WO1998010056A1 - Traitement de cellules presentant l'antigene pour moduler la fonction de cellules presentant l'antigene - Google Patents

Traitement de cellules presentant l'antigene pour moduler la fonction de cellules presentant l'antigene Download PDF

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WO1998010056A1
WO1998010056A1 PCT/US1997/015431 US9715431W WO9810056A1 WO 1998010056 A1 WO1998010056 A1 WO 1998010056A1 US 9715431 W US9715431 W US 9715431W WO 9810056 A1 WO9810056 A1 WO 9810056A1
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antigen presenting
tgf
cells
cell
presenting cells
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Stephen P. Brooks
Thomas B. Tomasi
Zale P. Bernstein
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Health Research Inc.
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Priority to AU41764/97A priority Critical patent/AU4176497A/en
Priority to EP97939745A priority patent/EP0889954A1/fr
Publication of WO1998010056A1 publication Critical patent/WO1998010056A1/fr

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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    • 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
    • C07K2317/74—Inducing cell proliferation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]

Definitions

  • the present invention relates to a novel mechanism by which the ability of antigen presenting cells (APCs) to stimulate T-cell function is inhibited. More particularly, the present invention is directed to methods and compositions for modulating the immunostimulatory function of APCs by regulating, on the surface of APCs, the formation or presentation of immuno- suppressive complexes comprising the membrane-associated proteoglycan ⁇ glycan and cytokine TGF-3.
  • APCs antigen presenting cells
  • the present invention relates to antigen presenting cells and their membrane-associated ⁇ glycan; to pathological conditions in which the expression of transforming growth factor ⁇ (TGF- ⁇ ) appears to be increased; and to the discovery illustrated herein, that in certain pathological conditions, TGF-/? binds to Sglycan on APCs thereby forming immunosuppressive complexes which suppress the T-cell stimulatory function of the APCs.
  • TGF- ⁇ transforming growth factor ⁇
  • Antigen presenting cells represent three populations of myeloid cells that are generated from a common CD34+ bone marrow precursor.
  • the three populations include: dendritic cells, found in tissues such as skin (Langerhans' cells) , spleen, lymph node, and Peyer's patch, and in peripheral blood; monocytes found primarily in peripheral blood; and macrophages, resident in both tissues and peripheral blood.
  • APCs are specialized accessory cells having functions that include presenting antigen in T-dependent immune responses, sensitization, and tolerance. Dendritic cells have been shown to be the primary allo-stimulating and antigen presenting cell in peripheral blood, and accounts for more than 80% of the T-cell stimulatory function in the mixed lymphocyte reaction.
  • dendritic cells like macrophages, are effective in stimulating memory responses.
  • the mixed lymphocyte reaction is an in vi tro assay in which irradiated antigen presenting cells from an individual (stimulator) are incubated with T lymphocytes from a genetically disparate individual (allogenic responder) resulting in the proliferation of the responder lymphocytes.
  • a reduced (less than normal) activity observed in the mixed lymphocyte reaction may represent a defect in the antigen presenting function of the stimulator APCs or a defect in the ability of the responder T lymphocytes to proliferate.
  • T-cell proliferation/ stimulation is suppressed, and in which the allo- stimulatory accessory cell function may be inhibited.
  • these conditions include, but are not limited to inflammation, inflammatory diseases (e.g., inflammatory joint disease, Crohn's disease), graft versus host disease, cancer (e.g., Hodgkin's disease, sarcomas), and microbial infections (e.g., HIV infection) .
  • inflammatory diseases e.g., inflammatory joint disease, Crohn's disease
  • graft versus host disease e.g., Hodgkin's disease, sarcomas
  • microbial infections e.g., HIV infection
  • T-cell stimulatory function of APCs has been reported by some groups to accompany HIV infection and has been hypothesized to be one of the primary mechanism by which the virus induces the suppression of systemic immunity which defines AIDS.
  • the defect in APCS remains to be elucidated (Mactonia et al . , 1992, Jnununolog 75:576-81).
  • individuals with multiple sclerosis have T cells that proliferate as well as T cells from healthy individuals.
  • monocytes have T cells that proliferate as well as T cells from healthy individuals.
  • monocytes have T cells that proliferate as well as T cells from healthy individuals.
  • APCs APCs from multiple sclerosis patients during active disease exhibit almost a complete inability to stimulate responder cell proliferation in the allo- or auto-mixed lymphocyte reaction.
  • monocytes from multiple sclerosis patients during the inactive stage of the disease exhibited some, but less than the normal range of, ability to stimulate responder cell proliferation in vi tro .
  • This APC stimulatory defect appears to fluctuate in close association with the disease (Baxevanis et al . , 1987, Clin . exp . Immunol .
  • TGF- ⁇ ⁇ glycan formerly known as the TGF-0 type III receptor
  • TGF-01, TGF-/32, TGF-03 The mammalian family of transforming growth factor ⁇ (TGF-01, TGF-/32, TGF-03) has been associated with the suppression of a number of immune reactions and are known to play a role in the regulation of T-cell function.
  • TGF-/3 has been shown to inhibit the proliferation response of T- cells to various in vi tro activation signals (see, e.g., Ellingsworth et al .
  • TGF-01. and TGF- ⁇ receptors are also synthesized by T- cells in response to T-cell receptor mediated activation signals as part of a negative feedback mechanism that regulates T-cell proliferation.
  • TGF-3 has also been well characterized as cell cycle inhibitors, and it has been suggested that TGF-0 inhibition of in vi tro T-cell proliferation is the result of cell cycle arrest (Ellingsworth et al., 1988, supra) .
  • TGF-/? acts as a potent inhibitor of the growth and functions of lymphoid cells are not known (Ruscetti et al . , 1993,
  • TGF- ⁇ inhibition of the growth and function of lymphoid cells is associated with the T-cell surface receptor expression of, or activation via, growth factor receptors such as interleukin- 1 (IL-l) or IL-2 (Ruscetti et al., 1993, supra) .
  • IL-l interleukin- 1
  • IL-2 IL-2
  • TGF-3 mediated immunosuppression is believed to play a role in several pathological conditions. Tumors that actively secrete TGF- ⁇ can inhibit or suppress CD4+ helper T cell activity, wherein such suppression can be overcome by the addition of neutralizing antibodies to TGF-0 (Ruscetti et al . , 1993, supra). A similar selective loss of T helper cell activity, as observed during tumorigenesis, has also been observed in symptomatic HIV- infected individuals. Candidates proposed to be somehow involved in the suppression observed in HIV infection include IL-4, IL-10 and TGF-/? (Ruscetti et al . , 1993, supra) . Additionally, TGF-/?1 expression is increased in the active stage of Crohn's disease (Babyatsky et al . , 1996, Gastroenterolog 110:975-84) .
  • the TGF-/? receptor complex on cells is made up of three component: /?glycan, TGF-/? receptor I (T/?R I) , and TGF-/? receptor II (T/?R II) which combine to form the heterodimeric TGF-/? transmembrane signaling receptor.
  • TGF- ⁇ has been shown to bind directly to the T/?R II receptor subunit allowing this receptor/ligand dimer to complex with and phosphorylate the T/?R I subunit. The formation of this trinary complex can then transduce a signal via the serine/threonine kinase function of the cytoplasmic tails of the two receptor subunits .
  • /?glycan has a high affinity for binding all isotypes of TGF- ⁇ to its protein core, and is the most numerous TGF-/? binding molecule found on the surface of most cells that bind to TGF-/?, with a density as high as 2 to 5 x 10 s molecules per cell. It is noted, however, that many cell types that respond to TGF-/? (e.g., hematopoietic cells) do not appear to have detectable amounts of /?glycan (Kaname and Ruoslahti, 1996, supra) . Because of its high affinity for binding TGF-/? and its high expression on the cell surface, ⁇ glycan functions as the primary capture molecule for TGF-/?
  • TGF- ⁇ bound to the ⁇ glycan can be presented to the T0RII subunit resulting in the formation of /?glycan/TGF-/?/T/?R II signalling complexes and transcription from TGF- ⁇ responsive genes.
  • TGF-/? forms long-term stable associations with the glycan molecule and its binding to the proteoglycan does not result in a down regulation of /?glycan expression on the cell membrane (Massague, 1985, Cancer Cells, 3:73). This permits a cell to "express" /?glycan/TGF-0 on its surface for prolonged periods of time in a configuration that makes the TGF- ⁇ available to bind to and signal through the TGF-/? signaling receptor.
  • the foregoing objects are achieved by providing methods, involving the novel use of one or more compositions, to treat an individual having a pathological condition, in which during the active stage of the condition occurs TGF-? mediated suppression or inhibition of antigen presenting cell function.
  • the methods comprise one or more of the following therapies for restoring antigen presenting cell function: a means to remove TGF-/?:/?glycan complexes from the antigen presenting cells' surfaces; a means to remove ⁇ glycan from the antigen presenting cells' surfaces; administration of anti -TGF-/? antibody; administration of anti-?glycan antibody; administration of a combination of anti -TGF-/? antibody and anti-/?glycan antibody; and administration of one or more cytokines which function to stimulate proliferation or function of antigen presenting cells, thereby overcoming TGF-/? suppression of APC function.
  • FIG. 1 is a bar graph illustrating the level of allo- stimulatory function of antigen presenting cells at different time periods of incubation with TGF-/?.
  • FIG. 2 is a bar graph illustrating the level of allo- stimulatory function in AIDS patients at different time periods following infusion with cytokine granulocyte/monocyte colony stimulating factor (GM-CSF) .
  • GM-CSF cytokine granulocyte/monocyte colony stimulating factor
  • pharmaceutically acceptable carrier is used herein, for purposes of the specification and claims, to mean a medium for facilitating the in vivo administration of a therapeutic compound.
  • pharmaceutically acceptable carrier media are known to those skilled in the art to include buffered saline solutions, buffered carbohydrate solutions, liposomes (Phillips et al . , 1994, J. Immunother . Emphasis Tumor Immunol . 15:185-93), and the like.
  • antigen presenting cells is used herein, for purposes of the specification and claims, to mean accessory cells which function to stimulate CD4+ helper T cell activity, which are generated from a common CD34+ bone marrow progenitor and which express differentiated cell surface markers as known in the art (Szablocs et al . , 1996, Blood 87:4520-4530), including: dendritic cells found in tissues such as skin (Langerhans' cells), spleen, lymph node, and Peyer's patch, and in peripheral blood; monocytes; and macrophages .
  • TGF-/?” is used herein, for purposes of the specification and claims, to mean either or collectively all, of the three mammalian isotypes including TGF-01, TGF-/?2, and TGF-/?3, as all three isotypes have been shown to suppress the APC function of stimulating CD4+ helper T cell activity.
  • PBL peripheral blood leukocytes contain various leukocyte subpopulations including antigen presenting cells, but which typically in the experimental conditions, neutrophils have been depleted.
  • the present invention relates to the discovery of a novel mechanism by which the ability of antigen presenting cells to stimulate T-cell function is inhibited. More particularly, it is disclosed that in vitro, and in vivo in diseases where T-cell proliferation/stimulation is suppressed, antigen presenting cells contain immunosuppressive complexes comprising the membrane-associated proteoglycan ⁇ glycan and cytokine TGF- ⁇ .
  • TGF-/? suppression of APC function a uni-que mechanism for the suppression of T-cell stimulation.
  • the TGF-/? suppression is not dependent upon a trans -signalling event mediated through the TGF- ⁇ receptor of the APC, nor is it due to the production of a soluble suppressive factor.
  • the TGF-/? suppression of APC function can be reversed by mild trypsin digestion of the APC.
  • TGF-/? mediated suppression where the cytokine forms immunosuppressive complexes with the membrane associated proteoglycan, /?glycan.
  • compositions of the present invention are targeted to either (i) blocking or inhibiting the binding of TGF-/? to /?glycan on the cell surface of APCs (e.g., administration of anti-TGF-/? antibody, or of anti-/?glycan antibody, or of a combination of anti-TGF-/?
  • TGF-? receptor on T- cells e.g., by administration of anti-TGF-/? antibody, of anti-/?glycan antibody, or of a combination of anti-TGF-/? antibody and anti-/?glycan antibody
  • This example illustrates that (a) the /?glycan on APCs binds to TGF-/?; and (b) the APC function of stimulating CD4+ helper T cell activity is suppressed or inhibited by the binding of TGF- ⁇ to ⁇ glycan.
  • the mechanism of TGF-/? suppression of APC function was characterized by using two well defined in vi tro assays: (1) the allo- stimulatory function of the dendritic cell component of spleen cells was assayed in a one-way mixed lymphocyte response assay; and (2) analyzed was the presentation of soluble protein antigen by purified peritoneal macrophage to antigen sensitized lymph node T- cells.
  • Spleen cells from BALB/c mice were harvested and washed using methods known in the art.
  • the stimulator cells were spleen cells isolated from BALB/c (H-2 d ) mice, washed with phosphate-buffered saline, and irradiated at 3500 rads before being resuspended in complete cell culture media (RPMI 1640 with 10% FBS , penicillin/ streptomycin and 5 x 10 "5 M /3-mercaptoethanol) .
  • Responder cells were isolated lymph node cells from CBA (H-2 b ) mice that were washed with phosphate-buffered saline and resuspended in cell culture medium.
  • the proliferation assay was carried out in 96 -well plates with a final volume of 200 ⁇ l/well, and 1 x 10 5 responder cells per well. The assay was incubated for five days at 37°C and pulsed for the last 18 hours with [ 3 H] thymidine. Cells were harvested onto glass wool filters and the filters are counted in 2 ml of scintillation fluid using a tritium window (expressed in counts per minute or "cpm”) .
  • one group of wells contained no spleen cells, but contained the responder cells.
  • 2 x 10 5 irradiated cells were used as stimulator cells and mixed with the responder cells.
  • spleen cells were first pre- treated with TGF- ⁇ for various periods of time, ranging from 30 minutes to 48 hours. For incubations of 24 hours and 48 hrs, 2 x IO 7 spleen cells in 10 ml of cell culture medium were pre- treated with 2 ng/ml of TGF-/? at 37°C.
  • TGF-/? As shown in Table 1, when the stimulator spleen cells were incubated with TGF-/? for as short a period of time as 30 minutes, observed is a statistically significant (p ⁇ 0.003) suppression of the spleen's antigen presenting cell function of stimulating allotypic lymph node cells (T- cells) . Since it is known in the art that the majority of allo- stimulatory function in the mixed lymphocyte response assay is due to dendritic cells, it may be concluded from this data that exposure of dendritic cells to TGF- ⁇ can suppress their T-cell stimulatory function.
  • SI stimulation index
  • Stimulator Cells mean cpm (SI)
  • the stimulator cells were peritoneal macrophages isolated by adherence from BALB/c (H-2 d ) mice. The isolated peritoneal macrophages were then washed with phosphate-buffered saline being resuspended in complete cell culture medium.
  • CBA (H-2 b ) mice were sensitized by injection of ovalbumin (OVA) in adjuvant into the hind foot pads, using methods known in the art (Krinzman et al . , 1996, Am. J. Physiol . 271:1476-83; Hogaboam et al . , 1996, Gastroenterology 110:1456-66).
  • OVA ovalbumin
  • Responder cells were lymph node cells isolated from the ovalbumin- sensitized mice, that were then washed with phosphate-buffered saline and resuspended in cell culture medium.
  • the proliferation assay was carried out in 96 -well plates with a final volume of 200 ⁇ l/well, and 5 x 10 5 responder cells per well, with 25 ⁇ g ovalbumin per well, and with 8x IO 4 of peritoneal macrophages per well. It is noted that ovalbumin- sensitized T- cells require the addition of APCs to proliferate in response to OVA.
  • the assay was incubated for five days at 37°C and pulsed for the last 18 hours with 3 H thymidine.
  • TGF- ⁇ treated pM ⁇ + OVA responder cells
  • Stimulator Cells mean cpm (SI)
  • EXAMPLE 2 This example illustrates that (a) antigen presenting cells express /?glycan on their cell surface; (b) that TGF-/? can be enzymatically removed from the glycan on the cell surface of APCs; and (c) that removal of TGF-/? bound to /?glycan on the cell surface of APCs resulted in restoration of the APC function of stimulating CD4+ helper T cell activity.
  • mouse spleen cells were pre- treated with TGF-/?. Before adding the TGF-/?
  • TGF-/? treated, irradiated mouse spleen cells were washed once in serum free cell culture medium, and then suspended in a preheated trypsin/EDTA solution (proprietary, and commercially available from Grand Island Biologies Co. ; GIBCO) at a concentration of 2 x IO 7 cells in 10 ml.
  • the suspension was incubated at 37°C for 10 minutes.
  • the cells were then washed twice in culture medium to remove the trypsin, and then assayed for their allo-stimulatory function in an mixed lymphocyte response assay.
  • to responder lymph node cells (1 x 10 5 cells/well) were added either 10 5 spleen cells (positive control) ; 10 5 spleen cells which had been pre-treated with 5 ng/ml TGF-/? for 24 hours, or 10 5 spleen cells which had been pre- treated with 5 ng/ml TGF-/? for 24 hours and then incubated with trypsin/EDTA for 10 minutes.
  • Table 3 represent the mean of six wells; and the data is representative of six different experiments.
  • the trypsin treatment of TGF- ⁇ preincubated spleen cells resulted in the complete recovery of their suppressed allo-stimulatory function.
  • the spleen cells had an SI of 12.2 that was suppressed to an SI of 4.6 following TGF- ⁇ incubation.
  • the allo-stimulatory function of TGF-/? suppressed spleen cells increased to an SI of 14.9 after trypsinization.
  • trypsinization protocol was used to remove /?glycan from the cell membrane.
  • the digestion of cells with low concentrations of trypsin has been reported to selectively remove the /?glycan molecule (Cheifetz et al . , 1984, J. Biol . Chem. 263:16) from cell surfaces.
  • the selective nature of the trypsin/EDTA digestion is demonstrated by the fact that trypsinization of TGF-/? pre- treated accessory cells restores a T-cell stimulatory function that is dependent on the expression of numerous cell surface glycoproteins (MHC class II, B7-2, ICAM-l, CD40 and others) .
  • TGF- ⁇ mediated suppression of APC function does not involve a soluble suppressive factor
  • TGF- ⁇ mediated suppression of APC function does not require a trans-signalling event on the antigen presenting cell.
  • mouse spleen cells were prepared as stimulator cells for the mixed lymphocyte response assay.
  • the spleen cells are fixed in paraformaldehyde .
  • the BALB/c spleen cells were cultured overnight in cell culture medium with 50 ⁇ g/ml of lipopoly-saccharide (LPS) .
  • LPS lipopoly-saccharide
  • LPS is known by those skilled in the art to amplify the antigen presenting function of APCs.
  • the LPS treatment was intended to improve, for purposes of assaying by the mixed lymphocyte response, the detection of antigen presenting cell function displayed by fixed cells.
  • the washed LPS- stimulated spleen cells were fixed in a solution of 0.4% paraformaldehyde in phosphate buffered saline for 5 minutes at room temperature. The reaction was stopped by adding an equal volume of 0.2 M lysine. Fixed cells were assayed for viability by exclusion of trypan blue dye, and were determined to be effectively killed by this fixation technique. The fixed cells were then washed three times in cell culture medium. The control fixed spleen cells were not treated with TGF-/?.
  • the test group comprised fixed spleen cells that were incubated for 30 minutes with 10 ng of TGF- ⁇ as described above.
  • TGF-/? mediated suppression of APC function.
  • TGF-/? pre- reated spleen cells (5 x 10 5 cells incubated with 10 ng TGF-? for 30 minutes at 37°C) were mixed with untreated (not treated with TGF- ⁇ ) spleen cells as stimulators for responder lymph node cells (1 x 10 5 cells per well) .
  • the mixture (1 x 10* cells per well) of TGF-/? pre- treated spleen cells and untreated cells was compared to control spleen cells (1 x 10 5 cells per well) and TGF-/?
  • pre- treated spleen cells (1 x IO 5 cells per well) in the mixed lymphocyte response assay.
  • Table 5 data is representative of four experiments
  • the presence of the TGF-/? pre- reated spleen cells did not inhibit the allo-stimulatory function of the untreated spleen cells, as would be expected if no soluble suppressor factor is present.
  • conditioned media from TGF-/? treated spleen cells was mixed with untreated (control) spleen cells were used in a mixed lymphocyte response assay. There was no suppression of APC function observed when the conditioned media from the TGF-/? treated spleen cells was used with the control spleen cells as stimulators in this assay.
  • TGF-/? pre- treated adherent macrophages as antigen presenting cells and lymph node T- cells as responding cells in a mixed lymphocyte response assay
  • TGF-/? treated accessory cells induced cell cycle arrest in the T- cells.
  • TGF-/? mediated suppression of APC function neither involves a soluble suppressive factor, nor requires a trans -signalling event on the antigen presenting cell; nor suggests that the presence of TGF-/? pre- treated accessory cells is inducing irreversible anergy (e.g., the presence of T-cells which, following contact with TGF-/?
  • the results support a mechanism that relates to intercellular presentation between the antigen presenting cell the T-cell which is responsible for the suppression of allo-stimulatory function mediated by TGF-/? (e.g., the presentation of complexed TGF-/? on the surface of the APC to the TGF-/? receptor of the T-cell) .
  • EXAMPLE 4 This ex.ample illustrates that TGF-/? mediated suppression of APC allo-stimulatory function can be overcome by treatment with one or more agents that induce homeotypic aggregation or conjugation of the antigen presenting cells with T-cells. It is noted that T-cell activation requires the cognate interaction between accessory cells and T-cells to be maintained for upwards of 20 minutes (Vallitutti et al . , 1995, J. Exp . Med. 181:577-584), and is dependent upon the activation of LFA-1 on the T-cell to maintain such contact (Dus in et al., 1989, Nature 341:619-630).
  • irradiated PBL from AIDS patients were used as allo- stimulator cells, and purified T-cells from an HIV-seronegative, healthy individual were used as responder cells.
  • Phorbol myristyl acetate was added a concentration per well of either 0.1 ⁇ g/ml or 0.001 ⁇ g/ml to induce homeotypic aggregation (human T-cells will proliferate in response to PMA at concentrations of 1-10 ⁇ g/ml).
  • the T-cells proliferated in response to the low concentrations of PMA and in the presence of PBLs from AIDS patients.
  • PMA alone did not stimulate T-cell proliferation.
  • the results demonstrate that the addition of low concentrations of PMA, which fail to induce T-cell proliferation but can induce homeotypic aggregation, will overcome the suppressive effects of TGF-? on antigen presenting cells. It can be concluded from these results that an agent which functions to promote maintenance of the conjugation between the antigen presenting cell and T-cell can overcome the TGF-/? suppressive effects.
  • EXAMPLE 5 This example illustrates that treating APCs with one or more cytokines can overcome TGF- ⁇ mediated suppression of APC function.
  • mouse spleen cells were prepared as stimulator cells for the mixed lymphocyte response assay.
  • GM-CSF was tested for its ability, in a mixed lymphocyte response assay, to restore the suppressed allo-stimulatory function of TGF- ⁇ treated spleen cells. Irradiated spleen cells (1 x 10 7 ) in 0.5 ml were incubated with 10 ng TGF-/? for 30 minutes.
  • GM-CSF was analyzed to overcome the TGF/?-mediated suppression of antigen presentation of ovalbumin by purified peritoneal macrophages to ovalbumin- sensitized lymph node T-cells (2 x 10 5 nylon wool enriched LN cells/well from OVA immunized BALB/c mice) .
  • T-cells 2 x 10 5 nylon wool enriched LN cells/well from OVA immunized BALB/c mice.
  • one group of wells contained peritoneal macrophages and responder T-cells (without OVA; Table 7, "pM ⁇ - OVA").
  • a positive control comprised TGF-/? treated peritoneal macrophages and responder cells with OVA (Table 7, "pM ⁇ + OVA") .
  • PBL Peripheral blood leukocytes
  • His opaqueTM a separation media and centrifugation methods known in the art
  • the PBL were then incubated overnight on plastic culture dishes, and the non-adherent cells were further separated on a 13.5% metrizamide gradient by centrifugation for 10 minutes at 400 x g.
  • Low density cells were plated for 20 minutes at 22 °C on culture dishes coated with 100 ⁇ g/ml human IgG to remove Fc- receptor (FcR) positive cells.
  • the FcR negative cells were then incubated for 30 minutes at 4°C with mouse antibodies made against human CD2 , CD3 , CD16, and CD19 (commercially available) .
  • the cells were then washed in phosphate buffered saline, and then absorbed onto magnetic beads crosslinked with anti-mouse IgG (commercially available) to remove contaminating T- cells, NK cells, and B- cells.
  • the resultant cells were determined by morphology and phenotype (HLA-DR hl ) to be >95% dendritic cells. This enriched population of dendritic cells was adhered to alcian blue- coated coverslips, and fixed in 2% formalin.
  • the fixed cells were first blocked with rabbit IgG (control antibody) in 1 mg/ml bovine serum albumin, and then incubated with either fluorescein isothiocyanate (FITC) -conjugated rabbit anti-human TGF-/?1 antibody (O'Conner et al . , 1987, J. Biol . Chem. 262:14090-14099), or FITC- conjugated rabbit anti -human /?glycan antibody, or FITC- conjugated isotype control antibody; and then analyzed for immunofluorescence using confocal laser scanning microscopy.
  • FITC fluorescein isothiocyanate
  • dendritic cells isolated from an HIV seronegative individual did not have detectable TGF- ⁇ on their surface;
  • dendritic cells isolated from AIDS patients (greater than 12 patients tested) stained positive for membrane associated /?glycan Three dimensional image analysis confirmed that in the cells staining positive TGF- ⁇ l or for ⁇ glycan, the signal is associated with the cell membrane, and not the cytoplasm, of the dendritic cells.
  • EXAMPLE 7 This example illustrates that one cause of the loss of T-cell stimulatory function of antigen presenting cells (e.g., dendritic cells, monocytes, and macrophages) observed in AIDS patients is due to TGF-/? mediated immunosuppression. This is in contrast to some reports (Cameron et al . , 1992, Clin . Exp. Immunol . 88:226-236; Weissman et al . , 1995, Proc. Natl . Acad. Sci . USA 92:826-830) that APC enriched PBL from HIV+ patients retained their ability to stimulate allo- responsive T-cells.
  • antigen presenting cells e.g., dendritic cells, monocytes, and macrophages
  • antigen presenting cells express j ⁇ glycan on their cell surface; that /?glycan on APCs binds to TGF-/?; that APC function of stimulating CD4+ helper T cell activity is suppressed or inhibited by the binding of TGF- ⁇ to /?glycan; that TGF-/? :/?glycan complexes can be enzymatically removed from the cell surface of APCs; that removal of TGF-/?:/?glycan complexes from the cell surface of APCs resulted in restoration of the APC function of stimulating CD4+ helper T cell activity; that TGF-/?
  • TGF- ⁇ mediated suppression of APC function does not involve a soluble suppressive factor; that TGF- ⁇ mediated suppression of APC function does not require a trans -signalling event on the antigen presenting cell; that TGF-/? mediated suppression of APC allo-stimulatory function can be overcome by treatment with one or more agents that induce homeotypic aggregation or conjugation of the antigen presenting cells with T-cells; and that treating APCs with one or more cytokines can overcome TGF-/? mediated suppression of APC function.
  • the stimulator PBL were irradiated (3,500 rads) , and then added to a 96 -well plate at a concentration of 1 X 10 s cells/well in cell culture medium.
  • Responder lymphocytes from a control HIV-1 sero-negative volunteer, were added to the assay in cell culture medium at a concentration of 1 X 10 5 cells/well. The assay was then incubated at 37°C for 7 days.
  • Responder T-cell proliferation is pulsed for the last 18 hours with 3 H thy idine.
  • Cells were harvested onto glass wool filters and the filters are counted in 2 ml of scintillation fluid using a tritium window (expressed in cpm) .
  • the antigen presenting cell function of PBL isolated from AIDS patients was then compared to PBL isolated from healthy individuals for allo-stimulatory function in the one-way mixed lymphocyte response assay.
  • the stimulation index (SI) is the fold increase in proliferation of T-cells in the presence of allo- stimulator cells in the mixed lymphocyte response assay, and is calculated by dividing the 3 H- thymidine incorporation (cpm) of unsti ulated cells into the 3 H- thymidine incorporation (cpm) from stimulated cells.
  • the stimulator cells were then washed four times in cell culture medium, and then irradiated with 3500 rads , before addition (10 5 spleen cells per well) to the human lymphocyte responder cells in the mixed lymphocyte response assay.
  • the APC function of TGF-/? treated PBL was compared to that of untreated PBL from the same individual .
  • the results, illustrated in Fig. 1, indicate that when the stimulator PBL were incubated with TGF-? for as short a period of time as 30 minutes, observed is a statistically significant suppression of the PBL antigen presenting cell function of stimulating allotypic T- cells.
  • the mean Stimulation Index (SI) decreased from a pre- incubation level of 9.3 to 3.5 following a 30 minute exposure to TGF-/?.
  • SI Stimulation Index
  • Control antigen presenting cells maintained full allo-stimulatory function in the presence of PBL from AIDS.
  • conditioned media from in vitro cultured PBL derived from AIDS patients showed no suppression of T-cell prolifera ion in a mixed lymphocyte response assay that was stimulated by normal PBL. Based on these results, the defect in allo-stimulatory function of antigen presenting cells from AIDS patients appears not to be the result of in vi tro production of any soluble suppressive factors.
  • This example illustrates that (a) antigen presenting cells in human PBL express /?glycan on their cell surface; (b) that ⁇ glycan and the TGF-/?:/?glycan complex can be enzymatically removed from the cell surface of these APCs; and (c) that removal of TGF- /?:/?glycan complex on the cell surface of these APCs resulted in restoration of the APC function of stimulating CD4+ helper T cell activity.
  • TGF-/? mediated suppression of APC function in human PBL is associated with the presence of /?glycan:TGF-/?
  • a mild trypsin digestion was used to remove /?glycan:TGF-/?l complexes from the membranes of both TGF- ⁇ l pre- treated normal PBL and PBL from AIDS patients.
  • /?glycan has a single accessible trypsin cleavage site in the membrane proximal region of its extra- cellular domain and is extremely sensitive to tryptic digestion.
  • a mixed lymphocyte response assay was performed. Irradiated human PBL were incubated for 30 minutes in cell culture medium either in the absence (Table 8, "none") or the presence of 10 ng of TGF-/?
  • TGF-/? mediated suppression of APC function in human PBL is associated with the presence of /?glycan:TGF-/? complexes.
  • the role of TGF-/? in the suppression of antigen presenting cell function in advanced AIDS patients was confirmed herein by using antibodies which recognize epitopes on TGF-/?1 when it is complexed with glycan.
  • the antibodies function to neutralize the intercellular interaction of the APCs with the responder T-cells (e.g., inhibit the presentation of the complexed TGF- ⁇ on the APC to the TGF-/? receptor of T-cells) .
  • antibodies which recognize epitopes on the ⁇ glycan component of /?glycan/TGF-/? complexes also function to neutralize the intercellular interaction of the APCs with the responder T-cells.
  • Example 8 Using the methods illustrated in Example 8 herein, a mixed lymphocyte response assay was performed wherein PBL isolated from AIDs patients were compared for antigen presenting function without antibody treatment ("untreated”); in the presence of anti -TGF- /Si antibody (lO ⁇ l/ml included for the duration of the assay; R&D Systems) ; in the presence of control antibody (lO ⁇ l/ml of anti-human IgA included for the duration of the assay), or in the presence of rabbit anti-?glycan antiserum (lO ⁇ l/ml included for the duration of the assay).
  • the results are illustrated in Table 9, wherein the stimulation index (SI) is the fold increase in proliferation of T-cells in the presence of allo- stimulator cells in the mixed lymphocyte response assay expressed with the standard error of the mean ( ⁇ SEM) .
  • SI stimulation index
  • This example illustrates that treating APCs in human PBL with one or more cytokines can overcome TGF-/? mediated suppression of APC function.
  • dendritic cells in AIDS patients have the cytokine TGF-/? present on their cell membranes, thereby forming immunosuppressive complexes with the membrane proteoglycan ⁇ glycan.
  • the enzymatic removal of, or blocking by antibodies of, these complexes restores T-cell stimulatory function to these APCs, indicating that accessory cell function is not lost.
  • illustrated is the in vi tro effect of GM-CSF on the suppressed accessory cell function of PBL from AIDS patients.
  • PBL from AIDS patients were prepared as stimulator cells for assaying the in vi tro T-cell stimulatory function of APCs in a one-way mixed lymphocyte response assay. Briefly, the separated PBL were irradiated (3,500 rads) and then added to a 96-well plate at a concentration of 1 X 10 cell/well in cell culture medium. Responder peripheral blood leukocytes (PBL), from an HIV-1 volunteer, were added to the assay in CM at a concentration of 1 X 10 5 cells/well.
  • PBL peripheral blood leukocytes
  • the assay was incubated at 37°C for 7 days, with responder T-cell proliferation being assayed by overnight [3, thymidine incorporation, and quantitation of the counts per minute from harvested cells.
  • responder T-cell proliferation being assayed by overnight [3, thymidine incorporation, and quantitation of the counts per minute from harvested cells.
  • recombinant human GM- CSF rh GM-CSF
  • irradiated PBL from AIDS patients were preincubated at 37°C for 30 minutes in lOng/ml rh GM-CSF, and then washed three times with culture medium before inclusion to the mixed lymphocyte response assay as stimulator cells.
  • pre-treating PBL with GM-CSF for 30 minutes before adding the PBL into a mixed lymphocyte response assay resulted in a statistically significant (p ⁇ 0.001) restoration of T-cell stimulatory function to the antigen presenting function of PBL in 7 of 9 AIDS patient samples assayed.
  • Allo-stimulatory function increased from a mean SI of 1.6 for untreated PBL, to a mean SI of 4.7 for PBL pre- treated with rh GM-CSF.
  • EXAMPLE 11 This example further illustrates that treating APCs in human PBL with one or more cytokines can overcome TGF-/S mediated suppression of APC function.
  • GM-CSF restores T-cell stimulatory function of PBL, from AIDS patients, which had been previously suppressed by TGF-/?.
  • GM-CSF signals through a transmembrane heterodimeric receptor composed of a cytokine specific a- chain and a common signal transducing ⁇ - chain (Sato et al., 1993, EMBO J. 12:4181).
  • Interleukin -3 is another member of this family of cytokines which shares this common ⁇ - chain as a signal transducer.
  • This family of cytokines may be classified as a group of antigen presenting cell activating factors.
  • PBL from AIDS patients were prepared as stimulator cells for assaying the in vi tro T-cell stimulatory function of APCs in a one-way mixed lymphocyte response assay. Briefly, the separated PBL were irradiated (3,500 rads) and then added to a 96-well plate at a concentration of 1 X 10 5 cell/well in cell culture medium. Responder PBL, from an HIV-l negative volunteer, were added to the assay in CM at a concentration of 1 X 10 s cells/ well.
  • the assay was incubated at 37°C for 7 days, with responder T-cell proliferation being assayed by overnight ,3, thymidine incorporation, and quantitation of the counts per minute from harvested cells.
  • responder T-cell proliferation being assayed by overnight ,3, thymidine incorporation, and quantitation of the counts per minute from harvested cells.
  • recombinant human IL-3 rh IL-3 was added at a concentration of lOng/ml for the duration of the assay.
  • Examples 10 and 11 provide evidence that the family of cytokines which share a common beta subunit for their receptors (e.g., GM-CSF, or IL-3) will restore in vi tro significant T-cell stimulatory function to PBL from AIDS patients.
  • cytokines which share a common beta subunit for their receptors (e.g., GM-CSF, or IL-3) will restore in vi tro significant T-cell stimulatory function to PBL from AIDS patients.
  • PBL from AIDS patients were tested as stimulator cells for assaying the in vi tro T-cell stimulatory function of APCs in a one-way mixed lymphocyte response assay, in the presence of or in the absence of PIXY321 (lOng/ml) for the duration of the assay.
  • PIXY321 is an IL- 3/GM-CSF fusion protein which demonstrates enhanced biological activity over GM-CSF or IL-3 alone or in combination (Mignacca et al . , 1996, Blood 88:848-854; Gillis, 1991, Breast Cancer Res . Treat . 20Suppl: S3-9); and which in clinical phasel/II studies has been shown to increase the number of circulating hematopoietic progenitors and differentiated hematopoietic cells (Gheilmini et al . , 1996, Sr. J. Haemtol . 93:6-12). PIXY321 is also a potent stimulator of T-cell proliferation.
  • PIXY321 may serve the dual role of restoring antigen presenting cell function and stimulating CD4+ T-cell regeneration.
  • antigen presenting cell activating factors other than the cytokines described above
  • the results indicate antigen presenting cell activating factors can restore in vi tro antigen presenting cell function of PBL from AIDS patients or other patients exhibiting TGF-/? mediated suppression of allo-stimulatory function of
  • antigen presenting cell activating factors may include, but are not limited to, CD40 ligand, fit- 3, B-7 family of cross-linking reagents, ICAM- l/ICAM-2 crosslinking reagents, MHC cross -linking reagents, and human monocyte chemotactic proteins (MCP) -2 and -3. Additionally, it is known that tumor necrosis factor alpha (TNF- ⁇ ) strongly potentiates the biological effects of GM-CSF and IL-3 (Caux et al . , 1996, Blood 87:2376-85).
  • TNF- ⁇ tumor necrosis factor alpha
  • therapeutically effective amounts of combinations of TNF- ⁇ and GM-CSF; TNF- ⁇ and IL-3; TNF- ⁇ , GM-CSF, and IL-3; and TNF- ⁇ and PIXY321, may be used to effect in vi tro or in vivo restoration of the allo- stimulatory function of PBL from AIDS patients or other patients exhibiting TGF-/? mediated suppression of allo- stimulatory function of APCs.
  • EXAMPLE 12 This example illustrates that treating APCs in human PBL with one or more cytokines can overcome TGF-/? mediated suppression of APC function in vivo .
  • In vivo administration of such cytokines, or other antigen presenting cell activating factors may effect significant restoration of T-cell stimulatory function to antigen presenting cells from most AIDS patients; and may be used as a component of comprehensive multi -modal treatment for AIDS patients (or other patients exhibiting TGF-/3 mediated suppression of allo- stimulatory function of APCs) designed to restore or improve immune function.
  • TGF- ⁇ complex formation
  • a group of twelve AIDS patients were infused intravenously with 125 ⁇ g/m 3 of recombinant human GM-CSF over a two hour period.
  • This regiment of GM-CSF treatment has been shown to produce serum levels of GM-CSF as high as lOng/ml.
  • patient volunteers had to conform to clinical guidelines that include 1) having CDC stage IV disease, 2) not receiving any other immunomodulating therapy at the time of trial, and 3) otherwise displaying reasonable good health.
  • the allo-stimulatory function of peripheral blood mononuclear cells was assayed in a mixed lymphocyte response assay before, and at various time points after, the completion of the single bolus infusion.
  • Venous blood was drawn from the contralateral arm 1 hour, 5 hours and 24 hours after cytokine treatment.
  • the allo- stimulator function of each patient was assayed against the PBL from a healthy individual (selected as being the best responder out of a group of four responders assayed) .
  • the pre-infusion proliferative response was normalized to l-unit of response and the mean proliferation of the post infusion time points were represented as fold increase compared to pre- infusion proliferation.
  • antigen presenting cells (a) must be able to recover T-cell stimulatory function after only a limited exposure to GM-CSF, and (b) appear to maintain that function after the cytokine exceeds its serum half -life.
  • This example illustrates a first embodiment of the method according to the present invention.
  • 3 complex formation comprising treating the APCs by the steps of:
  • a composition selected from the group consisting of a means to remove /?-glycan or /Sglycan complexed to TGF-/? from the cell surface of the APCs, a means to prevent presentation of complexed TGF- ⁇ (complexed to / Sglycan) , to TGF-/? receptors on T-cells, and one or more antigen presenting cell activating factors which function as co- stimulatory molecules on APCs, thereby resulting in overcoming the suppression of the T-cell stimulatory function of APCs.
  • a composition selected from the group consisting of a means to remove /?-glycan or /Sglycan complexed to TGF-/? from the cell surface of the APCs, a means to prevent presentation of complexed TGF- ⁇ (complexed to / Sglycan) , to TGF-/? receptors on T-cells, and one or more antigen presenting cell activating factors which function as co- stimulatory molecules on APC
  • This method may further comprise infusion (e.g., intravenously) of the treated APCs into back into the individual, wherein the treated cells are expected to migrate back into various body tissues (e.g., blood, spleen, lymph nodes, etc.).
  • the means to remove /?-glycan or /?glycan complexed to TGF-/? from the cell surface of the APCs involve contacting the cells with a trypsin/EDTA solution.
  • the means to prevent presentation of complexed TGF-/? to TGF- ⁇ receptors on T-cells include the use of anti-TGF-/? antibodies or anti-/?glycan antibodies or a combination thereof.
  • the one or more antigen presenting cell activating factors may include, but are not limited to, GM-CSF, IL-3, PIXY321, CD40 ligand, flt-3, B-7 family of cross-linking reagents, ICAM-l/ICAM-2 crosslinking reagents, MHC cross- linking reagents, MCP-2, MCP-3, TNF- a and GM-CSF, TNF-o. and IL-3, TNF- ⁇ ? and PIXY321, and a combination thereof.
  • the relative ratio and total amounts of the composition can be determined by techniques known to those skilled in the art, including the mixed lymphocyte response assay described in detail herein.
  • Isolating from an individual a population enriched in antigen presenting cells may be accomplished by any one of several techniques known in the art. As described in detail in Example 6 herein, and as further described previously (Macatonia et al . , 1992, Immunol . 75:576-581) peripheral blood leukocytes (PBL) can be separated from peripheral blood drawn from an individual. The PBL can be incubated overnight on plastic culture dishes, resulting in adherent cells and non-adherent cells. The adherent cells may be removed by gentle scraping, and represent a population of macrophages .
  • PBL peripheral blood leukocytes
  • the nonadherent cells are collected and then separated by gradient centrifugation, and low density cells are panned to remove FcR + macrophages, and antibody labelled non antigen presenting cells. Resultant cells may then be analyzed for morphology and phenotype (HLA-DR 1 ") to determine enrichment of a dendritic cell population. Similar panning techniques may be used for the enrichment of Langerhans cells (Koch et al., 1992, J. Invest . Derma tol . 99:803-807).
  • Another embodiment of the method according to the present invention is a method for restoring T-cell stimulatory function of APCs in an individual having such function suppressed by /?glycan-TGF-/? complex formation, wherein the method comprises treating the APCs in vivo by administering to the individual a therapeutically effective amount of a composition comprising one or more antigen presenting cell activating factors which function as co- stimulatory molecules on APCs, thereby resulting in overcoming the suppression of the T-cell stimulatory function of APCs.
  • the composition may comprise a therapeutically effective amount of antibody selected from the group consisting of an anti-TGF-/? antibody, an anti-/?-glycan antibody, or a combination thereof.
  • the antibody functions to inhibit (blocking) the presentation of the complexed TGF-/? on the surface of the APC to the TGF-/? receptor present on the T-cell, and/or may function to prevent TGF-/3 :/?glycan complexes from forming.
  • the composition may be given to the individual in any one of several routes of administration known in the art; however, a preferred route of administration is infusion intravenously.
  • the relative ratio and total amounts of the composition may be determined by a physician considering such factors as the age, weight, health, and possibly other clinical factors, of the individual.
  • the composition may further comprise a pharmaceutically acceptable carrier medium known in the art for use in facilitating the administration of therapeutic compositions (e.g., saline solution, parenterally acceptable carbohydrate or protein solutions, liposomes, etc.).
  • the one or more antigen presenting cell activating factors may include, but are not limited to, GM-CSF, IL-3, PIXY321, CD40 ligand, flt-3, B-7 family of cross-linking reagents, ICAM-l/ICAM-2 crosslinking reagents, MHC cross- linking reagents, MCP-2, MCP-3, TNF-o. and GM-CSF, TNF- ⁇ and IL- 3, TNF- ⁇ and PIXY321, and a combination thereof.

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Abstract

L'invention concerne un nouveau mécanisme grâce auquel la capacité de cellules présentant l'antigène à stimuler la fonction de lymphocytes T est inhibée par la formation de complexes immunodépresseurs comprenant le βglycane associée à la membrane cellulaire présentant l'antigène et la cytokine TGF-β. L'invention concerne également des procédés permettant de restaurer la fonction de stimulation des lymphocytes T de cellules présentant l'antigène d'un individu, une telle fonction étant supprimée par la formation de complexe βglycane-TGF-β; ces procédés consistent soit à enlever le TGF-β de la surface cellulaire des cellules présentant l'antigène, soit à enlever le β-glycane ou le β-glycane formant un complexe avec TGF-β de la surface cellulaire des cellules présentant l'antigène, soit à mettre les cellules présentant l'antigène en contact avec un ou plusieurs facteurs activant les cellules présentant l'antigène qui résistent à la suppression de la fonction de stimulation des lymphocytes T de cellules présentant l'antigène.
PCT/US1997/015431 1996-09-03 1997-09-02 Traitement de cellules presentant l'antigene pour moduler la fonction de cellules presentant l'antigene WO1998010056A1 (fr)

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