The present invention relates to the combination of a DNA vaccination strategy that makes use of a nucleic acid encoding an immunogenic polypeptide, with a non-immunosuppressive inducer of tumor cell differentiation and/or apoptosis or a tumor cell modifier, useful for cancer therapy. Vaccine compositions and kits are provided, as well as specific nucleic acid constructs that are particularly suitable for the preparation of such compositions.

Description of the Invention

The present invention relates to the combination of a DNA vaccination strategy that makes use of a nucleic acid encoding an immunogenic polypeptide, in particular a tumor antigen, with a non-immunosuppressive inducer of tumor cell apoptosis, useful for cancer therapy.

To date, many tumor-associated antigens have been identified and vaccination strategies to elicit immune response against these tumor antigens have been developed. Natural and recombinant cancer protein antigens contain defined immunogenic antigens at standardized levels and their efficacy depends on finding the right adjuvant and delivery system. DNA delivery, e.g. direct injection of gene expression cassettes into a living host, is a novel approach to vaccine and immune therapy. Expression of the delivered genes may result in the specific immune activation of the host immune defenses against the expressed antigen.

The effectiveness of a vaccine strategy relies on the acquisition of an immune response that can be both humoral and cytotoxic. DNA vaccines have been shown to meet these requirements, leading to a strong and persistent cell-mediated (generation of CD8+ cytotoxic and CD4+ helper T cells) and humoral immune responses to the antigen encoded by the plasmid. The application of this type of vaccination to cancers was used first on B-NHL using the idiotype of the surface immunoglobulin as the antigen against which the anti-tumoral response was elicited (Stevenson, F. K. et al., 1995; Syrengelas, A. D. et al., 1996). The protective immunity was also observed in other mouse models of lymphoma and myeloma.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal t(15;17) translocation fusing the Promyelocytic Leukemia gene (PML) to the retinoic acid receptor alpha gene (RAR.alpha.), and by an arrest of myeloid differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) mediated differentiation therapy is now the basis of standard treatment in patients with APL. However, despite prolonged survival obtained with the current trials combining ATRA with chemotherapy, around 10 to 20% of patients still relapse. Therefore, novel therapeutic strategies to eradicate residual disease are needed.

It has been shown that PML-RAR.alpha. junction peptides can be specifically recognized by CD4 T-lymphocytes (Dermime, S. et al., 1996). However, this approach was limited as no peptide specific T-cell line or clone could be generated from cells of patients with APL. This result is ascribed to the generalized impairment of the cellular immune system already reported in cancer patients. On this account, the poor immune status of APL patients is regarded as a major obstacle for immunotherapeutic approaches to APL.

The inventors' collaborators previously developed transgenic mice expressing a human PML-RAR.alpha. cDNA that provide an accurate animal model for human APL (Brown, D. et al., 1997). The inventors took advantage of this APL animal model to test the in vivo efficacy of a newly developed PML-RAR.alpha. DNA based vaccine linked to tetanus toxin fragment C (FrC) sequences. Surprisingly, their results demonstrate that ATRA acts as an adjuvant with PML-RAR.alpha.-FrC DNA vaccination to prolong survival. This was accompanied by an increase in CD4+ and CD8+ T-cells, RAR.alpha. antibody and IFN.gamma. production, suggesting the induction of relevant immune responses. When high dose of ATRA is administered, antibodies directed against FrC are also detected. Therefore, a particular subject of the present invention is a vaccine composition that comprises a nucleic acid PML (Promyelocytic Leukemia gene)--RAR.alpha. (retinoic acid receptor alpha gene)--FrC (tetanus toxin fragment C) fusion gene and all-trans retinoic acid (ATRA) that induces protective immunity and extends lifespan in an acute promyelocytic leukemia animal model.

More generally these results provide a novel targeted approach for APL therapy and may improve clinical outcome in human APL, by combining a DNA vaccination with conventional ATRA therapy.

Furthermore, the inventors found the ability of the DNA that contains a PML-RAR.alpha.-FrC gene to induce protection against challenge encouraging, and led them contemplate other oncogenic fusions. The inventors have shown indeed, for the first time, that the adjuvant property of the combination of ATRA and a strongly immunogenic DNA sequence may help maintain clinical remissions by boosting immune responses against tumor antigens generated by patients.

The inventors have further shown the ability of a DNA that contains a non-specific immunogenic sequence fused to a sequence encoding a polypeptide which enhances the immune response, such as a PML-RAR.alpha.AS-FrC or ScFvBCL1-FrC sequence, combined with ATRA to induce protection against challenge.

The present invention thus provides a vaccine composition comprising (i) a non-immunosuppressive inducer of tumor cell apoptosis and (ii) a nucleic acid comprising a sequence that encodes an immunogenic polypeptide, in particular a tumor antigen, in a pharmaceutically acceptable carrier. Said nucleic acid comprising a polynucleotide encoding an immunogenic polypeptide is present in an amount effective to suppress or attenuate tumor growth upon administration to a mammal, in particular to a human.

Vaccine Compositions

The vaccine compositions according to the invention comprise a nucleic acid that encodes an immunogenic polypeptide and optionally a nucleic acid that encodes a polypeptide that enhances the immune response. According to a particular embodiment, the sequence that encodes an immunogenic polypeptide and the sequence that encodes a polypeptide that enhances the immune response are operatively linked, preferably fused in-frame.

Advantageously, the sequence that encodes a polypeptide that enhances the immune response is selected from the group consisting of a tetanus toxin sequence, preferably the fragment C (FrC) sequence, the cholera toxin (CT) sequence, the E. coli heat-labile toxin (LT) sequence, the Clostridium difficile toxin A sequence and the pertussis toxin (PT) sequence, or fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.

According to a first embodiment, the immunogenic polypeptide may be "non-specific" of a cancer condition. Advantageously, the sequence that encodes said immunogenic polypeptide may be selected from the sequences PML-RAR.alpha.AS-FrC (SEQ ID no10) or ScFvBCL1-FrC (SEQ ID no11).

"PML-RAR.alpha.AS-FrC" denotes the PML-RAR.alpha.As sequence which has been cloned in frame to a BCL1 leader sequence at the 5' position and to the whole of fragment C at the 3' position (SEQ ID no10).

"ScFvBCL1-FrC", also called BCL1-Frc in the figure, denotes the ScFv construct disclosed in King et al 1998 that has been cloned from a lymphoma patient fused in frame to a BCL1 leader sequence at the 5' position and to the whole of fragment C at the 3' position (SEQ ID no11).

Any immunogenic sequence comprising a FrC sequence, or any equivalent immune response enhancer, even though the sequence does not include a tumor antigen, may be appropriate. The scope of this embodiment should not be limited to the sole PML-RAR.alpha.AS-FrC and ScFvBCL1-FrC sequences described above.

In another embodiment, the immunogenic polypeptide may comprise a tumor antigen. Thus, according to this embodiment, the vaccine compositions of the invention comprise a nucleic acid that codes for a tumor antigen.

Among tumor antigens that can be advantageously used, one can cite human PML (Promyelocytic Leukemia gene)--RAR.alpha. (retinoic acid receptor alpha gene), acute myeloid leukemia 1/Eight-Twenty one (AML1/ETO), core binding factor beta/muscle myosin heavy chain (CBF beta/MYH11), ets-like gene/plaletet derived growth factor receptor beta (Tel-PDGF), promyelocytic leukemia zing finger/retinoic acid receptor alpha (PLZF-RAR), myeloid/lymphoid (MLL) fusions, of which there are 40 potential partners, ets-like gene/acute myeloid leukemia 1 (TEL/AML-1), breakpoint cluster region/Abelson (BCR/ABL) (Yun et al., 1999). In a particular aspect of the invention, the tumor antigen is PML-RAR.alpha.. Accordingly, the polynucleotide encoding a tumor antigen of the vaccine composition may comprise sequence SEQ ID no1 (PML-RAR.alpha. fusion point):

5'-gag gtc ftc ctg ccc aac agc aac cac gtg gcc agt ggc gcc ggg gag gca g.rarw.PML|RAR.fwdarw.cc aft gag acc cag agc agc agt tct gaa gag ata gtg ccc agc cct ccc tcg-3'.

When the immunogenicity of the sole tumor antigen is not sufficient to insure efficient protection against tumor growth, it may be desired to provide a nucleic acid sequence that encodes a polypeptide that enhances the immune response to the tumor antigen. Such nucleic acid sequence may be carried on different nucleic acids or on a same nucleic acid.

The vaccine composition may thus further comprise a nucleic acid comprising a sequence that encodes a polypeptide that enhances the immune response to the tumor antigen. Alternatively the nucleic acid that encodes a tumor antigen may further comprise a sequence that encodes a polypeptide that enhances the immune response to the tumor antigen. According to a particular embodiment, the polynucleotide sequence encoding a tumor antigen and the polynucleotide sequence encoding a polypeptide that enhances the immune response to the tumor antigen are operatively linked, preferably fused in-frame.

The nucleic acid comprising a polynucleotide encoding a polypeptide which enhances the immune response to the tumor antigen may be advantageously selected from the group consisting of a tetanus toxin sequence, preferably the fragment C (FrC) sequence, the cholera toxin (CT) sequence, the E. coli heat-labile toxin (LT) sequence, the Clostridium difficile toxin A sequence and the pertussis toxin (PT) sequence, or fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.

Preferably, the nucleic acid used in the composition of the invention comprises a sequence that encodes a PML-RAR.alpha.-FrC antigen (SEQ ID no2 or 7).

The vaccine composition of the invention advantageously comprises a non-immunosuppressive inducer of tumor cell apoptosis or a non-immunosuppressive tumor cell modifier that has adjuvant activity towards the biological response elicited by said nucleic acid encoding the tumor antigen.

Preferably the non-immunosuppressive inducer of tumor cell apoptosis may be selected either from differentiation inducers or selected from the group consisting of arsenic and arsenic related compounds (Lallemand-Breitenbach et al., 1999), all-trans retinoic acid and other retinoid compounds which induce differentiation and apoptosis such as 9-cis RA N-(4-hydroxyphenyl)retinamide (4 HPR), 13 cis RA. CD437 and other differentiation and apoptosis inducers, activation of CD44 by antibodies or hyaluronic acid, hematopoietic growth and differentiation factors may also be effective.

The vaccine composition of the invention allows the simultaneous administration of a non-immunosuppressive inducer of tumor cell apoptosis and of a nucleic acid comprising a sequence that encodes an immunogenic polypeptide, in particular a tumor antigen. However this combined therapy may also be achieved by simultaneously or sequentially administering a composition comprising a non-immunosuppressive inducer of tumor cell apoptosis, and a composition comprising a tumor antigen encoding nucleic acid.

For that purpose the compositions may be in the form of a kit.

Kits

The present invention thus provides a kit comprising (i) a first pharmaceutical composition that comprises a non-immunosuppressive inducer of tumor cell apoptosis and (ii) a second pharmaceutical composition that comprises a nucleic acid comprising a sequence that encodes an immunogenic polypeptide, and preferably a tumor antigen.

The present invention also provides a kit comprising (i) a first pharmaceutical composition that comprises a non-immunosuppressive inducer of tumor cell apoptosis and (ii) a second pharmaceutical composition that comprises a nucleic acid comprising a sequence that encodes an immunogenic polypeptide, preferably a tumor antigen, fused in-frame or linked to a sequence that encodes a polypeptide that enhances the immune response.

The nucleic acid and the non-immunosuppressive inducer of tumor cell apoptosis are as defined above. Preferably, the inducer has adjuvant activity towards the biological response elicited by the nucleic acid that encodes the immunogenic polypeptide, and in particular the tumor antigen. It may be selected from the group consisting of arsenic, all-trans retinoic acid, 9-cis RA, 4 HPPR, 13 cis RA, CD437 and other differentiation and apoptosis inducers, antibodies or hyaluronic acid, hematopoietic growth and differentiation factors.

The components of the kit are preferably formulated in pharmaceutically acceptable carriers.

The nucleic acid and the non-immunosuppressive inducer of tumor cell apoptosis may be administered concurrently, i.e. simultaneously in time, or sequentially, i.e. at different times during the course of a common treatment schedule.

Specific Nucleic Acids

Specific isolated nucleic acids useful in the compositions and kits of the invention are also part of the present invention.

A particular subject of the invention is an isolated nucleic acid that comprises (a) a sequence encoding a PML-RAR.alpha. antigen and further comprises (b) a sequence encoding a polypeptide that enhances the immune response to said PML-RAR.alpha. antigen.

In a preferred embodiment, the sequence encoding a PML-RAR.alpha. antigen and the sequence encoding a polypeptide that enhances the immune response to the PML-RAR.alpha. antigen are operatively linked. Still preferably, said polynucleotides are fused in-frame. Preferably the sequence of the polynucleotide that encodes a PML-RAR.alpha. antigen is SEQ ID no1.

Advantageously, the sequence that encodes a polypeptide that enhances the immune response to the PML-RAR.alpha. antigen is selected from the group consisting of a tetanus toxin sequence, preferably the fragment C (FrC) sequence, the cholera toxin (CT) sequence, the E. coli heat-labile toxin (LT) sequence, the Clostridium difficile toxin A sequence and the pertussis toxin (PT) sequence, or fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. The isolated nucleic acid of the invention may thus comprise sequence SEQ ID no 2 or SEQ ID no 7).

Non-Specific Nucleic Acids

Non-specific isolated nucleic acids useful in the compositions and kits of the invention are also part of the present invention.

A particular subject of the invention is an isolated nucleic acid that comprises (a) a sequence encoding an immunogenic polypeptide, such as PML-RAR.alpha.AS, and further comprises (b) a sequence encoding a polypeptide that enhances the immune response to said immunogenic polypeptide.

In a preferred embodiment, the sequence encoding an immunogenic polypeptide and the sequence encoding a polypeptide that enhances the immune response to the immunogenic polypeptide are operatively linked. Still preferably, said polynucleotides are fused in-frame. Preferably, the sequence of the polynucleotide that encodes PML-RAR.alpha.AS is SEQ ID no8.

Advantageously, the sequence that encodes a polypeptide that enhances the immune response to the immunogenic polypeptide is selected from the group consisting of a tetanus toxin sequence, preferably the fragment C (FrC) sequence, the cholera toxin (CT) sequence, the E. coli heat-labile toxin (LT) sequence, the Clostridium difficile toxin A sequence and the pertussis toxin (PT) sequence, or fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. The isolated nucleic acid of the invention may thus comprise a sequence encoding a PML-RAR.alpha.AS-FrC polypeptide as shown in the sequence SEQ ID no10.

Therapeutics

The nucleic acids as defined according to the invention may be administered in a naked form, free from any delivery vehicles. To this end, the nucleic acid is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.

Alternatively, the isolated nucleic acid or the nucleic acid of the vaccine compositions or kits of the invention may be administered in association with agents that assist in cellular uptake. Examples of such agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes or viral particles for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides.

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

Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin.TM. also known as DOTMA (N-[I-(2,3-dioleyloxy) propyls N,N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DCChoi (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. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Pat. No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in WO 90/11092 as an example.

Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds. A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles may be used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al., (1992). The microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, and WO 94/24263. Otherwise, naked DNA can be directly injected, i.e. intramuscularly.

The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed tumor antigen, the condition of the mammal intended for administration (e.g., weight or age), the mode of administration, and the type of formulation. In general, a therapeutically effective dose from about 1 .mu.g to about 1 mg, preferably, from about 10 .mu.g to about 800 .mu.g and, more preferably, from about 25 .mu.g to about 250 .mu.g, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.

The route of administration is any conventional route used in the vaccine field. As general guidance, a nucleic acid of the invention may be administered via a parenteral route, e.g., by an intradermal, intraepidermal, or intramuscular route. The choice of administration route depends on the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscles. When a neutral or anionic liposome or a cationic lipid, such as DOTMA or DC-Chol, is used, the formulation can be advantageously injected via intramuscular or intradermal routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or subcutaneous routes. In addition electroporation can be developed to improve delivery of DNA to muscle (Mir et al., 1999).

The nucleic acid therapy is combined with administration of a non-immunosuppressive inducer of tumor cell apoptosis, such as arsenic, low dose chemotherapy or all-trans retinoic acid or other retinoic acid compounds--as 9-cis RA, 4 HPR, 13 cis RA, CD437 and other differentiation and apoptosis inducers, activation of CD44 by antibodies or hyaluronic acid, hematopoietic growth and differentiation factors.

A patient is administered with this inducer that is either present in the same vaccine composition as the nucleic acid of the invention, or is present in the form of a separate composition. In the latter, the route of administration may be identical or different to the route of administration used for the nucleic acid. For instance, one may deliver the nucleic acid composition through intradermal or intramuscular routes, whereas the inducer is administered orally.

Therapeutic Applications

The nucleic acids, kits and vaccine compositions of the invention are particularly useful for the treatment of tumor conditions, more particularly cancers. In particular, vaccine compositions comprising PML-RAR.alpha. as the tumor antigen are useful for the treatment of acute promyelocytic leukemia. Examples of antigens useful for cancer therapy include AML1/ETO for the treatment of acute myeloid leukemia (AML) type M2, CBF beta/MYH11 in AML type M4 Eosinophilia, Tel/PDGF for chronic myelomonocytic leukemia (CMML), PLZF-RAR.alpha. in variant acute promyelocytic leukemia, MLL fusions in various lymphoid or myeloid leukemia, TEL/AML-1 for childhood acute lymphoblastic leukemia and BCR/ABL for the treatment of chronic myelogenous leukemia.

A further subject of the invention is thus a method for treating a tumor condition, which method comprises administering to a patient in need of such treatment a therapeutically active amount of (i) a non-immunosuppressive inducer of tumor cell apoptosis and (ii) a nucleic acid comprising a sequence that encodes an immunogenic polypeptide, and in particular a tumor antigen.

Another subject of the invention is the use of a vaccine composition, a kit, or specific nucleic acids as defined above for the preparation of a medicament useful for the treatment of a tumor condition, e.g. cancers.
 

Claim 1 of 7 Claims

1. A vaccine composition comprising (i) a non-immunosuppressive inducer of tumor cell apoptosis and (ii) a nucleic acid comprising a sequence encoding PML-RAR.alpha., wherein the non-immunosuppressive inducer of tumor cell apoptosis is selected from the group consisting of arsenic, all-trans retinoic acid, 9-cis RA, 4 HPPR and 13 cis RA.

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