US20050153329A1 - Method for determining immune system affecting compounds - Google Patents

Method for determining immune system affecting compounds Download PDF

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US20050153329A1
US20050153329A1 US10/997,985 US99798504A US2005153329A1 US 20050153329 A1 US20050153329 A1 US 20050153329A1 US 99798504 A US99798504 A US 99798504A US 2005153329 A1 US2005153329 A1 US 2005153329A1
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production
factor
cancer
tumour
substances
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Leif Hakansson
Annika Hakansson
Birgitta Clinchy
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CANIMGUIDE THERAPEUTICS AB
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Leif Hakansson
Annika Hakansson
Birgitta Clinchy
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Priority claimed from SE0201563A external-priority patent/SE0201563D0/xx
Application filed by Leif Hakansson, Annika Hakansson, Birgitta Clinchy filed Critical Leif Hakansson
Priority to US10/997,985 priority Critical patent/US20050153329A1/en
Publication of US20050153329A1 publication Critical patent/US20050153329A1/en
Assigned to CANIMGUIDE THERAPEUTICS AB reassignment CANIMGUIDE THERAPEUTICS AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HAKANSSON, LEIF
Assigned to CANIMGUIDE THERAPEUTICS AB reassignment CANIMGUIDE THERAPEUTICS AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: THERIM DIAGNOSTICA AB
Priority to US12/822,013 priority patent/US8182983B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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 relates to method for treating cancer, method for determining immunoregulatory substances, kit for carrying out said determination, use of certain compounds for preparation of pharmaceutical compositions, as well as pharmaceutical compositions.
  • Immunosuppression of TIMC can, however, at least to some extent be overcome, either by washing, preincubation before stimulation, or culturing in interleukin-2.
  • the down-regulation of the immune system which relates to cancer, does not result in a seriously increased incidence of infectious diseases in these patients.
  • Extracts or supernatants from tumours are often immunosuppressive (Sulitzeanu, 1993).
  • TGF- ⁇ , PGE 2 , IL-10, IL-4 and others either being produced by the tumour cells as such or by tumour-infiltrating lymphocytes (TIL) or tumour associated macrophages (TAM) (e.g. Menetrier-Caux et al., 1999; Heimdal et al., 2000; Heimdal et al., 2001).
  • TIL tumour-infiltrating lymphocytes
  • TAM tumour associated macrophages
  • Immunostimulatory treatment of the dysregulated immune system of cancer patients might be counter-productive. If the immune system in cancer is directed to down-regulation of the chronic inflammatory reaction there is a risk that further therapeutic immunostimulation will enhance the immunosuppression and thereby further down-regulate the immune reactivity against the tumour cells. The strategy should therefore be to eliminate mediators of immunosuppression before the immune system is stimulated.
  • tumour For initiation of an immune response the tumour has to be recognised as non-self.
  • the initial induction of an immune response to tumour associated antigens takes place at an early stage of the malignant disease while the tumour burden is still reasonably small and tumour related immunosuppressive mechanisms are not yet activated. In this situation the immune reactivity to the tumour is beneficial and control the malignant growth for some time.
  • APCs antigen presenting cells
  • lymphocytes In order to get an immune response a proper interaction between antigen presenting cells (APCs) and lymphocytes has to take place with a well-orchestrated production of cytokines and expression/interaction of co-stimulatory molecules.
  • Tumour related immunosuppression takes place at four levels: Activation, recruitment of effector cells to the tumour, migration of these cells from stromal areas close to the tumour cells and cytotoxic activity.
  • Stimulation of the inhibited immune reactivity to the tumour can be achieved using several therapeutic strategies. However, several function parameters are down-regulated during the treatment, particularly in tumour areas with the most pronounced regressive changes.
  • the object of the present invention is thus to obtain a method for increasing the possibility of treating cancer, a method for determining factor(-s) related to prognosis and allowing prediction and monitoring therapy of cancer, and a kit for determining such factor(-s), use of substances to prepare pharmaceutical compositions, as well as pharmaceutical compositions.
  • any regulatory mechanism including inducing factor/s, of the production of immunoregulatory substances, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, is therapeutically controlled to minimise pathological production of such immunosuppressive immunoregulatory substances or to stimulate the production of immunosupportive immunoregulatory substances to enhance the performance status of a patient and/or to enhance the therapeutic control of a malignant tumour in a subject suffering from a cancer.
  • any dysregulatory mechanism of immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ and others, production in a patient suffering from cancer is modulated by therapeutically treating the patient to provide a FcR modulation by administering a therapeutically effective amount of a FcR modulating agent.
  • cytokines including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ and others
  • any dysregulatory mechanism of immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production in a patient suffering from cancer is modulated by therapeutically treating the patient to provide a FcR/Fc ⁇ R modulation by administering a therapeutically effective amount of a FcR/Fc ⁇ R blocking agent.
  • cytokines including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • any dysregulatory mechanism of immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production in a patient suffering from cancer is modulated by therapeutically treating the patient to provide a FcR/Fc ⁇ R modulation by administering a therapeutically effective amount of a FcR/Fc ⁇ R cross-linking agent.
  • cytokines including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • blocking or cross-linking of FcR is carried out using a therapeutically active amount of at least one immunoglobulin, FcR antibodies or fragments of antibodies or synthetic constructs including peptides directed to FcR.
  • At least one FcR-modulating substance minimising production of interleukin-1 receptor antagonist is administered in a therapeutically effective amount.
  • the modulation is carried out by blocking FcR by administering F(ab), or F(ab′)2-fragments.
  • the modulating substance is a soluble receptor, fragment, peptide or synthetic construct directed to the Fc-part of immunoglobulins.
  • Fc ⁇ R I, Fc ⁇ R II and/or Fc ⁇ R III is modulated to stimulate IL-2 stimulation of clonal expansion of lymphocytes.
  • any compound blocking the production or biological activity of a factor inducing pathological production of any dysregulatory mechanism of immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production, is administered in a therapeutically effective amount.
  • the blocking compound is a receptor blocking compound.
  • the blocking compound is a monoclonal antibody, fragments thereof, peptides or synthetic constructs.
  • a compound having the ability of inhibiting the activation or activity of enzymes generating immunomodulatory fragments is administered in a therapeutically effective amount.
  • the inhibiting compound is a monoclonal antibody, an anti-integrin antibody, peptides and/or synthetic constructs.
  • the modulating substance is an enzyme inhibitor.
  • the inhibiting compound is a matrix metalloproteinase inhibitor.
  • any compound blocking the factor inducing IL-6 production is administered in a therapeutically effective amount.
  • any compound blocking the factor inducing IL-1 ⁇ production is administered in a therapeutically effective amount.
  • any compound blocking the factor inducing IL-10 production is administered in a therapeutically effective amount.
  • any compound blocking the factor inducing TNF- ⁇ production is administered in a therapeutically effective amount.
  • any compound blocking the factor inducing IL-1Ra production is administered in a therapeutically effective amount.
  • monoclonal antibodies directed to enzymes generating immunomodulatory fragments are administered in a therapeutically effective amount.
  • At least one anti-integrin antibody, peptide or construct is administered in a therapeutically effective amount.
  • blocking cross-linking of FcR is carried out using a therapeutically active amount of FcR antibodies or fragments of monoclonal antibodies directed to FcR, preferably the FcR cross-linking is obtained by administering a therapeutically effective amount of at least one immunoglobulin, more preferably the FcR cross-linking is obtained by administering a therapeutically effective amount of IgG or complex bound IgG, and/or the FcR cross-linking is obtained by administering a therapeutically effective amount of IgA or complex bound IgA.
  • a therapeutically effective amount of Fc part of at least one immunoglobulin is administered, preferably a therapeutically effective amount of Fc part of IgG or complex bound IgG is administered, and/or a therapeutically effective amount of Fc part of IgA or complex bound IgA is administered.
  • any cross-linking of Fc ⁇ R I, Fc ⁇ R II and/or Fc ⁇ R III is carried out.
  • FcR is down-regulated using an inhibitor of its expression.
  • blocking of Fc ⁇ R is carried out by administering a therapeutically effective amount of anti-Fc ⁇ R I antibodies.
  • At least one FcR-blocking substance minimising production of interleukin-1 receptor antagonist is administered in an amount necessary to block the activity of interleukin-1.
  • a further aspect of the invention includes a method for analysing the amount and/or certain pattern of dysregulatory mechanism substances, including their mRNA, including any inducing factor, inducing the production of immunoregulatory substances, including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, whereby the amount of such dysregulatory mechanism substances is determined in a tissue sample, whereby the prognosis of a subject suffering from cancer can be determined and/or the therapeutic efficacy of any anti-cancer treatment can be predicted and monitored.
  • tissue is whole blood, serum, plasma, lymphatic fluid, saliva, urine, faeces, ascites, pleural effusion, pus, as well as any tissue, including inflammatory cells.
  • any compound having the ability of inhibiting the activation or activity of enzymes generating fragments of intra-tumoural tissue are determined.
  • any fragment or new epitopes generated by the activity of intratumoural enzymes is determined in any tissue from a cancer patient.
  • the activity of any compound having the ability of inhibiting the activation or activity of enzymes generating fragments is monitored by determining these fragments or new epitopes exposed by the enzymatic activity in any tissue from a cancer patient.
  • the inhibiting compound is a matrix metalloproteinase inhibitor.
  • any compound blocking production or biological activity of a factor inducing pathological production of any dys-regulatory mechanism of immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production, is determined.
  • the amount of any inducing factor or mRNA thereof inducing the production of immunoregulatory substances is determined by determining the production of immunoregulatory substances, including cytokines, including, including IL-1Ra, IL-6, IL-1 ⁇ and/or TNF- ⁇ and others produced by PBMC after exposure to these factors.
  • samples are obtained in a way, including tubes and syringes, not binding or inactivating immunoregulatory substances, including inducing factors.
  • the amount of any inducing factor inducing the production of immunoregulatory substances including one or more cytokines including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, is determined directly.
  • the amount/occurrence of any cell bound factor inducing the production of immunoregulatory substances is determined.
  • the amount of any urine present inducing factor inducing the production of immunoregulatory substances is determined by using an urine dip stick containing binding substance/s binding said inducing factor and/or colour developing reagents to said inducing factor.
  • the amount of cell bound immune complexes, CBIC is determined.
  • peripheral blood mononuclear cells PBMC being positive for any immunoglobulin staining are determined.
  • PBMC being positive for IgG staining are determined.
  • PBMC being positive for IgA staining are determined.
  • FcR positive to any immunoglobulin staining is determined, such positive for IgG staining and/or IgA staining.
  • the production of O 2 ⁇ is determined.
  • down-regulation of the ⁇ -chain of TCR is determined.
  • fine needle biopsy TIMC tumor infiltrating mononuclear cell
  • fine needle biopsy TIMC being positive to IgG are determined.
  • fine needle biopsy TIMC being positive to IgA are determined.
  • IL-1Ra is determined.
  • down-regulation of CD28 on CD4+ and/or CD8+ lymphocytes is determined.
  • down-regulation of CD80 and/or CD86 is determined.
  • the content of IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ is determined by using an assay, to determine the amount of an inducing factor inducing IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, activity.
  • the assay utilises any tissue and the determinations are made using any immuno-cyto-histochemical method, any immunoassay including, ELISA, Elispot, RIA and others any blotting technique, including Western blotting, Southern blotting and others, any bioassay, any tissue culture technique, RT-PCR, flow cytometry, cytometric bead array, DNA microarray and/or proteomics.
  • any immuno-cyto-histochemical method any immunoassay including, ELISA, Elispot, RIA and others any blotting technique, including Western blotting, Southern blotting and others, any bioassay, any tissue culture technique, RT-PCR, flow cytometry, cytometric bead array, DNA microarray and/or proteomics.
  • dys-regulatory mechanism substances in tissue from cancer patients, which substances suppress the immune mediated systemic protection resulting in establishment of micrometastases, are identified.
  • patients suffering from potential risk are candidates for adjuvant treatment.
  • the invention encompasses specific staining methods for IgG in PBMC or biopsies, whereby a first method for determining IgG/IC complexes using a staining, is characterized in that peripheral blood mononuclear cells are separated by centrifugation and spun onto microscope slides; the cells are pre-hydrated using Hank's balanced solution and Hepes solution and human serum albumin, are fixed in phosphate-buffered paraformaldehyde supplemented with glucose, are incubated with biotinylated protein G, followed by incubation with alkaline phosphatase-labelled streptavidin, are incubated in alkaline phosphatase substrate in Tris buffer with dimethylformamide, levamisole and Fast-Red TR salt, are counterstained in Mayer's haematoxylin and mounted in Glycergel; or alternatively, after fixation and a washing in Hank's balance solution containing goat serum, the cells are blocked in goat serum and are incubated
  • a further aspect of the invention includes a kit for quantitative and/or qualitative analysis of amount of and/or certain pattern of dys-regulatory factor and/or factors inducing the production and/or activation of immunoregulatory substances, whereby prognosis of cancer can be determined and the therapeutic efficacy of any anti-cancer treatment can be predicted and monitored, comprising an indicator for the presence of said dys-regulatory factor/s including inducing factor/s.
  • the kit comprises an indicator for the presence of any factor inducing immunoregulatory substances, including cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others, activity.
  • cytokines including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others, activity.
  • the kit comprises a nutrient for peripheral blood mononuclear cells and a determinant for inducing factor for inducing immunoregulatory substances including inducing factors of IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others.
  • a urine dip stick comprising binding substance/s and/or colour developing reagents.
  • the kit comprises determinants for enzymatic degradation products of tumour substances/extra cellular matrix, ECM.
  • a matrix metalloproteinaseinhibitor is monitored.
  • the kit comprises a determinant for dys-regulatory substances in tissue from cancer patients, which substances suppress the immune mediated systemic protection resulting in the establishment of micrometastases.
  • the amount of cell bound immune complexes, CBIC is determined.
  • a still further aspect of the invention includes use of at least one regulatory mechanism controlling factor of at least one immunoregulatory substance, including inducing factor of the production or biological activity of immunoregulatory substances including cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, for the production of a pharmaceutical preparation to be used for therapeutic control and for minimisation of pathological production of immunosuppresive immunoregulatory substances, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others or to stimulate production of immunosupportive immunoregulatory substances in a patient suffering from a cancer and to enhance the efficacy and/or possibility of therapeutic treatment of cancer or modulation of dys-regulatory factors to enhance performance status.
  • immunoregulatory substances including cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • a FcR modulating agent is used in the manufacture of a pharmaceuticai preparation for controlling immunoregulatory substances, including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others, production in a patient suffering from cancer by therapeutically modulating FcR activity.
  • immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others, production in a patient suffering from cancer by therapeutically modulating FcR activity.
  • a FcR modulating agent is used in the manufacture of a pharmaceutical preparation for controlling immunoregulatory substances, including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production in a patient suffering from cancer is modulated by therapeutically blocking FcR/Fc ⁇ R activity.
  • immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • a FcR modulating agent is used in the manufacture of a pharmaceutical preparation for controlling immunoregulatory substances, including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production in a patient suffering from cancer is modulated by cross-linking FcR/Fc ⁇ R activity.
  • immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • At least one immunoglobulin, FcR antibodies or fragments of antibodies or synthetic constructs including peptides directed to FcR is used in the manufacture of a pharmaceutical preparation for blocking or cross-linking FcR cross-linking.
  • an agent modulating Fc ⁇ R I, Fc ⁇ R II and/or Fc ⁇ R III used to stimulate IL-2 stimulation of clonal expansion of lymphocytes is used in the manufacture of a pharmaceutical preparation.
  • FcR antibodies or fragments of FcR antibodies directed to FcR is used in the manufacture of a pharmaceutical preparation for blocking cross-linking of FcR.
  • anti-Fc ⁇ R I, Fc ⁇ R II and/or Fc ⁇ R III antibodies is used in the manufacture of a pharmaceutical preparation for blocking of Fc ⁇ R.
  • a compound being able to down-regulate the expression of FcR is used in the manufacture of a pharmaceutical preparation for down-regulating FcR.
  • At least one FcR-blocking substance minimising production of interleukin-1 receptor antagonist is used in the manufacture of a pharmaceutical preparation for blocking the activity of interleukin-1.
  • At least one FcR-modulating soluble receptor, fragment, peptide or synthetic construct directed to the Fc-part of immunoglobulins is used in the manufacture of a pharmaceutical preparation.
  • At least one FcR-modulating enzyme inhibitor is used in the manufacture of a pharmaceutical preparation.
  • At least one FcR-inhibiting matrix metalloproteinase inhibitor is used in the manufacture of a pharmaceutical preparation.
  • any compound blocking production or biological activity of any factor inducing pathological production of one or more cytokines is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokines.
  • any factor inducing IL-6 production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • any factor inducing IL-1 ⁇ production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • any factor inducing IL-10 production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • any factor inducing IL-17 production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • any factor inducing TNF- ⁇ production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • any factor inducing IL-1Ra production is used in the manufacture of a pharmaceutical preparation for blocking a factor inducing pathological production of such cytokine.
  • a compound having the ability of inhibiting the activation and/or activity of enzymes generating immunomodulatory fragments is used in the manufacture of a pharmaceutical preparation for inhibiting the activation or activity of enzymes generating immunomodulatory fragments.
  • antibodies directed to enzymes generating immuno-modulatory fragments are used in the manufacture of a pharmaceutical preparation.
  • At least one monoclonal antibody, anti-integrin antibody, peptide and/or synthetic construct thereof is used in the manufacture of a pharmaceutical preparation.
  • a further aspect of the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a regulatory mechanism controlling factor of an inducing factor of the production or biological activity of immunoregulatory substances including one or more cytokines, for the production of a pharmaceutical preparation to be used for therapeutically control and minimise pathological production of immunosuppresive immunoregulatory substances including one or more cytokine, or or to stimulate production of an immunosupportive immunoregulatory substance in a patient suffering from a cancer to enhance the efficacy and/or possibility of therapeutic treatment of cancer, optionally in combination with therapeutically inert additive to enhance performance status.
  • a FcR modulating agent is present in a pharmaceutical preparation for controlling immunoregulatory substances, including one or more cytokine production in a patient suffering from cancer by therapeutically modulating FcR activity.
  • a FcR modulating agent is used in the manufacture of a pharmaceutical preparation for controlling immunoregulatory substances, including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others, production in a patient suffering from cancer is modulated by therapeutically blocking FcR/Fc ⁇ R activity.
  • immunoregulatory substances including one or more cytokines, including IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, TNF- ⁇ , and others
  • At least one immunoglobulin, FcR antibodies or fragments of antibodies or synthetic constructs including peptides directed to FcR is used in the manufacture of a pharmaceutical preparation for blocking or cross-linking FcR activity.
  • an agent modulating Fc ⁇ R I, Fc ⁇ R II and/or Fc ⁇ R III is present to stimulate IL-2 stimulation of clonal expansion of lymphocytes.
  • anti-Fc ⁇ R I antibodies is present in a pharmaceutical preparation for blocking of Fc ⁇ R.
  • a compound being able to down-regulate the expression of FcR is present in the pharmaceutical preparation for down-regulating FcR.
  • At least one FcR-blocking substance minimising production of interieukin-1 receptor antagonist is present in the pharmaceutical preparation for blocking the activity of interleukin-1.
  • At least one FcR-modulating soluble receptor is used.
  • At least one FcR-modulating enzyme innibitor is used.
  • At least one FcR-inhibiting matrix metalloproteinase inhibitor is used.
  • a compound having the ability of inhibiting the activation of enzymes generating immunomodulatory fragments is present in the pharmaceutical preparation for inhibiting the activation of enzymes generating immunomodulatory fragments.
  • any factor inducing IL-6 production is used.
  • any factor inducing IL-1 ⁇ production is used.
  • any factor inducing IL-10 production is used.
  • any factor inducing IL-17 production is used.
  • any factor inducing TNF- ⁇ production is used.
  • any factor inducing IL-1Ra production is used in the manufacture of a pharmaceutical preparation for
  • a compound blocking a factor inducing pathological production of immunoregulatory substainces, including one or more cytokine production is present in the pharmaceutical preparation for blocking a factor inducing pathological production of such immunoregulatory substances.
  • antibodies directed to enzymes generating immuno-modulatory fragments are present.
  • At least one monoclonal antibody, anti-integrin antibody, peptide and/or synthetic construct thereof, is used.
  • Flow-chart 1 shows a summary of the immunoregulatory mechanisms described in this invention. Connections between FcR mediated and dysregulatory factor mediated mechanisms are shown with Roman numericals.
  • IC can modulate the immune system
  • receptors for the Fc-part of immunoglobulins and complement receptors.
  • IC can activate complement and receptors for various complement factors are expressed on different types of cells of the immune system.
  • large ICs are more complement activating than small ones, which are more immunosuppressive.
  • binding of complement factors activated by ICs is not a major pathway for induction of immunosuppression.
  • Immune complexes can influence the activity of various types of cells of the immune system. The effect is highly dependent on the type of cell, which is involved and the characteristics of the immune complexes. In particular the size and whether the immune cells are exposed to soluble or solid phase IC seems to be of importance. Immunocomplexes can be either immunostimulatory or inhibitory depending on their sizes and interaction with different FcRs on immune cells. Large ICs have been demonstrated to be stimulatory whereby small complexes in antigen excess have been demonstrated to be inhibitory to mitogen induced proliferation (e.g. Gupta and Morton, 1981). It has been demonstrated that there is a correlation between the tumour burden and the immunosuppressive activity of immune complexes.
  • Serum blocking factors play a major role in immunosuppression in cancer. They have been shown to inhibit both cytotoxic and proliferative activity of lymphocytes from cancer patients (Baldwin, 1976; Bansal et al., 1976; Hellstrbm and Hellstrom, 1974). SBRs were demonstrated to be immunocomplexes (ICs), as their inhibitory activity was lost after dissociation at low pH, but reappeared after reconstitution at neutral pH (Sjögren et al.,1971). Tumour associated antigens can frequently be bound in IC (Kirkwood and Vlock, 1984; Vlock and Kirkwood, 1985). Removal of ICs from cancer patient sera, using protein A, also reduced the inhibitory effect of these sera.
  • ICs immunocomplexes
  • CICs circulating immunocomplexes
  • Th1 helper cells are considered to support the development of cytotoxic activity against the tumour. In malignant tumours, however, a predominance of Th2 over Th1 helper cells has been demonstrated repeatedly.
  • Cross-linking of Fc ⁇ Rs is of importance also for this diversion of the immune system, as cross-linking induces production of PEG2 (e.g. Berger et al. 1996), which favours a Th2 situation with production of IL-4, IL-10, etc.
  • IL-4 then inhibits the immune reactivity to the tumour partly by down-regulating monokine production in general, but also by stimulating the production of IL-1Ra .
  • IL-4 stimulates the expression of the inhibitory Fc ⁇ R II, CD32b, and inhibits expression of the stimulatory receptor CD32a (Pricop et al., 2001).
  • blockade of the Fc ⁇ R mediated triggering of the Th2 predominance in malignant tumours would certainly improve the immune reactivity to the tumour.
  • Cross-linking of Fc ⁇ R by IC can result in activation or inhibition.
  • the latter can be mediated in several ways:
  • interleukin-2 stimulated proliferation of PBMC and IL-1Ra production and was chosen as relevant markers for Fc ⁇ R mediated immunostimulation/suppression.
  • solid phase IgG results in a significantly increased proliferative response to PBMCs from healthy individuals, as well as cancer patients compared to cultures where solid phase binding was blocked by pre-incubation with human serum albumin (HSA).
  • HSA human serum albumin
  • This effect is most clearly shown when the culture plates were pre-coated with HSA/IgG allowing a high degree of cross-iinking of Fc ⁇ R.
  • the proliferative response to IL-2 could be normalised in cancer patients (compared to controls) using this technique.
  • a stimulatory effect of solid phase IgG was also seen when IgG from serum in the culture medium is allowed to bind to the surface of culture wells.
  • blockade of Fc ⁇ R I can be overcome by providing more extensive cross-linking similar to what is achieved in cultures with solid phase IgG.
  • the immunomodulatory role of cross-linking of Fc ⁇ R by solid phase IgG has furthermore been shown by analysing cytokine production in short term cultures of PBMCs with solid phase bound IgG, which in healthy individuals inhibited the production of IL-6.
  • IL-6 in PBMC cultures from both cancer patients and healthy individuals (less frequently) the production of IL-6, IL-1 ⁇ as well as TNF- ⁇ were frequently markedly increased when binding of serum IgG was blocked by pre-incubation of the culture wells with HSA.
  • IL-1Ra which is induced by IC or solid phase IgG, was reduced in cultures where solid phase binding of IgG was blocked by pre-incubation with HSA.
  • Interleukin-1 plays a fundamental role for the initiation of an immune response.
  • IL-1 Interleukin-1
  • soluble IL-1 receptors e.g., soluble IL-1 receptors, and IL-1 receptor antagonist (IL-1Ra)
  • IL-1Ra IL-1 receptor antagonist
  • the inhibitory role of IL-1Ra has been clearly demonstrated in studies on autoimmune diseases and rejection after allogen organ transplantation.
  • CBICs cell bound immuno-complexes
  • IL-1Ra obviously plays a central role in down-regulation of immune reactivity. As demonstrated here it is frequently expressed in large areas of malignant tumours. This finding is highly compatible with the occurrence of tissue bound IgG in the tumours as IC or solid phase IgG are the most potent inducers of this cytokine.
  • the inhibitory effect due to binding of IC to Fc ⁇ R can be treated using various techniques, e.g. by blocking the inhibitory receptors (see below), by down-modulating the sensitivity of receptors by protease inhibitors or by overcoming the inhibitory effects using more competitive binders/more extensive cross-linking of the receptors.
  • blocking the inhibitory receptors see below
  • down-modulating the sensitivity of receptors by protease inhibitors or by overcoming the inhibitory effects using more competitive binders/more extensive cross-linking of the receptors.
  • IL-6 an increased serum concentration of IL-6 is often found in cancer patients, especially in patients with advanced disease (Barton, 2001; Blay et al., 1992; Blay et al., 1997; Gadducci et al., 2001; Walther et al., 1998).
  • the source of serum IL-6 is still somewhat unclear and it is generally assumed to be derived from the tumour. It is shown herein that this cytokine is produced in large amounts, in short term cultures, by PBMCs from cancer patients and to a much lesser extent, or not at all, by unstimulated PBMCs from healthy individuals.
  • a high serum concentration of IL-6 is generally related to a paraneoplastic syndrome, poor response to immunotherapy and some types of chemotherapy, as well as a poor prognosis.
  • IL-6 large amounts of IL-6 is produced by PBMCs from about 50% of the cancer patients, as shown in malignant melanoma, renal cell carcinoma, and colorectal cancer.
  • An increased production of IL-6 is also found in patients with only a minimal tumour burden, as about 50% of the patients with radically resected stage III melanoma have a significantly increased production of IL-6.
  • IL-6 is a pleiotropic cytokine, which acts as an autocrine growth factor in for example renal cell carcinoma and multiple myeloma. It promotes the inflammatory response, induces acute phase reactants as well as IL-1Ra and is involved in the detrimental chronic inflammatory reaction in patients with malignant tumours. It is thus a good marker for dysregulation of the immune system in cancer patients and the paraneoplastic syndrome.
  • IL-6 can be induced in various ways, e.g., by IL-1 ⁇ , and TNF- ⁇ , and under certain circumstances also by cross-linking of FcR/Fc ⁇ R.
  • the degree of cross-linking is of importance for the regulatory effect on the production of IL-6.
  • the degree of cross-linking achieved by solid phase binding of serum IgG inhibits production of IL-6, as pre-coating the culture wells with HSA (thereby blockIng the binding of IgG) results in a significantly higher production of IL-6 in about 30% of cancer patients with various diagnoses.
  • IL-6 production was significantly inhibited in cultures of PBMCs from healthy individuals when the culture wells were coated with IgG instead of HSA alone.
  • This IL-6 inducing factor has been further characterised by fractionated ultrafiltration, which demonstrates a factor having a molecular weight of less than 50 kD. This factor has also been identified in the urine from cancer patients. The source of this factor was studied and various tumours were minced and extracted with physiological buffers. An IL-6 inducing factor, IL-6IF, is identified in about 60% of the analysed tumours. Similarly, an IL-6 inducing activity has been found in culture conditioned media from squamous cell carcinoma cell lines from the oral cavity.
  • IL-6IF which has been found in serum and urine, seems to be produced intratumourally. It is neither IL-1 ⁇ , TNF- ⁇ or interleukin-17, as these cytokines could not be found in fractions inducing IL-6.
  • IL-6IF in the urine opens interesting diagnostic possibilities. Based on the urine concentration of IL-6IF a simple diagnostic test can be developed which will give essential information about prognosis and the likelihood of therapeutic success, in particular for treatment where IL-6 is related to a poor response rate. This test will of course also be extremely valuable when it comes to treatment strategies dealing with the elimination of IL-6IF/treatment of the chronic inflammatory reaction in cancer patients.
  • TNF- ⁇ IL-1 ⁇ and IL-10 Similar to the situation with IL-6 we have also found inducing activity for TNF- ⁇ IL-1 ⁇ and IL-10 in serum and ultra-filtered urine. The number of inducing factors is for the moment unknown but TNF- ⁇ inducing activity was found in samples not inducing IL-6. Thus, reasonably several inducing factors are involved in dysregulation of the immune system in cancer.
  • the immunosuppressor factor in trauma patients was also found to inhibit migration of inflammatory cells. This mechanism might be of importance in immunosuppression in cancer as in untreated patients, inflammatory cells recruited to the tumour generally are found in the stromal areas surrounding the tumour nodules presumably because of inhibition of their migration close to the tumour cells.
  • fibronectin is of importance for migration it can be envisioned that if the cell receptors normally binding to fibronectin or other ECM-substances in the migration process are blocked by fragments of these substances the cells will no longer be able to migrate.
  • the phenomenon of leukocyte adherence inhibition is of interest as PBMCs from cancer patients under certain conditions demonstrate a highly reduced ability to adhere to plastic or glass surfaces.
  • IL-6 has by others been shown to be an inducer of IL-1Ra. However, in the present work based on the available malignant melanoma and renal cell carcinoma materials no correlation between IL-6 and IL-1Ra has been found rather the contrary. Fc ⁇ R cross-linking inhibits production of IL-6 as shown herein and stimulates IL-1Ra production.
  • IL-6IF containing sera in the immunoregulation in cancer is demonstrated by its effect on the proliferative response to IL-2.
  • the type of solid phase IgG binding is obviously of importance for the effect of IL-2.
  • Coating the wells with purified IgG mixed with HSA gives a more powerful stimulatory signal than binding of serum IgG from the culture medium (uncoated cultures).
  • This difference can be inhibited by sera containing IL-6IF, either by interfering with stimulatory mechanisms or by modulating monocytes to increased production of inhibitors, such as PGE 2 or IL-1Ra.
  • the inhibitory effect has not been shown in IgG coated cultures, probably because the more forceful effect on the proliferative response to IL-2 under the conditions used.
  • MMPs Matrixmetalloproteases
  • ECMs extra cellular matrix proteins
  • This activity is considered to be a pre-requisite for metastatic spread, and neo-angiogenesis. Occurrence of MMPs has, in several studies been shown to correlate to a poor prognosis.
  • proteolytic activity of MMPs will result in various types of degradation products, some of which are known to modulate angiogenesis and the activity of chemokines.
  • the low molecular weight fraction, IL-6IF, described herein can be a proteolytic fragment.
  • Factors derived from enzymatic degradation of ECM can most likely be both stimulatory and inhibitory as various cytokine patterns have been induced by various fibronectin fragments (Beezhold and Personius, 1992 ; López-Moratlla et al., 1995; Takizawa et al., 1995) and also it was recently demonstrated that the expression of various MMPs in malignant melanoma was related to therapeutic response and prognosis (Nikkola et al., 2001).
  • the generally found immunosuppressive effect of malignant tumours can thus very well be due to the increased enzymatic/proteolytic activity of the turnours resulting in various immunomoduiatory fragments/fragments of ECM or other tumour substances.
  • the proteolytic activity can also explain the frequent occurrence of a large number of soluble factors of importance for immune reactivity in cancer patients (Salih et al., 2001; Sheu et al., 2001), e.g. sIL-2 receptor, sTNF- ⁇ receptors, sCD8, sCD4, sICAM-1, sMHC I, sFcR etc.
  • Intra-tumoural enzymatic activity also seems to be of importance for activtion of pro-TGF- ⁇ to active TGF- ⁇ (Huber et al., 1992). Based on these considerations intra-tumoural proteolytic activity seems to be a fundamental mechanism by which the malignant tumour manage to divert the tumour bearers defence to the disease.
  • proteolytic fragments of ECM in serum and urine from cancer patients also opens the possibility to identify various types of fragments and analyse their prognostic significance.
  • therapeutic efficacy of matrixmetalloprotease inhibitors (MMPIs) resulting in a reduction/inhibition of the production of fragments of certain importance can be determined by analysing the amount of these fragments in serum or urine. Determination of these fragments will thus be proper surrogate endpoints for MMPI therapy and have the potential to significantly increase the therapeutic activity of these drugs by allowing monitoring whereby efficacious dose schedules can be developed.
  • MMPIs matrixmetalloprotease inhibitors
  • Dysregulation of the immune system by enzymatic/proteolytic fragments from various tumour substances has so far not been described.
  • a large number of inhibitory as well as stimulatory fragments can be produced as a result of the intra-tumoural enzymatic activity. This provides for a new understanding of immunosuppression in cancer patients and new therapeutic possibilities based on proper diagnostic tests.
  • the dysregulatory inducing factors were further characterised by using 2D-gel electrophoresis and identified after fragmentation using masspectrometry and N-terminal sequence analysis (proteomics technique).
  • the low molecular weight fraction ( ⁇ 30 kD) from urine was analysed by comparing samples from normal healthy controls with samples from patients; by comparing urine samples with and without IL-6 inducing activity; by comparing samples adsorbed and not adsorbed with a surplus of normal PBMCs (responding to IL-6IF).
  • proteins/fragments of potential immunoregulatory activity were thereby identified as described below, e.g. fragments of ⁇ 2-microglobulin, serum albumin and immunoglobulin.
  • IgG fragments possibly could have a modulatory activity on antibody dependent cellular cytotoxicity (ADCC), but the possibility that these fragments have an immunoregulatory activity in cancer has to our knowledge never been suggested.
  • ADCC antibody dependent cellular cytotoxicity
  • IL-6IF can be extracted from malignant tumours, e.g. malignant melanoma, renal cell carcinoma, colorectal cancer. These results were confirmed in a new series of experiments, where homogenised tumours were washed three times in PBS or RPMI in order to collect cytokine inducing factors already present in the tumours, so called “preformed inducing factors” (PIF).
  • malignant tumours e.g. malignant melanoma, renal cell carcinoma, colorectal cancer.
  • tumour sections/homogenates (after being thoroughly washed) were incubated for 24 hours at 37° C. together with MMP-2. IL-6 inducing activity could then be demonstrated in of supernatants from several tumours.
  • tumour tissue conditioned culture media from cancer cell lines, serum and urine.
  • Control of the intra-tumoural enzymatic/proteolytic activity is thus a fundamental mechanism to control the malignant tumour management of diverting the immune mediated defence system of the tumour bearer against the disease.
  • Fc ⁇ R cross-linking and dysregulatory inducing factors (ECM enzymatic/proteolytic fragments) play a fundamental role in immunosuppression in cancer patients.
  • Therapeutic control of these dys-regulatory mechanisms will thus improve quality of life, therapeutic response and increased over-all survival.
  • IL-6 is only one product of the dys-regulated inflammatory reaction mentioned herein and often is correlated to production of other cytokines such as IL-1 and TNF- ⁇ ; the strategy is to block the fundamental dys-regulatory mechanisms in order to down-regulate the detrimental chronic inflammatory reaction.
  • the present invention is thus not based on suppression of the activity of e.g., IL-1 ⁇ , IL-1Ra, IL-6, IL-10, IL-17, and/or TNF- ⁇ , and others, but to prevent or minimise their production.
  • tissue shall be understood to encompass whole blood, serum, plasma, lymphatic fluid, saliva, urine, faeces, ascites, pleural effusion, pus, as well as any tissue, as such including inflammatory cells.
  • cancer means: Any new and abnormal growth, specifically a new growth of tissue in which the growth is uncontrolled and progressive.
  • FcR By modulation of FcR either relief of immunosuppression or an enhanced immune activation can be achieved as demonstrated in the present document.
  • Several substances can be used to block FcRs e.g. antibodies or fragments thereof (e.g. F(ab′) 2 or Fab-fragments, of Mab directed to Fc ⁇ R) , peptides (e.g. from the Fc-part of IgG) or synthetic constructs.
  • the interaction between receptor binding ligands and the receptor can also be achieved by blocking the binding site of the ligand by using, soluble FcRs (recombinant) or peptides or synthetic constructs with a specific high binding affinity.
  • the signal transduction resulting from ligand binding to FcRs can be inhibited by signal transduction inhibitors.
  • their reactivity is influenced by proteolytic activity and can thus be modulated by protease inhibitors.
  • MMPIs can be of special interest.
  • the ligands of FcRs, immunoglobulins or complexed immunoglobulins can be eliminated and their immunosuppressive activity can thereby be avoided.
  • Recombinant FcRs or synthetic constructs with this reactivity of proper affinity and size can be administered in order that CIC (especially small CICs) are bound and eliminated.
  • CIC especially small CICs
  • binders of these substances can be linked to small microspheres (less than 3-5 mm if diameter, possibly degradable) which are rapidly taken up by the reticulendothelial system/monocyte macrophage phagocyte system.
  • Another therapeutic possibility is to reduce the number of FcRs which can have a negative effect of the immune reactivity to the malignant tumour, e.g. by increasing their shedding or by down-regulation of their expression.
  • FcRs which result in down-regulation of the immune reactivity
  • FcRs can, as has been demonstrated in this document, be to cross-link FcRs in order to overcome immunosuppression.
  • Several possibilities can be used for this pupose, e.g. complexed immunoglobulines/monoclonal antibodies or fragments (Fc) thereof binding to the receptors or synthetic FcR-binding constructs, cross-linking FcR to a degree (number of FcRs of one or several types) achieving optimal immune activation.
  • PBMC Peripheral blood mononuclear cells
  • HSA human serum albumin
  • PFA paraformaldehyde
  • the cells were blocked in 10% goat serum for 20 minutes and incubated with mouse anti-human IgG monoclonal antibody (Nordic Immunology) at 1 or 10 ⁇ g/ml, washed in BSS containing 2% goat serum, incubated with Envision (Dakopatts AB, SE) for 30 minutes, washed in TBS and incubated with the alkaline phosphatase substrate for 20 minutes after which the sections were again washed in TBS. They were then counterstained in Mayer s haematoxylin for 1 minute and mounted in Glycergel (Dakopatts AB,SE). All incubations were performed in a moist chamber and all antibody solutions contained 2% normal goat serum.
  • Biopsies from the resected metastases were immediately snap frozen and stored at ⁇ 700C until further processed. Frozen tissue sections, 6-7 ⁇ m thick, were fixed with 4% PFA for 5 minutes and then washed three times in BSS-HSA. For double-staining, sections were incubated with primary antibody, mouse IgGl anti-human CD3 (Dakopatts AB), at 1 ⁇ g/ml for 30 minutes, washed in BSS-HSA followed by incubation with goat-anti-mouse immunoglobulin (Dakopatts AB, SE) at a ⁇ fraction (1/25) ⁇ dilution in BSS-HSA.
  • primary antibody mouse IgGl anti-human CD3
  • Dakopatts AB goat-anti-mouse immunoglobulin
  • Monoclonal mouse IgG1 against an irrelevant antigen (Dakopatts AB, SE) was used as a negative control.
  • the sections were then incubated with PAP mouse monoclonal antibody (Dakopatts AB, SE) at a ⁇ fraction (1/25) ⁇ dilution in BSS-HSA for 30 minutes.
  • 3,3′-Diaminobenzidine (DAB, Sigma) was used as a substrate, which resulted in a brown colour.
  • IgG was then identified using 1 or 10 ⁇ g/ml biotinylated protein G (Sigma) which was incubated for 30 minutes, washed in BSS-HSA, followed by incubation with alkaline phosphatase-labelled streptavidin (Dakopatts AB, SE) at a ⁇ fraction (1/100) ⁇ dilution in BSS-HSA for 30 minutes. After washes in TBS and incubation with alkaline phosphatase substrate for 20 minutes, the sections were again washed in TBS. They were then counterstained in Mayer's haematoxylin for 1 minute and mounted in Glycergel (Dakopatts AB, SE).
  • IL.1Ra interleukin-1 receptor antagonist
  • the sections were first blocked with 10% normal human AB-serum before staining and were then incubated with biotinylated goat IgG antibodies directed either to interleukin-la or interleukin-1 receptor antagonist (both from R&D Systems, UK) at 10 ⁇ g/ml over night, washed in BSS-saponin, incubated with alkaline phosphatase-labelled streptavidin (DAKO, SE) at a ⁇ fraction (1/100) ⁇ dilution in BSS-saponin containing 2% human AB-serum for 30 minutes.
  • biotinylated goat IgG antibodies directed either to interleukin-la or interleukin-1 receptor antagonist (both from R&D Systems, UK) at 10 ⁇ g/ml over night, washed in BSS-saponin, incubated with alkaline phosphatase-labelled streptavidin (DAKO, SE) at a ⁇ fraction (1/100) ⁇ dilution in BSS
  • PBMC peripheral blood mononuclear cells
  • Mononuclear cells were then isolated by Ficoll-paque Plus (Pharmacia AB, SE) density gradient centrifugation after which the cells were washed twice in RPMI1640 Dutch's modification (RPMI) (Gibco BRL, Scotland) with 2% human serum albumin (HSA) (Pharmacia & Upjohn, SE). Cell viability was assessed by exclusion of 0.05% Trypan Blue and was always above 95%. The cell suspension was stained with Turks solution and the number of lymphocytes and monocytes in the PBMC preparation were counted in a hemocytometer. PBMCs were suspended in RPMI with 2% HSA and the cell concentration were adjusted to 5 ⁇ 10 5 lymphocytes/ml.
  • Mononuclear cells were isolated by Ficoll-Isopaque (Pharmacia, SE) density gradient centrifugation. 5 ⁇ 10 4 PBMC in a final volume of 200 ⁇ l were seeded into round-bottomed microtiter plates (Corning Inc. NY, US) in culture medium consisting of RPMI 1640 supplemented with 100 IU/ml Penicillin, 100 ⁇ g/ml Streptomycin (Flow laboratories) and 10% heat-inactivated, autologous fresh serum.
  • Phytohemagglutinin (PHA, Sigma Chemical Co, MO, US), at a final concentration of 20 ⁇ g/ml, and Chlorambucil (CHL, Sigma), at a final concentration of 1 ⁇ g/ml, were then added.
  • Cells were cultured for 3 days in a humidified 5% CO 2 atmosphere at 37° C. Proliferation was assayed by incorporation of 1.6 ⁇ Ci/well of [ 3 H]thymidine (Amersham Int, UK) during the last 18 hr. Mean values of dpm (disintegrations per minute) of triplicate cultures were used for the calculations.
  • TNF- ⁇ in PHA stimulated cultures PHA-stimulated cultures with or without chlorambucil (1 ⁇ g/ml) were set up in parallel as described above for mitogen-stimulated cultures. Supernatants were collected after 72 hours and cells were removed by centrifugation for 5 minutes at 4000 RPM. The SNs were frozen immediately and stored at ⁇ 70° C. The amount of TNF- ⁇ in the SNs was evaluated with an ELISA kit from Immunotech S.A., FR, according to the manufacturer's instructions. The lower limit of detection in this assay was 10 ⁇ g/ml.
  • HSA HSA/IgG Round-bottomed
  • 96 ⁇ well tissue culture plates (Costar, Corning Inc. NY, US) were pre-coated with HSA only or HSA and pooled human IgG for intravenous injection (Gammagard, Baxter AS, DK).
  • HSA was diluted in RPMI1640 without supplements to a concentration of 10 mg/ml.
  • 1 mg/ml IgG was mixed into a solution of 9 mg/ml HSA in RPMI (HSA/IgG). 200 ⁇ l of HSA or HSA/IgG were then added to each well of the plate. The plates were incubated at 4° C. for 30 minutes after which the wells were washed twice with 200 ⁇ l of RPMI1640. The coated plates were used immediately.
  • PBMC isolated from healthy individuals or patients with metastatic renal cell carcinoma, were diluted in RPMI/2% HSA at a concentration of 5 ⁇ 10 5 /ml and 100 ⁇ l were added to the microtiter wells.
  • Interleukin-2 (IL-2, Proleukin, Chiron, NL), at a final concentration of 120 IU/well, was added to some wells. Cells were cuitured for 7 days in a humidified, 5% CO 2 -atmosphere at 37° C. Proliferation was assayed by incorporation of 1.6 ⁇ Ci/well of [3H]-thymidine (Amersham Int., UK) during the last 18 hrs. Mean values of dpm (disintegrations per minute) of triplicates were used for the calculations.
  • PBMC peripheral blood mononuclear cells
  • F(ab′)2 mouse anti-human CD16 Fc ⁇ R III
  • CD 32 Fc ⁇ R II
  • CD 64 Fc ⁇ R I
  • SNs Supernatants were harvested after 24 hrs and residual cells were removed by centrifugation in a refrigerated centrifuge (Beckman) at 2600 ⁇ g for 5 minutes. SNs were frozen and stored at ⁇ 70° C. until monokine concentrations were measured by ELISA.
  • Monokines were assessed by ELISA using the DuoSet ELISA development system for human IL-6, TNF- ⁇ or IL-1 ⁇ (R&D Systems Europe, Ltd. UK) following the manufacturer's recommended procedures.
  • Lower limit of detection was 3.1 ⁇ g/ml for IL-6, 15.6 pg/ml for TNF- ⁇ and 3.9 ⁇ g/ml for IL-1 ⁇ .
  • IL-10 was detected with a kit from Diaclone Research, FR. The lower limit of detection was 5 ⁇ g/ml.
  • Human IL-1Ra was detected with a quantitative sandwich ELISA using a monoclonal mouse anti-human IL-1Ra as capture antibody and a biotinylated goat anti-human IL-1Ra as developing antibody (both from R&D Systems). Briefly, enhanced binding 96-well microtiter plates (Labsystems AB, SE) were coated overnight at room temperature with 10 ⁇ g/ml of capture antibody diluted in PBS. After washing in PBS with 0.05% Tween 20 (Sigma Chemical, MO, US) plates were blocked with a blocking buffer consisting of 1% bovine serum albumin (BSA, Sigma), 5% sucrose (Sigma) and 0.05% NaN 3 in PBS.
  • BSA bovine serum albumin
  • sucrose sucrose
  • PBMC culture SNs or recombinant human IL-1Ra standard were diluted in 0.1% BSA and 0.01% Tween 20 in PBS (dilution buffer) and incubated over night at room temperature. After washing, the biotinylated-developing antibody was added at 100 ng/ml in dilution buffer. This was incubated for 1 hr at room temperature. Plates were washed and alkaline phosphatase (ALP)-conjugated Extravedin (Sigma), at a dilution of 1:10000 in Tris buffered saline with 0.1% BSA was added.
  • ALP alkaline phosphatase
  • IL-1Ra was measured by hydrolysis of paranitrophenyl phosphate (Sigma). Optical density was read at dual wavelengths, 405 nm and 570 nm, respectively, in a Multiscan EX microplate reader (Labsystems). The lower limit of detection in this assay was 39 pg IL-1Ra/ml
  • IL-1Ra Production by PBMC with Antibodies against Fc ⁇ R II PBMCs isolated from healthy volunteers were pre-incubated at 1 ⁇ 10 6 /ml in RPMI1640+2% HSA with 5 or 50 ⁇ g/ml of azide-free, mouse anti-human CD32 Fab (Medarex Inc. NJ, US) for 1 hr at 37° C. under gentle agitation.
  • the cells (5 ⁇ 10 5 /well) were immediately seeded onto s uncoated or HSA/IgG coated tissue culture microtiter plates in RPMI1640 with 10% heat-inactivated, pooled human AB sera.
  • Supernatants were harvested after 24 hrs, as described under “generations of cell culture supernatants”, and the production of IL-1Ra was measured by ELISA.
  • PBMC peripheral blood mononuclear cells
  • ⁇ l of culture medium consisting of RPMI1640 supplemented with 200 IU/ml penicillin, 200 ⁇ g/ml streptomycin, 4 mM L-glutamine (all from Sigma) and 20% fresh heat-inactivated autologous serum were added to the HSA or HSA/IgG pre-coated microtiter plates followed by 100 ⁇ l of the PBMC suspensions (5 ⁇ 10 4 lymphocytes) in RPMI/2% HSA. Cells were cultured in a humidified, 5% CO 2 atmosphere at 37° C. Viability of cultured PBMCs were also assessed after 24 hrs by Trypan blue exclusion and found to be 100%.
  • SNs were harvested after 24 hrs and residual cells removed by centrifugation in a refrigerated centrifuge at 2600 ⁇ g for 5 minutes. SNs were frozen and stored at ⁇ 70° C. until IL-1Ra concentration was determined by ELISA.
  • Heat-inactivated human serum was passed over a HiTrap protein G-Sepharose HP affinity column (Amersham Pharmacia Biotech AB, SE). The non-binding fraction was eluted with RPMI1640, giving a final dilution effect of 1 ⁇ 5 (20% ) of the original serum. 200 IU/ml penicillin, 200 ⁇ g/ml streptomycin, 4 mM L-glutamine (all from Sigma) were added. The eluate was then sterile filtered with a 0.45 ⁇ m Millex syringe filter (Millipore Co. MA, US) and used immediately for culture with control PBMC.
  • Sera collected from cancer patients or from normal healthy individuals, were heat-inactivated for 30 minutes at 56° C. and frozen at ⁇ 70° C. After thawing, sera were diluted in RPMI1640 to a concentration of 20% and either used unfiltered or ultra-filtered, as described below, in co-culture experiments for monokine-induction with control PBMC.
  • Ultra filtered serum fractions consisted of filtrates from 100000 mw cut-off filters, retentates or filtrates from 50000 mw cut-off filters or retentates from serum fractions that had been sequentially spun on a 50000 mw cut-off filter followed by concentration on a 3000 mw cut-off filter.
  • Retentates were reconstituted in RPMI1640 with 200 IU/ml penicillin, 200 ⁇ g/ml streptomycin, 4 mM L-glutamine (Sigma) to their original volume. 100 ⁇ l of diluted sera (20% ) or ultra filtered serum fractions were added to uncoated or HSA-coated microtiter plates together with 100 ⁇ l PBMC suspension (5 ⁇ 10 5 /ml). Cell-free SNs were harvested after over-night incubation and tested for monokine activity by ELISA, as described above.
  • tumour biopsies from patients with renal cell carcinoma, malignant melanoma or colon carcinoma were embedded in glycergel (Dakopatts AB, SE) and frozen at ⁇ 70° C.
  • glycergel Dakopatts AB, SE
  • 50 ⁇ m cryostat sections were cut and transferred to 4 ml of cold RPM1640 with 200 IU/ml penicillin, and 200 ⁇ g/ml streptomycin (RPMI/PEST) (Gibco BRL) and kept on ice.
  • the sections were centrifuged and resuspended in 0.5 ml fresh RPMI/PEST after-which they were disaggregated in a Medimachine (Dako A/S, DK) using a sterile Medicon 50 ⁇ m unit.
  • the disaggregated sample was suspended in 2 ml RPMI/PEST, vortexed and kept on ice for 0.5 to 2 hrs.
  • the sample was centrifuged 10 minutes at 300 ⁇ g and the supernatant was harvested.
  • the supernatant was then filter sterilised through a 0.45 ⁇ m Millex-HV syringe filter (Millipore).
  • a newly excised biopsy (about 5 mm 3 ) was disaggregated in a Medimachine (Dako AS), using a sterile Medicon 50 ⁇ m unit and resuspended in a total volume of 5 ml TCN buffer (50 mM Tris, 50 mM NaCl, 10 mM CaCl 2 +200 IU/ml penicillin and 200 ⁇ g/ml streptomycin).
  • the cell suspension was incubated on ice for 1 hr.
  • the sample contained 2.7 ⁇ 10 6 cells/ml with a viability of 15%.
  • a cell-free supernatant was harvested after the cells had been pelleted for 10 minutes at 300 ⁇ g.
  • squamous cell carcinoma cell lines UT-SCC-10 and UT-SCC-20A (a gift from Dr. R. Grenman, University of Turku, Finland) were cultured in 5 ml media consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mM L-glutamine, 18 mM Hepes, 0.9% non-essential amino acid solution, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin (all from Gibco) and 100/% heat-inactivated human AB-serum (DMEM) in 25 cm 3 cell culture flasks (Costar) at 37° C. and 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM heat-inactivated human AB-serum
  • the medium was decanted off and replaced every 2 to 3 days. When the cells had reached confluence they were trypsinated and reseeded in new flasks. The decanted medium was centrifuged at is 1000 ⁇ g to remove residual cells and debris and then ultra filtered on Centriplus filters with a 50000 mw cut-off. The filtrate was then concentrated on a 3000 mw filter. In some experiments the retentate from the 50000 mw cut-off filter was also saved. Retentates were resuspended in RPMI1640 to 2.5 ml.
  • SNs 100 ⁇ l, were added to uncoated or HSA-coated microtiter plates together with 100 ⁇ l PBMC suspension (5 ⁇ 10 5 /ml). Cell-free SNs were harvested after over-night incubation and tested for monokine activity by ELISA, as described above.
  • Pre-swelled gelatine Sepharose 4B (Pharmacia Biotech AB, SE) was washed three times in PBS or RPMI1640. 0.3-0.5 ml Sepharose gel was incubated together with the 3-50 kD fractions of ultra filtered serum, diluted in 2-2.5 ml RPMI1640 for 30 minutes at room temperature. The gel was mixed gently by inversion approximately every 10 minutes. An equal portion of the ultra filtered serum sample was incubated in parallel without gelatine Sepharose, as a negative control. The gel was allowed to settle and the supernatant collected.
  • Buffer exchange to RPMI1640 with 200 IU/ml penicillin, 200 ⁇ g/ml streptomycin, 4 mM L-glutamine (Sigma) was performed by gel filtration over a Sephadex-G25 (PD-10) desalting column (Pharmacia) followed by filtration on a 0.45 ⁇ m Millex-HV syringe filter (Millipore).
  • SNs, 100 ⁇ l, were added to uncoated microtiter plates together with 100 ⁇ l of control PBMC suspension (5 ⁇ 10 5 /ml). Cell free SNs were harvested after over-night incubation and tested for monokine activity by ELISA as described above.
  • Urine samples (100-450 ml) from cancer patients or healthy controls were ultra centrifuged on Jumbosep centrifugal devices (Pall Life Science, MI, US) using a 30 K membrane insert or alternatively, with a Proflux M12 system using a 30 K Pellicon 2 mini filter (Millipore, Mass., US) followed by concentration on Jumbosep with a 3K membrane insert.
  • Samples were desalted over a Sephadex-G25 (PD-10) column (Amersham Biosciences, SE), lyophilised and dissolved in rehydration buffer (8M urea, 4% CHAPS, 10 mM DTT, 0.5% v/v IPG buffer and a trace of orange G). Samples were centrifuged to remove undissolved material.
  • rehydration buffer 8M urea, 4% CHAPS, 10 mM DTT, 0.5% v/v IPG buffer and a trace of orange G.
  • 2-DE was performed in a horizontal 2-DE set-up (Multiphore/IPGphore, Pharmacia Biotech, SE) as described (Lindahl M. et.al 1998) based on isoelectric focusing (IEF) in the first dimension and molecular mass in the second dimension. Briefly, samples (230 ⁇ g, 350 ⁇ g, 600 ⁇ g) were applied to IPG gels, pH 4-7, (Amersham Pharmacia Biotech, SE) and focused overnight for 48000 Vh. SDS-PAGE was then carried out with 16% T/1% C polyacrylamide casted slab gels. Molecular weight standards were included in each run. Separated proteins were detected by Coomassie blue staining or SYPRO Ruby staining.
  • the protein patterns in the gels were analyzed as digitised images using a CCD (Charged-Coupled Device) camera (1340 ⁇ 1040 pixels) in combination with a computerized imaging 12-bit system, PDQuest Version 6.1.0, in the case of fluorescent stained gels using UV scanning illumination mode (Fluor-S Multi-imager, Bio-Rad).
  • the amount of protein in a spot was assessed as background-corrected optical density, integrated over all pixels in the spot and expressed as integrated optical density (IOD).
  • Selected protein spots were electro transferred to PVDF membranes and subjected to N-terminal sequence analysis by Edman degradation in a Procise cLC or a Procise HT sequencer (PE-Applied Biosystems) at the Protein Analysis Center, Karolinska Institute, Sweden.
  • PBMC peripheral blood from normal controls as described above. Monokine production by the PBMCs in response to urine fractions from cancer patients was verified as described above. Remaining PBMC were frozen at ⁇ 70° C. until use. For adsorption of urine fractions, the PBMCs were thawed and washed carefully in cold phosphate buffered saline (PBS). Approximately 50 ⁇ 10 6 PBMC were added to 2.7 or 2.6 ml, respectively, of ultra centrifuged (3-30 KD) urine fractions pooled from two patients with renal cell carcinoma or from one patient with malignant melanoma.
  • PBS cold phosphate buffered saline
  • MMPs Matrix Metalloproteinases
  • MMP -1, -2, -13 (R&D Systems) and MMP-3 and -7 (Chemicon, UK) were activated according to instructions of the manufacturer. 1-50 ng/ml of the indicated MMPs were then incubated with 1 mg/ml of either human serum albumin (HSA, Octapharma, SE) or pooled human IgG for intravenous injection (IvIg, Gammagard, Baxter, DK) as substrate in RPMI or in 50 mM Tris-HCl, pH 7.5 (containing 0.15 M NaCl, 10 mM CaCl 2 and 0.05% Brij35). The mixtures were incubated for 5-20 hours at 37° C.
  • HSA human serum albumin
  • IvIg intravenous injection
  • the pelleted, washed tissue was cut into pieces of approximately the same size and each piece was homogenised using a Mikro-Dismembrator U (B. Braun Biotech International, GE).
  • the tissue was transferred to a PTFE shaking flask together with 1 ml RPMI/PEST or PBS/PEST and a tungsten carbide grinding ball and homogenised during 15-20 seconds with a shaking frequency of 1500-2000 RPM.
  • the homogenised tissue suspension was transferred to a test tube and kept on ice.
  • tumour cryo sections (3 ⁇ 300 ⁇ m) were carried out as described above.
  • the pelleted tumour tissue was suspended in 0.5 ml cold RPMI/PEST.
  • MMP-2 (R&D Systems, UK), activated according to instructions from the manufacturer, was added at a final concentration of 5 ng/ml.
  • the tumour tissue suspension was incubated with MMP-2 for 20 h at 37° C. and the supernatant collected after centrifugation. Parallel incubation of tumour tissue with no addition of MMP-2 was set up as a control.
  • the mixtures were buffer exchanged to RPMI by gel filtration through a Sephadex-G25 (PD-10) column (Amersham Biosciences, SE) and filtered through a 0.45 ⁇ m Millex-HV syringe filter (Millipore, Mass., US). Finally, the mixtures were analysed by gel-electrophoresis and tested for monokine inducing activity in fresh normal PBMC as described above.
  • PD-10 Sephadex-G25
  • Millex-HV syringe filter Millex-HV syringe filter
  • the pelleted homogenised tumour tissue formed during preparation of PIF was kept on ice and stored at ⁇ 70° C.
  • PIF were prepared as described above.
  • the homogenised sections were washed once more to collect a fourth supernatant, used as a control reflecting the IL-6 inducing activity in tumour sections before incubation.
  • the fourth supernatant was divided into two parts, to one of which was added 20 mg/ml of HSA and directly frozen at ⁇ 70° C.
  • tumour tissue with 20 mg/ml HSA or with 10 mg/ml pooled human IgG for intravenous injection (IvIg), as well as the mixtures of PIF with 20 mg/ml HSA or with 10 mg/ml IvIg, all in RPMI/PEST, were incubated for 18-21.5 h at 37° C.
  • the mixtures were centrifuged and supernatants collected.
  • the supernatants were buffer exchanged to RPMI as described previously and filtered through a 0.45 ⁇ m Millex-HV syringe filter (Millipore, Mass., US) and stored at ⁇ 70° C. until testing for monokine inducing activity in fresh, normal PBMC as described above.
  • Comparisons of the means of different patient groups or different test occasions were performed using an unpaired t-test. Time to progression and survival was analyzed using the Kaplan-Meier method and Logrank test.
  • MI modulation index
  • IL-1Ra is a potent inhibitor of immune stimulation/reactivity as it blocks the activity of IL-1. It is therefore of considerable importance that it is frequently expressed by tumour cells and tumour infiltrating mononuclear cells. Based on the results presented in the above, it is highly reasonable to assume that tissue bound IgG is the inducer of this cytokine. Thus either tissue bound antibodies or intra-tumourally precipitated ICs can play a major role in intra-tumoural down-regulation of the immune response.
  • FIG. 1 shows melanoma biopsies stained for the expression of IL1Ra. Different patterns are hereby found, viz. A) tumour cells are generally stained with some positive infiltrating mononuclear cells, and B) large numbers of infiltrating cells staining for IL-1Ra, tumour cells only faintly positive.
  • IL-1Ra Production of IL-1Ra can be induced by some cytokines (Tilg et al., 1994), but Fc ⁇ R cross-linking by solid phase IgG or ICs seems to be the most efficient inducer of this substance. It has been shown that in cultures where binding of IgG to the surface of the culture well is allowed (uncoated wells) the production of IL-1Ra is significantly enhanced (p ⁇ 0.0001) compared to cultures where binding of IgG has been reduced by pre-coating with HSA. This immunomodulatory effect has been studied in healthy individuals, patients with malignant melanoma and renal cell carcinoma ( FIG. 2 ). If purified human IgG is added to the HSA in coated wells (HSA/IgG), IL-1Ra production by control PBMC is restored. Based on these results it is obvious that solid phase IgG will play a major role in immune regulation.
  • tissue bound IgG exposing the Fc-parts, in tumour tissue inducing production of IL-1Ra as described herein is thus highly relevant to the down-regulation of the anti-tumour immune reactivity.
  • FIG. 4 shows inhibition of ILlRa production by Tosyl.
  • the effect of Tosyl on IL-1Ra production by PBMC from healthy individuals (n 9) in 10% autologous sera cultured in microtiter plates pre-coated with A) HSA, and B) HSA/IgG.
  • FIG. 5 shows IL-2 induced proliferation by PBMC from normal healthy individuals (first three bars) and PBMC from RCC patients (last three bars) cultured in 10% autologous sera on uncoated and pre-coated microtiter plates. Significant (p ⁇ 0.011) difference in proliferation between PBMC from healthy individuals and RCC patients on uncoated plates.
  • Solid phase IgG results in a broad cross-linking, which can elicit stimulation of the immune response in normal healthy individuals by eliciting a cytokine cascade including TNF- ⁇ and IL-1, which sensitises the response to IL-2 (increased numbers of receptors).
  • This model reflects the situation when opsonised antigens or large ICs elicits an immune response. In HSA coated wells this stimulatory cross-linking can not take place, which results in a significantly lower response to IL-2.
  • the difference in IL-2 induced proliferative response can be due to an immunomodulatory effect by the cross-linking per se and not a stimulatory cytokine cascade.
  • Inhibitory signals in monocytes/macrophages might be overcome by broad cross-linking of Fc ⁇ R.
  • Flow cytometry was performed to verify binding of the F(ab′)2 monoclonal antibodies to PBMC.
  • the inhibitory anti-CD64 antibodies bound to a higher percentage of monocytes that anti-CD16, but fewer monocytes than anti-CD32 antibodies, in both experiments. Furthermore, the mean fluorescent intensity of both anti-CD16 and anti-CD32 binding was higher than anti-CD64 in both experiments. Thus, increased binding of the anti-CD64 F(ab′)2 antibody, compared to anti-CD16 and anti-CD32 does not explain the inhibitory effect.
  • the culture model described above provides for excellent opportunities of studying immunosuppressive regulatory mechanisms in cancer patients.
  • CBIC cell-bound ICs
  • Immune complexes can be determined in different ways. Standard methods for determining circulating ICs (CIC) are by indirect non-functional parameters as complement binding and activation, PEG-precipitation, phagocytosis, platelet aggregation.
  • CIC Determination of CIC, to obtain prognostic information or to diagnose possible occurrence of ICs modulating the immune system, is likely to be irrelevant, as IC has to be bound to cellular receptors to have any immunomodulatory effect. Thus CBIC will have full immunomodulatory effect long before the amount of ICs is enough to saturate these receptors and ICs appear in the circulation. In the present case direct methods has been used to demonstrate the presence of cell/tissue bound IgG or ICs.
  • CBIC can be demonstrated using flow cytometry, or CBIC on blood cells has been demonstrated using immunocytochemistry (IHC) on cytospin preparations. Further CBIC has been demonstrated in tumour tissue (tumour cells, endothelium, tumour infiltrating inflammatory cells) using IHC.
  • the identified substance is considered being IC. Otherwise it is not possible to discriminate between IgG and IC as the monoclonal antibody and protein G might be able to bind to monomeric IgG as well. However, if IgG is bound to the tissue directly or in IC might be of minor importance as it will anyhow be recognised as “solid phase” IgG.
  • tumour biopsies stained for the presence of tissue bound IgG/IC red
  • T-lymphocytes brown
  • Different staining patterns are shown, viz. A) staining of vascular areas/endothelial cells and some lymphocytes for IgG. Some lymphocytes are not stained for IgG/IC.
  • tumour infiltrating mononuclear cells B) a diffuse staining of the tumour tissue for IgG/IC, whereby the majority of the lymphocytes are not stained for IgG/IC, C) staining tumour infiltrating macrophages and some lymphocytes for IgG/IC, but tumour cells are generally negative, D) extensive staining of vascular areas for IgG/IC with very low numbers of infiltrating mononuclear cells.
  • tissue bound IgG exposing Fc parts are inversely correlated to the presence of tumour infiltrating mononuclear cells and hence plays a major role in the down-regulation of the immune response to the tumour.
  • Interleukin-6 is a proinflammatory cytokine of importance for the initiation of immune reactivity (Barton, 1996; Barton, 2001). However, in cancer patients, its occurrence has a number of detrimental effects. Inflammatory cells, vascular endothelium and several types of tumour cells produce it. It has activity as autocrine growth factor in at least some malignancies, e.g., myeloma and renal carcinoma. It can interfere with the cytotoxic activity of cisplatinum (Borsellino et al., 1995; Mitzutani et al., 1995).
  • CBIC is of importance for the dys-regulation of the immune system in cancer.
  • IL-1 ⁇ IL-1 ⁇
  • IL-6 TNF- ⁇
  • PGE 2 TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • PGE 2 is well known to be a potent inhibitor of immune reactivity. Thus patients with high serum levels of PGE 2 before treatment or having an increased production of PGE 2 during the early treatment period will not respond to immunotherapy. (Deehan et al., 1994)
  • Therapeutic control of the dys-regulatory mechanisms of IL-6 production will thus improve quality of life, therapeutic response to immuno- and chemotherapy, and increase over- ⁇ ll survival.
  • IL-6 is only one product of the dys-regulated inflammatory reaction mentioned, and often correlates to production of the other cytokines such as IL-1 ⁇ and TNF- ⁇ , the strategy is to find the fundamental dys-regulatory mechanisms and block them in order to completely down-regulate the inflammatory reaction.
  • IL-6 production in cancer patients correlation to prognosis
  • the present analysis includes IL-6 production from PBMCs from three types of cancer patients. As shown in Table 3, 30 patients with radically resected stage III melanoma (MMO), 43 patients with previously untreated metastatic melanoma (MR1) 36 with previously untreated metastatic renal cell carcinoma (RCC1) and 46 patients with primary colorectal cancer (CRC) were studied. The cytokine production is, compared to that of healthy individuals (K), significantly increased in all categories.
  • IL-6 production is not restricted to patients with advanced disease as IL-6 is produced also by PBMCs from patients with primary colorectal cancers and radically resected stage III melanoma (MM 0), whereby in the latter group no metastatic lesions could be demonstrated by clinical or radiological investigations.
  • MM 0 stage III melanoma
  • the serum concentration of IL-6 was determined in melanoma, colon and renal cell carcinoma patients and was generally below the detection limit of the ELISA-technique used. In only a few cases measurable amounts of IL-6 were found but these were quite negligible compared to those found in cultures. TABLE 3 Production of IL-6 by PBMCs from healthy individuals and various types of cancer patients.
  • FIG. 9 shows the production of IL-6 by PBMCs from different subsets of colorectal cancer patients, viz. A), without LPS stimulation and B) with LPS stimulation.
  • IL-6 production is of prognostic significance as evident from FIG. 10 .
  • Production of IL-6 by PBMCs from in particular cancer patients in vitro means that either are the producing cells triggered in vivo and maintain this status when placed in culture or there is a serum factor which continuously stimulates production of IL-6.
  • sera from cancer patients, with a high IL-6 production in autologous PBMC cultures were used in the medium of five cultures with PBMCs from healthy individuals.
  • a high IL-6 production was induced in all these cultures demonstrating occurrence of an IL-6 inducing serum factor.
  • CIC are not involved in inducing IL-6 production, but either CIC or IgG obviously modulate the production as it increased when IgG was removed from the culture medium. This is compatible with the results above where Fc ⁇ R cross-linking by solid phase IgG inhibited IL-6 production.
  • IgG is removed from the culture medium, the inhibitory effect is relieved.
  • Affinity chromatography with proteinG-Sepharose had no effect on control sera not inducing IL-6 production. Determination of IgG after affinity chromatography did not find detectable amounts of IgG ( ⁇ 0.01 mg/ml).
  • IL-6 inducing factor (IL-6IF) by ultrafiltration
  • sera from cancer patients and healthy individuals were diluted (1:5) and ultrafiltered with a filter cut-off at 100, 50 and 3 kD.
  • IL-6IF was at least to some extent also found in the other fractions, but this activity could always be demonstrated in the less than 50 kD fraction.
  • IL-6 inducing activity was generally not found in the fraction with a molecular weight of less than 3 kD.
  • IL-6IF has a low molecular weight and thus IgG or CIC can not be involved.
  • the former case is compatible with the assumption that this factor is a proteolytic fragment of some large molecule, and that the activity is present in fragments of different sizes. If the IL-6IF were a small fragment it is certainly likely that it is bound to other serum proteins as it would otherwise immediately be excreted in the urine.
  • fractions obtained during ultrafiltration of sera described above were also analysed using PAGE electrophoresis of reduced proteins.
  • a band having a molecular weight of about 20 kD was identified. This was either very weak or not present in all fractions of control sera or patient sera with no IL-6 inducing activity.
  • IL-6IF As IL-6IF was found to have a molecular weight of less than 50 kD, it is assumed that this factor will at least, to some extent be excreted in the urine. Thus the less than 50 kD fraction was prepared and concentrated using the 3 kD filter. When these fractions from cancer patients were tested for IL-6 production in cultures with PBMCs from healthy individuals an IL-6 inducing activity was found.
  • IL-6IF or any other inducing factor of an immunoregulatory substance, in the urine opens interesting diagnostic and therapeutic possibilities.
  • concentration of IL-6IF of urine is related to the serum concentration a simple diagnostic test is made which provides essential information about prognosis and the likelihood of therapeutic success.
  • the diagnosis will also be of value to determine treatment strategies dealing with the elimination of IL-6IF—treatment of the chronic inflammatory reaction in cancer patients.
  • IL-6IF The origin of IL-6IF is so far unknown. However, it is related to the presence of a malignant tumour.
  • IL-6IF has a low molecular weight is compatible with its being an enzymatic fragment of some large molecule. It is assumed that this factor is produced in the tumour or by substances released from the tumour.
  • tumours were minced using a stainless steel mesh and extracted in physiological buffer during various times. In fact, 5 out of 7 analysed tumours produced the factor when the extracts were tested in cultures of PBMCs from healthy individuals.
  • Immunomodulating factors have been identified in large numbers at studies of either tumour extracts or conditioned culture media from tumour cell lines. It has now turned out that IL-6IF is produced by squamous cell carcinomas from the oral cavity. Conditioned media were collected and tested using PBMCs from healthy individuals as described. An IL-6 inducing activity was demonstrated and this will facilitate studying therapeutic possibilities such as reduction of IL-6IF by inhibiting different types of tumour related enzymes, such as MMPIs.
  • IL-6 inducing activity in ultrafiltered (3 to 50 kD) condition media from a squamous cell carcinoma (SCC) cell line grown in media with fetal calf serum (FCS) or pooled human AB-sera from two different batches AB7, and AB8, respectively.
  • SCC squamous cell carcinoma
  • FCS fetal calf serum
  • TNF- ⁇ and IL-1 ⁇ Similar to the situation with IL-6 we have also found inducing activity for TNF- ⁇ and IL-1 ⁇ (Table 10) and IL-10 in serum and ultrafiltered urine. IL-10 was found in four sera/serum fractions and one urine fraction also inducing IL-6. In addition, one serum and one urine fraction inducing IL-6 did not induce IL-10. TABLE 10 TNF- ⁇ and IL-1 ⁇ inducing activity in IL-6 inducing serum and ultrafiltered urine from cancer patients IL-6 TNF- ⁇ IL-1 ⁇ Source Control PBMC pg/ml pg/ml pg/ml Patient serum 1 None ⁇ 31.3 N.D. N.D.
  • control pool Comparison between healthy controls and cancer patients Comparisons were made between a pool of 11 healthy controls (control pool) and four individual controls and 1. a pool of one breast and one pancreas cancer patient, 2. a pool of two renal cell carcinoma patients and 3. three individual melanoma patients.
  • Proteins or fragments of proteins found in the urine fraction from different types of cancer patients but not in healthy controls are shown in Table 11. Some proteins/fragments were found in the urine of several different cancer patients, e.g. albumin/albumin fragments, CD59, plasma retinol binding protein and prostaglandin D2 synthase TABLE 11 Proteins/protein fragments found in urine fractions from cancer patients, but not in the pool of healthy controls Breast/pancreas Renal cell Malignant Proteins cancer carcinoma melanoma Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Albumin fragments X Alpha-a
  • Proteins over represented in urine fraction from patient with IL-6 inducing activity Alternative protein/s Protein according to spot on Protein/protein fragment analysed masspectormetry 2D gel by masspectometry analysis 1
  • Beta-microsemino protein 2 CD59 3
  • CD59 4 CD59 5
  • Colipase Inhibin beta 6 Extracellular superoxide dismutase 7
  • Heparan sulphate proteoglycan (Perlecan) 8
  • Immunoglobulin kappa light chain 9
  • Immunoglobulin superfamily LIR-D1 precursor member 8
  • IL-13 receptor alpha-1 chain Urokinase plasmin activator surface 11
  • Inter-alpha-trypsin inhibitor, chain 2 Endoplasmic reticulum protein ERP29 12 Lithostathine 1 alpha 13 Lithostathine 1 alpha
  • MMP Matrix Metalloproteinases
  • IgG or serum albumin was incubated with MMPs under well-defined conditions. Two different buffer systems were used in these experiments ( FIG. 16 ). The supernatants were analysed using gel-electrophoresis and their cytokine inducing activity was analysed in cultures with normal PBMCs. As demonstrated in FIG. 16 several MMPs released fragments of IgG and serum albumin, which induced or modulated IL-6 inducing production. In particular, MMP-1, -2, -3, -13 released active fragments from albumin and MMP-2, -3, -7 and ⁇ 13 from IgG.
  • IL-6IF can be extracted from malignant tumours, e.g. malignant melanoma, renal cell carcinoma, colorectal cancer. These results were confirmed in a new series of experiments, where homogenised tumours were washed three times in PBS or RPMI in order to collect cytokine inducing factors already present in the tumours, so called “preformed inducing factors” (PIF).
  • malignant tumours e.g. malignant melanoma, renal cell carcinoma, colorectal cancer.
  • cytokine inducing factors PEFs
  • tissue sections/homogenates were thoroughly washed before addition of proteolytic enzymes, such as MMPs.
  • the tumour sections/homogenates were then incubated for 20 hours at 37° C.
  • IL-6 inducing activity of the supernatants was then analysed in cultures of normal PBMCs.
  • FIG. 18 addition of MMP-2 modulated the release of IL-6 inducing activity.
  • the albumin band was found to be better preserved in incubations, to which MMP-2 was added. This is reasonably due to an effect of MMP-2 on the degradation of enzymes degrading albumin.
  • MMPs can degrade tumour tissue substances into fragments with cytokine inducing activity.
  • the effect of MMP-2 on the preservation of albumin in these incubations also demonstrates that proteolytic enzymes are involved in the regulation of the intra-tumoural proteolytic activity. A complex interaction of proteolytic enzymes is thus demonstrated and this results in generation and/or modulation of the production of dysregulatory inducing factors.
  • albumin was added to fresh PIF-fractions and incubated at 37° C. for 18 h.
  • the IL-6 inducing activity was lower when albumin was added, which shows that the markedly increased IL-6 inducing activity after adding albumin to washed homogenates is not due to any protective activity of albumin but rather that albumin acts as a substrate for intra-tumoural proteolytic enzymes.
  • immuno-modulating IgG and albumin fragments are produced in the intra-tumoural milieu.
  • FIG. 1 shows melanoma biopsies stained for the expression of IL1Ra. Different patterns are hereby found, viz. A) tumour cells are generally stained with some positive infiltrating mononuclear cells, and B) large numbers of infiltrating cells staining IL-1Ra, tumour cells only faintly positive.
  • FIG. 2 shows production of IL-1Ra in short term cultures of PBMC from healthy controls and cancer patients. The effect of pre-coating of the culture wells is shown.
  • FIG. 3 shows comparison of IL-1Ra production by PBMCs from healthy individuals and melanoma and renal cell carcinoma patients, cultured in A) HSA pre coated wells or B) uncoated wells.
  • FIG. 4 shows inhibition of IL1Ra production by Tosyl.
  • FIG. 5 shows IL-2 induced proliferation in healthy individuals and RCC patients.
  • IL-2 induced proliferation (mean ⁇ S.D.) by PBMC from normal healthy individuals (grey bars, 21-29 individuals) and PBMC from RCC patients (black bars, 12-18 individuals) cultured in 10% autologous sera on uncoated and pre-coated microtiter plates.
  • Significant (p 0.011) difference in proliferation between PBMC from healthy individuals and RCC patients on uncoated plates, but not on HSA coated or on HSA/IgG coated plates.
  • Significant (p 0.0045) difference in proliferation between PBMC from RCC patients cultured on uncoated and HSA/IgG coated plates.
  • FIG. 6 shows normal PBMC cultured in autologous sera (black bars) or sera from cancer patients (open bars) in the presence of IL-2. Proliferation was measured by 3H-uptake on day 7. Patients 88 and 124 had previously been shown to induce high levels of IL-6 production, whereas patient 112 did not.
  • FIG. 7 shows tumour biopsies stained for the presence of tissue bound IgG/IC and T-lymphocytes using a double staining technique with recombinant protein G (not binding to albumin). Different staining patterns are shown, viz. A) staining of vascular areas/endothelial cells and some lymphocytes for IgG. Some lymphocytes are not stained for IgG/IC.
  • tumour infiltrating mononuclear cells B) a diffuse staining of the tumour tissue for IgG/IC, whereby the majority of the lymphocytes are not stained for IgG/IC, C) staining tumour infiltrating macrophages and some lymphocytes for IgG/IC, but tumour cells are generally negative, D) extensive staining of vascular areas for IgG/IC with very low numbers of infiltrating mononuclear cells.
  • FIG. 8 shows cytospins of PBMC from two patients with malignant melanoma stained for the presence of IgG/IC, using recombinant protein-G (A) or monoclonal directed against the Fc-part of IgG.
  • FIG. 9 shows IL-6 production by PBMCs from different subsets of colorectal cancer patients. A) without LPS stimulation and B) with LPS-stimulation.
  • FIG. 10 shows survival in renal cell carcinoma patients according to IL-6 production by PBMCs in short term cultures where the microtitre plates were coated with HSA. High production is >2500 pg/ml.
  • FIG. 12 shows the effect of chlorambucil on PHA-induced proliferation of PBL from RCC patients before treatment with IL-2.
  • Modulation Index (MI) was calculated as described under Materials and Methods. The assays were performed at the following time points: 1) before start of treatment, 2) one week later, 48 hours after five days of IL-2 treatment, 3) after one week without IL-2 administration and 4) after an additional week, 48 hours after another five days of IL-2 treatment combined with chlorambucil.
  • FIG. 13 shows the effect of chlorambucil (CHL) on TNF- ⁇ production and proliferation of PHA-stimulated PBMC from RCC patients.
  • PBMC were cultured with PHA in the presence or absence of CHL for 72 hours and the cellular 3 H-uptake and the TN- ⁇ concentration in cell-free culture supernatants were assessed. Modulation Index was calculated as described under Materials and Methods. Each circle represents one patient.
  • FIG. 14 2D gel electrophoresis of PBMC-adsorbed and unadsorbed urine fractions pooled from two patients with renal cell carcinoma. 230 ug of protein was loaded per 2D gel. Separated proteins (based on isoelectric focusing in the first dimension and molecular mass in the second dimension) were detected by SYPRO Ruby staining.
  • FIG. 15 2D gel electrophoresis of PBMC- ⁇ dsorbed and unadsorbed urine fractions from a patient with malignant melanoma. 350 ug of protein was loaded per 2D gel. Separated proteins (based on isoelectric focusing in the first dimension and molecular mass in the second dimension) were detected by SYPRO Ruby staining.
  • FIG. 16 Generation of IL-6 inducing factors by incubation of IgG (A and C) and albumin (B and D) with MMPs. Two experiments using different buffer systems are shown (as described in Material and Methods). The substrates were incubated with MMPs for 20 hours in experiments A, B, C and for 5 hours in experiment D. The production of IL-6 was then tested in cultures of PBMCs from healthy controls.
  • FIG. 17 Effect of collagen/collagen fragments on the proliferative response (measured as incorporation of 3 H-TdR) of normal PBMCs to IL-2. As can be seen a strong inhibitory effect was demonstrated in two out of three experiments.
  • FIG. 18 Modulation of IL-6 inducing factors by incubation of homogenised, washed tumour tissue with MMP-2.
  • a and B represent two separate experiments with two different tumour tissues in each. The effect of this enzyme varies, dependent on the intra-tumoural milieu of different tumours, but it is clearly shown that MMP-2 has a modulatory effect on the production of IL-6 inducing factor/activity, which was tested in cultures of PBMCs from healthy controls.
  • FIG. 19 Generation of IL-6 inducing factors/activity by incubation of IgG and albumin with homogenised, washed tumour tissue.
  • a and B represent two separate experiments. As shown in these two experiments adding IgG and in particular albumin to tumour tissue markedly increases the production of IL-6 inducing factor/activity, which was tested in cultures of PBMCs from healthy controls.

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US20100323370A1 (en) 2010-12-23
CA2526950A1 (en) 2003-12-04
EP1507548A1 (de) 2005-02-23
US8182983B2 (en) 2012-05-22
EP1507548B1 (de) 2014-10-01
WO2003099312A8 (en) 2004-04-22
AU2003232872A8 (en) 2003-12-12
WO2003099312A1 (en) 2003-12-04
AU2003232872A1 (en) 2003-12-12

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