WO1992006117A1 - Inhibition de reponses immunes indesirees - Google Patents

Inhibition de reponses immunes indesirees Download PDF

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Publication number
WO1992006117A1
WO1992006117A1 PCT/US1991/007342 US9107342W WO9206117A1 WO 1992006117 A1 WO1992006117 A1 WO 1992006117A1 US 9107342 W US9107342 W US 9107342W WO 9206117 A1 WO9206117 A1 WO 9206117A1
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Prior art keywords
fragment
hybrid protein
diphtheria toxin
deleted
protein
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PCT/US1991/007342
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English (en)
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Richard Svrluga
John R. Murphy
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Seragen, Inc.
The University Hospital
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Publication of WO1992006117A1 publication Critical patent/WO1992006117A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention relates to the inhibition of unwanted immune responses, e.g., as occur in autoimmune diseases and in the rejection of allografts.
  • Autoimmune diseases are characterized by an immune response which is directed against constituents of a patient's own tissues and which damages those tissues. These disorders arise, at least in part, from the
  • T-cells play a central part in the propagation of an immune response.
  • Helper-inducer-T-cells specific for an antigen stimulate the appropriate B-cells to produce antibody and specific suppressor T-cells inhibit B-cells from
  • T-helper-cells The activity of T-helper-cells is dependent on interactions with monocytes, macrophages, B- cells, and other lymphocytes, Haynes et al. (1986), in Harrison's Principle of Internal Medicine, 11th Ed.
  • IL-2 derived lymphokine interleukin 2
  • T-lymphocytes The critical role of T-lymphocytes in the function of the immune system in autoimmune disease is suggested in experiments with the drug cyclosporine. Cyclosporine inhibits the proliferation of helper-inducer and
  • myasthenia gravis (induced by the injection of purified acetylcholine receptor) has been shown, in vitro, to decrease the severity of the induced autoimmune disease. Production of anti-acetylcholine receptor antibody, but not of unrelated antibodies, was inhibited when
  • lymphocytes from such rats were incubated with
  • Juvenile insulin-dependent diabetes is an autoimmune disease in which the immune system directs a response against the cells of the islets of Langerhans. These cells, which are located within the pancreas, are responsible for producing insulin. As a result of the autoimmune response the islet cells are impaired or destroyed, resulting in insulin deficiency.
  • Type 2 collagen is a structural protein found in the joints and in the
  • Rheumatoid arthritis is perhaps the most well studied autoimmune disease, in part because of the existence of two inducible animal models, collagen arthritis (CA) and adjuvant arthritis (AA).
  • CA collagen arthritis
  • AA adjuvant arthritis
  • mice 146:857, mice, Courtenay et al. (1980) Nature 283:666. or monkeys, Cathcart et al. (1986) Lab. Invest. 54:26, by an intradermal injection of native Type 2 collagen
  • AA which can only be established in rats, is induced by an injection of oil containing a preparation that possesses adjuvant activity, i.e., a preparation that contains heat killed and desiccated Mycobacterium tuberculosis.
  • T-lymphocytes play a prominent role in the progress of both CA and AA. In animals afflicted with CA the majority of the
  • T helper subset mononuclear cells infiltrating the synovium during the inceptual phase of arthritis belong to the T helper subset, based on their display of the W3/25 epitope, while T-non-helper-lymphocytes dominate in the later phases of the disease, Holmdahl et al. (1985) Scand. J. Immuno. 21:197. Furthermore, thymus deficient rats are refractory to the induction of CA, Klareskoy (1983) Clin. Exp. Immunol. 51:117, and CA can be prevented by
  • T-cells also play a dominant role in the progress of AA, as illustrated by the requirement of an intact thymus for the induction of the disease, Kohaski et al. (1981) Infect. Immun. 31:758, and by the suppressible nature of the disease by anti-lymphocyte antibodies, Currey et al. (1968) J. Exp. Med. 127:185.
  • T-cells A fundamental role for T-cells in the autoimmune response is also supported by observations that a T-cell response to Type 2 collagen can be demonstrated In vitro in many patients with rheumatoid arthritis, Trentham et al. (1978) New Eng. J. of Med. 299:327.
  • Allograft rejection is an immune response involving activated T-lymphocytes.
  • immunosuppressive protocols designed to inhibit rejection of allograft-tissue involve the administration of drugs such as azathioprine, cyclosporine, and corticosteroids, all of which cause toxic side-effects to non-lymphoid tissues.
  • drugs such as azathioprine, cyclosporine, and corticosteroids, all of which cause toxic side-effects to non-lymphoid tissues.
  • pan-T-lymphocyte monoclonal antibodies represents an important refinement in therapy, since only T-lymphocytes are targeted by the administration of such antibodies.
  • this therapy has the disadvantage of destroying, along with the T- lymphocytes involved in allograft rejection, those required for normal immune surveillance.
  • interleukin 2 interleukin 2
  • T-cells express cell- surface high-affinity IL-2 receptors. Targeted
  • the molecules and methods of the invention provide for the inhibition of an unwanted immune response.
  • the methods involve administering to a mammal an inhibiting amount of a hybrid protein including protein fragments joined together by covalent bonds, the protein fragments including: a fragment including a portion of IL-2, the portion including at least a portion of the binding domain of IL-2, the portion of the binding domain being effective to cause the hybrid protein to bind selectively to cells bearing the IL-2 receptor (examples of IL-2 deletions that are capable of binding to the IL-2 receptor are found in Genbauffe et al., U.S.S.N. 388,557, filed August 2, 1989, and hereby incorporated by reference); and, an enzymatically
  • diphtheria toxin which does not include a functional diphtheria toxin generalized
  • the hybrid protein is capable of stimulating the proliferation of peripheral blood mononuclear cells (PBMC) in vitro and of suppressing an immune response in vivo.
  • PBMC peripheral blood mononuclear cells
  • the fragment of the diphtheria toxin molecule has been enzymatically
  • a mutation inactivated by a mutation, preferably a mutation at position 53 of the diphtheria toxin.
  • diphtheria toxin which does not include a functional generalized eukaryotic binding site, and which when fused to IL-2 is capable of: 1) in vitro stimulation of PBMC; and 2) suppression of an immune response in vivo, can be used in the methods and
  • the inactive fragment includes residues 1-485 of diphtheria toxin, although not all of the amino acids upstream to residue 485 need be included.
  • domain 1 2 the hydrophobic transmembrane region;
  • Fragment A; Fragment B; or the protease sensitive domain is deleted.
  • at least 10%, more preferably at least 20%, and most preferably at least 30% of the amino acid residues between positions 1 and 485 of diphtheria toxin are deleted.
  • the invention includes a DNA sequence encoding the hybrid protein of the invention, an expression vector containing that DNA sequence, a cell transformed with that vector, and a method of producing the hybrid protein including culturing the cell and isolating the hybrid protein from the cultured cell or supernatant.
  • the methods of the invention also include: a method of inhibiting unwanted immune response in a mammal, including administering to the mammal an
  • inhibiting amount of the hybrid protein a method of inhibiting the T-lymphocyte- induced rejection of an allograft in a mammal, including administering to the mammal, following the allograft, and during the
  • a method of treating a patient having a disease e.g., an autoimmune disease, characterized by a
  • lymphocytes including lymphocytes
  • Enzymatically inactive diphtheria toxin means a diphtheria toxin molecule which has been modified, e.g., by mutation or by chemical modification, such that when present in the hybrid protein of the invention it no longer possesses sufficient ADP-ribosyl transferase activity to kill cells by virtue of ADP- ribosyl transferase activity.
  • In vitro stimulation of PMBC means an increase in any of: DNA synthesis; protein synthesis; or, of the secretion of a growth factor, e.g., a cytokine.
  • In vivo suppression of an immune response means any of: a decrease in delayed type
  • DTH hypersensitivity
  • Specific binding refers to the ability of a substance to bind virtually exclusively to a particular growth factor receptor, e.g., to the IL-2 has a specific affinity for the IL-2 receptor, it binds IL-2 receptor and not to other cell surface receptor proteins, e.g., insulin receptors.
  • Diphtheria toxin or native diphtheria toxin, as used herein, means the 535 amino acid residue mature form of diphtheria toxin protein secreted by Corynebacterium diphtheriae.
  • sequence of an allele of the gene which encodes native diphtheria toxin can be found in
  • Enzymatically active Fragment A means amino acid residues Gly 1 through Arg 193 of native DT, or an enzymatically active derivative or analog of the natural sequence.
  • Cleavage domain 1 1 means the protease sensitive domain within the region spanning Cys 186 and Cys 201 of native DT, Fragment B, as used herein, means the region from Ser 194 through Ser 535 of native DT.
  • the hydrophobic transmembrane region, or hydrophobic domain, of Fragment B means the amino acid sequence bearing a structural similarity to the bilayer-spanning helices of integral membrane proteins and located approximately at or derived from amino acid residue 346 through amino acid residue 371 of native diphtheria toxin.
  • Domain 1 2 as used herein, means the region spanning Cys 461 and Cys 471 of native DT.
  • the generalized eukaryotic binding site of Fragment B means a region within the C- terminal 50 amino acid residues of native DT responsible for binding DT to its native receptor on the surface of eukaryotic cells.
  • the generalized eukaryotic binding site of Fragment B is not included in the chimeric toxins of the invention.
  • the method of the invention inhibits an unwanted immune response as seen e.g., in autoimmune disease or in organ or tissue transplants, in a manner which does not cause general immune suppression, with its resulting risk of life-threatening infections.
  • the method spares donor-specific T suppressor cells, which can thus
  • molecules of the invention do not need to be tailored to individual patients; a single specific ligand-blocking agent can be used as a universal
  • therapy need not be continuous following allograft or following an acute stage of autoimmune diseases, but can be discontinued after a course of treatment.
  • Fig. 1 is a representation of the coding sequence, and the corresponding amino acid sequence, for the IL-2 gene portion of plasmid pDW15, following Sphl digestion Of pDW15.
  • Fig. 2 is a diagram illustrating the construction of plasmid pSI130, which contains DNA encoding the
  • Fig. 3 is a diagram of the structure of DT.
  • Fig. 4 is a graph of the effect of
  • Fig. 5 is a graph of the response of
  • mice treated with DA(197)B 486 -IL-2 treated mice to delayed hapten challenge.
  • Fig. 6 is a graph of the response of
  • mice treated with a hapten.
  • Fig. 7 is a graph of the response of
  • mice treated with a second hapten.
  • Fig. 8 is a graph of the effect of
  • diphtheria toxin molecule fused to IL-2.
  • the IL-2 gene used for these fusions was obtained from plasmid pDW15 (Williams et al. (1988) Nucleic Acids Res.
  • pDW15 contains a synthetic form of the IL-2 gene which, when cloned into E.coli JM101, expresses IL-2 protein at a rate about 16 times that of the native cDNA sequence cloned into the same strain of E.coli. DNA encoding
  • CRM197 was obtained from plasmids p ⁇ l97 and pABJ6508, as described below. The genetic fusion was made at the Sphl site of pDW15 so that the IL-2 domain of the fused gene would encode 133 amino acids of IL-2, plus one additional amino acid on its amino terminus encoded by the Sph site
  • Plasmid pABM6508, containing a gene coding for the N-terminal 485 amino acids of diphtheria toxin joined to ⁇ -melanocyte- stimulating hormone (Bishai et al. (1987) J.
  • Plasmid pDW15 was digested with Hindlll and with Sphl and a 0.5kB Sphl - Hindlll IL-2-gene containing fragment recovered.
  • the 6kB SpHI - Hindlll fragment from pAMB6508 was ligated to the 0.5 kB Sphl - Hindlll fragment from pDW15 to produce plasmid pABI6508.
  • pABI6508 was modified to include a laclq promoter, and a gene for canamycin resistance in place of a gene for ampicillin resistance, using standard techniques.
  • the modified pABI6508 was designated pSH00.
  • pS100 was digested with Accl and
  • Plasmid pßl97 (Bishai et al.) was digested with Accl and Xmnl and a 0.65kB fragment isolated.
  • pB197 carries the CRM197 mutant allele of DT, CRM197 is a full-length (535 amino acids) missense mutant (Gly 52 -> Glu 52 ) whose protein product is devoid of ADP-ribosyl transferase activity and is thus nontoxic (Uchida et al. (1973)
  • the missense mutation occurs within the 0.65kB Accl - Xmnl fragment of p ⁇ 197.
  • the 5.1kB Accl - Xmnl fragment from pS100 was ligated to the 0.65kB Accl - Xmnl fragment from pß197 to yield plasmid pSI130.
  • Expression of pSI130 in E.coli was induced as described by Bishai et al.; the CRM197/IL-2 gene product was purified using affinity chromatography (Williams et al.) followed by HPLC size exclusion chromatography.
  • Plasmid DNA was purified by the alkaline lysis/ cesium chloride gradient method of
  • DAB 486 -IL-2 is a chimeric toxin consisting of Met followed by amino acid residues 1 through 485 of mature DT fused to amino acid residues 2 through 133 of IL-2.
  • DAB 486 -IL-2 extends past the disulfide bridge linking Cys 461 with Cys 471. See Fig. 3 for the
  • the DT signal sequence was removed and fused to the sequence encoding mature DT which was fused to ATG using an oligonucleotide linker as described in Bishai et al. (1987) J. Bact. 169:5140-5151.
  • Selected methods and molecules of the invention provide therapy for autoimmune diseases, e.g.,
  • AA is a rat model of the human T-cell
  • AA appears as a subacute inflammation which progresses to a chronic polyarthritis. Although AA is triggered by an immune response to mycobacterial antigens, the disease is transferable to naive rats by injection of lymphoid cells from affected rats, Pearson (1964) J. Exp. Med.
  • AA is inducible in genetically susceptible rats (Lewis rats) by intradermal injection of Complete
  • CFA Freund's Adjuvant
  • Affected animals become progressively more disabled until approximately day 20 - day 25 post-immunization with CFA. Clinical symptoms gradually decrease to plateau at a level which is approximately 50% of the peak. They do not spontaneously remit. AA resembles rheumatoid
  • the progression of disease in the animals is shown in Fig. 4.
  • the severity of the disease was measured using a scoring system that assigned a score of zero to four for each of four paws.
  • the maximum index per animal is sixteen.
  • Each animal was scored by two individuals. The individual scores were averaged to give a final arthritis index for an animal.
  • a mean score per group is used to describe the condition of a group.
  • the TBS control animals were euthanized on day 18 because their arthritis had become so severe that it was very difficult for them to move to obtain food and water.
  • the DAB 486 -IL-2 and the DA(197)B 486 -IL-2 treated animals had only moderate disease. After the initial peak in mean arthritic score (day 20), the DAB486-IL-2 and
  • DA(197)B 486 -IL-2 treated groups displayed a mean score of between 2 and 5. On day 62 the DAB 486 -IL-2 and CRM197- IL2 treated groups continued to show similar clinical signs and similar scores.
  • DA(197)B 486 -IL-2 The ability of DA(197)B 486 -IL-2 to induce a state of immunologic unresponsiveness in vivo to a hapten introduced during DA(197)B 486 -IL-2 treatment was measured in a two phase delayed type hypersensitivity (DTH) response to TNBS. Concomitant with initial immunization with TNBS, mice were treated with either DA(197)B 486 -IL-2 or DAB 486 -IL-2, as described below.
  • DTH delayed type hypersensitivity
  • mice Treatment began on the day of priming with TNBS and continued through the sixth day. As shown in Fig. 5, untreated control mice (cross hatched bar) mounted a brisk DTH response to TNBS (39 + 6 U). Mice treated with DAB 486 -IL-2 (50 ug/d) (open bar) had a marked reduction (23% of positive control) in responsiveness to TNBS (9 + 2 U). Surprisingly, upon rechallenge of mice treated with DA(197)B 486 -IL-2 (50 ug/d) (checked bar), responsiveness to TNBS was suppressed to 54% (21 + 5 U), compared to untreated control.
  • DAB 486 -IL-2 (Seragen, Inc.) has been shown to be immunosuppressive in murine model of DTH using a single hapten (TNBS), Kelley et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3980-3984, hereby incorporated by reference.
  • DA(197)B 486 -IL-2 and DAB 486 -IL-2 were purified by the same method from cellular extracts of Escherichia coli. Both hybrid protein preparations were essentially free of contamination by endotoxins and were diluted in tris buffered saline (TBS), pH 7.9.
  • CRM197-I1-2 was not expected to suppress the DTH response, and therefore was intended to be a negative control molecule for DAB 486 -IL-2 (Bastos, M.G. et al., J. of Immunol, in press).
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • mice were primed with 10mM solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (ICN Pharmaceuticals, Inc., Cleveland, OH) in sterile PBS by subcutaneous injection, bilaterally into the dorsum.
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • mice were challenged with 30 ⁇ l of 10mM TNBS into the right footpad. Twenty-four hours after challenge, the thickness of both footpads was measured using a micrometer (Starrett, Athol, MA) .
  • Results were expressed in DTH units (U) defined as a difference of 0.001mm in thickness of injected and uninjected footpads. Mice received either no treatment, or daily subcutaneous injections of DA(197)B 486 -IL-2 or DAB 486 -IL-2, DTH responses were measured in a blinded fashion by individuals lacking knowledge of the treatment protocol in the test host.
  • mice that had been immunized with TNBS during DA(197)B 486 -IL-2 therapy were immunized with a second, non-crossreactive hapten, DNFB, after cessation of DA(197)B 486 -IL-2 therapy.
  • DNFB non-crossreactive hapten
  • DA(197)B 486 -IL-2 selectively targets antigen activated clones, then DA(197)B 486 -IL-2 treatment during the first phase response to TNBS should spare reactivity to DNFB during the second phase, since this hapten was introduced after cessation of DA(197)B 486 -IL-2 therapy.
  • mice were re- exposed to TNBS. Simultaneously, the mice were immunized with a second, non-cross-reactive hapten, 2,4-dinitro-1- fluorobenzene (DNFB) (Sigma Chemical Company, St. Louis, MO), by two consecutive daily abdominal paintings of 0.5% DNFB (25ul) diluted in a 4:1 acetone/olive oil mixture (vol:vol). Four days after the last abdominal painting, mice were re-exposed to 0.2% DNFB (20ul) diluted in acetone/olive oil applied to both ears. DTH response was determined as the difference in ear thickness as measured by micrometer before and 24 hours after ear painting. As shown in Fig. 6 responses to rechallenge by TNBS were much the same upon re-exposure as they had been
  • DA(197)B 486 -IL-2 like that seen in DAB 486 -IL-2-treated animals, was antigen-specific.
  • the cross hatched bar represents treatment with TBS (control)
  • the checked bar represents treatment with DA(197)B 486 -IL-2
  • the filled bar represents treatment with DAB 486 -IL- 2.
  • DAB 486 -IL-2 has been shown to significantly inhibit protein synthesis and proliferation in IL-2 receptor (IL-2R) bearing cells by virtue of its ability to catalyze the ADP-ribosylation of elongation factor 2, see Bacha et al. (1988) J. Exp. Med. 167:612. hereby incorporated by reference.
  • the gly-glu alteration in primary sequence at position 53 in DA(197)B 486 -IL-2 inactivates the ADP-ribosylating activity of the
  • PBMC peripheral blood mononuclear cells
  • DA(197)B 486 -IL-2 was capable of stimulating significant incorporation of [ 3 H]-thymidine in mitogen- activated PBMC.
  • Fig. 8 increasing doses of IL-2
  • DA(197)B 486 -IL-2 than IL-2 was required to haIf-maximally stimulate [ 3 H]-thymidine incorporation by the same cell population (IL-2 ED 50 ⁇ 3 X 10 -9 M).
  • PBMC peripheral blood of normal healthy volunteers by Ficoll-Hypaque density gradient centrifugation. For activation, cells were resuspended to 1 X 10 6 /ml in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 25mM HEPES, pH 7.4, 2mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml
  • the IL-2 receptor binding characteristics of DAB 486 -IL-2 and DA(197)B 486 -IL-2 were compared in a series of experiments using high affinity IL-2R bearing HUT 102/6TG cells and intermediate affinity IL-2R bearing YT2C2 cells.
  • the high affinity IL- 2R binding constant (K d ) of the DA(197)B 486 -IL-2 molecule was comparable to that determined for DAB 486 -IL-2;
  • DA(l97)B 486 -IL-2 displayed a K d for the intermediate affinity IL-2R on YT2C2 cells which again was comparable to that Of DAB 486 -IL-2, about 7 X 10 -7 M for CRM 197-IL-2 vs. 3.5 X 10 -7 M for DAB 486 -IL-2.
  • DAB 486 -IL-2, DA(197)B 486 -IL-2 and rIL-2 were iodinated using enzymobeads (BioRad, Richmond, CA) according to the instructions of the manufacturer. Six hundred to one thousand ⁇ Ci of [ 125 I] Na (Amersham,
  • the reaction was removed, diluted to 750 ⁇ l with RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 25mM HEPES, pH 7.4, 2mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 10% fetal calf serum (FCS, Gibco).
  • FCS fetal calf serum
  • the diluted reaction mixture was then applied to a 10ml Bio-Gel P-6 DG column (BioRad). Specific activities of [ 125 I] DAB 486 -IL-2, [ 125 I] rIL-2, and [ 125 I]
  • DA(197)B 486 -IL-2 were approximately 23-35 ⁇ Ci/ ⁇ g.
  • HUT 102/6TG or YT2C2 cells were harvested and washed three times with RPMI 1640 medium containing 25 mM HEPES, pH 7.4, 2mM L- glutamine, 100U/ml penicillin, 100 ⁇ g/ml streptomycin and 10% FCS. Cells were added at 5 X 10 6 - 14 X 10 6 /ml to [ 125 I]rIL-2 in the presence or absence of increasing concentrations of unlabeled rIL-2, DAB 486 -IL-2 or
  • DA(197)B 486 -IL-2 The cell suspension was then incubated for 30-120 min at 37° under 5% CO 2 .
  • cells were pretreated at 37°C for 60 min in phosphate buffered saline (PBS) containing 1 mg/ml bovine serum albumin (BSA) (Sigma, St. Louis, MO), 15 mM NaN 3 , and 50 mM 2-deoxy-D-glucose, pH 7.2, to inhibit internalization of radiolabeled ligand, see Cie ⁇ hanover et al. (1983) J. Biol. Chem. 258:9681. hereby incorporated by reference.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • DA(197)B 486 -IL-2 for extended periods of time.
  • DNA synthesis [ 3 H]-thymidine incorporation
  • protein synthesis [ H]-leucine incorporation were measured at 24 hour intervals for a period of 72-96h following the addition of DA(197)B 486 -IL-2 to the system.
  • stimulation indices [ 3 H]-thymidine incorporation) and protein synthesis.
  • ratio of cpm incorporated in treated vs. untreated cultures were typically > 1.00 (within experimental error) for DNA synthesis and protein synthesis.
  • the lack of a consistent downward trend in either stimulation index indicated no significant inhibition of either measured function.
  • HUT 102/6TG cells were treated with 10 -7 M or 10 -10 M CRM-197-IL-2 for 24h and DNA prepared for agarose gel electrophoresis and ethidium bromide staining. No obvious degradation of DNA was found in these preparations, indicating no evidence for a nuclease-like activity associated with the
  • the hybrid protein of the invention will be administered by intravenous infusion over a period of one to six hours, in a series, e.g., two to fifteen, more preferably five to ten infusions, given, e.g., once or twice daily or every two or three days, or in regular courses interrupted by periods of cessation of treatment.
  • a series e.g., two to fifteen, more preferably five to ten infusions, given, e.g., once or twice daily or every two or three days, or in regular courses interrupted by periods of cessation of treatment.
  • each dose preferably will be in the range of about 0.2-1.0 mg/kg. See Strom U.S.S.N. 772,893, filed September 5, 1985, hereby
  • treatment initiation can be delayed one or more days following the allograft, since therapy not only can prevent rejection, but can reverse it as well.

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Abstract

Protéine hybride comprenant des fragments de protéine joints par des liaisons covalentes, les fragments de protéine comprenant un fragment ayant une portion de IL-2, la portion comprenant au moins une partie du domaine de liaison de IL-2, la partie du domaine de liaison étant efficace pour provoquer la liaison de la protéine hybride de manière sélective sur des cellules portant le récepteur de IL-2; et un fragment inactif du point de vue enzymatique de la toxine de la diphtérie qui ne possède pas un site de liaison eukaryotique généralisé de la toxine de la diphtérie fonctionnelle. La protéine hybride peut stimuler la prolifération de PBMC in vitro et supprimer une réponse immune chez un mammifère $(in vivo).
PCT/US1991/007342 1990-09-28 1991-09-27 Inhibition de reponses immunes indesirees WO1992006117A1 (fr)

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EP0630383A1 (fr) * 1992-02-25 1994-12-28 Brigham And Women's Hospital Procedes de traitement des diabetes
EP0668774A4 (fr) * 1991-05-03 1995-04-25 Seragen Inc Molecules ciblees sur un recepteur de l'interleukine destinees a traiter l'arthrite inflammatoire.
US5621083A (en) * 1991-11-04 1997-04-15 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
EP0869176A1 (fr) * 1995-11-07 1998-10-07 Kaneka Corporation Autoantigenes
EP0871486A1 (fr) * 1995-11-30 1998-10-21 Regents Of The University Of Minnesota Epitopes de la toxine diphterique
US5837491A (en) * 1991-11-04 1998-11-17 Xoma Corporation Polynucleotides encoding gelonin sequences
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
US6165476A (en) * 1997-07-10 2000-12-26 Beth Israel Deaconess Medical Center Fusion proteins with an immunoglobulin hinge region linker
US6187564B1 (en) 1997-07-10 2001-02-13 Beth Israel Deaconess Medical Center DNA encoding erythropoietin multimers having modified 5′ and 3′ sequences and its use to prepare EPO therapeutics
US6242570B1 (en) 1997-07-10 2001-06-05 Beth Israel Deaconess Medical Center Production and use of recombinant protein multimers with increased biological activity
US6759385B1 (en) 1997-12-16 2004-07-06 Regents Of The University Of Minnesota Methods to treat undesirable immune responses
GB2451928A (en) * 2007-06-21 2009-02-18 Angelica Therapeutics Inc Modified diphtheria toxins
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation
US10059750B2 (en) 2013-03-15 2018-08-28 Angelica Therapeutics, Inc. Modified toxins

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EP0668774A1 (fr) * 1991-05-03 1995-08-30 Seragen, Inc. Molecules ciblees sur un recepteur de l'interleukine destinees a traiter l'arthrite inflammatoire
EP0668774A4 (fr) * 1991-05-03 1995-04-25 Seragen Inc Molecules ciblees sur un recepteur de l'interleukine destinees a traiter l'arthrite inflammatoire.
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
US5837491A (en) * 1991-11-04 1998-11-17 Xoma Corporation Polynucleotides encoding gelonin sequences
US5621083A (en) * 1991-11-04 1997-04-15 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US5744580A (en) * 1991-11-04 1998-04-28 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US5756699A (en) * 1991-11-04 1998-05-26 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US6146631A (en) * 1991-11-04 2000-11-14 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US6649742B1 (en) 1991-11-04 2003-11-18 Xoma Technology Ltd. Immunotoxins comprising ribosome-inactivating proteins
EP0630383A4 (fr) * 1992-02-25 1998-04-22 Brigham & Womens Hospital Procedes de traitement des diabetes.
EP0630383A1 (fr) * 1992-02-25 1994-12-28 Brigham And Women's Hospital Procedes de traitement des diabetes
EP0869176A1 (fr) * 1995-11-07 1998-10-07 Kaneka Corporation Autoantigenes
EP0869176A4 (fr) * 1995-11-07 2000-09-20 Kaneka Corp Autoantigenes
EP0871486A1 (fr) * 1995-11-30 1998-10-21 Regents Of The University Of Minnesota Epitopes de la toxine diphterique
EP0871486A4 (fr) * 1995-11-30 2000-08-09 Univ Minnesota Epitopes de la toxine diphterique
US6929796B1 (en) 1995-11-30 2005-08-16 Regents Of The University Of Minnesota Methods to treat undesirable immune responses
US6165476A (en) * 1997-07-10 2000-12-26 Beth Israel Deaconess Medical Center Fusion proteins with an immunoglobulin hinge region linker
US6242570B1 (en) 1997-07-10 2001-06-05 Beth Israel Deaconess Medical Center Production and use of recombinant protein multimers with increased biological activity
US6187564B1 (en) 1997-07-10 2001-02-13 Beth Israel Deaconess Medical Center DNA encoding erythropoietin multimers having modified 5′ and 3′ sequences and its use to prepare EPO therapeutics
US6759385B1 (en) 1997-12-16 2004-07-06 Regents Of The University Of Minnesota Methods to treat undesirable immune responses
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation
GB2451928A (en) * 2007-06-21 2009-02-18 Angelica Therapeutics Inc Modified diphtheria toxins
GB2451928B (en) * 2007-06-21 2011-03-16 Angelica Therapeutics Inc Modified Toxins
US8252897B2 (en) 2007-06-21 2012-08-28 Angelica Therapeutics, Inc. Modified toxins
US10059750B2 (en) 2013-03-15 2018-08-28 Angelica Therapeutics, Inc. Modified toxins

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