administering the inducing agent to the animal, may be generally referred to as "pre-priming". The prepriming of an animal can be achieved in a single dose or repeated at intervals.

As such, 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. One

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-7skilled in the art will appreciate that the dosage regime can be determinei 00 and/or optimized without undue experimentation.

The inducing agent and the antigen may be administered in varioui forms and combinations. For example, when either the inducing agent and/o the antigen is a protein they may be administered in the form of the protein o as a nucleic acid encoding the protein. Therefore, when either the inducin! Sagent and/or the antigen is a protein the term "administering an inducing Sagent" or "administering an antigen" includes both the administration of the C protein and the administration of the nucleic acid encoding the protein. Whei both the inducing agent and antigen are proteins they may be eaci administered as proteins, each administered as nucleic acids encoding thE protein or one may be administered as a protein and the other as a nucleii acid encoding the protein as well as various combinations or permutations o these.

In one example, the inducing agent may be administered as a proteir in both step and step of the method of the invention while the antiger may be administered as a nucleic acid encoding the antigen. In a furthe example, the inducing agent may be administered as a nucleic acid in bott step and step and the antigen can be administered as a protein. i another example, the inducing agent may be administered as either a proteir or a nucleic acid in step and as a nucleic acid in step and the antiger can be administered as a nucleic acid. In such an embodiment, 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 t( a second nucleic acid sequence encoding the antigen. As such, upor administration of the chimeric nucleic acid sequence to the animal, the inducing agent and the antigen will be expressed in vivo as a recombinan fusion protein. In another example, the inducing agent may be administerec as either a protein or a nucleic acid in step and as a protein in step anc the antigen may be administered as a protein. In such an embodiment, 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 b) 0 ci -8other means including chemical crosslinking as described in U.S. Patent No. 5,153,312. There are several hundred crosslinkers available that can PC 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 Sdesirable 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-N-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide.

In one embodiment of the invention, tetanus toxoid is used as an inducing agent. In another embodiment of the invention, 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. Generally, the production of the toxoid can be divided into 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. 5877298, which is incorporated herein by reference). As contemplated by this invention, 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.

In embodiments of the invention employing antigens that are relatively small polypeptides, the antigen may be synthesized in vitro using techniques well known to the person skilled in the art, By "polypeptide" or "protein" is meant any chain of amino acid, regardless of length or post-translational modification glycosylation or phosphorylation). Both terms are used interchangeably in the present application. The terms "polypeptide",or S"protein" as used herein are also intended to include analogs of antigen 00 containing one or more amino acid substitutions, insertions and/or deletion! Amino acid substitutions may be of a conserved or non-conserved nature Conserved amino acid substitutions involve replacing one or more amin acids with amino acids of similar charge, size, and/or hydrophobicit characteristics. Non-conserved substitutions involve replacing one or mor cN amino acids with one or more amino acids which possess dissimilar charge 0 size, and/or hydrophobicity characteristics. Amino acid insertions may consis c- of single amino acid residues or sequential amino acids. Deletions ma consist of the removal of one or more amino acids or discrete portions of thi polypeptide/protein. The deleted amino acids may or may not be contiguous.

As previously noted, one category of antigen is an antigen from pathogenic organism. Various peptides have been found to be significant ii stimulating a protective immune response in infectious diseases Immunotherapeutic antigens useful for the treatment of infectious diseasei may be obtained from pathogenic bacteria, viruses, and eukaryotes. Fo example, hepatitis viral peptides, HIV envelope peptides and plasmodiun yoeli circumsporozoite peptide are capable of protecting the host agains challenge with the infectious agent.

In other preferred embodiments, the antigen is a tumor antigen. Th< term "tumor antigen" as used herein includes both tumor associated antigen.

(TAAs) and tumor specific antigens (TSAs). A tumor associated antigei means an antigen that is expressed on the surface of a tumor cell in highe amounts than is observed on normal cells or an antigen that is expressed or normal cells during fetal development. A tumor specific antigen is an antiger that is unique to tumor cells and is not expressed on normal cells. The tem tumor antigen includes TAAs or TSAs that have been already identified anc those that have yet to be identified and includes fragments, epitopes and an) and all modifications to the. tumor antigens.

The tumor associated antigen can be any tumor associated antiger including, but not limited to, gp100 (Kawakami et al., J. Immunol. 154:3961, 3968 (1995); Cox et al., Science, 264:716-719 (1994)), MART 1/Melan A c-I (Kawakami et al., J. Exp. Med., 180:3.47-352 (1924): Castelli et al., J. Exp.

00 Med., 181:363-368 (1995)), gp75 (TRP-1) (Wang et al., J. Exp- Med., 186:1131-1140 (1296)). and Tyrosinase (Wotfel et al., Eur. J. Immunol., 24:759-764 (1994); Topalian et at., J. Exp. Med., 183:1965-1971 (1996))-; melanoma proteoglycan (H-elstrom et al., J. Immunol, 130:1467-1472 (1 S83); Ross et al., Arch. Biochem Biophys., 225:370-383 (1983)); tumor-specific, widely shared antigens, for example: antigens of MAGE family, for example, MAGE-1, 2,3, and 12 (Van der Bruggen et al., Science, 254:1643-1647 c-I (1991 Rogner et al., Genomics, 29:729-731 (1995)), antigens of BAGE family (Boel et al., immunity, 2:167-1 75 (1895)), antigens of GAGE family, for example, GAGE-1,2 (Van den Eynde et al., J. Exp. Med., 182:689-698 (1995)), antigens of RAGE family, for example, RAGE-1 (Gaugler et at., Immunogenetics, 44:323-330 (1996)), N-acetylglucosaminyltransferase-V (Guilloux et at., J. -Exp. Med., 183:1173-1183 (1996)), and p15 (Robbins et al., J. ImmunoLlS4:5944-5950 (1995)): tumor specific mutated antigens;, mutated 1-catenin (Robbins et al., J. Exp- Med., 183:1185-1192 (1996)), mutated MUM-i (Coulie et al., Proc. Natt Acad. Sd. USA, 92:7976-7980 (1995)), and mutated cyclin dependent kinases-4 (CDK4) (Wolfel et al.. Science, 269:1281- 1284 (1395)); mutated oncogene products: p21 ras (Fossum et at., Int. J.

Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85.2680-2684 (1995)), p53 (Theobald et al., Proc. Nati. Acad. Sci. USA, 02:11993-11997 (1995)), and p185 HER2Ineu (Fisk et al., J. Exp. Med., 181:2105-2117 (1195)); Peoples et al., Proc. Nat. Acad. Sd., USA, 92:432-436 (1985)); mutated epidermal growth factor receptor (EGFR) (Fujimoto et al., Eur. J.

254 Gynecol. Oncal., 16:40-47 (1995)); Harris et al., Breast Cancer Res. Treat, 29:1-2 (1934)); carcinoembryonic antigens (CEA) (Kwong et al., J. Nati.

Cancer Inst, 85:982-990 (1995)); carcinoma associated mutated mucins, fot example, MUC-1 gene products (Jerome et at., J. lmmunol., 151:1654-1662 (1993), loannides; et al., J. Immunol., 151:3653-3703 (1993), Takahashi et at., J. Immunol., 153:2102-2106 (1994)); EBNA gene products of EBV, fo' example, EBNA-1 gene product (Rickinson et al., Cancer Surveys, 13:53-8C (1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol 154:5934-5943 (1095)): prostate specific antigens (PSA) (Xue et al., The 00 Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMVA) (Israeli, et al., Cancer Res., 54:.1807-1811 (1984)) PCTA-11 (Sue et aL, Proc.

Nati. Acad Sci. USA, 93:7252-7257 (1996)); idiotypic epitopee or antigens, for C 5 example, immunoglobulin idiotypes or T cell receptor idiotypes, (Chen et al., J. Immunol., 153:4775-4787 (1994); Syrengelas et al., Nat. Mod., 2:1038c-i1040(1996)); KSA (US Patent 5348887); NY-ESO-11 (WO 98/14464).

Also included are modified tumor antigens and/or epitope/peptides derived therefrom (both unmodified and modified). Examples include, but are not limited to, modified and unmodified epitope/peptides derived from gpl 00 (WO 98/02558;, wO 95129193; WO 97/34613; WO 58/33810; CEA (WO 09/19478: S. Zaremba et al. (1997) Cancer Research 57:4570-7; K.T. Tsang et al. (1Q95) J. tnt. Cancer Inst 87:982-80); MART-I1 (WO 98/58951, WO 08/02538; D. Valmeri et al. (2000) J. Immunot- 164:1125-31); p53 Eura et al. (2000) Clinical Cancer Research 6:679-86); TRP-l and TRP-2 (WO 97/29195): tyrosinase (WVO 96/21734; WO) 97/11669, WO 97/34613; WO 98133810; WO 95/23234; WO 97/26535): KSA (WO 97/15507);, PSA (WO 96/40754); NY-ESO 1 (WO 99/18206); H-ER2/neu (US Patent #5869445); MAGE family related Heidecker et al. (2000) J. Immunol. 164:6041-5; WC 95/04542; WO 95/25530; WO 95/2573!; WO 96/26214; WO 97/31017; WO 98110780).

in a specific embodiment, the tumor-associated antigen is gpl 00, a modified gplOO or a fragment thereof. In one embodiment, the antigen is native gplOO, the sequence of which is known in the art or a modified gplOC having a nucleic acid sequence shown in Figure I and SEQ.ID.*NO.: I or ar amino acid sequence shown in Figure 2 or SEQ.ID.NO..2. The modifiec gpIOO antigen contains two mutations over the native gpioo. at position 21( the threonine was replaced by methionine and at position 288 the alanine wa-.

replaced by valene. The modified gplOO is more fully described in U.S application serial no. 09/693,755, filed on October 20, 2000, which i! incorporated herein by reference.

-12- In another specific embodiment, the tumor-associated antigen is oo carcinoembryonic antigen CEA, a modified CEA or a fragment thereof. The sequence of native CEA is known in the art. The sequence of a modified CEF is shown in Figure 3 or SEQ.ID.NO.:3 and SEQ.ID.NO.:4.

As noted above, the invention also encompasses administering nucleic acids coding for the antigen andlor the inducing agent. Accordingly, in one CN embodiment the antigen is administered as a nucleic acid sequence encoding

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0a native gp100 protein or encoding a modified gp100 protein having the aminc CN acid sequence shown in Figure 2 or SEQ.ID.NO.: 2. The nucleic acic sequence may preferably have the sequence shown in Figure 1 o SEQ.ID.NO.: 1. In another embodiment, the antigen is administered as nucleic acid sequence encoding a native CEA antigen or a modified CE/ 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 ii Figure 3 or SEQ.ID.NO.: 3. In one embodiment, the nucleic acid may bi administered as free or naked DNA or RNA. In a preferred embodiment, th nucleic acid sequence is contained in a vector or plasmid. In oni embodiment, the vectors of the invention may be viral such as poxviru, adenovirus or alphavirus. Preferably the viral vector is incapable c integration in recipient animal cells. The elements for expression from sai vector may include a promoter suitable for expression in recipient anims cells.

An example of an adenovirus vector, as well as a method fc constructing an adenovirus vector capable of expressing an immunogen i described in U.S. Patent No. 4,920,209 (incorporated herein by reference Poxvirus vectors that can be used include, for example, vaccinia and canal pox virus (as described in U.S. Patent Nos. 5364773, 4603112, 5762931 5378457, 5494807, 5505941, 5756103, 5833975 and 5990091-all of whic are herein incorporated by reference). Poxvirus vectors capable I expressing a nucleic acid of the invention can be obtained by homologol recombination as is known to one skilled in the art so that the polynucleotic

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C -13of the invention is inserted in the viral genome under appropriate conditions 00 C for expression in mammalian cells (as described below).

In one preferred aspect the poxvirus vector is ALVAC or ALVAC (2; (both of which have been derived from canarypox virus). ALVAC (oj 5 ALVAC does not productively replicate in non-avian hosts, a characteristic O thought to improve its safety profile. ALVAC is an attenuated canarypo> D virus-based vector that was a plaque-cloned derivative of the licensec 0 canarypox vaccine, Kanapox (Tartaglia et al., Virology 188:217-232 (1992); U.S. Patent Nos. 5505941, 5756103 and 5833975-all of which are incorporated herein by reference). ALVAC has some general properties which are the same as some general properties of Kanapox. ALVAC-based recombinant viruses expressing extrinsic antigens have also been demonstrated efficacious as vaccine vectors (Tartaglia et al, In AIDS Research Reviews (vol. 3) Koff Wong-Staol F. and Kenedy R.C. (eds.), Marcel Dekker NY, pp. 361-378 (1993a); Tartaglia, J. et al., J. Virol. 67:2370- 2375 (1993b)). For instance, mice immunized with an ALVAC (1) recombinant expressing the rabies virus glycoprotein were protected from lethal challenge with rabies virus (Tartaglia, J. et al., (1992) supra) demonstrating the potential for ALVAC as a vaccine vector. ALVAC-based recombinants have also proven efficacious in dogs challenged with canine distemper virus (Taylor, J. et al., Virology 187:321-328 (1992)) and rabies virus (Perkus, M.E. et al., In Combined Vaccines and Simultaneous Administration: Current Issues and Perspective, Annals of the New York Academy of Sciences (1994)), in cats challenged with feline leukemia virus (Tartaglia, J. et al., (1993b) supra), and in horses challenged with equine influenza virus (Taylor, J. et al., In Proceedings of the Third International Symposium on Avian Influenza, Univ. of Wisconsin-Madison, Madison, Wisconsin, pp. 331-335 (1993)).

ALVAC is a second-generation ALVAC vector in which vaccinia transcription elements E3L and K3L have been inserted within the C6 locus Patent No. 5990091, incorporated herein by reference). The E3L encodes a protein capable of specifically binding to dsRNA. The K3L ORF

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k -14has significant homology to ElF.2. Within ALVAC the E3L gene is under

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00 the transcriptional control of its natural promoter, whereas K3L has been placed under the control of the early/late vaccine H6 promoter. The E3L and K3L genes act to inhibit PKR activity in cells infected with ALVAC allowing enhancement of the level and persistence of foreign gene expression.

SAdditional viral vector systems involve the use of naturally host- \0 restricted poxviruses. Fowlpox virus (FPV) is the prototypic virus of the Avipox 0genus of the Poxvirus family. Replication of the avipox viruses is limited to avian species (Matthews. 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 hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant. After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor, J. et al,. Vaccine 6: 504-508 (1988)).

FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor, J. et al., J. Virol. 64:1441- 1450 (1990); Edbauer, C. et al., Virology 179:901-904 (1990); U.S. Patent No.

5766599-incorporated herein by reference).

A highly attenuated strain of vaccinia, designated MVA, have also been used as a vector for poxvirus-based vaccines. Use of MVA is described in U.S. Patent No. 5,185,146.

Other attenuated poxvirus vectors have been prepared by genetic modifications of wild type strains of virus, 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

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C proven useful as a recombinant vector in eliciting a protective immune 00 oO ci 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 5 poxviruses such as the vaccinia virus and avipox virus (described in U.S.

0 Patent Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; and 5,174,993-all of NO which are incorporated herein by reference).

SBacterial DNA useful in embodiments of the invention have been disclosed in the art. These include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette Gu6rin (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 attRS1 DNA sequences). These strains can be genetically engineered to express heterologous antigens, as described in WO 94/19482. (All of the aforementioned issued patent/patent applications are incorporated herein by reference.) 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 s to about 1x10 9 preferably about Ix10 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.

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-16- O0 As will be readily appreciated by those skilled in the art, other bacteria strains useful as vectors in embodiments of this invention include Shigell flexneri, Streptococcus gordonii, and Bacille Calmette Guerin (as described ir O WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 all of which are incorporated herein by reference). In bacterial vecto c embodiments of this invention, a polynucleotide of the invention may be

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0inserted into the bacterial genome, can remain in a free state, or be carried or C- a plasmid.

In another embodiment of the invention, plasmids and/or free/nakec DNA and RNA coding for the antigen can also be administered to an anima for immunogenic purposes (for example, US Patent No. 5589466; McDonnel and Askari, NEJM 334:42-45 (1996); Kowalczyk and Ertl, Cell Mol. Life Sci 55:751-770 (1999)). Typically, this nucleic acid is a form that is unable tc replicate in the target animal's cell and unable to integrate in said animal's genome. The DNA/RNA molecule is also typically placed under the control a a promoter suitable for expression in the animal's cell. The promoter car function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described ir U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter. The desmin promoter is tissue-specific and drives expression in muscle cells More generally, useful vectors have been described WO 94/21797).

For administration of nucleic acids coding for antigen, said nucleic acids can encode a precursor or mature form of the antigen. When it encodes a precursor form, the precursor form can be homologous or heterologous. Ir the latter case, a eucaryotic leader sequence can be used, such as the leadel sequence of the tissue-type plasminogen factor (tPA).

Standard techniques of molecular biology for preparing and purifying nucleic acids can be used in the preparation of aspects of the invention. Foi use as a source of an antigen, a nucleic acid of the invention can be formulated according to various methods known to those who are skilled ir the art.

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00 -17- First, a nucleic acid can be used in a naked/free form, free of any Sdelivery vehicles (such as anionic liposomes, cationic lipids, microparticles, gold microparticles), precipitating agents calcium phosphate)) or t- any other transfection-facilitating agent. In this case the nucleic acid can be t- 5 simply diluted in a physiologically acceptable solution (such as sterile saline or Ssterile buffered saline) with or without a carrier. When present, the carrier \0 preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low 0ionic strength (such as provided by a sucrose solution a solution containing 20% sucrose)).

Alternatively, a nucleic acid can be associated with agents that assist in cellular uptake. It can be, complemented with a chemical agent that modifies the cellular permeability (such as bupivacaine; see, for example, WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.

Cationic lipids are well known in the art and are commonly used for gene delivery. Such lipids include Lipofectin( also known as DOTMA (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2bis(oleyloxy)-3-(trimethylammonio) propane). DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as, for example, described in WO 90/11092.

Other transfection-facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, for example, WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. They include, spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO

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k -18- 93/18759) and membrane-permeabilizing compounds such as GALA o 00 Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for gene delivery (a, described in WO 91/359 and WO 93/17706). In this case, the microparticle coated polynucleotides can be injected via intradermal or intraepiderma 0routes using a needleless injection device ("gene gun"), such as those \0 described, for example, in U.S. Patent No. 4,945,050, U.S. Patent No S5,015,580, and WO 94/24263.

Anionic and neutral liposomes are also well-known in the art (see, foi example, Liposomes: A Practical Approach, RPC New Ed, IRL Press (1990) for a detailed description of methods for making liposomes) and are useful foi delivering a large range of products, including polynucleotides.

The amount of plasmid, naked/free DNA or RNA coding for an antiger to be administered to an animal generally depends on the strength of the promoter used in the DNA construct, the immunogenicity of the expressec gene product, the condition of the animal intended for administration the weight, age, and general health of the animal), the mode of administration and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 pg to about 1 mg, preferably, from about 10 pg to about 800 pg and, more preferably, from about 25 pg to about 250 pg, 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).

A nucleic acid encompassed by the invention can express one or several antigens, In addition, it can also express a cytokine (for example, such as interleukin-2 interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF)) and or co-stimulatory molecules (for example, such as the B7 family of molecules) and/or other lymphokines that enhance the immune response. Thus, for example, a nucleic acid can include an additional DNA sequence encoding, for example, at least one additional tumor associated antigen (and/or immunogenic fragment, homolog, mutant or derivative thereof) and a cytokine and/or lymphokine and/or co-stimulatory

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00 -19molecule placed under the control of suitable elements required fc C expression in an animal cell. Alternatively, embodiments of the invention ma include several nucleic acids, each being capable of expressing a immunogen of the invention.

In additional embodiments of the invention, the antigen per se (c 0 several antigens) can also be mixed with a cytokine and/or lymphokine and/c I0 co-stimulatory molecule, and/or nucleic acids coding therefor.

O An animal may be immunized with an antigen (or a nucleic acid codin! therefor) by any conventional route, as is known to one skilled in the art. Thi may include, for example, immunization via a mucosal oculai intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary traci surface, via the parenteral subcutaneous, intradermal, intramusculai intravenous, or intraperitoneal) route or intranodally. Preferred routes depeni upon the choice of the antigen and/or nucleic acid employed. The administration can be achieved in a single dose or repeated at intervals. Thi appropriate dosage depends on various parameters understood by skillei artisans such as the immunogen itself, the route of administration and the condition of the animal to be vaccinated (weight, age and the like).

In one embodiment, the administration of the inducing agent and thi antigen step of the method) may occur anywhere from about 2 to I weeks, preferably 3 to 6 weeks following the initial pre-priming with th< inducing agent step of the method). Most preferably, step occun from about 3 to 4 weeks after step The dose of the inducing agent is preferably from about 1 to about limit of flocculation units (Lfu), more preferably 4-10 Lfu. The dose of the antigen is preferably from about 10 ug/mg bodyweight to about 1 gg/m bodyweight, more preferably from about 50 jLg/mg to about 500 jg/mg. When the antigen is administered as a nucleic acid sequence in a recombinant vir vector it is preferably in an amount from about 106 to about 10" pfu/ml, mon preferably 5 x 106 to about 5 x 108 pfu/ml.

In one embodiment of the invention, the antigen is a tumor antigen an< the method can be used for the treatment of cancer. Accordingly, the presen

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oO invention provides a method of treating or preventing cancer in an anim.

comprising administering an effective amount of inducing agent to th, animal followed by administering an effective amount of the inducing ager and a tumor antigen to the animal. Preferably, the tumor antigen i administered as a nucleic acid sequence encoding the tumor antigen.

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N The immunization of an animal with the tumor antigen (or nucleic acil 0 coding therefor) in a cancer treatment of the invention may be for either cN prophylactic or therapeutic purpose. When provided prophylactically, thi tumor antigen (or nucleic acid coding therefor) is provided in advance of an evidence or in advance of any symptom due to cancer, or in patients rendere free of disease by conventional therapies but at significant risk fo reoccurrence. The prophylactic administration of the tumor antigen (or nuclei, acid coding therefor) serves to prevent or attenuate cancer in an animal When provided therapeutically, the tumor antigen (or nucleic acid codin! therefor) is provided at (or after) the onset of the disease or at the onset c any symptom of the disease. The therapeutic administration of the tumo antigen (or nucleic acid coding therefor) serves to attenuate the disease.

A particularly preferred method of immunizing an animal with thi antigen (or nucleic acid coding therefor) encompasses a prime-boost protocol Recent studies have indicated that this protocol prime-boost) it quite effective. Typically, an initial administration of an antigen or immunogel (or nucleic acid coding therefor) followed by a boost utilizing the antigen or.

fragment thereof (or alternatively, a nucleic acid coding therefor) will elicit ai enhanced immune response relative to the response observed following administration of either antigen (or nucleic acid coding therefor) or boostini agent. An example of a prime-boost methodology/protocol is described ii WO 98/58956, which is incorporated herein by reference.

Accordingly, in another embodiment the present invention provides method of enhancing an immune response to an antigen in an anima comprising administering an inducing agent to the animal followed by (b administering a first dose of the inducing agent and the antigen to the anima followed by administering a second dose of the inducing agent and th4

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c- -21antigen to the animal. Preferably, the second dose of the inducing agent and OO the antigen is administered anywhere from about 2 to about 8 weeks, preferably 3 to 6 weeks after the first dose administered in step Immunogenicity can be significantly improved if the antigens (or nucleic acids coding therefor) are, regardless of administration format poxvirus, Snaked/free DNA, protein/peptide), co-immunized with adjuvants. Commonly,

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adjuvants are used as an 0.05 to 1.0 percent solution in phosphate buffered saline. Adjuvants enhance the immunogenicity of an antigen but are not cnecessarily immunogenic themselves. Adjuvants may act by retaining the immunogen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system.

Adjuvants can also attract cells of the immune system to an immunogen depot and stimulate such cells to elicit immune responses.

Adjuvants (including the use of immunostimulatory agents as adjuvants) have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of killed or attenuatec bacteria used as vaccines: Extrinsic adjuvants are immunomodulators whic" are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified tha enhance the immune response to antigens delivered parenterally. Some o these adjuvants are toxic, however, and can cause undesirable side-effect making them unsuitable for use in humans and many animals. Indeed, onl! aluminum hydroxide and aluminum phosphate (collectively commonly referret to as alum) are routinely used as adjuvants in human and veterinary vaccines The efficacy of alum in increasing antibody responses to diphtheria ani tetanus toxoids is well established. Notwithstanding, it does have limitations For example, alum is ineffective for influenza vaccination and inconsistenti elicits a cell mediated immune response with other immunogens. Th antibodies elicited by alum-adjuvanted antigens are mainly of the IgG1 isotyp in the mouse, which may not be optimal for protection by some vaccin; agents.

-22- A wide range of extrinsic adjuvants can provoke potent immune 00 responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobactera and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.

N To efficiently induce humoral immune responses (HIR) and cello mediated immunity (CMI), antigens are often emulsified in adjuvants. Many Cl adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).

Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.

Desirable characteristics of ideal adjuvants include: 1) lack of toxicity; 2) ability to stimulate a long-lasting immune response; 3) simplicity of manufacture and stability in long-term storage; 4) ability to elicit both CMI and HIR to antigens administered by various routes, if required; 5) synergy with other adjuvants; 6) capability of selectively interacting with populations of antigen presenting cells (APC); ability to specifically elicit appropriate TH1 or TH2 cell-specific immune responses; and 8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens/immunogens.

U.S. Patent No. 4,855,283 granted to Lockhoff et al on August 8, 1989 which is incorporated herein by reference thereto, teaches glycolipic analogues including N-glycosylamides, N-glycosylureas and N.

glycosylcarbamates, each of which is substituted in the sugar residue by ar amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff et al (Chem. Int. Ed. Engl. 30:1611-1620 (1991)) reported that N-glycolipid analogs

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C -23displaying structural similarities to the naturally-occurring glycolipids, such as 00 0 glycophospholipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized (from long chainr 5 alkylamines and fatty acids that are linked directly with the sugars through the O anomeric carbon atom) to mimic the functions of the naturally occurring lipid ND residues.

0U.S. Patent No. 4,258,029 granted to Moloney and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Nixon-George et al. (J.

Immunol. 14:4798-4802 (1990)) have also reported that octadecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen enhanced the host immune responses against hepatitis B virus.

Adjuvant compounds may also be chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Preferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. 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 preferably with NaOH, 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.

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0 k -24- Persons skilled in the art can also refer to U.S. Patent No. 2,909,46: 00 S(incorporated herein by reference) which describes such acrylic polymer cross-linked with a polyhydroxylated compound having at least 3 hydrox) groups (preferably not more than the hydrogen atoms of the at least thre r- 5 hydroxyls being replaced by unsaturated aliphatic radicals having at least 0 carbon atoms. The preferred radicals are those containing from 2 to 4 carbol \0 atoms vinyls, allyls and other ethylenically unsaturated groups). Thi 0unsaturated radicals may themselves contain other substituents, such at methyl. The products sold under the name Carbopol (BF Goodrich, Ohio USA) are particularly appropriate. They are cross-linked with allyl sucrose o with allyl pentaerythritol. Among them, there may be mentioned Carbopol (fo example, 974P, 934P and 971P). Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto; which are copolymers of maleic anhydride and ethylene, linear or cross-linked, (fo example cross-linked with divinyl ether)) are preferred. Reference may bi made to J. Fields et al. (Nature, 1960, 186: 778-780) for a further descriptioi of these chemicals (incorporated (herein by reference).

In one aspect of this invention, adjuvants useful in any of the embodiments of the invention described herein are as follows. Adjuvants fo parenteral immunization include aluminum compounds (such as aluminun hydroxide, aluminum phosphate, and aluminum hydroxy phosphate). Thl antigen can be precipitated with, or adsorbed onto, the aluminum compouni according to standard protocols. Other adjuvants such as RIB (ImmunoChem, Hamilton, MT) can also be used in parenteral administration.

Adjuvants for mucosal immunization include bacterial toxins thi cholera toxin the E. coli heat-labile toxin the Clostridium difficili toxin A and the pertussis toxin or combinations, subunits, toxoids, o mutants thereof). For example, a purified preparation of native cholera toxil subunit B (CTB) can be of use. Fragments, homologs, derivatives, and fusiol to any of these toxins are also suitable, provided that they retain adjuvan activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants have been described in WO 95/17211 (Arg-7-Lys CT mutant)

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WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu- 00 N 129-Gly PT mutant)). Additional LT mutants that can be used in the methods and compositions of the invention include, for example Ser-63-Lys, Ala-69- Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (such as a 5 bacterial monophosphoryl lipid A (MPLA) of various sources E. coli, O Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, IN saponins, or polylactide glycolide (PLGA) microspheres) can also be used in C mucosal administration.

Adjuvants useful for both mucosal and parenteral immunization include polyphosphazene (for example, WO 95/2415), DC-chol (3 b-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol (for example, U.S. Patent No.

5,283,185 and WO 96/14831) and QS-21 (for example, WO 88/9336).

Antigens and inducing agents (or nucleic acids coding therefor) encompassed by embodiments of the invention may be formulated into pharmaceutical compositions in a biologically compatible form suitable for in vivo animal immunization. By "biologically compatible form suitable for in vivo animal immunization" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to animals in need thereof. immunization with a therapeutically active amount of the pharmaceutical compositions of the present invention, or an "effective amount", are defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result of enhancing an animal's immune response to the antigen. A therapeutically effective amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the animal, and the ability of immunogen to elicit a desired response in the animal. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the immunization context Additionally, the antigens and inducing agents (or nucleic acids therefor) and inducing agents may be in admixture with a suitable carrier

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k -26diluent, or excipient such as sterile water, physiological saline, glucose or the C like to form suitable pharmaceutical compositions. The compositions can also be lyophilized. The compositions may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the 0 like, depending upon the route of administration and the preparation desired.

\O Accordingly, the present invention provides a vaccine composition 0comprising an inducing agent and an antigen in admixture with a pharmaceutically acceptable diluent or carrier. The inducing agent and/or the antigen may be in the form of a protein or a nucleic acid encoding the protein.

In one embodiment, the vaccine composition comprises a recombinant fusion protein comprising an inducing agent linked to an antigen. In another embodiment, the vaccine composition comprises a chimeric nucleic acid sequence comprising a first nucleic acid sequence encoding an inducing agent linked to a second nucleic acid sequence encoding an antigen.

The present invention also includes a use of a vaccine composition of the present invention to enhance an immune response as well as a use of a vaccine composition of the present invention to prepare a medicament to enhance or immune response.

Animals may be immunized with the pharmaceutical compositions via a number of convenient routes, such as by injection (intradermal, intramuscular, subcutaneous, intravenous, intranodal etc.), or by oral administration inhalation, transdermal application, or rectal administration, or any other route of immunization that enables the modulation of an animal's immune system Depending on the route of immunization, the pharmaceutical composition ma) be coated in a material to protect the compound from the action of enzymes acids and other natural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se knowl methods for the preparation of pharmaceutically acceptable compositions witl which animals can be immunized, such that an effective quantity of thi antigen and inducing agent (or nucleic acid coding therefor) is combined in mixture with a pharmaceutically acceptable vehicle (for example, diluer

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-27and/or carrier). Suitable vehicles are described, for example, in Remington's 00 Pharmaceutical Sciences (Remington's Pharmaceutical Sciences (1985), Mack Publishing Company, Easton, Pa., USA). On this basis, the pharmaceutical compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable Scarriers and/or diluents, and may be contained in buffered solutions with a CI suitable pH and/or be iso-osmotic with physiological fluids. In this regard, Sreference can be made to U.S. Patent No. 5,843,456. Reference can also be N made to the textbook Vaccine Design: the Subunit and Adjuvant Approach, Michael F.Powell and Mark J. Newman, eds. Plenum Press, New York, 1995.

The following non-limiting examples are illustrative of the present invention:

EXAMPLES

Example 1 Enhancement of an immune Response to Gpl00 Antiqens Using TT as an Inducing Agent Summary The A2Kb transgenic mouse was used to assess the immunogenicity of recombinant ALVAC vectors expressing the native gpl00 gene and/or the modified gp100 gene (Figure 1 or SEQ.ID.NO.;1). HLA-A0201-restricted gp100-specific CTL (cytotoxic T cell) responses were assessed. The modified gp100 insert used to construct the ALVAC recombinants contained 2 point mutations, one at position 210 where threonine of the native gp100 was replaced by methionine and the other at position 288 where the native alanine was replaced by valine (as described in US Patent Application 09/693,755, filed October 20, 2000 incorporated herein by reference. See also Figure 2 and SEQ.ID.NO.: Mice were primed with vaccine quality tetanus toxoid (TT) in saline. The animals were then immunized and boosted with ALVAC recombinants in combination with TT, In parallel, control studies involving mice unprimed with TT, and boosted with ALVAC recombinants in the presence or absence of TT were also examined for their capability to generate gplOO-specific CTL responses.

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-28- The analysis of the specificity of ALVAC recombinant-induced CTL 00 was focused on HLA-A0201-restricted human CTL epitopes gp100 (209-217 amino acid sequence ITDQVPFSV, SEQ.ID,NO.:5) and gp100 (280-28E amino acid sequence YLEPGPVTA, SEQ.ID.NO.:6) of the native gplO0 molecule. For the transgenic mice that received the modified gp100 ALVA( Srecombinant vectors, effector responses directed against the mutate

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counterparts of these epitopes were examined (namely, gplO0 (209M) (i.e 0 amino acid sequence IMDQVPFSV, SEQ.ID.NO.:7) and gpI00 (280M) (i.e N amino acid sequence YLEPGPVTV, SEQ.ID.NO.:8)).

Materials and Methods Methods of the peptide synthesis, cell culture and cytotoxic T cell (CTL assay were conducted via well documented and standard methodologies, anc as such are well within the scope of those skilled in the art.

Vectors Recombinant ALVAC vectors were constructed via methodologies and/or processes well known to those skilled in the art.

ALVAC parent vector is described in US Patent Nos. 5505941 5756103, 5833975-all of which are incorporated herein by reference. ALVAC parent vector is described in US Patent No. 5990091, which is incorporated herein by reference. Modified gp100 is described in US Paten Application No. 09/693,755, filed on October 20, 2000-which is incorporatec herein by reference.

Synthesis of Peptides Solid phase peptide syntheses were conducted on an ABI 430A automated peptide synthesizer according to the manufacturer's standarc protocols, The peptides were cleaved from the solid support by treatmeni with liquid hydrogen fluoride in a presence of thiocresole, anisole, and methy sulfide. The crude products were extracted with trifluoroacetic acid (TFA) anc precipitated with diethyl ether. All peptides were stored in lyophilized form al The peptides synthesized were: CLP 168-ITDQVPFSV

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C -29- CLP 169-YLEPGPVTA (SEQ.ID.NO.:6) 00 0 CLP 572-IMDQVPFSV (SEQ.ID.NO.:7) CLP 573-YLEPGPVTV (SEQ.ID.NO.:8) CTL Assay 5 Mice of the B10 background (transgenic for the A2Kb chimeric geni 0 were purchased from the Scripps Clinic in California, USA. For tetanus toxo I\ (TT) priming, 20.0 Ag of Aventis Pasteur's TT vaccine prepared in 100.0 pt 0 sterile phosphate buffered saline (PBS, pH 7.2) was injected into th quadriceps and gluteus muscles of each mouse. 4 weeks later, the anima were boosted with an inoculum of 100.0 pi of PBS (pH 7.2) containing 1 x 1( plague-forming units of ALVAC recombinant with/without 20.0 Ig of 1 using the same intramuscular route. Mice were again boosted with tH respective inoculum 25 days later. 11 to 35 days after the final injectioi spleenocytes of the experimental mice were prepared and cultured to enri for CTLs before being assayed for effector activity. In vitro re-stimulation the in vivo generated CTLs was performed by co-culturing in a 25 cm 2 tissL culture flask 3 x 10 7 responder cells splenocytes) with 1.3 x 10 7 irradiate autologous LPS (lipopolysaccharide)-blasts which had been pulsed with ti appropriate peptide (100.0 ti per 108 cells). Cultures were kept in a 370( humidified CO 2 incubator for 7 days before being tested foreffector function a standard 5 hr in vitro s'Cr-release CTL assay as follows. The responde were harvested from the day 7 bulk cultures and washed twice with RPIV 1640 medium (without bovine serum). The positive target was created I incubating 3-5 x 106 P815-A2Kb transfectant cells with 100.0 t of tt specified peptide overnight in a 37 0 C COz incubator. The target cells we then labeled with 1 Cr at 250.0 p.Ci per 1 x 106 cells for 1 hr in the presence 15.0 p. of the same test peptides and 15.0 g of human P2-microglobulin. Aft washing twice with complete medium to remove excess free s"Cr, the targe were incubated at 2.5 x 10 3 with different numbers of the responders for in a 37"C COz incubator, Supematant aliquots were removed and counted I radioactivity.

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Results 00 oO Cl The results obtained for studies using ALVAC and ALVAC recombinants expressing modified gp100 are depicted in Figures 4 anc respectively. The results indicate that tetanus toxoid priming results i 5 clearly enhanced immune response to the immunogen modified gp100 wl O the vector encoding for the immunogen is administered as a mixture N IN tetanus toxoid. This was not vector specific since said enhancement observed with both vectors utilized.

Example 2 Enhancement of an Immune Response to Gp100 and CEA Antiqg Using TT or DT as an Inducinq Agent Summary The A2Kb transgenic mouse was used to assess the immunogeni of recombinant ALVAC vectors expressing native gpl00, modified gpl0C the full length carcinoembryonic antigen (CEA). HLA-A0201-restricted gp' or CEA specific reactive T cell responses were assessed using ELISP assays. The preparation of the gpl00 ALVAC recombinants are describe( Example 1. The full length CEA gene was incorporated into the ALV vector. ALVAC gp100 and ALVAC CEA immunized mice were primed v vaccine quality diphtheria toxoid (DT) and tetanus toxoid (TT) in sal respectively. The animals were then immunized and boosted with ALV recombinants in combination with TT or DT. In parallel, control stud involving mice unprimed with TT, and boosted with ALVAC recombinants the presence or absence of TT were also examined for their capability generate gp100 or CEA specific T cell responses.

The analysis of the specificity of ALVAC gp100 recombinant*inducei cell reactivity was focused on HLA-A0201-restricted human CTL epitof gpO00 (209-217) amino acid sequence ITDQVPFSV, SEQ.ID.NO.

(210M; i.e. amino acids sequence IMDQVPFSV, SEQ.ID.NO.:7), gpl00 (21 288) amino acid sequence YLEPGPVTA, SEQID.NO.:9) and (288V; amino acid sequence YLEPGPVTV, SEQ.ID.NO.:8) of the native and modif gp100 molecule. For the CEA analysis focus was given to the HLA-AO0

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k -31peptides CAP-1 amino acid sequence YLSGANLNL, SEQ.ID.NO.:10) and 00 its modified CAP-6D amino acid sequence (YLSGADLNL, SEQ.ID.NO.: 1) of the native CEA.

Materials and Methods Methods of the peptide synthesis, cell culture and ELISPOT assay were conducted via well documented and standard methodologies, and as such are well within the scope of those skilled in the art.

SVectors c Recombinant ALVAC vectors were constructed via methodologies and/or processes well known to those skilled in the art.

ALVAC parent vector is described in US Patent Nos. 5505941 5756103, 5833975-all of which are incorporated herein by reference, ALVAC parent vector is described in US Patent No. 5990091, which is incorporated herein by reference. Modified gp100 is described in US Paten Application No. 09/693,755, filed on October 20, 2000-which is incorporatec herein by reference.

Synthesis of Peptides Solid phase peptide syntheses were conducted on an ABI 430/ automated peptide synthesizer according to the manufacturer's standar( protocols. The peptides were cleaved from the solid support by treatmen with liquid hydrogen fluoride in a presence of thiocresole, anisole, and methy sulfide. The crude products were extracted with trifluoroacetic acid (TFA) ani precipitated with diethyl ether. All peptides were stored in lyophilized form a 0

C.

2 5 The peptides synthesized were: CLP 168-ITDQVPFSV CLP 169-YLEPGPVTA CLP 572-IMDQVPFSV CLP 573-YLEPGPVTV CLP 165 -YLSGANLNL CLP 1510 -YLSGADLNL

O

O

C -32- ELISPOT Assay SMice of the B10 background (transgenic for the A2Kb chimeric gent were purchased from the Scripps Clinic in California, USA. For tetanus toxoi (TT) and diphtheria toxoid priming, 20.0 jig of Pasteur Merieux Connaught TT and/or DT vaccine was prepared in 100.0 pl of sterile phosphate buffere O saline separately (PBS, pH 7.2) and was injected into the quadriceps an I0 gluteus muscles of each mouse. Three weeks later, the animals wer Sboosted with an inoculum of 100.0 pl of PBS (pH 7.2) containing 2 x 1( plague-forming units of either ALVAC recombinant with/without 20.0 P of TT or DT using the same intramuscular route. Mice were again booste with the respective inoculum 21 days later. After the final injectior splenocytes of the experimental mice were prepared and cultured to enrich fc either gp100 or CEA reactive T cells before being assayed for effector activity In vitro re-stimulation of the in vivo generated T cells was performed b culturing in a 25 cm 2 tissue culture flask 1 x 10 responder cells (i.e splenocytes) with peptide (100.0 pg per 108 cells). Cultures were kept in 37°C, humidified CO 2 incubator for 7 days before being tested for effectc function in a standard IFN gamma ELISPOT assay as follows. Th responders were harvested from the day 7 bulk cultures and washed twic with AIM-V medium (without bovine serum). The target cells were generate, by incubating 1 x 106 P815-A2Kb transfectant cells with 10ug of the specifie peptide 3-5 hours in a 37°C CO 2 incubator. The target cells were washei twice with complete medium to remove excess free peptide and plated on ai ELISPOT plate at 1 x 105 cells well. Responding T cells were harveste, from the tissue culture flasks, washed with excess AIM-V medium ani counted. The responding T cells were then co-cultured with the stimulator cells on the ELISPOT plate at 1x10 5 responders/well.

Results The results obtained for studies utilizing ALVAC CEA and ALVA( modified gp100 recombinants are shown in Figures 6 and 7, respectively The results obtained for studies using native or modified gp100 are shown ii Figure 8. The results indicate that diphtheria toxoid and tetanus toxoi k -33priming results in a clearly enhanced immune response to the modifi Sgpl00, gpO00 and CEA antigens when the vector encoding the antigen administered as a mixture with tetanus toxoid or diphtheria toxoid. This w.

not vector specific since said enhancement was observed with both vecto utilized.

Whereas the invention is susceptible to various modification and/ alternative forms, specific embodiments have been shown by way of examF and are herein described in detail. However, it should be understood that it not intended to limit the invention to the particular embodiments shown, but the contrary, the invention is to cover all modification, equivalents, and/ alternatives falling within the spirit and scope of the invention as defined I the appended claims.

All publications, patents and patent application referred to herein, a herein incorporated by reference in their entirety to the same extent as if ea individual publication, patent or patent application was specifically ai individually indicated to be incorporated by reference in its entirety.

Claims (21)

1. A method of enhancing an immune response to an antigen in an, O animal comprising administering an effective amount of an inducing agent to the animal followed by administering an effective amount of the inducing agent and the antigen to the animal.

2. A method according to claim 1 wherein the inducing agent is a bacterial O C- toxoid.

3. A method according to claim 2 wherein the bacterial toxoid is tetanus toxoid or diphtheria toxoid.

4. A method according to any one of claims 1 to 3 wherein the antigen is a protein. A method according to claim 4 wherein the antigen is selected from the group consisting of tumor antigens, autoimmune antigens and an antigen isolated from a pathogenic organism.

6. A method according to claim 5 wherein the tumor antigen is selected from the group consisting of gp100, carcinoembryonic antigen, tyrosinase, TRP-1, TRP-2, MART-1/Melan A, MAGE family, BAGE family, GAGE family, RAGE family, KSA, NY ESO-1, MUC-1, MUC-2, pS3, p185, HER2/neu, PSA and PSMA and modified forms thereof.

7. A method according to claim 5 wherein the tumor antigen is gpl00 or carcinoembryonic antigen or a modified form thereof.

8. A method according to claim 7 wherein the antigen is GP100 or modified gp100 having the sequence as shown in Figure 2 (SEQ.ID.NO.:2). I

9. A method according to claim 7 wherein the antigen is carcinoembryonic antigen (CEA) or modified CEA having the sequence shown in Figure 3 00 NC (SEQ.ID.NO.:4).

10. A method according to any one of claims 1-9 wherein the antigen is administered as a nucleic acid sequence encoding the antigen. O N 11. A method according to claim 10 wherein the nucleic acid sequence is Sin a vector, plasmid or bacterial DNA.

12. A method according to claim 11 wherein the vector is a viral vector.

13. A method according to claim 12 wherein the viral vector is selected from adenovirus, alphavirus, and poxvirus.

14. A method according to claim 13 wherein the poxvirus is selected from the group consisting of vaccinia, fowlpox and avipox. A method of claim 14 wherein the poxvirus is selected from the group comprising TROVAC. ALVAC, NYVAC, and MVA.

16. A method according to any one of claims 1 to .15 wherein step (b) occurs from about 3 weeks to about 6 weeks after step

17. A method according to any one of claims 1 to 15 wherein step (b) occurs from about 3 weeks to about 4 weeks after step

18. A method according to any one of claims 1 to 17 further comprising (c) administering a second dose of the inducing agent and the antigen.

19. A method according to claim 18 wherein step occurs from about 3 weeks to about 6 weeks after step -36 A method according to claim 18 wherein step occurs from about 3 weeks to about 4 weeks after step 00

21. A method according to any one of claims 1-20 wherein the antigen is administered in combination with at least one member selected from the group consisting of cytokines, lymphokines, co-stimulatory molecules, and nucleic acids coding therefor. O

22. A method according to any one of claims 1-21 wherein the antigen is Cl administered in combination with an adjuvant.

23. A method according to any one of claims 1-22 wherein the inducing agent is tetanus toxoid or diphtheria toxoid and the antigen is a tumor antigen.

24. A method according to claim 23 for the treatment of cancer. A vaccine composition comprising an inducing agent and an antigen.

26. A use of a vaccine composition according to claim 25 to enhance an immune response. Dated 28 April, 2006 Sanofi Pasteur Limited Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON