WO1998045430A1 - Modulation de l'immunite a l'aide de polypeptides apparentes au cr1 - Google Patents

Modulation de l'immunite a l'aide de polypeptides apparentes au cr1 Download PDF

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Publication number
WO1998045430A1
WO1998045430A1 PCT/GB1998/001012 GB9801012W WO9845430A1 WO 1998045430 A1 WO1998045430 A1 WO 1998045430A1 GB 9801012 W GB9801012 W GB 9801012W WO 9845430 A1 WO9845430 A1 WO 9845430A1
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scr1
soluble
cell
polypeptide
cells
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PCT/GB1998/001012
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English (en)
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Yuti Chernajovsky
Alex Annenkov
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Mathilda And Terence Kennedy Institute Of Rheumatology
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Priority to AU69281/98A priority Critical patent/AU6928198A/en
Publication of WO1998045430A1 publication Critical patent/WO1998045430A1/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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • This invention concerns previously undescribed effects and uses of polypeptides related to the human complement receptor CR1 , i.e. to the human C3b/C4b receptor; and to new polypeptides related to CR1 .
  • the invention further concerns their production in-vivo, ex-vivo and in-vitro; compositions containing and/or encoding them, uses of these, and compositions and methods related to those uses, particularly e.g. nucleic acid sequences encoding said polypeptides as DNA or RNA, recombinant nucleic acid vectors containing such sequences, and cells expressing said polypeptides.
  • Human complement receptor 1 is known as a protein expressed by certain cells of the immune system and certain other blood ceils, including B cells, a subpopulation of T cells also expressing CD4 and FC-gamma, follicular dendritic cells, neutrophils, macrophages, and erythrocytes.
  • CR1 is known to serve inter alia as a membrane-bound protease cofactor that enhances cleavage of complement component C3b.
  • the complement system has two distinct functions: the induction of an inflammatory response and the enhancement of the primary immune response.
  • the inflammation process is mediated via products of cleavage of C3 and C5 that cause vascular permeability, leukocyte adhesion and migration and direct membrane damage.
  • CR1 (CD35) is expressed in B cells, a subpopulation of T ceils ( 1 ) expressing CD4 and FC gamma (FC ) (2), follicular dendritic cells, neutrophils, macrophages and erythrocytes.
  • the immune enhancement function is mediated via the interaction of C3dg bound to antigen with CR2 (CD21 ), sig, TAPA- 1 and CD 1 9 on B lymphocytes (3, 4) .
  • CR1 serves as a membrane bound protease cofactor that enhances the cleavage of C3b into iC3b and C3dg (5) .
  • CR1 is also involved in the endocytosis of complexes and particles by neutrophils and macrophages, erythrocyte CR1 serves as a carrier for immune complexes (6) .
  • the role of complement in autoimmune diseases has been demonstrated in animal models. For example, collagen-induced arthritis and lupus-like autoimmune disease is inhibited by anti-C5 antibodies (7) (8) .
  • Collagen- induced arthritis fails to develop in T cell receptor transgenic animals lacking C5 (9) . Depletion of C3 with cobra venom ameliorates arthritis (10) and passive transfer of anti-collagen antibodies can cause arthritis in rats (1 1 ).
  • complement function such as factor H, C4bp, DAF, MCP and sCR1 . From all these only sCR1 is capable, at the nM range, to block both the alternative and classical pathways of complement activation by its function as cofactor dissociating both the C3 and C5 convertases ( 1 2) (1 3).
  • sCR1 has been used therapeutically to inhibit acute inflammation, and tissue injury in myocardial reperfusion injury ( 14), reversed passive Arthus reaction in rat skin (1 5), reperfusion injury/ischemia of skeletal muscle (1 6), burns ( 1 7) and transplantation ( 18).
  • CR1 is also disclosed as able to inhibit the classical and alternative pathway C3/C5 convertases, i.e.
  • WO 91 /05047 Uses described in WO 91 /05047 include treatment of patients with CR1 fragments, the patients having immune disorder or disorders involving undesirable or inappropriate complement activity, i.e. to inhibit complement activity.
  • WO 91 /05047 discloses the sequence of CR1 , indicates its presumed transmembrane segment, and discloses certain soluble fragments of CR1 .
  • the present invention is a first invention.
  • the invention described herein provides further uses and derivatives of sCR1 , and arises from the finding of previously undescribed activity in CR1 and its fragments, and from the identification of fragments with greater potency than previously described in relation to previously-described activities of CR1 and/or its fragments.
  • sCR1 produced by autologous cells and delivered constitutively by gene therapy is capable of inhibiting both B and T cell function in the context of an autoimmune disease model.
  • the present inventors' results point to a mechanism of action of sCR1 that was unpredicted from its known biochemical functions and indicate additional immune-modulatory functions of this molecule.
  • soluble polypeptide which has a binding functionality of soluble complement receptor 1 (sCR1 ) towards C4b and C3b, has activity and can be used in the inhibition of a T-cell-mediated or a B-cell- mediated immune response.
  • sCR1 soluble complement receptor 1
  • the activity of soluble CR1 fragments can usefully be assessed and measured by methods chosen from among, for example, inhibition of antigen-driven T-cell proliferative responses, of interferon-gamma secretion, and/or of antibody synthesis in the collagen-induced arthritis model; also usable is the reverse passive Arthus reaction in rat skin, analogous to an effect of full-length sCR1 .
  • a particular useful and potent example of a sCR1 fragment is one that has a binding functionality corresponding to that of a fragment of sCR1 comprising the N-terminal C4b and C3b binding sites; e.g. one that comprises a 95kD fragment of sCR 1 comprising the N-terminal C4b and C3b binding sites and truncated on the C-terminal side of the N-terminal C3b binding site.
  • the complete sequence of a useful example of such a fragment is given as Figure 1 of the appended drawings.
  • Such proteins and fragments can be made by expression of corresponding polynucleotides in a variety of cell types using per-se known expression systems, preferably in mammalian cells such as CHO cells or COS cells.
  • the polypeptides can be expressed using a per-se known viral-vector based expression system such as a baculovirus expression system. It can be preferable to express the polypeptides in cells which are of the same species as the subject to be treated, or a closely related species, e.g. in homologous or, where accessible, syngeneic cells; otherwise in primate cells for sCR1 to be used in human treatment. In such a way the glycosylation pattern of the expressed protein can be made usefully similar to the glycosylation pattern normally occurring in CR1 of the subject to be treated.
  • the expressed proteins can be purified by a variety of techniques such as for example the techniques described in WO 91 /05047 and/or USP 5 252 216.
  • Such a polypeptide can be used by administering the polypeptide (directly or by an expression vector) to a subject with autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, epidermis bullosa, immune destruction of pancreatic beta-cells leading to insulin-dependent diabetes, Hashimoto's disease; e.g. by administering the polypeptide to the locality of a site associated with inflammatory response in said autoimmune disease.
  • autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, epidermis bullosa, immune destruction of pancreatic beta-cells leading to insulin-dependent diabetes, Hashimoto's disease
  • Such administration can also be useful in some diseases which are not normally classified as autoimmune diseases, but where tissue destruction and atrophy occurs, such as Duchenne muscular dystrophy,
  • Alzheimer's disease or Parkinson's disease.
  • a method according to an example of the invention of inhibiting a T-cell-mediated or B-cell-mediated autoimmune inflammatory response, e.g. a response associated with rheumatoid arthritis, can comprise: directly or indirectly administering a soluble polypeptide which has a binding functionality of soluble complement receptor 1 (sCR1 ) towards C4b and C3b either systemically or to the locality of a tissue, e.g. a synovial joint at risk of such an inflammatory response, thereby inhibiting such an inflammatory response in the locality of such a tissue.
  • sCR1 soluble complement receptor 1
  • Such a method can for example include causing cellular secretion, in the locality of a tissue e.g. a synovial joint at risk of such an inflammatory response, of soluble complement receptor 1 (sCR1 ) or of a soluble polypeptide which has a binding functionality of soluble complement receptor 1 (sCR1 ) towards C4b and C3b, thereby inhibiting such an inflammatory response in the locality of such a tissue.
  • a tissue e.g. a synovial joint at risk of such an inflammatory response
  • soluble complement receptor 1 soluble complement receptor 1
  • sCR1 soluble polypeptide which has a binding functionality of soluble complement receptor 1 (sCR1 ) towards C4b and C3b
  • sCR1 proteins or protein fragments, or vectors encoding them can be directly injected into a suitable tissue in vivo of a subject to be treated, e.g. into muscle, brain, joints, pancreas, or into a transplanted tissue.
  • a suitable tissue in vivo of a subject to be treated e.g. into muscle, brain, joints, pancreas, or into a transplanted tissue.
  • the dose can be up to 100 mg per human adult to be treated; e.g. in divided doses; each dose can be for example from about 1 ng up to about 20 mg, e.g. of a protein concentrate in about 0.5 ml.
  • the dose can be calculated so as to produce a concentration in the treated tissue in the range about 0.1 pM to 100micro-M, e.g. up to about 100 nM or about 1 micro-M.
  • the dose can be suitably chosen in the range about 3ng to about 30micro-g per injection site.
  • Vectors encoding sCR1 or its fragments can be used according to examples of the invention for example by infecting or transfecting with the vectors, ex-vivo, cells which are autologous or syngeneic with the subject to be treated, or which are homologous and with similar MHC type, such as spleen cells or fibroblasts; and administering the treated cells to a suitable site in the subject to be treated.
  • the treated cells can be given at the site of a lesion or of a transplanted tissue which is to be treated.
  • autoimmune disease it can be useful to give cells such as syngeneic fibroblasts intraperitoneally after treatment of the cells to induce them to express sCR1 or its fragment, so as to generate systemic levels of the sCR1 or fragment chosen for example from one of the ranges mentioned above and sufficient to depress or abolish an autoimmune effect such as acute autoimmune inflammation or transplantation rejection.
  • Vectors according to examples of the invention can encode in addition another heterologous polypeptide intended to be expressed in vivo in a treated subject.
  • another polypeptide can for examples be chosen from among genes used or proposed to be used in (corrective) gene therapy (e.g.
  • ADA human adenosine deaminase
  • WO 92/1 0564 KW Culver et al: US Secretary for Commerce & Celico Inc
  • WO 89/1 21 09 & EP 0 420 91 1 IH Pastan et al
  • cystic fibrosis gene and variants described in WO 91 /02796 L-C Tsui et al: HSC Research & University of Michigan
  • WO 92/05273 FS Collins & JM Wilson: University of Michigan
  • WO 94/1 2649 RJ Gregory et al: Genzyme Corp
  • the sCR1 activity especially for example when expressed by the same cell which has been treated to express a heterologous protein for purposes of gene therapy, can provide a shelter for the expressed therapeutic polypeptide and lengthen the duration of expression of this in vivo where it would otherwise be limited by immune destruction.
  • fragments of CR1 as described herein can have greater potency than was described in connection with the full length soluble CR1 and its fragments in the complement-related activity, including inhibition of complement activation by both the classical and alternative pathways and the uses related thereto.
  • Suitability of fragments can be assessed and measured by the effect of the fragments in inhibition of complement cascade, e.g. by ability in vitro to inhibit neutrophil oxidative burst, complement mediated haemolysis, or C3a and C5a production.
  • Also provided by the invention are recombinant cells containing sequences of sCR1 , and uses of these, especially uses related to the finding that sCR1 can modulate T-cell mediated and B-cell mediated effects.
  • the forms of sCR1 which can be used according to examples of the invention include sub-sequences of sCR1 in which domain LHR-D is missing, e.g. subsequences of approximately the order of 95kDa in size, and fusion proteins of sub-sequences of sCR1 with other proteins, e.g. with an IgG heavy chain or constant domain(s) thereof.
  • Figure 1 shows a (deduced) protein sequence of a 95 kDa sCR1 (subsequence) made and used according to embodiments of the invention.
  • the regions spanning all of LHR-A and part of LHR-B are respectively indicated on the right.
  • the origin of each arrow indicates the beginning of each SCR.
  • the last four amino acids correspond to the open reading frame of the polylinker region of pBluescript before the artificial stop codon was introduced (as described elsewhere herein).
  • Figure 2 shows detection of 95 kDa sCR1 in supernatants of GP + E86 cells by mAb YZ-1 .
  • Serum free supernatants of GP + E86 clones selected in G418 were concentrated by precipitation and analyzed by western blot. Positions of molecular weight markers are shown on the left.
  • Slots 1 -3 relate to sCRI -MFG transfected cell clones, Slot 4 relates to untransfected control.
  • Figure 3 illustrates arthritis prevention by gene therapy with SCR1 .
  • Panels A and B show inhibition of arthritis development by arthritogenic splenocytes expressing sCR1 .
  • Panels C and D show inhibition of arthritis development by immortalized syngeneic fibroblasts expressing sCR1 .
  • Number of mice per group (n 10). Values shown are mean +_ SEM and were statistically significant (p ⁇ 0.05) from the twentieth day onwards as analysed by the Mann-Whitney non-parametric test.
  • Figure 4 illustrates inhibition of T cell proliferation to collagen type II in mice treated with SCR1 .
  • Figure 5 illustrates inhibition of IFN gamma secretion by T cells upon collagen type II presentation to lymphnode cells of mice treated with sCR1 .
  • IFN gamma levels (mean ⁇ _SO) were measured by BUSA in triplicates.
  • Figure 6 illustrates prevention of weight loss in mice treated with syngeneic fibroblasts expressing sCR1 .
  • Figure 7 illustrates protection from arthritis by fibroblast expressing sCR1 .
  • Panel A shows an arthritic paw showing fibrin deposition, empty chondrocyte lacunae, serrated degraded surface of cartilage, multi layered synovial membrane and inflammatory infiltrate.
  • Panel B relates to a sCR1 - treated animal showing the structure of a normal joint with well populated cartilage and single layered synovium.
  • Figure 8 illustrates amelioration of arthritis by SCR1 -expressing fibroblasts injected at different time points post-immunization.
  • A illustrates amelioration of paw swelling.
  • Number of mice per group (n 10) .
  • B illustrates inhibition of anti-collagen antibody levels by sCR1 -expressing fibroblasts injected at different time points post-immunization.
  • Number of mice per group (n 10).
  • Figures 10-1 1 respectively illustrate inhibition of collagen type II- dependent IL-2 production in sCR1 (95kDa)-expressing HCQ6 cells, and lack of effect of full-length CHO-derived sCR1 on collagen type II dependent IL-2 production.
  • Figure 1 2 illustrates inhibition of IL-2 secretion by COS-derived sCR1 (95kDa) and by a sCR1 (95kDa)-lg fusion protein.
  • Figures 13 and 14 illustrate detection, by various relevant antibodies, of sCR1 -lg and clones producing it.
  • Figure 1 5 shows a map of a rDNA construct capable of expressing a sCR1 -lg fusion protein, referred to in relation to earlier Figures.
  • a novel 95 kDa soluble form of human complement receptor 1 which includes the amino terminal C4b and C3b binding sites was produced using a retroviral vector.
  • DBA/1 syngeneic cells infected with- this retrovirus were implanted i.p. at different time points after immunization with bovine collagen. These genetically engineered cells were capable of preventing collagen-induced arthritis development and ameliorating established disease. This beneficial clinical outcome was accompanied by a significant decrease in anti-collagen antibody, inhibition of T cell proliferation and IFN gamma (IFN y) secretion by draining lymph-node lymphocytes or splenocytes presented with bovine collagen type II.
  • IFN gamma IFN gamma
  • This retroviral vector was used to transfect the ecotropic packaging cell line GP + E86 (Markowitz D et al, 1 988 (21 )) by the calcium phosphate co-precipitation method using pSV2neo (Southern PJ et al
  • sCR1 secreted soluble sCR1 was collected in serum-free media. sCR1 was concentrated from 5 ml from these samples after addition of SDS to 0.5%, boiling for 5 min, addition of 9 volumes methanol and incubation overnight at - 20°C followed by centrifugation at 1 3000 RPM in a Sorvall SS34 rotor for 30 min at 4°C. The pelleted proteins were resuspended in 200 microL Laemmli sample buffer (Laemmli, UK, 1 970 (23)) without #-mercaptoethanol.
  • SVU 1 9.5 (Jat PS et al, 1 991 (25), Almazan G et al 1 992 (26)) overnight in the presence of 0.8 //g/ml polybrene (Sigma, Poole, UK) .
  • Cells expressing SV40 tsTA58 antigen were selected in 1 mg/ml G41 8 (Geneticin, Gibco,
  • the packaging cell line GP + E86 (Markowitz D et al 1988 (21 )) was maintained in DMEM, 10% newborn calf serum with glutamine, penicillin and streptomycin.
  • DMEM 10% newborn calf serum with glutamine, penicillin and streptomycin.
  • cells were trypsinized and plated at 5x10 5 cells/9cm diameter dish.
  • the viral supernatants were collected overnight from confluent 1 5 cm plates containing 1 2 ml media with 10% fetal bovine serum
  • FBS fetal bovine serum
  • 10% newborn calf serum without the selective drug.
  • the supernatants were centrifuged at 1500 rpm for 10 minutes to remove the cellular debris, transferred to sterile tubes and snap frozen on dry ice. These supernatants were stored at -70°C.
  • Immortalized DBA/1 fibroblasts were plated at 10 ⁇ 5 cells in a 9cm dish, and infected three times overnight with supernatants from GP + E86 cells suplemented with 0.8 microg/ml polybrene. Between infections cells were let to recover for 24 hrs in fresh media. Using this procedure it has been established by immunohistology of coverslip-grown cells that at least 90% of the cells express the gene cloned in the retroviral vector.
  • mice Ten to 12 week old male DBA/1 mice were injected at the base of the tail with 100 microL of a solution containing 2 mg/ml native bovine type II collagen mixed 1 : 1 in Freund's complete adjuvant (27). Onset of arthritis was about three weeks after immunization. Mice were maintained according to approved Home Office protocols and following Institute guidelines. The number of mice used in these studies was the minimal required to achieve statistical significance in this study.
  • mice 2-3 days after onset of arthritis were incubated with collagen at 100 ⁇ g/ml for 48 hours at 37°C in a 10% C0 2 humidified incubator.
  • cells were infected with retrovirus as follows: for the last 4 hrs of incubation with collagen the cells were centrifuged at 1500 rpm for 5 min, resuspended at
  • Affected hind paws were removed at post mortem (day 45 after onset), fixed in 10% formalin in phosphate buffered saline (PBS) and decalcified in
  • EDTA 50% in PBS. After embedding in paraffin, the paws were sectioned and stained with haematoxyiin and eosin. The grading of the histological joint destruction was as follows: Mild: synovitis, cartilage loss and bone erosions limited to discrete foci.
  • the data was analysed statistically using the Mann-Whitney non-parametric test.
  • the mAb E-1 1 (30) was used as coating antibody.
  • the mAb YZ-1 (at 1 mg/ml) was biotinylated using suifosuccinimidyl-6-biotinamide hexanoate purchased from Pierce (Rockford, IL) and used as directed by the supplier. Biotinylated YZ-1 was used as detect antibody with biotinylated streptavidin conjugated to horseradish peroxidase (HRP) - ⁇ Amersham Life Science, Amersham, UK).
  • HRP horseradish peroxidase
  • E-1 1 - 200 kDa sCR1 complex from which excess unbound sCR1 was washed away with PBS containing 0.05% Tween 20 and binding of the free biotinylated YZ-1 mAb, allowed to occur for another 45 min. After several washes with PBS, 0.05 % Tween 20, the ELISA was finally developed with HRP-conjugated biotinylated streptavidin using O-phenylenediamine dichloride (Sigma, Poole, UK) at 0.4 mg/ml as a substrate. After 45 min incubation the reaction was halted by the addition of 4.5 N sulphuric acid. The plate was read at 490 nm on a Microplate autoreader 310, BioTek Instruments.
  • Clinical scoring of arthritis adjudicates a number from 1 to 3 to degree of redness, swelling and deformity of normal contours to each extremity. This subjective scoring system varies from 0 to 1 2. In addition, objective hind paw swelling was recorded with calipers every 3 to 4 days.
  • Splenocytes or draining lymphnode cells were activated with concanavalin A at 3 ⁇ g/ml or with bovine collagen type II at 50microg/ml. Supernatants were collected and IFN y secretion measured by ELISA using a commercial kit obtained from Pharmingen (Cambridge, U.K.) .
  • Splenocytes or draining lymplinode cells were activated with bovine collagen type II at 50 microg/ml. Three days later cells were pulse labeled with
  • SCR SCR and part of the twelfth SCR was cloned into the retroviral vector MFG.
  • the structure of the expected protein is shown in Fig.1 .
  • the polypeptide chain encodes 778 amino acids, has nine potential glycosylation sites and its four carboxy amino acids were derived from extended sequences part of the polylinker region of the intermediate plasmid used to introduce a stop codon.
  • This construct was used to permanently transfect the ectropic packaging cell line GP + E86 using pSV2neo as cotransfectant for selection purposes.
  • Several clones were isolated after selection in G41 8, grown to mass culture and their serum-free supernatants used in complement mediated red blood cell lysis assays and after concentration for detection of the protein by western blot analysis.
  • Fig.2 shows that sCR1 secreted by GP + E86 cells was recognised by the mAb YZ-1 as a protein of 95 kDa indicating that the mature protein with an expected molecular weight of 80.5 kDa was glycosylated.
  • GP + E86 cells is from 500 to 1000 fold more active than the full length soluble receptor.
  • the alternative pathway assay was performed with human serum diluted 1 : 1 5 whilst the classical used 1 :60 dilution.
  • the amount in ng of rsCRI/microL human serum necessary to inhibit lysis by 50% is indicated whilst the amount of sCR1 in serum-free media from GP + E86 cells producing sCR1 was calculated using 50-100 fold concentrated samples in the competitive ELISA described herein.
  • Syngeneic immortalized fibroblasts (4 x 10 ⁇ 6) or primary splenocytes (5 x 10 A 7) infected with sCR1 -MFG were implanted i.p. in DBA/1 mice on day two after immunization with bovine collagen type II. Mice were assessed for arthritis development by measuring with calipers hind paw swelling, clinical score of disease and anti-collagen antibody titers. Systemic administration with fibroblasts was more effective because these cells were infected repeatedly with the retrovirus whilst the primary splenocytes were only infected once, and the number of cells which are responsive to collagen is much lower (around 0.2%) .
  • Fig.3C and Fig.3D show that mice treated with fibroblasts or splenocytes (Fig.3A and 3B) expressing sCR1 fall to develop arthritis as assessed by paw swelling and clinical score.
  • Mice treated with non-infected fibroblasts developed the disease at a slower rate but by day 22 post-immunization all animals were arthritic (not shown).
  • Anti-collagen antibodies were clearly detected in the control arthritic mice or the mice implanted with non-infected fibroblasts by day 30 but animals treated with fibroblasts expressing sCR1 had no detectable levels.
  • Splenocytes expressing sCR1 had also an inhibitory effect on anti-collagen antibody production, as shown by the following Table 2.
  • Table 2 Inhibition of anti-collagen antibody production by gene therapy with SCR1 .
  • Anti-collagen antibodies were measured at day 30 post-immunization. Normally quantitation by ELISA used a starting dilution of the serum at 1 :200
  • mice sera were negative at this concentration, we also assessed a dilution at 1 :5 with negative results as well.
  • FIG. 4 shows that neither splenocytes or draining lymph node cells from sCR1 treated animals were capable of proliferating when presented with collagen type II whereas cells from mice implanted with non-infected fibroblasts proliferated well to collagen type II.
  • Fig.5 depicts the secretion of
  • lymph node cells incubated in vitro with concanavalin A or collagen type II. Whilst all these cells responded well to concanavalin A, only lymphnode cells from mice treated with non-infected fibroblasts secreted IFN y when presented with collagen type H. Lymph node cells from mice treated with sCR1 secreted fibroblasts did not secrete IFN y in response to the antigen.
  • sCR1 treatment was clearly demonstrated by a protection from body weight loss which normally accompanies the development of arthritis in this model (Fig.6). Histopathology of joints from control (arthritic) and sCR1 treated mice is shown in Fig.7.
  • FIG. 8A shows that sCR1 expressing fibroblasts ameliorated disease development also when implanted at onset (i.e. day 21 post-immunization). Anti-collagen antibody levels decreased very efficiently even when the fibroblasts were implanted just nine days before the serum samples were tested (i.e. 30 days post-immunization) (Fig.8B).
  • IL-2 production by sCR1 -MFG infected HCQ6 (collagen type ll-specific T cell hybridoma) cells (full line between points marked by open squares) (and uninfected control HCQ6 cells (dotted line between points marked by open diamonds)) was measured (vertical axis: pg/ml) in relation to exposure to type II collagen (horizontal axis: microg/ml) during culture for 1 7 hours in media containing collagen at the indicated concentrations 0-533 microg/ml. In each case 5 x 10 5 HCQ6 cells/ml
  • IL-2 production was estimated using an ELISA. Two-sample T-test indicates significant reduction (p ⁇ 0.05) between the compared IL-2 levels.
  • Figure 1 1 illustrates lack of effect of full-length CHO-derived sCR1 on collagen type II dependent IL-2 production.
  • the results show (vertical axis) IL- 2 concentration (as estimated using an ELISA sensitive down to 10 pg/ml) after 20 hours of incubation of splenocytes from DBA1 mice (5x10 * 5 cells/ml) which presented collagen type II (Cll) (50 microg/ml) to Cll-specific T cell hybridoma HCQ6 (5 x 10 * 6 cells/ml) in media containing various amounts of full length sCR1 (CHO derived) (horizontal axis: 0.1 -1000 microg/ml).
  • Figure 1 2 illustrates inhibition of IL-2 secretion by COS-derived sCR1 (95kDa) and by a sCR1 (95kDa)-lg fusion protein.
  • Cos7 cells on 9cm plates were transfected with 20 microg DNA (either sCR1 -pcDNA3, sCR1 -lg- pcDNA3 or pcDNA3) using the clacium phosphate co-precipitation method. Culture medium was harvested after a 24-hour incubation.
  • Collagen type II (Cll)-specific T cell hybridoma HCQ6 (5 x 10 * 5 cells/ml) was exposed to antigen presenting cells (APC) in the form of splenocytes (5 x 10 * 6 cells/ml) and antigen (50 microg/ml Cll) in the Cos7 supernatants (diluted 1 /2) . After 20 hours the cell culture medium was removed and analysed for IL-2 (vertical axis, 1 00-400 pg/ml) . The two constructs both down-modulated IL-2 production compared to supernatants from the mock-transfected (pcDNA3)
  • Figures 1 3 and 14 illustrate detection, by various relevant antibodies, of sCR1 and sCR1 -lg and clones producing them.
  • FIG. 1 there is shown the result of SDS-PAGE electrophoresis of proteins from several clones of sCR1 -lg/Psv2Hygro transfected DBA1 /tst cells.
  • Cells were selected for 3 weeks in hygromycin (200 microg/ml) and ten clones tested for CR1 -lgG expression.
  • the protein was immunoprecipitated using proteinA-sepharose. 3ml serum-free overnight culture was rotated at 4 deg.C overnight with 70 microL 10% protein A sepharose. After 10 minutes centrifugation, the pellet was resuspended in l OOmicroL sample buffer and boiled (5 min.
  • the detection antibody YZ1 was used at 1 microg/ml (incubated for 2 hrs) .
  • the sample tracks shown were numbered as follows: 1 - positive control (sCR1 -lg/Cos7); 2- 2ng 22kDa sCR1 ; 3- 50pg 220kDA sCR1 ; 4-1 2- samples from several DBA clones.
  • FIG 14 there is shown the detection of sCR1 from mouse DBA/1 fibroblasts with monoclonal antibodies J3D3, E1 1 and YZ1 .
  • 5ml serum-free overnight cultures from DBA1 /tst clones expressing sCR1 (in pcDNA3) were boiled 5 min. in 250 microL 10% SDS, precipitated in methanol (20 deg.) and centrifuged (3000 rpm, 1 5 min.) .
  • the pelleted proteins were taken up in 250microL Laemmli sample buffer (without beta-mercaptoethanol) 50microL was loaded onto the gel (9.5% polyacrylamide) separated for > 3 hr at 200 V, and transferred onto nitrocellulose for 18hr (80 mAmp) .
  • the detection antibodies were used at 1 microg/ml (incubated for 2 hours) .
  • Each of three antibodies was used with samples/tracks as follows: a- 2ng 220kDa sCR1 ; b- and c- negative controls; d through g- positive DBA1 /tst clones.
  • Figure 1 5 shows a map of a rDNA construct capable of expressing a sCR1 -lg fusion protein, referred to in experiments described above.
  • a TNF receptor-lgG chimeric protein was used as source for mouse IgG heavy chain (Peppel et al, (48)) .
  • a 696-bp fragment was isolated after digestion with
  • sCR1 -pCDNA3 was Asp71 8-linearised. The DNA contains 5 BamHI sites, but by partially digesting the DNA, the needed 2.4kb fragment (Asp718-BamHI) was isolated. pCDNA3 was restricted with Asp71 8 and Xba, the three DNA fagments were ligated, and the positive clone designated sCR1 - lg-pCDNA3 (c.8500bp) .
  • the complement system has evolved as a primary line of defence against infection. Lack of a functional complement system can lead to susceptibility to infections (4) and it has been also shown that it is responsible for the proper removal of immune complexes thus avoiding complications as seen in systemic lupus erythematosus (6). Whilst in rheumatoid arthritis there is no clear defect in the complement system, it could be considered as being constantly up-regulated by the chronic presence of pro-inflammatory cytokines (32).
  • sCR1 one of the most potent complement cascade inhibitors
  • one of the limitations of its use in therapy is its short half life.
  • Embodiments of the present invention overcome this obstacle by its constitutive expression by gene therapy.
  • Several soluble forms of sCR1 have been produced with parts or multiple copies of the C3b or C4b domains (37) or only the C3b (38) domains but all have retained the SCRs of the long homologous repeat D (LHR-D) which has not yet a defined biological activity and all are bigger than 1 50 kDa.
  • LHR-D long homologous repeat D
  • Examples of sCR1 partial sequences such as the example particularly described above can be for example only about 95 kDa in size and contain one copy each of the C3b and C4b binding domains.
  • the reduced size is considered by the rpesent inventors to be advantageous when penetration into tissues and the possible production of such protein in implanted microcapsules is considered (39).
  • sCR1 may block CR1 -mediated endocytosis of antigens by follicular dendritic cells or phagocytosis by complement-opsonized particles (42).
  • certain forms of sCR1 discussed herein can be produced in forms which have been glycosylated by autologous cells, and this may have a bearing on stability and complex formation with other complement components (murine, in the case of experiments particularly reported above) or other yet unidentified molecules.
  • complement components murine, in the case of experiments particularly reported above
  • glycosylation differences in the envelope protein of retroviruses packaged in murine cells renders them susceptible to inactivation by human complement (43) .
  • the monoclonal antibody CAMPATH-1 H has different biological activities when produced by different types of cell lines because its complement-mediated function is strongly dependent on its glycosylated side chains (44) . Glycosylation may also affect the kinetic parameters of an enzymatic reaction, as it has been shown for the lecithin:cholesterol acyltransferase (45).
  • sCR1 may Inhibit T/APC/B cell interactions if CR1 serves as an adhesion molecule in analogy to CR2 (CD21 ), which in addition to binding antigen-C3dg, interacts with CD23.
  • CD21 CR2
  • CD23 CR2
  • the sugar moieties in the CD21 -CD23 interaction are crucial (46). It is possible that sCR1 post-translationally modified in autologous cells has critical advantage.
  • CR1 acts as a membrane bound protease cofactor which may have important functions in the processing of antigens at the cell surface also by other proteases besides complement enzymes. sCR1 could block their action at the cell surface inhibiting antigen presentation. Proteases such as factor Xa have an important role in immune responses, inhibition of its cell bound receptor by a monoclonal antibody or blocking receptor expression with antisense oligonucleotides has been shown to be immunosuppressive also for
  • fragmentary sCR1 molecules e.g. the 95kDa example and other fragmentary examples illustrated and discussed herein, has a higher specific activity than the 200 kDa full- sequence recombinant sCR1 .
  • Using a competitive ELISA to assess the protein concentration of sCR1 it appears that our exemplified 95 kDa molecule is between 500 to 1000 fold more active that the full length receptor.
  • sCR1 can have additional unforeseen therapeutic benefits in autoimmune diseases such as SLE, rheumatoid arthritis, myasthenia gravis, epidermis bullosa and Hashimoto's disease, which have a clear B cell compartment component, and probably others such as multiple sclerosis.
  • Soluble human complement receptor 1 in vivo inhibitor of complement suppressing post- ischemic myocardial inflammation and necrosis. 1 990 Science 249: 146-1 51 .
  • Jasin HE Autoantibody specificities of immune complexes sequestered in articular cartilage of patients with rheumatoid arthritis and osteoarthritis. 1 985 Arth. Rheum. 28: 241 -248.

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Abstract

L'invention porte sur un polypeptide soluble qui présente la fonctionnalité de liaison du récepteur complémentaire soluble 1 (sCR1) vis à vis du C4b et du C3b, par exemple une sous-séquence de 95 kDa du CR1, et peut servir à inhiber une réponse immunitaire induite par une cellule T ou une cellule B. L'invention porte également sur l'obtention in-vivo, ex-vivo et in-vitro de polypeptides apparentés au CR1, sur leurs compositions et leurs utilisations, c.-à-d. sur les séquences d'acide nucléique codant pour lesdits polypeptides comme ADN et ARN, sur des vecteurs d'acide nucléique de recombinaison contenant lesdites séquences, et sur les cellules exprimant lesdits polypeptides.
PCT/GB1998/001012 1997-04-05 1998-04-06 Modulation de l'immunite a l'aide de polypeptides apparentes au cr1 WO1998045430A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020591A1 (fr) * 2000-06-12 2002-03-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, recepteur du complement 12, et polynucleotide codant ce polypeptide
WO2014006063A2 (fr) 2012-07-02 2014-01-09 Medizinische Universität Wien Produit de séparation du complément c4d pour le traitement d'affections inflammatoires
CN113365648A (zh) * 2018-06-22 2021-09-07 乌尔姆大学 补体抑制因子及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009220A1 (fr) * 1988-04-01 1989-10-05 The Johns Hopkins University LE RECEPTEUR HUMAIN DE C3b/C4b (CR1)
US5212071A (en) * 1988-04-01 1993-05-18 The Johns Hopkins University Nucleic acids encoding a human C3b/C4b receptor (CR1)
WO1994000571A1 (fr) * 1992-06-24 1994-01-06 Smithkline Beecham Plc Derives solubles de cr1 (recepteur du complement de type 1)
WO1995008343A1 (fr) * 1993-09-24 1995-03-30 Washington University Regulateurs modifies tronques du systeme du complement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009220A1 (fr) * 1988-04-01 1989-10-05 The Johns Hopkins University LE RECEPTEUR HUMAIN DE C3b/C4b (CR1)
US5212071A (en) * 1988-04-01 1993-05-18 The Johns Hopkins University Nucleic acids encoding a human C3b/C4b receptor (CR1)
WO1994000571A1 (fr) * 1992-06-24 1994-01-06 Smithkline Beecham Plc Derives solubles de cr1 (recepteur du complement de type 1)
WO1995008343A1 (fr) * 1993-09-24 1995-03-30 Washington University Regulateurs modifies tronques du systeme du complement

Non-Patent Citations (3)

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Title
D.T. FEARON: "anti-inflammatory and immunosuppressive effects of recombinant soluble complement receptors", CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 86, no. sup1, October 1991 (1991-10-01), pages 43 - 46, XP002071582 *
JR PRATT ET AL.: "Allograft immune response with sCR1 intervention", TRANSPLANT IMMUNOLOGY, vol. 4, no. 1, 1996, pages 72 - 75, XP002071581 *
PIDDLESDEN S J ET AL: "Soluble recombinant complement receptor 1 inhibits inflammation and demyelination in antibody-mediated demyelinating experimental allergic encephalomyelitis.", JOURNAL OF IMMUNOLOGY, vol. 152, no. 11, 1 June 1994 (1994-06-01), BALTIMORE US, pages 5477 - 5484, XP002071580 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020591A1 (fr) * 2000-06-12 2002-03-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, recepteur du complement 12, et polynucleotide codant ce polypeptide
WO2014006063A2 (fr) 2012-07-02 2014-01-09 Medizinische Universität Wien Produit de séparation du complément c4d pour le traitement d'affections inflammatoires
CN113365648A (zh) * 2018-06-22 2021-09-07 乌尔姆大学 补体抑制因子及其用途

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