WO2001015728A1 - Procede d'activation de lymphocytes t cytotoxiques (ctls) in vivo: composition comprenant un anticorps anti cd40 (ou cd40l ou une proteine liant le cd40) et un antigene - Google Patents

Procede d'activation de lymphocytes t cytotoxiques (ctls) in vivo: composition comprenant un anticorps anti cd40 (ou cd40l ou une proteine liant le cd40) et un antigene Download PDF

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Publication number
WO2001015728A1
WO2001015728A1 PCT/CA2000/000960 CA0000960W WO0115728A1 WO 2001015728 A1 WO2001015728 A1 WO 2001015728A1 CA 0000960 W CA0000960 W CA 0000960W WO 0115728 A1 WO0115728 A1 WO 0115728A1
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molecule
binding
antigen
cells
activating
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PCT/CA2000/000960
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English (en)
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Pamela S. Ohashi
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University Health Network
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the present invention relates to a novel method modulating the immune response in an individual by activating antigen specific cytotoxic T- lymphocytes (CTLs) in vivo to target and destroy antigen expressing cells.
  • CTLs cytotoxic T- lymphocytes
  • the present invention also relates to the use of this method to treat infectious diseases and cancer, and to improve the efficacy of vaccines.
  • T-helper T ⁇ cells
  • CTL cytotoxic T- lymphocytes
  • CTL cells on the other hand "kill” or lyse specific target cells, bacteria or viruses [Immunobiology: The Immune System in Health and Disease, supra]
  • APC professional antigen presenting
  • APCs A number of different cells have been identified as APCs including mononuclear phagocytes, dendritic cells (DCs), Langerhan cells of the skin, activated B and T lymphocytes and endothelial cells [Banchereau, J. and R.M. Steinman, supra].
  • DCs dendritic cells
  • Langerhan cells of the skin activated B and T lymphocytes and endothelial cells
  • DCs are about 5-10 fold more efficient at presenting allo-antigen than activated B cells.
  • DCs can be obtained as stem cell derived DCs either from bone marrow or peripheral blood stem cell populations, or peripheral blood derived DCs.
  • Dendritic cells prepared from bone marrow cells [ WO 98/01538; Inaba, K. et al, J. Exp. Med.
  • DCs are logical targets for manipulation in many clinical situations that involve T cells, such as transplantation, allergy, autoimmune disease, resistance to infection and to tumors, immunodeficiency, and vaccines.
  • DCs instead of inducing host resistance, provide a safe haven for several viruses.
  • DC cells may host latent cytomegalovirus, and Kaposi's virus (KSHV) may likewise be sheltered in DCs of patients with multiple myeloma.
  • KSHV Kaposi's virus
  • the consequences of DC infection are more overt, especially because upon interacting with memory T cells and activated T cells, they sustain the production of many HIV-1, SIV and measles particles.
  • CD80 and CD86 have reduced T-cell stimulatory activity.
  • tumors may secrete factors, such as IL-10, TGF- ⁇ and vascular endothelial growth factor (VEGF), that reduce DC development and function. Nonetheless, the more DCs that infiltrate the tumor, the better the prognosis.
  • VEGF vascular endothelial growth factor
  • the immune repertoire also carries tumor- reactive T cells, especially CTLs, but there is little evidence that these T cells are being activated in vivo. However, when tumor antigens are applied to DCs ex vivo and these DCs are then reinfused, specific immunity ensues.
  • DCs are the only cells that efficiently present inactivated virus, and therefore the efficacy of the new generation of attenuated vaccines could be improved by specific targeting to DCs.
  • the immune response can also be boosted by immunization with DNA vaccines; even though the DNA is primarily expressed in weak APCs, like dermal and muscle cells, DNA vaccines can activate both CD4- and CD8-bearing cells.
  • DCs isolated from vaccinated animals both express the vaccine DNA and present the corresponding peptides to specific T cells. The frequency of transfected DCs is low and greater efficiency should prove valuable.
  • APCs, especially DCs offer a tremendous potential for modulating immune responses to a variety of antigens.
  • a culture period of 35 days under optimized conditions using seven different cytokines is necessary to obtain 1.7xl0 7 DCs from a starting population of lxl 0 6 mononuclear cells of a peripheral stem cell preparation [WO 98/01538; Romani, N., J. Immunol. Meth.196:137-151(1994)].
  • the yield of DC in that study was about 3-8xl0 7 DCs from 40-100ml peripheral blood after 5-8 days of culture, but growth ceased at that time and no further expansion was possible.
  • Another limitation of generating DCs from bone marrow (BM) or peripheral blood (PB) is the decreased ability of long term cultured cells to function as APC.
  • Another a major drawback is that DCs cannot be cryopreserved. Similar problems are presented by other APCs.
  • APCs have been isolated and purified, they have to be activated in vitro before they can be used to prime resting CTL-cells in vivo to generate immune responses [WO 96/18409; WO 98/23728; WO 97/26000; WO 98/01145].
  • Typical models of generating immune reaction involve multiple injections of activated APCs over a course of administration over extended periods of time [WO 98/01538].
  • General protocols require an initial administration and subsequent boosts at intervals ranging from one to two weeks to up to several months.
  • One preferred protocol is to administer the APCs approximately at one to two week intervals, five to ten times.
  • the present invention provides a novel method of treating a variety of diseases by in vivo modulation of the immune response(s) in a subject.
  • This invention is based on the finding that administration of an immunogenic molecule, such as a peptide antigen, to a subject in vivo with a molecule that binds to and activates cell surface receptor molecule, CD40, that is present on the antigen presenting cell (APC), activates the APC into an efficient antigen presenting cell.
  • the activated APC prime cytotoxic T-lymphocyte (CTL) effector function in vivo turning it into efficient killer cells.
  • CTL cytotoxic T-lymphocyte
  • diseases which can be treated using the in vivo treatment methods of the invention include, but are not limited to prostate cancer, hepatitis A hepatitis B, hepatitis C, HSV, CMV, AIDS, bacterial diseases, parasitic diseases, renal cell carcinoma, cervical carcinoma, leukemias, lymphomas, condyloma accuminatum, breast cancer, ovarian cancer, and lung carcinoma.
  • One aspect of the invention contemplates methods for modulating the immune response in a subject by administering to the subject in vivo an immunogenically effective dose of an antigenic molecule along with therapeutically effective dose of a CD40 binding-CD40 activating molecule to activate the antigen presenting cell (APC) into priming cytotoxic T-lymphocyte (CTL) mediated antigen specific immune response.
  • Antigens may be selected from the proteins, polypeptides, peptide fragments, polysaccharides, lipopolysaccharides, and analogs thereof
  • Exemplary CD40 binding-CD40 activating molecules include anti-CD40 antibody, CD40 ligand (CD40L) and CD40 binding proteins.
  • Antigens may also be delivered using a recombinant vector containing a nucleic acid molecule encoding said peptide antigen, which vector is capable of expressing the peptide antigen in vivo.
  • a second aspect of the invention is directed to a method of priming activated cytotoxic T-lymphocyte (CTL) in vivo in a subject diagnosed with a tumor to specifically target and kill the tumor cells in the subject.
  • the method comprises: (a) administering to the subject diagnosed with the tumor an immunogenic dose of an antigenic molecule expressed by the tumor, thereby generating a population of APCs displaying said antigenic molecule on their cell surfaces; (b) administering a therapeutically effective dose of a CD40 binding-CD40 activating molecule that specifically binds to and activates the APC displaying the antigenic molecule, thereby activating in an antigen-specific manner the cytotoxic T- lymphocytes (CTLs) to target and destroy tumor cells displaying the antigen.
  • CTLs cytotoxic T- lymphocyte
  • the antigenic molecule is administered to the subject concomitantly with a therapeutically effective dose of a CD40 binding- CD40 activating molecule.
  • the subject is administered the antigenic molecule concomitantly with a CD40 binding-CD40 activating molecule, and an immunomodulatory dose of one or more cytokines or co-stimulatory molecules with the goal of optimizing the activation and proliferation of antigen-specific CTL cells in vivo in the individual.
  • the CD40 binding-CD40-stimulating molecules are selected from a group comprising anti-CD40 antibodies, CD40 Ligand (CD40L), and CD40-binding proteins.
  • Exemplary immunomodulatory cytokines and co-stimulatory molecules include, but are not limited to IL-1, IL-2,
  • LL-16 ⁇ L-17, EL- 18, G-CSF, GM-CSF, CSF-1, MGDF, interferons, TNF-family of ligands, LIF, SCF, EPO, TRANCE, Flt3 ligand (Flt3L), RANKL, and the B7 family of co-stimulatory molecules.
  • the antigenic molecule is administered to the subject prior to the administration of a therapeutically effective dose of a CD40 binding-CD40 activating molecule.
  • a therapeutically effective dose of CD40 binding-CD40 activating molecule is administered. This results in the optimal activation and proliferation of antigen-specific CTL cells in vivo in the subject.
  • the antigenic molecule is administered to the subject first, followed by the sequential administration of a CD40 binding-CD40 activating molecule, and one or more cytokines or co-stimulatory molecules.
  • the CD40 binding-CD40 stimulating molecules is selected from a group comprising anti-CD40 antibodies, CD40 Ligand (CD40L), and CD40-binding proteins.
  • CD40L CD40 Ligand
  • Exemplary immunomodulatory cytokines and costimulatory molecules are as set out above.
  • the antigenic molecule is delivered to the subject using a recombinant vector containing a nucleic acid encoding the tumor specific peptide antigen, which vector is capable of expressing said peptides in vivo.
  • the polypeptides synthesized from the tumor specific nucleic acid molecule are processed within the APC and presented on the cell surface to CTL to initiate "antigen specific" immune response.
  • Many tumor cell specific proteins, polypeptides, analogs, and subsets thereof are known in the art and may be used to practice the invention.
  • a second aspect contemplates a method of generating a population of antigen-specific cytotoxic T-lymphocyte (CTL) in vivo in a subject afflicted with an infectious disease caused by a virus, bacteria, fungus, parasite or other infectious agents.
  • CTL cytotoxic T-lymphocyte
  • the method comprises: (a) administering to the subject afflicted with an infectious disease, an immunogenic dose of an antigenic molecule specific to the infectious disease agent, thereby generating a population of APCs displaying said antigenic molecule on their cell surfaces; (b) administering a therapeutically effective dose of a CD40 binding-CD40 activating molecule(s) that specifically binds to and activates the APC displaying the antigenic molecule, thereby activating in an antigen-specific manner the cytotoxic T- cells (CTLs) to target and destroy cells or infectious agents displaying the antigen.
  • the antigenic molecule is administered to the subject concomitantly with a therapeutically effective dose of a CD40 binding- CD40 activating molecule.
  • the subject is administered the antigenic molecule concomitantly with a CD40 binding-CD40 activating molecule, and an immunomodulatory dose of one or more cytokines or co-stimulatory molecules.
  • a CD40 binding-CD40 activating molecule is selected from a group comprising anti-CD40 antibodies, CD40 Ligand (CD40L), and CD40-binding proteins.
  • immunomodulatory cytokines and co- stimulatory molecules include, but are not limited to EL-1, IL-2, IL-3, IL-4, D -5, ⁇ L-6, IL-7, ⁇ L-8, ⁇ L-9, ⁇ L-IO, ⁇ L-I I, iL-12, ⁇ L-13, ⁇ L-H, ⁇ L-15, ⁇ L-16, IL-I 7, IL-
  • G-CSF G-CSF
  • GM-CSF CSF-1
  • MGDF interferons
  • TNF-family of ligands SCF
  • EPO EPO
  • LIF LIF
  • TRANCE Flt3 ligand
  • RANKL RANKL
  • B7 family of co-stimulatory molecules B7 family of co-stimulatory molecules.
  • an immunogenically effective dose of an antigenic molecule is administered first to a subject prior to the administration of a therapeutically effective dose of a CD40 binding-CD40 activating molecule.
  • a therapeutically effective dose of CD40 binding-CD40 activating molecule is administered. This results in the enhanced activation and proliferation of antigen-specific CTL cells in vivo in the individual.
  • the antigenic molecule is administered to the subject prior to the administration of a CD40 binding-CD40 activating molecule, and an immunomodulatory dose of one or more cytokines or a costimulatory molecule(s).
  • the CD40 binding-CD40 activating molecules are selected from a group comprising anti-CD40 antibodies, CD40 Ligand (CD40L), and CD40-binding proteins.
  • Exemplary immunomodulatory cytokines and costimulatory molecules are selected from the group as set out above.
  • the tumor cell specific antigenic molecule can also be delivered to the subject using a recombinant vector containing a nucleic acid encoding the tumor specific peptide antigen, which vector is capable of expressing said peptides.
  • the polypeptides synthesized from the tumor specific nucleic acid molecule are processed within the APC and presented on the cell surface to CTL to initiate "antigen specific" immune response.
  • Many infectious disease agent specific antigens including proteins, polypeptides, polysaccharides, lipopolysaccharides analogs, and subsets thereof are known in the art and may be used to practice the invention.
  • a third aspect of the present invention is directed to a method for enhancing the efficacy of vaccines in vivo in a subject by activating a professional antigen presenting cell (APC) in vivo to prime cytotoxic T-lymphocyte (CTL) mediated antigen-specific immune response, the method comprising: a) administering to the subject an immunogenically effective dose of a vaccine; and b) administering a therapeutically effective dose of CD40 binding-CD40 activating molecule that specifically binds to and activates the antigen presenting cells (APCs) displaying in vivo the antigenic molecule of step a), thereby priming the activation of cytotoxic T-lymphocyte (CTL) mediated antigen-specific immune response.
  • the vaccine comprises all or part of an infectious disease agent.
  • the vaccine comprises one or more tumor specific antigens.
  • the vaccine molecule (e.g., protein, polypeptide, fragments and analogs thereof), may be administered to the subject by way of a recombinant vector containing a nucleic acid molecule encoding vaccine antigens, which vector is capable of expressing the antigens in vivo.
  • a fourth aspect of the invention discloses pharmaceutical compositions for efficient delivery of an immunogenically effective dose of an antigenic molecule, a therapeutically effective dose of a CD40 binding-CD40 activating molecule, and an immunomodulatory dose of a cytokine or co-stimulatory molecule in a pharmaceutically acceptable carrier, adjuvant or excipient that results in the optimal activation of antigenic-specific CTL.
  • the pharmaceutical compositions are suitable for parenteral, nasal, oral, and /or topical delivery.
  • Figure 1 shows that treatment of mice with peptide p33 and anti- CD40 in vivo leads to an increase in inflammatory CD8 + T cells.
  • FIG. 2 shows the activation of dendritic cells (DC) in vivo in response to administration of anti-CD40 antibody.
  • Figure 3 shows that dendritic cells (DCs) isolated from the LCMV-
  • GP, p33 and anti-CD40 antibody treated animals are potent antigen presenting cells (APCs).
  • FIG. 4 shows that anti-CD40 treatment enhances IFN- ⁇ production.
  • Figure 5 shows that treatment with anti-CD40 antibody prevents clonal deletion in anti CD40-antibody treated TCR transgenic mice.
  • Figure 6 shows that the CD40 stimulation by anti-CD40 antibody prevents the induction of tolerance.
  • the invention described herein is directed to a novel method of modulating the immune system response and activating cytotoxic T-lymphocytes (CTLs) in vivo. Also contemplated is the use of this novel method as a treatment regime for a variety of diseases including cancer, viral diseases, bacterial diseases, and parasitic diseases.
  • CTLs cytotoxic T-lymphocytes
  • cytotoxic T-lymphocytes refers to a subset of T-lymphocytes that have the ability to bind to a defined peptide/antigen and destroy the cell expressing the antigen. Such lymphocytes recognize target cells by engaging class I major histocompatibility molecules that present the specific peptide ligands..
  • helper T-lymphocytes refers to a subset of T-lymphocytes that amplifies the activity of B lymphocytes, other T lymphocytes, and other antigen presenting cells. Once a helper T lymphocyte recognizes an antigen, it divides and its progeny start to synthesize and secrete a variety of lymphokines. These cytokines cause B lymphocytes to divide and differentiate into plasma cells, enhance CTL function and cause antigen presenting cells to be recruited and activated.
  • B lymphocyte refers to a subset of lymphocytes that synthesize immunoglobulins.
  • the term "antigen presenting cells” or the term “professional antigen presenting cells” refers to a group of highly specialized cells that can process antigens and display the antigens as peptide fragments on the cell surface together with molecules required for lymphocyte activation.
  • the main antigen presenting cells for T-lymphocytes are dendritic cells, macrophages, and B- lymphocytes, while the main antigen presenting cells for B-cells are follicular dendritic cells.
  • protein is used to designate a molecule of one or more polypeptide chains, each composed of a linear chain of greater than 50 amino-acid residues connected one to another by peptide bonds between the alpha- amino and carboxy groups of adjacent residues.
  • peptides and “polypeptides” are used interchangeably to designate a series of no more than about 50 amino acid residues connected one to another as in a protein.
  • peptide fragments and “polypeptide fragments” are used to refer to short strands of amino acids generated by chemical synthesis or by proteolytic cleavage of proteins or polypeptides.
  • the term "antigen” refers to a substance primarily a protein, polypeptide, polysaccharides, or lipopolysaccharides that upon introduction into a vertebrate animal, stimulates an immune response.
  • epitope refers to the antigenic determinant recognized by T-cells and to the antigenic determinant to which the site within an immunoglobulin Fab binds.
  • allo-antigen refers to proteins or polypeptides that are products of different alleles of the same gene.
  • an "immunogenic molecule” refers to protein, polypeptides, peptide fragments, and their analogs that contain allele-specific motif such that the peptide will bind to the major histocompatibility complex "MHC" allele on APCs and be capable of inducing an immune response.
  • the term “vaccine” refers to a composition containing an antigenic molecule derived from an infectious disease agent or tumor cells, or is a suspension containing dead or attenuated infectious disease agent that is introduced into a subject for the deliberate induction of adaptive immunity to the antigenic molecule, tumor cells or infectious disease agent
  • the term “major histocompatibility complex (MHC)” refers to a cluster of genes that encode MHC class I molecules that present peptides generated in cytosol to CD8 + T cells , and the MHC class II molecules or proteins that present peptides degraded in cellular vesicles to CD4 + T cells. The MHC also encodes proteins involved in antigen processing and host defense.
  • HLA human leukocyte antigen
  • Individual loci are designated by upper case letters, as in HLA-A.
  • CD40 binding-CD40 activating molecule(s) refers to anti CD40-binding antibodies, CD40 ligand and CD40 binding molecules, which are capable of binding to CD40 molecules on the surface of "professional antigen presenting cells” (APCs) such as dendritic cells (DCs) and activating them by inducing their proliferation and effector function to optimize their ability to function as antigen presenting cells.
  • APCs professional antigen presenting cells
  • DCs dendritic cells
  • cytokine refers to a group of small proteins involved primarily in communication between cells of the immune system.
  • the term includes but is not limited to interleukins, interferons, lymphokines, and tumor necrosis factor and tumor necrosis factor receptor family of molecules.
  • antibody refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as F(ab), F(ab 2 ), Fv, and other fragments which retain the antigen-binding function of the parent antibody.
  • monoclonal antibody refers to immunoglobulins derived from a single clone of plasma cell or a clone thereof. Since all immunoglobulins produced by a given plasma cell are chemically and structurally identical, these antibodies constitute a pure population with highly specific antigen-binding properties Monoclonal antibodies to CD40 are known in the art, and the present invention contemplates a use for such antibodies in a novel method to activate CTLs as described below The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made
  • T-cell receptor TCR
  • CD40L T-cell receptor
  • CD40L CD40L
  • the gene for CD40L has been identified and characterized, and recombinant ligand produced
  • the present invention contemplated the use of recombinant CD40L or other CD40 binding proteins in the novel method to activate CTLs as described below
  • target cell refers to a syngeneic cell, a tumor cell or a cell infected by a pathogen (e g virus or bacteria)
  • the terms “effective amount”, “therapeutically effective amount”, and “immunologically effective amount” refer to the amount of the polypeptide/protein antigen necessary to support an observable level of CTL activation
  • the terms “effective dose”, “therapeutically effective dose”, and “immunologically effective dose” also refer to the amount of anti-CD40 antibody sufficient to bind to cell surface CD40 and induce activation of APCs
  • the terms “effective dose”, “therapeutically effective dose”, and “immunologically effective dose” also refer to the amount of CD40L or CD40 binding protein sufficient to bind to cell surface CD40 and induce proliferation of APCs and/or increase the production certain immunomodulatory molecules, for e.g., the B7 family of molecules.
  • the term "immunomodulatory amount” refers to the amount of cytokines or co-stimulatory molecules sufficient to modulate the activities of immune cells such as T-lymphocytes and B-lymohocytes.
  • the present invention provides a novel method of modulating the immune response in vivo in a patient by efficient activation of cytotoxic T- lymphocytes (CTLs).
  • CTLs cytotoxic T- lymphocytes
  • This invention is based on the discovery that co- administration or sequential administration of a peptide antigen with anti-CD40 antibody, CD40 ligand (CD40L) or CD40 binding protein serves as a potent activator of (CTL) function in vivo.
  • efficient methods for modulating immune response and treating a variety of diseases including bacterial diseases, viral diseases, cancer and autoimmune diseases are provided.
  • the procedures of the present invention depend in part upon the determination of epitopes recognized by CTLs capable of eliminating target infected cells.
  • One approach to identification of these epitopes is the identification of allele- specific peptide motifs associated with a particular disease for human Class I MHC allele subtypes.
  • the MHC class I antigens are encoded by the HLA-A B, and C loci. HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower. Each of these loci have a number of alleles.
  • a large number of cells with defined MHC molecules, particularly MHC Class I molecules, are known and readily available. These cells can be used to identify particular allele specific motifs associated with target diseases [see for e.g. WO 95/04817, incorporated herein by reference].
  • the allele-specific motifs can then be used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases or cancer. Potential epitopes on a number of target proteins can be identified in this manner.
  • suitable antigens include prostate specific antigen (PSA), hepatitis B core, surface and polymerase antigens (HBVc, HBVs, and HBVp), hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (e.g., MAGE- 1), human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, cytomegalovirus (CMV), herpes simplex virus (HSV), and other oncogene products (c-Erb B 2 , CE p53-breast/ovary, HER2/neu).
  • PSA prostate specific antigen
  • HBVc hepatitis B core
  • HBVs hepatitis C
  • the immunogenic peptides comprising the motif required for MHC binding and the epitope recognized by the CTLs can be synthesized.
  • the immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or isolated from natural sources such as whole viruses or tumors [see WO 95/04817, incorporated herein by reference], A skilled artisan will recognize that the immunogenic peptides can be of various lengths, either in their neutral (uncharged) forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modification, subject to the condition that the modification not destroy the biological activity of the polypeptides [see WO 95/04817, incorporated herein by reference].
  • the peptide will be as small as possible while still maintaining all of the biological activity or antigenicity of the large peptide.
  • Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g., at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T-cell.
  • the peptide may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue with another or one polar residue for another.
  • substitutions include combinations such as Gly, Ala, val, He, leu, met, Asp, Glu, Ser, Thr, Lys, Arg, Phe, and Tyr.
  • the effect of single amino acid substitutions may also be probed using D- amino acids.
  • modifications may be made using well known peptide synthesis procedures.
  • the proteins, polypeptides, peptide fragments and peptide analogs of the present invention can be prepared in a variety of ways. Because of their relatively short size, the polypeptides and peptide fragments can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984), Rockford, IL.
  • recombinant DNA technology may be employed wherein a nucleotide sequence encoding an immunogenic peptide or whole gene of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • Fusion proteins which comprise one or more peptide sequences of the invention can also be used to present the appropriate T-cell epitope.
  • the immunogenic peptides are then used to activate CTLs in vivo.
  • the in vivo therapy methods of the present invention and pharmaceutical compositions thereof are useful for treatment of mammals, particularly humans, to treat and/or prevent infectious dis eases, immune disorders and cancer.
  • the present invention contemplates the use of CD40 binding-CD40 activating molecules such as anti-CD40 monoclonal antibody, CD40L, CD40 binding protein and the like in conjunction with peptide antigen(s) to bypass the need for T H -cells in priming T H -cell dependent CTL activation.
  • CD40 binding-CD40 activating molecules such as anti-CD40 monoclonal antibody, CD40L, CD40 binding protein and the like in conjunction with peptide antigen(s) to bypass the need for T H -cells in priming T H -cell dependent CTL activation.
  • Anti-CD40-antibodies of the present invention are antibodies that are capable of binding to CD40 on the cell surface of APCs activating them.
  • the ant ⁇ -CD40 antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as F(ab), F(ab 2 ), Fv, and other fragments which retain the CD40 binding function of the parent antibody
  • CD40 mAbs A number of monoclonal antibodies directed against CD40 surface antigen (CD40 mAbs) are known in the prior art and have been shown to mediate various biological activities on human B cells.
  • CD40 monoclonal antibodies may be generated using conventional techniques (see WO 94/01547, WO 98/01538, U S Patent Nos
  • CD40 binding proteins may also be constructed utilizing recombinant DNA techniques
  • the variable regions of a gene which encodes an antibody to CD40 that binds in or near the ligand binding domain can be incorporated in to a useful CD40 binding protein [Reichman et al, Nature 332 333, (1988), Roberts et al, Nature 328.731, (1987), Verhoeyen et al, Science 239 1534, (1988), Chaudary et al, Nature 339 394, (1989)]
  • Suitable antibodies may be isolated or purified by many techniques well known to those of ordinary skill in the art [see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)] Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns or any combination of these techniques
  • CD40 is a member of the tumor necrosis factor (TNF) erve growth factor(NGF) receptor family, which is defined by the presence of cysteine rich motifs in the extracellular region [Smith et al, Science 248 1019, (1990), Mallett and Barclay, Immunology Today 12 220, (1991)]
  • CD40 is a cell surface molecule expressed on a variety of cells and interacts with a 30-33 kDa activation induced T cell counter receptor/ligand termed as CD40 ligand (CD40)
  • CD40 ligand CD40
  • the gene encoding CD40L has been cloned from a number of species including humans The cloning of murine CD40L is described in Armitage et al [Armitage et al, Curr. Opm.
  • CD40 + T cells were cloned from peripheral blood T-cells as previously described [Spriggs et al, J. Exp. Med 176 1543 (1992)] CD40L biological activity, mediated by its binding to CD40, includes the proliferation of CTLs
  • CD40L is believed to be important in feedback regulation of immune response to an antigen
  • a CD40 + antigen presenting cell APCs
  • APCs will present an antigen to a T-cell which will then become activated and express CD40L
  • the CD40L will, in turn, engage CD40 on the APC further activating the APC and increasing its efficiency at antigen presentation and secretion of a number cytokines and co stimulatory molecules [WO 98/01 145]
  • Useful forms of CD40L for the present invention also include soluble forms of CD40L (sCD40L), oligomeric CD40 ligand comprising a CD40- binding peptide and an oligomer-forming peptide
  • the CD40 binding portion of a CD40L may also be used to generate other CD40 binding proteins by methods known in the art with capability of transducing signal to a cell expressing biologically active CD40.
  • CD40 binding proteins that are substantially similar (i.e., those having an amino acid sequence at least 80% identical to a native amino acid sequence, most preferably at least 90% identical), to the previously described CD40 binding proteins will also be useful in the present invention
  • the percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 as previously described [Devereux et al, Nucl Acids Res. 12 387, (1984)]
  • the GAP program utilizes the alignment method of Needleman and Wunsch, [Needleman and Wunsch, J. Mol Biol 48 443,
  • substitutions of different amino acids from those in the native form of a useful CD40 binding protein should be made conservatively, i e the most preferred substitute amino acids are those that do not affect the ability of protein to bind to CD40 in a manner substantially equivalent to that of native CD40 ligand
  • conservative substitutions include substitutions of amino acids outside of the binding domain(s), and substitution of the amino acids that do not alter the secondary and/or tertiary structure of the CD40 binding proteins
  • substitutions of amino acids outside of the binding domain(s) substitution of the amino acids that do not alter the secondary and/or tertiary structure of the CD40 binding proteins
  • substitutions of amino acids outside of the binding domain(s) substitution of the amino acids that do not alter the secondary and/or tertiary structure of the CD40 binding proteins
  • the potential effect of deletion or insertion on the biological activity should be considered
  • Subunits of CD40 binding proteins may be constructed by deleting terminal or internal residues or sequences
  • Recombinant CD40L and CD40 binding proteins can be prepared according to standard methods, and tested for binding specificity to CD40 and biological activity utilizing assays known in the art Pharmaceutical Compositions and In Vivo Therapy
  • a preferred preparation of the antigenic molecule for use in the present invention, in whatever form, is as a pharmaceutical composition.
  • a preferred preparation of the CD40 binding-CD40 activating molecule(s), which is intended to be introduced into a host is also as a pharmaceutical composition.
  • a pharmaceutical composition of the present invention is comprised of one or more molecules which include an "immunogenic dose" of an antigenic molecule (e.g., peptide antigen) and a "therapeutically effective dose" of a CD40 binding-CD40 activating molecule (e.g., anti-CD40 antibody) combined optionally with a cytokine or a co-stimulatory molecule in a pharmaceutically acceptable carrier or excipient.
  • a mammal e.g., a human patient
  • an infectious disease viral, bacterial, or parasitic disease(s)
  • a malignant condition for example, which would be useful in the method of the present invention
  • a particular route may provide a more immediate and more effective reaction than another route. Accordingly, the described methods provided herein are merely exemplary and are in no way limiting.
  • the antigenic peptide(s), and/or the CD40 binding- CD40 activating molecule(s) of the present invention as described above will be administered as a pharmaceutical composition to an individual in need thereof as a therapeutic or prophylactic measure.
  • Those receiving treatment via the methods and compositions of the present invention may be treated with the presently disclosed compositions separately or in conjunction with other treatments, as appropriate.
  • compositions are administered to a patient in an amount sufficient to activate APC and to elicit an effective CTL response to a specific antigen(s), and to cure the individual of the diseases, or at least partially arrest the disease associated symptoms and/or complications.
  • An amount adequate to accomplish this is defined as a "therapeutically or preventatively effective dose” which may also be described as an "immune response provoking amount.”
  • Amounts effective for a therapeutic or prophylactic use will depend on a variety of factors. For example, such factors include the stage and severity of the disease being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician.
  • the dosage will also be determined by the antigenic molecule/CD40 binding-CD40 activating molecule/cytokine composition, method of administration, timing and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular compound, and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.
  • diseases which can be treated using in vivo methods and compositions of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV, breast, ovarian, lung, colon cancer, and various bacterial and parasitic diseases.
  • Suitable doses and dosage regimens can be determined by conventional techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is often increased by small increments until the optimum effect under the circumstances is achieved.
  • the present inventive method typically will involve the administration of about 0.1 ⁇ g to about 50 mg of one or more of the compounds described above per kg body weight of the individual. For a 70 kg patient, dosages of from about 10 ⁇ g to about 100 mg of peptide would be more commonly used, followed by booster dosages from about 1 ⁇ g to about 1 mg of peptide over weeks to months, depending on a patient's CTL response, as determined by measuring tumor-specific CTL activity in PBLs obtained from the patient.
  • compositions for therapeutic treatment as disclosed herein are generally intended for parenteral, topical, oral or local administration, and typically comprise a pharmaceutically acceptable carrier and an amount of the active ingredient sufficient to effectively treat an infectious disease or kill malignant cells.
  • the pharmaceutically acceptable carrier may be any of those conventionally used, and is limited only by physico- chemical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration.
  • pharmaceutically acceptable excipients described herein for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.
  • excipient will be determined in part by the particular epitope and epitope formulation chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of a pharmaceutical composition according to the present invention.
  • suitable formulations of a pharmaceutical composition according to the present invention are merely exemplary and are in no way limiting.
  • compositions for parenteral administration that comprise a solution of the cytotoxic T-lymphocyte stimulatory peptides dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions.
  • Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Compounds may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methyl cellulose, hydroxypropylmethyllcellulose, or caboxymethylcellulose
  • Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- ⁇ -aminopropionates, and 2-alkyl- imidazoline quaternary ammonium salts, and (e) mixtures thereof
  • suitable detergents include (a
  • compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17
  • HLB hydrophile-lipophile balance
  • Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan non-oleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • Parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried
  • sterile liquid excipient for example, water
  • injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Topical formulations including those that are useful for transdermal drug release, re well-known to those of skill in the art and are suitable in the context of the present invention for application to the skin.
  • Formulations suitable for oral administration require extra considerations considering the peptidyl nature of the epitopes and the likely breakdown thereof if such compounds are administered orally without protecting them from the digestive secretions of the gastrointestinal tract.
  • Such a formulation can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • the molecules and/or peptides of the present invention alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation.
  • the cytotoxic T- lymphocyte stimulatory peptides are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1 %-20% by weight of the composition, preferably 0.25-5%o.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included as desired, e.g., lecithin for intranasal delivery.
  • aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa.
  • the compounds and polymers useful in the present inventive methods may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Lipids have been identified that are capable of priming CTLs in vivo against viral antigens, e.g., tripalmitoyl-S-glycerylcysteinly-seryl-serine (P 3 CSS), which can effectively prime tumor-specific CTLs when covalently attached to an appropriate peptide [Deres et al, Nature 342.561-564 (1989)]
  • Antigenic molecules of the present invention can be coupled to P 3 CSS, for example and the lipopeptide administered to an individual to specifically prime a CTL response to a particular tumor cell or tissue
  • concentration of CTL-stimulatory antigenic molecules of the present invention in the pharmaceutical formulations can vary widely, i.e., from less than about 1%, usually at or at least about 10%) to as much as 20 to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected
  • a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of antigenic peptide or 100 mg of a CD40 binding-CD40 activating molecule, e.g.
  • the compounds of the present invention may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • Liposomes serve to target the antigenic peptides or the CD40 binding-CD40 activating molecule(s)to a particular tissue, such as lymphoid tissue or malignant cells or tissues. Liposomes can also be used to increase the half-life of the compositions
  • Liposomes useful according to the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide(s) or other molecules to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor (preferably one prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen), or with other therapeutic or immunogenic compositions.
  • a molecule which binds to e.g., a receptor (preferably one prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen), or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired antigenic molecule(s) or bioactive molecules of the invention can be directed to the site of APCs or lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic compositions.
  • Liposomes for use in the invention are typically formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871; 4,501,728, 4,837,028 and 5,019,369, the disclosures of which are incorporated herein by reference.
  • a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the peptide being delivered, the state of disease being treated, etc.
  • the present invention is directed to vaccines that contain as active ingredients an immunogenically effective amount of a CTL stimulating antigenic molecule and a CD40 binding-CD40 activating molecule, as described herein.
  • the antigenic molecule may be selected from the group consisting of a virus, a bacteria, a fungus, a parasite, a eukaryotic cell, DNA N protein, polypeptides, peptide fragments and analogs thereof.
  • the antigenic molecule or the CD40 binding-CD40 stimulating molecule may be introduced into a host — preferably a mammal, e.g., a murine species or a human — linked to its own carrier or as a homopolymer or heteropolymer of active peptide units.
  • a polymer has the advantage of increased immunological reaction and, where different antigenic molecules are used to make up the polymer, the additional ability to induce antibodies and/or prime CTL cells that react with different antigenic determinants of infectious disease agents or tumor cells.
  • Useful carriers are well known in the art, and include, e.g., keyhole limpet hemocyanin, thyroglobulin, albumins (e.g., human serum albumin), tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.
  • the vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum or materials well known in the art.
  • APC mediated CTL response(s) can be primed by conjugating peptides of the invention to lipids, such as P 3 CSS.
  • lipids such as P 3 CSS.
  • the immune system of the host responds to the vaccine by producing large amounts of CTL cells specific for viral, bacterial, parasitic antigens, or for a tumor-associated or tumor-specific antigen, and the host becomes at least partially immune to or resistant to the development of infectious diseases or malignancy to which the antigens relate.
  • Vaccine compositions containing the antigenic molecules of the invention may be administered to a patient susceptible to or otherwise at risk of developing the relevant infectious diseases or malignancy, to enhance the patient's own immune response capabilities.
  • a patient susceptible to or otherwise at risk of developing the relevant infectious diseases or malignancy to enhance the patient's own immune response capabilities.
  • Such an amount is defined to be a "immunogenically effective dose” or a “prophylactically effective dose.”
  • the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 ⁇ g to about 500 mg per 70 kilogram patient, more commonly from about 50 ⁇ g to about 200 mg per 70 kg of body weight.
  • the peptides of the invention can also be expressed by attenuated viral hosts or viral vectors, such as vaccinia virus, adenovirus etc.
  • viral vectors such as vaccinia virus, adenovirus etc.
  • This approach involves the use of the virus as a vector to express nucleotide sequences that encode the antigenic proteins/peptides of the vaccine.
  • the recombinant virus Upon introduction into a host, the recombinant virus expresses the antigenic peptide, and thereby elicits a host CTL response to an appropriate infectious disease- related or tumor-related antigenic molecule.
  • this immune response can be amplified and effectively modulated if the recombinant virus is introduced into a host in conjunction with a CD40 binding-CD40 activating molecule.
  • the vaccination strategy may be carried out in conjunction with cytokines and T-cell co-stimulatory molecules.
  • Viral vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
  • Another vector is BCG (Bacillus of Calmette Guerin).
  • BCG vectors are described in Stover et al, Nature 351 :456-460 (1991).
  • a wide variety of other vectors useful for therapeutic administration or immunization of the antigenic peptide(s) of the invention will be apparent to those skilled in the art from the description herein.
  • compositions of the present invention contain a physiologically tolerable carrier together with at least one species of therapeutic agent of this invention as described herein, dispersed therein as an active ingredient.
  • the therapeutic composition is not immunogenic when administered to a human patient for therapeutic purposes.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration upon a mammal or human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • physiological effects such as nausea, dizziness, gastric upset and the like.
  • compositions are prepared as sterile compositions either as liquid solutions or suspensions, aqueous or non-aqueous, however, suspensions in liquid prior to use can also be prepared.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, 2- ethyl amino ethanol, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, 2- ethyl amino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
  • additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water- oil emulsions.
  • a therapeutic composition contains a tumor-associated agent (e.g., a polypeptide) of the present invention, typically an amount ranging from at least .001 to 0.1 weight percent of agent per weight of total therapeutic composition.
  • a weight percent is a ratio by weight of tumor-associated agent to total composition.
  • 0.1 weight percent is 0.1 grams of agent per 100 grams of total composition.
  • a therapeutically effective amount of a tumor-associated agent- containing composition, or beneficial compound therein is a predetermined amount calculated to achieve the desired effect, i.e., to effectively benefit the individual to whom the composition is administered, depending upon the benefit to be conferred.
  • an effective amount can be measured by improvements in one or more symptoms associated with the condition of the lymphoproliferative disease occurring in the patient.
  • the dosage ranges for the administration of a tumor-associated agent (e.g., a polypeptide) of the invention are those large enough to produce the desired effect in which the condition to be treated is ameliorated.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient, and the extent of the disease in the patient, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any complication.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • a therapeutic amount of a disclosed composition of this invention is an amount sufficient to produce the desired result, and can vary widely depending upon the disease condition and the potency of the therapeutic compound.
  • the quantity to be administered depends on the subject to be treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosage ranges for systemic application are disclosed herein and depend on the conditions of administration Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent administration
  • administration should begin at the first sign of or diagnosis of an infectious disease or malignant condition, or shortly after diagnosis, and continue until the patient's symptoms are substantially abated and for a period thereafter.
  • loading doses followed by maintenance or booster doses may be required
  • Treatment of an affected individual with the compositions of the invention may hasten resolution of the condition in acutely affected individuals
  • One aspect of the invention contemplates modulating the immune response in an individual by administering to the individual in v vo an immunogenically effective dose of an antigenic molecule along with therapeutically effective dose of a CD40 binding-CD40 activating molecule to activate antigen presenting cells (APCs) into priming cytotoxic T-lymphocytes (CTLs) mediated antigen specific immune response
  • APCs antigen presenting cells
  • CTLs priming cytotoxic T-lymphocytes
  • Appropriate antigens may be selected from the proteins, polypeptides, analogs and sequential subsets thereof
  • Appropriate CD40 binding-CD40 activating molecules may be selected from anti-CD40 antibody, CD40 ligand (CD40L) and CD40 binding proteins
  • Appropriate antigens may also be delivered using a recombinant vector containing a nucleic acid molecule encoding said peptide antigen.
  • Cytokines including interleukins, interferons, and members of the TNF family of ligands have been shown to play a major role in activating APC and priming CTL cells.
  • T-cells require, in addition to cytokines and the primary signal provided by the T-cell receptors (TCR), co-stimulatory signals.
  • CD28 a cell surface molecule present on T-cells, is a major co stimulatory signal receptor for CD4 + and CD8 + T cells.
  • B7 family of proteins including B7-1 (CD80) and B7-2 (CD86) are its natural ligands on APCs.
  • a pharmaceutical composition comprising an immunogenically effective dose of an antigenic molecule is administered to an individual in need thereof in conjunction with a therapeutically effective dose of a CD40 binding, CD40 activating molecule, and optionally an immunomodulatory dose of one or more cytokines or a costimulatory molecules.
  • the pharmaceutical composition may also comprise of, in addition to an immunogenic dose of a peptide antigen, a therapeutically effective dose of a CD40 binding-CD40 stimulating molecule, and an immunomodulatory dose of one or more cytokines or co-stimulatory molecules selected from a group consisting of DL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, LL-10, IL-1 1, IL- 12, IL-13, IL-14, lL-15, IL-16, LL-17, IL-18, G-CSF, GM-CSF, CSF-1, MGDF, Interferons, TNF-family of ligands, SCF, LIF, EPO, TRANCE, Flt3 ligand (Flt3L), RANKL, and the B7 family of co-stimulatory molecules.
  • cytokines or co-stimulatory molecules selected from a group consisting of DL-l, IL-2,
  • the antigenic molecule is administered first as a pharmaceutical composition followed by the administration of a pharmaceutical composition consisting of a therapeutically effective dose of CD40 binding-CD40 activating molecule, and optionally an immunomodulatory dose of one or more cytokines or a co-stimulatory molecules.
  • a pharmaceutical composition consisting of a therapeutically effective dose of CD40 binding-CD40 activating molecule, and optionally an immunomodulatory dose of one or more cytokines or a co-stimulatory molecules.
  • the treatment regime will vary depending upon the specific disorder to be treated and the judgment of the treating physician.
  • the present invention also contemplates a method of treating an individual diagnosed with a tumor by priming cytotoxic T-lymphocyte (CTL) in vivo to specifically target and kill the tumor cells in the individual using the composition of the invention
  • CTL cytotoxic T-lymphocyte
  • the method contemplates in vivo activation of an antigen presenting cell (APC) by the in vivo delivery to the individual of an immunogenically effective dose of a tumor cell-derived antigenic molecule in conjunction with a therapeutically effective dose of a CD40 binding- CD40 activating molecule, to prime the proliferation, differentiation, and activation of CTL cells in vivo.
  • APC antigen presenting cell
  • the invention contemplates administering to the subject an immunogenically effective dose of a tumor-specific peptide antigen Preferably 6 to 96 hours, more preferably 12 to 72 hours, and most preferably 24 to 48 hours after the delivery of the tumor- specific peptide antigen(s) to the individual, an effective dose of CD40 binding- CD40 activation molecule is administered as a pharmaceutical composition resulting in the activation of antigen specific CTL cells in vivo in the individual
  • the pharmaceutical composition may also contain one or more cytokines or co-stimulatory molecules as described herinabove
  • the tumor specific antigenic molecule used to practice the invention may be derived from endogenously or exogenously displayed or processed proteins, polypeptides, peptide fragments and analogs
  • the tumor cell specific antigenic molecule can also be delivered via a nucleic acid molecule using an appropriate eukaryotic expression vector The nucleic acid molecule using an appropriate eukaryotic expression vector The nucleic acid molecule.
  • the method contemplates in vivo activation of an antigen presenting cell (APC) by the in vivo delivery to the individual an immunogenically effective dose of an infectious disease agent-derived antigenic molecule (e.g. viral, bacterial or parasitic proteins and/or peptides), in conjunction with a therapeutically effective dose of a CD40 binding-CD40 activating molecule, to prime the proliferation, differentiation, and activation of CTL cells in vivo.
  • the primed CTL cell then specifically targets and destroy the infectious disease agent or cells expressing antigens from the infectious disease agent.
  • the invention contemplates administering to an individual upon diagnosis of an infectious disease, a pharmaceutical composition comprising an immunogenically effective dose of a peptide antigen expressed specifically by the infectious disease agent.
  • a therapeutically effective dose of CD40 binding-CD40 activation molecule is administered as a pharmaceutical composition.
  • the pharmaceutical composition may also contain an immunomodulatory dose of one or more cytokines or co stimulatory molecules as described herinabove.
  • the infectious disease specific antigenic molecule used to practice the invention may be derived from endogenously or exogenously displayed or processed proteins, analogs, or portions thereof.
  • the infectious disease specific antigenic molecule can also be delivered via a nucleic acid molecule using an appropriate eukaryotic expression vector.
  • the nucleic acid molecule will be taken up by the APC in vivo, which then express the polypeptide(s) encoded by the nucleic acid.
  • the polypeptides synthesized from the nucleic acid are processed in vivo within the APC, and presented on the cell surface to CTL to initiate "antigen specific" immune response.
  • Many viral, bacterial and parasitic specific proteins, polypeptides, peptide fragments and analogs are known in the art and may be used to practice the invention.
  • Treatment of an infected individual with the methods of the invention may also hasten resolution of the infection in acutely infected individuals.
  • the methods are useful for preventing the evolution from acute to chronic infection.
  • the compositions can be targeted to them, minimizing the need for administration to a larger population.
  • the methods of the present invention can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers.
  • transgenic mice In the transgenic animal model used for these studies, induction of diabetes caused by an immunological mechanism involving CTLs that have not been tolerized to transgenic self antigens expressed on pancreatic islet cells was assessed.
  • transgenic mice Bln/TCR, generated as previously described were used [Ohashi et al. Cell 65:305-317 (1991)].
  • the transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), P33 in the pancreas were generated using the tissue specific rat insulin II promoter linked to the 5' end of the viral glycoprotein, cDNA sequence.
  • LCMV lymphocytic choriomeningitis virus
  • GP tissue specific rat insulin II promoter linked to the 5' end of the viral glycoprotein, cDNA sequence.
  • Exons 2 and 3 of the ⁇ - globin gene were ligated to the 3 ' end of the construct, providing splicing and polyadenylation signals. Tissue specific expression of the transgene was assessed by measuring RNA and protein levels. Evidence that GP protein was expressed by the ⁇ -islet cells was obtained from immunohistochemical analysis of the pancreas from the transgenic mice.
  • T cells specific for LCMV-GP, p33 are present in the T cell repertoire of these transgenic animals and are not tolerant to the identical antigen expressed in the pancreas.
  • these animals do not spontaneously become diabetic.
  • the "self reactive" T cells in these mice could be induced to proliferate when stimulated with LCMV-infected macrophages.
  • these T cells when activated, were able to lyse target cells expressing LCMV antigens. Rapid cytotoxic T cell response in these transgenic animals could be induced days after injection with high dose (1 x 10 6 pfu) and low dose (200 pfu) of LCMV.
  • mice were immunized intravenously with 5 ⁇ g of the LCMV GP p33 or control adenovirus peptide, AV, on day 0 followed by the administration of 100 ⁇ g of anti-CD40 antibody forty-eight hours later (day 2).
  • Induction of diabetes was assessed by monitoring the blood glucose levels in these animals as previously described [Ohashi et al, supra].
  • Al of the animals (100%>) developed diabetes with a mean onset time of 6 days (Table 1, row 1-3).
  • Subcutaneous or intraperitoneal administration of the antigenic peptide, p33 (100 ⁇ g/animal) followed by intravenous or intraperitoneal administration of the anti-CD40 antibody (100 ⁇ g/animal) also resulted in the induction of diabetes in a significant percentage of the animals with a mean onset time of diabetes induction of 6 days.
  • transgenic mice expressing p33 antigen of LCMV as a "transgenic self antigen with the p33 peptide antigen followed by administration of an appropriate dose anti-CD40 antibody within 48 hrs. of the administration of the peptide resulted in enhanced infiltration and subsequent T-cell mediated immunopathological destruction of the islet cells expressing the "transgenic self antigen.
  • DC Dendritic cells
  • APCs such as the Dendritic cells (DC) are essential for antigen presentation and activation of CTLs.
  • DCs capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses and activate CTLs.
  • DCs are usually present in the peripheral tissues as immature DCs. As a consequence of antigen deposition and inflammation, DCs begin to mature, expressing molecules that include molecules of the major histocompatibility complex (MHC), that ultimately lead to the binding and stimulation of T-cells.
  • MHC major histocompatibility complex
  • MHC class I and MHC class II stimulate CTLs and T H . cells respectively by forming a complex with peptide antigens and presenting them to the T-cells.
  • intracellular antigens cut into peptides in the cytosol of DCs bind to and complex with MHC class I molecules. This complex translocates to the cell surface where it is presented to and recognized by the CTLs, which, once activated, can directly kill target cells.
  • Extracellular antigens that are processed by DCs are presented by MHC class II, which in turn activates T H -cells.
  • FACS fluorescent activated cell sorter
  • DCs are normally present in an animal in a so-called “immature” state, unable to stimulate T-cells.
  • DCs capture the antigen, process it and become activated.
  • the fragments of the antigen complexed with MHC molecules are presented at the cell surface to CD8- expressing T-cells resulting in the activation of these cells.
  • This study demonstrates that DCs isolated from Bln TCR animals immunized with p33 peptide and subsequently treated with anti-CD40 antibody are activated into potent APCs as measured by their ability to stimulate the proliferation of T-cells in vitro.
  • T-cells were purified from naive Bln/TCR transgenic animals and plated in a 96 well flat bottom plate at a density of 5 x 10 4 cells/well. Dendritic cells were isolated from animals immunized with p33 antigen and treated anti-CD40 antibody or control antibody 24 hrs. after treatment with the antibody. Approximately 1.25 x 10 4 DCs were plated into the same well as the T-cells and cultured in Iscove' modified Dulbecco' s medium plus 10% FCS, 2 x 10 "5 2- mercaptoethanol, glutamine and penicillin-streptomycin (100 U/ml). The culture was exposed to increasing concentrations of p33 polypeptide ( 10 "10 to 10 "4 M).
  • mice were immunized with 5 ⁇ g of p33 peptide on day 0, followed by 100 ⁇ g of anti-CD40 antibody 48 hours later Quantitation of IFN- ⁇ levels by ELISA demonstrated an increase in serum IFN- ⁇ 3 days after peptide treatment.
  • IFN- ⁇ is known to increase class I MHC expression, and corresponding immunohistological analysis confirmed that increases in class I levels were seen in the pancreas. This may contribute to the enhanced T cell infiltration seen in p33 peptide/anti-CD40 treated mice (Figure 1)
  • TCR transgenic mice were immunized by intravenous administration of 5 ⁇ g of p33 peptide on days 0, 3, and 6 On day 2, 5, and 8, 100 ⁇ g of anti-CD40-antibody or control antibody were administered to the animals.
  • CD40 Stimulation Prevents the Induction of Tolerance and Enhances Immunity
  • mice were immunized with p33 (200 ⁇ g in IFA ip) and anti-CD 40 antibody (100 ug / iv) or p33 (200 ⁇ g in IFA ip) alone After 30 days, the mice were infected with LCMV Eight days after viral infection, the animals were sacrificed and their spleens removed to determine CTL activity Spleenocytes were cocultured with EL-4 target cells that were prepulsed with 51 - Chromium and lO ⁇ M peptide p33, or control adenovirus peptide, AV (10 '6 M)

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Abstract

L'invention concerne un nouveau procédé modulant la réponse immunitaire chez une personne en activant des lymphocytes T cytotoxiques spécifiques d'antigènes (CTL) in vivo pour cibler et détruire des cellules exprimant des antigènes. L'invention concerne également l'utilisation de ce procédé pour traiter des maladies infectieuses ainsi que le cancer, et pour améliorer l'efficacité de vaccins.
PCT/CA2000/000960 1999-08-27 2000-08-23 Procede d'activation de lymphocytes t cytotoxiques (ctls) in vivo: composition comprenant un anticorps anti cd40 (ou cd40l ou une proteine liant le cd40) et un antigene WO2001015728A1 (fr)

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Cited By (5)

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WO2002036141A2 (fr) * 2000-11-02 2002-05-10 Immunex Corporation Methode permettant d'ameliorer des reponses immunitaires induites par des lymphocytes
WO2004041866A1 (fr) * 2002-11-05 2004-05-21 Adjuvantix Limited Composition de conjugue comprenant des anticorps diriges contre cd40 ou cd28
WO2008107641A1 (fr) * 2007-03-02 2008-09-12 Adjuvantix Limited Préparation de liposome
US7455846B2 (en) 2000-05-17 2008-11-25 Stephen John Ralph Immune potentiating compositions of cancer cells
US7807155B2 (en) 2004-08-05 2010-10-05 Novartis Ag IL-17 antagonistic antibodies

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7455846B2 (en) 2000-05-17 2008-11-25 Stephen John Ralph Immune potentiating compositions of cancer cells
US7964400B2 (en) 2000-05-17 2011-06-21 Stephen John Ralph Immune potentiating compositions of cancer cells
WO2002036141A2 (fr) * 2000-11-02 2002-05-10 Immunex Corporation Methode permettant d'ameliorer des reponses immunitaires induites par des lymphocytes
WO2002036141A3 (fr) * 2000-11-02 2003-08-21 Immunex Corp Methode permettant d'ameliorer des reponses immunitaires induites par des lymphocytes
WO2004041866A1 (fr) * 2002-11-05 2004-05-21 Adjuvantix Limited Composition de conjugue comprenant des anticorps diriges contre cd40 ou cd28
US7807155B2 (en) 2004-08-05 2010-10-05 Novartis Ag IL-17 antagonistic antibodies
US8119131B2 (en) 2004-08-05 2012-02-21 Novartis Ag IL-17 antagonistic antibodies
US8617552B2 (en) 2004-08-05 2013-12-31 Novarts Ag IL-17 antibodies
US9765140B2 (en) 2004-08-05 2017-09-19 Novartis Ag IL-17 antagonistic antibodies
US10344084B2 (en) 2004-08-05 2019-07-09 Novartis Ag IL-17 antagonistic antibodies
WO2008107641A1 (fr) * 2007-03-02 2008-09-12 Adjuvantix Limited Préparation de liposome

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