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WO1997033612A1 - Dialdehydes servant d'adjuvants immunostimulants et de reticulants pour la production de preparations immunogenes, pour la generation de cellules soumises a pression et reticulation afin de renforcer et d'augmenter la reponse immunitaire contre le cancer, les tumeurs et les affections pathogenes - Google Patents

Dialdehydes servant d'adjuvants immunostimulants et de reticulants pour la production de preparations immunogenes, pour la generation de cellules soumises a pression et reticulation afin de renforcer et d'augmenter la reponse immunitaire contre le cancer, les tumeurs et les affections pathogenes Download PDF

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Publication number
WO1997033612A1
WO1997033612A1 PCT/US1997/003916 US9703916W WO9733612A1 WO 1997033612 A1 WO1997033612 A1 WO 1997033612A1 US 9703916 W US9703916 W US 9703916W WO 9733612 A1 WO9733612 A1 WO 9733612A1
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WIPO (PCT)
Prior art keywords
cells
antigen
dialdehyde
tumor
cell
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PCT/US1997/003916
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English (en)
Inventor
Amnon Gonenne
Avi Eisenthal
Meir Shinitzky
Cohava Gelber
Original Assignee
Immunotherapy, Inc.
Yeda Research And Development Co., Ltd.
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Application filed by Immunotherapy, Inc., Yeda Research And Development Co., Ltd. filed Critical Immunotherapy, Inc.
Priority to AU19910/97A priority Critical patent/AU1991097A/en
Publication of WO1997033612A1 publication Critical patent/WO1997033612A1/fr

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    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • DIALDEHYDES AS IMMUNOSTIMULATORY ADJUVANTS AND CROSSLINKERS FOR PRODUCING IMMUNOGENIC PREPARATIONS AND GENERATING PRESSURE AND CROSSLINK TREATED CELLS FOR ENHANCING AND AUGMENTING THE IMMUNE RESPONSE AGAINST CANCER, TUMORS AND PATHOGENIC DISEASES.
  • the present invention is generally in the field of enhancement and augmentation of the immune response to tumor or cancer cells and to foreign antigens of pathogenic and/or infectious microorganisms.
  • the present invention achieves enhancement and augmentation of the immune response by means of treating antigen presenting cells with crosslinker and hydrostatic pressure.
  • the invention also relates to novel costimulatory adjuvants and molecules for use in prophylactic and therapeutic vaccine formulations and immunogenic preparations, to processes for producing potent vaccine and immunogenic preparations comprising pressure- and dialdehyde crosslinker-treated accessory immune cells, such as antigen presenting cells, and to the use of dialdehyde crosslinkers as novel costimulatory adjuvants for increasing and enhancing specific in vivo and in vitro immunogenicity against pathogenic diseases, tumors and cancers.
  • Cancer and/or tumor cells frequently display on their external surfaces specific neo-antigens which are foreign to the immune system and immune cells of the host.
  • tumor or cancer cells often escape immune surveillance, and the immune system fails to develop an effective immune reaction against these cells.
  • Attempts have been made to immunize cancer patients with preparations that will stimulate their immune systems to develop a reaction against the neo-antigens, with the hope that such an immune reaction will destroy the residing cancer or tumor.
  • Transforming events can occur spontaneously by random mutations, by gene rearrangement, or, they may be induced by chemical, physical, viral, or microorganismal agents.
  • Major classes of chemical carcinogens are known to include polycyclic aromatic hydrocarbons, such as are found in tar and soot, and aromatic amines, such as are found in certain dyes. Examples of physical carcinogens are X-rays and ionizing and ultraviolet radiation.
  • both RNA and DNA viruses and viral oncogenes are capable of transforming cells.
  • the infected cells When viral genes are introduced into cells, the infected cells are then triggered to express on the cell surface virus-associated antigens that can be recognized by the immune system. Moreover, the abnormal maintenance of certain viral oncogenes in a transcriptionally active state can result in transformation. In a similar manner, the infection of cells by bacteria or parasites or other microorganisms may lead to the expression of antigens at the cell surface and to recognition by immune cells.
  • oncogenic, neo-antigenic, or "non- self" protein products should ideally render tumor, transformed, and infected cells sufficiently distinct from normal, unaffected cells so that the former can be detected by the immune system.
  • tumor and transformed cells including cells infected with microorganisms that can affect the composition of the plasma membrane of the infected cells, often have the ability to avoid immunologic surveillance and detection.
  • MHC major histocomptability complexes
  • Other escape mechanisms such as the down-regulation of costimulatory ligands on tumor cells or infected cells, may thwart the ability of responder T lymphocytes to be activated, thus resulting in an anergic response by the T lymphocytes.
  • the stimulation of suppressor T lymphocytes can further attenuate the immune response.
  • tumor antigens or proteins
  • TAA tumor associated antigens
  • the best-studied unique tumor antigens are the neoantigens expressed on tumors induced in inbred mice by oncogenic viruses and chemical carcinogens. In contrast, spontaneous tumors, such as those induced by exposure to environmental carcinogens, have no predictable antigenic markers.
  • the goal of therapies to treat and eradicate cancers and other types of transformed or infected cells is to provide efficient and safe vaccines and methods by which to increase the host's anti-tumor or foreign cell response against weakly immunogenic cell types, such as tumors and cells transformed by viruses or other pathogenic microorganisms.
  • Tumor and viral antigens elicit both humoral (antibody or B cell- mediated) and cell mediated immune responses in vivo, and virtually all of the effector components of the immune system have the potential to contribute to the eradication of tumor or virally infected cells.
  • the T cell response is a most important host response for the control of growth of antigenic tumor cells, and transformed or infected cells, via cell-mediated immunity.
  • the T cell response is effective for both the direct killing of tumor cells or infected (e.g., virus- or bacteria-infected) cells (by cytotoxic T cells) and the activation of other components of the immune system.
  • T cell immunity to tumors and infected cells involves the function of two T cell subsets: MHC class II-restricted T cells, which largely represent CD4 helper T cells (i.e., T caution), that mediate their effect by the secretion of lymphokines to activate other effector cells and to induce inflammatory responses; and MHC class I-restricted T cells, which represent CD8 cytotoxic T (T c ) cells, that also secrete lymphokines, but mediate their effect primarily by the direct lysis or killing of tumor cells. Because most tumor cells express class I, but not class II MHC molecules, the Tré cell subset cannot directly recognize these tumor cells.
  • TRON cell responses are dependent upon antigen-presenting cells or APC, such as macrophages, B lymphocytes and dendritic cells, to present the relevant tumor antigens in the context of class II MHC molecules for cell activation.
  • Antigen presenting cells capture, process, and present most proteinaceous immunogens to the CD4 helper T cell subset.
  • activated T Following antigen-specific triggering, activated T wa cells secrete lymphokines that, in turn, activate T c cells, macrophages, natural killer (NK) cells, and B cells; activated T H cells also produce other lymphokines, such as lymphotoxin or tumor necrosis factor (TNF) which may also be directly lytic to tumor cells.
  • lymphokines such as lymphotoxin or tumor necrosis factor (TNF) which may also be directly lytic to tumor cells.
  • T c cells cytotoxic T cells
  • T c cells cytotoxic T cells
  • T H 1 cells release both interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ), while T flesh2 cells release a combination of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), and interleukin-10 (IL-10). Both IL-4 and IL-10 have been shown to shut down the cell-mediated immune response. Conversely, IFN- ⁇ produced by T H 1 cells promotes cytotoxic T cell proliferation and inhibits antibody production.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • the various cytokines elaborated by the these two T cell subsets are cross-regulatory and can, when manipulated by a pathogen (e.g., virus, parasite or tumor cell) seriously suppress an individual's effective immune response status to the particular pathogen.
  • a pathogen e.g., virus, parasite or tumor cell
  • T H 1 T helper type 1
  • T H l-type cytokines e.g., IFN ⁇ , IL-2, TNF-S, IL-12, and the like
  • CD4+ T lymphocytes in a T deliberately2-type mode secrete characteristic cytokines (e.g., IL-10, IL-4, and the like) which preferentially stimulate clonal expansion of B lymphocytes, resulting in antibody production, and inhibit CD8+ T cell proliferation.
  • characteristic cytokines e.g., IL-10, IL-4, and the like
  • T leavel and T H 2 cytokines can have a profound effect on the clinical outcome in a number of parasitic, chronic bacterial, mycobacterial, viral diseases, as well as in cancer.
  • Leishmaniasis major infection in mice animals which heal spontaneously exhibit an augmented cellular immune response with a dominant T H 1 profile against the pathogen.
  • T H l-type response i.e., predominantly cell-mediated
  • T Tin2-type response i.e., primarily antibody production
  • class I- restricted T c cells are capable of directly recognizing and killing tumor target cells by disrupting the target cell membrane and nucleus (Bjorkman, P. et al., 1990. "Structure, function, and diversity of class I major histocompatibility complex molecules", Ann. Rev. Biochem. , 59:253). Only a minor fraction of class I- restricted T cells is capable of providing helper functions; thus, effective T c cell responses are generally dependent upon class Il-restricted T H cell responses to provide the necessary helper factors to activate and promote the proliferation of T c cells.
  • the T cell receptor of an antigen-specific T c cell clone recognizes class I MHC -peptide complexes which, after intracellular processing of viral or tumor antigens appear, at the surface of virally infected or transformed cells, for example.
  • Cytotoxic T cells become activated to eradicate foreign cells by releasing toxins or inducing the target cell to commit suicide, perhaps by physical contact with the foreign target cell.
  • the activated T c cells proliferate and give rise to additional T c cells having the same antigen specificity.
  • APC antigen-presenting cells
  • MHC molecules are obligatory components of the immunogenic complex recognized by T cells and play a central role in the immune response to foreign and tumor antigens.
  • the ability of T cells to recognize specific features of MHC proteins is crucial for the immune system to function properly and to discriminate "self from "non-self .
  • T Shap cells themselves first must be activated. Activation occurs when a T H cell recognizes a foreign antigen bound to a class II MHC glycoprotein on the surface of an APC.
  • APC are found in most tissues. They are derived from bone marrow and comprise a heterogeneous set of cells, such as dendritic cells in lymphoid organs, Langerhans cells in skin, and certain types of macrophages. Populations of APC can also be isolated and expanded for use in stimulating an immune response (with particular regard to dendritic cells, see, for example, WO 93/20185 to R.M. Steinman et al. and WO 91/13632).
  • APC thymus epithelial cells
  • fibroblasts which do not make class II MHC molecules and cannot present foreign antigens to T Formula cells
  • T H cells T H cells
  • thymus epithelial cells T H cells
  • fibroblasts which do not make class II MHC molecules and cannot present foreign antigens to T Formula cells
  • other cells that normally do not express class II MHC glycoproteins such as endothelial cells, can be induced to express these proteins following the production of interleukins, such as gamma interferon by activated T H cells.
  • interleukins such as gamma interferon by activated T H cells.
  • the sequence of events leading to the presentation of antigen to T cells at the cell surface of APC is known as antigen processing (see, Molecular Biology of the Cell, 2nd Ed., Eds. B. Alberts et al., 1989, pages 1045-1048).
  • Tumor cells, cancer cells, and otherwise transformed cells may lose their regulated proliferative state due to the acquisition of means by which to cause disease, grow uncontrollably, and invade distant tissue.
  • Such cells may also display unique or characteristic tumor associated antigens on the cell surface and the processed antigens may be complexed with class I or class II MHC structures for presentation to the effector cells of the immune system.
  • cells transformed by viruses, bacteria, parasites, and the like may also present processed antigens, peptides, proteins, or structures of the foreign pathogen, microorganism, or other infectious agent (e.g., prions), complexed with class I or class II MHC molecules for presentation to the effector cells of the immune system.
  • infectious agent e.g., prions
  • a potential solution to the problem of overcoming the ability of cancer, infected or transformed cells to avoid host immune surveillance is to manipulate and amplify the immune system and its cellular components in order to promote the eradication of the abnormal cells.
  • This solution is also conducive for eliminating cells infected or transformed by other exogenous agents, e.g. , viruses and other microorganisms such as parasites, protozoa, algae, yeast, fungi, and bacteria.
  • Provoking an effective immune response against malignant cells requires the elimination of control mechanisms utilized by cancer cells to conceal their identity antigens.
  • cancer antigens must expose and express themselves above a critical threshold in conjunction with MHC molecules.
  • a multivalent vaccine which comprises the widest repertoire of tumor antigens and which specifically augments the cellular immune response against a weakly immunogenic tumor. Such is afforded by the present invention.
  • adjuvants are agents, e.g., compounds, chemicals, proteins, molecules, lipids, and the like, which augment specific immune responses.
  • adjuvants can serve as immunostimulatory or immunopotentiating agents, alone or in combination with other compounds, to improve or increase the host's immune response and resistance to a tumor or invading pathogenic microorganism or virus.
  • Immunopotentiating or immunostimulatory agents are capable of stimulating, enhancing, or increasing an immune response to foreign antigen.
  • Adjuvants as immunopotentiators or immunostimulators can increase immune resistance to antigen in a vaccine preparation and to tumors and infected cells in a vaccine preparation employing tumor or infected cells.
  • alum and similar aluminum gels which are approved for use in humans; Freund's incomplete adjuvant; Freund's complete adjuvant; Quil A or saponin; muramyl dipeptide; iscoms or lipid micelles; non-metabolized synthetic molecules which are capable of binding proteins to cell surfaces; and nonionic block copolymer surfactants.
  • alum as adjuvant poses problems because it does not stimulate cell mediated immunity; it provides low yields of effective vaccines when small peptides are used as vaccines due to inadequate adsorption; and it may cause degradation of antigens via the induction of protease activity.
  • the materials other than alum are frequently not optimal for use in mammals, especially humans, due to their ability to cause chronic reactions and toxicity in the hosts.
  • Small Schiff-base forming molecules i.e., substituted benzaldehyde tucaresol, and monoaldehydes have been reported to provide costimulatory signals to cells by donating carbonyl groups through a mechanism that activates clofilium- sensitive potassium and sodium transport (J. Rhodes et al., 1995, Nature, 377:71- 75; X-M. Gao et al, 1990, J. Immunol. , 144:2883-2890).
  • a significant drawback to the use of tucaresol and other such molecules as costimulatory agents or adjuvants, as demonstrated by Rhodes et al., is that their functional usefulness in vivo is over an extremely narrow concentration range.
  • NAGO neuraminidase
  • GO galactose oxidase
  • NAGO treatment has been shown to result in lymphocyte blastogenesis and transformation in vitro (A. Novogrodsky and E. Katchalski, 1973, Proc. Natl. Acad. Sci. USA, 7Q: 1824-1827) and enhancement of immunogenicity in vivo (J. Rhodes et al., 1995, Ann.
  • NAGO may be toxic and may not be well tolerated in mammalian hosts.
  • this technique is likely to suffer from lack of specificity in vivo, especially in the narrow and high- level dose range required for efficacy and activity and after repeated administration in mammalian hosts.
  • the present invention provides the use of dialdehydes as stimulators, potentiators, and adjuvants to enhance and increase the immune response to antigens, particularly tumor antigens, antigens derived from viruses, bacteria, yeast, fungi and other pathogenic disease-causing microorganisms.
  • dialdehydes when reacted with cells or antigen, modify the cell membrane components or antigen by forming transient covalent chemical reactions such that one aldehyde group of the dialdehyde reacts as a monoaldehyde with a cell membrane component or antigen, and the other pendant monoaldehyde group is free to react with other cell membrane ligands or non-membrane bound ligands, such as protein or carbohydrate.
  • Dialdehydes function as adjuvanting molecules, immunostimulatory molecules and immunopotentiating molecules in the methods and compositions of the invention to achieve potent immunogens and vaccines comprising dialdehydes.
  • the dialdehydes also may provide the costimulatory signals that are required for activation and response by T cells; in this way the dialdehydes of the invention may replace the need for costimulatory molecular interactions traditionally supplied by antigen-presenting cells.
  • the dialdehyde functions as both a crosslinker to react with and crosslink cell membrane components within the cell surface, and as an adjuvant molecule wherein its two monoaldehydic groups are able to form reversible chemical covalent linkages with cell membrane components and/or with non-membrane-bound components.
  • the amount of monoaldehyde produced and available for further interaction with other components can be quantified after incubation of cells with dialdehyde.
  • the cells component of the composition is preferably PCL modified.
  • the antigen component of the composition is preferably not bound to a cell.
  • It is yet another object of the invention to provide vaccine and immunogenic formulations comprising dialdehyde and antigen, wherein one aldehyde group of the dialdehyde is linked to antigen and the other aldehyde group is linked to another adjuvant, immunopotentiating or immunostimulating agent, such as a cytokine, a lymphokine, or a chemokine.
  • It is a further object of the invention to provide a composition comprising dialdehyde coupled or linked to any immunogen, either cellular or noncellular, for example, an antigen such as protein, peptide, or carbohydrate, for use as therapeutic and prophylactic vaccines to enhance and augment the immune response to the immunogen.
  • any immunogen either cellular or noncellular, for example, an antigen such as protein, peptide, or carbohydrate, for use as therapeutic and prophylactic vaccines to enhance and augment the immune response to the immunogen.
  • antigen-presenting cells also called accessory cells
  • Such vaccines are also useful for the ex vivo generation of specific cytotoxic T lymphocytes (CTLs), which may be particularly employed in the treatment of cancers such as B-cell lymphomas, and in treating and preventing the growth of tumors and infected cells.
  • CTLs cytotoxic T lymphocytes
  • PCL treatment is performed on antigen-presenting cells or accessory cells to augment the capacity of these cells to elicit a vigorous immune response, ultimately activating T cells to target antigens, such as those associated with tumors, cancers and infectious microorganisms to reduce cancer, tumor or infected cell load and/or to eradicate the system of cancer, tumor and infected cells.
  • Fig. 1 shows a functional assay of 2', 3'-adenosine dialdehyde (AdA) determined by fluorescence and presented for three active and purified preparations (batches) of the crosslinker (see Example 1C).
  • AdA 2', 3'-adenosine dialdehyde
  • Fig. 2 shows an evaluation of the binding kinetics of AdA to B16 melanoma cells as a function of pH (see Example ID)
  • Fig. 3 shows the determination of the percent occupancy of available cell surface binding sites for AdA on B16 melanoma cell surfaces after reacting cells with this dialdehyde as a function of time (see Example IE).
  • Fig. 4 shows the results of analyses to determine the number of AmA molecules bound per cell and the effect of chemical reducing agents on lowering the amount of membrane-bound AdA (see Example 4).
  • Fig. 5 shows the results of IVS analyses to determine the immunological effects of preincubating cells in dialdehyde and then PCL-treating the preincubated cells by exposure to AdA crosslinker and hydrostatic pressure.
  • Fig. 6 demonstrates anti-tumor effectiveness in the presence of cell membrane bound adenosine monoaldehyde in vivo.
  • Fig. 7 demonstrates the effects of dialdehydes as adjuvants/immunopotentiators of the cellular response to immunoproliferating agents.
  • F ⁇ g. 8 shows the results of cytotoxic T cell assays using PCL- modified lymphoma cells and PCL-modified dendritic cells, in which cytotoxic T cells are stimulated to react against autologous human lymphoma cells. Increased precursor frequency of anti-tumor CTL is seen following stimulation with PCL- modified intact tumor cells or loaded (+ antigen) dendritic antigen presenting cells (APC).
  • Fig. 9 shows the augmentation of the proliferative response of primary T cell lines to intact, irradiated tumor cells (EBV-associated B cell lymphoma) following PCL-modification of the B lymphoma cells (Example 17).
  • EBV-associated B cell lymphoma irradiated tumor cells
  • closed squares autologous B cell lymphoma
  • closed diamonds HLA-A matched B cell lymphoma
  • open triangles K562.
  • Fig. 10 shows the secretion of higher levels of ⁇ lFN by primary T cell lines following stimulation with PCL-modified B cell lymphomas (Example 17).
  • closed squares autologous B cell lymphoma
  • closed diamonds HLA-A matched B cell lymphoma
  • closed triangles K562.
  • Fig. 11 shows the secretion of lower levels of IL-10 by primary T cell lines following stimulation with PCL-modified B cell lymphomas (Example 17).
  • closed squares autologous B cell lymphoma
  • closed diamonds HLA-A matched B cell lymphoma
  • closed triangles K562.
  • Fig. 12 shows the augmentation of the proliferative response of primary T cell lines to antigen presenting cells, i.e., dendritic cells (DC), pulsed with allogeneic B cell lymphoma extract following PCL-modification of the antigen presenting cells (Example 17).
  • DC dendritic cells
  • FIG. 12 shows the augmentation of the proliferative response of primary T cell lines to antigen presenting cells, i.e., dendritic cells (DC), pulsed with allogeneic B cell lymphoma extract following PCL-modification of the antigen presenting cells (Example 17).
  • DC dendritic cells
  • Fig. 13 shows the increased presentation of tumor-specific antigens to T cells, demonstrated by enhanced secretion of ⁇ lFN, following PCL- modification of APC (Example 17).
  • closed squares autologous B cell lymphoma
  • closed diamonds HLA-A matched B cell lymphoma
  • closed triangles K562.
  • Fig. 14 shows that primary T cell lines secrete decreased levels of LL-10 upon stimulation with dendritic cells pulsed with B cell lymphoma extract and modified by PCL (Example 17).
  • closed squares autologous B cell lymphoma
  • closed diamonds HLA-A matched B cell lymphoma
  • closed triangles K562.
  • Fig. 15 shows that PCL modification of intact tumor cells or dendritic cells (DC)-pulsed with tumor antigen elicits the proliferation of both CD4+ and CD8+ cells.
  • Figs. 16A-16D present profiles of the levels of chemokines and lymphokines produced by T cells during in vitro stimulation assays with BLCL that have been PCL modified or unmodified as controls.
  • Fig. 16A IFN-gamma profile
  • Fig. 16B MIP-1 -beta profile
  • Fig. 16C MIP-1 -alpha profile
  • Fig. 16D RANTES profile.
  • the present invention relates to an improved method of treating tumor or infected cells with hydrostatic pressure and a dialdehyde chemical crosslinker to result in immunogens having increased and heightened immunogenicity for stimulating a specific immune response in the host.
  • the immunogens may be whole tumor cells or infected cells, or they may be pathogenic disease-causing microorganisms or cells treated as described; they may also be plasma membranes derived from these cells; or specific immunogenic proteins obtained from the tumor, infected or pathogenic cells or cell membranes thereof.
  • Infected cells are those that are infected and/or transformed with viruses or other pathogenic microorganisms or agents as described herein.
  • the treatment of cells with pressure and crosslinker is termed PCL modification, PCL treatment, or PCL technology as used herein.
  • the invention further relates to the use of dialdehyde both as the aforementioned crosslinker and as an adjuvanting, immunostimulatory or immunopotentiating molecule, whether on the surfaces of tumor or infected cells or in compositions with antigen useful as immunogens and vaccines.
  • the invention further relates to therapeutic and prophylactic vaccines and immunogens comprising PCL-modified and unmodified antigen-presenting cells (APC) and PCL-treated tumor, cancer, or infected cells, and/or proteins and peptides derived therefrom.
  • APC may include, but are not limited to, dendritic cells, fibrocytes, astrocytes, macrophages, B cells, microglial cells and fibroblasts.
  • Fibrocytes are a preferred APC type and are characterized by their rapid entry from blood into subcutaneously implanted wound chambers and by their presence in connective tissue scars, as described by R. Bucala et al., 1994, Mol. Med. , 1:71-81.
  • the APC can be activated or unactivated, i.e., pulsed or not pulsed with antigen.
  • the APC may be genetically engineered to present antigen in the context of MHC glycoproteins, and as such, may be termed "synthetic" APC. Immortalized APC are also suitable for use in accordance with the invention.
  • APC can be immortalized via infection with viruses, e.g., Epstein Barr Virus (EBV), human papilloma virus (HPV) and the like); transfection of cells with plasmids containing oncogenes, e.g., SV40 and the like, and other sequences or elements which allow for appropriate transcription, translation, expression and processing of encoded polypeptides in host cells, such as promoters, enhancers, terminators, poly A sequence, and the like; or via mutagenesis, e.g. , using chemical mutagens alone or in combination with ultraviolet radiation.
  • PCL-modified populations of APC that are pulsed with antigen. More preferred are dendritic cells, which may be employed as an essentially homogeneous population of expanded cells, or as a more heterogeneous population isolated from the appropriate tissue source.
  • vaccines, vaccine formulations, compositions and preparations may be either prophylactic and therapeutic.
  • Immunogenic formulations, compositions and preparations may be considered to be vaccines by those having skill in the art.
  • PCL vaccines or immunogens are those comprising cells that have been modified by crosslinking and hydrostatic pressure treatment.
  • the improvement to PCL treatment of cells yields potent immunogens for use as anti-cancer and anti-infected cell vaccines both with and without exogenous adjuvants.
  • the application of pressure to cells exerts powerful biological and physical effects on cellular components. At the biological level, pressure results in a reversible dissociation of the cytoskeleton (B. Bourns et al. , 1988, Cell Motil. Cytoskel. , H ⁇ :380-390), as well as the subsequent transient induction of protein synthesis, in particular, the synthesis of stress-related proteins called pressure-induced proteins (PIPs) such as transporter or chaperone proteins, which participate in the recovery from pressure-induced stress.
  • PIPs pressure-induced proteins
  • HSP heat shock proteins
  • HSP are essential for the transport of MHC/antigen complexes from intracellular compartments to the cell surface and have been shown to be down-regulated in several tumor cells (D. Ang et al., 1991, J. Biol. Chem. , 26 ⁇ :24233-24236; P.K. Srivastava, 1993, Adv. Cancer Res. , £2: 153-177). It has also been recently shown that a number of HSP derived from tumor cells are potent immunogens by virtue of the tumor antigens which are adsorbed onto them (P.K. Srivastava et al., 1994, Immunogenetics, 22:93-98; H. Udono et al., 1994, Proc. Natl. Acad. Sci. USA, 22:3077-3081). Several of these heat shock proteins were found by the inventors to be induced during PCL of tumor cells (B16).
  • the murine B16 melanoma is known to lack the costimulatory ligand B7-1 , and since PCL-modified B16 cells become fully immunogenic, preincubation of cells in dialdehyde, PCL treatment, and the dialdehyde crosslinker and pressure components thereof, may cause the expression of this ligand and/or provide other costimulatory signals to cells, as is described further herein.
  • the elevated immunogenicity of tumor cells following subjection to PCL may result from (i) endogenous peptides forced into the class I and class II intracellular compartments where they are assembled with MHC molecules, routed, and transported through appropriate pathways to be expressed in an extended concentration on the cell membrane; (ii) removal of repressors utilized to inhibit the expression of cancer identity antigens and consequent alleviation of such inhibitory control; (iii) induced changes in the fluidity of the cell membrane, giving rise to an unusual display of self proteins; (iv) active induction of a wide range of proteins such as heat shock proteins, tumor specific antigens and MHC, with documented contribution to T-cell recognition of transformed or infected cells; and (v) co-stimulatory function of the free aldehyde generated by the dialdehyde crosslinker (AdA) and activation of T-cells through a Schiff base reaction, resulting in amplification of T-cell activation.
  • endogenous peptides forced into the class I and class II intracellular
  • tumor cells are modified by an improvement to the basic PCL method to produce an immunogen derived from modified tumor cells and capable of inducing an anti-tumor immune response, wherein the modified tumor cells have been prepared by first preincubating the tumor cells with dialdehyde crosslinking agent, and then exposing the preincubated cells to the dialdehyde crosslinking agent and to hydrostatic pressure at a concentration and for a time sufficient to cause a modification of proteins in the cells' plasma membranes. Preferred is exposure of the preincubated cells to crosslinking agent and to hydrostatic pressure at the same time, following the preincubation.
  • the preincubation was found to insure that the resulting immunogenicity of the PCL-modified tumor cells in the immunogenic vaccine preparation was fully controlled and afforded a consistent protective response in vivo and a consistent proliferation or stimulation response in in vitro stimulation assays.
  • infected or otherwise transformed cells are modified by the improved PCL method to produce an immunogen derived from the preincubated and PCL-modified infected or transformed cells, which is capable of inducing an immune response against the infecting or pathogenic disease-causing agent, wherein the modified cells have been prepared by first preincubating the infected cells with dialdehyde crosslinking agent, and then exposing the preincubated cells to crosslinking agent and to pressure at a concentration and for a time sufficient to cause a modification of proteins in the cells' plasma membranes.
  • after preincubation cells are exposed to crosslinking agent and to hydrostatic pressure at the same time.
  • An alternative embodiment encompasses direct PCL treatment, with and without dialdehyde preincubation, of bacterial cells, as well as other pathogenic disease causing cells, to augment relevant antigen expression and the use of the PCL-modified whole cell bacteria, and the like, in PCL vaccines.
  • PCL treatment of whole cell padiogenic bacteria and other cellular pathogenic organisms can result in d e expression of greater levels of the components that correlate with increased virulence indices.
  • APC are PCL-treated in accordance with the improved invention and the PCL-treated APC are used in vaccine and immunogenic preparations.
  • PCL-treated APC can also be used in IVS cultures to proliferate antigen-specific CTLs in vitro for use in immunoadoptive therapy.
  • the PCL-treated APC can be employed in combination with the proliferated CTLs and used as an active component in immunoadoptive therapy.
  • Non-limiting examples of APC suitable for use include dendritic cells, macrophages, astrocytes, fibroblasts, fibrocytes, Langerhans cells and endothelial cells. It is also envisioned that the aforementioned types of APC can be immortalized or genetically engineered to express exogenous genes to allow, enhance, or improve antigen presentation in the engineered cells.
  • antigen-specific T cells may be generated using professional APC (e.g. , DCs, fibrocytes, macrophages and the like) in the following manner.
  • the antigen(s) toward which the T cells are specifically activated can include those derived from tumors, cancers, viruses, parasites, bacteria, and the like.
  • T cells obtained from blood, lymph nodes, tumors or lesions are incubated with professional APC that have been previously pulsed with antigen or antigen extract, e.g. , cell extract, subcellular fractions, purified proteins or peptides.
  • the T cells are can be purified by conventional methods, such as FACS, magnetic beads, positive or negative selection and the like), or they can be stimulated as heterogeneous cell suspensions.
  • Professional APC can also be added to co-cultures of T cells with intact stimulator cells as "third party" cells.
  • the stimulator cells may be tumor or cancer cells, virus-infected cells, or bacteria- or parasite-infected cells, for example.
  • T cells and APC e.g., DC
  • APC e.g., DC
  • APC e.g., DC
  • DC antigen or T cells + stimulator cells + APC
  • TCR T cell receptor
  • T cell cultures can be kept for up to 21 days before requiring additional antigenic challenge.
  • T cell cultures can be rechallenged with APC (DC) and antigen at intervals of 14 to 21 days and for as many times as is required by the ongoing therapy.
  • This aspect of the invention also encompasses mixtures of several types of APC.
  • DC are extremely potent APC and must be used at the correct ratios, i.e., T cells: APC : antigen, to avoid the possibility of apoptosis upon additional restimulation or challenge with antigen.
  • the correct ratios are empirically determined as known to those in the art and may change according to culture conditions, state of the cells and type of antigen.
  • the effects of potent APC are attenuated by dilution of their numbers by the addition of less efficacious APC, such as B cells, fibrocytes, or macrophages, (also called filler cells, which are irradiated (3000 rad) leukocytes/lymphocytes) to the more potent APC cultures.
  • APC such as B cells, fibrocytes, or macrophages
  • the nature of the antigen may also dictate the use of a mixture of APC. If the antigen is soluble, fibrocytes and B cells may be sufficient for antigen processing and presentation. Accordingly, the addition of DC to cultures of B cells or fibrocytes increases the signals due to the high density of co ⁇ stimulatory molecules or carries out antigen processing of particular proteins found in an antigen preparation.
  • the preparation of non-PCL-treated B cells, macrophages and DC for use as APC is known to those having skill in the art.
  • the APC may be co-cultured with intact autologous cancer or tumor cells, or with intact autologous cells containing intracellular pathogens (e.g., cells infected with viruses, bacteria, parasites, yeast, algae, fungi, and the like, which express antigens associated with the pathogenic organism).
  • the autologous cancer, tumor or infected cells are preferably PCL-modified or PCL-modified and coupled to biological response modifiers (BRMs) as described herein.
  • BRMs biological response modifiers
  • the APC may be co-cultured with intact allogeneic cancer or tumor cells, or with intact allogeneic cells containing intracellular pathogens, as described above.
  • the allogeneic cancer, tumor or infected cells are preferably PCL-modified or PCL- modified and coupled to biological response modifiers as described herein.
  • the APC Before or after co-culture, the APC can be unmodified, PCL-modified, or PCL-modified and coupled to BRMs. Preferred are APC that have been PCL-modified or PCL- modified and coupled with BRMs in accordance with the improved methods of the invention.
  • the cancer or tumor cells may be derived from an intact cancer or tumor, an excised cancer or tumor, or may be immortalized or transformed cell lines.
  • APC can be co-cultured with disrupted cells, cell membranes, cell fragments and subcellular fractions prepared by methods known to those having skill in the art.
  • APC can be pulsed with a variety of immunogenic agents to allow the endogenous uptake of these agents, processing and antigen- presentation on the surface of the APC.
  • agents with which APC can be pulsed include, but are not limited to, autologous or allogeneic whole cell lysates or subcellular fractions thereof; defined peptides or proteins, combinations of different peptides, combinations of different proteins; antigens harvested from cancer or tumor cells wherein the cells are autologous, allogeneic or grown as cell lines; antigens harvested from cells infected with viruses and other pathogenic microorganisms; synthetic antigens, including peptides and polypeptides; and isolated RNA.
  • the APC Before or after pulsing, the APC can be unmodified, PCL-modified, or PCL-modified and coupled to BRMs. Preferred are APC that have been PCL-modified or PCL- modified and coupled with BRMs in accordance with the improved methods of the invention.
  • APC e.g., DC
  • APC e.g., DC
  • APC e.g., DC
  • the time of incubation is dependent upon the particular antigen.
  • Peptides bind to surface MHC within minutes.
  • Antigens which require processing i.e., peptides containing about 20 or more amino acids; proteins; membrane fragments or cell fragments, are usually incubated with APC for about 5 to 16 hours. The rate and final dose of antigen uptake are dependent upon a given APC.
  • the antigen is usually present in excess, for example, about 10 to 100 ⁇ g/ml of protein or peptide, or about 1-0.05 equivalent tumor cell extract per APC.
  • PCL treatment of APC is preferably carried out after pulsing APC with particular antigen or polypeptide.
  • APC frequently lose their capability to process antigen, e.g., proteins, cell fragments and the like, as described above. Therefore, the presentation of short peptides is not compromised.
  • infected cells or tumor cells are the antigen source, such cells can be PCL-treated, disrupted and used to pulse APC. These APC can then be PCL-modified following the completion of the incubation period with antigen.
  • the co-cultured or antigen-pulsed APC can also be used in immunogenic preparations comprising T lymphocytes which have been proliferated with cells bearing antigens associated with particular cancers, tumors or infectious organisms.
  • the method generally involves obtaining a tissue source of APC (e.g., adult, fetal or cord blood, bone marrow, spleen or afferent lymph); enriching, expanding and/or proliferating the APC by methods known in the art; exposing the APC via co-culture or antigen pulsing to cancer, tumor or infected cell-associated antigens to achieve antigen presentation and activation by the APC; incubating the antigen activated APC in dialdehyde crosslinker, particularly, 2', 3' nucleotide or nucleoside dialdehyde crosslinker from about 15 to 30 minutes, treating the crosslinker treated APC with hydrostatic pressure of about 500 to 1400 atmospheres, preferably 1000 to 1200 atm and washing the PCL-modified APC
  • T cells from a human peripheral blood mononuclear cell source are expanded and proliferated by IVS assay in which the stimulator cancer, tumor or infected cell antigens are displayed on intact cells or plasma membranes thereof.
  • the stimulator cells are preferably PCL-treated in accordance with the invention prior to their use as stimulators in the IVS assay.
  • the intact cancer or tumor cells, and/or antigen-pulsed APC demonstrate an increased immunogenicity and an immense capacity to elicit a vigorous anti-cancer, tumor or infected cell immune response.
  • cytotoxic T cells triggered by the above-PCL-modified cells secrete elevated levels of cytokines and lymphokines of the THl type to produce an active and specific cell-mediated response which targets and removes cancer, tumor or infected cells.
  • DC Dendritic cells
  • cell extract prepared from a pool of various allogeneic EBV-associated B-cell lymphoma were used as a cellular anti- cancer vaccine to generate tumor-specific T-cell lines.
  • DC-pulsed with tumor antigens (“loaded APC") derived from the cell extract were shown to trigger a strong T-cell activation against the B-cell lymphoma, as measured by proliferative response and cytokine secretion.
  • PCL-modification of DC pulsed with tumor extract added a dramatic increase to their immunogenic potential as compared with unmodified DC-pulsed with tumor antigen (Example 17 and Fig. 8).
  • 2', 3'-nucleoside and nucleotide dialdehydes such as 2', 3'-adenosine dialdehyde (AdA) act as chemical crosslinkers of cell surface proteins in the PCL process through the action of providing a linkage between newly exposed proteins, as well as anchoring these proteins to the - 24 -
  • AdA molecules that were not involved in active crosslinking of cell membrane structures could react with membrane proteins (or proteins and peptides in general) through a single aldehyde moiety as monoaldehydes, thereby leaving the remaining aldehyde moiety free, i.e., a pendant aldehyde, e.g., (2', 3'-adenosine monoaldehyde or AmA).
  • a pendant aldehyde e.g., (2', 3'-adenosine monoaldehyde or AmA
  • preincubation with dialdehyde allowed the cell membrane to be effectively derivatized with a monoaldehyde molecule, e.g., AmA, which was ultimately capable of forming a reversible chemical covalent bond (i.e., a Schiff base) with a CD4 + T cell.
  • a monoaldehyde molecule e.g., AmA
  • the chemical reaction is depicted, for example, as follows:
  • dialdehyde can function as both a crosslinker of cell membrane components and as a reagent for monoaldehyde derivatization of cellular surfaces.
  • This newly determined function of dialdehydes is achieved based upon the generation of two monoaldehyde moieties following the reaction of a cell (i.e., a cell surface protein or antigen (Ag)) with dialdehyde.
  • the dialdehyde can form a reversible chemical linkage with membrane protein, peptide or carbohydrate, and by means of its other pendant aldehyde group, the dialdehyde ultimately can also provide a costimulatory or immunopotentiating linkage and recognition between a monoaldehyde (e.g., AmA) on the surface of a modified tumor cell and the amine group of cell surface protein on a T cell, for example.
  • a monoaldehyde e.g., AmA
  • the invention encompasses the use of the monoaldehyde generated on the cell surface after treatment with dialdehyde, as a linking molecule for reaction with other biological molecules (e.g., cytokines, adjuvants, lymphokines) for slow or prolonged release of the attached molecule at a vaccination or immunization site.
  • Other biological molecules e.g., cytokines, adjuvants, lymphokines
  • the preincubation permits the formation of the critical concentration of monoaldehyde on the cell surface (i.e., not involved in intramembrane crosslinking) to provide a costimulatory signal to lymphocytes enhance and augment immunogenicity.
  • the dialdehyde crosslinking agent employed as a protein crosslinker in the improved method of PCL modification of cells also functions as a general reagent for derivatizing proteins, thereby yielding a preincubated and PCL-modified cellular immunogen in which a dialdehyde with which the cells are reacted has one of its aldehyde groups capable of interacting with lysine groups of a second molecule such as an antigen, e.g.
  • a protein antigen, or a carbohydrate antigen so as to form one monoaldehyde linkage with antigen, thereby leaving the other pendant monoaldehyde group capable of interacting with another suitable molecule, for example, a protein on the surface of a cell, a nonmembrane-bound cytokine or peptide or portion thereof, or another immunostimulatory molecule.
  • another suitable molecule for example, a protein on the surface of a cell, a nonmembrane-bound cytokine or peptide or portion thereof, or another immunostimulatory molecule.
  • Such an immunogen is able to stimulate the immune response, in all likelihood, by functioning as a costimulatory or immunopotentiating immunogen wherein the dialdehyde may act as an adjuvant for increasing the immune response.
  • a chemical crosslinking agent which is a dialdehyde, preferably a 2', 3'- nucleoside or nucleotide dialdehyde, which is referred to herein as a "crosslinker” or "crosslinking agent” or “crosslinking compound”.
  • the cells are subjected to a hydrostatic pressure in the range of from 800 to 1400 atmospheres, preferably about 1000 to about 1200 atm.
  • pressure of above about 1400-1600 atm or greater yields an immunogen having a far inferior anti-cancer immunization potency.
  • the application and release of pressure is preferably gradual, e.g., over a period of 5 to 15 minutes. Immunogenicity of the modified cells is even further augmented if cells are exposed to crosslinking agent and to hydrostatic pressure at the same time.
  • the preferred crosslinking agent is a 2', 3'-dialdehyde of a natural nucleotide or nucleoside, since non-naturally occurring, i.e. synthetic, nucleosides or nucleotides are very often highly toxic.
  • Such crosslinking agents are represented by the following formula I:
  • R is H, or a mono-, di- or tri-phosphate group
  • B is a nucleotide base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.
  • crosslinking agents are 2', 3'-adenosine dialdehyde (AdA) and 2', 3'-adenosine monophosphate dialdehyde (AMPdA).
  • the compound of formula I may be prepared by reacting a nucleoside or a nucleotide of the following formula II with an oxidizing agent, e.g. an alkali periodate:
  • an oxidizing agent e.g. an alkali periodate:
  • the improved PCL modification process is performed by preincubating isolated tumor cells or infected cells, and the like, with 2', 3'- nucleoside or nucleotide dialdehyde crosslinking agent in the range of about 1 mM to about 40 mM, preferably about 5 mM to 30 mM, and more preferably about 10 mM to about 20 mM, for a period of about 15 to 120 minutes, preferably about 20 to 60 minutes and more preferably for about 30 minutes, preferably at room temperature, i.e., about 25°C.
  • a dialdehyde concentration of 10 mM for 30 minutes at room temperature was found to be especially effective for the preincubation of cells.
  • cells were PCL modified as described herein and in Example 2 by exposure to dialdehyde crosslinker (e.g. , 10 mM) and hydrostatic pressure (e.g., 1200 atm), with exposure to dialdehyde crosslinker and to pressure preferably being carried out at the same time at room temperature.
  • dialdehyde crosslinker e.g. 10 mM
  • hydrostatic pressure e.g. 1200 atm
  • optimal immunogens are generated from the improved PCL method when cell viability following preincubation and PCL modification is greater than or equal to about 65 to 75%, as determined by Trypan Blue dye exclusion.
  • Another embodiment of the invention encompasses in general the formulation of compositions and products comprising dialdehyde as costimulatory adjuvanting molecules linked to antigens via one aldehyde group and having a pendant aldehyde group for chemical interaction with other suitable molecules, as described herein.
  • the pendant monoaldehyde moiety which is unreacted with antigen in the composition, serves as a linker for attachment of other biological response molecules, e.g., cytokines, or peptides thereof, which may than act as slow or prolonged release adjuvanting agents at the site of vaccination or immunization, as further described hereinbelow.
  • compositions comprising dialdehydes reacted with antigenic protein or peptides or carbohydrate (either on cell surfaces or isolated antigens, preferably isolated antigens) provide an immunogen with enhanced or increased antigenic specificity and function.
  • the compositions of the invention provide a controlled immunostimulatory signal to activated T cells to maximize the activation of responder T cells in a T culinaryl mode.
  • the compositions may be used both therapeutically and prophylactically.
  • Another embodiment of the invention comprises a dialdehyde component reacted with an antigenic component, e.g., a protein, a peptide, or a portion thereof, or a carbohydrate, for use in a vaccine or immunogenic formulation to afford specific immunological protection against the antigen as well as adjuvanting/costimulatory functions afforded by the monoaldehydic reacting groups generated by the dialdehyde.
  • the antigen component may be considered to be "modified” via its chemical interaction with dialdehyde; thus, the composition can be considered to contain a "modified antigen".
  • the resulting dialdehyde and antigen composition comprises a compound having one monoaldehyde group of the dialdehyde component attached to the antigenic component and a pendant monoaldehyde group of the dialdehyde component available for complexing with cell surface protein amine groups or the amine groups of non cell-surface-bound protein molecules.
  • the modified antigen and a pendant aldehyde group can be schematically depicted as follows: A-X-A + Immunogen «• Immunogen-mA-X-mA; wherein A-X-A represents the dialdehyde that chemically reacts with an immunogen; Immunogen represents an antigen, either cellular or noncellular to which the dialdehyde is chemically complexed; mA-X-mA represents the monoaldehyde group of the dialdehyde which chemically reacts with immunogen, and the pendant monoaldehyde group that is capable of complexing with another protein or carbohydrate molecule.
  • one of the monoaldehydes derived from the dialdehyde is coupled to Ag or immunogen, while the other pendant monoaldehyde is free to combine with the appropriate chemical groups (e.g., amine groups) on other protein or carbohydrate structures.
  • antigens such as proteins or carbohydrates, that are suitable for interaction with the pendant aldehyde group of the dialdehyde may be present on the surface of cells, or they may be unbound to cell membranes.
  • dialdehydes for example, the 2', 3'- nucleoside and nucleotide dialdehydes, and in particular, AdA, as costimulatory adjuvanting molecules in the methods and compositions described herein is that the dialdehydes are operative and effective over an unexpectedly wide range of concentrations (e.g., from nanomolar to millimolar concentrations) as demonstrated by in vitro sensitization experiments. (Fig. 7).
  • the present invention provides a number of other advantages to the art for increasing and enhancing the immune response to foreign or non-self antigens, namely, eliciting the presentation of immunologically important molecules on the cell surface for enhanced recognition by cells of the immune system; generating costimulatory molecules for T cells in a T H 1 mode, which is crucial for the cytotoxic response to eradicate tumor and infected cells; and providing anchors for the attachment of biological response modifiers to control environmental events at the site of immunization.
  • the invention also addresses a daunting problem in the art concerning ways in which to stimulate, increase, and improve a host animal's immune response to tumor cells or otherwise transformed, infected, or foreign cells, by providing modified immunogenic cells which present tumor and foreign antigens to a host in the context of MHC (or HLA) molecules for appropriate recognition and destruction by the effector cells of the immune system.
  • the modified cells, or membranes or proteins derived from the cells are used either prophylactically or therapeutically as immunogens or in immunogenic preparations to immunize both naive animals, including humans, as well as to treat rumored, infected, or diseased animals, including humans, which have previously encountered the tumor or foreign antigens.
  • the immunogens containing antigen- bound monoaldehyde and free monoaldehyde resulting from the exposure of antigen to dialdehyde are further able to enhance a recipient's immune response via costimulatory or adjuvanting functions.
  • Preparation of the dialdehyde and cellular or noncellular immunogen/antigen composition comprises mixing and incubating the immunogen with dialdehyde at a concentration of between about 1 mM to 40 mM, preferably about 5 mM to 30 mM, and more preferably about 10 mM to 20 mM, for a period of about 15 to 120 minutes, preferably about 20 to 60 minutes and more preferably for about 30 minutes, preferably at room temperature, i.e., about 25 °C.
  • a dialdehyde concentration of 10 mM for 30 minutes at room temperature is effective for saturating the available sites of the immunogen, thereby allowing for the binding of a monoaldehyde group of the dialdehyde molecule to the immunogen and for providing a free monoaldehyde group for binding to other cell structures, or to proteins, peptides, carbohydrates, and the like.
  • the dialdehyde and antigen compositions of the invention may be formulated into a pharmaceutically acceptable preparation comprising suitable carrier, excipient, or buffer and injected into a mammal in an amount effective to restore immune function to normal or near-normal levels or to increase the immune response to the particular antigen above normal levels to control, reduce, or eradicate a tumor or infection.
  • the carrier is typically a conventional carrier for an injectable vaccine.
  • the dialdehyde and antigen composition is used in an aqueous diluent which may also contain other soluble or particulate antigens, either alone or associated with a lipid carrier.
  • dialdehyde and antigen composition may be incorporated into water-in-oil emulsions, in liposomes, or in vehicles containing additional immunostimulatory or adjuvanting elements such as muramyl dipeptide for release at the site of administration, if such additional elements are necessary or desired.
  • the vaccine formulation comprises the antigenic component and the dialdehyde component, which may be considered to afford adjuvanting properties to the formulation.
  • suitable amounts of vaccine formulation for injection into a host are from about 50 ⁇ l to about 5 ml, preferably about 100 ⁇ l to about 2 ml, more preferably about 200 ⁇ l to about 500 ⁇ l.
  • the smaller amounts are generally more suitable for subcutaneous or intradermal routes of administration, while the larger amounts are generally more suitable for intramuscular routes of administration.
  • dialdehyde and antigen components of the composition can be mixed prior to immunization, and then injected into a host or mammalian recipient, or each component may be injected independently at the site of immunization to provide the dialdehyde and antigen-containing immunogen, which is capable of stimulating and/or augmenting the recipient's immune response to the specific antigen, essentially in situ at the inoculation site.
  • the dialdehyde and antigen composition may be "further modified" by reacting the unreacted pendant aldehyde group of the dialdehyde with an immunostimulatory, adjuvanting, or immunopotentiating compound.
  • the composition can be made more highly immunogenic or can afford further adjuvanting or immunostimulatory properties after immunization in a recipient host.
  • the modified composition can also be formulated into a pharmaceutically acceptable preparation comprising suitable carrier, excipient, or buffer and injected into a mammal, including humans, to increase the immune response to antigen.
  • dialdehyde and antigen composition may be admixed with other dialdehyde and antigen compositions to form a multivalent adjuvanting immunogen in which various antigens or elements thereof are combined.
  • the dialdehyde molecule may be linked to two antigen molecules or elements thereof, via each of its aldehyde groups, or to two adjuvant molecules, e.g., lymphokines or cytokines, and used and administered separately or combined with other dialdehyde and antigen, or other dialdehyde, antigen, and adjuvant-containing compositions of the invention.
  • dialdehydes suitable for use in the compositions of the invention include, but are not limited to, 2', 3 '-nucleoside or nucleotide dialdehydes, as described hereinabove, particularly, 2', 3'-adenosine dialdehyde and 2', 3'-adenosine monophosphate dialdehyde, malonic dialdehyde, and glutaric dialdehyde, with 2', 3 '-nucleoside or nucleotide dialdehydes being preferred.
  • 2', 3 '-nucleoside or nucleotide dialdehydes being preferred.
  • antigenic components suitable for use in the compositions include, but are not limited to, any molecular structure that is antigenic and which contains amino groups, or can be induced, treated, or modified to contain amino groups, and which can be reacted with dialdehyde, such as proteins, polypeptides, peptides, and carbohydrate antigens.
  • the antigens may be tumor or cancer antigens or peptides, antigenic proteins or components of bacteria, RNA and DNA viruses, particularly envelope polypeptides for enveloped viruses, parasites, protozoa, mycoplasma, fungi, and yeast.
  • cancer or tumor antigens which may be used include those of various origins, for example, those derived from pancreatic tumors, ovarian tumors, cervical tumors, melanomas, sarcomas, breast cancers, gastric cancers, colon cancers, lung cancers, cancers of the head and neck, brain tumors, laryngeal cancers, rectal cancers, small lung carcinomas, non- small lung carcinomas, renal cancers, epitheliomas, bladder cancers, prostate cancers, lymphomas, non-Hodgkin's lymphoma, and leukemias.
  • the antigen for formulation in the antigen and dialdehyde composition may be a protein or peptide that is synthetically or recombinantly produced, and isolated and purified as required prior to reacting with dialdehyde.
  • the antigen may be a tumor associated antigen (TAA), or portion thereof, for which the gene has been cloned and the nucleic acid and amino acid sequences determined.
  • TAAs tumor associated antigen
  • Examples of TAAs that may be used in the compositions include, but are not limited to, melanoma TAAs which include, but are not limited to, MART-1 (Kawakami et al., 1994, J. Exp. Med.
  • the TAAs can also be CA-19-A (pancreatic cancer), CA-125 (ovarian cancer), PSA (prostate cancer), erb-2, (breast cancer), HER-2neu (breast and ovarian cancer), CA-171A and the like (Boon et al., 1994, Ann. Rev. Immunol. 12:337).
  • the antigens for use in the dialdehyde and antigen composition are not intended to be limited to the above listed TAAs.
  • Viral antigens for use in the invention include subunit antigens from influenza virus, from human immunodeficiency virus, e.g., gp 120 and gp 160, and from hepatitis viruses. Attenuated, e.g., heat-killed or detergent-killed, whole organism vaccines are suitable for use with the dialdehyde component in the formulation of more highly immunogenic vaccine preparations.
  • Other examples of viral antigens include antigens from cytomegalovirus, polio virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus, Epstein Barr virus, adenovirus, rhinovirus.
  • Animal viruses whose antigens are suitable for use in the compositions of the invention include rabies virus, foot and mouth disease virus, equine flu, equine encephalitis, feline immunodeficiency and feline leukemia viruses.
  • Bacterial antigens for use in the compositions of the invention include among the non-limiting examples, B. pertussis, from which the following antigenic components may be used with dialdehydes as adjuvanting compounds to provide effective vaccines: pertussis toxin, filamentous haemagglutinin, and pertactin, P69; £. tetani. N_. meningitidis. N. gonorrhea. £. aureus. S_. pneumoniae. H. influenzae, £.
  • Parasites e.g. , P. falciparum: - major
  • yeast e.g., Aspergillus: Q. albicans and the like
  • fungi or algae also offer protein or carbohydrate antigenic components for use with dialdehydes in the compositions of the invention.
  • Pathogenic protozoans having antigenic components that may be used include malaria, Babesia. Schistosoma. Toxiplasma. Toxocara canis. and the like. Protein or antigenic components from these and other viruses and bacteria may be used in the dialdehyde-containing formulations of the invention to provide vaccines and immunogenic preparations against pathogenic infections.
  • immunostimulatory, adjuvanting, or immunopotentiating molecules that can be reacted with the pendant monoaldehyde group in the above- described compositions of the invention include, but are not limited to, cytokines such as interleukins IL-1 (i.e., IL-l ⁇ and IL-l ⁇ ) to IL-16; interferons such as alpha, beta or gamma interferon; hematopoietic cell growth and proliferation factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), human growth hormone (hGH); TNF- ⁇ and TNF- ⁇ ; RANTES (Regulated upon Activation, Normal T Expressed and presumably Secreted cytokines, Promega, G5661), and the like; leukotrienes; T cell proliferation factors, e.g., OKT3, and the like, and as described herein
  • immunostimulatory molecules are B7-1 , B7-2, ICAM-1, ICAM-2, LFA-1 , LFA-3, CD72 and the like.
  • Preferred cytokines include IL-2, IL-12, IFN- ⁇ (gamma interferon), and TNF- ⁇ .
  • chemokines that may also be used in the present invention include, but are not limited to, CTAP III, ENA-78, GRO, 1-309, PF-4, IP-10, LD-78, MBSA, MlP-l ⁇ , MIP-1B, and the like. It is to be understood that combinations and mixtures of the foregoing examples of immunostimulatory, adjuvants, or immunopotentiating molecules may be attached to an antigen or immunogen through the dialdehyde of the compositions in accordance with the invention.
  • B7-1 and B7-2 are both members of the Ig gene superfamily. These molecules are present on macrophages, dendritic cells, and monocytes, i.e., antigen presenting cells (APC). If a lymphocyte encounters an antigen alone, without costimulation by B7-1 or an equivalent stimulatory event or signal, it will respond with either anergy or apoptosis (programmed cell death); if the costimulatory signal is provided, it will respond with clonal expansion against the target antigen.
  • APC antigen presenting cells
  • the dialdehyde component of the present invention is capable of providing costimulatory signaling via its monoaldehyde functionality and its interaction(s) with cell surface molecules, particularly those on T cells, more particularly T cells of the T H 1 subclass.
  • the B7-2 costimulatory molecule is generally associated with the stimulation of a T hinder2 type immune response; thus, B7-2 linked via the pendant monoaldehyde in the dialdehyde and antigen composition could stimulate T cells of the T H 2 subclass, if necessary or desired.
  • compositions of the invention may be used alone or in combination with other therapeutic agents for the treatment of cancers and pathogenic diseases caused by the above infections and conditions.
  • combination therapies may comprise the administration of at least one composition of the invention or a physiologically functional derivative thereof, and at least one other pharmacologically active ingredient.
  • the pharmacologically active ingredient(s) may be administered separately or together. When administered separately, this may occur at the same time or sequentially in any order. Based on conventional methods and knowledge, those skilled in the art will select the amounts of the active ingredient(s) and pharmacologically active agent(s) and the relative timing, schedule, and mode of administration to achieve the desired combination therapeutic effect.
  • such combination therapy comprises the administration of at least one dialdehyde and antigen composition of the invention and another therapeutic agent.
  • therapeutic agents are those employed, for example, in the treatment of HIV or hepatitis virus infections or associated conditions, such as the 2', 3'-dideoxynucleosides, e.g., 2', 3'-dideoxycytidine, 2', 3'-dideoxyadenosine, and 2', 3'-dideoxyinosine; 3'-azido- 3'-deoxythymidine (zidovudine); acyclic nucleosides, e.g., acyclovir; interferons, e.g., ⁇ -interferon; TAT inhibitors; protease inhibitors; ribavirin; nucleoside transport inhibitors, and the like.
  • the dialdehyde component of the invention can be considered to serve as adjuvant in combination with antigen in a vaccine or immunogenic preparation.
  • a vaccine or immunogenic preparation may be formulated comprising the dialdehyde component and the antigen component in admixture with one or more acceptable carriers and other therapeutic agents, if desired.
  • Acceptable carriers are those which are compatible with other ingredients in the formulation and not deleterious to the host or patient.
  • the dialdehyde and the antigen components may be prepared in suitable buffer or diluent and admixed or combined prior to or at the time of administration as immunogen.
  • the route of administration may include parenteral, including subcutaneous, intradermal, intramuscular, and intravenous; intranasal, oral, rectal, inhalation, or topical.
  • Effective amounts of the compounds in the composition and an effective dose of the composition will depend upon a number of factors known to those having skill in the art, including the nature of the recipient host, i.e., human, non-human primates, other mammalian species; the type and size of the host; the severity of the disease, cancer, or condition being treated; the route of administration; and general and specific responses by the patient.
  • the total daily dosage may be divided and administered in portions throughout the day, if desired.
  • An effective dose of the compositions of the invention via the above- mentioned routes of administration for human use for the purposes of vaccines and to provide costimulatory signals can be conventionally determined by the practitioner. Effective doses and amounts will depend on a number of factors mentioned hereinabove, known to those in the art. As a general guide, doses may range from 0.01 to 100 g/M 2 in a human recipient, preferably in the range of 0.05 to 50 g/M 2 , more preferably in the range of 1 to 10 or 20 g/M 2 . An exemplary dose range is about 1 to 3 g/M 2 , for example, for two to five, preferably three, days per month.
  • the proper dosage and administration schedule for a particular situation is within the skill of the medical practitioner, and significantly lower doses (i.e., in the ng and mg ranges) may be administered, given the potency and large effective concentration range of action of the dialdehyde as adjuvant or immunostimulator.
  • the effective dose as desired may be administered at multiple appropriate intervals throughout the day, e.g., as between two and five sub-doses, or for a period of several days monthly.
  • Formulations containing a daily dose or unit daily subdose as described or known to those in the art, or an appropriate fraction thereof, of the compounds of the invention, or physiologically acceptable salts thereof, are preferred unit dose formulations.
  • parenterally-administered drugs may have much greater bioavailability than the oral forms, parenteral doses may be only a fraction of what would be given orally.
  • Pharmaceutical preparations containing the dialdehyde and antigen-containing compositions as described and conventional pharmaceutical carriers may be employed in unit dosage forms such as solids or liquids.
  • the methods for preparing unit dosage formulations are well known and practiced in the art of pharmacy. All of the methods involve bringing into association the compounds of the compositions, i.e., the active ingredients, with the carrier which comprises one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient(s) with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.
  • solid form preparations include, for example, tablets, pills, capsules, powders, dispersible granules, cachets, and suppositories.
  • Liquid form preparations include isotonic solutions, suspensions, or elixirs for oral administration or liquid solutions, suspensions, and emulsions for parenteral use.
  • Suspensions may be in an aqueous or non-aqueous liquid, as an oil-in- water liquid emulsion, or as a water-in-oil liquid emulsion.
  • the active ingredient(s) may be presented as a bolus, electuary, or paste.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these in packaged form. Tablets contain the active ingredient in admixture with non-toxic, pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating or disintegrating agents, for example, maize starch or alginic acid; binding agents, for example, starch, gelatin, or acacia; and lubricating agents, for example, magnesium stearate or stearic acid.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, and thereby provide a sustained action for a longer period of time.
  • the composition may be formed by dispersing the components in a suitable pharmaceutically-acceptable liquid or solution such as sterile physiological saline or other injectable aqueous liquids.
  • a suitable pharmaceutically-acceptable liquid or solution such as sterile physiological saline or other injectable aqueous liquids.
  • the composition may be administered parenterally, including subcutaneous, intravenous, intramuscular, or intrasternal routes of injection or infusion techniques, although conventionally via subcutaneous or intramuscular routes of injection.
  • the composition is in sterile solution or suspension or may be emulsified in pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives and material for rendering the solution or suspension isotonic with body fluids (i.e. blood) of the recipient.
  • Such formulations may be used in unit-dose or in multi-dose containers (e.g., sealed ampules and vials) for convenience.
  • Excipients suitable for use are water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof.
  • the sterile solutions for injection may also contain antioxidants, buffers, and bacteriostatic agents.
  • the unit or multi-dose formulations may be stored in a freeze-dried or lyophilized condition requiring only the addition of sterile liquid carrier, e.g., water, for dissolving the injectable material prior to use.
  • extemporaneous injection solutions may be prepared from sterile powders, granules, and tablets of the kind as described herein.
  • the composition may be presented as a draught in water or in a syrup, in capsules, cachets, boluses, or tablets, as an aqueous or oleaginous solution or suspension, or a suspension in a syrup.
  • suspensions optionally may include suspending agents, or may be presented as an oil-in- water or water-in-oil emulsion.
  • sweetening agents are glycerol, sorbitol, or sucrose.
  • Such formulations may also contain a demulcent and coloring agents.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules, wherein the active ingredient is mixed with an oil medium, for example, arachis oil, liquid paraffin, or olive oil. Additional formulations suitable for other modes - 39 -
  • suppositories may include binders and carriers, for example, polyalkylene glycols or triglycerides.
  • Tablets may contain the preparation as a powder or granules, for example, a lyophilized powder or granules optionally mixed with binders, lubricants, inert diluents, or surface-active or dispersing agents, and may be formed by compression or by mouling in inert liquid diluent. Such tablets may be optionally scored and/or coated.
  • Capsules and cachets may contain the active compound(s) alone or in admixture with one or more accessory ingredients. Capsules may also contain the active ingredients in aqueous or oleaginous solution, suspension, or emulsion, optionally in association with accessory ingredients.
  • each dose may be conveniently contained in, but is not limited to being contained in, volumes of from about 0.1 mL to about 1.0 mL, preferably about 0.5 mL.
  • Aqueous suspensions contain the active ingredients in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example, sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, poly
  • the aqueous suspensions may also contain one or more preservatives, for example, ethyl or n- propyl p-hydroxy benzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin, or sodium or calcium cyclamate.
  • preservatives for example, ethyl or n- propyl p-hydroxy benzoate
  • coloring agents for example, ethyl or n- propyl p-hydroxy benzoate
  • flavoring agents such as sucrose, saccharin, or sodium or calcium cyclamate.
  • sweetening agents such as sucrose, saccharin, or sodium or calcium cyclamate.
  • Suppository formulations for rectal administration may be presented with the usual carriers such as cocoa butter. It is also to be understood that the compounds of the invention or physiologically acceptable salts thereof may be presented as slow release or depot formulations as known in the art from which the active ingredient is released over a prolonged period of time, once the formulation is in place in the body of the recipient, e.g., transdermal patch or subdermal injectables, and the like, as known in the art.
  • the improved method generates cellular immunogens having surface proteins crosslinked by dialdehyde, as well as monoaldehydic functionalities on the cell surface suitable for interacting in an costimulatory capacity with immune cells, for example, or in an adjuvanting capacity.
  • the dialdehyde-preincubated and PCL-modified cellular immunogen may consist of whole modified tumor cells, membranes derived from such cells, as well as protein obtained from such cells or membranes, which substantially retain the capability of the modified tumor cells to induce the anti-tumor immune response.
  • the immunogen may be cells infected with bacteria, parasites, protozoans, yeast, or fungi.
  • Also encompassed by the invention is direct PCL- treatment of bacterial cells, and the like, for enhancing the presentation of antigen of the pathogen to the cells of the immune system to increase the specific immune response against the invading pathogenic microorganisms.
  • Such direct PCL modification of isolated pathogenic cells may serve to present to the cells of the immune system previously unexpressed antigens, or to increase the relative numbers of antigens, which are normally expressed at low or undetectable levels, on the surfaces of the pathogenic cells.
  • the modified cells are exposed to high intensity radiation in order to destroy their genetic material.
  • This is particularly important where the whole modified tumor cells are used for immunization, but may not necessarily be required where said immunogen consists of membrane preparations or membrane- derived proteins.
  • the desired immunogen consists of the whole modified tumor 41 -
  • the product of the above process may be used per se or after several purification treatments, e.g., consisting of centrifugation and removal of the supernatant.
  • the preparation consists of membranes of such modified tumor cells
  • the modified tumor cells are subjected to further treatment in which the cells are disrupted, e.g. by exposure to a hypotonic medium or by sonication, and then the membrane fragments are collected e.g. by centrifugation in a sucrose gradient, as known in the art and as generally described in Example 13.
  • the whole modified cells or the plasma membranes are subjected to further treatment, for example, dissolving or solubilizing the membranes using detergents, separating the proteinaceous material by one of various methods conventionally known, e.g., gel filtration, and then determining which of the separated proteins and/or proteinaceous material fragments possesses the desired immunogenicity.
  • the immunogen may be used for the immunization of cancer patients against their tumor or may be used for the sensitization and proliferation of immune cells in vitro (i.e., in in vitro sensitization or "TVS" assays).
  • the immunogen may be injected into a patient together with a pharmaceutically acceptable carrier or adjuvant in an amount sufficient to achieve an anti-cancer or tumor immune response.
  • peripheral blood mononuclear cells including immune cells, e.g., leukocytes or lymphocytes, are withdrawn from the patient by known methods and are then cultured together with the immunogen until a population of such immune cells reactive against said immunogen is obtained (see Example 10).
  • Such a stimulated population of immune cells may then be reinjected into a cancer patient in order to treat his/her tumor.
  • an allogeneic immunogen While the immunization of patients in accordance with the present invention can be performed by employing an allogeneic immunogen, it is preferably performed by employing an autologous immunogen.
  • the use of an autologous immunogen provides significant advantages in that the immune response which occurs is primarily directed against the neo-antigen of the tumor. When an allogeneic immunogenic preparation is used, the resulting immune response will be against all of the "non-self or foreign antigens of such an immunogen.
  • the use of an autologous immunogen has the further advantage in that the neo-antigens associated with a specific tumor may differ from one patient to another.
  • allogeneic immunogens also provide significant immune responses against PCL- modified allogeneic cells in humans.
  • Methods in which an autologous PCL modified cell preparation is used to prepare an immunogen comprise: withdrawing a tumor growth or infected cell population from a patient by biopsy or surgery; dissociating intact tumor, cells or infected cells, if necessary, by mechanical or enzymatic means; dispersing the cells in a medium; preincubating the cells with 2', 3 '-nucleoside or nucleotide dialdehyde, e.g., 2', 3'-adenosine dialdehyde (AdA); exposing the cells to the 2', 3'-nucleoside or nucleotide dialdehyde, e.g., 2', 3'-adenosine dialdehyde (AdA), and to hydrostatic pressure in a concentration and for a time sufficient to modify proteins in the cells' plasma membranes; preparing a tumor-specific immunogen derived from the preincubated and modified cells obtained; injecting the immunogen into the patient, whereby an anti
  • a preferred embodiment comprises exposing the cells to a hydrostatic pressure between about 800 and about 1400 atmospheres, preferably about 900 and about 1200 atmospheres, more preferably, 1000 or 1200 atmospheres, at the same time that the cells are exposed to the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinker, at a concentration of about 5 to 40 mM, preferably 10 to 20 mM.
  • the method may involve the step of using the modified tumor or infected cells in an in vitro sensitization assay with immune cells to generate stimulated, sensitized immune cells, i.e., leukocytes and lymphocytes, which will react against and ultimately destroy the tumor cells following injection in vivo.
  • Another alternative of the method involves washing the cells following preincubation in dialdehyde and PCL modification, e.g. , three times in HBSS, resuspending the cells in medium containing serum, e.g., human AB serum, and cryopreserving the cells for future use.
  • serum e.g., human AB serum
  • an immunogen derived from modified tumor or infected cells obtained from a defined tumor or infected cell line or cell type may be used.
  • modified tumor cells obtained from the same tumor type from another source or donor individual may be used with equal success.
  • infected cells for PCL modification may be from another source or donor individual having the same infection.
  • the modified cells or cell preparations may include pharmaceutically acceptable carriers, excipients, or formulations, such as normal or buffered saline and the like.
  • PCL- modified cell immunogens may further include non-classical adjuvants which are co-injected in vivo or are formulated into the immunogenic preparation or vaccine comprising PCL-modified cells, cell preparations, plasma membranes, to enhance the immune response. It is to be understood that these types of adjuvants can also be used in the above described compositions comprising dialdehydes and proteins.
  • Nonlimiting examples of such non-classical adjuvants, or mixtures and combinations thereof, that can also be injected with PCL-modified cells or membranes include human growth hormone (hGH), hematopoietic cell stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), and the like, Bacillus calmette guerin or BCG, saponins, T cell stimulating or activating factors, such as OKT3, TNF- ⁇ , and the like, interleukins, e.g., IL-1 to IL-16, and interferons, e.g., alpha, beta, and gamma interferons.
  • hGH human growth hormone
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • BCG Bacillus calmette guerin or BCG
  • saponins such as OKT3, TNF- ⁇ ,
  • adjuvant such as GM-CSF (100 ⁇ g) in conjunction with cellular immunogen (e.g., 3x10* cells) on day 1, and subsequent injections of GM-CSF (75 ⁇ g) alone on days 2 and 3, allow a reduction in the number of immunogen cells required for use in a subsequent immunization by as much as one third to one half the number of cells originally used, without sacrificing vaccine potency.
  • cellular immunogen e.g., 3x10* cells
  • GM-CSF 75 ⁇ g
  • combinations or mixtures of adjuvants may be used in conjunction with PCL-based immunogens to augment the immune response.
  • Non-classical adjuvants may be administered in doses which one skilled in the art may determine by known methods and protocols to be efficacious in augmenting the immune response.
  • non- classical adjuvants may be administered in the range of about 1 ⁇ g to 1 mg per injection, preferably about 100 ⁇ g to about 400 ⁇ g, and more preferably about 50 ⁇ g to 200 ⁇ g.
  • GM-CSF in particular may be administered in the range of about 1 to 500 ⁇ g per injection, preferably about 100 to 200 ⁇ g per injection, and more preferably about 50 to 100 ⁇ g per injection.
  • human growth hormone may be administered in the range of about 0.01 to 3.0 mg/kg of body weight, preferably about 0.05 to 1.25 mg/kg.
  • an immunogenic formulation is prepared comprising PCL-modified cells in the range of at least about 1.0 x 10 s to 1.0 x 10* or more (e.g., to about 20.0 x 10 6 ) per immunizing dose, in admixture with saline or other excipients known in the art.
  • a suitable number of cells is about 1-2 x 10* for an immunizing dose.
  • Those skilled in the art can routinely determine the appropriate cell number for an immunizing dose, depending on the type of tumor or infection under treatment.
  • a greater number of modified cells formulated in the immunogenic preparation may increase or heighten an individual's immune response to his/her tumor or infection.
  • Normal modes of administration e.g., intravenous, subcutaneous, intradermal, intramuscular, sublingual, intraperitoneal, percutaneous, intrathecal, intracutaneous, or enteral, may be used with the immunogenic compositions afforded by the invention; the preferred routes of immunization are intradermal and intravenous.
  • local administration to the afflicted site may be accomplished through means known in the art, including injection and implantation.
  • PCL-modified means cells treated in accordance with the PCL methods of the invention.
  • tumor cell, transformed cell, cancer cell, and infected cell refer to cells which contain, display or present on their surfaces foreign protein or peptide antigens to the cells of the immune system, regardless of whether the presented antigens are autologous or allogeneic to the host.
  • antigen can refer to a protein or peptide structure, molecule, complex, or component thereof, or a carbohydrate that is generally recognized as foreign, non-self, and immunogenic by cells of the immune system. The antigen may be present on the surface of a cell, or it may be unbound or unassociated with a cell surface.
  • an antigen may be an epitope or determinant comprised of a series of amino acid residues, e.g., comprising from about 3 to about 7 residues, or from about 5 to about 10 or more residues, that are recognized or bound by immune cells due to their particular configuration and/or conformation characteristics.
  • the method of the invention encompasses cells which have in some way become distinct from normal cells due to genetic or in vivo events, or to exogenous events or agents, resulting in cancer cells, tumor cells, non-normal or non-self cells, or cells causing another type of pathogenic or disease condition in an animal, including humans.
  • nonlimiting examples of cancer or tumor cells and infected or transformed cells which may be preincubated with dialdehyde and modified by the improved PCL method and used in PCL vaccines of the invention are all types of tumor or cancer cells of various origin, including, but not limited to, cells derived from pancreatic tumors, ovarian tumors, melanomas, sarcomas, breast tumors, colon cancers, lung cancers (e.g., mesotheliomas), small-cell lung carcinomas, non-small cell lung carcinomas, liver or renal cancers, bladder cancers, prostate cancers, tumors and cancers of hematopoietic origin (e.g., lymphomas and leukemias), and the like; as well as virally-infected cells of different origins, including those of the lymph
  • Virus-infected cells include those containing DNA and RNA viruses which infect and/or reside in cells of various types and which may be oncogenic.
  • Non-limiting examples of the types of virally-infected cells that are suitable for PCL modification and used in the invention are human immunodeficiency virus (H ⁇ V)-infected cells of various strains (e.g., HIV-1 and HIV-2), human T-lymphotrophic virus-infected cells, Herpes simplex virus-infected cells, Epstein-Barr virus-infected cells, Hepatitis A, B, C, D, and E-infected cells, and the like.
  • H ⁇ V human immunodeficiency virus
  • HIV-1 and HIV-2 human T-lymphotrophic virus-infected cells
  • Herpes simplex virus-infected cells Herpes simplex virus-infected cells
  • Epstein-Barr virus-infected cells Hepatitis A, B, C, D, and E-infected cells, and the
  • the various cell types may also be transformed, infected, or otherwise altered by a variety of endogenous or exogenous means, such as by genetic mutations or abnormalities, exposure to chemical or physical carcinogens, infection by viruses, intracellular bacteria, parasites, or other disease- causing agents, thereby resulting in cells, e.g., tumor cells or cancer cells or infected cells, that are transformed or morphologically changed away from their normal cell counterparts.
  • Examples of the types of bacterially-infected cells for use in the invention include, but are not limited to cells infected with the mycobacteria that cause leprosy, E. c ⁇ /i-infected cells, S.
  • Non-limiting examples of types of parasites that can infect cells and that can be treated and used in accordance with the invention are malaria, leishmania, and schistosomes, and the like.
  • Example 1 The examples as set forth herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the invention in any way.
  • Example 1 The examples as set forth herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the invention in any way.
  • ARadLV 136 which is a radiation-induced leukemogenic variant of ARadLV, were maintained in vitro as described previously (Haran-Ghere et al., 1977, J. Immunol. 118:600).
  • B16-BL6 melanoma tumor cells syngeneic (i.e., autologous) to C57BL/6 mice were serially passaged in mice by subcutaneous inoculation of 2-5 x 10* cells in 1.0 mL Hanks Balanced Salt Solution (HBSS) or phosphate buffered saline (PBS) at physiological pH.
  • HBSS Hanks Balanced Salt Solution
  • PBS phosphate buffered saline
  • AdA which is a biologically compatible chemical crosslinker
  • AdA was synthesized by a modification of the procedure previously described (Hansske et al., 1974, Bioorg. Chem. 3:367). The procedure was as follows: Adenosine (Sigma Chemical Co., St. Louis, MO) and sodium metaperiodate (Fluka Chemie AG, Buchs. FRG) were mixed in 100 mL aqueous solution to a final concentration of 10 mM of each of these substances, stirred in the dark, and cooled with ice water for 1 hour, and then concentrated to 5 mL under vacuum at 30°C.
  • the resulting concentrate was then incubated for 12 hours at 4°C and the crystalline product which was obtained was separated and found to be homogeneous in thin- layer chromatography (silica gel G plate, 0.2 mm thickness, Merck Darmstadt; running solvent; acetonitrile/water, 4:1 v/v, R F 0.80).
  • the crystals were filtered, washed three times with cold water and dried over silica gel in vacuum (12 mm Hg: 1.6 kPa).
  • the yield of the above preparation procedure was found to be approximately 90% .
  • the obtained product had a melting point of 110°C and melting was accompanied by decomposition, this being in agreement with previous reports (Hansske et al., 1974, supra).
  • the final AdA product as prepared and used in the PCL modification of the invention should be active in crosslinking membrane proteins in accordance with the invention, should be free of iodate impurities, and should be soluble at the pH in which crosslinking is performed (i.e., about neutral) in accordance with the invention.
  • the molar concentration of AdA can be determined by measuring the adenosine concentration or the dialdehyde concentration of the AdA preparation.
  • an AdA concentration of about 20 mM, as determined by measurement of the adenosine concentration of the AdA preparation is equivalent to an AdA concentration of about 10-13 mM, as determined by measurement of the dialdehyde concentration of the same AdA preparation. Accordingly, in vitro and in vivo results obtained using about 20 mM AdA as measured by its adenosine content and those obtained using about 10-13 mM AdA as measured by its dialdehyde content are essentially equivalent.
  • the 2', 3 '-nucleoside and nucleotide dialdehydes have several features which make them advantageous for use in the invention.
  • These crosslinkers are biocompatible and are virtually non-toxic to cells when used in accordance with the invention; they are membrane impermeant; they have a slow rate of uptake into the cell, and thus are retained longer in the cell membrane where they can effect their crosslinking functions; they do not interfere with solubilizing plasma membranes prior to membrane isolation; they are non- immunogenic by themselves; they also possess a relatively long shelf life.
  • the kinetics of AdA binding was determined using approximately 1 x 10 7 B16-BL6 cells and a fH] -adenosine tracer present at a final concentration of 10 mM AdA.
  • the B16-BL6 cells were prepared from freshly excised tumors in mice; however, B16 cells grown in culture may also be used. The cells were incubated in 10 mM AdA for various periods of time, and the amount of AdA bound was determined after washing the cells after incubation for the appropriate time period. All measurements were performed on a beta-counter in duplicate. In Fig. 2, the percent bound AdA was determined as a function of pH. Three pHs were used (pH 7.0; pH 7.5; and pH 8.0).
  • PBMC peripheral blood mononuclear cell
  • x 10 7 to 1 x 10 s freshly harvested or previously cryopreserved cells e.g., tumor cells, infected cells, PBMCs
  • HBSS or PBS preferably sterile
  • AdA 10 mM adenosine dialdehyde
  • PCL treatment was carried out as follows: up to about 5 x 10 7 to 1 x 10* of the preincubated cells were suspended in sterile HBSS containing 10 mM AdA and were immediately exposed to 1,200 atmospheres of hydrostatic pressure for 15 minutes using a pressure bomb, as described below, or more preferably, a computer controlled pressure device (APP, Inc., Ithaca, NY). Following decompression the cells were preferably allowed to remain in AdA for an additional 15 to 50 minutes, usually 45 minutes, prior to washing and further use or storage.
  • a pressure bomb as described below, or more preferably, a computer controlled pressure device (APP, Inc., Ithaca, NY).
  • PCL-modified cells were washed two times in sterile HBSS, irradiated at 10,000 R (10 Gy) and either used immediately (e.g., in an IVS assay) or were cryopreserved at -70°C until further use.
  • cells may be preincubated in dialdehyde, crosslinked with dialdehyde, and then subsequently subjected to pressure as described.
  • the exposure of preincubated cells to dialdehyde crosslinker and to hydrostatic pressure at the same time after dialdehyde preincubation in accordance with the invention comprises an improvement and is the preferred mode of PCL- modification of the present invention.
  • cell surface monoaldehyde e.g.. AmA
  • dialdehyde e.g.. 3' nucleoside dialdehyde AdA
  • B16-BL6 cells were serially passaged in vitro every 3 to 4 days. Cells were cultured as adherent monolayers in complete medium (CM) containing RPMI 1640, 10% heat- inactivated fetal calf serum, 0.03% L-Glutamine, antibiotics (Beit Haemek Biological Industries, Israel), and 50 ⁇ M /3-mercaptoethanol (Fluka, Switzerland, cat. #63690). Cells were preincubated in 10 mM AdA for either 0 or 30 minutes at the indicated temperatures and were then subjected to PCL at 1200 atm for 15 minutes.
  • CM complete medium
  • AdA AdA
  • PCL 30' 4°C 38.9(total)/95.7(38%) 36,000(95,000)
  • B16-B6 cells were preincubated in 10 mM AdA for either 0 or 30 minutes at the indicated temperatures and were then subjected to PCL at 1200 atm for 15 minutes. Following this, cell surface aldehydes were fluorescence-labeled as described in this examples and FACS analysis was performed.
  • k Untreated refers to cells that were not preincubated with AdA and were not subjected to PCL treatment. During the time of these treatments, untreated cells were suspended in HBSS" and maintained at RT.
  • MFC Mean Fluorescence Channel
  • the number of AmA molecules/cell was calculated using a standard quantification kit composed of fluorescent beads loaded with a known number of fiuorescein molecules (Flow Cytometry Standards Corp., San Juan, PR, USA cat. # 826). A calibration plot was calculated using QuickCal ® (Flow Cytometry Standards Corp., San Juan, PR, USA) and the fluorescent values of unknown samples were then calculated from this calibration curve.
  • B16-BL6 cells were serially passaged in vitro every 3 to 4 days.
  • Cells were cultured as adherent monolayers in complete medium (CM) containing RPMI 1640, 10% heat-inactivated fetal calf serum, 0.03% L-Glutamine, antibiotics (Beit Haemek Biological Industries, Israel) and 50 ⁇ M j ⁇ -mercaptoethanol (Fluka, Switzerland, cat. # 63690).
  • CM complete medium
  • AdA 10 mM AdA for various periods of time (i.e., 0, 10, 20, and 60 minutes) at 4°C and then were subjected to PCL at 1,200 atm for 15 minutes. The last two columns in Fig.
  • HA and SBH are aldehyde reducing compounds which convert aldehydes to alcohols.
  • human PBMC were prepared as described by diluting about 30 mL of heparinized blood 1:2 in sterile HBSS and separating the cell populations using Ficoll-Paque cell density gradient centrifugation (1.077 g/cm 3 , Pharmacia). PBMC were harvested from the interphase of the gradient, washed two times in HBSS and stored at 4°C for up to 12 hours until use. IVS cultures were established essentially as described (see Example 10).
  • 2 x 10 5 freshly prepared PBMC served as responder cells in the culture and were incubated at 37°C for 5 days with 1 x 10 3 to 4 x 10 5 sensitizing B16-BL6 tumor cells (i.e., dialdehyde-preincubated, PCL-modified cells or unmodified cells (B16-UNM) as control) in 200 ⁇ L of culture medium in a 96-well microtiter plate (Nunc).
  • Preincubation of tumor cells in AdA dialdehyde was as described in Example 2 and was carried out for either 0 or 30 minutes.
  • 1 ⁇ Ci/10 ⁇ L of [ ⁇ ]-thymidine was added to each well for an additional 4 hours, followed by cell harvesting using an automated cell harvester (Filtermate 196, Packard).
  • the amount of radioactivity which reflected the amount of PBMC cell proliferation in response to the stimulating cells was measured as cpm in a liquid scintillation analyzer (Packard).
  • B16-PCL-30' Virtually no proliferation was observed using unmodified responder B16-B6 cells (B16-UNM) or using responder cells that had not been preincubated in AdA prior to PCL treatment (i.e., B16-PCL-0').
  • B16-BL6 cells were serially passaged in vitro every 3 to 4 days.
  • Cells were cultured as adherent monolayers in complete medium (CM) containing RPMI 1640, 10% heat-inactivated fetal calf serum, 0.03% L-Glutamine, antibiotics (Beit Haemek Biological Industries, Israel) and 50 ⁇ M /3-mercaptoethanol (Fluka, Switzerland, cat #63690).
  • CM complete medium
  • AdA AdA
  • mice The preincubated and PCL-modified cells were then washed two times in HBSS" and were used as immunogens to inoculate two month old C57BL/6J female mice. Two inoculations were given s.c, 1 week apart, with 2 x 10 7 irradiated cells (10,000 R) per inoculation. One week after the second inoculation, the animals were challenged with 10 s viable B16 cells and the tumor diameter was scored as a function of time on day 12, day 15, and day 19. Each group contained 6 mice.
  • UT represents B16 immunogen cells that were neither preincubated in AdA nor PCL-treated, "untreated”;
  • PCL represents B16 immunogen cells that were not preincubated in AdA but were PCL-treated as described;
  • 30' PCL represents B16 immunogen cells that were preincubated in AdA for 30 minutes and were PCL- treated as described.
  • Dialdehydes as adjuvants/immunopotentiators of the cell response to proliferating a ents
  • the dialdehyde AdA as immunopotentiator in culture with PBMC enhanced the proliferation of PBMC to OKT3 as well as or better than the presence of 700 ⁇ M of DHA in the cultures with OKT3 (compare column 4 with columns 6-10). These results demonstrate that AdA causes immunopotentiation or costimulation of the proliferation response of PBMC to OKT3 at an approximately ten thousand fold lower concentration than does the compound DHA (i.e., 0.01 ⁇ M versus 700 ⁇ M, respectively).
  • Example 8 Preparation of human tumor cells from resected human tumors
  • Freshly resected tumors (usually 1-3 x 1-3 x 1-2 cm in size) were transferred in the cold (approximately 4°C) within 1 hour from the operating room (OR). Tumors were transferred to a 100 mm petri dish, rinsed 3-5 times in 10 mL cold PBS, and then transferred to a second petri dish where necrotic and fatty tissue were removed.
  • the tumor tissue was cut into small pieces (approximately 1- 5 mm 2 each), transferred to a 500 mL plastic flask containing: 100 mL of RPMI medium, 50 mg collagenase (Sigma), 1500 units DNase type IV, 5 mg Hyaluronidase type V, 0.01 M Hepes (Biological Industries, Israel), 0.03% L- glutamine (Biological Industries, Israel), Pen 5000 units/Strep 5 mg (Biological Industries, Israel), 1:20 dilution of Fungizone (Biomycin-2, Biological Industries, Israel), and 25 mg gentamycin (Biological Industries, Israel).
  • the tumor cell suspension was transferred to a 50 mL plastic centrifuge tube through a 120 micron nylon mesh, and washed one time in phosphate buffered saline (PBS) or Hank's Balanced Salt Solution (HBSS). The cell pellet was then resuspended in 35 mL PBS and layered gently on top of 15 mL of Ficoll gradient (1.077 g/cm 5 ).
  • PBS phosphate buffered saline
  • HBSS Hank's Balanced Salt Solution
  • cells in the interphase were collected, washed twice in PBS or HBSS by centrifuging at room temperature at 400 x g for 10 minutes, counted for viability using trypan blue exclusion, and diluted or concentrated to obtain the desired cell number.
  • the tumor in patients bearing a tumor, the tumor may be wholly or partially excised by biopsy or surgery employing techniques and practices known to the skilled practitioner. Appropriate precautions are taken for safety and sterility. Individual tumor cells may be dissociated into single cell suspensions or dispersions using conventional enzymatic, chemical, or mechanical means.
  • the tumor cell suspension obtained as described herein from freshly resected tumor tissue and used in accordance with the invention represents a heterogeneous population of cells of which about 20% to about 70% is comprised of tumor cells and about 30% to about 80% is comprised of a mixture of mononuclear cell types from peripheral blood, including accessory cells (e.g., macrophages, monocytes, or antigen-presenting cells) and lymphocytes, that reside within the tumor tissue (for example, see Table 5).
  • accessory cells e.g., macrophages, monocytes, or antigen-presenting cells
  • lymphocytes that reside within the tumor tissue
  • PCL modification of the non-tumor accessory cells or antigen-presenting cells and lymphocytes, which are part of the tumor preparation may affect or enhance the presentation of antigenic molecules, either alone or in combination with MHC proteins and the like, when these PCL-modified cells are used in association with PCL-modified tumor cells in an immunogenic preparation, thereby allowing a better presentation of immunogenic structures by these cells to the lymphocytes, particularly, the T lymphocyte subsets, of the immune system.
  • Example 9 Preparation of cells from solid tumor or ascites
  • HBSS (Ca 2+ -and Mg 2+ -free) as described by Lafreniere, R. and S.A. Rosenberg. 1986. "A novel approach to the generation and identification of experimental hepatic metastases in a murine model", JNCI, 7 ⁇ :309. The suspension was then collected and passed through 100- ⁇ m nylon mesh, washed three times in HBSS, and resuspended at the appropriate concentration.
  • the cells were either frozen in aliquots at -70°C for 24 h and then transferred to liquid nitrogen and stored for later use, or serially passaged in vitro every 3-4 days.
  • Cells were cultured as adherent monolayers in tissue-culture flasks (Falcon 3024), seeded at approximately 3x10 s cells/75-cm 2 flask in 20 mL complete medium containing RPMI-1640, heat-inactivated fetal calf serum (10% v/v), penicillin (100 U/mL), streptomycin (100 ⁇ g/mL), 0.03% fresh L-glutamine, 0.1 mM non-essential amino acids, 0.1 ⁇ M sodium pyruvate, 50 ⁇ g gentamicin/mL, 0.5 ⁇ g solubilized amphotericin B (Sigma Chemical Co., St.
  • the proliferative response of effector cells, i.e., PBMCs or lymphocytes, in the presence of tumor cells was carried out as follows: For human experiments using human PBMCs, 2 x 10 5 viable PBM cells in 0.1 mL of conditioned medium were co-cultured with irradiated tumor cells (10,000 R) in the wells of a 96-well microtiter plate (final concentration of tumor cells in 0.1 mL was 5 x 10 4 to 5 x 10 5 ). Tumor cells were either PCL-treated or PCL-untreated.
  • Tumor cells were prepared by cutting the tumors into small pieces (approximately 1 mm), followed by enzymatic digestion for 2-3 hours, and then separating viable cells on a cell separation gradient (450 x g for 20 minutes at room temperature), or such as is described in Examples 2 and 4.
  • PBMC were isolated from 20 mL of blood taken from the patient, followed by dilution (1:1) in sterile PBS or HBSS and separation on a cell separation gradient as described for tumor cells.
  • PCL- modification was carried out by exposing 5 x 10* to 1 x 10 7 cells (either tumor cells or PBMCs) to 1200 atmospheres of hydrostatic pressure in the presence of 40 mM AdA. Thereafter, cells were irradiated at 10,000 R.
  • the endpoint of the IVS assay was the measurement of cell proliferation which correlates directly with the extent of stimulation.
  • Cell proliferation was measured by adding [ ⁇ J-thymidine for the final 6 hours of incubation of a 5 day IVS assay. Cells were then harvested using a cell harvester (Packard) and radioreactivity retained on the filters was counted using a beta counter (Packard).
  • samples containing 2 x 10 s viable cells i.e., splenocytes
  • 1 x 10 5 irradiated (50 Gy) PCL-treated or untreated B16-BL6 cells in a 96- well flat-bottomed microplate (Nunc Denmark), for 48 h at 37°C in a humidity-controlled incubator under a 5% COj atmosphere.
  • the culture medium consisted of RPMI-1640 medium plus 10% heat-inactivated fetal calf serum supplemented with penicillin (100 U/mL) and streptomycin (100 ⁇ g/mL).
  • Cultures of effector cells and "stimulator" tumor cells were pulsed with [methyl- ⁇ lthymidine (Amersham) after about 42 h of culture, and after 6 h, the cells were harvested and the incorporated radioactivity was measured by conventional methods.
  • in vitro sensitization assays may also be described as mixed lymphocyte culture (MLC) assays.
  • MLC mixed lymphocyte culture
  • T cells respond to foreign histocompatibility antigens on unrelated lymphocytes or monocytes.
  • the test may be performed as either a "one way” or a "two way” assay.
  • the stimulating cells are treated with either irradiation (approximately 1500-2000 R) or with mitomycin to prevent DNA synthesis without killing the cells.
  • the magnitude of the response is the result of DNA synthesis measured in the non-irradiated or non-mitomycin treated cells.
  • DNA synthesis of both stimulating and responding cells represents the net response of both sets of cells.
  • Controls include co-culture of syngeneic irradiated and nonirradiated pairs (to determine baseline DNA synthesis) and co-culture of allogeneic irradiated pairs (to deteimine adequate inactivation by irradiation).
  • responder peripheral blood lymphocytes are mixed 1 : 1 with irradiated stimulator cells and incubated at 37°C in a humidified atmosphere with 5% CO 2 . After 5 days, the culture is pulsed with fHj-thymidine to label the nucleic acid in the responder cells. After 18 hours, the cells are harvested and counted for internalized radioactivity. For example, if the MHC or HLA antigens of the stimulator cells differ from those of the responder cells, the responder cells undergo blastogenesis, synthesize DNA, and proliferate; increased sample radioactivity is the result. If there are no MHC or HLA difference, the cells remain quiescent and no increase in radioactivity is measured at the end of the assay.
  • tissue culture medium e.g., RPMI (Gibco)
  • Anti-CD3 antibody was obtained in culture supernatant prepared from hybridoma cell line number 454 (Dr. J. Lawrence, Cornell Medical Center, N.Y.).
  • Other sources of anti-human CD3 antibody can be used.
  • OKT3 a hybridoma cell line which produces anti-CD3 monoclonal antibody directed against human peripheral T cells, is available through the ATCC, e.g., ATCC CRL 8001.
  • Another anti-human CD3 monoclonal antibody-producing hybridoma cell line is also available through the ATCC, ATCC HB231.
  • both anti-CD3 monoclonal antibody and PHA were diluted 1:20 in culture medium.
  • the DTH response was measured by skin reaction in the ear as described by Vadas et al., 1975. Int. Arch. Allergy Appl. Immunol. 49:670.
  • unmodified or modified tumor cells were irradiated (10,000 rad) and then injected i.p. into C57BL/6J female mice at a dose of 10 7 viable cells in 1 mL of PBS/mouse (cell counts were determined before irradiation). After 8 days, immunization was repeated as above with a fresh batch of unmodified or modified tumor cells.
  • a sample of 10 s unmodified and irradiated tumor cells (an empirically determined optimal dose) in 10 ⁇ L PBS was injected intradermally 8 days later in the right ear (0.5 in., 1.27 cm, 30G needle, Becton Dickinson, N.J.).
  • the left ear (control ear) was injected with 10 ⁇ L PBS.
  • the mice were injected i.p. with 0.1 mL of a 1.0 mM 5-fluoro-2' deoxyuridine (FdUrd, Sigma) solution.
  • the mice were injected i.v. with 2 ⁇ Ci of 5- ,23 I- labeled 2'-deoxyuridine ( l25 IdUrdR, sp. act. 5 Ci/ng, Amersham, UK) in the lateral tail vein.
  • mice were sacrificed after 24 h from the time of challenge with tumor cells. The ears were then cut out carefully at the rims and the amount of radioactivity determined in a gamma counter (Gammamatic, Kontron). The results were expressed as the ratio of radioactivity in the right ear to that in the left ear (R/L ,23 IdUrd index). Five mice were included in each group. Control groups included unprimed mice, as well as those primed with unmodified tumor cells.
  • the development of the skin test reaction or immune reaction in humans was scored at 24, 36, 48, and 72 hours after injection of the cells. 24-48 hours were frequently optimal and normal for observing the peak of a reaction.
  • An immune reaction elicited by the injected cells was evidenced by a swollen, red area which appeared in the skin at the inoculation site.
  • the degree of a patient's skin reaction was determined by the diameter of the redness (erythema) and the degree of swelling (induration), as described by Scornick et al. 1981.
  • DTH responses can be scored numerically, such that a score of "3" indicates a very strong response (and corresponds to the above-described " + + + " response); a score of "2" indicates a moderate response, corresponding to " + + "; and a score of "0-1” indicates a minimal or poor DTH response, corresponding to " + ", above.
  • Plasma membranes were prepared from cells, e.g., tumor cells, essentially as described by Maeda, T. et al. 1983. Biochim. Biophys. Acta. ,
  • HBSS Hank's Balanced Salt Solution
  • the interface band was carefully aspirated using a pasteur pipet and was centrifuged at 100,000 x g for 90 minutes. The pellet was then resuspended in a small volume of HBSS. Protein content was determined by Lowry's Folin-
  • Ciocalteau assay Lignocalteau assay (Lowry, O.H. et al. 1951. J. Biol. Chem. , 122:265).
  • cytosolic and membrane proteins Methods to prepare cytosolic and membrane proteins from cells.
  • Cells are centrifuged in cell medium and the pelleted cells are washed once in PBS.
  • tumor cells are dispersed in cell medium by mincing with a scalpel prior to centrifuging.
  • the washed cells are resuspended in a hypotonic buffer A (Buffer A: 10 mM KC1, 10 mM HEPES, pH 8.0, 1 mM EDTA/EGTA, protease inhibitors and phosphatase inhibitors) at a final cell density of about 10 million per mL in buffer A for about 15 to 30 minutes on ice.
  • Buffer A 10 mM KC1, 10 mM HEPES, pH 8.0, 1 mM EDTA/EGTA, protease inhibitors and phosphatase inhibitors
  • NP- 40 or Triton-X ® 100 is added per mL of cell suspension (i.e. , about 0.6% nonionic detergent final concentration).
  • the cell and detergent mixture is vortexed for about 15-30 seconds, and centrifuged in an microcentrifuge (Eppendorf) for about 1 minute.
  • the resulting cell pellet contains cell debris (i.e., connective tissue) and nuclei.
  • the resulting cell supernatant contains cytosolic proteins and solubilized plasma membrane proteins.
  • cytosolic cell protein fraction To isolate the cytosolic cell protein fraction only, cells are resuspended in buffer A, quick frozen in liquid nitrogen, thawed, and centrifuged for about 30 minutes in a microcentrifuge (Eppendorf). The resulting supernatant contains predominantly cytosolic proteins.
  • the cell pellet resulting from the above-described 30 minute centrifugation is extracted on ice for about 30 minutes in buffer A containing 0.5% NP-40 or Triton-X* 100 and is then centrifuged for 5 minutes in a microcentrifuge (Eppendorf). The soluble fraction contains predominantly plasma membrane proteins and residual cytosol.
  • cells are treated with crosslinker and pressure in accordance with the invention (i.e., about 10 to 20 mM 2', 3' nucleoside or nucleotide dialdehyde, at the same time that the cells are exposed to about 800 to 1400 atm hydrostatic pressure; most preferably 10 mM crosslinker and 1200 atm pressure).
  • the PCL-treated cells are then subjected to hydrostatic pressure of greater than or equal to about 1600 atm and the cells are centrifuged to pellet cell debris.
  • the resulting cell supernatant is applied to a G100 or G250 column, whereby the fractions of crosslinked proteins are isolated.
  • Example 14 In vivo immunization and survival studies of animals vaccinated with an immunogenic preparation comprising modified B16/B16 melanoma cells and non- classical adjuvant to enhance the immune response
  • immunogenic preparations comprising B16/BL6 tumor cells that are preincubated in 10 mM dialdehyde AdA and then PCL- modified by exposing the tumor cells to AdA crosslinker at 10 mM at the time that the cells are also subjected to hydrostatic pressure of 1200 atmospheres.
  • the immunogenic preparations also contain either GM-CSF or HGH as a non-classical adjuvant. Unmodified or untreated cells are used as controls as described.
  • BL6/BL6 melanoma cells are a very invasive variant of the B16 cell line (Hart 1979, Am. J. Pathology, 22:587) and are obtained from B16/BL6 tumors that are serially passaged in syngeneic C57BL/6 mice by subcutaneous (s.c.) inoculation of 2 - 5 x 10* cells.
  • the viability of C57BL mice challenged with 1 x 10 5 viable non-PCL-treated B16/BL6 tumor cells is tested following immunizations with the immunogenic preparations comprising B16/BL6 cells, either unmodified or PCL-modified in accordance with the invention.
  • the cells used for vaccination are of the same kind as the cells used to challenge the mice.
  • the immunization protocol comprises two vaccinations, i.e., injections of an immunogenic preparation comprising about 20 x 10* PCL-modified B16/BL6 tumor cells formulated with either HGH or GM-CSF at concentrations of 1 ⁇ g/mL to 100 ⁇ g/mL per injection, administered one week apart.
  • the dose concentrations used for each adjuvant are typically 1 , 5, 10, 20, 50, and 100 ⁇ g/mL.
  • One week after the last vaccination animals are challenged with tumor cells.
  • Control immunogens contain unmodified cells (e.g., in medium such as Hank's Balance Salt Solution, HBSS) with and without the presence of adjuvant, as well as PCL-modified cells without adjuvant.
  • HBSS Hank's Balance Salt Solution
  • 6-10 mice are immunized to test the controls and each immunogenic preparation containing PCL-modified cells and the various doses of adjuvant.
  • the immunized animals are challenged by injection with viable B16/BL6 tumor cells and the ability of immunized animals to survive the challenge is assessed over a period of about 45 days or longer post-challenge.
  • DTH analyses were performed in a human patient to assess the DTH response to PCL-immunogens administered in combination with non-classical adjuvants, such as GM-CSF.
  • a melanoma patient was immunized with an immunogen comprising PCL-modified allogeneic melanoma cells in conjunction with non-classical adjuvant, i.e., GM-CSF. It is to be understood that autologous melanoma cells may also be used.
  • the patient Prior to the immunization protocol using PCL- modified immunogen and adjuvant, the patient was screened or pretested in a baseline DTH assay to select the optimal adjuvant dose of GM-CSF to use during the immunization protocol.
  • PCL-treated allogeneic melanoma cells were injected subcutaneously (SC) at sites 2 inches apart in the patient's forearm.
  • PCL-treated cells were injected alone (0.5 cc) or mixed with 20 ⁇ g (low dose) or 100 ⁇ g (high dose) of GM-CSF (Leukine, available from Immunex Corp.) in 0.1 cc.
  • GM-CSF Leukine, available from Immunex Corp.
  • Cells for immunization were PCL modified as described using 10 mM AdA and 1200 atm pressure.
  • the GM-CSF injections were repeated using the respective low and high doses at 24 and 48 hours at the DTH immunization sites.
  • the development of the DTH response was scored at 24 and 48 hours after immunization by measuring the diameter or size of the area of induration at the immunization site using callipers and as known by those in the art. For example, if the DTH response area was essentially circular, a single diameter was measured and reported; if the response area was non-circular or irregular in shape, the area was measured in two dimensions (at two independent positions) and the mean of the measurements was determined. Based on the patient's response to the DTH baseline assay, a given dose of GM-CSF (i.e., 100 ⁇ g) was selected for use in the PCL immunization protocol.
  • GM-CSF i.e. 100 ⁇ g
  • the immunization protocol was generally carried out about 48 hours following the baseline DTH assay and comprised a course of three subcutaneous injections, most preferably at sites near a draining lymph node.
  • the patient was immunized SC (e.g., in the forearm or in the thigh) with 1 x 10 7 PCL-treated cells, together with 100 ⁇ g of GM-CSF (0.6 cc total).
  • the immunizing dose of PCL-modified cells as immunogen was on the order of about, or greater than, ten times the number of cells used in the DTH screening assay (e.g., 1 x 10 7 cells versus 1 x 10 5 cells in the DTH screen).
  • the second injection at 24 hours comprised GM-CSF adjuvant alone (100 ⁇ g) at the same site and the third injection at 48 hours comprised adjuvant alone (100 ⁇ g).
  • IVS assays were performed at several intervals (e.g., at four and six weeks) following the immunization protocol to assess the patient's immune response status and potency level.
  • DTH assays were again performed as described to evaluate the patient's immune response to both PCL modified and unmodified cells.
  • GM-CSF as adjuvant revealed a clear and significant augmentation of the patient's DTH immune response in a dose-dependent manner, as evidenced by the size of the area of the induration (i.e., the DTH response area) at the immunization site after injections with 10 PCL-treated melanoma cells and either 20 or 100 ⁇ g of GM-CSF administered as adjuvant as described.
  • the results are presented in Table 2: TABLE 2
  • DTH response evaluations such as those described in this example are performed using the dialdehyde and antigen compositions of the invention, wherein melanoma cells as immunogen are replaced with a protein or peptide antigen, or portion thereof, such as a tumor associated antigen (TAA) derived from melanoma cells as described hereinabove.
  • TAA tumor associated antigen
  • Dialdehyde AdA and melanoma antigen are incubated in a 10: 1 or 20: 1 ratio for 30 minutes at room temperature, the resulting composition is admixed with carrier or excipient, as necessary or desired, and used to immunize a patient to test the resulting DTH response.
  • AdA is used at 10 to 20 mM.
  • the composition may contain a non- classical adjuvant component; alternatively, the adjuvant, e.g., GM-CSF or hGH, may be co-administered.
  • Example 16 Dialdehyde and protein compositions of the
  • compositions comprising dialdehyde and protein antigen are prepared for use as immunogens for enhancing, stimulating or potentiating the immune response to antigen.
  • the antigen used in the composition is baculovirus- produced polyoma virus middle T protein antigen and the dialdehyde is AdA.
  • AdA baculovirus- produced polyoma virus middle T protein antigen
  • Other antigens are also suitable for use as described herein.
  • 10 mM AdA is admixed and reacted with antigen in HBSS, and the mixture is allowed to incubate for 30 minutes at room temperature.
  • 10 to 20 mM dialdehyde incubated with antigen for 30 minutes at room temperature provides a dialdehyde and antigen composition in accordance with the invention for use as immunogen.
  • dialdehyde to antigen e.g., protein or peptide
  • ratio of dialdehyde to antigen may be on the order of 10: 1 to 20:1.
  • the dialdehyde may be mixed with the antigen to produce the dialdehyde and antigen preparation immediately prior to use, or the two components may be mixed and cryopreserved for subsequent use, depending upon the stability of the particular antigen.
  • the AdA and antigen immunogenic preparation is injected into mice.
  • mice are injected s.c. either with antigen alone, with the composition comprising antigen plus the dialdehyde 2 ',3 '-adenosine dialdehyde (AdA), or with antigen plus 100 ⁇ g of a suitable adjuvant or carrier. Additional injections of the aforementioned test compounds are provided subsequently for up to four days at the same injection site. After a week to 10 days inguinal (regional) lymph nodes are removed and the lymph node cells are restimulated with antigen alone. Antigen specific proliferation of lymphocytes is measured after a 5 day IVS assay. Tests for humoral immunity are performed by administering antigen and test compounds in the same way.
  • AdA dialdehyde 2 ',3 '-adenosine dialdehyde
  • mice a are immunized subcutaneously with composition of AdA (10 ⁇ g) plus keyhole limpet hemocyanin (50 ⁇ g) as antigen and are boosted with the same composition 3 to 5 weeks later. After a further 6 to 8 weeks, and a final boost with the same composition, spleens are harvested. Harvested spleen cells may be further stimulated in vitro with antigen for 5 days prior to performing the cytotoxic assay using conventional procedures.
  • Target cells P815 cells, a mastocytoma
  • an irrelevant antigen e.g., the NP peptide of influenza virus
  • Spleen and target cells are incubated for 4 to 6 hours and killing is measured by the release of 51 Cr expressed as a percentage of the total released from detergent lysed cells. Spontaneous release is also measured. Percent killing is measured as a function of the effector (spleen) to target (labeled P815) cell ratio.
  • Cytotoxic T cells are expected to be activated in response to immunization with the dialdehyde and antigen compositions of the invention.
  • lymphoma e.g., EBV-associated lymphoma
  • EBV-transformed B lymphocyte cell lines were established as described in A. Toubert et al., 1984, "Identification of several functional subgroups of HLA-B27 by restriction of the activity of antiviral T killer lymphocytes", Immunogenetics, 22:513.
  • B95.8 cells which are a marmoset tumor cell line used to propagate EBV (ATCC Accession No. CRL 1612), were grown to confluence in RPMI supplemented to contain 20% FBS. Cells were centrifuged and resuspended at 1.2 x 10* cells/ml. Cells were incubated at 37°C, 5% CO 2 in a humidified incubator for three days, after which time supernatant was harvested to use for infection.
  • PBMC peripheral blood mononuclear cells
  • DC lines were initiated as follows: 400 x 10* PBMCs were plated in T225 flasks in maintenance medium (described above) and were incubated for 2 hours in a 37°C, 5% CO 2 humidified incubator. Two-hour supernatant was removed and non-adherent cells were frozen and stored for future use. Additional maintenance medium was added containing GM-CSF (800 U/ml) and IL-4 (500 U/ml). Cells were cultured for 8 days, and then were harvested using cell dissociation medium. Cells were frozen for future use or used immediately. T Cell Lines:
  • T-cell lines were initiated as follows: 4 x 10* BLCL were irradiated (6000 RADS) and combined with 4 x 10* DC for each T cell group. Cells were incubated for 2 hours in low volume maintenance medium. 80 x 10* cells from 2- hour supernatant (as generated above) were added to each flask with BLCL and DC (4 x 10* cells). Maintenance medium was added to a total volume of ⁇ 15 ml and the flasks were incubated for 3 days. Thereafter, nonadherent cells were transferred to new flasks. As the maintenance medium turned a light orange color, cell lines were split and additional medium was added. Pressure and Cross-Linking (PCL) Modification
  • BLCLs and K562 cells were counted and irradiated (6000 RADS). Cells were serially diluted in maintenance medium and plated in 96-well U-bottom plates in concentrations indicated for the individual experiment. Identical plates containing BLCLs and K562 cells were plated for each T cell group. T cells were counted and plated over BLCLs at 50,000 cells/well. Additional control plates containing BLCLs alone and T cells alone were established. The total volume of each well was 200 ⁇ l maintenance medium. Plates were incubated for 72 hours at 37oC, 5% CO 2 in a humidified incubator.
  • cytokine assays were to be performed, 100 to 150 ⁇ l of supernatant was removed and stored at 4°C. Thereafter, approximately 1 ⁇ Ci 3 H-Thymidine was added per well in a small volume of maintenance medium. Cells were incubated in radiolabel overnight and were harvested using the Tomtec Cell Harvester (Wallac, Gaithersburg, MD). 3 H-Thymidine incorporation was determined using the Wallac Microbeta plate reader (Wallac, Gaithersburg, MD). Net counts were determined by subtracting background counts obtained from plates housing BLCLs and T cells alone. A stimulation index was determined by dividing net counts by counts of T cells alone. Cytokine as ay
  • ELISA kits (Endogen and R&D Systems) were used to measure cytokine release from cells in the cell cultures. Supematants from all T cell groups and BLCL concentrations were plated in duplicate on pre-coated ELISA plates. Cytokine standards were also plated in duplicate. Biotinylated antibody was plated over supernatant. Plates were covered and incubated at room temperature for 2 hours and then were washed three times using wash buffer as provided by the supplier. Streptavidin-HRP concentrate was diluted in dilution buffer and 100 ⁇ l were added to each well. The plates were covered and incubated at room temperature for 30 minutes. The plates were again washed three times using wash buffer. Premixed TMB substrate solution was added to each well.
  • PBMC from a healthy donor buffy coat were isolated, washed, and irradiated (6000 RADS). Cells were again washed and were resuspended at 2 x 10* cells/ml maintenance medium + 50 U/ml rhIL-2. Cells were plated in one 96-well U-bottom plate for each T cell group.
  • effector cells T cells from each experimental group were counted and resuspended at 1 x 10* cells/ ml in maintenance medium. Cells were serially diluted 10-fold to produce a series of 6 dilutions (10 5 , 10 ⁇ 10 ⁇ 10 2 , 10, and 1 cell(s) per well). T cells were plated over the feeder cell layer.
  • Anti-CD3 200 ng/ml was added to all of the wells.
  • the total well volume was 200 ⁇ l/well in maintenance medium.
  • the plates were incubated in a 37°C, 5% CO, humidified incubator for 14 days.
  • 50 ⁇ l/well cell-free supernatant was removed and replaced with 50 ⁇ l/well fresh maintenance medium containing 100 U/ml rhIL-2.
  • 50 ⁇ l/well cell-free supernatant was removed and replaced with fresh maintenance medium containing 1 x 10 5 PBMC irradiated feeder cells (6000 RADS) and 100 U/ml rhIL-2.
  • the cells in each well were resuspended and distributed uniformly into one 96-well plate for each target cell group. Additional maintenance medium was added to bring the volume in each well to 100 ⁇ l/well.
  • Cells were assayed on day 14. 100 ⁇ l/well target cells were plated over T cells to be assayed. A control cell plate was set up containing cells for obtaining spontaneous and maximum release values for each target. The plates were incubated for 5-7 hours at 37°C, 5% CO 2 in a humidified incubator. 100 ⁇ l/well were removed and combined with an equal volume of scintillation fluid. The plates were read using a Wallac Microbeta plate reader, and the frequency of CTL precursors was determined.
  • Dendritic cells pulsed with cell extract prepared from a pool of various allogeneic EBV-associated B-cell lymphoma were used as a cellular cancer immunogen/vaccine to generate tumor-specific T cell lines.
  • DC-pulsed with tumor antigen were shown to trigger a strong T cell activation against the B cell lymphoma, as measured by proliferative response and cytokine secretion (Fig. 12 and Fig. 13).
  • whole cell extract (WCE) was prepared from tumor cells by repeated freeze-thaw. The concentrations or levels of the proteins in the WCE was measured using a commercially available kit for this purpose (Pierce). Approximately 1 x 10 7 DC were incubated with 100 ⁇ g of WCE for about 5 to 16 hours.
  • the ratio of responders to stimulators was about 10-20: 1.
  • PCL modification of DC-pulsed with tumor extract added a dramatic increase to their immunogenic potential compared with unmodified DC-pulsed with tumor antigen.
  • LDA limiting dilution analysis
  • tumor antigens to T cells by professional APC were shown to be highly effective.
  • Treatment of loaded DC to PCL modification further enhanced the potency of APC presentation to T cells.
  • the PCL treatment reduced the number of T cells required to lyse 50% of target cells to approximately 2 cells per well compared with 100 cells per well achieved with T cells generated following stimulation with unmodified APC (Fig. 8).
  • tumor cell presentation by APC combined with PCL treatment created a powerful cell immunogen which exhibited the capacity to elicit high affinity tumor-specific CTLs from peripheral blood lymphocytes. It is likely that such an immunogen can be used to break tolerance to cancer cell antigens in order to provoke a highly specific and protective T-cell response, to abrogate metastatic dissemination, thereby halting disease progression.
  • PCL-modification was demonstrated to augment the presentation of tumor antigens to T cells by professional APC (DC), thereby creating a potent anti-tumor immunogen with exceptional capacity to elicit highly efficient CTLs to eradicate cancers, tumors and infected cells.
  • DC professional APC

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Abstract

La présente invention concerne un procédé d'incubation préparatoire de cellules avec des dialdéhydes, de préférences des 2',3'-nucléoside ou nucléotide dialdéhydes servant de réticulants avant la modification des composants des membranes des cellules, s'agissant des cellules ayant subi une incubation préparatoire par exposition à une pression hydrostatique en vue de la production de préparations immunogènes puissantes. On appelle modification PCL ce traitement de pressurisation et réticulation. La modification PCL d'incubation préparatoire permet aux dialdéhydes de servir, non seulement de réticulant pour les composants de la membrane, mais aussi de source de groupes monoaldéhydes sur la surface de la cellule, ce qui confère aux cellules une fonction d'adjuvant et de costimulant. L'invention concerne également des compositions immunogènes comprenant un dialdéhyde et un antigène et permettant d'augmenter et de renforcer la réponse immunitaire vis à vis d'un antigène spécifique. Entre le composant dialdéhyde et le composant antigène, il se forme une liaison chimique covalente réversible utilisant un monoaldéhyde du dialdéhyde. L'autre monoaldéhyde peut réagir avec une molécule ou un adjuvant de réponse biologique permettant un ralentissement ou une prolongation du déblocage et du renforcement de la réponse immunitaire après immunisation. L'invention concerne en outre de puissants immunogènes et stimulants de réponse immunitaire obtenus par traitement PCL des cellules de présentation d'antigènes (APC) qui ont été impulsés ou co-cultivés avec des antigènes associés à des cellules cancéreuses, tumorales ou infectées. Les APC modifiés PCL et pulsées par antigènes conviennent particulièrement dans des préparations immunogènes et vaccinales destinées à accroître le caractère immunogène et à provoquer des réponses de médiation cellulaire vigoureuses ainsi qu'une production de cytokine TH1. De telles cellules ont été utilisées avec des lymphocytes T spécifiques d'antigènes se trouvant dans des préparations immunogènes et vaccinales, et pour des thérapies d'adaptation immunitaire destinées à traiter ou prévenir les cancers, tumeurs et infections. Pour accroître encore plus la réponse immunitaire, il est possible de coupler les compositions pulsées par antigènes et modifiées PCL ainsi que les APC avec des molécules de réponse biologiques telles que des cytokines immunostimulantes, des lymphokines et des facteurs de croissance.
PCT/US1997/003916 1996-03-15 1997-03-13 Dialdehydes servant d'adjuvants immunostimulants et de reticulants pour la production de preparations immunogenes, pour la generation de cellules soumises a pression et reticulation afin de renforcer et d'augmenter la reponse immunitaire contre le cancer, les tumeurs et les affections pathogenes WO1997033612A1 (fr)

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AU19910/97A AU1991097A (en) 1996-03-15 1997-03-13 Dialdehydes as immunostimulatory adjuvants and cross-linkers for producing immunogenic preparations and generating pressure and cross-linked treated cells for enhancing and augmenting the immune response against cancers, tumors and pathogenic diseases

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US61683496A 1996-03-15 1996-03-15
US08/616,834 1996-03-15
US80376497A 1997-02-21 1997-02-21
US08/803,764 1997-02-21

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US6139844A (en) * 1997-04-02 2000-10-31 Bioniche, Inc. Method for treating or eliminating a parasitic disease
WO1998043656A1 (fr) * 1997-04-02 1998-10-08 Bioniche Inc. Utilisation d'extrait de paroi cellulaire bacterienne afin de prevenir, traiter ou eliminer une protozoose ou une maladie parasitaire
US7196073B2 (en) 1997-10-03 2007-03-27 Adjuvantys, Inc. Imine-forming polysaccharide adjuvants and immunostimulants
US6960344B2 (en) 1997-10-03 2005-11-01 Galenica Pharmaceuticals, Inc. Use of imine-forming polysaccharides as adjuvants and immunostimulants
WO1999055715A3 (fr) * 1998-04-28 1999-12-29 Galenica Pharmaceuticals Inc Conjugues d'antigene et de polysaccharide
US6573245B1 (en) 1998-04-28 2003-06-03 Galenica Pharmaceuticals, Inc. Modified polysaccharide adjuvant-protein antigen conjugates, the preparation thereof and the use thereof
US7125858B2 (en) 1999-12-28 2006-10-24 Bioniche Life Sciences Inc. Hyaluronic acid in the treatment of cancer
WO2002060390A3 (fr) * 2001-01-04 2002-10-03 Univ Yale Induction de reponses immunitaires a des antigenes modifies a l'isoaspartyle
US7625567B1 (en) 2001-01-04 2009-12-01 Yale University Induction of immune responses to isoaspartyl-modified antigens
WO2002053176A3 (fr) * 2001-01-08 2002-12-19 Hadasit Med Res Service Vaccin anti-cancer autologue
EP1465657A4 (fr) * 2001-12-18 2006-02-01 Health Research Inc Methode permettant de renforcer l'immunogenicite par liaison covalente d'antigenes a des proteines a la surface de cellules dendritiques
EP2073011A1 (fr) * 2007-12-20 2009-06-24 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Moyens et procédés pour le traitement d'échantillons biologiques
WO2009080308A1 (fr) * 2007-12-20 2009-07-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Moyens et procédés de traitement d'échantillons biologiques
WO2009111849A1 (fr) * 2008-03-10 2009-09-17 Universidade Federal Do Rio De Janeiro - Ufrj Procédé pour la production de vaccin stabilisé
ITTO20110652A1 (it) * 2011-07-20 2013-01-21 Medestea Res & Production S P A Composto derivato dall'adenina avente azione immuno-modulante, antinfiammatoria e antidolorifica
WO2013011489A1 (fr) * 2011-07-20 2013-01-24 Medestea Research & Production S.P.A. Dérivés d'adénine ayant une activité immunomodulatrice, anti-inflammatoire et analgésique
CN116437950A (zh) * 2020-07-24 2023-07-14 四川大学华西医院 一种靶向ebv的同种异体b细胞疫苗及其制备方法
WO2024207942A1 (fr) * 2023-04-06 2024-10-10 中山大学孙逸仙纪念医院 Adjuvant de vaccin antitumoral et son utilisation

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