EP1171612A2 - Vaccin anticancereux specifique de la telomerase - Google Patents

Vaccin anticancereux specifique de la telomerase

Info

Publication number
EP1171612A2
EP1171612A2 EP00920996A EP00920996A EP1171612A2 EP 1171612 A2 EP1171612 A2 EP 1171612A2 EP 00920996 A EP00920996 A EP 00920996A EP 00920996 A EP00920996 A EP 00920996A EP 1171612 A2 EP1171612 A2 EP 1171612A2
Authority
EP
European Patent Office
Prior art keywords
telomerase
peptide
antigen
amino acids
length
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00920996A
Other languages
German (de)
English (en)
Inventor
Babita Agrawal
Bryan Michael Longenecker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncothyreon Canada Inc
Original Assignee
Biomira Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biomira Inc filed Critical Biomira Inc
Publication of EP1171612A2 publication Critical patent/EP1171612A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464457—Telomerase or [telomerase reverse transcriptase [TERT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • telomeres the DNA at the chromosome ends, are made up of simple tandem repeats. In most somatic cells, telomere sequences are lost during DNA replication due to the need of DNA-dependent DNA polymerases for an RNA primer annealed to the template strand. Because the RNA primer cannot anneal beyond the 5' end of the DNA strand, each time a cell's DNA replicates, short bits of telomeric DNA are lost with each generation. Cells displaying such telomeric shortening go into senescence after a fixed number of population doublings, and senescence correlates directly with the erosion of telomeres to a critical minimum length.
  • telomere length is maintained in these cells by the action of telomerase, a ribonucleoprotein enzyme that uses a short endogenous RNA as a template for telomere addition.
  • telomerase a ribonucleoprotein enzyme that uses a short endogenous RNA as a template for telomere addition.
  • cancer ceHs express high levels of telomerase, whereas somatic cells express little, if any.
  • telomerase enzyme presents an attractive therapeutic target. Due to the fact that telomerase is a normal "self antigen, however, conventional vaccination strategies are unavailable. Thus, the focus of telomerase- based therapeutics has been enzyme inhibitors of various sorts, rather than vaccine-based approaches.
  • telomerase antigens are provided that are capable of marshalling the immune system against telomerase-expressing cells.
  • telomerase antigens are provided, which are based on peptide sequences of the protein portion of telomerase.
  • telomerase antigens are provided as nucleic acids that are capable of being used to express peptide-based telomerase antigens.
  • the invention provides vaccine compositions which include at least one telomerase antigen.
  • the invention provides vaccine compositions containing telomerase peptide antigens.
  • polynucleotides are provided, which encode protein-based telomerase antigens.
  • the telomerase antigens of the invention may be directly administered in beneficial amounts to a patient.
  • the present telomerase antigens may be administered to a patient encoded in a nucleic acid.
  • ex vivo methods that involve contacting a cell with a telomerase antigen, and administering that cell to a patient.
  • the contacted cell may be an antigen presenting cell, which may be used to generate a primed T-cell ex vivo, at which time the primed T-cell may be administered to a patient.
  • FIG. 1 shows fluorescence activated cell sorting (FACS) analysis of T-cells activated by telomerase-specific antigens.
  • Panels A and B are negative and positive controls, respectively, and panels C, D and E are antigen candidates.
  • FACS fluorescence activated cell sorting
  • the present invention relates to a telomerase-specific vaccines, which are useful generating telomerase-directed activated T-cells. More particularly the inventive vaccines are useful in generating antigen-specific major histocompatability- (MHC-) restricted T-cell responses against telomerase presented by antigen presenting cells. It is believed that the present vaccines act to relieve the tolerance or anergy induced through self- tolerance mechanisms to telomerase in normal individuals. Since telomerase represents a cancer-specific therapeutic target, in one embodiment, the vaccines of the invention are useful in the treatment or prevention of a variety of cancers.
  • MHC- major histocompatability-
  • an "activated T-cell” is one that is in the following phases of the cell cycle: the G, phase, the S phase, the G 2 phase or the M (mitosis) phase.
  • an “activated T-cell” is undergoing mitosis and/or cell division.
  • An activated T- cell may be a T helper (T H ) cell or a cytotoxic T-cell (cytotoxic T lymphocyte (CTL or T c )).
  • Activation of a naive T-cell may be initiated by exposure of such a cell to an antigen- presenting cell (APC) (which contains antigen/MHC complexes) and to a molecule such as IL-1, IL-2, IL-12, IL-13, ⁇ -IFN, and similar lymphokines.
  • APC antigen- presenting cell
  • the antigen/MHC complex interacts with a receptor on the surface of the T-cell (T-cell receptor (TCR)).
  • T-cell receptor TCR
  • CD4 and CDS " T-cell responses against a target antigen are usually dependent upon in vivo priming, either through natural infection or through deliberate immunization.
  • a “naive" T-cell is one that has not been exposed to foreign antigen (non-autologous) antigen or one that has not been exposed to cryptic autologous antigen.
  • a “naive” T-cell is sometimes referred to as an "unprimed” T-cell.
  • a “resting” cell is in the G 0 phase of the cell cycle and hence is not dividing or undergoing mitosis.
  • an "anergic" T-cell is one that is unable to function properly; i.e.. such as a cell that lacks the ability to mediate the normal immune response.
  • T-cells from diseased patients may contain T-cells that have been primed, but are anergic.
  • memory T-cells also known as “memory phenotype” T-cells, is used to designate a class of T-cells that have previously encountered a peptide antigen but are now resting and are capable of being activated.
  • Memory T-cells are T- cells which have been exposed to antigen and then survive for extended periods in the body without the presence of stimulating antigen. However, these memory T-cells respond to
  • memory T-cells are more responsive to a "recall” antigen, when compared with the naive T-cell response to peptide antigen.
  • Memory cells can be recognized by the presence of certain cell-surface antigens, such as CD45R0, CD58, CD1 l ⁇ , CD29, CD44 and CD26, which are markers for differentiated T-cells.
  • an "telomerase-specific" T-cell response is a T- cell response (for example, proliferative, cytotoxic and/or cytokine secretion) to telomerase antigenic stimulus, for example a peptide. which is not evident with other stimuli, such as peptides with different amino acid sequences (control peptides).
  • the responsiveness of the T- cell is measured by assessing the appearance of cell surface molecules that are characteristic of T-cell activation, including, but not limited to CD25 and CD69. Such assays are known in the art.
  • treating in its various grammatical forms in relation to the present invention refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent or other abnormal condition.
  • Telomerase antigens share the characteristic ability to generate a specific T-cell response.
  • This response may be either class I- or class ll-specific.
  • this response is MHC class l-specific, and will comprise antigen- and MHC- restricted cytotoxicity.
  • Class I molecules include HLA-A, HLA-B and HLA-C.
  • preferred antigens bind class I molecules, e.g., HLA-A1, HLA-A2, HLA-A3 or HLA-A11. More preferred antigens bind all class I molecules.
  • class ll-specific (e.g., helper functions) responses are desired, class-II-binding antigens will be used.
  • Class II molecules include HLA-DR, HLA-DQ and HLA-DP. Useful antigens can be determined as set out below.
  • Telomerase antigens are typically derived from the sequence of the protein portion of telomerase, which is disclosed in U.S. Patent No. 5,837,857 (1998) and at GenBank Accession Nos. AF015950 and AF018167, which sequences are hereby incorporated by reference. They may be made, for example, by proteolytic digestions of the telomerase protein and/or by recombinant DNA means. Generally, the relatively short peptide versions will be prepared by synthetic means.
  • telomerase antigens are not limited by size, and they may be a portion or even all of the telomerase protein, they are usually small peptide antigens. A small size is preferred, due to ease of manufacture and greater specificity. Accordingly, unless they are multimeric (i.e., multiple copies of the same epitope) most telomere antigens will be less than about 50 amino acids in length. Preferred antigens are less than about 25 amino acids in length, with other preferred antigens being between about 8 to about 12 amino acids long, although sequences as short as 6 or 7 amino acids are contemplated.
  • telomerase antigens Nine-mers are typical of class I antigens, since they usually retain the requisite functional character; they include Ile-Leu-Ala-Lys-Phe-Leu-His-Trp-Leu (ILAKFLHWL) as a preferred species. Variants of telomerase antigens are also contemplated. It is only important that any variants retain the functional characteristics of a telomerase antigen: (1) the ability to bind an MHC molecule, e.g., HLA-A2, and (2) the ability to induce a telomerase specific T-cell response. Amino acid substitutions, i.e.
  • “conservative substitutions” that yield “conservative variants,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline. phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine. tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine. lysine. and histidine;
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d).
  • glycine and proline may be substituted for one another based on their relatively small sizes and lack of side-chains.
  • amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in ⁇ -helices, while valine, isoleucine, phenylalanine, tyrosine. tryptophan and threonine are more commonly found in ⁇ -pleated sheets.
  • Glycine, serine. aspartic acid, asparagine, and proline are commonly found in turns. The importance of substitution groups based on structure, of course, increases with the length of the antigen.
  • Some preferred conservative substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A. V, L and I.
  • S and T S and T
  • P and G P and G
  • A. V, L and I A preferred conservative substitutions
  • the skilled scientist readily can construct DNAs encoding the conservative amino acid variants. Of course, smaller variants may be synthesized.
  • One such genus of conservative HLA-A2-binding variants includes peptides of the structure: (A/V/L/I)(A/V/L/I)(A/V/L/I)(A/V L/I)KF(A/V/L/I)HW(A/V/L/I).
  • some preferred conservative va ⁇ ants include LLAKFLHWL, ILAKFLHWI, IIAKFLHWL, IIA FLHWI, ILARFLHWL, and ILVKFLHWL, and permutations thereof, so long as the requisite functional characteristics are retained.
  • one or more of the amino acids of the foregoing HLA-A2-binding peptides may be replaced with glycine.
  • Parker et al, J. Immunol. 149:3580-87 (1992) disclose that up to six amino acids in a nine-mer may be replaced with glycine; thus,
  • GLFGGGGGV can bind HLA-A2.
  • the only real conservation observed in 9-mer HLA-A2- binding peptides was an He or a Leu at about position 2 (counting N- to C-terminal) and a Val or a Leu at about position 9.
  • Some simple variants therefore, include GLAKFLHWL, ILAGFLHWL, ILAKGLHWL, ILAKFGHWL.
  • ILAKFLGWL and ILAKFLHGL subject to the presence of the requisite functional characteristics.
  • the anisan will appreciate that, while the anchor residues and auxiliary residues are relatively conserved in HLA binding, the remainder of the antigen can vary widely, and is probably responsible for the particular antigenic character of the antigen, i.e., it differentiates telomerase from non-telomerase.
  • substitutions include replacing L-amino acids with the corresponding D-amino acids.
  • This rationale moreover can be combined with the foregoing conservative substitution rationales.
  • D-leucine may be substituted for L-isoleucine.
  • these D- amino acid-containing peptides may be prepared which have an inverse sequence, relative to the native sequence.
  • ILAKFLHWL becomes LWHLFKALI.
  • Such "retro-inverso" peptides are expected to have improved properties, such as increased in vivo half-life. This translates into smaller doses and more economically viable production.
  • Multimers can contain multiple copies of the same peptide, or they can be mixed and matched.
  • the multimers can be direct tandem repeats, and may contain short spacers sequences of amino acids (e.g., 2-5 residues) like Gly and/or Pro, or other suitable spacers.
  • Multimers may be any length, but typically will be less than about 100 amino acids.
  • Preferred multimers are less than about 60 amino acids and have between about 2 and 5 copies of peptides of about 8 to about 12 amino acids long. Multimers may also comprise several different telomerase antigens.
  • telomerase antigens may be glycosylated or partially glycosylated according to methods known in the art. They also can be modified with large molecular weight polymers, such as polyethylene glycols. In addition, lipid modifications are preferred because they may facilitate the encapsulation or interaction of the derivative with liposomes. Exemplary lipid moieties useful for this purpose include, but are not limited to, palmitoyl, myristoyl, stearoyl and decanoyl groups or. more generally, any C 2 to C 30 saturated, monounsaturated or polyunsaturated fatty acyl group.
  • telomerase antigen For convenience in making chemical modifications, it is sometimes useful to include in a telomerase antigen one or more amino acids having a side chain amenable to modification.
  • a preferred amino acid is lysine. which may readily be modified at the ⁇ -amino group.
  • Side-chain carboxyls of aspartate and glutamate are readily modified, as are serine, threonine and tyrosine hydroxyl groups, the cysteine sulfhydryl group and the histidine amino group.
  • the introduction of two cysteine residues, at spaced locations in a peptide antigen, may serve to form a structural constraint through a disulfide bridge, which may improve binding to MHC molecules.
  • telomerase antigen within the present invention is a non- peptide "mimetic," i.e., a compound that mimics one or more functional characteristics of the telomerase antigen.
  • Mimetics are generally water-soluble, resistant to proteolysis, and non- immunogenic. Conformationally restricted, cyclic organic peptides which mimic telomerase antigens can be produced in accordance with known methods described, for example, by Saragovi, et al. Science 253: 792 (1991).
  • Telomerase antigens may also be constructed as hybrids (and/or formulated as distinct molecules together in liposomes, as described below) with immune-stimulatory molecules, like cytokines and adjuvants.
  • Interleukin-2 IL-2
  • Other cytokines include GM-CSF, IL-12 and flt-3 ligand.
  • Telomerase antigens may be made as fusion proteins with IL-2, for example, by recombinant DNA or chemical synthetic means, or they may made as chemical conjugates using bi-functional chemical linkers. It is anticipated that such chimeric proteins would possess an increased ability to generate a T-cell-specif ⁇ c response against telomerase.
  • Adjuvants include monophosphoryl lipid A (MPLA), and derivatives thereof, which also may be attached to a telomerase antigen by conventional linkers.
  • Other conventional immune stimulatory molecules include keyhole limpet hemocyanin (KLH).
  • telomerase antigens which would be expected to generate a more specific response, associated with a particular epitope for example. Moreover, these small antigens may be more economically produced.
  • telomerase antigen it is advantageous to identify additional telomerase antigen and further to refine the T-cell antigenicity of telomerase. even down to the epitopic level.
  • One classic method involves proteolytic treatment of the large antigen to derive smaller antigens.
  • fragments of protein antigens can be produced by recombinant DNA techniques and assayed to identify particular epitopes.
  • small peptides can be produced by in vitro synthetic methods and assayed.
  • phagocytic antigen presenting cells or any APC
  • macrophages may be fed large antigens (or portions thereof) and thus act as the starting material for these methods.
  • the MHC class I or class II molecules can be isolated from these starting cells using known methods, such as antibody affinity (MHC-specific antibodies) and chromatographic techniques.
  • Isolated MHC molecules are then treated to release bound peptides. This may be accomplished by treatment with agents that disrupt the interactions between the bound peptide and the MHC molecule, for example, detergent, urea, guanidinium chloride, divalent cations, various salts and extremes in pH.
  • agents that disrupt the interactions between the bound peptide and the MHC molecule for example, detergent, urea, guanidinium chloride, divalent cations, various salts and extremes in pH.
  • the peptides released can be further purified using conventional chromatographic and antibody affinity (using antigen-specific antibody) methodologies.
  • the purified peptides may then be subjected to sequence and structural determinations, using for example peptide sequencing, gas chromatography and/or mass spectroscopy.
  • sequences structures of the most prevalent peptide epitopes associated with class I and/or class II molecules may be determined. Supplied with this sequence/structural information, permutations of the determined sequence can be made, as detailed above, and assayed using known T-cell assays. Rammensee et al., supra, provides extensive methods and guidance related to identifying both class I and class II motifs.
  • Yet another method of generating telomerase antigens may utilize algorithms known in the art for predicting binding sequences. Publicly available comparison programs using these algorithms to compare known peptide sequences to different HLA-binding motifs may be found, for example, at http://www-bimas.dcrt.nih.gov/hla_bind. Different class I and class II binding motifs may be found at that site or in publications like Rammensee et al. and Parker et al, both supra.
  • telomerase-specific antigen as described above, will be useful in formulating telomerase-specific vaccines.
  • Preferred antigens may be associated with lipids, usually either by direct lipid modification of the antigen and/or by liposomal association, as described below.
  • the antigens may be administered as peptides or peptide mimetics, or they may be administered in nucleic acid form.
  • the telomerase antigen is associated with a liposome.
  • Techniques for preparation of liposomes and the formulation of various molecules, including peptides, with liposomes ⁇ e.g., encapsulation or complex formation) are well known to the skilled artisan.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993) and Kim, Drugs 46: 618 (1993).
  • Liposomes are similar in composition to cellular membranes and as a result, liposomes generally can be administered safely and are biodegradable.
  • liposomes may be unilamellar or multilamellar, and can van' in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • agents can be encapsulated in liposomes. Hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s). See, for example, Machy et al, LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et al, (1989) American J. Hosp. Pharm. 46: 1576.
  • Liposomes can adsorb to virtually any type of cell and then release the encapsulated agent.
  • the liposome fuses with the target cell, whereby the contents of the liposome empty into the target cell.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents. Scherphof et al., (1985) Ann. N Y. Acad. Sci. 446: 368.
  • Anionic liposomal vectors have also been examined. These include pH sensitive liposomes which disrupt or fuse with the endosomal membrane following endocytosis and endosome acidification. Among liposome vectors, however, cationic liposomes are the most studied, due to their effectiveness in mediating mammalian cell transfection in vitro.
  • Cationic lipids are not found in nature and can be cytotoxic, as these complexes appear incompatible with the physiological environment in vivo which is rich in anionic molecules. Liposomes are preferentially phagocytosed into the reticuloendothelial system. However, the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means. Classen et al, (1984) Biochim. Biophys. Acta 802: 428.
  • Cationic liposome preparations can be made by conventional methodologies.
  • Suitable liposomes that are used in the methods of the invention include multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUN), medium-sized unilamellar vesicles (MUN), large unilamellar vesicles (LUV), giant unilamellar vesicles ( GUV), multivesicular vesicles (MW), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion
  • the present vaccine formulations may be formulated advantageously with some type of adjuvant.
  • adjuvants are substances that act in conjunction with specific antigenic stimuli to enhance the specific response to the antigen.
  • MPLA for example, has been shown to serve as an effective adjuvant to cause increased presentation of liposomal antigen by the APCs to specific T Lymphocytes. Alving, C.R. 1993. Immunobiol. 187:430-446.
  • adjuvants such as Detox, alum, QS21, complete and/or incomplete Freund's adjuvant, MDP, LipidA and derivatives thereof, are also suitable.
  • Another class of adjuvants include stimulatory cytokines, such as IL-2.
  • the present vaccines may be formulated with IL-2 or IL-2 may be administered separately for optimal antigenic response.
  • IL-2 is beneficially formulated with liposomes.
  • Vaccines may also be formulated with a pharmaceutically acceptable excipient.
  • excipients are well known in the art. but typically should be physiologically tolerable and inert or enhancing with respect to the vaccine properties of the inventive compositions. Examples include liquid vehicles such as sterile, physiological saline. When using an excipient, it may be added at any point in formulating the vaccine or it may be admixed with the completed vaccine composition.
  • Vaccines may be formulated for multiple routes of administration. Specifically preferred routes include intramuscular, percutaneous, subcutaneous, or intradermal injection, aerosol, oral or by a combination of these routes, at one time, or in a plurality of unit dosages. Administration of vaccines is well known and ultimately will depend upon the particular formulation and the judgement of the attending physician.
  • Vaccine formulations can be maintained as a suspension, or they may be lyophilized and hydrated later to generate a useable vaccine.
  • inventive compositions In order to provide greater specificity, thus reducing the risk of toxic or other unwanted effects during / ' /; vivo administration, it is advantageous to target the inventive compositions to the cells through which they are designed to act, namely antigen-presenting cells. This may conveniently be accomplished using conventional targeting technology.
  • Targeting molecules have the characteristic of being able to distinguish to some degree, target APCs over background. non-APCs. Targeting molecules include mannose and the Fc portion of antibodies, and the like, which will target antigen presenting cells. Targeting molecules may be directly associated with telomerase antigens, for example, by chemical conjugation or by fusion protein production, in the case of protein-based targeting sequences.
  • Antibodies and their derivatives include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies including single chain Fv (scFv) fragments, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, epitope-binding fragments, and humanized forms of any of the above.
  • mAbs monoclonal antibodies
  • scFv single chain Fv
  • Fab fragments fragments
  • F(ab') 2 fragments fragments produced by a Fab expression library
  • epitope-binding fragments fragments produced by a Fab expression library
  • Affinity of the antisera for the antigen may be determined by preparing competitive binding curves, as described, for example, by Fisher, Chap. 42 in: Manual of Clinical Immunology, second edition, Rose and Friedman, eds., Amer. Soc. For Microbiology, Washington, D.C. (1980).
  • Fragments or derivatives of antibodies include any portion of the antibody which is capable of binding an APC target molecule, typically a surface antigen.
  • Antibody fragments specifically include F(ab')-,, Fab, Fab' and Fv fragments. These can be generated from any class of antibody, but typically are made from IgG or IgM. They may be made by conventional recombinant DNA techniques or. using the classical method, by proteolytic digestion with papam or pepsm. See CURRENT PROTOCOLS IN IMMUNOLOGY, chapter 2, Coligan et al . eds., (John Wiley & Sons 1991 -92).
  • F(ab')2 fragments are typicalh about 1 10 kDa (IgG) or about 150 kDa (IgM) and contain two antigen-bmding regions, joined at the hmge by disulfide bond(s) Virtually all, if not all, of the Fc is absent in these fragments.
  • Fab' fragments are typically about 55 kDa (IgG) or about 75 kDa (IgM) and can be formed, for example, by reducing the disulfide bond(s) of an F(ab')2 fragment. The resulting free sulfhydryl group! s) may be used to conveniently conjugate Fab' fragments to other molecules, such as telomerase antigens or adjuvant molecules.
  • Fab fragments are monovalent and usually are about 50 kDa (from any source).
  • Fab fragments include the light (L) and heavy (H) chain, vanable (V_ and VJJ, respectively) and constant (C_ CH, respectively) regions of the antigen-bmding portion of the antibody.
  • the H and L portions are linked by one or more intramolecular disulfide b ⁇ dges
  • Fv fragments are typically about 25 kDa (regardless of source) and contain the variable regions of both the light and heavy chains (V_ and VJJ, respectively)
  • V_ and VJJ chains are held together only by non-covalent interactions and, thus, they readily dissociate. They do, however, have the advantage of small size and they retain the same binding properties of the larger Fab fragments. Accordingly, methods have been developed to crosslink the V_ and VJJ chains, using, for example, glutaraldehyde (or other chemical crosshnkers), lntermolecular disulfide bonds (by incorporation of cystemes) and peptide linkers. The resulting Fv is now a single chain (i.e., scFv).
  • chime ⁇ c antibodies also include "chime ⁇ c antibodies” (Morrison et al , Proc. Natl. Acad. Sci., 81 :6851-6855 ( 1984), Neuberger et al , Nature, 312 604-608 (1984); Takeda et ⁇ /., Nature, 314:452-454 (1985)).
  • chimeras are made by splicing the DNA encoding a mouse antibody molecule of appropriate specificity with, for instance, DNA encoding a human antibody molecule of appropriate specificity.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Recombinant molecules having a human framework region and murine complementarity determining regions also are made using well-known techniques. These are also known sometimes as "humanized” antibodies and they and chimeric antibodies or antibody fragments offer the added advantage of at least partial shielding from the human immune system. They are, therefore, particularly useful in therapeutic in vivo applications.
  • naked DNA vaccines or vaccines comprising RNA have broad applicability. They may be employed, for example, as anti-cancer vaccines (Scheurs et ai, Cancer Res. 58: 2509-14 (1998); Hurpin et al, Vaccine 16: 208-15 (1998)) and anti-viral vaccines (Bohm et al. Vaccine 16: 949-54 (1998); Lekutis et al, J. Immunol. 158: 4471-77 (1997)), among others. Naked DNA vaccines have been shown to elicit both class I- (Scheurs , et al, supra; Bohm et al, supra; Hurpin et al. supra) and class Il-restricted responses (Lekutis et al, supra; Manickan et al., J. Leukoc. Biol. 61 : 125-32 (1997))).
  • any of the foregoing telomerase antigens may be administered as a naked DNA vaccine.
  • These vaccines will comprise a nucleic acid vector that encodes a telomerase antigen under the control of transcription and translation signals that operate in a mammal, preferably a human. They may be administered associated with or encapsulated by (usually cationic) liposomes, as detailed above, or they may be administered in any other physiologically tolerable excipient.
  • telomerase antigens may be used as a conventional vaccine directly to induce an immune response against telomerase.
  • the antigen may be liposome-associated, as indicated above.
  • telomerase is expressed at high levels in cancer cells
  • the present antigens and vaccines are particularly suited for methods of treating and/or preventing cancer.
  • a representative method involves administering to a cancer patient an effective amount of one or more of the foregoing telomerase antigens, which may be formulated as a vaccine. Again, smaller peptide antigens are preferred.
  • telomerase antigens and vaccines will be particularly useful in ex vivo techniques.
  • these techniques entail isolating cells from a patient, contacting them with a telomerase antigen (or a vaccine, including nucleic acid vaccines) and administering the contacted cells back to the patient.
  • a telomerase antigen or a vaccine, including nucleic acid vaccines
  • the cells may be taken from one patient for administration to another.
  • autologous or compatible antigen presenting cells usually dendritic cells or peripheral blood lymphocytes
  • telomerase antigen usually dendritic cells or peripheral blood lymphocytes
  • telomerase-primed cells may then be transferred in beneficial amounts into a patient in need of therapy or prophylaxis.
  • the ex vivo priming step may be accomplished using lipid- and or liposome-associated small peptide antigen.
  • T-cells so generated may be adoptively transferred in beneficial amounts to a patient in need.
  • art-recognized techniques for adoptive T-cell transfer therapy see Bartels, et al. Annals of Surgical Oncology, 3(1):67 (1996), which is hereby incorporated by reference.
  • IL-2 Co-treatment with other immunostimmulatory, listed above, is also contemplated.
  • IL-2 for example, may be administered concurrently, separately or in a combined formulation, or it may be administered in an alternative dosing regime with the telomerase antigen or vaccine.
  • the IL-2 is formulated with liposomes.
  • any of the foregoing antigens or vaccines may be used in conjunction with known anti-cancer agents.
  • One example includes MUC-1-based therapeutics. Numerous additional examples of these are well-known in the art
  • Conventional chemotherapeutic agents include alkylating agents, antimetabolites, various natural products (e.g., vinca alkaloids, epipodophyllotoxins, antibiotics, and amino acid- depleting enzymes), hormones and hormone antagonists.
  • Specific classes of agents include nitrogen mustards, alkyl sulfonates, nitrosoureas.
  • triazenes triazenes, folic acid analogues, pyrimidine analogues, purine analogs, platinum complexes, adrenocortical suppressants, adrenocorticosteroids, progestins, estrogens, antiestrogens and androgens.
  • Some exemplary compounds include cyclophosphamide, chlorambucil, methotrexate, fluorouracil, cytarabine, thioguanine, vinblastine, vincristine, doxorubincin, daunorubicin, mitomycin, cisplatin, hydroxyurea, prednisone, hydroxyprogesterone caproate, medroxyprogesterone, megestrol acetate, diethyl stilbestrol, ethinyl estradiol. tomoxifen, testosterone propionate and fluoxymesterone .
  • a therapeutically or prophylactically beneficial or effective amount is an amount sufficient to induce a clinically relevant telomerase-specific T-cell response, as defined above. Clinical relevance can be determined by clinician.
  • Administration may be by any number of routes, including parenteral and oral.
  • Cell-based vaccines are advantageously administered intravenously.
  • Other vaccines typically will be administered intramuscularly, intradermally, subcutaneously or orally.
  • the skilled artisan will recognize that the route of administration will vary depending on the nature of the vaccine formulation. Determining the optimal route of vaccination may be determined empirically and is well within the level of ordinary skill in the art.
  • Nucleic acid vaccines may also be administered by a variety of routes, the optimal route being determined empirically. For instance, some antigens have been found to elicit a superior cytotoxic response when administered intravenously. Hurpin et al, supra. For a superior immune response to oral administration, it may be advantageous to co- administer with the vaccine a mucosal adjuvant, like cholera toxin or cationic lipids. Ethchart et al., J. Gen. Virol. 78: 1577-80 (1997). Intramuscular, intradermal and subcutaneous administration are also preferred.
  • This example illustrates the use of the telomerase-specific peptides to generate an antigen-specific cytotoxic T-cell response.
  • Peptides were selected using the HLA peptide search program at http://www-bimas.dcrt.nih.gov. HLA-A2 specificity was selected, with default parameters. Three of the top twenty scores were synthesized and tested. These peptides has the sequences: RLVDDFLLV, ELLRSFFYV and ILAKFLHWL.
  • the bulk liquid composition of liposomes consisted of dipalmitoyl phosphatidyl choline (DPPC), cholesterol (Choi) and dimyristoyl phosphatidyl glycerol (DMPG) in a molar ratio of 3 : 1 :0.25 and contained Lipid A at a concentration of 1 % (w/w) of bulk lipid.
  • Synthetic telomerase peptides were present in the aqueous phase during liposome formation at a concentration of 0.7 mg/ml, and approximately 28% of the input peptide was captured within the liposome structures.
  • the formulated product contained 2 mg of bulk lipid, 20 ⁇ g Lipid A and about 20 ⁇ g of peptide per injected dose of 100 ⁇ l.
  • lipids and Lipid A were dissolved in chloroform/methanol (methanol was used initially to solubilize DMPG).
  • the lipid mixturefor each 4 ml preparationof liposomes consisted of 64 mg DPPC, 1 1 mg Choi, 5 mg DMPG, 0.8 mg Lipid A in 12 ml of chloroform/methanol in amolar ratio of 3: 1 :0.25 at a final lipid concentration of 30 mM.
  • Each 12 ml of the lipid mixture was dried to a film by rotary evaporation at 53°C in a 250 ml round bottom flask, and residual solvent was removed under high vacuum.
  • the lipid film was hydrated by addition of 4 ml PBS containing the peptide and slow rotation of the flasks at 53°C followed by 5 cycles of vortexing and warmthing to 53°C.
  • Liposome structures were reformed to a more uniform size by a series of 5 freeze/thaw cycles consisting of freezing in a dry ice bath, thawing, warming to 41 °C and vortexing before beginning the next cycle. Liposomes then were collected by ultracentrifugation at 1500,000 x g at 4°C for 20 minutes, washed twice by addition of PBS and ultracentrifugation again. Liposomes were finally reconstituted to the desired volume.
  • Cytokines In order to promote CTL generation, human recombinant cytokines, IL-12 (R&D Systems, Minneapolis. MN), IL-7 (Intermedico, Markham, Ontario) were diluted in serum-free AIM-V media (Life Technologies) just prior to use.
  • Cvtotoxic T lymphocyte assays For the CTL assay, three (HLA.A2 + ) normal donors' PBLs were used. The T-cells were grown for five weeks in bulk cultures as described above. At the end of two weeks, live T-cells were harvested from flasks and counted. The targets were mutant T2 cells. Houbiers et al, Eur. J. Immunol 23:2072-2077 (1993); Stauss et al., Proc. Natl. Acad. Sci. U.S.A. 89:7871-7875 (1992).
  • telomerase peptide-mediated upregulation of HLA.A2 expression on T2 cells was examined using the HLA-A2-associated peptides ILAKFLHWL (BPl-187), RLVDDFLLV (BPl-190), and ELLRSFFYV (BPl-191) using known methods. Townsend et al, Nature 346:476 (1989). T2 cells were loaded overnight at 37°C in 7% CO 2 with various the telomerase synthetic peptides at 200 ⁇ M in presence of 8 ⁇ g exogenous ⁇ 2 microglobulin. Houbiers et al, supra; Stauss et al, supra.
  • the peptide-loaded T2 target cells were loaded with Cr (using NaCrO 4 ) for 90 minutes and washed. CTL assays were performed as previously described. Agrawal et al, J. Immunol. 156:2089 (1996). Percent specific killing was calculated as: experimental release -
  • the peptide- fed T2 cells were washed once in cold PBS containing 1% BSA followed by addition of 1 ⁇ g of anti-A2 monoclonal antibody, MA2.1 or a control antibody and incubated for 45 minutes on ice. Cells were then washed and a secondary antibody, goat anti-mouse IgG (H+L)-FITC labeled (Southern Biotech) was added for 30 minutes on ice.
  • T-cells stimulated with autologous APCs pulsed with liposomal telomerase peptide were determined.
  • the source of T-cells was PBLs from three HLA.A2 * donors.
  • Target T2 cells HLA.A2 + ) were loaded with the telomerase peptide indicated above.
  • the negative control was T2 cells, and the positive control was the 10-mer flu peptide FLPSDYFPSV, which strongly upregulates HLA.A2 expression on T2 cells.
  • Figure 1 shows FACS analysis of cells treated according to the foregoing methods
  • panel A shows the negative control
  • Panel B is the positive control, showing a 227% increase in median channel intensity of A2 expression as compared to the negative control
  • Panel C is the experimental with BP1-187, and shows a 396% increase in median channel intensity of A2 expression as compared to the negative control.
  • Panels D and E show the results with BPl-190 and BPl-191, respectively yielding 0% and 6% increases over the negative control.
  • Table I shows specific killing of targets by telomerase antigen-primed (BPl-
  • cytotoxic T-cells The negative control was SIINFEKL.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des antigènes des lymphocytes T spécifiques de la télomerase utiles dans la génération de réponses des lymphocytes T à la télomérase. Les formulations d'antigènes de la télomérase comme vaccins servent à traiter et prévenir le cancer grâce à des techniques in vivo ou ex vivo.
EP00920996A 1999-04-09 2000-04-07 Vaccin anticancereux specifique de la telomerase Withdrawn EP1171612A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US12853999P 1999-04-09 1999-04-09
US128539P 1999-04-09
PCT/IB2000/000610 WO2000061766A2 (fr) 1999-04-09 2000-04-07 Vaccin anticancereux specifique de la telomerase

Publications (1)

Publication Number Publication Date
EP1171612A2 true EP1171612A2 (fr) 2002-01-16

Family

ID=22435817

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00920996A Withdrawn EP1171612A2 (fr) 1999-04-09 2000-04-07 Vaccin anticancereux specifique de la telomerase

Country Status (5)

Country Link
EP (1) EP1171612A2 (fr)
JP (1) JP2002541811A (fr)
AU (1) AU781376B2 (fr)
CA (1) CA2368967A1 (fr)
WO (1) WO2000061766A2 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
AU734089B2 (en) 1996-10-01 2001-06-07 Geron Corporation Human telomerase catalytic subunit
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
CA2347067C (fr) * 1998-03-31 2013-09-17 Geron Corporation Vaccin a base de cellule dendritique contenant de la transcriptase inverse de la telomarase pour le traitement du cancer
EP1257284A4 (fr) * 2000-02-15 2005-12-21 Univ California Vaccin universel et methode de traitement du cancer utilisant la transcriptase inverse de la telomerase
JP2004527449A (ja) 2000-02-15 2004-09-09 リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア テロメラーゼ逆転写酵素を用いる癌を治療するための万能ワクチンと方法
GB0031430D0 (en) 2000-12-22 2001-02-07 Norsk Hydro As Polypeptides
JP2005525992A (ja) * 2001-06-25 2005-09-02 イサム・リサーチ・デベロツプメント・カンパニー・オブ・ザ・ヘブルー・ユニバーシテイ・オブ・エルサレム 生物学的物質を充填した小胞の調製法およびそれらの様々な使用
DE102006025146A1 (de) * 2006-05-30 2007-12-06 Charité - Universitätsmedizin Berlin Peptide zur differentiellen Modulation der proteasomalen Aktivität
EP1887084A1 (fr) 2006-08-10 2008-02-13 International Investment and Patents SA Plasmides avec l'action immunologique
WO2014079464A1 (fr) * 2012-11-21 2014-05-30 Sherif Salah Abdul Aziz Nouvelles compositions enzymatiques pour le traitement d'une infection par le virus de l'immunodéficience humaine (vih)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU734089B2 (en) * 1996-10-01 2001-06-07 Geron Corporation Human telomerase catalytic subunit
ATE394474T1 (de) * 1997-05-08 2008-05-15 Biomira Inc Verfahren zur gewinnung von aktivierten t-zellen und mit antigen inkubierten antigenpräsentierenden zellen
CA2347067C (fr) * 1998-03-31 2013-09-17 Geron Corporation Vaccin a base de cellule dendritique contenant de la transcriptase inverse de la telomarase pour le traitement du cancer
US7030211B1 (en) * 1998-07-08 2006-04-18 Gemvax As Antigenic peptides derived from telomerase
EP1126872B1 (fr) * 1998-10-29 2006-12-13 Dana-Farber Cancer Institute, Inc. IMMUNOTHERAPIE ET DIAGNOSTIC DU CANCER AU MOYEN D'ANTIGENES UNIVERSELS ASSOCIES A DES TUMEURS TELS QUE hTERT

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0061766A2 *

Also Published As

Publication number Publication date
WO2000061766A2 (fr) 2000-10-19
WO2000061766A3 (fr) 2001-03-08
JP2002541811A (ja) 2002-12-10
CA2368967A1 (fr) 2000-10-19
AU781376B2 (en) 2005-05-19
AU4139400A (en) 2000-11-14

Similar Documents

Publication Publication Date Title
US20220041655A1 (en) Class i mhc phosphopeptides for cancer immunotherapy and diagnosis
US6600012B1 (en) Lipid-modified muc-1 derivatives
CA2665816C (fr) Vaccins a multiples epitopes specifiques a un antigene
EP3421047B1 (fr) Procédé d'amélioration de l'efficacité d'un vaccin à base de survivine pour le traitement du cancer
AU781376B2 (en) Telomerase-specific cancer vaccine
JP2003521245A (ja) ペプチドおよび核酸組成物を使用する、前立腺癌抗原に対する細胞性免疫応答の誘導
EP0749315A1 (fr) Induction de lymphocytes t cytotoxiques antitumoraux chez l'homme au moyen d'epitopes de peptides synthetiques
JP2003521243A (ja) ヘテロクリティックアナログおよび関連方法
JP2011502484A (ja) Ｈｌａ−ｄｒ結合性ペプチドおよびそれらの使用
Berger et al. Tumor‐specific peptides in cutaneous T‐cell lymphoma: Association with class I major histocompatibility complex and possible derivation from the clonotypic T‐cell receptor
JP2003516344A (ja) Ｈｌａクラスｉａ２腫瘍関連抗原ペプチドおよびワクチン組成物
JP2010090167A (ja) Ｈｌａ結合ペプチドおよびそれらの用途
US6312718B1 (en) Vaccine for B-cell malignancies
CA2404327A1 (fr) Adjuvants lipopeptidiques
MXPA03006581A (es) Vacunas subunitarias con superporciones a2.
WO1997029769A9 (fr) Vaccin contre des malignites affectant les lymphocytes b
CA3124905A1 (fr) Vaccins ciblant des neo-epitopes
WO2022074098A1 (fr) Méthode d'identification de néoantigènes du cancer
JP2004529881A (ja) 合成ワクチン剤
JP2018508181A (ja) Ｔｈ１細胞のためのＧＰＣ３エピトープペプチドおよびこれを含有するワクチン
AU4111999A (en) Induction of anti-tumor cytotoxic T lymphocytes in humans using synthetic peptide epitopes
MXPA98007512A (en) Peptides with increased affinity by molecules from locus to leucocitus huma

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20011107

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20050406

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060325