WO1996025664A1 - Procedes et compositions pour le depistage, le pronostic et le traitement en matiere d'immunotherapie - Google Patents

Procedes et compositions pour le depistage, le pronostic et le traitement en matiere d'immunotherapie Download PDF

Info

Publication number
WO1996025664A1
WO1996025664A1 PCT/US1996/001876 US9601876W WO9625664A1 WO 1996025664 A1 WO1996025664 A1 WO 1996025664A1 US 9601876 W US9601876 W US 9601876W WO 9625664 A1 WO9625664 A1 WO 9625664A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
tumor
infected
cell
response
Prior art date
Application number
PCT/US1996/001876
Other languages
English (en)
Inventor
Avi Eisenthal
Meir Shinitzky
Amnon Gonenne
Original Assignee
Immunotherapy, Inc.
Yeda Research And Development Corporation, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunotherapy, Inc., Yeda Research And Development Corporation, Ltd. filed Critical Immunotherapy, Inc.
Priority to AU49778/96A priority Critical patent/AU4977896A/en
Publication of WO1996025664A1 publication Critical patent/WO1996025664A1/fr

Links

Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is generally in the field of immunotherapy and treatment of cancer and infection, and relates to novel immunogens, anti-tumor and anti-infected or transformed cell immunogenic preparations, and vaccines for use in animals, including humans.
  • the invention also relates to processes for the preparation of the immunogens, and methods of utilizing the immunogenic preparations as anti-tumor cell and anti-infected or transformed cell immunogens both in vitro and in vivo.
  • the immunogens of the present invention may be derived from tumor cells which are treated as described herein to increase their specific immunogenicity.
  • the immunogen may be whole cells treated as described, plasma membranes derived from these cells, or tumor-specific immunogenic proteins obtained from the cells or cell membranes.
  • the immunogens may be derived from cells infected and/or transformed with viruses or other microorganisms or agents that are treated as described herein to increase their specific in vivo and in vitro immunogenicity.
  • 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. Nevertheless, for reasons which are not entirely clear, 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.
  • U.S. Patent No. 4,931,275 discloses anti-tumor vaccines which contain as an active ingredient tumor cells, plasma membranes, or specific membrane proteins obtained from these cells or membranes, which have been treated to augment their immunogenic properties.
  • the treatment described to augment cells' immunogenicity in accordance with this patent consists of either treatment with cholesteryl hemisuccinate, which rigidifies the lipid layer of the plasma membrane, or application of hydrostatic pressure up to about 1500 atmospheres (atm), or a combination of the two treatments.
  • 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 found in tar and soot, and aromatic amines, such as found in certain dyes. Examples of physical carcinogens are X-rays and ionizing and ultraviolet radiation.
  • 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.
  • 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.
  • tumors In the case of tumors, the task of host immune surveillance is especially daunting because tumor cells and other abnormal cells have many similarities to normal cells, in spite of their abnormal propensities to fail to respond to normal regulatory signals, to proliferate and spread throughout the host, and to interfere with normal organ function. Further, although foreign antigens or mutated proteins may be expressed on the surfaces of transformed cells and tumor cells, the cells of the immune system frequently develop an ineffective or weak immune response against such tumor cells. Alternatively, the emergence of tumors in an animal may reflect the failure or inefficiency of immune surveillance and the virtual absence of an immune response during progressive growth of the tumor.
  • 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. However, it is pointed out that even if unique antigens are not found on human tumor cells, it may not be because such antigens do not exist, but because such antigens are difficult to detect, given the methods available in the art at the present time. Accordingly, needed in the art are ways to stimulate a strong and sustained immune response against tumor cells, which may have great potential for being highly immunogenic, but can successfully evade immune destruction.
  • MHC proteins on tumor cells is believed to be critical for the immunologic recognition and destruction of the tumor cells. This is especially true if T lymphocytes are required for the cognitive and/or effector stages of specific anti-tumor immune responses, since T cells can recognize antigens only in association with MHC molecules (Goodman, J.W. , 1994.. "Antigen Presentation & the Major Histocompatibility Complex” , In: Basic and Clinical Immunology. Eds. D.P. Stites, A.I. Terr, and T.G. Parslow, Appleton & Lange, Norwalk, Connecticut, pp.58-65).
  • tumors which can stimulate protective immune responses express adequate amounts of MHC molecules, while other tumors which are not immunogenic fail to express enough MHC proteins, or fail to express any MHC proteins.
  • the lack of expression of sufficient amounts of MHC molecules may be particularly important in the case of certain chemically- and virally-induced tumors.
  • murine and human spontaneously arising tumors including neuroblastomas, basal cell carcinomas, small-cell lung tumors, choriocarcinomas, and B cell lymphomas, the expression of class I MHC antigens may be significantly reduced or virtually absent (Ramakrishna, V. et al. 1993.
  • any abnormal cellular protein, and not just proteins derived from the cell membrane, is capable of being a potential immunogen for presentation to the immune system.
  • the presence in a tumor cell of a nonfunctional or truncated protein product of a mutated allele could potentially result in the immunogenicity of that product.
  • the presence in a virally-transformed or infected cell of a viral protein product in conjunction with MHC molecules may also potentially result in the immunogenicity of that viral product expressed by the cell.
  • the goal of therapies to treat and eradicate cancers and other types of transformed cells is to provide efficient and safe methods by which to increase the host's anti-tumor or foreign cell response against weakly immunogenic cell types, such as tumors and transformed cells.
  • Tumor 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 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 H-restricted T cells, which largely represent CD4 helper T cells (i.e., T H ) 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 ⁇ MHC molecules, the T H cell subset cannot directly recognize these tumor cells.
  • APCs antigen-presenting cells
  • APCs such as macrophages, B lymphocytes and dendritic cells
  • Antigen presenting cells capture, process, and present most proteinaceous immunogens to the CD4 helper T cell subset.
  • activated T H 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 H cells two functional subsets of T H cells exist.
  • the first subset the type 1 or T H 1 subset, appears to facilitate and then to reinforce primarily a cell-mediated immune response by cytotoxic T cells, i.e. , T c cells (see below);
  • the second subset the type 2 or T H 2 subset, appears to help B lymphocytes to mature and then to produce antibodies (C. Ezzell, 1993, The J. of NIH Research, 5:59-64).
  • the two T cell subsets are also distinguished by the types of cytokines that they produce.
  • T H 1 cells release both interleukin-2 (IL-2) and interferon-7 (IFN-7), while T H 2 cells release a combination of interleukin-4 (IL-4), interleukin-5 (I -5), interleukin- 6 (IL-6), and interleukin-10 (IL-10). Both IL-4 and IL-10 has been shown to shut down the cell-mediated immune response. Conversely, IFN-7 produced by T H 1 cells promotes cytotoxic T cell proliferation and inhibits antibody production. Thus, the various cytokines elaborated by the these two T cell subsets are cross- regulatory and can, when manipulated by a pathogen (e.g.
  • T H l-type response i.e., predominantly cell-mediated
  • T H 2-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 H-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 mtracellular 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.
  • the role of antigen-presenting cells is to offer antigenic peptides complexed with MHC molecules to the available repertoire of T 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".
  • a potential solution to this problem is to manipulate and amplify the immune system and its cellular components in order to promote tumor eradication. This solution is also conducive for eliminating cells infected or transformed by other exogenous agents, e.g., drugs, carcinogens, viruses, other microorganisms.
  • the present invention provides immunotherapy methods and applications which are prognostic, therapeutic, and prophylactic.
  • the invention fulfills the grave needs of ameliorating and augmenting the immune response to foreign or non-self antigens in and/or on cells in both a therapeutic and a prophylactic manner, as detailed hereinbelow.
  • the present invention provides a method and treatment to modify tumor or infected or otherwise transformed cells to augment the immune response so that the cells of the immune system are stimulated toward more efficient recognition and eradication of the tumor or infected or otherwise transformed cells, particularly after immunotherapy involving the use of such modified cells.
  • Cells to be modified or treated are non-normal or pathogenic cells, for example, tumor cells or infected or transformed cells. Transformed cells may also include some virus- infected cells, bacteria-infected cells, parasite-infected cells, or cells otherwise altered away from their normal state due to cancer or to tumorigenic or malignant transformation by a foreign pathogen or other endogenous or exogenous transformation-inducing agents.
  • the cells treated in accordance with the invention may be used as immunogens to elevate the immune response to the nonmodified counterparts of the modified cells both in vitro and in vivo.
  • the modification resulting in heightened immune response is the treatment of cells with a crosslinking effect amount of a crosslinking agent and with hydrostatic pressure in a particular pressure range, preferably at the same time, as described herein.
  • a crosslinking effect amount of a crosslinking agent preferably at the same time, as described herein.
  • hydrostatic pressure in a particular pressure range, preferably at the same time, as described herein.
  • PCL-treated or PCL- modified cells means "pressure and crosslinker treatment or modification" of cells in accordance with the invention.
  • the invention also provides the above-described modification process to render tumor or infected cells more recognizable and immunogenic to the cells of the immune system.
  • Another object of the invention is to provide modified whole cells, or components thereof, i.e., plasma membranes and membrane proteins, to increase and stimulate the immune response to tumor antigens and other foreign antigens presented by cells at the cell surface.
  • the immune cell stimulation may occur ex vivo; the effector immune cells may be isolated from an individual, incubated with the modified cells, or components thereof, to activate the effector immune cell populations, and the activated effector cells may be re-introduced into the individual to carry out an immune response in vivo.
  • a further object of the invention is to provide a method for treating a tumor in a tumor-bearing individual, comprising sensitizing immune cells to the cell surface antigens presented by the tumor and introducing the sensitized immune cells to the individual to eradicate the tumor.
  • Yet another of the invention is to provide a method for treating a tumor in a tumor-bearing individual, comprising immunizing the individual with cells modified in accordance with the invention to augment the individual's immune response to the tumor cells.
  • Another object of the invention is to use the above-described immunogen comprising modified whole tumor cells, immunogenic plasma membranes or proteins derived therefrom, as a prophylactic or a therapeutic vaccine capable of inducing a specific anti-tumor immune response.
  • a further object of the invention is to screen and monitor patients by the pressure-crosslinking technique in combination with in vitro sensitization assays.
  • candidates for in vivo immunotherapy can be screened, if desired, for the ability of their PCL-modified tumor cells or infected cells or transformed cells to stimulate autologous mononuclear blood cells in an in vitro sensitization assay, prior to immunoadoptive therapy or vaccination.
  • Yet another object of the invention is to use the results obtained from the in vitro sensitization assays combined with cytokine secretion by a patient's peripheral blood mononuclear cells as a method of determining or resolving whether a cancer or tumored patient should undergo chemotherapy or immunotherapy treatment regimens to eradicate the cancer or tumor.
  • Another object of the invention is to provide a reliable and objective clinical application of PCL-modified tumor or infected cells in cancer or infected patients by determining the immunogenicity of PCL-modified human tumor or infected cells in a functional test performed ex vivo.
  • Fig. la-le depicts histology of the delayed-type hypersensitivity reaction (DTH) in the ear of mice challenged with 10 3 irradiated syngeneic EL4 leukemia cells in the ear following priming with an immunogenic preparation: a. unprimed: b. primed with unmodified EL4 cells; c. primed with AdA treated EL4 cells; d. primed with pressure treated ⁇ 1 ⁇ cells; and e. primed with AdA and pressure treated EL4 cells.
  • DTH delayed-type hypersensitivity reaction
  • mice I non-immunized mice
  • mice II mice immunized with untreated EL4 cells
  • mice HI mice immunized with AdA-treated EL4 cells
  • mice immunized with pressure and AdA-treated EL4 cells mice immunized with pressure and AdA-treated EL4 cells
  • Fig. 3 is a graphic representation of a similar experiment to that shown in Fig 2. in which the cytotoxicity of anti- ARadLV 136 effector lymphocytes against ARadLV 136 target cells at various effector: target (E:T) ratios has been tested using various anti-ARadLV 136 effector lymphocyte preparations obtained from mice treated as follows: non-immunized mice (Treatment I): mice immunized with untreated ARadLV 136 cells (Treatment II); mice immunized with AdA-treated ARadLV 136 cells (Treatment HI); and mice immunized with pressure and AdA-treated ARadLV 136 cells.
  • Fig. 4 is a graphic representation of results of a reciprocal assay in which the cross- reactivity of anti-tumor effector lymphocytes has been tested: effector cells: from mice immunized with pressure and AdA-treated ARadLV 136 cells; target cells: EL4 cells (filled circle). Effector cells: obtained from mice immunized with untreated EL4 cells; target cells: ARadLV 136 cells (empty triangle). Effector cells: obtained from mice immunized with AdA-treated EL4 cells; target cells: ARadLV 136 cells (filled triangle). Effector cells: AdA-treated ARadLV 136 cells: target cells: ⁇ 1A cells (filled squares). Effector cells: AdA and pressure-treated EL4 cells; target cells: ARadLV 136 cells (empty squares).
  • Fig. 5 is a graphic representation of an experiment in which the survivability of mice challenged with tumor cells after immunization with one of the following preparations was tested: EL4 cells treated with various levels of hydrostatic pressure in the presence of 40 mM AdA (filled squares); EL4 cells treated with increasing level of hydrostatic pressure and then with 40 mM AdA (empty squares); B16 melanoma cells treated with various levels of hydrostatic pressure in the presence of 40 mM AdA (filled circles) and B16 melanoma cells treated with hydrostatic pressure and then with 40 mM AdA.
  • Fig. 6 shows the survivability of mice challenged with tumor cells following immunization with one of the following preparations: EL4 leukemia cells treated for 10 min with hydrostatic pressure of 1350 atm in the presence of various concentrations of AdA (filled squares); B16 melanoma cells treated in the same manner (filled circles); B16 melanoma cells treated in the same manner with AMPdA (empty circles).
  • Fig. 7a and 7b show a three-dimensional overlay of antigen expression on B16-BL6 melanoma cell plasma membrane surface as analyzed on FACScan by indirect immunofluorescence: A & B - negative controls (A - autofluorescence; B -cells reacted only with secondary antibody); C - unmodified cells; D & X - cells exposed to 20 Mm AdA; E & Z - cells exposed to hydrostatic pressure of 1 ,200 atm for 15 minutes; F & Y - cells exposed simultaneously to both 20 Mm AdA and 1 ,200 atm pressure.
  • Fig. 7a fluorescence results using anti-MHC class I (Kb + Db) antibody; Fig.
  • FIG. 7b fluorescence results using anti-B16 antibody.
  • Fig. 8 shows the effect (as per cent of positive cells counted by FACScan instrument out of 10,000 events) of graded levels of hydrostatic pressure (applied for 15 minutes) combined with a constant dose of 20 Mm AdA on antigen expression in B16-BL6 melanoma cells; filled bars - class I antigen, gray bars -B16 antigen.
  • Fig. 9 shows the effect (per cent positive as determined on FACScan out of 10,000 events) of varying concentrations of AdA combined with a constant level of hydrostatic pressure (1200 atm, 15 minutes) on antigen expression in B16- BL6 melanoma cells: black bars - MHC class I antigen, gray bars - B16 antigen.
  • Fig. 10 shows the results of delayed type hypersensitivity assays performed in humans using both PCL-modified and unmodified autologous tumor cells as immunogens.
  • Fig. 11 shows the results of delayed type hypersensitivity assays performed in humans using both PCL-modified and unmodified allogeneic tumor cells as immunogens.
  • crosslinker or “crosslinking agent” or “crosslinking compound”
  • tumor-specific immunogenicity was augmented by subjecting the cells to a combined treatment of exposure to both crosslinking agent and hydrostatic pressure. It was also found that this immunogenicity was even further augmented if exposure of cells to crosslinking agent and to hydrostatic pressure was done simultaneously. Simultaneous refers to the crosslinking of cells at the time that they are exposed to hydrostatic pressure.
  • One aspect of the present invention provides 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 exposing tumor cells to the crosslinking agent at a concentration and for a time sufficient to cause crosslinking of proteins in the cells' plasma membranes.
  • the modified tumor cells are prepared by exposing tumor cells to both the crosslinking agent and to hydrostatic pressure at a level and for a time sufficient to cause displacement of proteins in the cells' plasma membranes.
  • exposure to the crosslinking agent and to hydrostatic pressure is done at the same time.
  • concentration of the crosslinking agent is in the range of about 1 Mm to about 40 Mm, preferably about 5 Mm to 20 Mm, and more preferably about 10 Mm to about 15 Mm.
  • the hydrostatic pressure is within the range of about 800 to about 1400 atmospheres (atm), preferably about 1000 atm to about 1200 atm.
  • pressure above about 1400-1500 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 about 5 to 15 minutes.
  • the crosslinking agent is preferably 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.
  • the preferred 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
  • the compound of formula I may be prepared by reacting a nucleoside or a nucleotide of the following formula II:
  • R and B have the meanings given above for formula I, with an oxidizing agent, e.g. an alkali periodate.
  • the immunogen may consist of the whole modified tumor cells, membranes derived from such cells, as well as proteinaceous material (e.g. , membrane proteins and fragments thereof) obtained from such cells or membranes which substantially retain the capability of the modified tumor cells to induce the anti-tumor immune response.
  • proteinaceous material e.g. , membrane proteins and fragments thereof
  • the modified tumor cells are exposed to high intensity radiation in order to destroy their genetic material.
  • said immunogen consists of cell membrane preparations or proteins or membrane and protein fragments.
  • the present invention provides a process for preparing an immunogen derived from modified tumor cells and capable of inducing an anti-tumor immune response, said process comprising the steps of: a) providing tumor cells; b) incubating the cells with said crosslinking agent at a concentration and for a time sufficient to obtain crosslinking between membrane proteins; and c) removing the crosslinking agent from the treated cells.
  • the above process preferably comprises also exposing the tumor cells to hydrostatic pressure at a level and for a time sufficient to cause displacement of proteins in the plasma membranes of the cells.
  • the hydrostatic pressure is preferably applied during the incubation step of the cells with said crosslinking agent (step b).
  • 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 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 11.
  • the desired immunogen consists of protein material (e.g., proteins or protein fragments)
  • 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 "IVS" 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 Examples 6 and 14).
  • immune cells e.g., leukocytes or lymphocytes
  • Such a stimulated population of immune cells may then be reinjected into a cancer patient in order to treat his/her tumor.
  • the present invention thus provides a vaccine composition comprising the immunogen and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treatment of a cancer or tumor comprising injecting a cancer or tumored patient with the immunogen or with the sensitized immune cells as described. While the immunization of patients in accordance with the present invention can be performed by the use of an allogeneic immunogen, it is preferably performed by the use of an autologous immunogen.
  • an autologous immunogen provides significant advantages in that the immune response which occurs is primarily directed against the neo-antigen of the tumor.
  • an allogeneic immunogenic preparation 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 provide significant immune responses against PCL- modified allogeneic cells in humans as demonstrated in Example 7 and shown in Fig. 10.
  • the method comprises the steps of: a) withdrawing tumor growth from a patient by biopsy or surgery; b) dissociating intact tumor cells by mechanical or enzymatic means; c) dispersing the cells in a medium; d) incubating the cells with 2' , 3'-adenosine dialdehyde (AdA) in a concentration and for a time sufficient to cause crosslinking of proteins in the cells' plasma membranes; e) removing the AdA and preparing a tumor-specific immunogen derived from the modified cells obtained; and f) injecting the immunogen into the patient, whereby an anti-tumor immune response in the patient is induced.
  • AdA 2' , 3'-adenosine dialdehyde
  • step d) also includes exposing the cells to hydrostatic pressure between about 800 and about 1400 atmospheres, preferably about 900 and about 1200 atmospheres, more preferably, about 1000 atmospheres, at the same time that the cells are exposed to the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinker.
  • step f) may involve providing the modified tumor 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.
  • an immunogen derived from modified tumor cells obtained from a defined tumor cell line may be used.
  • modified tumor cells obtained from the same tumor type from another source or donor individual may be used with equal success.
  • the PCL-modification technique 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 cells which present tumor and foreign antigens to a host in the context of MHC 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 as immunogens or in immunogenic preparations to immunize both na ⁇ ve animals, including humans, as well as to treat tumored, infected, or diseased animals, including humans, which have previously encountered the tumor or foreign antigens.
  • the modified cells or cell preparations may include pharmaceutically acceptable carriers, excipients, or formulations, such as normal or buffered saline and the like; 'classical' adjuvants and emulsions, such as alum, incomplete Freund's adjuvant, and the like; and additional immunostimulants, such as 3-deacylated monophosphoryl lipid A (3-D-MPL), should these be needed or desired.
  • pharmaceutically acceptable carriers, excipients, or formulations such as normal or buffered saline and the like
  • 'classical' adjuvants and emulsions such as alum, incomplete Freund's adjuvant, and the like
  • additional immunostimulants such as 3-deacylated monophosphoryl lipid A (3-D-MPL), should these be needed or desired.
  • Monophosphoryl lipid A can be suspended in either saline or in oil- water emulsion.
  • Additional components of the immunogenic preparations can include, for example, sugars such as lactose, dextrose, and the like, and merthiolate, provided that the immunogenicity of the modified cells is not adversely affected.
  • another aspect of the invention comprises the use of other, 'non-classical' adjuvants which are co-injected in vivo or are formulated into the immunogenic preparation or vaccine comprising PCL-modified cells, cell preparations, or plasma membranes to enhance the immune response (see Examples 16 and 17).
  • 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-or, 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-or
  • one or more adjuvants in amounts determined to provide an effective and enhanced immune response, for example, as dete ⁇ nined by DTH assay, Example 17.
  • adjuvants e.g. , GM-CSF together with hGH, as a nonlimiting example
  • 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
  • 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 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 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
  • 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 6 or more (e.g., to about 20.0 x 10°) per immunizing dose, in admixture with saline or other excipients known in the art. Although fewer and more than about 10° cells may be used, a suitable number of cells is about 1-2 x 10 6 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.
  • the term modified as used herein means cells treated in accordance with the 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, that is generally recognized as foreign or non-self by cells of the immune system. It will be appreciated by those in the art that 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.
  • Examples of cells which may be modified by the PCL methods of the invention include, but are not limited to, 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.
  • Viruses include 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 (HIV)- infected cells of various strains (e.g., HIV-1 and HIV-2), human T-lymphotropic 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.
  • HIV human immunodeficiency virus
  • 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 mo ⁇ hologically changed away from their normal cell counte ⁇ arts.
  • 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. co/Hnfected 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.
  • 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 ⁇ MHC structures for presentation to the effector cells of the immune system. Similarly, 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 ⁇ MHC molecules for presentation to the effector cells of the immune system.
  • infectious agent e.g. , prions
  • PBMCs peripheral blood mononuclear cells
  • PBLs peripheral blood lymphocytes
  • this aspect of the invention provides a functional ex vivo assay for determining the immunogenicity of PCL-modified human tumor cells and the ability of an individual's mononuclear blood cells to respond to or immunoreact against the modified tumor cells.
  • a correlative parameter that is used to determine an individual's immune response status is the cytokine profile or pattern secreted by stimulated versus unstimulated mononuclear cells.
  • the cytokine profile as determined by measuring the levels of cytokine secreted by PBMCs indicates if an individual has produced a cell mediated immune response or a T H 2 to T H 1 switch.
  • the cytokine pattern can also be determined by analyzing the cytokine messenger RNA levels by methods known in the art, i.e. , PCR and RT-PCR.
  • a successful or positive in vitro immune cell response is meant that immune cells are sensitized and are immunoreactive against the PCL-modified target cells (e.g., PCL-modified tumor cells or infected cells) in an IVS assay, such that the immune cells proliferate or otherwise react against the modified cells.
  • a high stimulation or response index is achieved (i.e. , greater than or equal to about 1.5 to 2.0, or above, preferably greater than or equal to 2.0).
  • a successful or positive immune cell response is also determined by the ability of sensitized immune cells to react against the appropriate target cells in a cytotoxic assay.
  • this aspect of the invention provides an assay or protocol the results of which are predictive of in vivo efficacy and are indicative of the likelihood of success in using PCL-modified cells as immunogens in vivo in human patients who will be most likely to mount an immune response to their tumor or infection.
  • This is an essential step toward the clinical application of PCL-modified tumor cells in cancer patients presenting with various tumor types, for example, or in infected patients presenting with infections caused by a variety of viruses or microorganisms.
  • the invention provides a prognostic application and allows the evaluation of potential selection criteria for determining those individuals who may be responders and those who may be non-responders to the PCL-modification treatment, IVS assay, and eventual immunotherapy or immunoadoptive therapy using PCL-modification of tumors, and the stimulation, sensitization, and immunoreactivity of human immune cells (or PBMCs) in vivo.
  • PCL- immunotherapy refers to the immunization or vaccination of a patient with PCL-modified cells, e.g. , tumor cells or infected cells, to elicit a specific immune response (i.e., an infected cell- or tumor cell- inhibiting or reducing response) against the tumor cells or the infected cells.
  • the technique of immunoadoptive therapy is known among those having skill in the art; a description of the technique is found in U.S. Patent No. 5,192,537 to Michael E. Osband.
  • the peripheral blood mononuclear cells of a patient afflicted with a cancer or tumor or infection are treated to activate their responsiveness to one or more antigens associated with the tumor, cancer, or infecting agent.
  • Mononuclear cells are first obtained from the patient's blood, e.g., peripheral blood. If necessary or desired, suppressor T lymphocytes are then removed and the remaining cells are suspended in a tissue culture medium containing a non-specific lymphocyte activator (e.g., phytohemagglutinin).
  • a non-specific lymphocyte activator e.g., phytohemagglutinin
  • the mononuclear cells may also be incubated with an extract of the patient's tumor or infected cells, preferably tumor or infected cells that have been PCL-modified in accordance with the invention, and with the patient's own serum.
  • the cells are then incubated, preferably under hyperthermic conditions (e.g. , about 38°C to about 41°C) for a period of time so that they are activated against the patient's modified autologous tumor or infected cells (i.e. , in an IVS assay).
  • hyperthermic conditions e.g. , about 38°C to about 41°C
  • the activated mononuclear cells may be subjected to one or more additional procedures to re-deplete various kinds of suppressor cells, including macrophages, to additionally boost their activity.
  • Such depletion methods include the use of gamma radiation (e.g., in the range of 50-400 rad) to remove radiosensitive suppressor cells.
  • the activated cells are then re-infused or administered to the patient using established procedures known to the skilled practitioner in order to reduce or eliminate the tumor or infected cells, to inhibit tumor growth, or to reduce or eliminate recurrences of cancer or infection.
  • Additional anti-T cell suppressor drugs or agents e.g. , cimetidine may be used in carrying out the immunoadoptive therapy protocol, if necessary or desired.
  • Another embodiment of the invention provides selection criteria for the screening and monitoring of patients for immunocompetence and immuno- reactivity to tumors, infection, and to foreign or pathogenic cells using a combination of PCL-modification of cells and IVS techniques.
  • candidates for PCL-immunotherapy undergo screening for the ability of their PCL-modified tumor cells to stimulate their autologous peripheral blood mononuclear cells to become sensitized and activated against, and to proliferate in response to, PCL-modified antigens in an IVS assay.
  • PCL-modified autologous and allogeneic cells e.g., tumor cells or infected cells, may be used to stimulate an individual's PBMCs in the IVS assay.
  • the allogeneic cells are tumor cells, they should ideally be of the same tumor type as the patient's tumor. Similarly, if the allogeneic cells are infected cells, they should infected with the same infective agent as the patient's infected cells, and preferably be of the same cell type.
  • the IVS response or stimulation ratio also called the response or stimulation index (RT or SI) herein (see Example 14).
  • the response ratio is determined from the quantitative measurement of a patient's PBMC proliferative response to PCL-modified tumor cells (numerator) in relation to the measurement of the patient's PBMC proliferative response to non- modified or "native" tumor cells (denominator).
  • patients whose IVS response ratio is less than about 1.5 to about 2.0 are less likely to respond to subsequent PCL-immunotherapy than patients whose response ratio is equal to or exceeds about 1.5 to about 2.0.
  • Patients who have a response or stimulation index value greater than or equal to about 1.5 to about 2.0 represent a patient population showing a high level or positive response to the PCL-modified tumor or infected cells.
  • an SI value of 2.0 or above generally correlates with an exceptionally good immune activation response, and an SI value of less than 1.5 to 2 generally correlates with a poor immune activation response.
  • the ability to determine whether an individual will have a good or a poor cellular immune response and/or a switch from T H 2 to T H 1 cell production is improved upon by also analyzing the corresponding cytokine synthesis and secretion of the peripheral blood cells in the IVS assay.
  • those patients having a low stimulation or response ratio and a cytokine profile indicating a lack of a T H 2 to T H 1 switch using PCL-modified and unmodified cells as stimulators are considered to be poor responders and, consequently, such patients are not likely to benefit from subsequent PCL- immunotherapy, based on the determined response or stimulation ratio and the cytokine pattern.
  • patients having a high ratio and a cytokine pattern that is indicative of a T H 2 to T H 1 switch are considered to be good responders to the tumor or infected cells and are determined to be likely to benefit from subsequent immunotherapy, particularly PCL-based immunotherapy.
  • control studies to determine the general immune responsiveness or immune status of a patient's PBMCs can be performed in accordance with the method using mononuclear cells isolated from the patient's blood.
  • these control IVS assays which are optimally performed in accordance with the invention, the responsiveness of a patient's PBM cell population to stimulation by mitogenic agents, nonspecific activators, or cytokines such as phytohemagglutinin (PHA), pokeweed mitogen, or anti-CD3 antibody (e.g., OKT3) is evaluated (see Example 14).
  • a baseline IVS analysis is performed as described herein using a patient's PBMCs and his or her autologous tumor cells within about one to two weeks following tumor surgery (see Example 14). Based upon the immune response level of the patient's own PBMCs to his or her PCL-modified tumor cells in the IVS assays performed as described, reliable and objective determinations can be made concerning whether or not an individual patient would be likely to benefit from subsequent PCL-immunotherapy, and whether or not an individual will or will not be enrolled in subsequent PCL-immunotherapy procedures.
  • a protocol used for tumor patient assessment in accordance with the invention is as follows: after tumor surgery, a patient may undergo either chemotherapy or radiation regimens, or combinations thereof. During this time, the IVS assays as described herein are carried out using as stimulators the tumor cells processed from the patient's tumor, and as responders the patient's PBMCs. About a month after recovery from either the chemotherapy or irradiation post- surgery adjunct treatments, PCL-immunotherapy is begun, if the IVS assays which were performed following surgery showed a high level of response by the patient's PBMCs toward the autologous PCL-modified tumor cells.
  • selection criteria used for determining those patients to include in tumor treatment involving PCL-modification of tumors, IVS assay, and PCL-immunotherapy or adoptive immunotherapy techniques include, but are not limited to, selecting patients having certain cancers or tumors, such as colorectal carcinomas, non-small cell lung carcinomas, renal carcinomas, and ovarian carcinomas, who undergo tumor resection surgery.
  • Nonlimiting exclusion criteria for excluding patients from subsequent PCL-immunotherapy or immunoadoptive therapies include those patients undergoing chemotherapy or radiotherapy for up to four weeks prior to surgery; patients undergoing any experimental therapy known or intended to improve immune status; and patients greater than or equal to 80 years of age.
  • PCL-modified tumor cells are potent stimulators of immunoreactivity by an individual's autologous lymphocytes.
  • PCL-modified cells e.g., tumor cells
  • IVS assays can be used to clonally expand discrete and specifically immunoreactive populations of peripheral blood mononuclear cells, in particular, lymphocytes, e.g., T cells, which can be harvested and used in immunoadoptive therapy protocols.
  • T cell enrichment and separation procedures include, but are not limited to, flow cell cytometry or cell sorter methods, nylon wool column separation, antibody or lectin column chromatography designed to isolate or separate discrete T cell populations based on surface antigen or receptor specificities, and B lymphocyte removal using specific antibodies and complement.
  • the activated T cell population can then be cultured in the presence of T cell specific or nonspecific cytokines or activators, or mixtures thereof, (and additional modified tumor cells as in vitro "boosting" antigen) to cause the proliferation and growth of the so-selected immunoreactive and enriched activated T cell population. Thereafter, the proliferating and immunoreactive T cell population can be used to immunize a patient against his or her tumor in immunoadoptive therapy techniques. It is to be understood that the above- described methods, with modifications known to those in the art, may be used to enrich for or separate other types or populations of specifically immunoreactive mononuclear blood cells.
  • patients who receive such immunotherapy should optimally demonstrate a progressive increase in their IVS response ratios as a function of their immunization with PCL-modified, autologous cells as immunogens or vaccines, such that an increase in the IVS response ratio correlates with a patient's therapeutic outcome using the PCL- immunotherapy technique.
  • modified cells e.g. tumor cells or infected cells, and the like
  • the mean IVS response ratio should remain at a value of at least about 1.5 to 2.0, or above, and should optimally increase during the progression of the immunotherapy schedule or immunizing regimen.
  • Another aspect of the invention relates to a method of determining whether or not a cancer or tumored patient should receive immunotherapy or other treatment regimens, such as chemotherapy or radiation therapy, following surgery and excision of his or her tumor.
  • the invention also relates to a method for determining whether or not a tumored or cancer patient will have a successful clinical outcome after immunotherapy, particularly, PCL immunotherapy, and whether or not that patient should enter an immunotherapy or adoptive immunotherapy protocol as a treatment regimen for his or her cancer or tumor.
  • RT stimulation or response index outcome
  • T H 1 immune response i.e., cell-mediated immunity
  • T H 1 cells produce IL-2, which causes the continued proliferation of specific T lymphocytes or CD8-I- lymphocytes, and also produce IFN-7, which activates the anti-tumor and anti-virus properties of cytotoxic T lymphocytes.
  • T H 2 cells i.e., CD4+ cells
  • IL-6, and IL-10 which stimulate the proliferation and maturation of antibody-producing B lymphocytes, which are active participants in the humoral immune response to antigen.
  • the synthesis and release of IFN-7 by T H 1 cells during a strong cell-mediated response may suppress T H 2 activation of B lymphocytes that are responding to the same immunogenic stimulus.
  • IL-4 and IL-10 released by T H 2 cells during a strong humoral immune response may actually suppress the ability of T H 1 cells to carry out a successful cell mediated immune response.
  • Such cross-regulatory interactions during a patient's in vivo immune response may actually suppress an effective, cell-mediated response to a tumor or viral infection after antibody production is triggered.
  • the generation and maintenance of a strong cell mediated immune response, a T H 1 response, or a T H 2 to T H 1 switch in patients receiving PCL treatment for their tumor or infection is an optimum situation and endpoint for those patients who will benefit from PCL immunotherapy and/or who will best respond to PCL immunotherapy and treatment as a clinical therapeutic protocol.
  • Such a cell-mediated immune response is best orchestrated by the production of levels of secreted T H 1 cytokines that are stimulatory for or directly linked to a cell-mediated immune response (e.g.
  • T H 2 cytokines that are inhibitory for a cell- mediated immune response, or are stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10).
  • IL-4, IL-5, IL-6, and IL-10 a humoral immune response
  • high levels of T H 1 cytokines secreted by PBLs in response to PCL-modified stimulator cells relative to the levels of these cytokines secreted by PBLs in response to unmodified stimulator cells are indicative of a cell mediated immune response.
  • T H 2 cytokines that are inhibitory for a cell-mediated immune response, or that are stimulatory for a humoral immune response secreted by PBLs in response to PCL- modified stimulator cells relative to the levels of these cytokines secreted by PBLs in response to unmodified stimulator cells are indicative of a cell mediated immune response.
  • a patient may also convert from primarily a T H 2 immune response to a T H 1 response (i.e. , a T H 2 to T H 1 "switch" or conversion) during the course of in vivo or in vitro PCL treatment involving immunization with PCL-modified cells (e.g., tumors or virus-infected cells).
  • a T H 2 to T H 1 switch is accompanied by a cytokine secretion pattern that reflects a T H 1 response and is indicative that the patient will respond appropriately (i.e. , by producing a cell mediated immune response) to destroy tumor cells or virus-infected cells during the course of PCL immunotherapy.
  • an optimum finding which indicates that a patient will respond to tumor or infection in a positive way by generating a cell mediated response, is that the patient is producing low (or virtually no) levels of the IL-10 cytokine in response to PCL- modified stimulator cells (e.g. , tumor or infected cells); thus, the T H 1 response is not likely to be affected or suppressed.
  • PCL- modified stimulator cells e.g. , tumor or infected cells
  • IFN-7 is released by T H 1 cells and is indicative that a cell mediated reponse has been generated against a tumor or infected cell
  • the finding that a patient is producing high levels of IFN- 7cytokine in response to PCL-modified stimulator cells is also indicative that a positive and successful clinical response to PCL treatment and immunotherapy has been achieved by the patient.
  • PCL-modified stimulator cells e.g. , tumor or infected cells
  • Example 15 demonstrate that in vitro determinations of an individual's IVS response index and the analysis of the individual's cytokine profile parameters after assaying the response of a patient's PBMC cell populations to PCL-modified tumor or infected stimulator cells relative to a response to unmodified stimulator cells offer to the practitioner a way to predict, determine, or support a decision concerning whether or not a patient should undergo (or continue) immunotherapy, in particular.
  • PCL immunotherapy whether or not a patient will have a positive or successful clinical outcome as a result of PCL treatment and immunotherapy; and whether or not an alternative type of treatment regimen, e.g., chemotherapy or radiation therapy, should be instituted for a patient instead of PCL immunotherapy.
  • a positive PBMC proliferative response as measured by IVS assays (i.e. , a positive response or stimulation index), is necessary to determine whether or not an individual is responding to vaccination or immunogens comprising PCL-modified cells by producing a cell mediated response and/or a T H 1 response against tumors or infected cells, and the like.
  • the determination of the cytokine profile or pattern that is produced by an individual's activated PBMCs provides the indication and is capable of demonstrating that an appropriate immune response is occurring and is also indicative of a T H 2 to T H 1 pattern or conversion.
  • the proliferating PBMCs or PBLs cells can be phenotyped using methods known in the art (e.g., B and/or T cell specific antibody markers and assays such as cell sorting, immunolabeling, or immunochemistry protocols) to distinguish the discrete populations and/or subsets of lymphocytes that are responding in the IVS assays.
  • B and/or T cell specific antibody markers and assays such as cell sorting, immunolabeling, or immunochemistry protocols
  • monoclonal anti-CD8 antibody and monoclonal anti-CD4 antibody preparations can be used in the immunophenotyping analyses.
  • immunophenotyping of the proliferating cells can determine if a CD4+ or a CD8+ cell population is being stimulated to respond to PCL-modified tumor or infected cells.
  • in vitro cytotoxic T cell assays e.g., see Example 8
  • CTLs cytotoxic T cells
  • the present invention allows the determination and prediction of a positive or negative clinical outcome against tumors or infection by using the IVS analysis, the resulting stimulation or response index, and the correlation of these parameters with cytokine profiles and patterns as detailed in Example 15 hereinbelow.
  • the invention provides a method of determining if an individual beset with a cancer, a tumor, or an infection will or will not be likely to respond to in vivo immunotherapy treatment by mounting a cell mediated response to reduce, inhibit, or destroy the tumor or infection, comprising: a) crosslinking autologous or allogeneic cancer, tumor or infected cells with a 2', 3' nucleoside or nucleotide d.aldehyde crosslinker at a concentration and for a time effective to crosslink proteins in the cells' plasma membranes and treating the cells with hydrostatic pressure for a time sufficient to cause a modification of proteins in the cells' plas ma membranes, thereby resulting in a crosslinked and pressure-treated modified cell preparation; b) measuring the ability of the modified cells of step a) to stimulate the proliferation and immunoreactivity of the individual's mononuclear blood cells compared with the ability of native or unmodified cells to stimulate the proliferation and immunoreactivity of the mononuclear blood cells; and c)
  • the cytokine pattern as mentioned above in step c) and used in conjunction with the response or stimulation index obtained from step b) as disclosed herein can be used to determine that an individual is likely to respond in a positive manner to immunotherapy treatment (i.e. , by mounting a cell mediated immune response and/or a T H 1 response) based on the following exemplary parameters: 1) a high level of mononuclear blood cell proliferation as determined in accordance with step c), and 2) at least one or more of: (i) a high level of stimulatory T H 1 cytokine secretion; (ii) a low level of T H 1 inhibitory cytokine secretion; (iii) a low level of stimulatory T H 2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, as determined according to step c).
  • the determination that an individual will respond in a positive manner to immunotherapy treatment by mounting a cell mediated immune response can also be concluded based on 1) a low level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of less than about 1.5), and 2) at least one or more of: (i) a high level of stimulatory T H 1 cytokine secretion; (ii) a low level of T H 1 inhibitory cytokine secretion; (iii) a low level of stimulatory T H 2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, as measured according to step c).
  • the cytokine pattern indicates that a T H 2 to T H 1 switch has occurred in the individual.
  • the determination that an individual will not respond in a positive manner to immunotherapy treatment and is not likely to mount a successful cell mediated immune response can be concluded based on 1) a low level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of less than about 1.5), and 2) at least one or more of: (i) a low level of stimulatory T H 1 cytokine secretion; (ii) a high level of T H 1 inhibitory cytokine secretion; (iii) a high level of stimulatory T H 2 cytokine secretion; and/or (iv) a high level of cytokines stimulatory for the humoral immune response, as measured according to step c).
  • This cytokine pattern indicates that a T H 2 to T H 1 switch has not occurred in the individual.
  • the determination that an individual will not respond in a positive manner to immunotherapy treatment and is not likely to mount a successful cell mediated immune response can also be concluded based on 1) a high or relatively high level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of about 1.5 to 2 or greater), and 2) at least one or more of: (i) a low level of stimulatory T H 1 cytokine secretion; (ii) a high level of T H 1 inhibitory cytokine secretion; (iii) a high level of stimulatory T H 2 cytokine secretion; and/or (iv) a high level of cytokines stimulatory for the humoral immune response, as measured according to step c).
  • This cytokine pattern combined with a positive proliferation index indicates that a T H 2 to T H 1 switch has not occurred.
  • this aspect of the invention allows several clinically-relevant determinations to be made with respect to the results of performing the IVS assays, including determining a stimulation index or response index, determining the associated cytokine profile or pattern, and if desired or necessary, immunophenotyping the PBMCs to determine which cell subsets are stimulated and proliferating in the in vitro IVS assays in response to PCL-modified cells, using a tumored or infected patient's PBMCs as responders, PCL-modified tumor or infected cells as stimulators, and unmodified tumor or infected cells as control stimulators.
  • a given patient's clinical outcome or prognosis with respect to immunotherapy treatment can be directly correlated with the results of the above-mentioned determinations and can be concluded to be either positive (i.e. , a high likelihood of success) or negative (i.e., a low likelihood of success) based on the following four exemplary conditions:
  • a positive or high proliferative response to PCL-modified stimulator cells in the IVS assay e.g., a response index greater than 1.5 or 2
  • a positive cytokine profile or pattern determined by the types of cytokines secreted by responder PBMCs is a predictor of a positive or successful clinical outcome or a positive prognosis for immunotherapy treatment, particularly PCL immunotherapy, for the patient.
  • a positive cytokine profile or pattern is one in which the tested PBMCs show a production of high levels of secreted T H 1 cytokines that are stimulatory for, directly linked to, or associated with a cell-mediated immune response (e.g., IL-2 or IFN-7), and/or a production of low (or virtually no) levels of secreted T H 2 cytokines that (i) are inhibitory for a cell-mediated immune response, or (ii) are stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10 and the like).
  • a cell-mediated immune response e.g., IL-2 or IFN-7
  • a humoral immune response e.g., IL-4, IL-5, IL-6, and IL-10 and the like.
  • the levels of secreted cytokines are determined by comparing the stimulation or response values obtained in the in vitro proliferation assays for PBMCs cultured in the presence of PCL-modified stimulator cells (e.g. , tumor or infected cells) versus PBMCs cultured in the presence of unmodified stimulator cells (e.g., tumor or infected cells).
  • PCL-modified stimulator cells e.g. , tumor or infected cells
  • a positive or successful prognosis for immunotherapy treatment is based upon results showing secretion of low levels of IL-10 (and/or IL-4) and secretion of high levels of IFN- 7(and/or IL-2), thus indicating a T H 1 profile or a T H 2 to a T H 1 profile) of a patient's PBMCs in response to stimulation by PCL-modified stimulator cells (e.g. , Example 15 and the IVS(+) panels of Tables 12 and 13, and Table 14); (2) a negative or low proliferative response to PCL-modified stimulator cells in the IVS assay (e.g.
  • a response index of less than 1.5 or 2) combined with a positive cytokine profile or pattern as determined by the secretion of T H 1 stimulatory cytokines by responder PBMCs, detailed in (1) above, is also a predictor of a positive or successful clinical outcome or a positive prognosis for immunotherapy treatment, particularly PCL immunotherapy, for a patient. That the patient is secreting T H 1 cytokines which indicate the production of a cell mediated immune response against tumor or infected cells, in spite of the low proliferation of PMBCs, serves as a positive indicator of successful immunotherapy.
  • Example 15 and the IVS(-) panel of Table 12 demonstrate that after stimulation by PCL-modified stimulator cells, the secretion of IFN-7 by responder PBLs increases and is indicative that the PCL-modification protocol has forced a cytokine profile change and a corresponding T H 2 to T H 1 pattern change; thus, the patient is likely to respond to PCL immunotherapy during the course of treatment.
  • repeated immunizations or vaccinations of a patient with PCL-modified cell- containing immunogenic preparations may increase the patient's response index and/or may aid in maintaining the T H 1 pattern or the T H 2 to T H 1 pattern or can result in the conversion from a T H 2 to a T H 1 response in a patient.
  • the T H 2 to T H 1 conversion then allows a corresponding positive response to immunotherapy treatment regimens and a positive clinical outcome during future PCL- immunotherapy treatment; (3) a negative or low proliferative response to PCL-modified stimulator cells in the IVS assay (e.g. , a response index of less than 1.5 or 2) combined with a negative cytokine profile or pattern determined by the types of cytokines secreted by responder PBMCs is a predictor of a negative or unsuccessful clinical outcome or a negative prognosis for immunotherapy treatment, particularly PCL immunotherapy, for the patient.
  • a negative or low proliferative response to PCL-modified stimulator cells in the IVS assay e.g. , a response index of less than 1.5 or 2
  • a negative cytokine profile or pattern determined by the types of cytokines secreted by responder PBMCs is a predictor of a negative or unsuccessful clinical outcome or a negative prognosis for immunotherapy treatment, particularly PCL immunotherapy
  • a negative cytokine profile or pattern is one in which there is a production of high levels of secreted T H 2 cytokines that are either inhibitory for a cell-mediated immune response or stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10).
  • a negative or unsuccessful prognosis for immunotherapy treatment is based upon results showing secretion of higher levels of IL-10 (and/or IL-4) and secretion of no or low levels of IFN-7 (and/or IL-2).
  • the negative cytokine profile may be due to several factors that may be tested, if necessary or desired, such as the stimulation and proliferation of suppressor T cells.
  • the fourth condition prognoses a negative clinical outcome of immunotherapy treatment for the patient.
  • the present invention provides the clinician or skilled practitioner with the tools and methods to predict and determine the clinical outcome of immunotherapy, particularly PCL immunotherapy, for a given patient based on the in vitro analyses performed using PCL-modification technology.
  • the skilled practitioner is able to make informed and substantiated decisions regarding further and future treatment for cancer patients and patients harboring microbial or viral infections that can dramatically impact on the outcome of their diseases in a beneficial and humane manner.
  • cytokine treatment may be combined with the PCL-modification methodology of the invention to further augment or improve the directed response of immune cells against tumor cells, infected cells, and transformed cells.
  • cytokines are not completely specific for anti-tumor directed effector cells (e.g. , PBMCs), these factors have the ability to augment and enhance one or more components of cellular immune function.
  • cytokines such as gamma interferon, or other activators, such as phytohemagglutinin, pokeweed mitogen, or combinations thereof, prior to crosslinking and pressure modification as described herein is likely to cause a further increase or enhancement in the expression of MHC antigens, in addition to a stabilization of the presentation of MHC structures as a consequence of crosslinking.
  • the cytokines may be purified or recombinantly produced; highly purified and recombinant cytokines are also commercially available.
  • cytokines include, but are not limited to, interleukins, e.g., interleukin-2 (IL-2) and interleukin-12 (IL-12), tumor necrosis factor (TNF), alpha interferon (IFN- ⁇ ), beta interferon (IFN- ⁇ ), gamma interferon (IFN-7), and hematopoietic factors such as granulocyte-macrophage stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF).
  • GM-CSF granulocyte-macrophage stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • cytokines in the invention is the transfection of tumor cells in vitro with genes encoding cytokines, and modifying the tumor cells in accordance with the invention.
  • the transfected, modified tumor cells when used as immunogens to stimulate the immune response after immunization in vivo are thus capable of producing immunostimulatory cytokines in abundance, particularly at the site of tumor growth.
  • Genes encoding IL-2, GM- CSF, and IFN-7 are particularly useful for stimulating a particular immune effector function when the cytokine is produced and secreted by the modified and transfected cells.
  • T cell activator compounds which cause stimulation or proliferation such as anti-T cell receptor compounds, anti-CD3 compounds, or OKT3, can be used in conjunction with PCL-modified tumor cells or infected cells in an immunogenic preparation or vaccine to augment or enhance a patient's immune response to the tumor or infected cells (for example, see Example 15,
  • 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
  • mice were serially passaged in mice by subcutaneous inoculation of 2-5 x
  • HBSS Hanks Balanced Salt Solution
  • AdA AdA
  • AdA which is a biologically compatible chemical crosslinker
  • 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 as used in accordance with the invention; they are membrane impermeable; 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.
  • Example 2 Example 2
  • 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). 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 3 ).
  • PBS phosphate buffered saline
  • 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.
  • Anti-CD3 and PHA stimulation of PBMCs In the wells of a 96-well microtiter plate, 2x10 s cells were incubated in 0.2 mL tissue culture medium, e.g. , RPMI (Gibco), supplemented to contain 20 ⁇ L of either (a) anti-CD3 monoclonal antibody or PHA in a 96-well microplate.
  • 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.
  • OKT 3 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 HB 231. For these assays, both anti- CD3 monoclonal antibody and PHA were diluted 1 :20 in culture medium.
  • a subcutaneous tumor was carefully excised, minced with scissors into fragments 1-2 mm in size, and stirred in a triple-enzyme mixture of hyaluronidase, deoxyribonuclease and collagenase (Sigma Chemical Co. , St. Louis, Mo.) for 30-60 min in 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, 76:309. 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 cells were washed twice with PBS (pH 7.4) and then were subjected to one of the modification treatments described hereinbelow.
  • the viability of the modified tumor cells was assessed by trypan blue dye exclusion.
  • Modification I Crosslinking of proteins on the tumor cell surface. About 10 8 cells/mL were inoculated into a 50 mL tube (Falcon, Becton Dickinson Labware, N.J.) holding a PBS solution containing 0.5 % AdA (about 20 mM to 40 mM, preferably less than 40 mM) and were incubated in this solution for 1 hour at room temperature with occasional mixing. Unbound AdA was removed by three cycles of centrifugation at 1500 ⁇ for 5 minutes, followed by gentle resuspending of the pellet in PBS (in the final resuspension, PBS was added to obtain the desired cell concentration).
  • Modification IH Hydrostatic pressure and crosslinking in sequence. Cells were pressurized and then immediately crosslinked by the two modification procedures outlined above. Modification IV: Hydrostatic pressure and crosslinking simultaneously. Cells were pressurized and crosslinked at the same time by the two modification treatments described above. The exposure of cells to the crosslinking compound and to hydrostatic pressure at the same time in accordance with the invention is a preferred PCL-modification.
  • 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 s 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* 10 5 x 10 s ). 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 PBS 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 [ 3 H]-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 s 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 % CO 2 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- 3 H]thymidine (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 determine 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 [ 3 H]-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.
  • DTH Delayed-tvpe hypersensitivity
  • 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- 12s I- labeled 2 '-deoxyuridine ( 12S 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 I2S IdUrd index). Five mice were included in each group. Control groups included unprimed mice, as well as those primed with unmodified tumor cells.
  • the ears were fixed with Bouin's fixative for 48 h at room temperature. Excess fixative was removed thereafter by extensive washing with 70% ethanol. Any hair present was carefully shaved off and the ears were cut to uniform size for embedding and subsequent sectioning on a rotary microtome (Spencer model No. 820; American Optical Co.). The slides were stained with eosin and in some cases with toluidine blue (Vadas et al., 1975. , supra).
  • 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. Cancer Immunother. Immunol. , 11 :93.
  • DTH skin reactions were scored as follows: “-”, “ + “, “ + + “, or “ + + + “, where “-” indicates no reaction and " + + + “ indicates maximal redness and swelling reactions.
  • 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.
  • tuberculin a lipoprotein of Mycobacterium tuberculosis
  • the DTH test allowed the determination of the immunogenic potential of the tested cells.
  • a high immunogenic potential correlated with a severe DTH reaction.
  • 23 out of 25 patients had DTH responses of "2" and above when PCL-modified cells were used to as immunogens in DTH analyses, compared with unmodified cells.
  • each patient was treated with an PCL-treated or untreated allogeneic tumor preparation which corresponded to his or her tumor type (e.g. , a melanoma patient was treated with a donor's allogeneic PCL-modified melanoma tumor cells, and a lung cancer patient was treated with a donor's allogeneic PCL- modified lung tumor cells, and so forth, depending on the type of cancer with which a given patient was afflicted).
  • an PCL-treated or untreated allogeneic tumor preparation which corresponded to his or her tumor type (e.g. , a melanoma patient was treated with a donor's allogeneic PCL-modified melanoma tumor cells, and a lung cancer patient was treated with a donor's allogeneic PCL- modified lung tumor cells, and so forth, depending on the type of cancer with which a given patient was afflicted).
  • the patients were given two separate subcutaneous injections, side by side, of unmodified (i.e., no PCL treatment) and of PCL-modified allogeneic tumor cells at 1 x 10 s cells per injection.
  • the tumor cells were treated with AdA crosslinker at about 40 mM at the same time that hydrostatic pressure in the range of between 1200 to 1400 atm was being applied.
  • the development of the DTH skin test reaction was scored at 48 hours after injection.
  • patient #13 and patient #14 had lung cancer
  • patient #15 and patient #20 had melanoma
  • patient #17 and patient #18 had colorectal cancer.
  • All six cancer patients had a demonstrably significant increase in their DTH responses to the PCL-modified allogeneic tumor cells relative to the unmodified tumor cells, consistent with the results obtained using PCL-modified autologous tumor cell vaccines.
  • chromium release cytotoxicity assays were performed. A five hour 5 'chromium release cytotoxicity assay was carried out as described by Brunner et al. 1976. "The 51 Cr release assay as used for the quantitative measurement of cell-mediated cytolysis in vitro", In: In vitro methods in cell mediated immunity. Eds. B.R. Bloom and J.R. David, Academic Press, London, p. 423. Target cells used in these assays were EL4 (EL4 originated from a chemically-induced T cell leukemia; Gorer, P. A. 1961. "The isoantigens of malignant cells", In: Biological approaches to cancer immunotherapy. Ed.
  • ARadLV 136 is a radiation- induced leukemogenic variant of ARadLV and is maintained in vitro as described in N. Haran-Ghera et al. 1977. J. Immunol. , 118:600.
  • EL4 as well as ARadLV 136 target cells were washed once in RPMI-1640 medium and the supernatant was aspirated to leave 0.1 mL with the cell pellet.
  • the pellet was gently dispersed and resuspended in PBS with 5 % fetal calf serum and centrifuged at low speed (1000 ⁇ m for 5 min).
  • 0.1 mL Na 2 51 CrO 4 solution (Amersham, UK; 1 mCi/mL, sp. act. 200 mCi/mg) per 2x10 s target cells was added to the pellet in 0.1 mL buffer and the suspension was gently vortexed.
  • T cells were then washed, centrifuged, and resuspended as before in PBS containing 5 % fetal calf serum. Enrichment of T cells was done by incubation of cells with nylon fibers (Fenwall Laboratories, Deerfield, 111.) Aliquots of 0.2 mL 51 Cr-labeled target cell suspension containing 10 s cells were pipetted into round-bottomed plastic tubes (12x55 mm, Falcon); equal volumes of various dilutions of spleen effector cells were then added to the target cells to yield ratios of lymphocytes to target cells of 50, 10, 2.5 and 1.25.
  • the tubes were then centrifuged at 1000 ⁇ m for 2 min before incubation at 37°C in a humidified incubator flushed with 5 % CO 2 . After 5 hours, 0.6 mL of PBS were added, the tubes were centrifuged at 1000 ⁇ m for 5 min. , and 0.5 mL of supernatant was collected for counting in a well-type gamma counter (Gammamatic, Kontron). Maximum release was determined by adding 0.6 mL 0.5 % NP-40, and spontaneous release was counted from tubes containing labeled target cells alone. The percentage of specific lysis was defined and calculated by the following formula:
  • Figs. 2 and 3 The results from the cytotoxicity tests as described are presented in Figs. 2 and 3. As seen in these figures, the cytotoxic ability of anti-EL4 effector cells, isolated from spleens of mice and primed with AdA-treated, or pressure -I- AdA-treated tumor cells, to lyse sl Cr-EI_4 targets remained high (about 60%) at all effector: target ratios. In contrast, the ability of anti-(ARadLV 136) effector cells to lyse 5I Cr-ARadLV 136 target cells was generally low (about 15 %) and varied at the different lymphocyte-to-target cell ratios.
  • mice immunized, i.e. vaccinated, with tumor cells modified by both AdA crosslinking and the application of hydrostatic pressure were immunized, i.e. vaccinated, with tumor cells modified by both AdA crosslinking and the application of hydrostatic pressure.
  • a marked increase in DTH reactivity was also seen in mice vaccinated with tumor cells treated with AdA alone.
  • DTH response after priming with cells modified by pressure treatment only was essentially the same as that observed after priming with unmodified tumor cells.
  • ARadLV 136 cells Against the relatively strong DTH response obtained with EL4 cells, only a very weak response, compared with control, was obtained with the ARadLV 136 cells, although the priming with cells modified by exposure to both pressure and AdA showed the strongest response.
  • Fig. la-le Histological examination of the ears is depicted in Fig. la-le and as can be seen in this figure, there is a predominant infiltration of monocytes or macrophages 24 hours after the challenge. In ear sections stained with toluidine blue, it was not possible to detect many cells with granules typical of basophils. This indicated that the inflammatory reaction was distinct from cutaneous basophil hypersensitivity.
  • Lymphocyte proliferation assay The assay was modified from a procedure described earlier (Vanky et al. ,
  • Tumor cells (5x10 s ), EL4 cells as well as ARadLV 136 cells, were heavily irradiated (10,000 rad) prior to incubation with an equal number of effector cells. Effector cells were prepared from spleen, cleared of erythrocytes, and enriched for T cells. The proliferative responses of effector lymphocytes in the presence of inactivated stimulators (tumor cells) were assayed in triplicate in round-bottomed 96-well microtiter plates (Greimer,
  • the plates were centrifuged at 1500 ⁇ m for 10 minutes and washed once with cold PBS. 0.2 mL ice-cold 10% trichloroacetic acid was then added to the cell pellets. The cells were then harvested using a cell harvester (Titertek, Flow Laboratories, UK) and automatically transferred to glass-fiber filters. Excess acid was aspirated and filters were washed with 70% ethanol. Washing was repeated twice and the discs punched out in the machine were placed at the bottom of scintillation vials and left to dry overnight at room temperature.
  • Scintillation counting was performed by adding 5 mL of scintillation fluid (Instamix/xylene, 4: 1) to the vials. Inco ⁇ orated [ 3 H]thymidine was expressed as cpm ⁇ SEM, and values that were at least double the controls were considered to be positive.
  • Plasma membranes were prepared from cells, e.g., tumor cells, essentially as described by Maeda, T. et al. 1983. Biochim. Biophys. Acta. , 731 : 115.
  • HBSS Hank's Balanced Salt Solution
  • PMSF DNAse using a polytron homogenizer with three cycles of homogenization, at 5 seconds per cycle.
  • the crude homogenate was first centrifuged at low speed (i.e. , about 800 0 ⁇ ) to remove debris and nuclei.
  • the supernatant was collected and layered on top of a 41 % sucrose solution and centrifuged at 91 ,000 x g for 60 minutes.
  • 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 (Lowry, O.H. et al. 1951. J. Biol. Chem. , 193:265).
  • 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 0 prior to centrifuging.
  • the washed cells are resuspended in a hypotonic buffer A (Buffer A: 10 mM KCl, 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 KCl, 10 mM HEPES, pH 8.0, 1 mM EDTA/EGTA, protease inhibitors and phosphatase inhibitors
  • NP- 5 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 Q 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 5 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; 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 G 100 or G250 column, whereby the fractions of crosslinked proteins are isolated.
  • Such a high pressure method allows the collapse of the cell membrane structure and the corresponding release and isolation of soluble proteins or protein complexes, some or all of which have their hydrophilic portions in association with membrane lipids.
  • the pretreatment consisted of two vaccinations (or immunizations), one at three weeks and the other at one week prior to the tumor cell challenge.
  • the immunizations were performed using an immunogenic preparation comprising cells subjected to one of the following modification treatments: exposure to AdA, application of hydrostatic pressure, or a combination of the two treatments, following essentially the same procedure as described in Example 1 modifications.
  • the cells used for vaccination were of the same kind as the cells used to challenge the mice.
  • the cells used in this experiment were either EL-4 or BL6 melanoma cells which are a very invasive variant of the B16 cell line (Hart 1979, Am. J. Pathology, 97:587).
  • the B16-BL6 tumor was serially passaged in syngeneic C57BL mice by subcutaneous (s.c.) inoculation of 2 - 5 x 10° cells. Three test were performed as described: Test No. 1 :
  • mice Four groups of mice were used, each pretreated with one of following preparations. Molar concentrations of AdA were based on adenosine concentration.
  • Group 1 EL-4 leukemia cells treated for 10 minutes with various levels of hydrostatic pressure, in the presence of 40 mM AdA.
  • Group 2 EL-4 leukemia cells treated for 10 minutes with various levels of hydrostatic pressure and then with 40 mM AdA.
  • Group 3 B16 melanoma cells treated as described for Group 1 cells.
  • Group 4 B16 melanoma cells treated as described for Group 2 cells. The results are shown in Fig. 5 (each point represents an average of 10 animals): Group 1 - filled squares; Group 2 - empty squares; Group 3 - filled circles; Group 4 - empty circles.
  • mice were pretreated by one of the following preparations:
  • Treatment 1 EL-4 leukemia cells were treated for 10 minutes with hydrostatic pressure of 1350 atm in the presence of various increasing concentrations of AdA; Treatment 2: B16 melanoma cells treated as described for Treatment 1. Treatment 3: B16 melanoma cells treated for 10 minutes with hydrostatic pressure of 1350 atm in the presence of various concentrations of AMPdA.
  • mice Five groups of C57BL mice (6 mice in each group) were immunized subcutaneously and then challenged with B16-BL6 cells.
  • the preparation used for immunization comprised plasma membranes isolated from untreated or treated cells by discontinuous sucrose gradient centrifugation (Maeda et al., 1983, Biochim. Biophys. Acta 731 : 115). Details of the immunizing preparation are provided in Table 4.
  • c antigens H-2k b
  • tumor-specific retroviral antigen on B16-BL6 melanoma cells derived from s.c. tumors, either directly after obtaining single cell suspensions, or after passaging the cells in culture 6 to 8 times over a period of 3-4 weeks.
  • PCL-modified and unmodified cells were labeled and analyzed on either FACS 440 (Becton-Dickinson, Mountainview, CA) or on the FACScan instrument (Becton-Dickinson). Cell populations that were determined to be positive on dual parameter (forward and orthogonal light scatter) analysis were gated, and data were acquired on live gates. Histograms were generated using either Consort 40 software on FACS 440 or consort 30 with LYSYS Software available with FACScan. Appropriate controls were introduced in order to apply logic threshold values.
  • Amelanotic cells which appeared in the population were gated out in the present analysis by light scatter gating. Approximately 10,000 events were tested in each sample. Exposure to AdA, AMPdA and/or to hydrostatic pressure was carried out in a manner similar to that described in Example 5.
  • B16-BL6 cells were exposed to hydrostatic pressure in the range of 600 to 1 ,200 atm and to a constant concentration of AdA of 20 mM. The results are shown in Fig. 8.
  • B16-BL6 cells were exposed to a constant level of 1,200 atm of hydrostatic pressure during incubation with AdA at concentrations from 0.002 mM to 20 mM, and the results are shown in Fig. 9.
  • maximum fluorescent intensity for both class I and B16-BL6 tumor antigen was observed following PCL- modification with a combination of a level of pressure of about 1 ,200 atm and an AdA protein crosslinker concentration of about 20 mM.
  • Tumor cells 10 carcinomas (primary and metastatic), renal cell carcinomas (primary), non- small cell lung carcinomas (primary), lung carcinomas (primary), and ovarian carcinomas.
  • Tumor cells were obtained and PCL-modified as described in Examples 2 and 5 shortly after tumor resection. Tumor cells may be freshly
  • J5 prepared and PCL-modified (i.e., within one hour after surgery) or they may be kept in the cold (i.e., 4°C) for up to three days prior to PCL treatment.
  • the resected tumor may be frozen for further or subsequent use or PCL treatment of the tumor cells.
  • cells such as murine melanoma (i.e., B16 cells) can be successfully stored in the cold for at least one month prior to 0 PCL, especially while data collection via experimentation is ongoing.
  • the immunogenicity of the human tumor cells was measured in a 5-day IVS assay (see Example 6) in which irradiated PCL-modified or unmodified tumor cells served as stimulators and autologous, PBMCs obtained from the patient about 7 to $ 10 days post surgery served as the responders. Autologous, irradiated, PCL- modified PBMCs served as control stimulators. In addition, the non-specific stimulation of PBMCs with anti-CD3 antibody and PHA was used to assess the patient's general immune status.
  • Non-responders 2/7 (29%), mean : 0.8 ⁇ 0.4
  • RT indicates the proliferation index of responder PBMCs, with PCL- modified tumor cells serving as stimulators in the IVS assay.
  • RT signifies the ratio between the proliferation of responder cells (PBMCs) in the presence of PCL- modified tumor cells and in the presence of unmodified tumor cells.
  • RP indicates the proliferation index of responder PBMCs, with PCL- modified autologous PBMCs serving as stimulators.
  • RP signifies the ratio between the proliferation of responder cells (PBMCs) in the presence of PCL-modified autologous PBMCs and unmodified PBMCs.
  • RT/RP shows the specificity of the response of the PBM cells against the PCL-modified tumor cells and is correlated with eventual outcome of PCL-immunotherapy.
  • the RP value serves as a control value, which shows that specific foreign or non-self antigen(s), and not autoantigens, causes specific stimulation and immunoreactivity of the PBM cells in the IVS assay.
  • Adenocarcinoma is a histological type of non-small cell lung carcinoma (NSCC).
  • NSCC non-small cell lung carcinoma
  • Table 7 presents the PHA and CD3 (assayed using monoclonal anti-CD3 antibody) responses of the PBMCs of patients whose lung carcinomas were analyzed (as presented in Table 6 above).
  • non-specific cell stimulating agents such as PHA and/or anti-T cell antibody CD3
  • PCL-modified cells e.g. , tumor cells
  • the results of these studies showed that the response of PBMCs to either PHA or OKT 3 cannot necessarily be used alone to predict whether or not a patient's PBMCs will be stimulated to react against PCL-modified tumor cells.
  • RCC Renal cell carcinoma
  • Table 9 presents the PHA and OKT3 responses of the PBMCs of patients whose renal cell carcinomas were analyzed (see Table 8 above): TABLE 9
  • assays similar to those presented using tumor tissue and cells from colon, lung, and renal cell carcinomas were performed using tumor cells derived from tumor tissue and cells from one patient's ovarian carcinoma.
  • the response ratio was less than one, indicating that this individual was a nonresponder (i.e. , under the conditions of the IVS assays described herein, the individual's PBMCs were unable to mount an immunoreactive response against autologous PCL-modified ovarian tumor cells) and would not be likely to respond in a positive fashion to PCL-modification of cells and subsequent PCL-immunotherapy.
  • renal cell carcinomas and melanomas may be more immunogenic types of tumors than non- small cell lung carcinomas and colon carcinomas; however, response ratios greater than or equal to 2 were demonstrated against all of the tumor types presented in Tables 5, 6, and 8.
  • the highest response ratio values determined by the novel methods of the invention may be directly correlated with the highest response rates (or more aggressive immunoreactivity) against the relevant tumor types in vivo.
  • a positive clinical outcome of PCL immunotherapy potentially correlates highly with a patient's stimulation or response index of greater than about 1.5 or 2, obtained via IVS assays using the patient's peripheral blood mononuclear cells (PBMCs) as responders and PCL-modified or control (unmodified) tumor cells (or infected cells) as stimulators in these in vitro stimulation and proliferation assays.
  • PBMCs peripheral blood mononuclear cells
  • the experiments set forth in this example utilized a correlation between the stimulation or response index and cytokine synthesis and secretion by a patient's PBMCs to determine whether or not a patient should undergo an alternative treatment regimen (i.e., chemotherapy), or whether or not a patient would be a successful or an unsuccessful candidate for prolonged immunotherapy protocols.
  • the secretion of cytokines by PBMCs was tested by in vitro protocols such as enzyme linked immunosorbent assays (ELISAs).
  • ELISAs enzyme linked immunosorbent assays
  • IL-4 Mouse anti-human IL-4 monoclonal antibody (at a concentration of 2 ⁇ g/mL); IL- 10: 2) Rat anti-human IL-10 monoclonal antibody (at a concentration of 4 g/mL); 3) IFN- ⁇ : rabbit polyclonal anti-human IFN- ⁇ (at a concentration of 1 :250); and 4) IL-2: Mouse anti-human IL-2 monoclonal antibody (at a concentration of 2.5 ⁇ g/mL). 50 ⁇ L of antibody were added to the wells of a 96-well microtiter plate (Nunc). The plates were incubated overnight at 2-8°C or for 8 hours at room temperature and then washed three times with phosphate buffered saline (PBS) containing 0.05 % Tween, pH 7.4.
  • PBS phosphate buffered saline
  • the wells of the plates were blocked with PBS supplemented to contain 10% fetal calf serum (FCS) (250 ⁇ L/well); the plates were incubated for 1 - 2 hours at room temperature; and then were washed two times with PBS containing 0.05 % Tween. To the blocked wells of the plates were added 40 ⁇ L per well of the standards (i.e..
  • recombinant human IL-4 at concentrations of 15 pg/mL-2 ng/mL: recombinant human IL-10 at concentrations of 15 pg/mL-2 ng/mL; recombinant human IFN- ⁇ at concentrations of 60 pg/mL-4 ng/mL; and recombinant human IL-2 at concentrations of 15 pg/mL-1 ng/mL), or about 40 to 100 ⁇ L of the samples to be tested (i.e. , the culture supernatants from the IVS assays), diluted in complete cell culture medium.
  • the plates containing the standards and test samples were incubated for 1 hour at 37 °C and then were washed three to four times with PBS containing 0.05% Tween.
  • the anti-cytokine secondary monoclonal antibodies were diluted in PBS containing 10% FCS.
  • the monoclonal antibodies used were as follows: for IL-4 detection, biotinylated rat anti-human IL-4 mAb at a concentration of 1 ⁇ g/mL; for IL-10 detection, biotinylated rat anti-human IL-10 mAb at a concentration of 2 ⁇ g/mL; for IFN- ⁇ detection, mouse anti-human IFN- ⁇ mAb at a concentration of 2 ⁇ g/mL; and for IL-2 detection, biotinylated mouse anti-human IL-2 mAb at a concentration of 1.25 ⁇ g/mL.
  • a detection reagent comprising avidin-peroxidase diluted 1 :500 in PBS containing 10% FCS (1 mg/mL solution; Jacson ImmunoResearch Lab. , Inc.) for detection of the IL-2, IL-4 and IL-10 cytokines.
  • IFN- ⁇ dilute peroxidase-conjugated goat anti- mouse IgG (H+L) (Jacson ImmunoResearch Lab., Inc.) was diluted 1 :500 in the same buffer.
  • the detection antibodies 100 ⁇ L were added to the wells of the plates and were incubated at room temperature for 1 hour. Thereafter, the plates were washed four times with PBS containing 0.05 % Tween.
  • the substrate for the peroxidase reaction development was prepared by dissolving 1 mg of tetramethyl benzidine (TMB) (Sigma) in 1 mL DMSO and adding 9 mL of 0.05 M phosphate-citrate buffer, pH 5.0. Immediately prior to use, 2 ⁇ L of 30% hydrogen peroxide (Sigma) were added per 10 mL of substrate buffer solution. All components for human IL-4 and IL-10 cytokine detection were obtained from PharMgen; the Duoset ELISA Development System for human IL-2 detection and all components used for human IFN- ⁇ were purchased from Genzyme Diagnostics (Cambridge, MA).
  • Tables 12, 13, and 14 are divided into two panels each: one panel shows the results of a patient whose PBMCs responded positively to PCL-modified colon carcinoma cells in the IVS assay (i.e., a positive responder, IVS(+)); the other panel shows the results of a patient whose PBMCs did not proliferate in response to PCL-modified colon carcinoma cells in the IVS assay (a negative responder, IVS(-)).
  • Table 14 shows the results of a positive responder to PCL-modified non- small cell lung carcinoma cells in the IVS assay.
  • the proliferation and cytokine secretion of tumor sensitizer cells, responder PBMCs, and sensitizer and responder cells cultured together are presented.
  • the sensitizer cells were either unmodified or PCL-modified PBLs or tumor cells.
  • Other controls included PBL responder cells cultured together with only complete medium or with the T cell stimulators PHA or OKT3.
  • the PBL responders (R) + tumor cell sensitizers (S) were cultured together and tested in the in vitro assay and included unmodified PBLs with PBL responder cells (i.e. , PBL + R); PCL- modified PBLs + PBL responder cells (i.e.
  • PBL-PCL 4- R unmodified tumor cells + PBL responder cells (i.e. , Tu + R); and PCL-modified tumor cells -I- PBL responder cells (i.e., Tu-PCL + R).
  • Tu + R unmodified tumor cells + PBL responder cells
  • PCL-modified tumor cells -I- PBL responder cells i.e., Tu-PCL + R
  • results indicate that the patients have produced a cell mediated response to their tumors, that a T H 1 response was produced, and/or a T H 2 to T H 1 conversion had occurred. These results are also indicative and predictive of a positive clinical response or outcome to PCL immunotherapy (i.e. , the patient can be expected to maintain a cell mediated response to fight and destroy his or her tumor cells during the course of immunotherapy).
  • the response by this patient may be boosted by repeated vaccinations or immunizations with PCL-modified tumor cells to maintain the T H 1 response and/or to activate and stimulate more of the appropriate cell types to respond.
  • the IVS(-) patient shows secretion of IL-10 and a complete turn off of IFN- ⁇ secretion after culture and stimulation of PBLs by PCL-modified tumor cells. This indicates that a T H 2 to T H 1 conversion has not occurred; therefore, in the absence of a proliferation response and no production of IFN- ⁇ , it can be determined that this patient will have neither a successful cell mediated immune response nor a successful clinical response, and thus is unlikely to benefit from immunotherapy.
  • the invention also encompasses a more rapid or quick screening assay for determimng the cytokine pattern secreted by a patient's peripheral blood cells and/or a T H 2 to T H 1 conversion in response to PCL modified stimulator cells (e.g., tumor or infected cells) relative to nonmodified stimulator cells.
  • the more rapid assay method comprises the use of the tumor preparation containing both tumor cells and resident PBMC populations as described in Example 2.
  • the cytokines secreted by the PBLs localized within the tumor can be assayed as described to determine the T H profile; the results obtained are expected to be representative of the response determined by assaying the patient's PBLs isolated from blood.
  • the cytokine pattern of the tumor-associated PBLs can be analyzed without the need to rely on a patient's freshly drawn blood sample. In this manner, the determination of the cytokine pattern can be determined more rapidly, thereby increasing the efficiency of these types of analyses.
  • the cytokine pattern analysis can still be performed using PMNs isolated from a patient's blood sample using the disclosed IVS assays, as necessary or desired.
  • the tumor cell preparation containing the various types of mononuclear blood cells as described in Example 2 can also be used to analyze the proliferation of a patient's mononuclear blood cells against tumor cells having particular surface molecules, as well as to detect the presence or absence of particular cell surface molecules which are on these PBMCs and which are associated with the generation of an appropriate immune response against antigens, e.g. , tumor and infected cell surface molecules, and the like.
  • the suspended admixture of tumor cells and peripheral blood cells derived from the tumor tissue preparation may ultimately used in the IVS assays as described herein to measure proliferation, cytokine secretion, and the like, and also to substitute for or to supplement the use of a patient's freshly drawn blood sample and the cells obtained therefrom in these types of assays.
  • the determinations as described above can be used to decide if an individual should undergo chemotherapy or radiation treatment(s) rather than immunotherapy treatment(s) . If a patient has a low or negative response index and shows no indication that his or her cytokine pattern supports a cell mediated or a T H 1 response, the medical provider can offer chemotherapy or radiation treatment regimens to the patient which will ultimately benefit the patient and impact in a significant way on his or her survival outcome.
  • Example 12 Experiments similar to those as described in Example 12 are performed to test the survivability of C57BL 6 mice immunized prophylactically with immunogenic preparations comprising B16/BL6 tumor cells that are PCL- modified by exposing the tumor cells to AdA crosslinker at about 10 mM at the time that the cells are also subjected to hydrostatic pressure of about 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, 97:587) 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 6 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 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.
  • the immunization protocol comprises two vaccinations, i.e. , injections of an immunogenic preparation comprising about 20 x 10 6 PCL-modified 83
  • the dose concentrations used for each adjuvant are typically 1, 5, 10, 20, 50, and 100 ⁇ g/mL.
  • 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.
  • 6-10 mice are immunized to test the controls and each immunogenic preparation containing PCL-modified cells and the various doses of adjuvant.
  • 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, as described hereinabove and in Example 16.
  • 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.
  • a baseline DTH assay 1 x 10 s 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 GM-CSF
  • 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 the area of erythema at the immunization site using callipers and as known by those in the art (Table 15). 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.
  • 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.
  • the use of 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 erythema (i.e. , the DTH response area) at the immunization site after injections with 10 5 PCL-treated melanoma cells and either 20 or 100 ⁇ g of GM-CSF administered as adjuvant as described.
  • PBMC proliferation associated with cell surface molecule presentation e.g.. co- stimulatory molecules and cell adhesion molecules
  • Experiments are performed to analyze cell surface molecules present on tumor cells, infected cells, or on PBMCs in order to determine the immunogenic potential of antigen presenting cells and the ability of PBLs to respond to antigen.
  • the analysis of cell surface molecules complements the proliferation and cytokine secretion measurements as described and offers additional markers for determining and predicting if a patient will produce an effective immune response and have a successful clinical outcome of immunotherapy treatment.
  • IVS assays are performed to determine the potential immune responsiveness of a patient's mononuclear blood cells to PCL- modified versus unmodified tumor cells by testing for the ability of the PBMCs to proliferate in response to co-stimulatory molecules, such as B7-1 and B7-2, as well as other co-stimulatory molecules, which are present on the surfaces of some tumor cells (see, for example, June, CH. et al. , 1994, Immunol. Today, 15:321-331 ; Freeman, G.J. et al. , 1993, Science, 262:909-911; Nabavi, N. et al., 1992, Nature, 36Q:266-268; Freeman, G.J. et al.
  • B7-1 and B7-2 glycoprotein molecules are not present on many types of tumor cells. Further, a tumor cell may have the proper MHC or HLA presentation, but it also requires the proper co- stimulatory signals provided by molecules such as B7-1 or B7-2 to stimulate an appropriate immune response. Thus, tumor cells lacking these co-stimulatory molecules may not stimulate an appropriate immune response by a patient's lymphocytes.
  • tumor cells are subjected to PCL modification and are tested in IVS assays with a patient's PBMCs. along with unmodified tumor cell controls.
  • the presence or absence of B7-1 or B7-2 co- stimulatory molecules on tumor cells is determined by ELISA assay using commercially-available anti-B7 monoclonal antibodies (e.g., Valle, A. et al. , 1990, Immunology, 69:531-535) and protocols similar to those described for the cytokine analyses.
  • PCL-modification of tumor cells may enhance the presentation of and/or force the appearance of B7-1 and B7-2 molecules on the tumor cell surface, if the levels of these molecules are low or suboptimal on the native or unmodified cells.
  • these types of analyses can also be performed on cells from surgically-removed tumor tissue preparations containing PBMCs and tumor cells before and after PCL treatment.
  • CAM cell adhesion molecules
  • ICAM-1, ICAM-2, ICAM-3 de Fougerolles, A.R. et al. , 1994, J. Exp. Med. , 179:619-629; Kirchhausen, T. et al. , 1993, J. Leukoc. Biol. , 53:342-346) which are displayed on the surfaces of B cells, T cells, and macrophages, and which interact with the LFA-1, 2, or 3 ligand molecules present on T cells, may be augmented and improved by PCL modification in accordance with the invention.
  • PCL-modified PBMCs may have increased or augmented ICAM presentation as a result of the crosslinking and pressure treatment of the invention, and thus, will be expected to interact more successfully with (or to adhere more effectively to) target cells to effect their ultimate destruction.
  • ICAM molecules that may impact in a positive way on the patient's ability to generate a successful immune response against tumor or infected cells or foreign antigens
  • the presence of ICAM molecules is assessed by antibody assays, e.g. ELISA, following co-culture of PBLs with PCL-modified tumor or infected cells to measure the proliferative response (i.e. , in an IVS assay).
  • anti-ICAM antibodies are used in E ISAs to determine the levels of ICAM molecules on the cell surfaces of the PBMCs in these assays.
  • anti-ICAM antibodies are added to the
  • IVS assay cell samples and the levels of PBMC proliferation is measured. Control samples receive no anti-ICAM antibody. If the addition of anti-ICAM antibodies results in a decrease in cell proliferation relative to the controls without antibody in these assays, it is concluded that ICAM molecules are present on the responding cells and that the added anti-ICAM antibody has inhibited proliferation by binding to ICAM molecules on the cell surface. The presence of detectable ICAM molecules on a patient's PBMCs is indicative that the patient is likely to respond positively to tumor or infected cells and will be able to mount an effective cell mediated immune response against the specific target cells. These types of analyses can also be performed on cells obtained from surgically-removed tumor tissue preparations containing PBMCs and tumor cells before and after PCL treatment. The contents of all patents, patent applications, published articles, books, and abstracts cited herein are hereby incorporated by reference in their entirety.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une préparation immunogène obtenue à partir de cellules, de membranes cellulaires ou de leur protéines, qui ont été modifiées par traitement au moyen d'un agent de réticulation dialdéhyde de 2',3'-nucléoside ou nucléotide et par exposition à une pression hydrostatique. Le traitement des cellules par pression et réticulant exécuté selon la présente invention s'appelle une 'modification PCL' (pression-réticulant). On obtient une amélioration de l'immunogénicité si l'on traite les cellules avec la pression en même temps qu'on les soumet à des composés de réticulation des protéines. Les cellules utilisables pour la modification PCL selon la présente invention, et destinées à être utilisées comme immunogènes, peuvent être des cellules tumorales ou cancéreuses, des cellules transformées, des cellules infectées par virus ou des cellules infectées par micro-organismes par exemple des bactéries, des parasites, des levures et analogue. Les cellules tumorales ou infectées ayant subi une modification PCL sont notamment capables d'induire et de déclencher une réponse immunitaire spécifique et puissante contre, suivant le cas, les cellules tumorales, les cellules infectées ou tout autre type de cellules modifiées, tant chez un patient animal que chez un patient humain. Les cellules mononucléaires de sang périphérique humaines (PBMC) sont sensibilisées et stimulées spécifiquement pour provoquer une réaction immunitaire contre les cellules tumorales ou infectées ayant subi une modification PCL en fonction de ce qui a été déterminé par des dosages de sensibilisation in vitro. De tels dosages, seuls ou combinés à des analyses de cytokines de PBMC, constituent des moyens de diagnostic permettant de déterminer ou d'identifier, par examen in vitro et in vivo, les patients qui vont réagir ou ne pas réagir aux traitements et immunothérapies PCL.
PCT/US1996/001876 1995-02-17 1996-02-12 Procedes et compositions pour le depistage, le pronostic et le traitement en matiere d'immunotherapie WO1996025664A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49778/96A AU4977896A (en) 1995-02-17 1996-02-12 Immunotherapy screening, prognosis, and treatment methods and compositions

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US39255395A 1995-02-17 1995-02-17
US08/392,553 1995-02-17
US48185795A 1995-06-07 1995-06-07
US08/481,857 1995-06-07

Publications (1)

Publication Number Publication Date
WO1996025664A1 true WO1996025664A1 (fr) 1996-08-22

Family

ID=27013925

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/001876 WO1996025664A1 (fr) 1995-02-17 1996-02-12 Procedes et compositions pour le depistage, le pronostic et le traitement en matiere d'immunotherapie

Country Status (3)

Country Link
AU (1) AU4977896A (fr)
IL (1) IL117159A0 (fr)
WO (1) WO1996025664A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033612A1 (fr) * 1996-03-15 1997-09-18 Immunotherapy, Inc. 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
WO2000011476A1 (fr) * 1998-08-25 2000-03-02 The Immune Response Corporation Procedes d'evaluation de la fonction immunitaire
WO2001051082A1 (fr) * 2000-01-14 2001-07-19 Allergy Therapeutics Limited Administration sublinguale d'une composition contenant des antigenes et un adjuvant glycolipidique
EP1777523A1 (fr) * 2005-10-19 2007-04-25 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthode pour pronostiquer la progression d'un cancer et les résultats du patient et les moyens pour performer la méthode
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
US7718178B2 (en) 1997-04-05 2010-05-18 Allergy Therapeutics Limited Allergen formulation
US7815920B2 (en) 1998-09-21 2010-10-19 Allergy Therapeutics (UK) Ltd Method of preparing an antigen-containing formulation
EP2410334A1 (fr) * 2007-02-15 2012-01-25 IRX Therapeutics, Inc. Mitogène de lymphocyte T pour une utilisation dans un test cutané
WO2023121362A1 (fr) * 2021-12-22 2023-06-29 씨제이제일제당 (주) Composition antivirale comprenant des analogues nucléosidiques dérivés d'un acide nucléique et d'un sel pharmaceutiquement acceptable de celui-ci
KR20230096817A (ko) * 2021-12-22 2023-06-30 씨제이제일제당 (주) 핵산 유래 뉴클레오사이드 아날로그 및 이들의 약학적으로 허용가능한 염을 포함하는 항바이러스용 조성물
WO2024144320A1 (fr) * 2022-12-29 2024-07-04 씨제이제일제당 (주) Composition antivirale comprenant des analogues nucléosidiques dérivés d'acide nucléique et sels pharmaceutiquement acceptables de ceux-ci

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4931275A (en) * 1985-12-02 1990-06-05 Yeda Research & Development Co., Ltd. Anti-tumor vaccines and their preparation
EP0615758A1 (fr) * 1988-05-24 1994-09-21 Yeda Research And Development Co. Ltd. Vaccins antitumoraux
WO1994028113A1 (fr) * 1993-05-31 1994-12-08 Biotop S.A.S. Di Rita Cassarin Immortalisation de cellules dendritiques avec l'oncogene v-myc
WO1996003523A1 (fr) * 1994-07-22 1996-02-08 United States Of America, Represented By The Secretary, Department Of Health And Human Services Procede d'identification de patients presentant un etat immunitaire modifie

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4931275A (en) * 1985-12-02 1990-06-05 Yeda Research & Development Co., Ltd. Anti-tumor vaccines and their preparation
EP0615758A1 (fr) * 1988-05-24 1994-09-21 Yeda Research And Development Co. Ltd. Vaccins antitumoraux
WO1994028113A1 (fr) * 1993-05-31 1994-12-08 Biotop S.A.S. Di Rita Cassarin Immortalisation de cellules dendritiques avec l'oncogene v-myc
WO1996003523A1 (fr) * 1994-07-22 1996-02-08 United States Of America, Represented By The Secretary, Department Of Health And Human Services Procede d'identification de patients presentant un etat immunitaire modifie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A. EISENTHAL ET AL.: "Th2 to Th1 cytokine shift in autologous peripheral blood mononuclear cells responding to autologous human tumor cells modified by hydrostatic pressure and crosslinking (PCL) in vitro.", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 37, no. 0, 1996, WASHINGTON, USA, pages 474, XP002008471 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033612A1 (fr) * 1996-03-15 1997-09-18 Immunotherapy, Inc. 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
US7718178B2 (en) 1997-04-05 2010-05-18 Allergy Therapeutics Limited Allergen formulation
US8105605B2 (en) 1997-04-05 2012-01-31 Allergy Therapeutics (Uk) Ltd. Allergen formulation
WO2000011476A1 (fr) * 1998-08-25 2000-03-02 The Immune Response Corporation Procedes d'evaluation de la fonction immunitaire
US7815920B2 (en) 1998-09-21 2010-10-19 Allergy Therapeutics (UK) Ltd Method of preparing an antigen-containing formulation
US8470331B2 (en) 2000-01-14 2013-06-25 Allergy Therapeutics (Uk) Limited Composition of antigen and glycolipid adjuvant for sublingual administration
WO2001051082A1 (fr) * 2000-01-14 2001-07-19 Allergy Therapeutics Limited Administration sublinguale d'une composition contenant des antigenes et un adjuvant glycolipidique
EP2100616A3 (fr) * 2000-01-14 2009-09-23 Allergy Therapeutics (UK) Limited Composition d'antigène et administration sublinguale d'adjuvant de glycolipide
WO2007045996A1 (fr) * 2005-10-19 2007-04-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Procede in vitro pour le pronostic de la progression et du resultat d'un cancer chez un patient, et systeme de mise en oeuvre
EP1777523A1 (fr) * 2005-10-19 2007-04-25 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthode pour pronostiquer la progression d'un cancer et les résultats du patient et les moyens pour performer la méthode
US8481271B2 (en) 2005-10-19 2013-07-09 Institut National De La Sante Et De La Recherche Medicale (Inserm) Vitro method for the prognosis of progression of a cancer and of the outcome in a patient and means for performing said method
US10191059B2 (en) 2005-10-19 2019-01-29 Institut National De La Sante Et De La Recherche Medicale (Inserm) In vitro method for the prognosis of progression of a cancer and of the outcome in a patient and means for performing said method
EP2410334A1 (fr) * 2007-02-15 2012-01-25 IRX Therapeutics, Inc. Mitogène de lymphocyte T pour une utilisation dans un test cutané
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
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
WO2023121362A1 (fr) * 2021-12-22 2023-06-29 씨제이제일제당 (주) Composition antivirale comprenant des analogues nucléosidiques dérivés d'un acide nucléique et d'un sel pharmaceutiquement acceptable de celui-ci
KR20230096817A (ko) * 2021-12-22 2023-06-30 씨제이제일제당 (주) 핵산 유래 뉴클레오사이드 아날로그 및 이들의 약학적으로 허용가능한 염을 포함하는 항바이러스용 조성물
KR102584927B1 (ko) * 2021-12-22 2023-10-06 씨제이제일제당 (주) 핵산 유래 뉴클레오사이드 아날로그 및 이들의 약학적으로 허용가능한 염을 포함하는 항바이러스용 조성물
WO2024144320A1 (fr) * 2022-12-29 2024-07-04 씨제이제일제당 (주) Composition antivirale comprenant des analogues nucléosidiques dérivés d'acide nucléique et sels pharmaceutiquement acceptables de ceux-ci

Also Published As

Publication number Publication date
IL117159A0 (en) 1996-06-18
AU4977896A (en) 1996-09-04

Similar Documents

Publication Publication Date Title
Dhodapkar et al. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells
Panelli et al. Phase 1 study in patients with metastatic melanoma of immunization with dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100
US7670611B2 (en) Cancer immunotherapy with semi-allogeneic cells
JP2004512030A (ja) 特異的細胞溶解性ｔ細胞応答を誘導するための組成物および方法
JP2010222352A (ja) 腫瘍の治療のための細胞治療方法
AU7112098A (en) Dendritic cell hybrids
WO2005115447A2 (fr) Immunotherapie anticancereuse humaine
AU2003261546A1 (en) A cell therapy method for the treatment of tumors
US5582831A (en) Anti-tumor vaccines
EP2939690A1 (fr) Vaccins
US20180085398A1 (en) Compositions and methods of treating cancer
WO1996025664A1 (fr) Procedes et compositions pour le depistage, le pronostic et le traitement en matiere d'immunotherapie
US20090081127A1 (en) Dendritic-like cell/tumor cell hybrids and hybridomas for inducing an anti-tumor response
WO2006026746A2 (fr) Procedes de separation et extension de cellules t specifiques d'antigenes
EP0839044B1 (fr) Cellules dendritiformes/cellules tumorales hybrides deservant a provoquer une reponse antitumorale
Yei et al. Novel membrane-bound GM-CSF vaccines for the treatment of cancer: generation and evaluation of mbGM-CSF mouse B16F10 melanoma cell vaccine
WO1997033612A1 (fr) 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
Engleman Dendritic cells: potential role in cancer therapy
JP5054875B2 (ja) 樹状細胞ハイブリッドによって活性化される細胞傷害性ｔリンパ球
US6063375A (en) Semiallogeneic cell hybrids and related methods for treating cancer
JP2002502880A (ja) ハプテン改変化腫瘍細胞膜およびその使用
US20040022813A1 (en) Shed antigen vaccine with dendritic cells adjuvant
JP2003521936A5 (fr)
Ishii et al. In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances
Hayashi et al. A preclinical model to assess the antigenicity of an HLA‐A2 melanoma cell vaccine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US US UZ VN AZ BY KG KZ RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase