EP0386059A1 - Anticorps contre le recepteur complementaire de type 3 (cr3) et composition therapeutique - Google Patents

Anticorps contre le recepteur complementaire de type 3 (cr3) et composition therapeutique

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Publication number
EP0386059A1
EP0386059A1 EP88909567A EP88909567A EP0386059A1 EP 0386059 A1 EP0386059 A1 EP 0386059A1 EP 88909567 A EP88909567 A EP 88909567A EP 88909567 A EP88909567 A EP 88909567A EP 0386059 A1 EP0386059 A1 EP 0386059A1
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Prior art keywords
cells
antibody
recruitment
myelomonocytic cells
antibodies
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German (de)
English (en)
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Hugh Rosen
Siamon Gordon
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the field of immunology.
  • the invention relates to antibodies to cell surface receptors of myelomonocytic cells and to the therapeutic use of these antibodies to inhibit recruitment of myelomonocytic cells in response to inflammatory stimuli.
  • Myelomonocytic cells include monocytes, neutrophils and macrophages and are types of white blood cell. These cells constitute major components of the cell-mediated immune response system, providing the principal means of defence against bacterial and fungal infections. In addition, however, myelomonocytic cells are implicated in the pathogenesis of tissue damage in certain noninfectious diseases, including in particular some inflammatory and autoimmune diseases. These cells circulate within the vascular system and are recruited to sites of inflammation egressing from the vascular system and migrating towards the site of the inflammatory stimulus.
  • LFA Leukocyte Functional Antigen
  • the LFA family of cell surface receptors is made up of three structurally related heterodimeric glycoproteins, each having a unique higher molecular weight ⁇ subunit noncovalently linked to a common ⁇ subunit, which is structurally identical for all three family members .
  • LFA cell surface receptors are LFA-1 (comprising the CD11a ⁇ subunit and the common CD18 ⁇ subunit), CR3 (also known as Mac-1 or Mo 1) (CD11 b -/CD18) and p 150, 95
  • the antibody used in these studies (60.3) appears to have specificity for an epitope present on the common ⁇ chain polypeptide of the CD w 18 complex, or an epitope formed by the quaternary structure of the ⁇ - ⁇ heterodimers (Wallis et al (1986) Blood 67: 1007).
  • the experimental model used in this study was based upon determination of endothelial cell damage at the vascular-endothelium surface interface not upon damage as the result of recruitment of neutrophils to extra-vascular sites of inflammatory stimuli.
  • rat monoclonal antibody having specificity for an epitope on the ⁇ chain polypeptide of the type 3 complement receptor (CR3) component of the LFA family has been described (Springer, T., G, Galfre, D.S. Secher and C. Milstein, Eur. J Immunol. (1979) 9:301).
  • this antibody has not proven to be useful for inhibiting in vivo recruitment of myelomonocytic cells to inflammatory stimuli, nor does it pass the in vitro screening assay which we have used to identify the new sub-class of anti-CR3 antibodies.
  • an anti-Mol antibody anti-Mol monoclonal antibody 44
  • Ismail et al ibid
  • the invention provides an anti CR3 specific antibody for use in the inhibition of recruitment of myelomonocytic cells to inflammatory stimuli.
  • the antibodies of the invention are capable of inhibiting the attachment of myelomonocytic cells to bacteriologic plastic in vitro in a screening assay as hereinafter described.
  • a screening assay as hereinafter described.
  • anti-CR3 specific antibodies do not inhibit the attachment of myelomonocytic cells to bacteriologic plastic in the in vitro screening assay and thus this assay defines a new sub-class of anti-CR3 specific antibodies.
  • the invention provides an anti-CR3 specific antibody characterised in that the antibody inhibits the attachment of myelomonocytic cells to bacteriologic plastic in an in vitro screening assay as hereinafter described.
  • the in vitro screening assay which is used to identify and define the antibodies of the invention may be substantially as hereinafter described in detail. However, important features of the screening assay are as follows:
  • the cells used in the screening assay are characteristically myelomonocytic cells which have the CR3 receptor on their surfaces.
  • the cells used are macrophages and conveniently thioglycollate-elicited peritoneal macrophages (TPM) may be used.
  • the surface used in the in vitro screening assay is typically bacteriologic plastic or any other surface which has similar adhesion properties for CR3 bearing myelomonocytic cells.
  • the surface is an unmodified polystyrene plastic surface.
  • unmodified polystyrene bacteriologic plastic micro-titre plates may be used in the screening assay. Tissueculture treated plastic surfaces are generally not suitable for use in the screening assay.
  • the screening method used may involve incubation of cells with the bacteriologic plastic surface followed by fixation and then staining of the cells.
  • the extent of adhesion may then be quantified by recovery of stain from the cells and measurement by spectrophotometry.
  • the inhibition of adhesion may be determined by comparison of the results obtained for untreated cells and cells which have been pretreated with antibody prior to adhesion.
  • Pretreatment with antibody may be carried out conveniently at a relatively low temperature, for instance, in the bacteriologic plastic micro-titre plate wells, e.g. 4oC for 30 minutes.
  • the adhesion normally requires incubation at relatively higher temperature e.g. 37oC for 30 minutes.
  • the incubation medium may comprise Iscove's modification of Dulbecco's Medium containing 20%
  • FCS foetal calf serum
  • the antibodies of the second aspect of the invention inhibit adhesion of myelomonocytic cells to bacteriologic plastic to the extent of at least 85%, preferably at least 90% especially at least 95%, and most preferably substantially completely when tested in an in vitro screening assay substantially as herein described.
  • the antibodies of the second aspect of the invention are capable of causing detachment of myelomonocytic cells from bacteriologic plastic, for instance, in an in vitro assay as hereinafter described.
  • the cells are attached to the bacteriologic plastic by suitable incubation e.g. 60 minutes incubation at 37oC, and are then incubated with medium containing a saturating concentration of the antibody for a suitable period of time, e.g. 5ug/ml for 30 minutes at 37oC.
  • the extent of detachment may be determined using a fixing, staining and spectrophotometric measurement method as for the adhesion assay.
  • Preferred antibodies are typically capable of detaching at least 50%, especially about 65%, of thioglycollate- elicited peritoneal macrophages or at least 75%, especially at least 85% of polymorphonucleur leukocytes in an in vitro detachment assay as herein specifically described, e.g. after exposure to a saturating concentration (5ug/ml) of antibody for a period of 30 minutes at 37°C.
  • the antibodies of the invention are capable of inhibiting the in vivo recruitment of myelomonocytic cells to inflammatory stimuli.
  • the inhibition of in vivo recruitment of myelomonocytic cells may be determined by an in vivo assay as hereinafter described.
  • the antibodies are capable of inhibiting in vivo recruitment of myelomonocytic cells to an extent similar to the specific antibody 5C6 as hereinafter described.
  • Particularly preferred antibodies are those having characteristics similar to or the same as the specific antibody 5C6 as hereinafter described.
  • the antibody has binding specificity for an antigenic epitope which is specific for CR3; for example an epitope of the ⁇ subunit of CR3.
  • the epitope may be one requiring the presence of a ⁇ -chain, though is characteristically not one on the common ⁇ -chain polypeptide of the LFA family.
  • the antibody is characteristically a CR3 specific antibody not an LFA family specific antibody.
  • the antibodies may be of any suitable immunoglobulin class or subclass, preferably they may be IgG, e.g. IgG2, or IgG2b antibodies.
  • the antibodies are of relatively high affinity; for instance of affinity sufficient to inhibit attachment of myelomonocytic cells to bacteriologic plastic surfaces, e.g. as herein specifically described.
  • the antibodies of the invention may comprise monospecific antisera, or more preferably monoclonal antibodies or recombinant antibodies i.e. antibodies produced by recombinant DNA techniques, including chimeric, humanised, and CDR-grafted antibodies. Methods for the preparation of recombinant antibodies, chimeric antibodies, humanised antibodies and CDR-grafted antibodies are described in published International Patent Applications Nos. WO 84/03712,
  • the antibodies preferably comprise complete or substantially complete antibody molecules; though may comprise antibody fragments e.g. F(Ab') 2 fragments.
  • the antibodies are obtained as monoclonal antibodies by cell fusion or other antibody-producing cell line immortalisation techniques.
  • the antigen used to raise the antibodies characteristically comprises CR3.
  • Myelomonocytic cells e.g. thioglycollate-elicited peritoneal macrophages, may be used as the antigen, though purified and/or synthetic CR3 antigens e.g. purified CR3 ⁇ subunit glycoprotein or polypeptide or fragments thereof, may be used.
  • the techniques for preparation of monoclonal antibodies are well known and understood by workers skilled in the art.
  • Recombinant antibodies may be obtained by suitable manipulation of immunoglobulin genes and preparation of transformed host cells, also, by techniques well known in the art. Genes for recombinant DNA manipulation may be obtained conveniently from suitable monoclonal antibody producing cell lines. Both monoclonal antibody and recombinant DNA techniques provide for establishment of cells which may be used to produce the antibodies of the invention.
  • the invention also includes a method for the preparation of a cell line which produces the antibodies of the second aspect of the invention comprising preparing a plurality of different cells which produce CR3 specific antibodies, screening the different cells for antibodies which inhibit adhesion of myelomonocytic cells to bacteriologic plastic and selecting cells which produce antibodies which inhibit adhesion.
  • the invention includes cell lines which produce antibodies according to the second aspect of the invention.
  • the antibodies of the invention may be used for therapeutic treatment of humans or animals.
  • the antibodies used are typically not capable of causing aggregation, lysis or clearance of myelomonocytic cells, e.g. they do not fix complement or lyse cells.
  • the antibodies are preferably human, or humanised antibodies.
  • the antibodies may be used for treatment or prophylaxis of diseases or disease states which arise as the result of recruitment of myelomonocytic cells to inflammatory stimuli or as the result of complications of the adhesion of myelomonocytic cells to endothelium, such as increased vascular permeability.
  • Diseases in which the recruitment of myelomonocytic cells appear to be involved in their development and or pathogenesis include inflammatory, acute hypersensitivity and autoimmune diseases.
  • the antibodies of the invention may be used in the treatment and prophylaxis of diseases involving recruitment of myelomonocytic cells in delayed type hypersensitivity reactions mediated by T-lymphocytes such as chronic inflammation and drug induced hypersensitivity reactions.
  • Such diseases include rheumatoid arthritis, immune vasculitis, glomerulonephritis, and inflammatory bowel disease.
  • diseases which may be treated using the antibodies of the invention are: endotoxin toxicity, gout, immune complex diseases, multiple sclerosis and other inflammatory demyelinating diseases, neutrophil dermatoses, the after effects of myocardial infarction, adult respiratory distress syndrome, disseminated intravascular coagulation syndrome, emphysema, asthma, and the Arthus phenomenon.
  • the antibodies of the invention may be used in treatment and prophylaxis in relation to these and similar diseases. It will be appreciated, however, that it may not be desirable to use the antibodies of the invention in disease states caused by rapidly proliferating acute infectious agents such as some types of bacteria.
  • the invention provides a therapeutic composition
  • a therapeutic composition comprising an antibody according to the first aspect of the invention in conjunction with a suitable excipient, diluent or carrier.
  • Therapeutic compositions for use according to the present invention may be formulated in conventional manner, optionally with one or more physiologically acceptable carriers diluents or excipients.
  • the antibody compositions may be formulated for oral, buccal, parenteral or rectal administration or in a form suitable for nasal administration or administration by inhalation or insufflation.
  • the antibody compositions are formulated for parenteral administration by injection e.g. by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
  • suitable vehicle e.g. sterile pyrogen-free water
  • depot compositions may be administered by implantation or by intramuscular injection.
  • the invention provides a process for the preparation of a therapeutic composition comprising admixing an antibody according to the first or second aspect of the invention with a suitable excipient, diluent or carrier.
  • the excipient, diluent or carrier is a physiologically acceptable excipient, diluent or carrier.
  • the invention includes the use of an anti-CR3 specific antibody in the preparation of a medicament for inhibiting the recruitment of myelomonocytic cells to inflammatory stimuli.
  • the invention provides a therapeutic method of inhibiting recruitment of myelomonocytic cells to inflammatory stimuli in a human or animal subject by administering to the subject an effective amount of an antibody of the first aspect of the invention.
  • the amount of antibody which is administered to the human or animal subject will depend upon the properties of the antibody and the type of subject. Thus we have found in the case of the 5C6 antibody, as hereinafter specifically described, that a dose of about 0.5mg is sufficient to inhibit recruitment of myelomonocytic to inflammatory stimuli in a mouse for a period of up to 4 days. On this basis the dose required for a human subject if likely to be about lg, typically in the range from 0.1 up to about 10g.
  • anti-CR3 specific antibodies may be used to inhibit the recruitment of myelomonocytic cells to inflammatory stimuli in vivo.
  • CR3 alone is critical for egress of myelomonocytic cells through the vascular endothelial cell monolayer and their subsequent migration to sites of inflammation.
  • the use of anti-CR3 sepcific antibodies is particularly advantageous in comparison with use of antibodies to other members of the LFA family, such as antibodies to the common ⁇ subunit of LFA.
  • CR3 is a surface antigen which is specific to meylomonocytic cells whereas other members of the LFA family are present on the surfaces of other types of cells of the immune system, including B lymphocytes and T lymphocytes.
  • an anti-CR3 specific antibody specifically targets myelomonocytic cells and does not significantly effect other cells of the immune system which may lead to undesirable, generally comprising effects on the overall immune system.
  • use of an anti-CR3 specific antibody leaves the remainder of the immune system substantially intact and functioning and capable of warding-off infectious and other invasive stimuli.
  • Figure 1 is a graph showing the results of quantitative adhesion assays after preincubation of cells in medium alone (C), 5C6 or Ml/70 mAb (10 Thioglycollate elicited peritoneal macrophages (TPM) or bone marrow PMN (BM) were plated per well in 96 well plates on Bacterial (BP) or Tissue culture plastic (TCP). Results show the mean ⁇ SD of quadruplicates in three separate experiments after incubation for 30 mm at 37 oC, washing and Giemsa staining. Cell number was calculated from an internal standard of 10 adherent cells/well);
  • Figure 2 is a graph showing the results of quantitative assays of detachment by mAb 5C6 of TPM adherent to BP, or of bone marrow PMN (BM) adherent to TCP (Assay as described in Materials and Methods.
  • Medium alone (C) or M1/70 supernatant (M1/70) failed to detach either TPM from BP or BM from TCP (results expressed as mean ⁇ SD of triplicates in two separate experiments));
  • Figure 3 is an autoradiograph of a 10% SDS-PAGE gel of S-met labelled immunoprecipitate of cultured TPM (control preparation was precipitated with the rat anti-guinea pig PMN mAb 1A10.4 whilst both
  • Figure 4 is a graph showing the effect of i.v. administration of 5C6 on the time course of increased footpad thickness in sensitised mice challenged with SRBC;
  • Figure 5 is a graph showing the time course of footpad swelling seen after challenge of actively immunised mice in the presence or absence of 5C6, and
  • Figure 6 is a graph showing the effects of 5C6 on the inflammatory recruitment of myelomonocytic cells following transfer of sensitised lymphocytes and tuberculin to the peritoneal cavity of naive, syngeneic mice.
  • This Example describes the preparation, in vitro screening, characterisation and in vivo testing of an antibody according to the invention.
  • Murine thioglycollate peritoneal macrophages (TPM) and polymorphonuclear leucocytes (PMN) were harvested from the peritoneal cavity by lavage 4 and 1 days respectively after the intraperitoneal (ip) injection of 1ml Brewer's complete thioglycollate broth. Cytospin preparations stained with Giemsa revealed that ⁇ 80% of the 4 day exudate (3 ⁇ 10 7 cells) were PMN. Bone marrow cells, ⁇ 45% PMN, were obtained by flushing the femoral cavity with phosphate-buffered saline (PBS). BCG activated and resident peritoneal M ⁇ and Kupffer cells were isolated as described (1).
  • TPM Murine thioglycollate peritoneal macrophages
  • PMN polymorphonuclear leucocytes
  • mice C57/B1 or Pathology Oxford, PO
  • AO rats specific pathogen free mice were from the MRC Cellular Immunology Unit in our department.
  • BP flat-bottomed bacteriologic plastic
  • TCP tissue-culture treated plastic
  • Stain was quantified by measuring absorbance at 460nm in an automatic plate reader (Dynatech, Alexandria, Va.). This assay was linear between 5 ⁇ 10 3 and 10 5 adherent cells per well.
  • antibody (ab) inhibition experiments hybridoma conditioned media were added to microtitre plates and cooled to 4oC before addition of cells in the same medium. After preincubation at 4oC for 30 min, neither ab treated nor control cells had adhered and the plates were then warmed to 37o for 30 min before washing and processing.
  • detachment assays 10 5 M ⁇ were plated in serum containing medium and allowed to adhere for 60 min at 37oC.
  • FCS with either 5mM Ethylenediaminetetracetic acid (EDTA) or 5mM
  • Ethylglycolbisaminoethylethertetracetic acid to restrict Ca 2+ and Mg 2+ or Ca 2+ alone.
  • the absolute cation requirements were determined by performing adhesion assays in Ca 2+ and Mg 2+ free Hanks balanced salt solution (HBSS) , with 10% FCS dialysed to remove divalent cations and defined quantities of added CaCl or MgCl 2 .
  • HBSS Hanks balanced salt solution
  • Antibody production mAb 5C6 was the product of a fusion between spleen cells from an AO rat, immunised four times with TPM, and the Y3 rat myeloma line. The fusion was performed as described by Galfre et al (2). Hybridoma supernatants were screened for inhibition of attachment and for detachment of TPM.
  • F(ab') 2 fragments were prepared by pepsin digestion of IgG at pH 4.0 in 0.1M acetate after Rousseaux et al (4) and purified by gel filtration chromatography.
  • Fab fragments were produced by papain digestion of IgG in 75mM Na phosphate pH 7.0/75nM NaCl and purified by anion exchange chromatography on DEAE-sephacel.
  • 5C6 is a rat IgG2b as typed by Ouchterlony gel diffusion using antisera directed against rat IgG subclasses supplied by Dr. H. Bazin (Catholic University, Louvain). 5C6 was fluoresceinated by the method of Mason (5).
  • TPM 4 day TPM were plated at 10 cells/10cm culture dish and cultivated overnight in methionine-free medium with 200uCi- 35 S-methione.
  • A-sepharose The beads were washed by standard methods and boiled in sample buffer containing 2%. SDS. The eluates were analysed by electrophoresis on 10% polyacrylamide gels and autoradiography.
  • Control preparations contained a mAb to determinants not found on mouse M ⁇ .
  • 5C6 and M1/70 immunoprecipitates on protein-A-sepharose beads were digested for varying times at 37oC with 100 ⁇ g/ml crystalline trypsin. The reaction was stopped by boiling the beads in SDS sample buffer and the resultant cleavage patterns analysed by SDS-PAGE.
  • Fibronectin-coated surfaces were prepared by coating tissue-culture plastic surfaces with gelatin followed by fresh mouse serum for an hour at room temperature. The wells were then extensively washed with PBS. Poly-L-lysine coated, glutaraldehyde activated surfaces were derivatised with Dinitrophenyl (DNP) Bovine serum albumin (BSA)-anti-DNP immune complexes as described (6).
  • DNP Dinitrophenyl
  • BSA Bovine serum albumin
  • mice were injected intravenously (iv) with PBS, 5C6 IgG F(ab') 2 or the anti-L3T4 subclass matched (IgG2b) YTA mAb, 4 hours prior to injection of 1ml thioglycollate broth ip. All ab injections were 0.5mg unless stated otherwise.
  • mice were killed and peritoneal exudate cells, blood, livers, spleens and bone marrows removed. Total and differential counts of exudate cells, bone marrow cells and blood leukocytes (after sedimentation of erythrocytes with 1% Dextran T500) were obtained using May-Grunwald-Giemsa stain.
  • Inhibition of recruitment by mAb was calculated as total exudate cells - resident cells in Ab-treated mice divided by recruited cells from control mice. Cells were then tested for adhesion to BP or TCP. Livers and spleens were examined histologically as follows. In some experiments tissues were fixed in glutaraldehyde and stained with haematoxylin and eosin. In experiments where fluoresecein isothiocyanate (FITC)-5C6 had been injected in vivo, organs were examined for the presence of mAb by direct immunofluorescence of frozen sections (Sum) embedded in Tissuetek (Miles Laboratories, Naperville, II). Alternatively, animals were perfusion fixed as described (8) and frozen sections of tissues analysed by the immunoperoxidase technique without adding exogenous first ab to the sections.
  • FITC fluoresecein isothiocyanate
  • Sheep erythrocytes (E) were opsonised with IgM ab and iC3b or C142 (9), or rabbi-anti-sheep E IgG and used as a 5% v/v suspension for rosetting at 4oC or phagocytosis at 37oC by adherent TPM (10). Binding was quantified by counting the number of attached erythrocytes to 100 TPM by phase contrast microscopy following washing and glutaraldehyde fixation.
  • Fluorescence microscopy was performed using a Zeiss Axiphot epifluorescence microscope. Scanning electron microscopy was performed as described (10).
  • a rat anti-CR3 mAb was used either as IgG or hybridoma supernatant from laboratory stocks.
  • the cell line was the gift of Dr. T. Springer (Dept. Pathology, Harvard Medical School, Boston).
  • YTA mAb was a gift of Dr. H. Waldmann (Dept. Immunology, University of Cambridge) .
  • Rabbit-anti-E IgM (EA) and EA iC3b were provided by Dr. R. Sim (MRC Immunochemistry, University of Oxford), Reagents were obtained from the following sources: Iscove's medium (Flow); FCS (Gibco, Paisley, Scotland); Colcemid (Dr. E.P. Evans, Sir William Dunn School of Pathology, Oxford); Deoxyglucose, cytochalasin B, EDTA and EGTA (Sigma, Poole, Dorset); Protein-A-Sepharose (Pharmacia).
  • the adhesion of M ⁇ and exudate PMN to BP was Mg -dependent, pronase senstitive and required elevated temperature as well as cytoskeletal stabilisation for efficient binding. Exposure of TPM to 0.5mg/ml pronase at 37oC for 30 min completely abolished their ability to adhere to BP. This effect persisted for at least 6 hours and cells recovered fully by 24 hours in culture. Resting PMN and tissue M ⁇ such as Kupffer cells were unable to adhere to BP. Adhesion to BP required at least 100 ⁇ M extracellular Mg 2+ and Ca 2+ alone was unable to substitute for this requirement. The adhesion of PMN (whether resting or exudate) to TCP had the same qualities as M ⁇ adhesion to
  • TPM Cation requirement
  • TPM Protease sensitivity
  • TPM TPM Factors
  • Results of quadruplicates in three separate experiments ar expressed as the mean absorbance and the mean ⁇ SD of adherent cell number calculated from an internal standard. ⁇ 1 ⁇ 10 5 myelomonocytic cells were plated/well. The adherence of M ⁇ to BP appeared to be a useful means of identifying a pronase-sensitive, cation-dependent surface component involved in cell adhesion. We thus used this assay to screen for functional mAb capable of, first, inhibiting attachment of TPM to BP and, second, detaching adherent TPM from BP.
  • mAb 5C6 inhibits adhesion to BP and detaches adherent TPM
  • one hybridoma secreting a mAb capable of inhibiting attachment of TPM to BP was isolated. Both identify the same antigen and this report is confined to the first of these mAb designated 5C6.
  • the effects of mAb 5C6 on adhesion of TPM to bacterial and tissue-culture treated plastic was investigated. We found that untreated 4 day TPM adhered and spread on BP after 30 minutes at 37oC in serum-containing medium.
  • 5C6 IgG, F(ab') 2 and Fab were used at 1, 10 and 100 ⁇ g/ml. Rosetting was quantified as described in Methods. Results reflect the mean 1 SD of at least 3 separate experiments.
  • TPM were digested in suspension (30 mm, 37oC) with 0.5mg/ml pronase, fixed in 0.25% glutaraldehyde, quenched in FCS and 1 ⁇ 10 6 cells assayed in quadruplicate by trace indirect binding assay (2 experiments). Untreated cells bound 3903 ⁇ 47 cpm of second ab after binding of 5C6 and 3336 ⁇ 51 cpm after pronase digestion. Untreated cells bound 3292 ⁇ 35 cpm using
  • FIG. 1 and Table II summarise quantitative assays of adhesion in the presence or absence of mAb and show that 5C6 IgG at 0.5 ⁇ g/ml inhibited attachment of TPM to BP by 92 ⁇ 1% whilst untreated cells or cells treated with saturating concentrations of the anti-CR3 mAb M1/70, used for comparison, displayed unaltered adhesive capacities.
  • the increased adherence of M1/70-treated cells as compared to untreated cells reflected aggregation of cells by this ab and adhesion of these clusters.
  • 5C6 In addition to its effects on M ⁇ adhesion, 5C6 also abolished the adhesion of PMN to TCP, which was unimpaired in control or M1/70-treated PMN. Whilst M1/70 treatment led to aggregation of myelomonocytic cells in suspension, 5C6-treated PMN or M ⁇ remained discrete.
  • the CSAT ag or Fibronectin receptor of avian fibroblasts was identified by the ability of ab to detach cells adherent to a fibronectin-coated substratum (13). Following this precedent, we examined the ability of 5C6 to detach TPM from BP or bone marrow PMN from TCP. After allowing cells to attach and adhere for 60 minutes at 37oC, plates were washed and medium containing saturating concentrations (5 ⁇ g/ml) of 5C6 or M1/70 or no ab was added.
  • mAb 5C6 binds to CR3 and inhibits iC3b binding
  • the ag recognised by 5C6 was characterised by the cell binding profile of the mAb and the molecular species identified by immunoprecipitation.
  • Indirect binding radioimmunoassays with fixed cells and direct immunofluorescence studies of live cells revealed that 5C6 bound to a methanol- and glutaraldehyde- stable epitope on TPM, RPM, BCG-activated M ⁇ and PMN, that was absent from the surface of Kupffer cells (as gauged by immunoperoxidase staining) and from resting or BCG-activated lymphocytes.
  • the 5C6 epitope is distinct from the M1/70 epitope
  • the disparity between 5C6 and ml/70 in their ability to inhibit adhesion of TPM to BP led us to explore the possibility that the two ab identify distinct epitopes on the CR3 molecule. We therefore compared the sensitivity of the binding of these ab to proteolytic digestion of the TPM cell surface. Digestion of the cell surface with pronase (0.5mg/ml for 30 mm at 37oC) destroyed most of the
  • the 5C6 antigen like that of M1/70, is probably an ⁇ -chain epitope as it was restricted to myelomonocytic cells and was absent on lymphocytes, that are rich in LFA-1 which shares a common ⁇ chain with CR3.
  • the 5C6-epitope might require ⁇ chain together with the common ⁇ chain since after dissociation of chains by pH 11.5 in solution, 5C6 failed to precipitate any ag.
  • the 5C6-epito ⁇ e was also dissociated from the adhesive domain of CR3 because pronase digestion abolished adhesion of TPM to BP whilst binding of 5C£ was largely intact (of Table I).
  • mice with purified 5C6 IgG were followed four hours later by an intraperitoneal challenge of sterile thioglycollate broth.
  • a total of 22 5C6 IgG-injected mice and 20 control mice were examined in this series of experiments (Table III).
  • mice 0.5mg 5C6 IgG iv 9.0+2 35P 51M 14 17 ⁇ 2 77P 13M 10L (18 mice)
  • mice 0.5mg YTA IgG iv 22 65P 33M 2L 14 75P 10M 15L (2 mice)
  • mice The blood, bone marrow, liver and spleen of control and mAb-treated mice were examined to identify its site of action. There was no difference in number or differential counts of femoral marrow cells, between control and 5C6-treated mice. Marrow plugs were lightly fixed in paraformaldehyde, embedded and stained by the immunoperoxidase method after frozen section to detect mAb which had bound in vivo. Marrows from 5C6-treated mice showed heavy labelling of most mature myelomonocytic cells. Blast cells, lymphocytes and resident bone marrow M ⁇ s remained unlabelled, as expected. There was no evidence of cell death.
  • IgG-treated mice were not aggregated. Blood PMN from control animals adhered well to BP and TCP in vitro whereas blood leukocytes from 5C6-IgG-treated mice failed to adhere to either substratum.
  • This example describes investigations of the extent to which T-cell dependent inflammation induced by specific antigen (ag) challenge in sensitised mice, or by adoptive transfer in naive, syngeneic mice is inhibitable by a monoclonal antibody (5C6) according to the invention, directed specifically to CR3.
  • mice 8 week old C57/BL6 female mice were obtained either from the Sir William Dunn School of Pathology, Oxford, or the specific pathogen-free unit of the Institut Pasteur, Paris.
  • SRBC Sheep erythrocytes
  • mice Four days after immunisation mice were tested for DTH by an antigenic challenge of 10 7 or 10 8 SRBC injected subcutaneously in a volume of 50 ⁇ l into the right hind footpad. Footpad swelling was measured at a range of times with a dial gauge caliper and the measurement of the uninjected footpad was subtracted from that of the challenged footpad. Footpads were subsequently removed and fixed in formol-saline. The wax-embedded footpads were then sectioned and stained with haematoxylin and eosin. The local adoptive transfer of the DTH reaction in syngeneic naive mice was performed as described (15). Briefly, four days after immunisation, mice received 50 IU of heparin. Heparinised blood was collected fifteen minutes later, diluted and used as a source of
  • SRBC-sensitised T-cells This was then mixed with either SRBC or unrelated ag and injected into one hind footpad of naive, syngeneic recipients and the increase in footpad thickness examined 14-18 hrs later. Footpads were processed for histological examination as above.
  • M ⁇ macrophages
  • a rat IgG2b mAb directed against the murine CR3 was prepared and purified as described in Example 1. lmg of IgG was injected i.v. either at the time of ag challenge or up to 6 hrs later and the effects compared with controls in which buffer alone, a control rat IgG2a mAb 7/4 (19) restricted to murine myelomonocytic cells and with a similar site number to 5C6 or the F(ab') 2 fragment of 5C6 that has no in vivo activity, was injected. In experiments where local injection of sensitised T-cells and ag was used to adoptively transfer DTH, 5C6 or a control preparation was injected i.v.
  • fetal bovine serum and 4-day thioglycollate-elicited M ⁇ were then incubated in the diluted serum before measurement of adhesion to bacterial plastic as described in Example 1. 4 days after injection, a 1:10 dilution of the sampled serum still inhibited adhesion to plastic by > 85%.
  • the degree of mAb binding to blood leukocytes, peritoneal cells and bone marrow cells was assessed at 1, 4, 24, 48 and 72 hrs after a single i.v. injection of FITC-5C6, by fluorescence analysis on a Becton-Dickinson FACS II apparatus. Saturation binding to myelomonocytic cells alone was seen, and persisted for the duration of the experiment.
  • the reciprocal of the dilution of antibody that gives rise to 50% maximal binding of a fixed quantity of iodinated second antibody is designated the number of binding units per ml.
  • lmg/ml of pure 5C6 IgG contains 6300 binding units.
  • SRBC-specific response can be transferred systemically or locally to naive, syngeneic mice only by transfer of cell suspensions containing Thy 1 + , CD 4+ lymphocytes from SRBC-sensitised mice
  • T-lymphocytes are transferred to mice that have been lethally irradiated 30 hrs before transfer.
  • the DTH response is restored only by the i.v. injection of bone marrow cells
  • Figure 4 shows the increase in footpad thickness seen after local ag challenge with SRBC 4 d after active systemic immunisation. Naive animals (bar A) showed a negligible increase in footpad thickness of
  • the ability of 5C6 to inhibit the DTH at 20 hrs was confirmed on histological examination of the footpad sections.
  • the 20 hrs response to injected SRBC in a naive mouse shows intact SRBC in the extravascular tissue spaces.
  • the host response to these erythrocytes was minimal with an occasional infiltrating neutrophil or monocyte.
  • the actively sensitised mouse challenged with SRBC in the absence of 5C6 mAb shows an obvious inflammatory infiltrate at 20 hrs, comprised of a mixture of monocytes and neutrophils.
  • intact SRBC are seen dispersed in the extravascular tissue space without any accompanying myelomonocytic inflammatory infiltrate.
  • Fig. 5 illustrates the time course of the footpad swelling seen after ag challenge of actively immunised mice in the presence or absence of 5C6.
  • the control sensitised mice showed significant increases in footpad thickness within 12 hrs of ag challenge. This swelling then reached a peak at 24 hrs, subsided to 42% of the peak level at 48 hrs and returned to the baseline at 72 hrs.
  • Mice injected with 5C6 at the time of ag challenge had an increase in footpad size of 0.08 10.07 mm at 24 hrs compared to 0.90 1 0.17 mm of the sensitised controls.
  • the 5C6-treated mice had an increase in footpad size of 0.39 1 0.07 mm which was not different to the declining increase of 0.3810.05 in the untreated sensitised mice. From 48 hrs onwards, the curves of the 5C6-treated and the control mice were virtually superimposable.
  • a second injection of 5C6 20hrs after ag challenge failed to alter appreciably the late increase in footpad size seen at 48 hrs in the 5C6 treated mice. Naive animals injected with SRBC in the footpad showed no increase in footpad size at any time.
  • Circulating levels of 5C6 IgG following a single injection of lmg IgG were analysed by serial dilution of plasma samples.
  • the anti-adhesive titre of the mAb was assayed in vitro using either bone marrow neutrophils or elicited M ⁇ as described in Example I and functionally saturating blood levels were maintained for at least 72 hrs.
  • FACS analysis of 5C6 binding to blood, bone marrow and peritoneal cavity cells after a single i.v. injection of 5C6 showed that saturation binding to blood myelomonocytic cells was achieved within 1 hr of injection and maintained for 72 hrs.
  • mice were injected i.v. with 5C6 and then immunised with SRBC.
  • sensitised T-cells were harvested, washed by centrifugation, mixed with SRBC and injected into the footpad of naive, syngeneic mice.
  • Table V shows that the footpad swelling seen after passive transfer of ag and blood leukocytes was similar using donor leukocytes recovered from mice immunised with SRBC in the presence or absence of systemic 5C6.
  • mAb 5C6 inhibits T-cell-dependent myelomonocytic recruitment but not ag-dependent T-cell priming or activation
  • Treatment Transfer to recipients Increase footpad thickness (recipients) at 15 hrs (mm)
  • mice Performed as described in Materials and Methods. Results reflect the mean + SD of 4 mice per group. Equivalent results were obtained in each of 2 separate experiments. We also examined the effect of 5C6 injection in recipient mice on hypersensitivity following passive local transfer of SRBC-reactive T-cells and SRBC. Local transfer led to acute onset of DTH and reached a peak at 15 hrs after ag challenge.
  • Table II shows that local transfer to naive, syngeneic recipients of sensitised blood leukocytes mixed with SRBC led to an increase in footpad size of 0.52 1 0.05 mm in control mice at 15 hrs whilst leukocytes or ag alone failed to elicit any response. If mice were injected with 5C6 i.v. at the time of local transfer of sensitised cells and ag, increase in footpad size was significantly diminished at 0.10 10.08 mm at 15 hrs. This result was confirmed by histologic observation.
  • the effect of 5C6 was not restricted to inhibiting T-cell-dependent recruitment of inflammatory cells to one ag alone at a single anatomical site.
  • Fig. 6 shows the effects of 5C6 on the inflammatory recruitment following transfer of sensitised lymphocytes and tuberculin to the peritoneal cavity of naive, syngeneic mice.
  • mice treated by the i.v. injection of 5C6 at the time of transfer of sensitised cells and tuberculin had only 4.28 1 2.22 ⁇ 10 6 peritoneal leukocytes at 48 hrs of which 47%: (2.01 ⁇ 10 6 ) were M ⁇ , 37, neutrophils and 50% lymphocytes.

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Abstract

L'anticorps décrit sert à inhiber le recrutement de cellules myélomonocytiques en réponse à des stimuli inflammatoires, c'est-à-dire dans le traitement ou la prophylaxie de maladies ou d'états pathologiques, telles que des maladies inflammatoires, d'auto-immunité et d'hypersensibilité, qui résultent du recrutement de cellules myélomonocytiques en réponse à des stimuli inflammatoires. Ledit anticorps est un anticorps présentant une spécificité pour le récepteur complémentaire de type 3 (CR3), tel que notamment sa sous-unité alpha, et est généralement un anticorps pouvant inhiber la fixation de cellules myélomonocytiques sur du plastique bactériologique.
EP88909567A 1987-11-10 1988-11-10 Anticorps contre le recepteur complementaire de type 3 (cr3) et composition therapeutique Ceased EP0386059A1 (fr)

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WO1998039659A1 (fr) * 1997-03-06 1998-09-11 Bion Diagnostic Sciences, Inc. Criblage et traitement au moyen d'un regulateur du complement ou de proteines receptrices du complement
US6221621B1 (en) 1997-03-06 2001-04-24 Bard Diagnostic Sciences, Inc. Methods of screening for colorectal cancers in which a complement Factor I or related protein is associated
US6663863B2 (en) 2000-03-17 2003-12-16 Millennium Pharmaceuticals, Inc. Method of inhibiting stenosis and restenosis
US11072661B2 (en) 2015-12-23 2021-07-27 Sorbonne Universite Agents that inhibit the binding of CFH to CD11 b/CD18 and uses thereof

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