WO2008037080A1 - Methods and compositions for immune response modulation and uses thereof - Google Patents
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- WO2008037080A1 WO2008037080A1 PCT/CA2007/001731 CA2007001731W WO2008037080A1 WO 2008037080 A1 WO2008037080 A1 WO 2008037080A1 CA 2007001731 W CA2007001731 W CA 2007001731W WO 2008037080 A1 WO2008037080 A1 WO 2008037080A1
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
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- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the present invention relates to immunology and more specifically to methods and compositions for modulating the immune response and uses thereof in prophylactic and therapeutic anti-tumor applications.
- the immune system acts as a defense against a variety of internal and external conditions which include, for example, infections, cancer, mutations, injuries and the like, and is mediated by two interconnected systems: the humoral and cellular immune systems.
- the humoral system is mediated by the action of soluble molecules termed antibodies or immunoglobulins which, through their properties of specifically combining with a target (e.g., an antigen) recognized as being foreign to the body (or non-self), can inactivate same.
- a target e.g., an antigen
- the cellular immune system also involves the mobilization of cells, termed T cells. T cells are responsible for what is called cell-mediated immunity. This immunity involves the destruction of foreign cells, infected cells or the like by the action of cells of the immune system.
- T cells can be subdivided into different subsets based on surface markers or based on function. For example, “helper”, “regulatory” and “killer” T cell subsets have been described.
- a T cell which recognizes and binds to a particular antigen displayed on the surface of another cell (often termed antigen presenting cell or APC) can become activated.
- An activated T cell can multiply, produce cytokines and, if it is a killer T cell, can kill the cell to which it is bound.
- Helper T cells generally produce cytokines and activate other cells of the immune system. Killer T cells recognize infected, foreign or altered cells, such as cancer cells and eliminate them. Regulatory T cells can modulate or suppress certain immune responses.
- T cell activation involves the recognition of an MHC-peptide complex by the antigen-specific T cell receptor together with other receptor ligand interactions, known as costimulatory interactions.
- additional means can be used to stimulate T cells, such as antibodies to T cell surface receptors or mitogenic lectins.
- the induction of proliferation is only but one marker of T cell activation, since other markers include: increase in lymphokine or cytokine production, cytotoxic activity and a change in the basal or resting state of the cell.
- T cell activation involves a variety of receptor/ligand interactions between T cells and antigen presenting cells.
- a component of T cell activation is the T cell receptor (TCR), a disulfide-linked heterodimer which contains two glycoprotein chains ( ⁇ / ⁇ ) uncovalently associated with a complex of low molecular weight invariant proteins which are commonly designated as CD3. While the TCR ⁇ and ⁇ chains (or Y and ⁇ ) determine the antigen specificities of the T cell, the CD3 structures of the TCR are thought to be responsible for transducing the activation signal upon binding of the ⁇ and ⁇ chains to its ligand.
- the TCR typically interacts with small peptidic antigens which are presented by the major histocomptability complex (MHC) proteins.
- MHC proteins are a highly polymorphic set of molecules which are randomly dispersed throughout the species and further increase the complexity of the T cell activation phenomenon.
- T cell activation usually involves a trimolecular interaction between a TCR, a peptidic antigen and MHC proteins which bind to this antigen.
- this signal alone is usually not sufficient to activate a T cell, rather, other receptor-ligand interactions, called costimulatory interactions are usually also required.
- the CD28 receptor on T cells, binding to B7 molecules on antigen presenting cells can provide such a costimulatory signal.
- CD28 is a cell surface glycoprotein constitutively expressed on most mature T-cells and thymocytes, while the CTLA-4 receptor, which triggers an inhibitory signal in the T cell, is not present on resting T cells and is only detectable 48 to 72 hours after T cell activation.
- the primary ligands for these molecules are
- CD80 and CD86 are members of the Ig gene superfamily. Both molecules have a cytoplasmic tail (T domain), a trans-membrane spanning domain (TM) as well as two extracellular Ig-like domains: the N-terminal domain most closely resembles the Ig variable-like region (V-domain) and the membrane proximal domain shows homology with the Ig constant-like domain (C-domain) (Freeman, G. J. et al., 1989. J. Immunol. 143:2714; Freeman, G. J. et al., 1993. Science 262:909).
- CD86 is constitutively expressed on APC and after activation of APC, the expression of CD86 is quickly up-regulated followed by a gradual return to baseline levels.
- the expression of CD80 is delayed compared to CD86 and its expression is maximal 48 to 72 hours after the initiation of an immune response.
- Vaccines are useful to immunize individuals against target antigens such as pathogen antigens or antigens associated with cells involved in human diseases.
- Antigens associated with cells involved in human diseases include tumor- associated antigens (TAA) and antigens associated with cells involved in autoimmune diseases.
- TAA tumor- associated antigens
- Cancer immunotherapy aims at destroying tumor cells by immunological mechanisms, for example by inducing an antitumor immune response using a vaccine comprising a tumor antigen.
- Immunotherapy compared to conventional methods of cancer therapy like surgery, radiation, and chemotherapy, is much less toxic, and no serious complications have been described so far.
- immunotherapy has the potential to work at different stages of disease. At initial stages it could be a good supplementary treatment to surgical removal of primary tumors, aiming to prevent development of disseminated disease. At advanced stages of disease it could be the only means of treatment, as conventional methods are often ineffective.
- the goal of immunotherapy is to increase the ability of the immune system to recognize tumor cells and to develop effective mechanisms of tumor elimination.
- tumor-associated antigens typically induce weak immune responses. Therefore there is a continuous need to develop new cancer immunotherapies and new strategies to improve/enhance the immune response against TAA.
- the invention relates to products (compositions, nucleic acids and/or polypeptides) and uses thereof e.g., for inducing an immune response against a tumor/cancer-related antigen.
- the invention provides a composition comprising: a) a first recombinant nucleic acid comprising a nucleic acid sequence encoding a chimeric co-stimulatory molecule, said chimeric co-stimulatory molecule comprising; i) a functional V region of CD80; ii) a functional C region of CD86; and iii) transmembrane and cytoplasmic domains of CD86 or functionally active fragments thereof; b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
- the above-mentioned composition is an immunogenic or vaccine composition.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the above-mentioned composition and a pharmaceutically acceptable carrier or excipient.
- the above-mentioned composition further comprises an adjuvant.
- the above-mentioned chimeric co-stimulatory molecule comprises the amino acid sequence of SEQ ID NO:2.
- the above-mentioned first recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO:1.
- the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of:
- the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned composition.
- the above-mentioned first nucleic acid is present in a vector comprising said nucleic acid sequence encoding a chimeric co-stimulatory molecule operably linked to a transcriptional regulatory sequence.
- the above-mentioned second nucleic acid is present in a vector comprising said nucleic acid sequence encoding said tumor- associated antigen polypeptide operably linked to a transcriptional regulatory sequence.
- the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of:
- a tumor-associated antigen polypeptide or a recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
- the above-mentioned subject is a mammal, in a further embodiment, a human.
- the above-mentioned vector is a viral vector.
- the viral vector is an adenoviral vector.
- the above-mentioned tumor-associated antigen is STEAP, or an antigenic variant or fragment thereof.
- the above-mentioned cancer is characterized by
- the above-mentioned cancer is selected from prostate cancer, pancreatic cancer, colon cancer, breast cancer, testicular cancer, cervical cancer, bladder cancer, ovarian cancer, leukemia and Ewing sarcoma.
- the above-mentioned cancer is prostate cancer.
- the above-mentioned cell is an antigen-presenting cell.
- the invention provides a vector comprising: a) a nucleic acid sequence encoding the chimeric co-stimulatory molecule defined in claim 1 operably linked to a transcriptional regulatory sequence; and b) a nucleic acid sequence encoding a tumor-associated antigen polypeptide operably linked to a transcriptional regulatory sequence.
- the invention provides a recombinant cell comprising the above-mentioned vector.
- the above- mentioned cell is an antigen-presenting cell.
- the invention provides a composition comprising the above-mentioned cell and a pharmaceutically acceptable carrier or excipient.
- the invention provides a method of activating a tumor-specific human T cell, said method comprising contacting said T cell with the above-mentioned cell or the above-mentioned composition.
- the above-mentioned contacting step is carried out ex vivo or in vitro.
- the above-mentioned contacting step is carried out in vivo and said cell or composition is administered to a subject comprising the above-mentioned human T cell.
- the above-mentioned human T cell is derived from a subject suffering from a cancer.
- the invention provides a commercial package or kit comprising:
- the invention provides a use of an agent selected from (i) the above-mentioned composition (ii) the above-mentioned vector (iii) the above-mentioned cell and (iv) any combination of (i) to (iii), for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
- the invention provides a use of an agent selected from (i) the above-mentioned composition (ii) the above-mentioned vector (iii) the above-mentioned cell and (iv) any combination of (i) to (iii), for the preparation of a medicament for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
- Figure 1 shows an analysis of A) STEAP and B) CD80 protein expression in different 293 cells infected with different adenovirus constructs
- Figure 2 shows the results of a Limulus amebocyte lysate (LAL) test in Adenovirus stocks. Adenovirus samples were compared to a standardized curve of E. coli isotype 01 11 :B4 endotoxin (0,50 to 0,03 EU/ml);
- Figure 3 shows the expression of CD80 in different adenovirus constructs-infected monocytes 24h after infection;
- Figure 4 shows the prophylactic tumor vaccination model used. Mice were vaccinated with an adenovirus vector expressing STEAP and the chimeric
- mice Four weeks later, mice were injected with CT26 tumor cells expressing
- Figure 5 shows the detection by RT-PCR of STEAP expression in different CT26 tumor cell clones transfected with the nucleic acid encoding STEAP;
- Figure 6 shows tumor growth in BALB/c mice injected with different numbers (10 3 , 10 4 and 10 5 ) of CT26 tumor cells expressing STEAP. Six mice were injected in each group. Each point represents the size of a tumor in a given mouse;
- FIG 7 shows the assessment of tumor growth (CT26 cells expressing STEAP) following vaccination with Adv control, Adv-STEAP, Adv- CD ⁇ Owt, Adv-CD80wt/STEAP and Adv-V1C2/STEAP. Tumor growth was determined 21 days after injection of 10 5 tumor cells; Figure 8 shows tumor growth at different time points (day 9, 13, 15,
- Figure 9 shows the phenotypic and functional properties of antigen- specific T cells following vaccination with Adv-V1C2/STEAP.
- CD8 + T cells Unstimulated CD8 + T cells.
- Figure 10 shows the response (expressed as the percentage of IFN- and IL-2-positive CD4 + and CD8 + T cells) of T cells isolated from mice vaccinated with a control Adv, Adv-CD80wt/STEAP and Adv-V1 C2/STEAP following stimulation with different pools of STEAP overlapping peptides;
- Figure 11 sets forth the nucleotide (SEQ ID NO: 1 ) and polypeptide (SEQ ID NO: 2) sequences of V1C2CD80/CD86 (also referred to as V1C2) used in the studies described herein. Nucleotide and amino acid residues corresponding to CD80 V1 region are in bold; and
- Figure 12 sets forth the nucleotide (SEQ ID NO: 3) and polypeptide (SEQ ID NO: 4) sequences of human STEAP (derived from Genbank accession No. NM_012449).
- the present invention relates to a use of a chimeric costimulatory molecule or variant thereof and a tumor-associated antigen (or nucleic acids encoding such a molecule and antigen, as well as corresponding recombinant cells comprising one or both components) for inducing or modulating an immune response in respect of a tumor or tumor antigen, and in turn for prophylactic and therapeutic antitumor/anticancer applications.
- activation refers to any change induced in the basal or resting state of an immune cell, for example a T cell.
- Non-limiting examples of such changes include any increase in at least one of the following: cell proliferation, cell division, cytokine production (e.g., IFN, TNF, IL-2), enhanced response to an antigen or MHC, DNA synthesis, lymphokine, cytotoxic activity, intracellular rise in calcium, increased expression of receptors (e.g., IL-2-receptor).
- cytokines refers to a diverse group of soluble proteins which are released by one cell type to mediate a biological effect in a second cell type. Biological effects are varied and include cell proliferation, differentiation, growth. Non-limiting examples of cytokines include interleukins (e.g., IL-12), interferons (e.g., IFN- ⁇ , ⁇ and Y) tumor necrosis factor (e.g., TNF- ⁇ , ⁇ and the like). The biological effect of a cytokine is generally mediated by the binding thereof to its receptor.
- the cytokine is often referred as a "ligand" of a receptor.
- ligand is well-known in the art of immunology and other ligands include, for example, antibodies which bind a receptor.
- ligand as used herein is used in its broad sense to refer to a molecule which can bind specifically to another one.
- the terms "antigen” or “antigenic determinant” or (“antigenic fragment”) are very well-known in the art.
- the strength of an antigen is often referred to as the antigenicity or immunogenicity and relates to the property (which is often quantifiable) in eliciting or inducing an immune response.
- the invention provides a composition comprising: a) a first recombinant nucleic acid comprising a nucleic acid sequence encoding a chimeric co-stimulatory molecule, said chimeric co-stimulatory molecule comprising;
- transmembrane and cytoplasmic domains of CD86 or functionally active fragments thereof b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
- CD86 (B7.2) was first described in Azuma, M. et al. 1993.
- Figure 2B of that publication discloses the nucleotide and predicted amino acid sequence of the B7.2 protein.
- the sequence information is also available in the Genbank database as U04343.
- Human CD80 is expressed as a 288 amino acid protein (1-288) which is processed to a mature protein (35-288).
- CD80 is divided into four regions: the variable (V) region, the constant (C) region, the transmembrane region (tm) and the cytoplasmic tail region (ct).
- Amino acids 35-242 make up the extracellular domain of the protein.
- Amino acids 43-123 make up the V region, also referred to as the Immunoglobulin like V-type domain.
- Amino acids 155-223 make up the C region, also referred to as the Immunoglobulin like C2-type domain.
- Amino acids 243-263 make up the transmembrane region.
- Amino acids 264-288 make up the cytoplasmic tail.
- V1C2CD80/CD86 or “V1C2” are meant to refer to molecules which comprise a functional V region of CD80, a functional C region of CD86 such that, through the absence of all or part of the C region of CD80, such molecules do not transmit the negative signal associated with wild type CD80 C region interactions with CTLA4.
- a "functional V region of CD80” or a “functional C region of CD86” are meant to refer to complete protein regions from CD80 or CD86 as well as partial regions that retain the activity of the complete region.
- a functional V region of CD80 refers to amino acids 43-123 of CD80 or a fragment thereof, including proteins which include other sequences including but not limited to other CD80 sequences, which retains the ability to bind to CD28.
- the above-mentioned V1C2CD80/CD86 molecule comprises the amino acid sequence of SEQ ID NO:2.
- the above-mentioned V1 C2CD80/CD86 molecule is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1.
- the present invention relates to a composition (e.g. a pharmaceutical or vaccine/immunogenic composition) comprising the above- mentioned nucleic acid encoding a chimeric costimulatory molecule or variant thereof and a tumor-associated antigen, and a pharmaceutically acceptable carrier.
- a composition e.g. a pharmaceutical or vaccine/immunogenic composition
- the present invention also relates to a vector comprising a nucleic acid sequence encoding the chimeric co-stimulatory molecule according to the present invention and a chosen tumor-associated antigen.
- polypeptides and nucleic acids which are homologous to or substantially identical with, based on sequence, to a polypeptide or nucleic acid of the invention (e.g. a chimeric co-stimulatory molecule or antigen) and retain the relevant function.
- Homology and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness).
- nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs.
- sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
- a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.
- An "unrelated" or “non-homologous" sequence shares less than 40% identity, though preferably less than about 25 % identity, with a nucleic acid sequence of the present invention.
- Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule.
- Two nucleic acid or protein sequences are considered “substantially identical” if, when optimally aligned, they share at least about 70% sequence identity.
- sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
- Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2. 482, the homology alignment algorithm of Needleman and
- Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/).
- the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold.
- Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs.
- the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- W word length
- B BLOSUM62 scoring matrix
- E expectation
- M 5
- P(N) the smallest sum probability
- nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.
- hybridize to each other under moderately stringent, or preferably stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in 0.2 x SSC/0.1% SDS at 42 0 C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1 , Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
- hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% SDS, 1 mM EDTA at 65 0 C, and washing in 0.1 x SSC/0.1 % SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
- Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
- stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
- vector is commonly known in the art and defines e.g., a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a vehicle into which the nucleic acid of the present invention can be cloned.
- the vector is a viral vector which can introduce a molecule, e.g. a chimeric co-stimulatory molecule, in a cell or in a living organism.
- the viral vector is an adenoviral vector or an adeno-associated vector (AAV).
- genes and nucleic acid sequences of the invention may be recombinant sequences.
- the term "recombinant” means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques.
- the term “recombinant” when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
- Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as 'recombinant 1 therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation.
- Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.
- expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
- expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation or transfection into a host.
- the cloned gene (inserted sequence) is usually placed under the control of control element or transcriptionally regulatory sequences such as promoter sequences.
- control element or transcriptionally regulatory sequences such as promoter sequences.
- the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
- a first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
- operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
- enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.
- Transcriptional regulatory element is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked.
- Operably-linked sequences may also include two segments that are transcribed onto the same RNA transcript.
- two sequences such as a promoter and reporter sequence are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
- two sequences In order to be "operably-linked" it is not necessary that two sequences be immediately adjacent to one another.
- Expression control sequences will vary depending on whether the vector is designed to express the operably-linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
- Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest.
- This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e. g. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography, etc.).
- the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies or a specific ligand. The purified protein can be used for therapeutic applications.
- Prokaryotically expressed eukaryotic proteins are often not glycosylated.
- the DNA (or RNA) construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
- Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
- the promoter is preferably bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site
- RNA polymerase RNA polymerase
- Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CCAT” boxes.
- Prokaryotic promoters contain -10 and-35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences, which serve as ribosome binding sequences during translation initiation.
- vectors which can be used in accordance with the present invention include adenoviral vectors, poxviral vectors, VSV-derived vectors and retroviral vectors. Such vectors and others are well-known in the art.
- the designation "functional derivative” or “functional variant” denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
- This functional derivative or equivalent may be a natural derivative or may be prepared synthetically.
- Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
- derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained.
- the substituting amino acid When relating to a protein sequence, the substituting amino acid generally has chemico-physical properties which are similar to that of the substituted amino acid.
- the similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like.
- the term “functional derivatives” is intended to include “fragments”, “segments”, “variants”, “analogs” or “chemical derivatives” of the subject matter of the present invention.
- the above-mentioned derivative, variant or fragment is an "antigenic derivative, variant or fragment” (e.g., which has the capacity to induce/elicit an immune response against the parental antigen).
- variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention but is not limited to a variant which retains all of the biological activities of the parental protein, for example.
- the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.
- chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (e.g. solubility, absorption, half-life, decrease of toxicity and the like). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide or nucleic acid sequence are well known in the art.
- purified refers to a molecule having been separated from a cellular component.
- a “purified protein” has been purified to a level not found in nature.
- a compound is “substantially pure” when it is separated from the components that naturally accompany it.
- a compound is substantially pure when it is at least 60%, more generally 75% or over 90%, by weight, of the total material in a sample.
- a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components.
- a nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived.
- a substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
- the terms "molecule”, “compound”, or “agent” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds.
- molecule therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
- Non-limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents.
- the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling.
- the terms "rationally selected” or “rationally designed” are meant to define compounds which have been chosen based on the configuration for example of interacting domains of the present invention (4-1 BB and 4-1 BBL for example).
- macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule”.
- peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above.
- polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain.
- the molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by a defect in T cell activation.
- the term "ligand” also encompasses molecules such as peptides, antibodies and carbohydrates.
- Non-limiting examples of fusion proteins include hemaglutinin fusions and glutathione-s-transferase (GST) fusions and Maltose binding protein (MBP) fusions.
- GST glutathione-s-transferase
- MBP Maltose binding protein
- Bacterial OmpA and yeast Suc2 are two non- limiting examples of proteins containing signal sequences.
- eukaryotic signal sequences include myelin-associated glycoprotein.
- linker commonly known
- Such fusion protein finds utility in the assays of the present invention as well as for purification purposes, detection purposes and the like.
- sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It will be clear to the person of ordinary skill that whether an interaction domain of the present invention, variant, derivative, or fragment thereof retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
- nucleic acid of the invention is administered to a subject, such as gene therapy methods.
- Nucleic acids may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors.
- Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991 ) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).
- a delivery apparatus e.g., a "gene gun" for injecting DNA into cells in vivo may be used.
- Such an apparatus may be commercially available (e.g., from BioRad).
- Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621 ; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis.
- a cation such as polylysine
- a DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991 ) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
- Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76:271 ).
- retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
- suitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am.
- Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.
- Adeno-associated virus may be used as a gene therapy vector for delivery of DNA for gene therapy purposes.
- AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129).
- AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. MoI. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.
- An AAV vector such as that described in Tratschin et al. (1985) MoI. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) MoI. Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) MoI. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
- Lentiviral gene therapy vectors may also be adapted for use in the invention.
- the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents.
- the DNA segments or proteins according to the present-invention can be introduced into individuals in a number of ways.
- peripheral T cells can be isolated from an individual afflicted or at risk of suffering from a disease or condition, transfected with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways, including intravenous injection.
- the DNA construct can be administered directly to the afflicted individual, for example, by injection in the thymus.
- the DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes.
- a vehicle such as a liposome
- proteins or peptides can also be administered.
- a person of ordinary skill can adapt the transfection method, type of cells transfected, type of disease or condition, co- stimulus (general or specific) etc to meet particular needs.
- compositions within the scope of the present invention should contain the active agent (e.g. fusion protein, peptide, nucleic acid, and molecule, or antigen, or antibody, or APC) in an amount effective to achieve the desired prophylactic and/or therapeutic T cell activation while avoiding adverse side effects.
- the nucleic acids in accordance with the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated.
- Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16 th ed., Mack ed.).
- the invention therefore further provides a composition comprising an active agent and a pharmaceutically acceptable carrier.
- a composition comprising an active agent and a pharmaceutically acceptable carrier.
- the amount administered should be chosen so as to avoid adverse side effects.
- the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
- T cells may be removed from a patient (e.g. cancer patient, or virally affected patient [or susceptible of being infected by a virus]), activating these T cells in accordance with the present invention and re- administering these activated T cells to the patient.
- T cells could be carried-out prior to assaying an activated T cell function or re-injecting same into a patient.
- cytokines or other mitogens or molecules could be added to the culture medium.
- the immunogenic or vaccine compositions of the invention are administered by conventional routes known the vaccine field, in such as to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g. via a patch).
- a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
- parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
- topical administration e.g. via a patch.
- the choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intran
- lipid formulation or an aluminum compound is used, the parenteral route is preferred with the subcutaneous or intramuscular route being most preferred.
- the choice also depends upon the nature of the vaccine agent.
- a polypeptide or derivative thereof may be formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
- liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
- VLPs virus-like-particles
- Adjuvants other than liposomes and the like are also used and are known in the art.
- Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system.
- An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below.
- a polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.
- a further aspect of the invention provides (i) a vaccine vector such as an adenovirus, containing a nucleic acid molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically and/or prophylactically effective amount of a vaccine vector of the invention; (iv) a cell (e.g.
- an antigen-presenting cell transfected or transformed with the nucleic acid or vector of the present invention
- a method for inducing an immune response against a tumor in a mammal e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing tumor growth or cancer in non-human animals
- the method can be used in veterinary applications for treating or preventing tumor growth or cancer in non-human animals
- the mammal e.g., a human
- the method can be used in veterinary applications for treating or preventing tumor growth or cancer in non-human animals
- a method for preventing and/or treating cancer which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an individual having, or at risk of developing, cancer.
- the invention further provides a use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating cancer.
- a vaccine vector expresses one or several polypeptides or derivatives of the invention.
- the vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), which enhances the immune response (adjuvant effect).
- IL-2 interleukin-2
- IL-12 interleukin-12
- the invention further provides a composition comprising several polypeptides or derivatives thereof of the invention or vaccine vectors (each of them capable of expressing a polypeptide or derivative thereof of the invention).
- a composition may also comprise an additional tumor antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12 (or vaccine vector(s) capable of inducing their expression).
- Vaccine refers to a composition or formulation comprising one or more polypeptides/peptides of the invention, or a vaccine vector of the invention.
- Vaccination methods for treating or preventing an infection or a disease in a mammal comprises use of a vaccine or vaccine vector of the invention to be administered by any conventional route.
- Treatment may be effected in a single dose or repeated at intervals.
- the appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine or vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like).
- Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette-Guerin (BCG), and Streptococcus.
- adenovirus vector An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a nucleic acid molecule of the invention, are described in U.S. Patent No. 4,920,209.
- Poxvirus vectors include vaccinia and canary poxvirus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, e.g., Tartaglia et al., Virology (1992) 188:217) for a description of a vaccinia virus vector and Taylor et al., Vaccine (1995) 13:539 for a description of a canary pox vector).
- Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
- the dose of vaccine viral vector can be of from about 1x10 4 to about 1x10 11 , advantageously from about 1x10 7 to about 1x10 10 , preferably of from about 1x10 7 to about 1x10 9 plaque-forming units per kilogram.
- viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
- Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known.
- Mekalanos et al., Nature (1983) 306:551 and U.S. Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced.
- WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations.
- WO 94/01533 describes a deletion mutant lacking functional ctxA and attRSI DNA sequences.
- An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1x10 5 to about 1x10 9 , preferably about 1x10 6 to about 1x10 8 , viable bacteria in a volume appropriate 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 vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361.
- Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
- the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.
- composition comprising a polypeptide or vaccine vector of the present invention may further contain an adjuvant.
- adjuvants are known to those skilled in the art. Examples of adjuvants are described below.
- a further aspect of the invention provides (i) a composition comprising a polypeptide or nucleic acid of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polypeptide or polynucleotide of the invention; (iii) a method for inducing an immune response against a tumor in a mammal by administration of an immunogenically effective amount of a polypeptide or polynucleotide of the invention to elicit a protective immune response against a tumor; and particularly, (iv) a method for preventing and/or treating a cancer, by administering a prophylactic or therapeutic amount of a polypeptide or polynucleotide of the invention to an individual having, or at
- the invention further provides a use of a polypeptide or polynucleotide of the invention in the preparation of a medicament for preventing and/or treating cancer.
- Use of the nucleic acids of the invention include their administration to a mammal as a vaccine or an immunogenic agent, for therapeutic or prophylactic purposes.
- Such nucleic acids are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
- a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue-specifically.
- non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, MoI. Cell Biol. (1985) 5:281 ).
- CMV Cytomegalovirus
- Rous Sarcoma Virus promoter described in Norton & Coffin, MoI. Cell Biol. (1985) 5:281
- An example of a tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991 ) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403).
- Use of promoters is well-known to those skilled in the art.
- polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide.
- the signal peptide is either homologous or heterologous.
- a eukaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred.
- Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention.
- a polynucleotide of the invention is formulated according to various methods outlined below.
- One method utilizes the polynucleotide in a naked form, free of any delivery vehicles.
- a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier.
- 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.
- An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake.
- agents include (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides.
- Liposomes A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes
- Cationic lipids are also known in the art and are commonly used for gene delivery.
- Such lipids include LipofectinTM also known as DOTMA (N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1 ,2-bis(oleyloxy)- 3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (3 beta-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol).
- 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.
- DOPE dioleyl phosphatidylethanolamine
- Formulations containing cationic liposomes may optionally contain other transfection-facilitating compounds.
- 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,
- Gold or tungsten microparticles are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152.
- the microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in
- 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 gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation.
- a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
- the administration can be achieved in a single dose or repeated at intervals.
- such a composition can also contain an adjuvant.
- a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21 , which is described in U.S. Patent No. 5,057,546.
- Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals.
- An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art.
- a polypeptide of the invention, administered as a vaccine is administered by a mucosal route in an amount from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
- polypeptides and polynucleotides of the invention may be used sequentially as part of a multistep immunization process.
- a mammal is initially primed with a vaccine vector of the invention such as an adenovirus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route.
- liposomes associated with a polypeptide or derivative of the invention are also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT).
- a mucosal adjuvant e.g., LT
- adjuvants useful in any of the vaccine/immunogenic compositions described above are as follows.
- Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
- the antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.
- Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT), are used in parenteral administration.
- Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used.
- CT cholera toxin
- LT E. coli heat-labile toxin
- PT pertussis toxin
- CTB native cholera toxin subunit B
- Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
- a mutant having reduced toxicity is used.
- Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
- Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112-Asp mutants.
- Other adjuvants such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
- MPLA bacterial monophosphoryl lipid A
- Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b ⁇ N ⁇ N'.N'-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO
- composition of the invention containing a polypeptide, a polypeptide derivative or a polynucleotide of the invention is manufactured in a conventional manner.
- a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution such as phosphate buffer saline.
- a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
- pharmaceutically acceptable carrier or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the carrier is suitable for parenteral administration.
- the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.
- Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.
- Supplementary active compounds can also be incorporated into the compositions.
- the composition for cancer treatment of the present invention can be used in conjunction with existing chemotherapy drugs or be made as a mixture with them.
- a chemotherapy drug include, for example, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, alkaloids derived from plant, topoisomerase inhibitors, hormone therapy medicines, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, other immunotherapeutic drugs, and other anticancer agents.
- hypoleukocytosis e.g. a chemotherapeutic agent
- a chemotherapeutic agent can be administered concomitantly or sequentially.
- the composition for cancer treatment of the present invention can be used together with immunopotentiative substance(s) or be made as a mixture with them.
- immunopotentiative substances include, for example, various cytokines and a tumor antigen, etc.
- Cytokines that stimulate immune reactions include, for example, GM-CSF, M-CSF, G-CSF, interferon- ⁇ and Y, IL-1 , IL-2, IL-3, and IL-12, anti-CD3 antibodies can also improve the immune reactions.
- the composition of the present invention and immunopotentiative substance(s) e.g. a cytokine or a chemokine
- treating cancer or “treatment of cancer” as used herein includes at least one of the following features (prophylactic/therapeutic results): alleviation of the symptoms associated with the cancer, a reduction in the extent of the cancer (e.g. a reduction in tumor growth), a stabilization of the state of the cancer (e.g. an inhibition of tumor growth), a prevention of further spread of the cancer (e.g. a metastasis), a prevention of the occurrence or recurrence of a cancer, a delaying or retardation of the progression of the cancer (e.g. a reduction in tumor growth) or an improvement in the state of the cancer (e.g. a reduction in tumor size).
- a reduction in the extent of the cancer e.g. a reduction in tumor growth
- a stabilization of the state of the cancer e.g. an inhibition of tumor growth
- a prevention of further spread of the cancer e.g. a metastasis
- a prevention of the occurrence or recurrence of a cancer
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the above-mentioned desired therapeutic result.
- a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
- a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting onset or progression of cancer and associated symptoms and disease. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
- the cancers to which the compositions and methods of the present invention can be applied include swellings and true tumors including benign and malignant tumors.
- tumors include gliomas such as astrocytoma, glioblastoma, medulloblastoma, oligodendroglioma, ependymoma and choroid plexus papilloma; cerebral tumors such as meningioma, pituitary adenoma, neurioma, congenital tumor, metastatic cerebral tumor; squamous cell carcinoma, lymphoma, a variety of adenomas and pharyngeal cancers resulted from these adenomas such as epipharyngeal cancer, nasopharyngeal cancer and hypopharyngeal cancer; laryngeal cancer, thymoma; mesothelioma such as pleural mesothelioma, peritoneal mes
- lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, myeloma, plasmacytoma, acute myelocytic (myeloid) leukemia and chronic myeloid leukemia, leukemia such as adult T-cell leukemic lymphoma and chronic lymphocytic leukemia, chronic myeloproliferative diseases such as true plethora, essential thrombocythemia and idiopathic myelofibrosis, lymph node enlargement (or swelling), tumor of pleural effusion, ascitic tumor, other various kinds of adenomas, lipoma, fibroma, hemangeoma, myoma, fibromyoma and endothelioma.
- the cancer/tumor is characterized by or associated with STEAP expression.
- "Characterized by or associated with STEAP expression” as used herein refers to an alteration of STEAP expression in a tumor cell relative to a non-tumor cell for a given tissue. For example, such an alteration may be an increase in STEAP expression in a tumor cell relative to a corresponding non-tumor cell exhibiting lower or no STEAP expression.
- STEAP (see Figure 12) has been shown to be expressed or overexpressed in human prostate cancer and also in several tumors cell lines of various origins, such as prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma (Hubert RS et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 14523-14528).
- tumor-associated antigen refers to an antigen (e.g. a polypeptide or an antigenic fragment or variant thereof) that is overexpressed in a tumor cell/tissue as compared to a corresponding normal cell/tissue.
- Overexpression can be, for example, an increase in expression of a given antigen in a tumor cell/tissue as compared to a normal cell/tissue, but also the expression of an antigen in a tumor cell/tissue that do not express it in a normal state (i.e. when the cell or tissue is not cancerous).
- TAA include known oncoproteins such as HER-2/Neu and c-myc, survival proteins such as survivin and lens epithelium-derived growth factor (LEDGF/p75), cell cycle regulatory proteins such as Cyclin B1 , differentiation and cancer-testis antigens such as NY-ESO-1 , colorectal cancer antigen such as carcinoembyronic antigen (CEA), most antigens of the MAGE family, melanA/MART-1 , MUC1 , Wilms' tumor protein (WT-1 ), STEAP and others (see Novellini et al., 2005. Cancer Immunol Immunother. 54(3): 187-207 for a list of known TAA).
- CEA carcinoembyronic antigen
- the above-mentioned TAA is STEAP, or an antigenic variant or fragment thereof.
- Antigenic fragment or variant of STEAP are well known in the art.
- antigenic STEAP fragments/epitopes have been described in Machlenkin A. et al., 2005, Cancer Res., 65:6435-6442; Rodeberg D.A. et al., 2005, Clin. Cancer Res., 11 : 4545-4552; Alves P.M. et al., 2006, Cancer Immunol. Immunother., 55: 1515-1523; and Kobayashi H. et al., 2007. Cancer Res. 67: 5498-5504.
- the invention provides a recombinant cell comprising the above-mentioned vector.
- the above- mentioned cell is an antigen-presenting cell (e.g. a dendritic cell).
- the invention provides a composition comprising the above-mentioned cell and a pharmaceutically acceptable carrier or excipient.
- the present invention provides a method of activating a tumor-specific human T cell, said method comprising contacting said T cell with the above-mentioned recombinant cell.
- the above- mentioned contacting is performed ex vivo or in vivo.
- cells e.g. host cells
- Methods for transforming/transfecting host cells with nucleic acids/vectors are well-known in the art and depend on the host system selected as described in Ausubel et al. (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994).
- the terms "transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection.
- Host cells transfected or transformed with the chimeric nucleic acid or vector of the invention can be used as a vaccine (e.g., autologous cell vaccine) in order to induce or increase an immune response against an antigenic epitope or antigen (e.g., STEAP) in a subject.
- a vaccine e.g., autologous cell vaccine
- host cells e.g., APCs, dendritic cells
- APCs e.g., dendritic cells
- known steps for further cultivating or modifying these cells could be carried- out prior to re-injecting/transplanting them into a subject.
- cytokines/chemokines or mitogens or molecules could be added to the culture medium.
- T cells may be removed from a subject (e.g. cancer patient), activated in accordance with the present invention (e.g., by contacting them with a host cells transfected with a chimeric nucleic acid of the invention) and re-administered to the patient.
- a subject e.g. cancer patient
- activated in accordance with the present invention e.g., by contacting them with a host cells transfected with a chimeric nucleic acid of the invention
- known steps for further cultivating or proliferating these T cells could be carried-out prior to re-injecting them into a subject.
- cytokines or other mitogens or molecules could added to the culture medium.
- the above-mentioned T cell is derived from a subject suffering from cancer or at risk of suffering from cancer.
- the present invention provides a kit or package comprising:
- Example 1 Confirmation of STEAP and mutant CD80 expression by the adenoviral vaccines of large preparation.
- Five adenoviral vector vaccines (Adv) were produced with different combination of STEAP and CD80 mutant expression:
- V1 C2/STEAP chimeric V1 C2 ( Figure 11 ) and STEAP
- 293HEK cells were infected with the viral vaccines obtained from large preparations.
- Adherent 293 cells were plated and cultured until confluence.
- cells were infected with 100 pfu Adv/cell during 24 hours. After infection, infected cells were washed and analyzed for CD80 expression by flow cytometry detected with mouse anti-CD80 monoclonal antibody (reference P91853F, Biodesign, Saco, ME) and for STEAP expression by western blot detected with rabbit anti-STEAP polyclonal antibody (reference 187385, Zymed laboratories, San
- FIG. 1 panel A shows a Western blot that demonstrates STEAP expression in all samples infected with adenoviral vaccines containing the STEAP transgene (lane 3,4 and 5), but not in the control (lane 1 ) and CD ⁇ Owt (lane 2) adenovirus.
- FIG. 1 panel B shows FACS analysis of CD80 mutant expression.
- CD80 expression was detected in all samples infected with the adenoviral vaccine having CD80 expression cassette: ADV CD ⁇ Owt, Adv-CD80wt/STEAP and Adv-
- V1C2/STEAP are expressed at comparable levels.
- Human adherent monocytes are used as the main antigen presenting cells in our in vitro analysis of human T cell response to STEAP, we also tested the CD80 mutant expression in the adenoviral vaccines infected human monocytes.
- Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus gradient centrifugation (Amersham Biosciences). Fresh PBMC were plated at 3.5X10 6 cells per well in a 48-well plate for 2 hours to allow the adherent fraction to attach to the plastic wells.
- Adv control Adv-CD80wt, Adv-STEAP, Adv-CD80wt/STEAP and Adv- V1C2/STEAP were added at a multiplicity of infection of 100 per cell. After 24 hours of incubation at 37 0 C with adenovirus, monocytes were washed and stained for CD80 expression.
- Figure 3 shows expression of CD80 molecules (wild-type or mutant) detected on 37% to 56% human adherent monocytes at considerable high level, 24 hours after the cells were infected with adenoviral vaccines.
- Example 2 Testing of the endotoxin contamination of our adenovirus preparations.
- Figure 2 shows that the adenovirus preparations contain less than
- E. coli 0,125 endotoxin units (EU) per ml of adenovirus preparations.
- Example 3 Tumor experiments in BALB/c mice: prophylactic vaccination model. The adjuvant efficacy of human CD80 mutants was next evaluated in Adenoviral vaccines using STEAP-expressing CT26 cells as tumor model; a prophylactic vaccination model was developed (Figure 4).
- mice Six (6) mice were injected in each group, except for group 4 and 5 (seven (7) mice). Mice received sub-cutaneous injection of 10 5 CT26-STEAP cells. The tumor length (L) and width (W) were measured and the volume was determined by W 2 X L X 0,52 (mm 3 ).
- CT26 is a colon cancer line of BALB/c origin (Wang et al., J. Immunol., 154(9), p.4685- 92).
- CT26 cells were transfected with the plRESneo3 plasmid (Invitrogen) in which a full length human STEAP cDNA was inserted at the poly-linker site. After a two week selection with G418, the drug-resistant cells were further cloned by limiting dilution.
- the G418-resistant clones i.e. those stably transfected with neomycin- resistant gene) were analyzed by Western blot for STEAP expression.
- Figure 5 shows that G418-resistant CT26 clones expressed human
- FIG. 6 shows the tumorigenicity of CT26 cells clone expressing
- STEAP in BALB/c mice using three different dosages of CT26-STEAP injected to mice (10 3 to 10 5 cells/mouse).
- the expression of STEAP by tumors was tesyed by RT-PCR.
- Figure 5 shows the detection of STEAP gene in CT26-STEAP tumoral cell biopsy (lane 4).
- Figure 7 shows the tumor size at day 21 post-injection in the different vaccinated groups of mice. There is a significant difference (p ⁇ 0.05) in the average tumor size between mice vaccinated with Adv-STEAP or Adv V1C2-STEAP and mice vaccinated with CD80wt-STEAP.
- Figure 8 shows the tumor growth (tumor size in mm 3 ) over a time course of 3 weeks in 2 different vaccinated groups of mice. This figure shows that injection of Adv V1 C2CD80/CD86-STEAP induces a delay in tumor growth as compared to mice injected with Adv CD80wt-STEAP. This difference is significant at day 21 (p ⁇ 0.05). At day 24, 5/7 mice immunized with the V1 C2CD80/CD86-STEAP were still alive, as compared to 4/7 and 3/6 in mice immunized with Adv CD80wt- STEAP and Adv control, respectively.
- Example 4 Assessment of mouse T cell phenotype and effector functions following vaccination with Adenoviral constructs expressing STEAP.
- ICS Cytokine Staining
- IFN- ⁇ and IL-2 secretion were assessed in T cells from vaccinated mice after 6h of stimulation with STEAP peptides.
- Fresh spleen cells were obtained from mice immunized in the prophylactic vaccination protocol (at day 58) and stimulated during 6 hours in the presence of 1 DgAnI of each overlapping STEAP peptide in order to determine the frequency and functionality of the responding mouse T cells.
- Each pool contains ten
- the non-stimulated condition i.e. no peptide
- background response After 6 hours of stimulation, cells were fixed, permeabilized and stained for CD3, CD4 or CD8, IL-2 and IFN- ⁇ as previously described (Younes et a/., 2003. J Exp Med 198: 1009-1022).
- Figure 9 shows a flow cytometric analysis of phenotypic and functional properties of the responding immune T cells.
- A 52% lymphocytes, 15% CD3 + T cells, 15% CD3 + CD4 + T cells, 6% CD3 + CD8 + T cells.
- B unstimulated CD3 + CD8 + T cells
- D and E Production of IFN- ⁇ by CD3 + CD8 + and IL-2 by CD3 + CD4 + T cells following stimulation with STEAP peptides.
- Figure 10 is a summary of the percentages of responding T cells following activation with the 8 different pools of STEAP peptides (p1 to p8).
- the percentage represents the proportion of cytokine-(IFN- ⁇ or IL-2) positive cells within total CD8 + or CD4 + T cells.
- the percentage of CD3 + CD4 + IL-2 + and CD3 + CD8 + IFN- ⁇ + T cells is about 0.2 and 0.5, respectively.
- the percentage of CD3 + CD4 + IL-2 + and CD3 + CD8 + IFN- ⁇ + T cells is about 0.2 and 0.5, respectively.
- the percentage of CD3 + CD8 + T cells is about 0.2 and 0.5, respectively.
- 9.5, 7.9 and 6.8% of CD3 + CD8 + T cells produces IFN- ⁇ in response to p4, p5 and p8 respectively.
- 2.9, 3.2 and 3.5% of CD3 + CD4 + T cells produces IL-2 in response to the same pools (p4, p5 and p ⁇ , respectively).
- Adv V1 C2CD80/CD86-STEAP vaccinated group 1.5 to 3.2% of CD3 + CD8 + T cells stained positive for IFN- ⁇ in response to p1 , p2, p3, p4, p5 and p8. This response correlates with the frequency (1.9 to 3%) of CD3 + CD4 + cells secreting IL-2 following stimulation with the same pools. Therefore, Adv V1 C2CD80/86-STEAP induces a strong and diversified T cell response to STEAP antigen.
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Abstract
Methods and compositions are described relating to the use of a chimeric co-stimulatory molecule having CD80 variable and CD86 constant regions or functional portions thereof for inducing an immune response against a tumor/cancer-related antigen. Corresponding prophylactic and therapeutic methods for the prevention and/or treatment of cancer are also described.
Description
METHODS AND COMPOSITIONS FOR IMMUNE RESPONSE MODULATION
AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit, under 35 U. S. C. § 119(e), of U.S. provisional application Serial No. 60/827,465 filed on September 29, 2006, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION The present invention relates to immunology and more specifically to methods and compositions for modulating the immune response and uses thereof in prophylactic and therapeutic anti-tumor applications.
BACKGROUND OF THE INVENTION The immune system acts as a defense against a variety of internal and external conditions which include, for example, infections, cancer, mutations, injuries and the like, and is mediated by two interconnected systems: the humoral and cellular immune systems. Briefly, the humoral system is mediated by the action of soluble molecules termed antibodies or immunoglobulins which, through their properties of specifically combining with a target (e.g., an antigen) recognized as being foreign to the body (or non-self), can inactivate same. The cellular immune system also involves the mobilization of cells, termed T cells. T cells are responsible for what is called cell-mediated immunity. This immunity involves the destruction of foreign cells, infected cells or the like by the action of cells of the immune system.
T cells can be subdivided into different subsets based on surface markers or based on function. For example, "helper", "regulatory" and "killer" T cell subsets have been described. A T cell which recognizes and binds to a particular antigen displayed on the surface of another cell (often termed antigen presenting cell or APC) can become activated. An activated T cell can multiply, produce cytokines and, if it is a killer T cell, can kill the cell to which it is bound. Helper T cells generally produce cytokines and activate other cells of the immune system. Killer T cells recognize infected, foreign or altered cells, such as cancer cells and
eliminate them. Regulatory T cells can modulate or suppress certain immune responses.
The physiological mechanism of human T cell activation involves the recognition of an MHC-peptide complex by the antigen-specific T cell receptor together with other receptor ligand interactions, known as costimulatory interactions. However, a number of additional means can be used to stimulate T cells, such as antibodies to T cell surface receptors or mitogenic lectins. Of note, the induction of proliferation is only but one marker of T cell activation, since other markers include: increase in lymphokine or cytokine production, cytotoxic activity and a change in the basal or resting state of the cell.
The complex phenomenon of T cell activation involves a variety of receptor/ligand interactions between T cells and antigen presenting cells. A component of T cell activation is the T cell receptor (TCR), a disulfide-linked heterodimer which contains two glycoprotein chains (α/β) uncovalently associated with a complex of low molecular weight invariant proteins which are commonly designated as CD3. While the TCR α and β chains (or Y and δ) determine the antigen specificities of the T cell, the CD3 structures of the TCR are thought to be responsible for transducing the activation signal upon binding of the α and β chains to its ligand. As discussed above, the TCR typically interacts with small peptidic antigens which are presented by the major histocomptability complex (MHC) proteins. The MHC proteins are a highly polymorphic set of molecules which are randomly dispersed throughout the species and further increase the complexity of the T cell activation phenomenon.
In summary, therefore, T cell activation usually involves a trimolecular interaction between a TCR, a peptidic antigen and MHC proteins which bind to this antigen. Although the recognition of antigen/MHC by the antigen-specific T cell is necessary for T cell activation, this signal alone is usually not sufficient to activate a T cell, rather, other receptor-ligand interactions, called costimulatory interactions are usually also required. The CD28 receptor on T cells, binding to B7 molecules on antigen presenting cells can provide such a costimulatory signal.
CD28 is a cell surface glycoprotein constitutively expressed on most mature T-cells and thymocytes, while the CTLA-4 receptor, which triggers an inhibitory signal in the T cell, is not present on resting T cells and is only detectable
48 to 72 hours after T cell activation. The primary ligands for these molecules are
B7.1 (CD80) and B7.2 (CD86) expressed on the surface of professional antigen presenting cells (APC). CD80 and CD86 are members of the Ig gene superfamily. Both molecules have a cytoplasmic tail (T domain), a trans-membrane spanning domain (TM) as well as two extracellular Ig-like domains: the N-terminal domain most closely resembles the Ig variable-like region (V-domain) and the membrane proximal domain shows homology with the Ig constant-like domain (C-domain) (Freeman, G. J. et al., 1989. J. Immunol. 143:2714; Freeman, G. J. et al., 1993. Science 262:909). CD86 is constitutively expressed on APC and after activation of APC, the expression of CD86 is quickly up-regulated followed by a gradual return to baseline levels. The expression of CD80 is delayed compared to CD86 and its expression is maximal 48 to 72 hours after the initiation of an immune response.
Vaccines are useful to immunize individuals against target antigens such as pathogen antigens or antigens associated with cells involved in human diseases. Antigens associated with cells involved in human diseases include tumor- associated antigens (TAA) and antigens associated with cells involved in autoimmune diseases.
Despite numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive or because of recurrence due to regrowth at the original site and/or metastases. Also, no effective methods of treatment have been suggested so far for advanced cancers. Cancer immunotherapy aims at destroying tumor cells by immunological mechanisms, for example by inducing an antitumor immune response using a vaccine comprising a tumor antigen. Immunotherapy compared to conventional methods of cancer therapy like surgery, radiation, and chemotherapy, is much less toxic, and no serious complications have been described so far. In addition, immunotherapy has the potential to work at different stages of disease. At initial stages it could be a good supplementary treatment to surgical removal of primary tumors, aiming to prevent development of disseminated disease. At advanced stages of disease it could be the only means of treatment, as
conventional methods are often ineffective. The goal of immunotherapy is to increase the ability of the immune system to recognize tumor cells and to develop effective mechanisms of tumor elimination. However, tumor-associated antigens typically induce weak immune responses. Therefore there is a continuous need to develop new cancer immunotherapies and new strategies to improve/enhance the immune response against TAA.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The invention relates to products (compositions, nucleic acids and/or polypeptides) and uses thereof e.g., for inducing an immune response against a tumor/cancer-related antigen. In an aspect, the invention provides a composition comprising: a) a first recombinant nucleic acid comprising a nucleic acid sequence encoding a chimeric co-stimulatory molecule, said chimeric co-stimulatory molecule comprising; i) a functional V region of CD80; ii) a functional C region of CD86; and iii) transmembrane and cytoplasmic domains of CD86 or functionally active fragments thereof; b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
In an embodiment, the above-mentioned composition is an immunogenic or vaccine composition.
In a further aspect, the invention provides a pharmaceutical composition comprising the above-mentioned composition and a pharmaceutically acceptable carrier or excipient.
In an embodiment, the above-mentioned composition further comprises an adjuvant.
In an embodiment, the above-mentioned chimeric co-stimulatory molecule comprises the amino acid sequence of SEQ ID NO:2.
In another embodiment, the above-mentioned first recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO:1. In a further aspect, the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of:
(a) the first recombinant nucleic acid defined above; and (b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor- associated antigen polypeptide.
In a further, the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned composition.
In an embodiment, the above-mentioned first nucleic acid is present in a vector comprising said nucleic acid sequence encoding a chimeric co-stimulatory molecule operably linked to a transcriptional regulatory sequence.
In an embodiment, the above-mentioned second nucleic acid is present in a vector comprising said nucleic acid sequence encoding said tumor- associated antigen polypeptide operably linked to a transcriptional regulatory sequence.
In a further aspect, the invention provides a method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of:
(a) a recombinant cell expressing the chimeric co-stimulatory molecule defined above; and
(b) a tumor-associated antigen polypeptide or a recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
In an embodiment, the above-mentioned subject is a mammal, in a further embodiment, a human.
In an embodiment, the above-mentioned vector is a viral vector. In a further embodiment, the viral vector is an adenoviral vector.
In an embodiment, the above-mentioned tumor-associated antigen is STEAP, or an antigenic variant or fragment thereof. In an embodiment, the above-mentioned cancer is characterized by
STEAP expression. In a further embodiment, the above-mentioned cancer is selected from prostate cancer, pancreatic cancer, colon cancer, breast cancer, testicular cancer, cervical cancer, bladder cancer, ovarian cancer, leukemia and Ewing sarcoma. In a further embodiment, the above-mentioned cancer is prostate cancer.
In an embodiment, the above-mentioned cell is an antigen-presenting cell.
In a further aspect, the invention provides a vector comprising: a) a nucleic acid sequence encoding the chimeric co-stimulatory molecule defined in claim 1 operably linked to a transcriptional regulatory sequence; and b) a nucleic acid sequence encoding a tumor-associated antigen polypeptide operably linked to a transcriptional regulatory sequence. In a further aspect, the invention provides a recombinant cell comprising the above-mentioned vector. In a further embodiment, the above- mentioned cell is an antigen-presenting cell.
In a further aspect, the invention provides a composition comprising the above-mentioned cell and a pharmaceutically acceptable carrier or excipient. In a further aspect, the invention provides a method of activating a tumor-specific human T cell, said method comprising contacting said T cell with the above-mentioned cell or the above-mentioned composition. In an embodiment, the above-mentioned contacting step is carried out ex vivo or in vitro.
In a further embodiment, the above-mentioned contacting step is carried out in vivo and said cell or composition is administered to a subject comprising the above-mentioned human T cell.
In an embodiment, the above-mentioned human T cell is derived from a subject suffering from a cancer.
In a further aspect, the invention provides a commercial package or kit comprising:
(a) an agent selected from (i) the above-mentioned composition, (ii) the above-mentioned vector, (iii) the above-mentioned cell and (iv) any combination of (i) to (iii); and
(b) instructions for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
In a further aspect, the invention provides a use of an agent selected from (i) the above-mentioned composition (ii) the above-mentioned vector (iii) the above-mentioned cell and (iv) any combination of (i) to (iii), for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
In a further aspect, the invention provides a use of an agent selected from (i) the above-mentioned composition (ii) the above-mentioned vector (iii) the above-mentioned cell and (iv) any combination of (i) to (iii), for the preparation of a medicament for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which:
Figure 1 shows an analysis of A) STEAP and B) CD80 protein expression in different 293 cells infected with different adenovirus constructs; Figure 2 shows the results of a Limulus amebocyte lysate (LAL) test in Adenovirus stocks. Adenovirus samples were compared to a standardized curve of E. coli isotype 01 11 :B4 endotoxin (0,50 to 0,03 EU/ml);
Figure 3 shows the expression of CD80 in different adenovirus constructs-infected monocytes 24h after infection; Figure 4 shows the prophylactic tumor vaccination model used. Mice were vaccinated with an adenovirus vector expressing STEAP and the chimeric
CD80. Four weeks later, mice were injected with CT26 tumor cells expressing
STEAP and tumor growth was monitored for 2-3 weeks;
Figure 5 shows the detection by RT-PCR of STEAP expression in different CT26 tumor cell clones transfected with the nucleic acid encoding STEAP;
Figure 6 shows tumor growth in BALB/c mice injected with different numbers (103, 104 and 105) of CT26 tumor cells expressing STEAP. Six mice were injected in each group. Each point represents the size of a tumor in a given mouse;
Figure 7 shows the assessment of tumor growth (CT26 cells expressing STEAP) following vaccination with Adv control, Adv-STEAP, Adv- CDδOwt, Adv-CD80wt/STEAP and Adv-V1C2/STEAP. Tumor growth was determined 21 days after injection of 105 tumor cells; Figure 8 shows tumor growth at different time points (day 9, 13, 15,
17, 21 and 24 post-injection of 105 CT26 cells expressing STEAP) in a prophylactic vaccination model using an adenoviral vector expressing CD80wt/STEAP and chimeric V1C2/STEAP;
Figure 9 shows the phenotypic and functional properties of antigen- specific T cells following vaccination with Adv-V1C2/STEAP. A) Gating strategy. B)
Unstimulated CD8+ T cells. C) Unstimulated CD4+ T cells. D) CD8+ T cells stimulated with a pool of overlapping STEAP peptides. E) CD4+ T cells stimulated with a pool of overlapping STEAP peptides;
Figure 10 shows the response (expressed as the percentage of IFN- and IL-2-positive CD4+ and CD8+ T cells) of T cells isolated from mice vaccinated with a control Adv, Adv-CD80wt/STEAP and Adv-V1 C2/STEAP following stimulation with different pools of STEAP overlapping peptides;
Figure 11 sets forth the nucleotide (SEQ ID NO: 1 ) and polypeptide (SEQ ID NO: 2) sequences of V1C2CD80/CD86 (also referred to as V1C2) used in the studies described herein. Nucleotide and amino acid residues corresponding to CD80 V1 region are in bold; and
Figure 12 sets forth the nucleotide (SEQ ID NO: 3) and polypeptide (SEQ ID NO: 4) sequences of human STEAP (derived from Genbank accession No. NM_012449). Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawings which is
exemplary and should not be interpreted as limiting the scope of the present invention.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a use of a chimeric costimulatory molecule or variant thereof and a tumor-associated antigen (or nucleic acids encoding such a molecule and antigen, as well as corresponding recombinant cells comprising one or both components) for inducing or modulating an immune response in respect of a tumor or tumor antigen, and in turn for prophylactic and therapeutic antitumor/anticancer applications.
Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, transfection, molecular biology methods and the like, antibody purification and the like, are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratories), Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York), Campbell 1984, in "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands), in Harlow et al. 1988 (in: Antibody-A Laboratory Manual, CSH Laboratories), Klein 1982 (in: Immunology: The Science of Self-Nonself Discrimination, Wiley & Sons, N.Y.), in Immunology Today 10: 254 (1989), and in Kanoff, M. E. 1991 : Immunological studies in humans. In Current protocols in Immunology, Vol. 1. Wiley & Sons, New York, p. 7.1.
In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided herein below.
As used herein, the term "activation" or "stimulation" in the context of an immune cell, refers to any change induced in the basal or resting state of an immune cell, for example a T cell. Non-limiting examples of such changes include any increase in at least one of the following: cell proliferation, cell division, cytokine production (e.g., IFN, TNF, IL-2), enhanced response to an antigen or MHC, DNA
synthesis, lymphokine, cytotoxic activity, intracellular rise in calcium, increased expression of receptors (e.g., IL-2-receptor).
The term "immunopotentiation" as used herein refers to an enhanced ability of the immune system to respond to an antigen. The term "cytokines" as used herein refers to a diverse group of soluble proteins which are released by one cell type to mediate a biological effect in a second cell type. Biological effects are varied and include cell proliferation, differentiation, growth. Non-limiting examples of cytokines include interleukins (e.g., IL-12), interferons (e.g., IFN-α, β and Y) tumor necrosis factor (e.g., TNF-α, β and the like). The biological effect of a cytokine is generally mediated by the binding thereof to its receptor. The cytokine is often referred as a "ligand" of a receptor. The term "ligand" is well-known in the art of immunology and other ligands include, for example, antibodies which bind a receptor. For certainty, the term "ligand" as used herein is used in its broad sense to refer to a molecule which can bind specifically to another one.
The terms "antigen" or "antigenic determinant" or ("antigenic fragment") are very well-known in the art. The strength of an antigen is often referred to as the antigenicity or immunogenicity and relates to the property (which is often quantifiable) in eliciting or inducing an immune response. In an aspect, the invention provides a composition comprising: a) a first recombinant nucleic acid comprising a nucleic acid sequence encoding a chimeric co-stimulatory molecule, said chimeric co-stimulatory molecule comprising;
(i) a functional V region of CD80; (ii) a functional C region of CD86; and
(iii) transmembrane and cytoplasmic domains of CD86 or functionally active fragments thereof; b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
The nucleotide and amino acid sequences of human CD80 are well known and set forth in Freeman et al. (1989) J. Immunol. 143 (8): 2714-2722, Selvakumar et al. (1992) lmmunogenetics 36 (3): Freeman et al. J. Exp. Med. 174
(3): Lanier et al. (1989) J. Immunol. 154: 97-105, and Genbank accession code
P33681. CD86 (B7.2) was first described in Azuma, M. et al. 1993. Figure 2B of that publication discloses the nucleotide and predicted amino acid sequence of the B7.2 protein. The sequence information is also available in the Genbank database as U04343.
Human CD80 is expressed as a 288 amino acid protein (1-288) which is processed to a mature protein (35-288). CD80 is divided into four regions: the variable (V) region, the constant (C) region, the transmembrane region (tm) and the cytoplasmic tail region (ct). Amino acids 35-242 make up the extracellular domain of the protein. Amino acids 43-123 make up the V region, also referred to as the Immunoglobulin like V-type domain. Amino acids 155-223 make up the C region, also referred to as the Immunoglobulin like C2-type domain. Amino acids 243-263 make up the transmembrane region. Amino acids 264-288 make up the cytoplasmic tail. As used herein, the term "V1C2CD80/CD86" or "V1C2" are meant to refer to molecules which comprise a functional V region of CD80, a functional C region of CD86 such that, through the absence of all or part of the C region of CD80, such molecules do not transmit the negative signal associated with wild type CD80 C region interactions with CTLA4. As used herein, a "functional V region of CD80" or a "functional C region of CD86" are meant to refer to complete protein regions from CD80 or CD86 as well as partial regions that retain the activity of the complete region. For example, a functional V region of CD80 refers to amino acids 43-123 of CD80 or a fragment thereof, including proteins which include other sequences including but not limited to other CD80 sequences, which retains the ability to bind to CD28. In an embodiment, the above-mentioned V1C2CD80/CD86 molecule comprises the amino acid sequence of SEQ ID NO:2. In an embodiment, the above-mentioned V1 C2CD80/CD86 molecule is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1.
In a further aspect, the present invention relates to a composition (e.g. a pharmaceutical or vaccine/immunogenic composition) comprising the above- mentioned nucleic acid encoding a chimeric costimulatory molecule or variant thereof and a tumor-associated antigen, and a pharmaceutically acceptable carrier.
The present invention also relates to a vector comprising a nucleic acid sequence encoding the chimeric co-stimulatory molecule according to the present invention and a chosen tumor-associated antigen.
Also within the context of the invention are polypeptides and nucleic acids which are homologous to or substantially identical with, based on sequence, to a polypeptide or nucleic acid of the invention (e.g. a chimeric co-stimulatory molecule or antigen) and retain the relevant function.
"Homology" and "homologous" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than about 25 % identity, with a nucleic acid sequence of the present invention.
Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered "substantially identical" if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 ,
Adv. Appl. Math 2. 482, the homology alignment algorithm of Needleman and
Wunsch, 1970, J. MoI. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915- 10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001 ), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , preferably less than about
0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.
An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 650C, and washing in 0.2 x SSC/0.1% SDS at 420C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1 , Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 650C, and washing in 0.1 x SSC/0.1 % SDS at 68°C (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
The term "vector" is commonly known in the art and defines e.g., a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a vehicle into which the nucleic acid of the present invention can be cloned. Numerous types of vectors exist and are well known in the art. In a particular embodiment, the vector is a viral vector which can introduce a molecule, e.g. a chimeric co-stimulatory molecule, in a cell or in a living organism. In an embodiment the viral vector is an adenoviral vector or an adeno-associated vector (AAV).
Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term "recombinant" means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a
protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term "recombinant" when made in reference to genetic composition refers to a gamete or progeny or cell or genome with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as 'recombinant1 therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.
The term "expression" defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation or transfection into a host. The cloned gene (inserted sequence) is usually placed under the control of control element or transcriptionally regulatory sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading
frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked.
Operably-linked sequences may also include two segments that are transcribed onto the same RNA transcript. Thus, two sequences, such as a promoter and reporter sequence are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence. In order to be "operably-linked" it is not necessary that two sequences be immediately adjacent to one another.
Expression control sequences will vary depending on whether the vector is designed to express the operably-linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest. This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e. g. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography, etc.). In addition, the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies or a specific ligand. The purified protein can be used for therapeutic applications. Prokaryotically expressed eukaryotic proteins are often not glycosylated.
The DNA (or RNA) construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule. "Promoter" refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of the
present invention, the promoter is preferably bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site
(conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain -10 and-35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences, which serve as ribosome binding sequences during translation initiation. Non-limiting examples of vectors which can be used in accordance with the present invention include adenoviral vectors, poxviral vectors, VSV-derived vectors and retroviral vectors. Such vectors and others are well-known in the art. As used herein, the designation "functional derivative" or "functional variant" denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid generally has chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "fragments", "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention.
In an embodiment, the above-mentioned derivative, variant or fragment is an "antigenic derivative, variant or fragment" (e.g., which has the capacity to induce/elicit an immune response against the parental antigen).
Thus, the term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention but is not limited to a variant which retains all of the biological activities of the parental protein, for example.
The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.
As used herein, "chemical derivatives" is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (e.g. solubility, absorption, half-life, decrease of toxicity and the like). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide or nucleic acid sequence are well known in the art.
As used herein, the term "purified" refers to a molecule having been separated from a cellular component. Thus, for example, a "purified protein" has been purified to a level not found in nature. A compound is "substantially pure" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75% or over 90%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
As used herein, the terms "molecule", "compound", or "agent" are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term "molecule" therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non-limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling. The terms "rationally selected" or "rationally designed" are meant to define compounds which have been chosen based on the configuration for example of interacting domains of the present invention (4-1 BB and 4-1 BBL for example). As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above. Similarly, in a preferred embodiment, the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain. The molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by a defect in T cell activation. As defined above, the term "ligand" also encompasses molecules such as peptides, antibodies and carbohydrates.
Non-limiting examples of fusion proteins include hemaglutinin fusions and glutathione-s-transferase (GST) fusions and Maltose binding protein (MBP) fusions. In certain embodiments, it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused. Such protease cleavage sites between two heterologously fused polypeptides are well known in the art. In certain embodiments, it might also be beneficial to fuse the interaction domains of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell. Signal peptides from diverse organisms are well known in the art. Bacterial OmpA and yeast Suc2 are two non-
limiting examples of proteins containing signal sequences. Examples of eukaryotic signal sequences include myelin-associated glycoprotein. In certain embodiments, it might also be beneficial to introduce a linker (commonly known) between the interaction domain and the heterologous polypeptide portion. Such fusion protein finds utility in the assays of the present invention as well as for purification purposes, detection purposes and the like.
For certainty, the sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It will be clear to the person of ordinary skill that whether an interaction domain of the present invention, variant, derivative, or fragment thereof retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
Also within the context of the present invention is the in vivo administration of a nucleic acid of the invention to a subject, such as gene therapy methods.
Nucleic acids may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991 ) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621 ; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991 ) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76:271 ). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381 ; Chowdhury et al. (1991 ) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. MoI. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that described in Tratschin et al. (1985) MoI. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) MoI. Cell. Biol. 4:2072-2081 ;
Wondisford et al. (1988) MoI. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). Lentiviral gene therapy vectors may also be adapted for use in the invention.
From the specification and appended claims, the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents. Further, the DNA segments or proteins according to the present-invention can be introduced into individuals in a number of ways. As exemplified herein, peripheral T cells can be isolated from an individual afflicted or at risk of suffering from a disease or condition, transfected with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways, including intravenous injection. Alternatively, the DNA construct can be administered directly to the afflicted individual, for example, by injection in the thymus. The DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes. Of course, proteins or peptides can also be administered. A person of ordinary skill can adapt the transfection method, type of cells transfected, type of disease or condition, co- stimulus (general or specific) etc to meet particular needs.
Compositions within the scope of the present invention should contain the active agent (e.g. fusion protein, peptide, nucleic acid, and molecule, or antigen, or antibody, or APC) in an amount effective to achieve the desired prophylactic and/or therapeutic T cell activation while avoiding adverse side effects. Typically, the nucleic acids in accordance with the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th ed., Mack ed.). The invention therefore further provides a composition comprising an active agent and a pharmaceutically acceptable carrier. For the administration of polypeptides, antagonists, agonists and the like, the amount administered should be chosen so as to avoid adverse side effects. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be
administered to the mammal. In accordance with one embodiment of the present invention, and as exemplified herein, T cells may be removed from a patient (e.g. cancer patient, or virally affected patient [or susceptible of being infected by a virus]), activating these T cells in accordance with the present invention and re- administering these activated T cells to the patient. Of course, known steps for further cultivating or proliferating these T cells could be carried-out prior to assaying an activated T cell function or re-injecting same into a patient. For example, cytokines or other mitogens or molecules could be added to the culture medium.
As used herein, the immunogenic or vaccine compositions of the invention are administered by conventional routes known the vaccine field, in such as to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g. via a patch). The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the subcutaneous or intramuscular route being most preferred. The choice also depends upon the nature of the vaccine agent. For use in a composition of the invention, a polypeptide or derivative thereof may be formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990).
Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below.
A polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery
vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.
Accordingly, a further aspect of the invention provides (i) a vaccine vector such as an adenovirus, containing a nucleic acid molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically and/or prophylactically effective amount of a vaccine vector of the invention; (iv) a cell (e.g. an antigen-presenting cell) transfected or transformed with the nucleic acid or vector of the present invention; (v) a method for inducing an immune response against a tumor in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing tumor growth or cancer in non-human animals), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response against a tumor; and particularly, (v) a method for preventing and/or treating cancer, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an individual having, or at risk of developing, cancer. Additionally, the invention further provides a use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating cancer.
As used herein, a vaccine vector expresses one or several polypeptides or derivatives of the invention. The vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), which enhances the immune response (adjuvant effect). It is understood that each of the components to be expressed is placed under the control of elements required for expression in a mammalian cell.
The invention further provides a composition comprising several polypeptides or derivatives thereof of the invention or vaccine vectors (each of them capable of expressing a polypeptide or derivative thereof of the invention). A composition may also comprise an additional tumor antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12 (or vaccine vector(s) capable of inducing their expression).
"Vaccine" as used herein refers to a composition or formulation comprising one or more polypeptides/peptides of the invention, or a vaccine vector of the invention. Vaccination methods for treating or preventing an infection or a disease in a mammal comprises use of a vaccine or vaccine vector of the invention to be administered by any conventional route.
Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine or vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like). Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette-Guerin (BCG), and Streptococcus.
An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a nucleic acid molecule of the invention, are described in U.S. Patent No. 4,920,209. Poxvirus vectors include vaccinia and canary poxvirus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, e.g., Tartaglia et al., Virology (1992) 188:217) for a description of a vaccinia virus vector and Taylor et al., Vaccine (1995) 13:539 for a description of a canary pox vector). Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of vaccine viral vector, for therapeutic or prophylactic use, can be of from about 1x104 to about 1x1011, advantageously from about 1x107 to about 1x1010, preferably of from about 1x107 to about 1x109 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551 and U.S.
Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced. WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations. WO 94/01533 describes a deletion mutant lacking functional ctxA and attRSI DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1x105 to about 1x109, preferably about 1x106 to about 1x108, viable bacteria in a volume appropriate 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 vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
Other bacterial strains used as vaccine vectors in the context of the present invention are described for Shigella flexneri in High et al., EMBO J.(1992) 11 :1991 and Sizemore et al., Science (1995) 270:299; for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. ScL USA (1995) 92:6868; and for Bacille Calmette Guerin in Flynn J.L., Cell. MoI. Biol. (1994) 40 (suppl. l):31 , WO 88/06626, WO 90/00594, WO 91/13157, WO 92/01796, and WO 92/21376. In bacterial vectors, the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.
The composition comprising a polypeptide or vaccine vector of the present invention may further contain an adjuvant. A number of adjuvants are known to those skilled in the art. Examples of adjuvants are described below. Accordingly, a further aspect of the invention provides (i) a composition comprising a polypeptide or nucleic acid of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polypeptide or polynucleotide of the
invention; (iii) a method for inducing an immune response against a tumor in a mammal by administration of an immunogenically effective amount of a polypeptide or polynucleotide of the invention to elicit a protective immune response against a tumor; and particularly, (iv) a method for preventing and/or treating a cancer, by administering a prophylactic or therapeutic amount of a polypeptide or polynucleotide of the invention to an individual having, or at risk of developing, cancer. Additionally, the invention further provides a use of a polypeptide or polynucleotide of the invention in the preparation of a medicament for preventing and/or treating cancer. Use of the nucleic acids of the invention include their administration to a mammal as a vaccine or an immunogenic agent, for therapeutic or prophylactic purposes. Such nucleic acids are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, such a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, MoI. Cell Biol. (1985) 5:281 ). An example of a tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991 ) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al., Human Gene Therapy ( 1996) 7:1205. Polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide. In the precursor form, the signal peptide is either homologous or heterologous. In the latter case, a eukaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred. Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention. For use as a vaccine, a polynucleotide of the invention is formulated according to various methods outlined below.
One method utilizes the polynucleotide in a naked form, free of any delivery vehicles. Such a polynucleotide 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.
An alternative method utilizes the polynucleotide 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 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™ also known as DOTMA (N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1 ,2-bis(oleyloxy)- 3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (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 described in WO 90/11092 as an example.
Formulations 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 are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152. 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. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
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 gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg and, more preferably, from about 25 μg to about 250 μg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.
Although not absolutely required, such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21 , which is described in U.S. Patent No. 5,057,546.
Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In an embodiment, a polypeptide of the invention, administered as a vaccine, is administered by a mucosal route in an amount from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 μg-
When used as vaccine agents, polypeptides and polynucleotides of the invention may be used sequentially as part of a multistep immunization process. For example, a mammal is initially primed with a vaccine vector of the invention such as an adenovirus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or derivative of the invention are also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT). Examples of adjuvants useful in any of the vaccine/immunogenic compositions described above are as follows.
Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), are used in parenteral administration.
Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b^N^N'.N'-dimethyl
aminomethane)-carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO
96/14831 ) and QS-21 (WO 88/09336).
Any pharmaceutical composition of the invention containing a polypeptide, a polypeptide derivative or a polynucleotide of the invention, is manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. The composition for cancer treatment of the present invention can be used in conjunction with existing chemotherapy drugs or be made as a mixture with them. Such a chemotherapy drug include, for example, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, alkaloids derived from plant, topoisomerase inhibitors, hormone therapy medicines, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, other immunotherapeutic drugs, and other anticancer agents. Further, they can be used together with hypoleukocytosis (neutrophil) medicines that are cancer treatment adjuvant, thrombopenia medicines, antiemetic drugs, and cancer pain medicines for
patient's QOL recovery or be made as a mixture with them. The composition of the present invention and the other agent(s) (e.g. a chemotherapeutic agent) can be administered concomitantly or sequentially.
The composition for cancer treatment of the present invention can be used together with immunopotentiative substance(s) or be made as a mixture with them. Such immunopotentiative substances include, for example, various cytokines and a tumor antigen, etc. Cytokines that stimulate immune reactions include, for example, GM-CSF, M-CSF, G-CSF, interferon-β and Y, IL-1 , IL-2, IL-3, and IL-12, anti-CD3 antibodies can also improve the immune reactions. The composition of the present invention and immunopotentiative substance(s) (e.g. a cytokine or a chemokine) can be administered concomitantly or sequentially.
The term "treating cancer" or "treatment of cancer" as used herein includes at least one of the following features (prophylactic/therapeutic results): alleviation of the symptoms associated with the cancer, a reduction in the extent of the cancer (e.g. a reduction in tumor growth), a stabilization of the state of the cancer (e.g. an inhibition of tumor growth), a prevention of further spread of the cancer (e.g. a metastasis), a prevention of the occurrence or recurrence of a cancer, a delaying or retardation of the progression of the cancer (e.g. a reduction in tumor growth) or an improvement in the state of the cancer (e.g. a reduction in tumor size).
A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the above-mentioned desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting onset or progression of cancer and associated symptoms and disease. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific
dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
The cancers to which the compositions and methods of the present invention can be applied include swellings and true tumors including benign and malignant tumors. Specific examples of such tumors are gliomas such as astrocytoma, glioblastoma, medulloblastoma, oligodendroglioma, ependymoma and choroid plexus papilloma; cerebral tumors such as meningioma, pituitary adenoma, neurioma, congenital tumor, metastatic cerebral tumor; squamous cell carcinoma, lymphoma, a variety of adenomas and pharyngeal cancers resulted from these adenomas such as epipharyngeal cancer, nasopharyngeal cancer and hypopharyngeal cancer; laryngeal cancer, thymoma; mesothelioma such as pleural mesothelioma, peritoneal mesothelioma and pericardial mesothelioma; breast cancers such as thoracic duct cancer, lobular carcinoma and papillary cancer; lung cancers such as small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma and adenosquamous carcinoma; gastric carcinoma; esophageal carcinomas such as cervical esophageal carcinomas, thoracic esophageal carcinomas and abdominal esophageal carcinomas; carcinomas of large intestine such as rectal carcinoma, S-like (sigmoidal) colon carcinoma, ascending colon carcinoma, lateral colon carcinoma, cecum carcinoma and descending colon carcinoma; hepatomas such as hepatocellular carcinoma, intrahepatic hepatic duct carcinoma, hepatocellular blastoma and hepatic duct cystadenocarcinoma; pancreatic carcinoma; pancreatic hormone-dependent tumors such as insulinoma, gastrinoma, VIP-producing adenoma, extrahepatic hepatic duct carcinoma, hepatic capsular carcinoma, perial carcinoma, renal pelvic and uretal carcinoma; urethral carcinoma; renal cancers such as renal cell carcinoma (Grawitz tumor), Wilms1 tumor (nephroblastoma) and renal angiomyolipoma; testicular cancers or germ cell tumors such as seminoma, embryonal carcinoma, vitellicle tumor, choriocarcinoma and teratoma; prostatic cancer, bladder cancer, carcinoma of vulva; hysterocarcinomas such as carcinoma of uterine cervix, uterine corpus cancer and solenoma; hysteromyoma, uterine sarcoma, villous diseases, carcinoma of vagina; ovarian germ cell tumors such as dysgerminoma, vitellicle tumor, premature teratoma, dermoidal cancer and ovarian tumors such as ovarian
cancer; melanomas such as nevocyte and melanoma; skin lymphomas such as mycosis fungoides, skin cancers such as endoepidermal cancers resulted from skin cancers, prodrome or the like and spinocellular cancer, soft tissue sarcomas such as fibrous histiocytomatosis, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, synovial sarcoma, sarcoma fibroplasticum (fibrosarcoma), neurioma, hemangiosarcoma, fibrosarcoma, neurofibrosarcoma, perithelioma
(hemangiopericytoma) and alveolar soft part sarcoma, lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, myeloma, plasmacytoma, acute myelocytic (myeloid) leukemia and chronic myeloid leukemia, leukemia such as adult T-cell leukemic lymphoma and chronic lymphocytic leukemia, chronic myeloproliferative diseases such as true plethora, essential thrombocythemia and idiopathic myelofibrosis, lymph node enlargement (or swelling), tumor of pleural effusion, ascitic tumor, other various kinds of adenomas, lipoma, fibroma, hemangeoma, myoma, fibromyoma and endothelioma. In an embodiment, the cancer/tumor is characterized by or associated with STEAP expression. "Characterized by or associated with STEAP expression" as used herein refers to an alteration of STEAP expression in a tumor cell relative to a non-tumor cell for a given tissue. For example, such an alteration may be an increase in STEAP expression in a tumor cell relative to a corresponding non-tumor cell exhibiting lower or no STEAP expression. For example, STEAP (see Figure 12) has been shown to be expressed or overexpressed in human prostate cancer and also in several tumors cell lines of various origins, such as prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma (Hubert RS et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 14523-14528).
As used herein, the expression "tumor-associated antigen" or "TAA" refers to an antigen (e.g. a polypeptide or an antigenic fragment or variant thereof) that is overexpressed in a tumor cell/tissue as compared to a corresponding normal cell/tissue. Overexpression can be, for example, an increase in expression of a given antigen in a tumor cell/tissue as compared to a normal cell/tissue, but also the expression of an antigen in a tumor cell/tissue that do not express it in a normal state (i.e. when the cell or tissue is not cancerous). For example, TAA include known oncoproteins such as HER-2/Neu and c-myc, survival proteins such as
survivin and lens epithelium-derived growth factor (LEDGF/p75), cell cycle regulatory proteins such as Cyclin B1 , differentiation and cancer-testis antigens such as NY-ESO-1 , colorectal cancer antigen such as carcinoembyronic antigen (CEA), most antigens of the MAGE family, melanA/MART-1 , MUC1 , Wilms' tumor protein (WT-1 ), STEAP and others (see Novellini et al., 2005. Cancer Immunol Immunother. 54(3): 187-207 for a list of known TAA).
In an embodiment, the above-mentioned TAA is STEAP, or an antigenic variant or fragment thereof. Antigenic fragment or variant of STEAP are well known in the art. For example, antigenic STEAP fragments/epitopes have been described in Machlenkin A. et al., 2005, Cancer Res., 65:6435-6442; Rodeberg D.A. et al., 2005, Clin. Cancer Res., 11 : 4545-4552; Alves P.M. et al., 2006, Cancer Immunol. Immunother., 55: 1515-1523; and Kobayashi H. et al., 2007. Cancer Res. 67: 5498-5504.
In a further aspect, the invention provides a recombinant cell comprising the above-mentioned vector. In a further embodiment, the above- mentioned cell is an antigen-presenting cell (e.g. a dendritic cell).
In a further aspect, the invention provides a composition comprising the above-mentioned cell and a pharmaceutically acceptable carrier or excipient.
In another aspect, the present invention provides a method of activating a tumor-specific human T cell, said method comprising contacting said T cell with the above-mentioned recombinant cell. In an embodiment, the above- mentioned contacting is performed ex vivo or in vivo.
Within the scope of the invention are cells (e.g. host cells) transfected or transformed with the chimeric nucleic acid or the vector of the invention. Methods for transforming/transfecting host cells with nucleic acids/vectors are well-known in the art and depend on the host system selected as described in Ausubel et al. (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Host cells transfected or transformed with the chimeric nucleic acid or vector of the invention can be used as a vaccine (e.g., autologous cell vaccine) in order to induce or
increase an immune response against an antigenic epitope or antigen (e.g., STEAP) in a subject. For example, host cells (e.g., APCs, dendritic cells) may be removed from a subject (e.g., a cancer patient), transfected or transformed in accordance with the present invention and re-administered to the patient. Of course, known steps for further cultivating or modifying these cells could be carried- out prior to re-injecting/transplanting them into a subject. For example, cytokines/chemokines or mitogens or molecules could be added to the culture medium.
In accordance with another embodiment of the present invention, T cells (e.g., CD4+ and/or CD8+ T cells) may be removed from a subject (e.g. cancer patient), activated in accordance with the present invention (e.g., by contacting them with a host cells transfected with a chimeric nucleic acid of the invention) and re-administered to the patient. Of course, known steps for further cultivating or proliferating these T cells could be carried-out prior to re-injecting them into a subject. For example, cytokines or other mitogens or molecules could added to the culture medium.
In an embodiment, the above-mentioned T cell is derived from a subject suffering from cancer or at risk of suffering from cancer.
In another aspect, the present invention provides a kit or package comprising:
(a) an agent selected from (i) the above-mentioned composition, (ii) the above-mentioned vector, (iii) the above-mentioned cell, and (iv) any combination of (i) to (iii); and
(b) instructions for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The following examples are illustrative of
various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
EXAMPLES The present invention is illustrated in further detail by the following non-limiting examples.
Example 1 : Confirmation of STEAP and mutant CD80 expression by the adenoviral vaccines of large preparation. Five adenoviral vector vaccines (Adv) were produced with different combination of STEAP and CD80 mutant expression:
- Adv control
- Adv STEAP
- Adv CD80wt (Wild-type CD80) - Adv CD80wt/STEAP (wild-type CD80 and STEAP)
- Adv V1 C2/STEAP (chimeric V1 C2 (Figure 11 ) and STEAP)
A series of experiments were performed to confirm the expression of
STEAP and CD80 mutants by the large preparation of the vaccines. 293HEK cells were infected with the viral vaccines obtained from large preparations. Adherent 293 cells were plated and cultured until confluence.
Then, cells were infected with 100 pfu Adv/cell during 24 hours. After infection, infected cells were washed and analyzed for CD80 expression by flow cytometry detected with mouse anti-CD80 monoclonal antibody (reference P91853F, Biodesign, Saco, ME) and for STEAP expression by western blot detected with rabbit anti-STEAP polyclonal antibody (reference 187385, Zymed laboratories, San
Francisco, CA).
Figure 1 , panel A shows a Western blot that demonstrates STEAP expression in all samples infected with adenoviral vaccines containing the STEAP transgene (lane 3,4 and 5), but not in the control (lane 1 ) and CDδOwt (lane 2) adenovirus.
Figure 1 , panel B shows FACS analysis of CD80 mutant expression.
CD80 expression was detected in all samples infected with the adenoviral vaccine
having CD80 expression cassette: ADV CDδOwt, Adv-CD80wt/STEAP and Adv-
V1C2/STEAP are expressed at comparable levels. Besides the confirmation of STEAP and CD80 expression, we also determined the viral titers of the large preparations by plaque forming assay. Human adherent monocytes are used as the main antigen presenting cells in our in vitro analysis of human T cell response to STEAP, we also tested the CD80 mutant expression in the adenoviral vaccines infected human monocytes. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus gradient centrifugation (Amersham Biosciences). Fresh PBMC were plated at 3.5X106 cells per well in a 48-well plate for 2 hours to allow the adherent fraction to attach to the plastic wells. The non adherent fraction was removed. After washing, Adv control, Adv-CD80wt, Adv-STEAP, Adv-CD80wt/STEAP and Adv- V1C2/STEAP were added at a multiplicity of infection of 100 per cell. After 24 hours of incubation at 370C with adenovirus, monocytes were washed and stained for CD80 expression.
Figure 3 shows expression of CD80 molecules (wild-type or mutant) detected on 37% to 56% human adherent monocytes at considerable high level, 24 hours after the cells were infected with adenoviral vaccines.
Example 2: Testing of the endotoxin contamination of our adenovirus preparations.
As quality control, the endotoxin concentrations was evaluated in samples using Limulus amebocyte lysate (LAL) test (reference 50-500U-12,
Cambrex, Walkersville, MD). Samples from Adenovirus preparation were incubated with the limulus lysate sample during 1 hour at 37°C. The presence of endotoxin is characterized by the formation of a solid gel.
Figure 2 shows that the adenovirus preparations contain less than
0,125 endotoxin units (EU) per ml of adenovirus preparations. Dilution series of E. coli isotype 0111 :B4 endotoxin (0,50 to 0,03 EU/ml) was used as positive control.
Example 3: Tumor experiments in BALB/c mice: prophylactic vaccination model.
The adjuvant efficacy of human CD80 mutants was next evaluated in Adenoviral vaccines using STEAP-expressing CT26 cells as tumor model; a prophylactic vaccination model was developed (Figure 4).
• DO: Immunization of mice with Adv (109 pfu/mouse) expressing
STEAP and wild-type CD80 (CDδOwt) and chimeric CD80/86 (V1C2CD80/CD86). • 4 weeks later: Tumor challenge with STEAP-expressing CT26 cells (Figures 5 and 6). • Over the next 3 weeks: assessment of tumor size every 2 to 3 days using a digital calibrator. In this experiment, the different constructions tested were:
- group 1 : Adv control (empty Adv vector),
- group 2: Adv-STEAP - group 3: Adv-CD80wt
- group 4: Adv-CD80wt/STEAP,
- group 5: Adv-V1C2CD80/CD86-STEAP,
Six (6) mice were injected in each group, except for group 4 and 5 (seven (7) mice). Mice received sub-cutaneous injection of 105 CT26-STEAP cells. The tumor length (L) and width (W) were measured and the volume was determined by W2 X L X 0,52 (mm3).
In order to perform STEAP-specific tumor protection and eradication on mice, a mouse tumor cell line expressing human STEAP was established. CT26 is a colon cancer line of BALB/c origin (Wang et al., J. Immunol., 154(9), p.4685- 92). CT26 cells were transfected with the plRESneo3 plasmid (Invitrogen) in which a full length human STEAP cDNA was inserted at the poly-linker site. After a two week selection with G418, the drug-resistant cells were further cloned by limiting dilution. The G418-resistant clones (i.e. those stably transfected with neomycin- resistant gene) were analyzed by Western blot for STEAP expression. Figure 5 shows that G418-resistant CT26 clones expressed human
STEAP for more than 5 weeks after G418 selection. The STEAP-positive clones will be used as a tumor model for in vivo tumorigenicity and tumor protection tests.
Figure 6 shows the tumorigenicity of CT26 cells clone expressing
STEAP in BALB/c mice (n=6) using three different dosages of CT26-STEAP injected to mice (103 to 105 cells/mouse). The expression of STEAP by tumors was tesyed by RT-PCR. Figure 5 shows the detection of STEAP gene in CT26-STEAP tumoral cell biopsy (lane 4).
Figure 7 shows the tumor size at day 21 post-injection in the different vaccinated groups of mice. There is a significant difference (p<0.05) in the average tumor size between mice vaccinated with Adv-STEAP or Adv V1C2-STEAP and mice vaccinated with CD80wt-STEAP. Figure 8 shows the tumor growth (tumor size in mm3) over a time course of 3 weeks in 2 different vaccinated groups of mice. This figure shows that injection of Adv V1 C2CD80/CD86-STEAP induces a delay in tumor growth as compared to mice injected with Adv CD80wt-STEAP. This difference is significant at day 21 (p<0.05). At day 24, 5/7 mice immunized with the V1 C2CD80/CD86-STEAP were still alive, as compared to 4/7 and 3/6 in mice immunized with Adv CD80wt- STEAP and Adv control, respectively.
Therefore, these results demonstrate that the Adv V1 C2CD80/CD86- STEAP vaccine induces a protective anti-tumor response that inhibits tumor growth in the CT26-STEAP tumor model.
Example 4: Assessment of mouse T cell phenotype and effector functions following vaccination with Adenoviral constructs expressing STEAP.
In order to test the immunogenicity of STEAP antigen in vaccinated BALB/c mice, an ex vivo immune monitoring assay was performed: Intracellular
Cytokine Staining (ICS) by flow cytometry. STEAP-specific T cell activation (IFN-γ and IL-2 secretion) was assessed in T cells from vaccinated mice after 6h of stimulation with STEAP peptides.
Fresh spleen cells were obtained from mice immunized in the prophylactic vaccination protocol (at day 58) and stimulated during 6 hours in the presence of 1 DgAnI of each overlapping STEAP peptide in order to determine the frequency and functionality of the responding mouse T cells. Each pool contains ten
15-mer peptides derived from STEAP. 8 pools (p1 to p8) have been tested. The
positive control for stimulation was PMA (10 ng/ml) and ionomycin (0.5 mM). NS:
The non-stimulated condition (i.e. no peptide) for background response. After 6 hours of stimulation, cells were fixed, permeabilized and stained for CD3, CD4 or CD8, IL-2 and IFN-γ as previously described (Younes et a/., 2003. J Exp Med 198: 1009-1022).
Figure 9 shows a flow cytometric analysis of phenotypic and functional properties of the responding immune T cells.
A: 52% lymphocytes, 15% CD3+ T cells, 15% CD3+CD4+T cells, 6% CD3+CD8+ T cells. B: unstimulated CD3+CD8+ T cells
C: unstimulated CD3+CD4+ T cells
D and E: Production of IFN-γ by CD3+CD8+ and IL-2 by CD3+CD4+ T cells following stimulation with STEAP peptides.
Figure 10 is a summary of the percentages of responding T cells following activation with the 8 different pools of STEAP peptides (p1 to p8). The percentage represents the proportion of cytokine-(IFN-γ or IL-2) positive cells within total CD8+ or CD4+ T cells.
In the splenocytes of Adv control vaccinated mice, the percentage of CD3+CD4+IL-2+ and CD3+CD8+IFN-γ+ T cells is about 0.2 and 0.5, respectively. In the Adv CD80wt-STEAP, 9.5, 7.9 and 6.8% of CD3+CD8+ T cells produces IFN-γ in response to p4, p5 and p8 respectively. Similarly, 2.9, 3.2 and 3.5% of CD3+CD4+ T cells produces IL-2 in response to the same pools (p4, p5 and pδ, respectively).
In the Adv V1 C2CD80/CD86-STEAP vaccinated group, 1.5 to 3.2% of CD3+CD8+ T cells stained positive for IFN-γ in response to p1 , p2, p3, p4, p5 and p8. This response correlates with the frequency (1.9 to 3%) of CD3+CD4+ cells secreting IL-2 following stimulation with the same pools. Therefore, Adv V1 C2CD80/86-STEAP induces a strong and diversified T cell response to STEAP antigen. Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
Claims
1. A composition comprising: a) a first recombinant nucleic acid comprising a nucleic acid sequence encoding a chimeric co-stimulatory molecule, said chimeric co- stimulatory molecule comprising; i) a functional V region of CD80; ii) a functional C region of CD86; and iii) transmembrane and cytoplasmic domains of CD86 or functionally active fragments thereof; and b) a tumor-associated antigen polypeptide or a second recombinant nucleic acid comprising a nucleic acid sequence encoding said tumor-associated antigen polypeptide.
2. The composition of claim 1 , wherein said composition is an immunogenic or vaccine composition.
3. The composition of claim 1 or 2, further comprising a pharmaceutically acceptable carrier or excipient.
4. The composition of any one of claims 1-3, further comprising an adjuvant.
5. The composition of any one of claims 1-4, wherein said chimeric co- stimulatory molecule comprises the amino acid sequence of SEQ ID NO:2.
6. A method of preventing or treating cancer, said method comprising administering to a subject a prophylactically or therapeutically effective amount of the composition of any one of claims 1 -5.
7. The method of claim 6, wherein said first nucleic acid is present in a vector comprising said nucleic acid sequence encoding a chimeric co-stimulatory molecule operably linked to a transcriptional regulatory sequence.
8. The method of claim 6, wherein said second nucleic acid is present in a vector comprising said nucleic acid sequence encoding said tumor-associated antigen polypeptide operably linked to a transcriptional regulatory sequence.
9. The method of any one of claim 6-8, wherein said chimeric co-stimulatory molecule is expressed in a recombinant cell.
10. The method according to any one of claim 6-9, wherein said subject is a human.
11. The method according to claim 6, wherein said vector is a viral vector.
12. The method according to claim 11 , wherein said viral vector is an adenoviral vector.
13. The method according to claim 6, wherein said tumor-associated antigen polypeptide is STEAP, or an antigenic variant thereof, or a fragment thereof.
14. The method of claim 13, wherein said cancer is characterized by STEAP expression.
15. The method according to claim 14, wherein said cancer is prostate cancer.
16. The method according to claim 9, wherein said cell is an antigen-presenting cell.
17. A vector comprising: a) a nucleic acid sequence encoding the chimeric co-stimulatory molecule defined in claim 1 operably linked to a transcriptional regulatory sequence; and b) a nucleic acid sequence encoding a tumor-associated antigen polypeptide operably linked to a transcriptional regulatory sequence.
18. A recombinant cell comprising the vector of claim 17.
19. The cell according to claim 18, wherein said cell is an antigen-presenting cell.
20. The cell according to claim 18, further comprising a pharmaceutically acceptable carrier or excipient.
21. A method of activating a tumor-specific human T cell, said method comprising contacting said T cell with the recombinant cell of claim 18.
22. The method according to claim 21 , wherein said contacting step is carried out ex vivo.
23. The method according to claim 21 , wherein said contacting step is carried out in vivo and said recombinant cell is administered to a subject comprising said tumor-specific human T cell.
24. The method according to claim 21 , wherein said human T cell is derived from a subject suffering from a cancer.
25. A kit comprising:
(a) an agent selected from (i) the composition according to any one of claims 1-5 and (ii) the vector according to claim 17, (iii) the cell according to any one of claims 18-20, and (iv) any combination of (i) to (iii); and (b) instructions for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
26. Use of an agent selected from (i) the composition according to any one of claims 1-5, (ii) the vector according to claim 17, (iii) the cell according to any one of claims 18-20, and (iv) any combination of (i) to (iii), for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
27. Use of an agent selected from (i) the composition according to any one of claims 1-5, (ii) the vector according to claim 17, (iii) the cell according to any one of claims 18-20, and (iv) any combination of (i) to (iii), for the preparation of a medicament for preventing or treating cancer or for inducing an immunological or protective immune response against a tumor.
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