A DNA vaccine effective for eliciting an immune response against cells that present a carcinoembryonic antigen (CEA) comprises a DNA operably encoding a CEA and a DNA operably encoding a CD40 ligand, SEQ ID NO:1 and SEQ ID NO: 2, respectively, or its homotrimer, CD40LT. The DNA vaccine can be incorporated in a delivery vector such as an attenuated live bacterium or virus, or a liposome carrier. In a method embodiment, the DNA vaccine is administered orally to a mammal, such as a human, to elicit an immune response against CEA presenting cells such as colon cancer cells. A preferred method embodiment includes the additional step of treating the mammal with recombinant antibody fusion protein huKS1/4-IL2 to enhance the immune response effectiveness of the vaccine.

SUMMARY OF THE INVENTION

A vaccine that is effective against CEA presenting cells such as colon cancer cells is provided. The vaccine comprises a plasmid DNA encoding CEA (e.g., DNA sequence SEQ ID NO: 1) and a plasmid DNA encoding a CD40 ligand such as human CD40L, DNA sequence SEQ ID NO: 2 or its homotrimer CD40LT. The CEA and CD40L DNA, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, can be incorporated in the same plasmid or in different plasmids. The combination of plasmid DNA encoding both CEA and a CD40 ligand in a single vaccine promotes activation of both naive T cells and antigen presenting cells such as dendritic cells, thus stimulating two different immune response systems.

The plasmid DNA of the vaccines of the present invention can be operably incorporated in an efficient carrier such as an attenuated bacterium, a non-replicating virus, or a liposome particle.

In a method aspect of the present invention, a DNA vaccine comprising a plasmid DNA encoding CEA (SEQ ID NO: 1) and a plasmid DNA encoding a CD40L (SEQ ID NO: 2) or its homotrimer CD40LT, is administered to a mammal, such as a human, in an amount effective for eliciting an immune response against CEA presenting cells such as colon cancer cells.

In another method aspect of the invention, a mammal such as a human is sequentially administered (a) a DNA vaccine comprising a plasmid DNA encoding CEA (SEQ ID NO: 1) and a CD40 ligand (SEQ ID NO: 21) or its homotrimer CD40LT, in an amount effective for eliciting an immune response against CEA presenting cells such as colon cancer cells, and (b) an effective immune response enhancing amount of a recombinant antibody-IL2 fusion protein (huKS1/4-IL2). The huKS-1/4-IL2 fusion protein enhances the immune responsiveness of the mammal treated with the DNA vaccine so that the immune system more effectively attacks CEA presenting cells, such as colon cancer cells. The vaccine and fusion protein can be administered orally or parenterally. Preferably the vaccine is administered orally, and the fusion protein is administered intravenously.

The vaccines of the present invention provide a preventative treatment for cancers such as, for example, colon cancer, by eliciting an immune response against cells that present CEA, including colon cancer cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A DNA vaccine effective against CEA presenting cells such as certain cancer cells comprises a plasmid DNA construct encoding CEA (SEQ ID NO: 1) and a plasmid DNA encoding a CD40 ligand (SEQ ID NO: 2) or its homotrimer, CD40LT. The CEA and CD40L DNA, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, can be incorporated in the same plasmid or in different plasmids. The DNA plasmid(s) can be operably incorporated into a carrier such as an attenuated bacterium, a non-reproducing virus, or a liposome particle. The DNA vaccine can also comprise "naked" DNA.

The DNA vaccines of the present invention stimulate formation of CTLs that are active against CEA presenting cells, such as colon cancer cells. Because CEA is a specific marker for colon cancer cells, a CTL that forms in response to the vaccine will substantially target only such cancer tissues. CD40 ligand stimulates dendritic cells, which are the most effective type of APCs that aid in producing a cellular immune response. A vaccine comprising a combination of DNA encoding CEA and a CD40 ligand, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, can promote activation of naive T cells both directly and indirectly through the intervention of dendritic cells. Such a combination vaccine simultaneously stimulates two different immune response mechanisms, thus increasing the efficiency of the treatment.

As used herein, and in the appended claims, the term "DNA" refers to deoxyribonucleic acid in both the singular and plural grammatical forms. The term "immunity", as used herein, refers to long term immunological protection against the virulent form of the infectious agent. The term "immunization", as used herein, refers to prophylactic exposure to an antigen of a pathogenic agent derived from a non-virulent source and which results in immunity to the pathogen in the treated subject.

A DNA useful in the vaccines of the present invention preferably comprises a nucleotide sequence that encodes CEA (SEQ ID NO: 1) and/or a CD40 ligand (SEQ ID NO: 2), operably linked to regulatory elements needed for gene expression. Preferably the CD40 ligand is CD40LT. The CEA and CD40 ligand DNA, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, are preferably incorporated in the vaccine as a single plasmid, designated herein as pCEA-CD40LT (i.e., plasmid CEA-CD40 LT). Alternatively, the vaccine can comprise a plasmid DNA encoding CEA (SEQ ID NO: 1) and a separate plasmid DNA encoding CD40L (SEQ ID NO: 2).

When taken up by a cell, a DNA molecule can remain present in the cell as a functioning extrachromosomal molecule and/or can integrate into the cell's chromosomal DNA. DNA can be introduced into cells in the form of a plasmid which can remain as separate genetic material. Alternatively, a linear DNA that can integrate into the chromosome can be introduced into the cell. When introducing DNA into a cell, reagents which promote DNA integration into chromosomes can be added, as is known in the art. DNA sequences that promote integration can also be included in the vaccine. DNA encoding CEA and a CD40 ligand, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, can remain part of the genetic material in an attenuated live microorganism or recombinant microbial vector form of the vaccine, which can live in the cells of the patient.

Useful DNA vaccines preferably include regulatory elements necessary for expression of a nucleic acid molecule. Such elements include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for expression of a sequence that encodes an immunogenic target protein. As is known in the art, these elements are preferably operably linked to the sequence that encodes the desired protein. Regulatory elements are preferably selected that are operable in the species to which they are to be administered.

Initiation codons and stop codons are preferably included as part of a nucleotide sequence that encodes the CEA and CD40 ligand protein in a genetic vaccine of the present invention. The initiation and termination codons must be in frame with the coding sequence.

Promoters and polyadenylation signals included in a vaccine of the present invention are preferably selected to be functional within the cells of the subject to be immunized.

Examples of promoters useful in the vaccines of the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein. Other useful promoters include tissue specific promoters such as tumor endothelium-directed promoters, as well as tumor-selective promoters such as CEA promoters, and treatment-responsive promoters such as early growth response promoters.

Examples of polyadenylation signals useful in the vaccines of the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.

In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. Enhancers include the promoters described hereinabove. Preferred enhancers/promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.

An additional element can be added to the vaccine to serve as a target for cell destruction if it is desirable to be able to eliminate cells receiving the genetic construct for any reason. For example, a herpes thymidine kinase (tk) gene, in an expressible form, can be included in the vaccine. The drug gancyclovir can be administered to the immunized subject, which will cause the selective killing of any cell producing tk. Such means for introducing genetic targets for selective destruction of cells are known and are described in U.S. Pat. No. 5,817,637 to Weiner et al.

Regulatory sequences and codons are generally species dependant. In order to maximize protein production, the regulatory sequences and codons are preferably selected to be effective in the species to be immunized. One having ordinary skill in the art can produce DNA constructs that are functional in a given subject species.

DNA useful in the vaccines of the present invention also includes "naked" DNA as defined in Restifo et al. Gene Therapy, 2000, 7:89-92, the pertinent disclosure of which is incorporated by reference. Alternatively, the DNA can be operably incorporated in a carrier or delivery vector. Useful delivery vectors include biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers such as viruses or bacteria.

Examples of suitable attenuated live bacterial carriers/delivery vectors include Salmonella typhimurium, Salmonella typhi, Listeria monocytogenes, Shigella, Bacillus, Lactobacillus, Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae, Campylobacter, and any other suitable bacterial vector, as is known in the art. Preferred bacterial delivery vectors include attenuated Salmonella typhimurium and attenuated Listeria monocytogenes; particularly preferred is attenuated Salmonella typhimurium. Methods of transforming live bacterial vectors with an exogenous DNA construct are well described in the art. See, for example, Joseph Sambrook and David W. Russell, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

Preferred attenuated viral carriers include Herpes viruses, Adenoviruses, Vaccinia virus, and Avipox virus. Methods of transforming a viral vector with an exogenous DNA construct are also well described in the art. See Sambrook and Russell, above.

Liposome carriers are unilamellar or multilamellar vesicles, having a membrane portion formed of lipophilic material and an interior aqueous portion. The aqueous portion is used in the present invention to contain the polynucleotide material to be delivered to the target cell. It is generally preferred that the liposome forming materials have a cationic group, such as a quaternary ammonium group, and one or more lipophilic groups, such as saturated or unsaturated alkyl groups having about 6 to about 30 carbon atoms. One group of suitable materials is described in European Patent Publication No. 0187702, and further discussed in U.S. Pat. No. 6,228,844 to Wolff et al., the pertinent disclosures of which are incorporated by reference. Many other suitable liposome-forming cationic lipid compounds are described in the literature. See, e.g., L. Stamatatos, et al., Biochemistry 27:3917-3925 (1988); and H. Eibl, et al., Biophysical Chemistry 10:261-271 (1979). Alternatively, a microsphere such as a polylactide-coglycolide biodegradable microsphere may be utilized. A DNA is encapsulated or otherwise complexed with the liposome or microsphere for delivery of the DNA to a tissue, as is known in the art.

The inventive vaccine can also be administered in conjunction with a facilitating agent that improves the uptake of the genetic material of the vaccine by the treated cells. In some preferred embodiments, the DNA can be formulated with or administered in conjunction with a facilitator selected from the group consisting of benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those of the family of local anesthetics, such as disclosed in U.S. Pat. No. 6,248,565 to Williams et al., the pertinent disclosures of which are incorporated herein by reference.

In a method aspect of the present invention, a DNA vaccine can be utilized to provide long term protection against CEA presenting cells such as colon cancer cells, in a vaccinated patient. In one preferred method embodiment a DNA vaccine comprising a plasmid DNA operably encoding both CEA and a CD40 ligand, SEQ ID NO: 1 and SEQ ID NO: 2, respectively (e.g., pCEA-CD40LT), is administered to a mammal in need of protection against CEA presenting cells, in an amount that is sufficient to elicit an immune response against CEA presenting cells.

In another preferred method embodiment of the present invention, tumor growth is inhibited by vaccination of a mammal with the pCEA-CD40LT vaccine of the present invention. In such a method embodiment, an immune response eliciting effective amount of a vaccine comprising a plasmid DNA construct operably encoding both CEA and CD40L, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, is administered to a mammal having a growing tumor comprising CEA presenting cells. The vaccination results in tumor growth arrest and minimizes formation of new tumors by immunizing the mammal against the tumor cells.

In yet another method aspect of the present invention, a mammal is sequentially administered (a) a DNA vaccine comprising a plasmid DNA encoding CEA and a plasmid DNA encoding a CD40 ligand, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, in an amount effective for eliciting an immune response against CEA presenting cells such as colon cancer cells, and (b) an immune response enhancing effective amount of recombinant, humanized KS-1/4 antibody-IL2 fusion protein (huKS-1/4-IL2). Hu KS-1/4-IL2 is described in detail by Gillies et al. J. Immunol., 1998, 160:6195-6203, the relevant disclosure of which is incorporated herein by reference.

IL2 is a complex cytokine produced by activated T cells, which stimulates growth of both B cells and T cells. IL2 activation of T cells also stimulates the production of CD40 ligand on the T cell surface (see generally, Chapter 7 of Charles A. Janeway, Jr. and Paul Travers, Immunobiology The Immune System in Health and Disease, Second Edition, Garland Publishing Co., New York, 1996). The role of IL2 targeted to a tumor microenvironment by huKS-1/4-IL2 fusion protein is to boost anti-tumor T cell responses either by acting as a second costimulatory signal in the activation of CTLs or by further activating pre-activated DCs expressing IL2 receptors. The huKS-1/4-IL2 fusion protein thus enhances the immune responsiveness of the mammal treated with the DNA vaccine so that the immune system more effectively attacks CEA presenting cells, thereby enhancing the effectiveness of the vaccination.

In the method embodiments of the present invention, the vaccines preferably are administered enterally, such as by oral administration, or parenterally, such as by intravenous infusion. In some preferred embodiments, the vaccine is administered intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally, topically, or orally. Most preferably the vaccine is administered orally, incorporated in an attenuated bacterial delivery vector. Preferably, the vaccines are provided in a pharmaceutically acceptable carrier, such as physiological a saline solution, dextrose solution, and the like, as is well known in the art.

Patients suffering from epithelial cancers, such as cancers of the colon, pancreas, lung and breast, can benefit from immunization by the vaccines of the present invention.

Vaccines of the present invention are preferably formulated with pharmaceutically acceptable carriers and exipients such as water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The vaccines can also contain auxiliary substances such as wetting agents, emulsifying agents, buffers, and the like.

The vaccines of the present invention are preferably administered orally to a mammal, such as a human, as a solution or suspension in a pharmaceutically acceptable carrier, at a DNA concentration in the range of about 10 micrograms per milliliter to about 100 micrograms per milliliter. The appropriate dosage will depend upon the subject to be vaccinated, and can depend upon the capacity of the subject's immune system to express the nucleic acids contained in the vaccine. The exact dosage chosen may also depend, in part, upon the judgment of the medical practitioner administering or requesting administration of the vaccine.

Another embodiment of the present invention is a kit comprising the vaccines of the present invention packaged in suitably sterilized containers such as ampules, bottles, vials, and the like, either in multi-dose or in unit-dosage forms. The containers are preferably hermetically sealed after being filled with a vaccine preparation. Preferably, the vaccines are packaged in a container having a label affixed thereto, which label identifies the vaccine, and bears a notice in a form prescribed by a government regulatory agency such as the United States Food and Drug Administration reflecting approval of the vaccine under appropriate laws, dosage information, and the like. The label preferably contains information about the vaccine that is useful to a health care professional administering the vaccine to a patient. The kit also preferably contains printed informational materials relating to the administration of the vaccine, instructions, indications, and any necessary required warnings.

The kit of the present invention can also contain recombinant antibody fusion protein huKS1/4-IL2 packaged in suitably sterilized containers such as ampules, bottles, vials, and the like, either in multi-dose or in unit-dosage forms. Preferably, the fusion protein is packaged in a container having a label affixed thereto, which label identifies the vaccine, and bears a notice in a form prescribed by a government agency such as the United States Food and Drug Administration reflecting approval of the fusion protein under appropriate laws, dosage information, and the like. The label preferably contains information about the fusion protein that is useful to a health care professional administering the fusion protein to a patient. The printed informational materials present in the kit, also preferably contains information relating to the administration of the fusion protein, instructions, indications, and any necessary required warnings.

In particularly preferred embodiments of the vaccine, the plasmid DNA encoding a CD40 ligand encodes CD40 ligand trimer (CD40LT). It is particularly preferred that the plasmid DNA encoding both CEA and CD40LT is operably incorporated in an attenuated bacterial delivery vector. Preferred bacterial delivery vectors are attenuated Salmonella typhimurium and attenuated Listeria monocytogenes, most preferably attenuated Salmonella typhimurium. Vaccines of the present invention comprising a nucleic acid encoding CD40LT in combination with a DNA encoding CEA can simultaneously stimulate two different immune response systems (i.e. cellular and humoral immunity).

The nucleotide sequences of some members of the carcinoembryonic antigen family are known in the art. The nucleotide sequence encoding a human CEA gene has been disclosed by Schrewe et al., in the EMBL database of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK (EMBL accession number is EMBL:HSCEA01), the disclosure of which is incorporated herein by reference (FIG. 7, SEQ ID NO: 1).

Human CD40 ligand (CD40L) is a 154 amino acid protein that plays a central role in regulation of humoral immunity. The DNA sequence encoding human CD40L (also known as CD154) has been published by Grammar et al., in the EMBL database of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK (EMBL accession number is EMBL:HACD40L), the disclosure of which is incorporated herein by reference (FIG. 8, SEQ ID NO: 2). The DNA sequence encoding murine CD40L has been published by Marra et al., in the EMBL database of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK (EMBL accession number is EMBL:AI385482), the disclosure of which is incorporated herein by reference (FIG. 9, SEQ ID NO: 3).

Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same, or a functionally equivalent amino acid sequence to CEA and/or CD40 ligand can be used in the practice of the present invention. Such DNA sequences also include those which are capable of hybridizing to the CEA and/or CD40 ligand sequences.

Altered DNA sequences that can be used in accordance with the present invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the CEA and/or CD40 ligand sequences, which result in a silent change, thus producing functionally equivalent CEA and/or CD40 ligand proteins. Such amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine, asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

As used herein, "a functional equivalent" of CD40 ligand refers to a ligand that binds to CD40 or fragments thereof, but not necessarily with the same binding affinity as native CD40 ligand. In like manner, a functional equivalent of CEA refers to a protein that will bind to antisera raised against human CEA, but not necessarily with the same binding affinity as native human CEA.

As used herein, and in the appended claims, the term carcinoembryonic antigen (CEA) includes the natural antigen found in humans and functional equivalents thereof; and the term "CD40 ligand" includes monomers, dimers, and trimers of the natural ligands found in mammals and functional equivalents thereof. Preferably the functional equivalents of the CEA and/or CD40 ligand DNA share at least about 80% homology with the DNA encoding the aforementioned CEA and/or CD40 ligand proteins.

The DNA sequences of the invention can be engineered in order to alter the CEA and/or CD40 ligand coding sequence for a variety of ends including, but not limited to, alterations that modify processing and expression of the gene product. For example, mutations can be introduced using techniques that are well known in the art, e.g. site-directed mutagenesis, to insert new restriction sites, and the like.
 

Claim 1 of 30 Claims

1. A DNA composition effective for eliciting an immune response against cells that present a carcinoembryonic antigen (CEA) comprising: a plasmid DNA operably encoding a CEA and a CD40 ligand; together with a pharmaceutically acceptable carrier.

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