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WO1994008620A1 - SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE - Google Patents

SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE Download PDF

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Publication number
WO1994008620A1
WO1994008620A1 PCT/US1993/009777 US9309777W WO9408620A1 WO 1994008620 A1 WO1994008620 A1 WO 1994008620A1 US 9309777 W US9309777 W US 9309777W WO 9408620 A1 WO9408620 A1 WO 9408620A1
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antibody
mac
atcc
binding
cbrm1
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PCT/US1993/009777
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English (en)
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Michael Diamond
Timothy A. Springer
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Center For Blood Research, Inc.
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Priority to EP93924919A priority Critical patent/EP0670734A4/fr
Priority to CA002144738A priority patent/CA2144738A1/fr
Priority to JP6510234A priority patent/JPH08502956A/ja
Priority to AU54423/94A priority patent/AU674556B2/en
Publication of WO1994008620A1 publication Critical patent/WO1994008620A1/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
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—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]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of cellular adhesion and the use of anti-adhesion molecules as a means of mediating biological processes which are facilitated by myeloid cell adhesion, migration and activation.
  • the present invention discloses that specific subpopulations of Mac-1 are activated when myeloid cells are stimulated and that upon activation, activation specific epitopes appear on the Mac-1 molecule.
  • a new class of anti-Mac- 1 antibodies are disclosed which are capable of binding to an activated form of Mac-1 present on stimulated myeloid cells but are substantially incapable of binding to non-activated Mac-1 and resting myeloid cells.
  • the integrin family of heterodimeric glycoproteins mediate cell adhesion and attachment to the extracellular matrix (Hynes, Cell •48:549-554 (1987); Hynes, Cell 69:11-25 (1992)).
  • the leukocyte integrin subfamily includes three members, LFA-1 (CDlla/CD18), Mac-1 (CDllb/CD18) and p 150,95 (CDllc/CD18) that share a common ⁇ subunit that is noncovalently associated with unique but closely related a chains (Kishimoto et al, Adv. Immunol. 46:149-182 (1989); Springer, Nature 546:425-433 (1990)).
  • glycoproteins share a common CD18 ⁇ subunit (95,000 M r ) but have individual unique CD11 ⁇ subunits (175,000, 160,000, 150,000 M r ) respectively, that are structurally homologous (Larson, et al, J. Cell Biol 108:703-712 (1989)). All three members share two prominent features in the extracellular region of the molecule, a putative divalent cation binding region consisting of three tandem repeats of an EF-hand motif, and a 200 amino acid inserted or "I" domain (Arnaout, et al., J. Cell Biol 106:2153-2158 (1988); Corbi, et al, J. Biol Chem.
  • the I domain is absent in all other known integrin ⁇ subunits except for the ⁇ , and ⁇ 2 subunits of the VLA subfamily of integrins (Hemler, Annu. Rev. Immunol 8:365-400 (1990); Hynes, Cell 69:11-25 (1992)).
  • the leukocyte integrins mediate several adhesive events that are crucial for immune system function. They promote the adhesion that is required for T lymphocyte target cell lysis (Davignon, et al. , Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)), T lymphocyte proliferation (Davignon, et al, Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)), natural killing (Krensky et al, J. Immunol 757:611-616 (1983)), leukocyte adhesion to, and migration through endothelial cells (Dustin et al, J. Cell Biol.
  • Mac-1 is expressed in an intracellular, vesicular compartment in circulating neutrophils and monocytes which is mobilized to the cell surface by inflammatory mediators (Todd, et al, J. Clin. Invest. 74:1280-1290 (1984); Springer, etal, In: Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 (1986); Lanier, et al, Eur. J. Immunol 75:713-718 (1985); Yancey, et al., J. Immunol 755:465-470 (1985)). This mobilization correlates with increased adhesiveness (Anderson, et al., Ann. Rev. Med. 58:175-194 (1987)).
  • Mac-1 ⁇ -subunit message was detected in blood monocytes and PMA-induced myeloid cell lines, but not in most cells of the T or B lineages, correlating with Mac-1 protein surface expression.
  • Some cytotoxic T lymphocyte clones have been found to express similar quantities of pl50,95 and LFA-1.
  • Monoclonal antibodies to the LFA-1 and pl50,95 alpha-subunits inhibit killing by such CTL clones to similar extent and are additive in their inhibitory effects (Keizer, et al, J. Immunol 758:3130-3136 (1987)).
  • pl50,95 alpha-subunits have been shown to inhibit monocyte attachment to endothelium (Keizer, et al, Eur. J. Immunol 77:1317-1322 (1987)).
  • Monoclonal antibodies to Mac-1 or p 150,95 inhibit neutrophil aggregation and adherence to endothelial cells, protein-coated surfaces, bacteria, protozoan parasites, and fungi (Harlan, et al., Blood 66: 167-178 (1985); Springer, et al , In: Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 (1986); Dana, et al, J. Immunol. 757:3259 (1986); Bullock, etal, J. Exper. Med. 765:195-210 (1987); Mosser, et al, J. Immunol. 755:2785-2789 (1985)).
  • Mac-1 (CDllb/CD18) is a leukocyte adhesion glycoprotein that has been demonstrated to bind multiple ligands including iC3b (Beller, et al., J. Exper. Med. 756:1000-1009 (1982)), fibrinogen (Altieri, et al., J. Cell. Biol 707:1893-1900 (1988); Wright, et al, Proc. Nat'l Acad. Sci. (U.S.A.) 85:7734-7738 (1988)),and Factor X (Altieri, etal., J. Biol Chem. 865:7007- 7015 (1988)) in addition to its role in cell-cell and cell-substrate adhesive interactions. Detergent-soluble Mac-1 and pl50,95 have been shown to bind to iC3b-Sepharose (Micklem, et al, Biochem. J. 257:233-236 (1985)).
  • the ⁇ -subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail.
  • the extracellular domain contains 3 putative divalent cation- binding sequences and 19 potential N-glycosylation sites.
  • the amino acid sequence of Mac-1 ⁇ shows that it is a member of the integrin superfamily; Mac-1 ⁇ shows 63% identity to the ⁇ -subunit of the leukocyte adhesion glycoprotein p 150,95 and 25% to the ⁇ -subunits of the extracellular matrix receptor platelet glycoprotein Ilb/IIIa, the fibronectin receptor and the vitronectin receptor.
  • the Mac-1 ⁇ -subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree 'of identity both to the p 150,95 ⁇ -subunit (87% identity at the amino acid level) and to the rest of the integrin ⁇ -subunits (38%).
  • the ⁇ -subunit of Mac-1 like the pl50,95 ⁇ - subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This inserted or "I" domain is homologous to the A domains of van Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2.
  • Mac-1 The functional role of Mac-1 was first illustrated by the ability of anti- Mac- 1 ⁇ -subunit monoclonal antibodies (MAb) to block the rosetting of iC3b- coated erythrocytes to macrophages and polymorphonuclear leukocytes (Beller, et al, J. Exper. Med. 756:1000-1009 (1982)), demonstrating that Mac-1 is indistinguishable from the complement receptor type three (CR3). Subsequently, the involvement of Mac-1 in inflammatory processes was evidenced by the inhibition of neutrophil aggregation and adhesion to endothelial cells by anti-Mac- 1 ⁇ -subunit and anti-/3-subunit-specific MAb (Anderson, et al., J.
  • MAb monoclonal antibodies
  • Mac-1 The expression of functional activity of Mac-1 is regulated during leukocyte differentiation and activation. Differentiation and maturation of myelomonocytic cell lines results in increased Mac-1 expression (Miller, et al. , J. Immunol. 137:2891-2900 (1986)), while blood monocyte differentiation into tissue macrophages is accompanied by a considerable decrease in the amount of Mac-1 on all cell surfaces (Hogg, et al, Eur. J. Immunol. 76:240- 248 (19886)).
  • Mac-1 The expression of Mac- 1 on the surface of circulating neutrophils and monocytes is upregulated by inflammatory stimuli; Mac-1 is stored in an intracellular vesicular compartment which is rapidly mobilized to the cell surface by chemoattractants (Todd, et al., J. Clin. Invest. 74:1280- 1290 (1984)); Miller, et al, J. Clin. Invest. 80:535-544 (1987)).
  • chemoattractants Todd, et al., J. Clin. Invest. 74:1280- 1290 (1984)
  • Miller, et al, J. Clin. Invest. 80:535-544 (1987) Although the augmented expression of Mac-1 can lead to increased adhesiveness, qualitative changes after cell activation may also be important in regulation ligand binding (Detmers, et al., J. Cell Biol. 705:1137-1145 (1987)). Both the qualitative and quantitative changes may be important in regulation of leukocyte binding to post
  • leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells) and proteins (such as iC3b). This adherence has been found to require contacts which involve specific receptor molecules present on the surface of the leukocytes. Some of these cell surface receptor molecules have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms.
  • Mac-1 on neutrophils does not bind its cellular or soluble ligands including ICAM-1 (Diamond, et al, Cell 65:961-971 (1991)), fibrinogen (Altieri, et al, J. Cell. Biol 707:1893-1900 (1988); Wright, et al., Proc. Natl. Acad. Sci. USA 85:7734-7738 (1988)), factor X (Altieri et al , J. Biol Chem. 265:7007- 7015 (1988)) or iC3b (Wright etal, J.
  • Mac-1 is hypothesized to undergo additional, conformational changes that facilitate adhesion (Buyon, et al., J. Immunol 740:3156-3160 (1988); Lo et al., J. Exp. Med. 769:1779-1793 (1989); Philips, et al, J. Clin. Invest. 82:495-501 (1988); Vedder et al., J. Clin. Invest. 87:676-682 (1988)).
  • Studies that show that divalent cations (Altieri, /. Immunol. 747: 1891-1898 (1991); Dransfield, et al, J. Cell Biol.
  • the platelet adhesion molecule ⁇ -m,-/ ⁇ (CD41/CD61) is the integrin family member that is best characterized for the subunit domains which contribute to ligand recognition.
  • Mac-1 is one of the most interesting of the integrins that contain an I domain as it promiscuously binds several soluble and cell surface ligands. Because previous studies had demonstrated that distinct Mac-1-dependent adhesive functions could be blocked by disparate groups of MAbs to Mac-1 a (Anderson, et al, J. Immunol. 137: 15-27 (1986); Dana, et al., J.
  • the present invention is based on the novel observation that when resting myeloid cells are stimulated, not all Mac-1 molecules which are present on the cell surface become activated. Only select sub-populations of Mac-1 molecules become activated and gain the ability to bind ligand.
  • the present invention further discloses that upon activation, activation specific epitopes appear on the Mac-1 molecule. These activation specific epitope can be used to differentiate activated Mac-1 molecules and stimulated myeloid cells from non-activated Mac-1 molecules and resting myeloid cells.
  • the present invention discloses the generation of a novel class of monoclonal antibodies, Mabs, which bind to activated Mac-1 molecules present on stimulated myeloid cells. These antibodies were also found to possess the novel ability to bind to purified Mac-1 substrates, as well as detergent solubilized cell lysates.
  • CBRM1/5 A member of this class of antibodies, hereinafter CBRM1/5, is described.
  • CBRM1/5 binds to a subset (10-30%) of Mac-1 molecules present on stimulated neutrophils (those that are activated and are capable of binding ligand).
  • CBRMl/5 was further found to be capable of blocking the binding of activated neutrophils to the Mac-1 ligands, fibrinogen and ICAM-1.
  • Another member of this class of antibodies, hereinafter CBRM1/19 was additionally isolated. This antibody, though binding to an activation specific epitope, recognizes a different subpopulation then that bound by CBRM1/5.
  • the present invention further provides methods of isolating other members of this class of antibodies.
  • antibodies which react with the same subpopulation of stimulated myeloid cells or the same subpopulation of activated Mac-1 can be identified by their ability to bind the same subpopulation of cells, or the same subpopulation of activated Mac-1 molecules as does CBRM1/5 or CBRM 1/19.
  • the present invention further provides methods of selectively inhibiting the binding of stimulated myeloid cells to a ligand of Mac-1.
  • stimulated myeloid cells can be blocked from binding to Mac-1 ligands by supplying to the myeloid cells an antibody, antibody fragment, or antibody derivative which is (1) capable of binding to activated Mac-1 present on stimulated myeloid cells, (2) is substantially incapable of binding to non- activated Mac-1 and resting myeloid cells, and (3) is capable of inhibiting the ligand/Mac-1 interaction which is being targeted.
  • CBRM1/5 when supplied to a myeloid cell, blocks ICAM-1 and fibrinogen binding.
  • the present invention further provides methods of treating various pathological conditions which are mediated by myeloid cell adhesion, migration, and ligand interaction.
  • an inflammatory response which is mediated by the non-specific defense system can be treated by administering an effective amount of an antibody based agent to a patient in need of such treatment.
  • the antibody based agent used in the present method is an antibody, an antibody fragment, or an antibody derivative which is capable of binding to activated Mac-1 present on stimulated myeloid cells but is substantially incapable of binding to non-activated Mac-1 and resting myeloid cells.
  • the present invention further provides methods of localizing the presence of stimulated myeloid cells within a subject. Specifically, the presence and location of cells expressing an activated form of Mac-1 can be determined within a subject by administering to the subject a detectably labeled antibody, or antibody fragment, which is capable of binding to activated Mac- 1 present on stimulated myeloid cells but is substantially incapable of binding to non-activated Mac-1 and resting myeloid cells. The location of the labeled antibodies or antibody fragments can then be detected using known methods.
  • the present invention further provides methods of identifying agents capable of activating Mac-1 present on resting myeloid cells. Specifically, resting myeloid cells, or deactivated Mac-1 molecules are contacted with an agent which is to be tested.
  • the cells are then contacted with an antibody which is capable of binding to activated Mac-1 present on stimulated myeloid cells but is substantially incapable of binding to non-activated Mac-1 and resting myeloid cells.
  • the cells, or purified Mac-1 are then examined to determine whether the antibody binds to the cells.
  • the present invention further provides methods of identifying agents capable of deactivating activated Mac-1 which is present on stimulated myeloid cells. Specifically, stimulated myeloid cells, or purified activated Mac-1 molecules are contacted with an agent which is to be tested. The cells are then contacted with an antibody which is capable of binding to activated Mac-1 present on stimulated myeloid cells but is substantially incapable of binding to non-activated Mac-1 and resting myeloid cells. The cells, or purified Mac-1 molecules are then examined to determine whether the cells bind the antibody.
  • the present invention further provides methods of selectively killing cells expressing activated Mac-1. Specifically, cells which have activated Mac-
  • toxin derivatized antibody which comprises a toxin moiety and an antibody, or antibody fragment which is capable of binding to activated Mac-1 surface but is substantially incapable of binding to non-activated Mac-1.
  • the present invention further provides methods of selectively removing cells expressing activated Mac-1 from a solution or fluid such as blood.
  • stimulated myeloid cells which contain activated Mac-1 on their cell surface can be selectively removed from fluid by passing the fluid over an immobilized antibody, or antibody fragment, which is capable of binding to activated Mac-1 but is substantially incapable of binding to non-activated Mac- 1.
  • Such a method can be used in standard leukophoresis type procedures to remove stimulated myeloid cells or myeloid tumor cells which express activated Mac-1 from a patients blood without effecting non-stimulated myeloid cells.
  • FIGURE 1 A. Immunofluorescence flow cytometry of peripheral blood neutrophils that were unstimulated or treated with PMA (100 ng/ml) for ten minutes at 37°C. Neutrophils were stained with either a negative control (X63), a MAb to Mac-1 ⁇ subunit (LM2/1), or CBRM1/5.
  • FIGURE 2 Saturation binding of CBRM1/5 IgG and Fab.
  • Neutrophils were incubated with 125 I-labelled CBRM1/5 either in the absence (open circles) or presence of excess unlabelled CBRM1/5 (open triangles) for 4 hours on ice.
  • Specific binding was measured by subtracting the non-specific from the total binding.
  • Panel B Inset A Scatchard plot of the specific binding from the curve in Panel B.
  • the calculated K, is 15 nM.
  • the x-intercept (1010 pM) is equivalent to 60,800 sites per neutrophil.
  • FIGURE 3 Kinetics of expression of Mac-1 epitopes. Neutrophils that were activated at 37°C for the indicated times with no stimuli (open triangles), fMLP (10" 7 M, solid squares), IL-8 (25 ng/ml, solid circles), or PMA (100 ng/ml, open circles) were immunostained with A, CBRM1/5 or B, LM2/1 and subjected to immunofluorescence flow cytometry. Data are expressed as relative linear fluorescence units and represent the mean of three experiments. The bars indicate the standard error of the mean.
  • FIGURE 4 The effect of temperature on Mac-1 expression on A, peripheral blood monocytes and B, neutrophils. Cells were incubated at 4°C, 25°C, and 37°C in the absence or presence of stimuli (fMLP, 10"TM PMA, 100 ng/ml) for ten minutes, and then immunostained with either a negative control (X63), a MAb to Mac-1 ⁇ subunit (LM2/1), or CBRM1/5. Representative histograms show the log fluorescence intensity versus cell number.
  • stimuli fMLP, 10"TM PMA, 100 ng/ml
  • X63 negative control
  • MAb to Mac-1 ⁇ subunit LM2/1
  • CBRM1/5 Representative histograms show the log fluorescence intensity versus cell number.
  • FIGURE 5 Schematic representation and flow cytometric analyses of Mac-l/pl50,95 chimeric molecules.
  • MAbs used for the flow cytometry were X63 (non-binding control), LM2/1 (anti-Mac-1 ⁇ ), OKM1 (anti-Mac-1 ⁇ ), BLY6 (anti-p 150,95 ⁇ ) and CBRM1/5. Representative histograms show the log fluorescence intensity versus cell number.
  • B Chimeric molecules were constructed by the ligation of fragments from either pl50,95 or Mac-1 cDNA, and expressed stably in CHO cells. Those parts of the chimers from Mac-1 are represented by open bars, those from pl50,95 are represented by shaded bars.
  • FIGURE 6 The region from the EcoRV to the Bglll restriction site constitutes the I domain.
  • FIGURE 6 Divalent cations affect expression of Mac-1 MAb epitopes.
  • A Neutrophils were incubated at 4°C (resting) and 37°C (fMLP, 10 "7 M) for ten minutes in HBSS that was supplemented by the indicated divalent cation or chelating agent.
  • Cells were immunostained with either a negative control (X63), a MAb to Mac-1 ⁇ subunit (LM2/1), or CBRM1/5. All subsequent washes were performed in the presence of the indicated divalent cation. Representative histograms show the log fluorescence intensity versus cell number.
  • FIGURE 7 SDS-5% PAGE analysis of immunoprecipitates from neutrophil lysates. Iodinated neutrophils were lysed in 1 % Triton-X-100 and aliquots (60 ⁇ l, 1:4 dilution) were incubated with directly coupled Sepharose (Lane 1, LM2/1; Lane 4, CBRM1/5; Lanes 7 and 10, Mouse IgG) for 2h at 4°C. The beads were pelleted, the pre-cleared supernatants were retrieved and reprecipitated with additional directly coupled Sepharose (Lane 2, LM2/1; Lane 5, CBRM1/5; Lanes 8 and 11, Mouse IgG) for 2h at 4°C.
  • sICAM-1 Neutrophils were allowed to bind to purified sICAM-1 in 6 cm Petri dishes for four minutes at room temperature in the presence of fMLP (10" 7 M) after a ten minute preincubation with the following MAbs: TS1/22 (anti-LFA-1 ⁇ , 1/250 dilution of ascites), LPM19c (anti-Mac-1 ⁇ , 1/250 dilution of ascites), CBRM1/5 (anti-Mac-1 ⁇ , 25 ⁇ g/ml IgG and Fab' 2 ; 50 ⁇ g/ml Fab), CBRM1/23 (anti-Mac-1 ⁇ , 25 ⁇ g/ml).
  • MAbs MAbs: TS1/22 (anti-LFA-1 ⁇ , 1/250 dilution of ascites), LPM19c (anti-Mac-1 ⁇ , 1/250 dilution of ascites), CBRM1/5 (anti-Mac-1 ⁇ , 25 ⁇ g/ml IgG and Fab'
  • Neutrophils were allowed to bind to purified fibrinogen in 6 cm Petri dishes for four minutes at room temperature in the presence of fMLP (10" 7 M) after a ten minute preincubation increasing concentrations of CBRM1/5 IgG or Fab.
  • the binding was determined by light microscopy (40X) after the unbound cells were removed by serial washes with a Pasteur pipette. Data is expressed as the percent inhibition of cell binding relative to a media control. Datapoints are the average of five microscopic fields and the bars indicate the standard error of the mean.
  • the graphs are representative experiments that were repeated several times.
  • FIGURE 9 Schematic representation of the leukocyte integrin a chain and Mac-l/pl50,95 chimeras. Restriction sites (e, EcoRV; b, Bglll; a, AflH) that facilitated reciprocal exchanges are indicated with arrows or in the name of each chimera.
  • Restriction sites e, EcoRV; b, Bglll; a, AflH
  • FIGURE 10 SDS-PAGE of Mac-1, pl50,95 or Mac-l/pl50,95 chimeras immunoprecipitated from 125 I-COS cell detergent lysates.
  • COS cells were cotransfected with the ⁇ subunit and (A) Mac-1, (B) pl50,95, (C) M-e- X-b-M, or (D) X-e-M-b-X cDNA, surface labelled with 125 I, and immunoprecipitated with the X63 (Lane 1, negative control), TS1/18 (Lane 2, anti-CD18), LM2/1 (Lane 3, anti-CDllb), Mn41 (Lane 4, anti-CDllb), VIM12 (Lane 5, anti-CDllb), OKMl (Lane 6, anti-CDl lb), OKM10,*, (Lane 7, anti-CDl lb), SHCL3 (Lane 8, anti-CDllc), BLY6 (Lane 9, anti
  • FIGURE 11 Flow cytometry profiles of CHO cells expressing Mac-1, pl50,95 and Mac-l/pl50,95 chimeras.
  • CHO cell transfectants (indicated on right) were immunostained with either a negative control (X63), a MAb to the I domain (LM2/1) or C-terminal (OKMl) region of the Mac-1 ⁇ subunit, a MAb to the C-terminal region of the pl50,95 ⁇ subunit (CBRpl50/4Gl), or a MAb to the CD18 ⁇ subunit (TS1/18).
  • X63 negative control
  • MAb to the I domain LM2/1
  • CBRpl50/4Gl C-terminal region of the pl50,95 ⁇ subunit
  • TS1/18 MAb to the CD18 ⁇ subunit
  • FIGURE 12 SDS-PAGE of Mac-1, pl50,95 or Mac-l/pl50,95 chimera immunoprecipitated from , 5 I-CHO cell detergent lysates.
  • CHO cells transfectants A) Mac-1, (B) X-e-M, (C) M-e-X, (D) X-a-M, (E) X-b-M, (F) X-e-M-b-X, (G) M-b-X-a-M, (H) pl50,95, (I) M-b-X, (J) X-b-M-a-X, (K) M-a-X, and (L) M-e-X-b-M were surface labelled with 125 I, and immunoprecipitated with a negative control MAb (X63, Lane 1), a MAb to the I domain of Mac-1 (LM2/1, Lanes A2, B2, F2, 12 and K2, a MAb to the C- terminal region of Mac-1 (OKMl,
  • FIGURE 13 Summary of MAb reactivity as determined by immunofluorescent flow cytometry with CHO cells transfected with wild type or chimeric Mac-1 and p 150,95 molecules. (+ + +) indicates that 100% of cells stained the MAb with a pattern similar to that shown in Figure 3. (+) indicates that 100% of cells stained positively with the MAb but with a significantly lower fluorescence intensity. (+/-) indicates that the MAb stained a subpopulation of cells of cells positively. (-) indicates that the MAb staining was not significantly different from the negative control (X63).
  • FIGURE 14 Summary of the functional effects of MAbs on Mac-1 interaction with ligands.
  • the assays for the interaction with the four ligands are described in the Methods.
  • the inhibition data with OKM 10 old was obtained from iC3b binding to transfected COS cells instead of neutrophils.
  • the data is expressed as the percent inhibition by each MAb and is the average of at least three independent experiments. Standard error of the means are indicated after the ⁇ _ sign. ND indicates that the value was not determined.
  • FIGURE 16 CHO cell transfectant binding to ICAM-1 (A) and iC3b-E
  • Erythrocytes (iC3b-E, 50 ⁇ l of 2 x 10 8 cells/ml) were added and incubated with transfectants for 60 minutes at 37°C. Non-adherent erythrocytes were removed after eight washes with a transfer pipette and rosettes (> 10 erythrocytes/CHO cell, > 100 cells examined) were scored by light microscopy at 100X magnification. The data is the average of three experiments. Bars indicate the standard error of the means.
  • the present invention is based on the novel observation that when resting myeloid cells are stimulated, not all of the Mac-1 molecules present on the cell surface become activated and capable of binding ligand. Only select sub-populations of Mac-1 molecules as well as subpopulation of myeloid cells become activated and capable of binding to ligands of Mac-1. Upon activation, activation specific epitopes appear on the Mac-1 molecule which differentiate the activated Mac-1 molecules and stimulated myeloid cells from the non- activated Mac-1 molecules and resting myeloid cells.
  • the present invention discloses the generation of a novel class of monoclonal antibodies, MAbs, which bind to activated Mac-1 molecules present on stimulated myeloid cells but are substantially incapable of binding to non-activated Mac-1 molecules on stimulated and resting myeloid cells. These antibodies were also found to possess the novel ability to bind to purified Mac-1 substrates, as well as detergent solubilized cell lysates.
  • Anti-Mac-1 MAbs are generated using routine procedures known in the art (Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)) using either purified Mac-1 or cells expressing activated Mac-1 as an immunogen.
  • antibodies which selectively bind to activated Mac-1 can be identified in several fashions.
  • antibodies which selectively bind stimulated myeloid cells can be identified by screening the antibody for the ability to bind to stimulated myeloid cells such as neutrophils, and the inability to bind to resting myeloid cells.
  • antibodies which bind stimulated myeloid cells can be identified by screening the antibody for the ability to bind to purified activated Mac-1, and the inability to bind to purified deactivated Mac-1.
  • a monoclonal antibody is said to "selectively bind" to activated Mac-1 or stimulated myeloid cells when the antibody binds to stimulated myeloid cells or activated Mac-1 but is substantially incapable of binding to non-activated Mac-1 or resting myeloid cells.
  • An antibody is said to be “substantially incapable” of binding to resting myeloid cells if the level of binding to the resting myeloid cells is less than 10% of the level of binding to stimulated cells.
  • a myeloid cell is said to be stimulated when the cell is capable of binding to a ligand of Mac-1 or is capable of participating in a Mac-1 dependent biological function.
  • a stimulated myeloid cell is a cell which is capable of binding to ICAM-1, iC3b, fibrinogen, and/or is capable of participating in Mac-1 dependent fractions such as homotypic aggregation.
  • Treatments which stimulate myeloid cells include, but are not limited to agents such as fMLP and PMA.
  • a myeloid cell is said to be resting when the cell is incapable of binding to a ligand of Mac-1 or is incapable of participating in a Mac-1 dependent biological function. Therefore, a resting myeloid cell is a cell which is incapable of binding to ICAM-1, iC3b, fibrinogen, and/or is incapable of Mac-1 dependent homotypic aggregation.
  • differential screening is performed using two populations of myeloid cells, or purified Mac-1 molecules. Myeloid cells, such as neutrophils, and Mac-1 molecules (activated and de-activated forms) are isolated using known methods. When using purified cells, the purified cells are separated into two populations.
  • the two populations are preincubated under conditions which keeps Mac-1 on these cells in a resting conformation, for example at about 37'C for about five minutes in HHMC.
  • One of the population of cells is incubated under conditions which activate Mac-1, for example, in a media containing lOOng/ml PMA, while the other population is maintained in the resting medium.
  • the antibodies which are to be screened are then incubated with a sample of the two populations of cells, or a sample of purified activated and de-activated Mac-1, and antibodies are selected which selectively bind to the activated population of myeloid cells, or purified activated Mac-1 molecules.
  • Any method for screening antibody binding to a cell or molecule can be used. These include, but are not limited to, flow cytometry, ELISA, and RIA.
  • it may be preferable to fixed the myeloid cells prior to incubation with the antibody for example by incubating the cell in 2% paraformaldehyde.
  • the above procedures can be performed with two populations of cells expressing recombinant Mac-1.
  • Any cell capable of expressing Mac-1 can be used. These include, but are not limited to yeast, bacteria such as E. coli, mammalian cells and insect cells.
  • CBRM1/5 or CBRM1/19 as a marker of a distinct subpopulation of stimulated myeloid cells and activated Mac-1 molecules, other members of this class of antibodies can be identified.
  • Antibodies which bind to the same activation specific epitope, or to a sterically overlapping activation specific epitope on the activated Mac-1 molecule as that bound by CBRM1/5 or that bound by CBRM1/19 can be identified using a two antibody binding assay or a competitive binding assay. Such antibodies may recognize the same sub-population of Mac-1 molecules as that bound by CBRM1/5 or CBRM1/19 or may bind to a different subpopulation of activated Mac-1 molecules.
  • activation specific epitope refers to an epitope which is present on activated Mac-1 molecules but is not present on non-activated Mac-1 molecules. Such epitopes are often referred to as a neoepitope.
  • two epitopes are said to be the "same” or “sterically overlapping” if antibodies to the two epitopes compete with and exclude each other's ability to bind to the antigen.
  • Antibodies which bind to different or non-sterically overlapping activation specific epitopes can be identified by their ability to non- competitively bind to the same sub-population of stimulated myeloid cells or the ability to non-competitively bind to the same sub-population of activated Mac-1 as that bound by CBRM1/5 or that bound by CBRM1/19.
  • any of the known method for testing whether two antibodies bind to the same population of cells or the same molecule can be modified by one skilled in the art to identify antibodies which bind to the same population of cells or molecules in either a competitive or non-competitive fashion, as that bound by CBRM1/5 or that bound by CBRM1/19.
  • assays include, but are not limited to, flow cytometry and ELISA analysis.
  • the present invention further provides the antibodies CBRM1/5 and CBRM1/19 in substantially purified form.
  • an antibody is said to be “substantially purified” if it purified to the level necessary in order to be effectively administered to a subject or to be of use for a desired purpose. In some cases the antibody will be substantially ftee from all cellular components while in other instances the antibody will only be free from other immunoglobulin like proteins.
  • the present invention further provides a hybridoma capable of producing the CBRMl/5 antibody.
  • the hybridoma producing this antibody has been deposited at the ATCC and designated .
  • the present invention further provides a hybridoma capable of producing the CBRM1/19 antibody.
  • the hybridoma producing this antibody has been deposited at the ATCC and designated .
  • the present invention further includes fragments of the antibodies of the present invention which maintain their ability to bind to stimulated myeloid cells or to activated Mac-1. Such fragment include, but are not limited to, the Fv, the Fab, and the Fab 2 fragments. One skilled in the art can readily generated antibody fragments which maintain binding ability using routine methodology.
  • the present invention further includes derivatives of the antibodies of the present invention (antibody derivative). As used herein, an "antibody derivative" contains an antibody of the present invention, or a fragment of said antibody, as well as an additional moiety which is not normally a part of the antibody.
  • Such moieties may improve the antibodies' solubility, absorption, biological half life, etc, or may alternatively decrease the toxicity of the antibody, eliminate or attenuate any undesirable side effect of the antibody, etc.
  • Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).
  • Toxin-derivatized antibodies constitute a special class of antibody derivatives.
  • a "toxin-derivatized” antibody is an antibody, or antibody fragment which contains an additional toxin moiety. The binding of such an agent to a cell brings the toxin moiety into close proximity to the cell and thereby promotes cell death. Any suitable toxin moiety may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the diphtheria toxin, radioisotopic toxins, membrane-channel-forming toxins, etc. Procedures for generating such toxin derivatives are well known in the art.
  • Detectably labeled antibodies constitute another special class of the antibody derivatives of the present invention.
  • An antibody is said to be detectably labeled if the antibody, or fragment thereof, is attached to a molecule which is capable of identification, visualization, or localization using known methods.
  • the detectable labels of the present invention include, but are not limited to, radioisotopes labels, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc. Procedures for accomplishing such labeling are well known to the art.
  • the present invention further includes humanized forms of the antibodies of the present invention.
  • Humanized forms of the antibodies of the present invention may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Cabilly, et al, European Patent Application 125,023; Better, et al, Science 240:1041-1043 (1988); Liu, et al, Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, et al., J. Immunol 759:3521-3526 (1987); Wood, et al, Nature 574:446-449 (1985)); all of which references are incorporated herein by reference).
  • the present invention further provides methods of selectively inhibiting the binding of stimulated myeloid cells to a ligand of Mac-1.
  • a ligand of Mac-1 is defined as a non-antibody molecule which is capable of binding to Mac-1.
  • Ligands of Mac- 1 include, but are not limited to, iC3/b, ICAM-1, and fibrinogen.
  • stimulated myeloid cells can be blocked from binding to a Mac-1 ligand by supplying to the myeloid cells an antibody, antibody fragment, or antibody derivative which is capable of binding to activated Mac- 1 present on stimulated myeloid cells and is substantially incapable of binding to non-activated Mac-1.
  • CBRM1/5 when supplied to a stimulated myeloid cell, will bind to the activated Mac-1 molecules present on the cell and block the binding of the cell to ICAM-1 and fibrinogen.
  • activation specific anti-Mac- 1 antibodies which inhibit other Mac-1/ligand interactions, such as Mac-l/iC3b binding, can be isolated.
  • the adhesion, migration, and biological activity of circulating myeloid cells results from interactions involving
  • Mac-1 and it's ligands have been found to be required for neutrophil and monocyte migration to sites of inflammation and for various neutrophil and monocyte effector functions contributing to inflammation.
  • Non-activation specific anti-Mac- 1 antibodies which inhibit such cellular adhesion have been demonstrated to be effective at preventing an inflammatory response when administered to a human subject.
  • anti-Mac- 1 antibodies have been demonstrated to inhibit phagocytosis of foreign material by monocytes.
  • antibodies which bind to Mac-1 have been demonstrated to be effective at mediating other monocyte and neutrophil activities such as chemokinesis, and chemotaxis both in vitro as well as in vivo.
  • the antibodies, the antibody fragments, and the antibody derivatives of the present invention agents provide an improvement over previously disclosed anti-Mac- 1 antibodies for use as an anti-inflammatory agent, an anti-malarial agent, an anti-HIV agent, and an anti-asthma agent, for example, see Fisher et al. , Lancet 2: 1058 (1986), Perez et al , Bone Marrow Transp. 4:379 (1989), Patarroyo et al. , Scan J. Immunol 50:129-164 (1989), Lindbom et al, Clin. Immun. Immuno Patholo. 57:105- 119 (1990), Butcher, Amer. J. Pathol.
  • inflammation is meant to include only reactions of the non-specific defense system.
  • a "reaction of the non-specific defense system” is a response mediated by myeloid cells incapable of immunological memory. Such cells include neutrophils and macrophages.
  • inflammation is said to result from a response of the non-specific defense system, if the inflammation is caused by, mediated by, or associated with a reaction of the non-specific defense system.
  • inflammation which result, at least in part, from a reaction of the non-specific defense system include inflammation associated with conditions such as: adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to septicemia or trauma; reperfusion injury of myocardial or other tissues; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis; granulocyte trans- fusion associated syndromes; and cytokine-induced toxicity.
  • ARDS adult respiratory distress syndrome
  • multiple organ injury syndromes secondary to septicemia or trauma reperfusion injury of myocardial or other tissues
  • acute glomerulonephritis reactive arthritis
  • dermatoses with acute inflammatory components acute purulent meningitis or other central nervous system inflammatory disorders
  • thermal injury hemodialysis
  • leukapheresis ulcerative
  • the activation specific anti-Mac-1 antibodies of the present invention, fragments of said antibodies, and derivatives thereof, are administered to a patient as an anti-inflammatory agent to treat the above recited condition as well as others conditions which are mediated by the non-specific defense system.
  • agents differ from general anti-inflammatory agents in that they are capable of selectively targeting stimulated myeloid cell populations and inhibit Mac-1/specific ligand interaction. Such specificity will only affect specific biological function of the myeloid cell and will not cause side effects such as nephrotoxicity and myeloid cell depletion which are found with conventional and less specific anti-inflammatory agents. Further, such agents differ from other previously known anti-Mac- 1 antibody agents in that they do not affect non-activated myeloid cell populations. Therefore, the antibodies, antibody fragments, and antibody derivatives of the present invention provide an improvement over all the previously recognized usage of anti-Mac- 1 antibodies such as the treatment of inflammation resulting from a reaction of the non-specific defense system.
  • activation specific anti-Mac- 1 antibodies to inhibit the interactions necessary for an inflammatory reaction provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes Reynaud's syndrome, rheumatoid arthritis, etc.
  • Such antibodies may also be employed as a therapy in the treatment of psoriasis.
  • the monoclonal antibodies capable of binding to activated Mac-1 may be employed in the treatment of those diseases currently treatable through steroid therapy and in which previous know non-activation specific anti-Mac- 1 antibodies have been suggested.
  • antibodies to activated Mac-1 which recognize the same subpopulation of stimulated myeloid cells or activated Mac-1 as that bound by the antibody CBRM1/5.
  • Such antibodies can be used to block myeloid/ICAM-1 and myeloid/fibrinogen binding and hence can be used in vivo as a means of treating an inflammatory response of the non-specific defense system without affecting the reactions of the specific defense system and without the potential side effects of depleting non-activated neutrophils.
  • the present invention further provides methods of determining the location and distribution of expressing activated Mac-1 cells within a subject.
  • the administration of detectably labeled activation specific anti-Mac- 1 antibodies to a patient provides a means for imaging or visualizing the location, migration, and aggregation of cells expressing activated Mac-1 such as stimulated myeloid cells and myeloid tumor cells.
  • Clinical application of antibodies in diagnostic imaging are reviewed by Grossman, Urol. Clin. North Amer. 75:465-474 (1986)), Unger, etal., Invest. Radiol. 20:693-700 (1985)), and Khaw, et al, Science 209:295-297 (1980)).
  • the antibodies of the present invention are administered to a patient in detectably labeled form.
  • antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc using procedures known to the art for use in the present invention.
  • the present invention further provides methods of selectively killing expressing activated Mac-1 cells.
  • cells expressing activated Mac-1 can be selectively killed by contacting them with a "toxin derivatized antibody" which is capable of binding to activated Mac-1 but is substantially incapable of binding to non-activated Mac-1.
  • the antibody derivatives of the present invention include antibodies which are conjugated to toxic molecules such as the ricin A chain.
  • the present procedure provides a means of both treating Mac-1 dependent biological process by selectively killing the myeloid cells which mediate the process as well as a means of selectively killing tumor cells expressing activated Mac-1.
  • the dosage of the agent which is to be administered will vary depending upon such factors as whether the antibody agent is being administered as a diagnostic agent or a therapeutic agent. Factors such as the patient's age, weight, height, sex, general medical condition, previous medical history, etc will also affect the dosage administered.
  • one compound is said to be additionally administered with a second compound when the administration of the two compounds is in such proximity of time that both compounds can be detected at the same time in the patient's serum.
  • the activation specific anti-Mac- 1 antibodies of the present invention, or fragment thereof, whether for use as a diagnostic or therapeutic agent may be administered to patients intravenously, intranasally, intramuscularly, subcutaneously, enterally, or parenterally. When administering such molecules by injection, the administration may be by continuous infusion, or by single or multiple boluses.
  • the anti-inflammatory agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to suppress the binding of activated Mac-1 to the Mac-1 ligand which mediates the biological function which is to be inhibited.
  • antibodies used to treat inflammation are administered to a patient in sufficient concentration so as to inhibit ICAM-l/Mac-1 binding.
  • An amount is said to be sufficient to "suppress" Mac-1/ligand interaction if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent Mac- 1 /ligand interaction.
  • Such attenuation or prevention can be assayed by examining whether the biological function mediated by the targeted interaction is occurring in vivo, for example by examining neutrophil infiltration into a site of inflammation, or by correlating in vitro blocking studies with predicted in vivo efficacy.
  • the activation specific anti-Mac- 1 antibodies of the present invention may be administered either alone or in combination with one or more additional immunosuppressive agents (especially to a recipient of an organ or tissue transplant).
  • the administration of such compound(s) may be for either a "prophylactic" or "therapeutic" purpose.
  • the compound(s) are provided in advance of any inflammatory response or symptom.
  • the prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent inflammatory response.
  • the compound(s) is provided at (or shortly after) the onset of a symptom of actual inflammation.
  • the therapeutic administration of the compound(s) serves to attenuate any actual inflammation.
  • the anti-inflammatory agents of the present invention may, thus, be provided either prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient. Such an composition is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the antibody based agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g.
  • compositions suitable for effective ad ⁇ ministration will contain an effective amount of anti-Mac- 1 antibody.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the therapeutic agents of the invention.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate anti-Mac- 1 antibodies into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcelluloseorgelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the present invention further provides methods of identifying agents capable of activating Mac-1 present on resting myeloid cells. Agents can be assayed for their ability to stimulate resting myeloid cells or activate Mac-1 by examining treated cells, or purified Mac-1 molecules which have been treated with the agent for the appearance of activation specific epitopes.
  • isolated myeloid cells such as neutrophils or isolated de ⁇ activated Mac-1 molecules are first contacted with the agent which is to be tested.
  • the treated cells or Mac-1 molecules are then contacted with an antibody which is capable of binding to activated Mac-1 but is substantially incapable of binding to non-activated Mac-1 and resting myeloid cells.
  • the cells or Mac-1 molecules are then examined to determine whether the cells or molecules binds to the activation specific antibody. If the cells or Mac-1 molecules are able to bind such an antibody, the agent which was placed in contact with the cells Mac-1 or molecules are said to be a "stimulating agent.”
  • the present invention further provides methods of identifying agents capable of deactivating Mac-1.
  • stimulated myeloid cells such as
  • PMA treated neutrophils, or isolated activated Mac-1 molecules are first contacted with the agent which is to be tested.
  • the treated cells or Mac-1 molecules are then contacted with an antibody which is capable of binding to activated Mac-1 but is substantially incapable of binding to non-activated Mac- 1 or to resting myeloid cells.
  • the cells or Mac-1 molecules are then examined to determine whether the cells or Mac-1 molecules binds to the antibody. If the cells or molecules are no longer able to bind to the antibody, the agent which was first placed in contact with the cells or Mac-1 molecules are said to be a "deactivating agent. "
  • the present invention further provides methods of identifying agents capable of blocking the activation of Mac-1 present on resting myeloid cells.
  • resting myeloid cells such as neutrophils, or isolated de-activated Mac-1 molecules
  • agent capable of stimulating myeloid cells, or activating Mac-1 molecules, for example PMA.
  • These cells or Mac-1 molecules are then incubated with an antibody which is capable of binding to activated Mac-1 but is substantially incapble of binding to non-activated Mac-1 or to resting myeloid cells.
  • the cells or Mac-1 molecules are then examined to determine whether the cells or Mac-1 molecules binds to the antibody.
  • the present invention further provides methods of selectively removing cells from fluids or tissues which contain activated Mac-1 on their cell surface.
  • stimulated myeloid cells which contain activated Mac-1 can be selectively removed from a fluid or tissue, such as a patient's blood, by passing the fluid over an immobilized antibody which is capable of binding to activated Mac-1 present on stimulated myeloid cells but is substantially incapable of binding to non-activated Mac-1 or resting myeloid cells.
  • stimulated myeloid cells or tumor cells expressing activated Mac-1 can be removed from a patient's blood by subjecting the patient to leukophoresis in which the leukophoresis contacts the patient's blood with an immobilized, activation specific anti-Mac- 1 antibody.
  • the present invention is further based on the novel observation that a majority of the antibodies which block Mac-1/ligand interactions bind to regions within the I domain of the Mac-1 molecule (see example 2). Further, it was found that antibodies could be generated which block specific subsets of Mac-1/ligand interaction, for example CBRMl/1 completely blocked Mac- 1/ICAM-l interaction but had no effect on Mac-1/neutrophil binding. Based on this observation, the present invention provides methods of identifying agents which are capable of blocking specific Mac-1/ligand interaction.
  • the antibodies described in Figures 13 and 14, or an equivalent set of antibodies can be used to map the peptide residues within the Mac-1 molecule which bind to specific Mac-1 ligands.
  • a similar approach to epitope mapping is described in co-pending U.S. Ser. No. 07/618,286, filed November 28, 1990, herein incorporated by reference.
  • the present invention discloses that the sites of interaction between Mac-1 and the various Mac-1 ligands (iC3b, ICAM-1, and fibrinogen) are located within the I domain of Mac-1 and are distinct and mostly do not overlap. Based on this disclosure short peptide fragments of the Mac-1 sequence can be generated which are substantially capable of blocking specific subsets of Mac-1/ligand interaction.
  • specific subsets of Mac-1/ligand interaction refers to an agent's ability to block one or more Mac-1/ligand interactions.
  • a peptide may block a specific subset of Mac-1/ligand interactions by blocking Mac-1 binding to a single ligand such as ICAM-1.
  • peptides which bind to the Mac-1 contact site on the Mac-1 ligand which comprises amino acid sequences derived from the amino acid sequence of Mac-1, as well as peptide which bind to the ligand binding site present on
  • Mac-1 which comprise amino acid sequences derived from the Mac-1 ligand.
  • the monoclonal antibody blocking data presented herein can be combined with routine mutagenesis studies in order to further map MAb epitopes to distinct regions within the I domain of Mac-1.
  • amino acid substitution and domain deletion mutagenesis of Mac-1 can be performed, for example by oligonucleotide directed mutagenesis as described by Kunkel, ⁇ Proc. Natl Acad. Sci. USA, 82:488-492, (1985)), as modified by Peterson et al. (Nature, 529:842-846 (1987)), in order to generate Mac- 1 deletion and substitution mutants which lack the ability to bind to a specific Mac-1 ligand or antibody.
  • short peptide sequences which serve as the ligand contact site on Mac-1 can be determined.
  • the present invention further provides chimeric molecules comprising a fusion between Mac-1 and pl50,95, see Example 2.
  • Such chimeric molecules are useful in mapping epitopic domains and ligand contact regions within the Mac-1 and pl50,95 molecules.
  • chimeric molecules can be generated which maintain the ability to associate with the 0-subunit (CD11) of the protein dimer and participate in a select set of ⁇ -subunit ligand binding.
  • a chimeric Mac-l/pl50,95 ⁇ subunit can be generated which maintains the ability to form a dimer with CD18 as well as bind ICAM-1 and fibrinogen, but is incapable of neutrophil aggregation or Mac-1 dependent homotypic aggregation. Methods of generating such chimeras, as well as suggestions of their use are described in co-pending U.S. Ser. No. 07/618,286, filed November 28, 1990, herein incorporated by reference.
  • TS 1/22 anti-CD 11a, IgGl, ascites
  • LM2/1 anti-CDllb, IgGl, protein A-purified
  • CBRM1/23 anti-CDllb, IgG2a, protein-A purified
  • OKMl anti-CDllb, IgG2b, protein-A purified
  • LPM19c anti-CDllb, IgG2a, a gift of Dr. K. Pulford, Oxford, UK (Uciechowski et al , In Leukocyte Typing TV: White Cell Differentiation Antigens, Knapp editor, Oxford University Press, Oxford, pages 543-551 (1989)); BLY6 (anti-CDllc, igGl, ascites (Uciechowski et al., In Leukocyte Typing TV: White Cell Differentiation Antigens, Knapp editor, Oxford University Press, Oxford, pages 543-551 (1989)); %6.5 (anti-CD54, IgG2a, generous gift of Dr. R.
  • Mac-1 was purified from leukocyte lysates by immunoaffinity chromatography after detergent solubilization as described (Diamond et al. , Cell 65:961-971 (1991)). Soluble IC AM- 1 (sICAM-1, generous gift of Dr. S. Marlin, Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT) was purified from supernatants of ICAM-1 transfected CHO cells by immunoaffinity chromatography as described (Marlin et al, Nature 544:70-72 (1990)).
  • Neutrophils were isolated from the whole blood of healthy volunteers by dextran sedimentation at room temperature, with Ficoll gradient centrifugation and hypotonic lysis at 4 * C as described (English et al, J. Immunol. Methods 5:249 (1974), Miller et al., J. Clin. Invest. 80:535-544 (1987)).
  • neutrophils (2 x 10 7 cells/ml) were stored at 4 * C at in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl 2 , 1 mM CaCl 2 (HHMC) in polypropylene tubes (Falcoln 2097, Becton Dickinson, Lincoln Park, NJ).
  • the leukocyte rich supernatant was used after dextran sedimentation.
  • Cell suspensions were placed on ice immediately and washed four times to remove platelets in HBSS, 10 mM HEPES pH 7.3, and resuspended at 5 x 10° cells/ml in HHMC.
  • Neutrophils were identified during flow cytometry by forward and 90° scatter, and confirmed by immunostaining with a MAb to CD16.
  • Mononuclear cells were isolated from the interface of the Ficoll gradient and washed five times in RPM1 1640, 5 mM EDTA, 2.5% FCS (heat-inactivated, low endotoxin, Hyclone, Utah), and resuspended in L15, 2.5% FCS, 1 mg/ml human ⁇ - globulin (ICN ImmunoBiologicals, Costa Mesa, CA) at 5 x 10 6 cells/ml at 4°C. Mononuclear cells were immunostained according to the protocol for neutrophils (see below). The monocyte subpopulation was determined by the flow cytometric forward and 90° scatter pattern and confirmed by immunostaining with the a MAb to the monocyte-specific CD14 antigen.
  • CHO cells expressing wild type and chimeric forms of Mac-1 and pl50,95 will be described. They were maintained in ⁇ -MEM, 10% dialyzed FCS, 16 ⁇ M thymidine, 0.05 ⁇ M methotrexate, 2 mM glutamine, and 50 ⁇ g/ml gentamicin.
  • hybridomas were prepared from BALB/c mice that were immunized with immunoaffinity purified Mac-1.
  • Hybridomas 500 were screened as follows. Purified neutrophils were isolated, separated into two groups, and preincubated at 37°C for five minutes in HHMC. One population was treated with phorbol esters (PMA, 100 ng/ml) for ten minutes while the other remained untreated. Cell suspensions were placed on ice for five minutes, incubated with hybridoma supernatants, and processed by flow cytometry. In some cases, neutrophils were fixed with 2% paraformaldehyde prior to incubation with MAbs.
  • PMA phorbol esters
  • CBRM1/5 was identified for its ability to bind PMA stimulated but not resting neutrophils. It was cloned twice by limiting dilution, and purified from culture supernatant by protein-A Sepharose affinity chromatography after NH 4 SO 4 precipitation (Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor: Cold Spring Harbor Laboratory (1988)).
  • CBRMl/5 was determined to be of the IgGl subclass (ImmunoPure Monoclonal Antibody Isotyping Kit, Pierce, Rockford, IL)
  • Fab fragmentation of CBRM1/5 was performed by pepsin digestion followed by cysteine reduction (Parham, P., In Immunological Methods in Biomedical Sciences, Weir et al editors, Blackwell, Oxford (1983)).
  • Fab' were separated by 1.5 cm x 75 cm S-200 Sephacryl (Pharmacia, Piscataway, NJ) size exclusion chromatography. Fab' eluted from the column as a single homogeneous peak with an apparent M r of 40,000.
  • MAbs (100 ⁇ g) were labelled with Na 125 I (1 mCi) using 1,3,4,6- tetrachloro-3 ⁇ ,6 ⁇ -diphenylglycouril (Pierce Chemical Co, Rockford, IL)
  • Purified neutrophils (2 x 10 7 cells/ml in HHMC) were either kept on ice or warmed to 37°C for five minutes and stimulated (PMA, 100 ng/ml; fMLP, 10" 7 M) for ten minutes.
  • Activated neutrophils were immediately placed on ice and 500,000 cells were aliquotted into U-bottom plates (96 well non- tissue culture treated microtiter plates, Linbro-Titertek, Flow Laboratories, McLean, VA) containing increasing concentrations of iodinated MAb (0.109 nM - 112 nM) in the absence or presence of 20-100 fold excess cold MAb.
  • the binding media contained L15, 2.5% FCS, 1 mg/ml human -globulin.
  • Neutrophils were incubated with iodinated MAbs for 4h on ice, washed five times with L15, 2.5% FCS by 21 gauge needle aspiration, lysed with L15, 2.5% FCS, 0.2N NaOH, and counted for 7-emission. Specific binding was determined by subtracting the CPM in the presence of excess cold MAb. Scatchard plots were generated (Scatchard, G. Ann N. Y. Acad. Sci. 57:660- 672 (1949)) and site densities were determined using the data point at which there was no additional increase in specific binding of labelled MAb. Surface Labelling. Immunoprecipitation. and Gel Electrophoresis
  • Immunoprecipitations were performed one of two ways: (i) indirectly by incubating 250 ⁇ l of MAb (neat supernatant or 20 ⁇ g ml purified IgG) with 1 ⁇ l of 1 mg/ml purified rabbit anti-mouse IgG (Zymed, San Francisco CA) for 4 hours at 4°C. Protein- A-Sepharose (20 ⁇ l of a 1:1 slurry) was added for overnight incubation.
  • Immunoaffinity purified human Mac-1 was diluted 1/20 in 25 mM Tris- HC1, 150 mM NaCl, 2 mM MgCl 2 and adsorbed to individual wells of a non- tissue culture treated microtiter plate (Linbro-Titertek, Flow Laboratories, McLean, VA) for 1 h at 37 ° C. Plates were washed five times and blocked for one hour at 37°C in HHMC supplemented with 1 % human serum albumin.
  • Enzyme linked second antibody 50 ⁇ l of 1/400 dilution of horseradish peroxidase goat anti-mouse IgG, Zymed Laboratories, San Francisco, CA was added in HHTcation, incubated for 1 h at 4°C, and the plates were washed five times with HHTcation at 4°C.
  • Substrate (2,2-azino- di(3-ethylbenzthiazoline) sulfonic acid, Zymed Laboratories, San Francisco, CA) was added in 0.1 M citrate with 0.05% H 2 O 2 , and the results quantitated at 414 nm on an ELISA plate reader (Titertek Multiscan MCC340).
  • Purified fibrinogen 2.0 mg/ml, in PBS, Sigma Chemical Co., St. Louis, MO
  • purified sICAM-1 200 ⁇ g/ml in PBS
  • sICAM-1 200 ⁇ g/ml in PBS
  • Plates were blocked with the detergent Tween 20 as described (Diamond et al. , Cell 65:961-971 (1991)).
  • Neutrophils (4 x 10° cells in 1 ml) were resuspended in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl 2 and preincubated with MAbs for 10 minutes at room temperature.
  • fMLP 10 "7 M, final volume of 3 ml
  • Non-adherent cells were removed by twelve washes with a Pasteur pipette after gentle swirling with same buffer supplemented with 0.5% BSA. Binding was quantitated by scoring the number of adherent cells of at least four different fields using an ocular grid at 100X magnification. The percent inhibition by MAb was determined upon comparison with the media control.
  • the binding of ICAM-1 + L cells is a modification of previously described protocols (Diamond et al., J. Cell Biol. 777:3129-3139 (1990)).
  • Mac-1 was diluted and adsorbed (30 ⁇ l) to 6 cm Petri dishes. After a 90 minute incubation at room temperature, non-specific binding sites were blocked with 0.5% heat-treated BSA.
  • ICAM-1 + L cells after removal from tissue culture plates with trypsin-EDTA (GIBCO, Long Island, NY), were washed twice and resuspended in PBS, 2 mM MgCl 2 , 0.5% heat-treated BSA (0.5-1.0 x 10 6 cells/ml).
  • the Petri dishes or cells were preincubated at room temperature with MAb (1/200 dilution of ascites or 20 ⁇ g/ml of purified MAb) in PBS, 2 mM MgCl 2 , 0.5% heat-treated BSA (2 mis). Cells (1 ml) were added to Petri dishes, and incubated for 60 minutes at 37°C. The Petri dishes then were swirled and non- adherent cells were removed by tipping the plate at an angle, removing the binding buffer with a Pasteur pipette, and adding fresh media (3 ml) to the edge of the dish. This procedure was repeated five times. Subsequently, the number of adherent cells was determined by light microscopy at 100X using an ocular grid. The percentage binding was normalized to a media control.
  • CBRM1/5 One MAb, CBRM1/5, showed little binding to resting neutrophils but bound to neutrophils that were stimulated with phorbol esters (Figure la). CBRM1/5 bound to activated cells at a significantly lower level compared to previously characterized MAbs to Mac-1. Immunoprecipitation from detergent lysates of surface labelled neutrophils ( Figure lb) or transfected COS cells (data not shown) confirmed that CBRM1/5 recognized Mac-1.
  • Neutrophils stimulated with fMLP or PMA had a total Mac-1 site density of 139,000 and 197,000 sites/cell (determined with LM2/1) but a CBRM1/5 neoepitope site density of 13,000 to 57,000 sites/cell, respectively (Table 1). Bivalent binding of CBRM1/5 to Mac-1 was not strictly required, because monovalent Fab' fragments of CBRM1/5 bound specifically to activated neutrophils and displaced (K, of Fab' 200 nM) bivalent CBRM1/5 IgG ( Figure 2c, and data not shown).
  • CBRM 1/5 localized specifically to the I domain of the Mac-1 ⁇ chain as it, like LM2/1, bound to CHO cells that expressed the wild type Mac-1 and chimera X-e-M-b-X which contains the I domain of Mac-1 on a p 150,95 frame.
  • CBRM 1/5 did not bind to wild type pl50,95 or the reciprocal chimera, M-e-X-b-X which contains a pl50,95 I domain on a Mac-1 backbone ( Figure 5).
  • divalent cations are required for integrins to bind to their ligands. Adhesion is abolished uniformly by the presence of EDTA and altered quantitatively by the type of divalent cation that is present (Gailit et al, J. Biol. Chem. 265:12927-12932 (1988); Diamond et al, In Leukocyte Typing IV, Knapp et al editors, London, Oxford University, pp. 570-574 (1989); Altieri, D.C. J. Immunol. 747: 1891-1898 (1991) and Dransfield et al , J. Cell. Biol. 776:219-226 (1992)).
  • Mac-1 that was purified by iC3b- Sepharose chromatography (Hermanowski-Vosatka et al, Cell 68:341-352 (1992)) expressed the LM2/1 epitope but lacked the CBRM1/5 epitope, and was unable to sustain ligand binding (MS Diamond, KPM van Kessel, SD Wright, and TA Springer, unpublished observations).
  • Bivalent IgG, Fab 2 , or monovalent Fab fragments of CBRM 1/5 inhibited greater than 80% of the Mac-1- ependent binding of fMLP- stimulated neutrophils to ICAM-1 (Figure 9a).
  • CBRM 1/5 was comparable in effectiveness, to LPM19c, a MAb to Mac-1 that binds a similar number of sites/cell as LM2/1 and completely blocks Mac-l-ICAM-1 interaction ( Figure 8a, (Diamond, et al, J. Cell Biol 777:3129-3139 (1990)), and data not shown).
  • a third MAb to Mac-1, CBRM 1/23 had no significant inhibitory effect on neutrophil adhesion to ICAM-1.
  • CBRM 1/5 a novel MAb that binds to a subpopulation of Mac-1 molecules on activated, but not resting peripheral blood neutrophils and monocytes.
  • This MAb recognizes an activation-dependent neoepitope and acts as a reporter for the activation state of Mac-1.
  • Our results demonstrate that the subset which expresses the CBRM 1/5 neoepitope contains the structurally active Mac-1 molecules that sustain adhesion to at least two of its characterized ligands, ICAM-1 and fibrinogen.
  • Evidence for this includes the following: (i) CBRM 1/5 does not react by immunofluorescence with Mac-1 on the surface of resting neutrophils or monocytes.
  • Lysates that are precleared of the CBRM 1/5 epitope contain residual Mac-1 molecules that are immunoprecipitated by LM2/1 but lysates that are precleared of Mac-1 containing the LM2/1 epitope do not contain residual quantities of CBRM 1/5 precipitable material, (iv) Functional studies reveal that despite binding to less than 15% of Mac-1 molecules on neutrophils stimulated with fMLP, CBRM 1/5 (IgG or Fab fragments) inhibits almost completely (> 85%) the Mac- 1 -dependent adhesion to fibrinogen and ICAM-1. Additional experiments confirmed the blocking capacity of CBRM 1/5, as it abrogates adhesion of ICAM-I+ L cells to purified Mac-1.
  • CBRM 1/5 is unique as it recognizes the subset of Mac-1 molecules that mediates ligand interaction.
  • CBRM 1/5 maps to the I domain on Mac-1 which appears to contain the major recognition site for ICAM-1 and fibrinogen (described below).
  • MAbs against activation-dependent neoepitopes on integrin superfamily members have been described recently.
  • MAbs against the platelet integrin gpIIb-IIIa have been reported to monitor structural changes that are induced either by cellular activation or ligand binding (Du et al , Cell 65:409 (1991)).
  • the initial evidence for a structural change in integrins came from the development of the PAC-1 MAb that recognizes gpIIb-IIIa on ADP, epinephrine, or thrombin-treated but not resting platelets (O'Toole, et al, Cell Reg. 7:883-893 (1990); Shattil, et al, J. Biol.
  • PAC-1 binds to a subset (20-50%) of gpIIb-IIIa molecules after stimulation and inhibits fibrinogen-mediated platelet aggregation (Shattil, et al., J. Biol. Chem. 260:11107-11114 (1985)). Furthermore, CHO cells that express gpIIb-IIIa are unable to bind soluble fibrinogen or the PAC-1 MAb until a structural change has occurred (O'Toole, et al, Cell Reg. 7:883-893 (1990)).
  • the MAb 7E3 that was raised originally against gpIIb-IIIa (Coller, J. Clin. Invest. 76:101-108 (1985)), binds to all Mac-1 molecules on ADP-stimulated but not resting monocytes or monocyte cell lines (Altiere et al, J. Immunol 747:2656-2660 (1988)). Unlike PAC-1 or CBRM1/5, it has no reported functional effects.
  • the NKI-L16 MAb recognizes the leukocyte integrin LFA-1 on resting T lymphocytes only weakly (Larson et al. , Cell Reg.
  • NKI-L16 induces homotypic adhesion of B cell lines and T cell clones and restores partially binding to purified ICAM-1 of COS cells that have been transfected with a wild type LFA-1 chain and a defective CD18 ⁇ subunit (Hibbs et al, Science 257:1611-1613 (1991)).
  • the NKI-L16 epitope is not sufficient for cell binding since cloned T cells express high levels constitutively but do not aggregate spontaneously (van Kooyk, et al, J. Cell Biol. 772:345-354 (1991)).
  • the MAb KIM 127 recognizes the common CD 18 ⁇ subunit of the leukocyte integrins and promotes LFA-1 and Mac-1 dependent adhesive events (Robinson, et al, J. Immunol. 748:1080-1085 (1992)). Binding of this MAb may trigger a conformational change that mimics activation, and increases avidity for ligand.
  • CBRM 1/5 appears unique as it binds to a subset of molecules on stimulated cells, blocks rather than promotes adhesion, and has an epitope whose presence correlates with, but does not induce, the highly avid form of Mac-1 on neutrophils and monocytes.
  • CBRM 1/5 is the first example of an activation-specific MAb against an integrin a subunit whose expression requires divalent cations but is known to bind to a region outside the divalent cation binding repeats.
  • Mg 2+ or Ca 2+ is sufficient and EDTA abolishes the expression of the CBRM 1/5 epitope; on neutrophils, Mn 2+ increases the CBRM 1/5 epitope expression in the absence of stimulation, and thus may impose structural alterations that mimic cell activation (Altieri, J. Immunol. 747:1891-1898 (1991); Dransfield, et al, J. Cell Biol. 776:219-226 (1992)). Although these studies suggest that divalent cations modulate integrin function, it remains unclear if they are permissive for ligand binding or whether divalent cations are involved actively in the regulation of adhesion.
  • leukocyte trafficking is a protective response of the host defense system against foreign pathogens or tissue repair
  • leukocytes can mediate significant degrees of vascular and tissue injury.
  • Neutrophils have been implicated in the pathogenesis of a number of clinical disorders including adult respiratory distress syndrome, ischemia-reperfusion injury following myocardial infarction, vasculitis, and shock (for review see Carlos et al, Immunol Rev. 774:1-24 (1990)).
  • inflammatory injury mediated by leukocytes can be attenuated by antibodies against the leukocyte integrins (Mileski, et al, Surgery 708:206-212 (1990); Rosen, et al , J. Exp. Med.
  • MAbs like CBRM 1/5 which recognize activated, and not resting leukocytes, are useful clinically to target, monitor or deplete the highly adhesive cells that effect inflammatory injury.
  • TS1/22 anti-CDlla, IgGl
  • CD 11 in Leucocyte Typing TV: White Cell Differentiation Antigens, Knapp et al. , eds., Oxford University Press, Oxford.543-551 (1989)), TMG-65 (anti- CDllb, IgGl, a gift of Dr. I. Ando, Szeged, Hungary) (Uciechowski and
  • VIM12 anti-CDl lb, IgGl
  • Mac-1 was prepared for the first immunization by combining 10 ⁇ g of purified Mac-1 with trehalose dimycolate from Mycobacterium phlei, monophosphoryl lipid A from Salmonella minnesota R595, PBS, 0.2% Tween 80, and squalene as described in the manufacturer's instructions (RIBI Immunochemical Research, Hamilton MT). The second immunization was performed in PBS. Mice were test bled on day 8 prior to fusion and the titer of the antisera by ELISA against purified Mac-1 was 1/5000. The protocol for fusion and hybridoma maintenance has been described (Springer, et al, Eur. J. Immunol. 9:301-306 (1979)).
  • oligonucleotide directed mutagenesis was used to introduce restriction sites into homologous regions of the cDNA without altering the coding sequence of ⁇ M (Mac-1) (Corbi, et al, J. Biol. Chem. 265:12403-12411 (1988)) and ⁇ x (pl50,95) (Corbi, et al, EMBO J. 6:4023- 4028 (1987)) in pCDM ⁇ .
  • the Bglll and Aflll restriction sites define a region that contains exons 11 to 13 and the three divalent cation binding repeats (exons 14 to 16): for simplicity this will be referred to as the divalent cation binding region: the Bgl II site is 10 nucleotides before the end of exon 10 and the Aflll site is precisely at the end of the last exon (exon 16) of the divalent cation binding repeats (Corbi, et al, J. Biol. Chem. 265:2782-2788 (1990)).
  • Mac-1 ⁇ subunits that contained silent mutations were intact antigenically and functionally as they bound iC3b-E and appropriate ⁇ chain specific MAbs after transfection into COS cells (data not shown).
  • M represents a region of the Mac-1 ⁇ subunit
  • X represents a region of the pl50,95 ⁇ subunit
  • e, b, and a refer to the restriction sites EcoRV, Bglll, and Aflll.
  • M-e-X and X-e-M were constructed by ligating the EcoRV/Not I fragments of pCDM8- ⁇ M (4.7 kb) or pCDM8- ⁇ x (4.1 kb) with the Notl/EcoRV fragments (3.5 or 4.7 kb) of pCDM8- ⁇ x or pCDM8- ⁇ M .
  • M-b-X and X-b-M, or M-a-X and X-a-M were constructed similarly using the Bglll/NotI or the Aflll/NotI fragments of the corresponding plasmids.
  • M-b-X-a-M and X-b-M-a-X were constructed by ligating the 0.8 kb Bglll/Aflll of ⁇ M or ⁇ * with the Aflll/BgHI fragment of pCDM8- ⁇ (6.7 kb) or pCDM8- ⁇ M (7.2 kb).
  • M-e-X-b-M was constructed by ligating a Sall/EcoRV (4.5 kb) fragment of pCDM8- ⁇ M with a BgHI/Sall fragment (3.0 kb) of pCDM8- ⁇ M and a EcoRV/BgHI (0.5 kb) fragment of ⁇ x .
  • X-e-M-b-X was constructed by ligating a EcoRV/BgHI fragment (0.5 kb) of ⁇ M with the Bglll/EcoRV fragment (7.0 kb) of pCDM8- ⁇ x .
  • the construction of each chimera was confirmed by restriction digest analysis, hybridization with appropriate segments of cDNA, and by sequencing through the junctions (Maniatis, et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).
  • Neutrophils were isolated from whole blood of healthy volunteers by dextran sedimentation, Ficoll gradient centrifugation, and hypotonic lysis as described (English et al, J. Immunol. Methods 5:249 (1974); Miller, et al,
  • COS cells were grown on 15-cm tissue culture treated plates in RPMI 1640, 10% FCS (JRH Biosciences, Lenexa, KS), 2 mM glutamine, and 50 ⁇ g/ml gentamicin, and then co-transfected with CsCI purified ⁇ M , ⁇ x or chimeric ⁇ M x and the common ⁇ (Kishimoto, et al., Cell 48:681-690. (1987)) cDNA in pCDM ⁇ by the DEAE-Dextran method as described (Staunton, et al, Cell 67:243-254 (1990)). Surface expression was monitored three days after transfection by flow cytometry as described (Diamond, et al, J. Cell Biol 777:3129-3139 (1990)).
  • CHO DG 44 cells which have a deletion of the DHFR gene were obtained from Dr. L.Chasin (Columbia University, New York NY) and maintained as described (Qu, et al, J. Exp. Med. 767:1195-1210 (1988)). Cells were cotransfected by electroporation at 380 V and 960 ⁇ F (Chan et al. , Mol Biol Cell 5:157-166 (1992)) with 25 ⁇ g of wild type or chimeric ⁇ in pCDM ⁇ , 25 ⁇ g of wild type ⁇ in pCDM8, and 5 ⁇ g of pDCHIP vector that contains a CHO DHFR minigene.
  • CHO cells expressing a domain deleted form of ICAM-1 (Domain 3 deleted, F185) (Staunton, D.E., et al., Cell 56:849-853 (1989)) were generated by cotransfection of F185 cDNA in pCDM8 with pDCHIP followed by selection and immunopanning as described. For all transfectants, positive surface expression was monitored by immunofluorescent flow cytometry.
  • the ICAM-1 + L cell stables were maintained on 15 cm tissue culture plates in DME, 10% heat inactivated FCS (Low endotoxin serum (defined), Hyclone Laboratories, Logan, UT), 2 mM glutamine, and 50 ⁇ g/ml gentamicin as previously described (Diamond, et al, J. Cell Biol 777:3129- 3139 (1990)). Prior to adhesion assays, cells were removed from tissue culture plates with trypsin-EDTA, and washed twice in PBS, 2 mM MgCl 2 , 0.5 % heat-treated BSA (Diamond, et al, J. Cell Biol 777:3129-3139 (1990)).
  • Transfected COS or CHO cells (1-2 X 10° cells) were detached with HBSS, 5 mM EDTA, washed four times with PBS, and resuspended in 2 mis. The cells were iodinated, lysed, and precleared as described (Diamond, et al , Cell 65:961-971 (1991)).
  • Immunoprecipitations were performed as follows: Immune complexes were formed by incubating MAb (250 ⁇ l volume of neat supernatant, 1/100 dilution of ascites, 25 ⁇ g/ml purified MAb) with purified rabbit anti-mouse antibody (5 ⁇ l of 1 mg/ml, Zymed Laboratories, San Francisco, CA) for 4 hours at 4°C. Protein-A Sepharose (Pharmacia KLB, Piscataway, NJ) (25 ⁇ l of a 1: 1 slurry) was added for overnight incubation.
  • Mac-1 was purified from leukocyte lysates after detergent solubilization by LM2/1-Sepharose immunoaffinity chromatography as described (Diamond, et al, J. Cell Biol 777:3129-3139 (1990)). ICAM-1 was purified from detergent lysates of the erythroleukemic cell line K562 by RRl/1-Sepharose immunoaffinity chromatography as described (Marlin et al. Cell 57:813-819 (1987)). Fibrinogen was obtained from Sigma Chemical Company (St. Louis, MO)
  • IgM and iC3b-coated erythrocytes (IgM-E, iC3b-E) were prepared as described previously (Rothlein et al , J. Immunol. 755:2668-2672 (1985)) with the following modifications.
  • sheep erythrocytes (Colorado Serum Company, Denver, CO) were washed once in PBS, and labelled for 2 h at 37°C with a 0.22 ⁇ m filtered solution of FTTC (0.8 mg/ml in PBS after being dissolved in 30 ⁇ l DMSO).
  • Erythrocytes were washed thrice in HBSS, 10 mM HEPES pH 7.3, 2 mM MgCl 2, resuspended (6 x 10 8 cells in 10 mis) and incubated with a 1/128 dilution of heat inactivated anti- Forssman IgM MAb (Ml/87) for 60 minutes at room temperature while shaking gently.
  • IgM coated erythrocytes (IgM-E) were washed thrice, resuspended (1 ml) in HBSS, 10 mM HEPES, 1 mM MgCl 2, 1 mM CaCI 2 supplemented with C5 deficient human serum (100 ⁇ l) (Sigma Chemical Co, St.
  • iC3b coated erythrocytes (iC3b-E) and IgM-E were washed thrice and resuspended (4 x 10 7 cells/ml) in HBSS, 1 mM MgCl 2 , ImM CaCI 2 , 10 mM HEPES pH 7.3. Neutrophils and transfected CHO cells were assayed for binding to iC3b-E.
  • Peripheral blood neutrophils (1.25 x 10 s cells/well in 50 ⁇ l) were seeded onto 96-well tissue culture treated plates (Corning Glass Works, Corning, NY) in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl 2 , ImM CaCl 2 for 20 minutes at 37°C in a 5% CO 2 incubator; non-adherent cells were removed after two washes with the same buffer.
  • Adherent neutrophils were preincubated with MAb (addition of 50 ⁇ l of diluted ascites or purified MAb in PBS, 1 mM MgCl 2 , 0.5% BSA) for 20 minutes; each condition was performed in quintuplicate.
  • iC3b-E or IgM-E (2 x 10 6 in 50 ⁇ l) and PMA (65 ng/ml final concentration) were added; the erythrocytes were centrifuged (300 RPM, 5 minutes, 4°C) onto the neutrophils and the plates were incubated in a 37°C water bath for 15 minutes. Non-adherent erythrocytes were removed by flicking the plates six times after addition of PBS, 1 mM MgCl 2 , 0.5% BSA (175 ⁇ l/well). Bound cells were quantitated in the 96 well plates using a Pandex fluorescence concentration analyzer (Baxter Healthcare Corp., Mundelein, IL). The percentage of inhibition was normalized as follows:
  • Transfected CHO cells (2 x 10 5 cells/ml in 250 ⁇ l) were seeded onto 6-well tissue culture treated plates in L15, 10 mM HEPES pH 7.3, 5% heat- inactivated FCS and adhered for greater than three hours at 37°C. Subsequently, iC3b-E or IgM-E (2 x lOVml, 50 ⁇ l) and PMA (100 ng/ml final concentration) were added and cells were bound for 60 minutes at 37°C. Non- adherent erythrocytes were removed after six to eight washes and rosettes (> 10 erythrocytes/CHO cell, > 100 cells examined) were scored by light microscopy at 200X magnification. Neutrophil Homotypic Adhesion Assays
  • the number of particles (single cells or cell aggregates) in a fixed volume was determined with a Model S-Plus Coulter counter (Coulter, Hialeah, FL). The average of two readings was used as an individual datapoint. Each condition was repeated at least three times with different donors. The percent aggregation was determined according to the following equation:
  • X Aggregation 100 X ⁇ 1 ( # of particles in a sample stimulated with PHA) (# of particles in unstimulated sample) ⁇
  • Neutrophil aggregometry was performed as described (Hammerschmidt, et al, Blood 55:898-902 (1980)). Briefly, purified neutrophils were washed twice and resuspended (10 7 cells/ml) in HHMCH (without phenol red). Aliquots of cells (0.5 ml) were preincubated with MAbs for at least 25 minutes at room temperature. Neutrophils (0.4 ml) were stirred (700 RPM) at 37°C in a siliconized cuvette in a standard platelet aggregometer (Model DP-247F, Sienco, Morrison, CO). A baseline was recorded for three minutes at which time PMA (100 ng/ml) was added and the change in light transmission was monitored on a strip chart recorder.
  • PMA 100 ng/ml
  • Non-adherent cells were removed by twelve washes with a Pasteur pipette after gentle swirling with the same buffer supplemented with 0.5 % BSA. Binding was quantitated by scoring the number of adherent cells in at least four different fields using an ocular grid at 100X magnification. The percent inhibition by MAb was determined upon comparison with the media control.
  • the plates were preincubated at room temperature with MAb (1/200 dilution of ascites or 20 ⁇ g/ml of purified MAb) in PBS, 2 mM MgCl 2 , 0.5% heat- treated BSA (PMBSA, 2 mis).
  • MAb 1/200 dilution of ascites or 20 ⁇ g/ml of purified MAb
  • PMBSA heat- treated BSA
  • ICAM-1 + L cells were removed from tissue culture plates with trypsin-EDTA (GIBCO, Long Island, NY), washed twice and resuspended in PMBSA (0.5-1.0 x 10° cells/ml). Some aliquots were preincubated for 25 minutes at room temperature with control or anti-ICAM-1 MAbs.
  • the binding assay was performed as described (Micklem, et al, J. Immunol. 744:2295-2303 (1990)). The percent inhibition by MAb was determined upon comparison with the media control.
  • ICAM-1 The binding of transfected CHO cells to ICAM-1 is a modification of a previously described protocol for COS cell transfectants (Diamond, et al, J. CellBiol. 777:3129-3139(1990)). Briefly, purified ICAM-1 was diluted and adsorbed (25 ⁇ l) to 6 cm Petri dishes. After a 90 minute incubation, non-specific binding sites were blocked with 0.5% heat-treated BSA.
  • CHO cell transfectants after removal from tissue culture plates with HBSS, 10 mM HEPES pH 7.3, 5 mM EDTA, were washed twice and resuspended (8 x 10 5 cells ml) in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl 2 , 0.5% heat treated BSA, and bound to ICAM-1 coated Petri dishes for 90 minutes at room temperature.
  • Non-adherent cells were removed after five washes with a Pasteur pipette and the number of adherent cells was determined by light microscopy at 100X using an ocular grid.
  • MAbs Epitope Mapping of MAbs to Mac-1 Because an improper association of ⁇ and ⁇ subunits in a subset of chimeras could affect functional assays and our subsequent analyses, we used MAbs to generate a structure-function map of the ligand binding domains on Mac-1. MAb epitopes were localized on the Mac-1 ⁇ subunit by their ability to bind to a particular subset of chimeras and the results correlated with the ability of the MAb to block distinct adhesive functions of the native Mac-1 molecule.
  • the transfected CHO cells contained a functional form of Mac-1 as stimulation with phorbol esters prompted Mac-1 transfectants to bind to purified ICAM-1 (Figure 16A).
  • Figure 16A In contrast to previous studies with transfected COS cells (Diamond, et al. , J. CellBiol. 777:3129-3139 (1990)), little binding to ICAM-1 was observed in the absence of stimulation (data not shown).
  • Two chimeras (M-a-X, M-b-X) which contained the I domain of Mac-1 bound to ICAM-1 , but more weakly than anticipated.
  • the CHO cells that expressed wild type pl50,95 also bound to ICAM-1.
  • pl50,95 acts as a counter-receptor for an uncharacterized adhesion molecule on stimulated endothelial cells (Stacker et al, J. Immunol. 746:648-655 (1991)c), and L cell transfectants that express ICAM-1 bind weakly to purified pl50,95 (Stacker and Springer, unpublished data).
  • the N- terminal and perhaps divalent cation binding regions may contribute to recognition of several of the ligands.
  • MAb epitopes were used to localize the ligand recognition sites because interpretation of the results from functional assays with the Mac- 1/p 150,95 chimeras was not straightforward.
  • the I domain appears important in the recognition of all four ligands by Mac-1 because in each of the experimental systems tested, several different MAbs that mapped to the I domain blocked Mac-1-dependent binding strongly ( > 80 %). Furthermore, in all cases, the individual MAb that abrogated binding most completely mapped to the I domain. Two-thirds of the MAbs that mapped to the I domain blocked Mac-1 interaction with iC3b, fibrinogen, and ICAM-1 by greater than 70%, and all of the MAbs that blocked neutrophil homotypic adhesion strongly, mapped to the I domain.
  • the I domain is immunodominant; it is more homologous (81 % identity) and smaller in size (200 compared to 500 amino acids) than the C-terminal region.
  • the strong identity between I domains of murine and human Mac- 1 conserves sites of ligand recognition as both species of Mac- 1 bind to human and murine iC3b coated particles (Beller, et al., J. Exp. Med. 756:1000-1009 (1982)) and to COS cells transfected with murine ICAM-1 (Diamond, et al. unpublished).
  • Human Mac-1 and pl50,95 have a 63% overall protein sequence identity with a 55% identity between corresponding I domains (Corbi, et al, J. Biol. Chem. 265:12403-12411 (1988); Corbi, et al , EMBO J. 6:4023-4028 (1987)). None of the MAbs studied cross react between the subunits of Mac-1 and pl50,95.
  • LFA-1 and Mac-1 which are 36% identical at the amino acid level share ICAM-1 as a ligand, although the binding appears distinct as LFA-1 binds to the first Ig-like domain (Staunton, et al., Cell 67:243-254 (1990)) and Mac-1 binds to the third Ig-like domain on ICAM-1 (Diamond, et al. , Cell 65:961-971 (1991)). More refined mutagenesis studies are required to determine whether Mac-1, pl50,95, and LFA-1 utilize related recognition sites to bind their shared ligands.
  • the structure of the leukocyte integrins thus differs from the linear array of domains that is proposed for members of the immunoglobulin and selectin families of adhesion molecules (Bevilacqua, et al., Science 245:1160-1165 (1989); Driscoll, et al, Nature 555:762-765 (1991); Kansas, et al, J. Cell. Biol. 774:351-358 (1991); Williams et al, Annu. Rev. Immunol. 6:381-405 (1988)).
  • pl50,95 Although others have reported complement receptor activity for pl50,95 (Micklem et al., Biochem. J. 257:233-236 (1985); Myones, et al, J. Clin. Invest. 82:640-651 (1988); Ross, et al, J. Leukoc. Biol 57: 109-117 (1992)), in our experiments, iC3b coated erythrocytes did not bind to transfected CHO cells expressing p 150,95 ( Figure 16B, and data not shown). Possibly, in the transfected cells, the I domain of pl50,95 may contain the structural information to bind iC3b but lack proper interactions with other parts of the ⁇ or ⁇ subunits.
  • CBRM 1/5 recognizes a subpopulation of functionally active Mac-1 molecules on neutrophils and monocytes (Diamond and Springer, submitted), and is expressed on other chimeras (X-e-M-b-X, M-b-X-a-M) that contain the I domain of Mac-1 and bind ligand (data not shown).
  • M-a-X and M-b-X may not be in a conformation that sustains ligand binding; the molecular packing of the I domain may be disrupted sufficiently by the improper context of surrounding domains so that it is unable to recognize ligand.
  • Mac-1 and LFA-1 On leukocytes, to recognize ligands, Mac-1 and LFA-1 must convert from a low avidity to high avidity state (Buyon, et al. , J. Immunol 740:3156-3160 (1988); Dustin et al. Nature 547:619-624 (1989); Lo, et al, J. .Exp. Med. 769:1779-1793 (1989); van Kooyk, et al. Nature 542:811-813 (1989)). It is plausible to speculate that integrins require a physical "opening or closing" of the molecule that enables ligand binding and that the juxtaposition of homologous, but non-native domains in some of the chimeras prevents this change from occurring.
  • the ⁇ subunit could modulate the structural change in the ⁇ subunit. This could explain why deletion or substitution of residues in the cytoplasmic domain of the ⁇ subunit abolishes LFA-1 binding to ICAM-1 (Hibbs, et al, J. Exp. Med.

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Abstract

La présente invention est fondée sur l'observation récente selon laquelle la stimulation de cellules myéloïdes au repos n'entraîne pas l'activation de toutes les molécules Mac-1 exprimées par la cellule. Seules des sous-populations sélectionnées de molécules Mac-1 sont activées et deviennent capables de lier des ligands. En outre, on a trouvé que les molécules Mac-1 activées possèdent des épitopes à spécificité d'activation qui les distinguent des molécules Mac-1 non activées. Sur la base de ces observations, des anticorps qui se lient sélectivement à des molécules Mac-1 activées mais sont pratiquement incapables de se lier à des Mac-1 non activées sont également décrits.
PCT/US1993/009777 1992-10-09 1993-10-12 SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE WO1994008620A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP93924919A EP0670734A4 (fr) 1992-10-09 1993-10-12 SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE.
CA002144738A CA2144738A1 (fr) 1992-10-09 1993-10-12 Sous-population de molecules mac-1(cd11b/cd18), intermediaires dans l'adherence des agents neutrophiles a icam-1 et au fibrinogene
JP6510234A JPH08502956A (ja) 1992-10-09 1993-10-12 ＩＣＡＭ‐１及びフィブリノーゲンへの好中球接着を媒介するＭａｃ‐１（ＣＤ１１ｂ／ＣＤ１８）分子のサブ集団
AU54423/94A AU674556B2 (en) 1992-10-09 1993-10-12 A subpopulation of MAC-1 molecules which mediate neutrophil adhesion to ICAM-1 and fibrinogen

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US95890492A 1992-10-09 1992-10-09
US07/958,904 1992-10-09
US96415692A 1992-10-22 1992-10-22
US07/964,156 1992-10-22

Publications (1)

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WO1994008620A1 true WO1994008620A1 (fr) 1994-04-28

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PCT/US1993/009777 WO1994008620A1 (fr) 1992-10-09 1993-10-12 SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE

Country Status (5)

Country Link
EP (1) EP0670734A4 (fr)
JP (1) JPH08502956A (fr)
AU (1) AU674556B2 (fr)
CA (1) CA2144738A1 (fr)
WO (1) WO1994008620A1 (fr)

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WO1996012742A1 (fr) * 1994-10-25 1996-05-02 Glaxo Group Limited Agents de fixation pour le traitement des maladies inflammatoires, auto-immunes ou allergiques
US5811227A (en) * 1995-10-09 1998-09-22 Fuji Photo Film Co., Ltd. Method for dispersing droplet type emulsified material within liquid feeding system and coating method using the dispersing method
US5914112A (en) * 1996-01-23 1999-06-22 Genentech, Inc. Anti-CD18 antibodies in stroke
WO2007048186A1 (fr) * 2005-10-25 2007-05-03 Baker Medical Research Institute Polypeptides de liaison à des leucocytes et leurs utilisations
US7361344B2 (en) 1996-01-23 2008-04-22 Genentech, Inc. Co-administration of a thrombolytic and an-anti-CD18 antibody in stroke
WO2010017083A1 (fr) * 2008-08-04 2010-02-11 Wayne State University Procédés de traitement du cancer par des anticorps cd11b
WO2013025944A1 (fr) 2011-08-17 2013-02-21 Genentech, Inc. Inhibition de l'angiogenèse dans les tumeurs réfractaires
US20150337039A1 (en) * 2014-05-22 2015-11-26 Case Western Reserve University Mac-1 antibodies and uses thereof

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012742A1 (fr) * 1994-10-25 1996-05-02 Glaxo Group Limited Agents de fixation pour le traitement des maladies inflammatoires, auto-immunes ou allergiques
US5811227A (en) * 1995-10-09 1998-09-22 Fuji Photo Film Co., Ltd. Method for dispersing droplet type emulsified material within liquid feeding system and coating method using the dispersing method
US5914112A (en) * 1996-01-23 1999-06-22 Genentech, Inc. Anti-CD18 antibodies in stroke
US7361344B2 (en) 1996-01-23 2008-04-22 Genentech, Inc. Co-administration of a thrombolytic and an-anti-CD18 antibody in stroke
US7655230B2 (en) 1996-01-23 2010-02-02 Genentech, Inc. Co-administration of a tissue plasminogen activator, anti-CD11b antibody and anti-CD18 antibody in stroke
WO2007048186A1 (fr) * 2005-10-25 2007-05-03 Baker Medical Research Institute Polypeptides de liaison à des leucocytes et leurs utilisations
WO2010017083A1 (fr) * 2008-08-04 2010-02-11 Wayne State University Procédés de traitement du cancer par des anticorps cd11b
WO2013025944A1 (fr) 2011-08-17 2013-02-21 Genentech, Inc. Inhibition de l'angiogenèse dans les tumeurs réfractaires
US20150337039A1 (en) * 2014-05-22 2015-11-26 Case Western Reserve University Mac-1 antibodies and uses thereof

Also Published As

Publication number Publication date
JPH08502956A (ja) 1996-04-02
AU674556B2 (en) 1997-01-02
EP0670734A4 (fr) 1998-01-14
AU5442394A (en) 1994-05-09
EP0670734A1 (fr) 1995-09-13
CA2144738A1 (fr) 1994-04-28

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