JPH08502956A - Subpopulation of Mac-1 (CD11b / CD18) molecules that mediate neutrophil adhesion to ICAM-1 and fibrinogen - Google Patents

Abstract

  (57) [Summary] The present invention is based on the new observation that stimulation of resting myeloid cells does not activate all of the Mac-1 molecules expressed by the cells. Only selective subpopulations of Mac-1 molecules are activated and are able to bind ligand. Furthermore, activated Mac-1 molecules were found to have activation-specific epitopes that distinguish them from non-activated Mac-1 molecules. Based on these observations, antibodies have been described that selectively bind to activated Mac-1 molecules, but are substantially unable to bind to non-activated Mac-1.

Description

Detailed Description of the Invention       Neutrophils to ICAM-1 and fibrinogen       Mac-1 (CD11b / CD18) molecule that mediates adhesion       Subgroup of BACKGROUND OF THE INVENTION   This application is filed on October 9, 1992 in US Application No. 07 / 958,90. It is the continuation of No. 4. Field of the invention   The present invention is facilitated by the field of cell adhesion and by bone marrow-like cell adhesion, migration and activation. To the use of anti-adhesion molecules as a means of mediating biological processes. Specifically The present invention shows that specific subpopulations of Mac-1 are active when myeloid cells are stimulated. Is activated and upon activation, an activation-specific epitope appears on the Mac-1 molecule. Disclosure. Stimulated myeloid cells based on confirmation of activation-specific epitopes Capable of binding to the active form of Mac-1 present above, but inactive Mac-1 and resting bone A new class of anti-Mac-1 antibodies that are substantially incapable of binding to medullary cells have been disclosed .Description of related technology   Under normal conditions, circulating neutrophils flow through the vasculature and are mostly found in basal endothelial cells. Does not adhere. Tyranny of inflammation Within minutes of dew, neutrophils spin along nearby blood vessel walls and tightly engage endothelial cells. Adhere and then enter the parenchyma (Atherton et al., J. Physiol. 222: 447-474 (1 972)). Recent experiments have identified molecules on the surface of neutrophils and endothelial cells that promote transmigration. I accepted. Members of the selectin family (Springer et al., Nature, 349: 196-197) (1991)) mediates early rolling and is a member of the integrin family (Hyne S., et al., Cell, 69: 11-25 (1992)) enhances neutrophil attachment to endothelial cells (Lawrence et al. , Cell, 65: 859-873 (1991)) and then promote the transition to basal tissues (Anderson Et al., Blood, 76: 2613-2621 (1990); Smith et al., J. Chem. Clin. Invest. 83: 2008-2017 ( 1989)).   The integrin family of heterodimeric glycoproteins enters the extracellular matrix Mediate cell adhesion and attachment of the cells (Hynes et al., Cell, 48: 549-554 (1987); Hynes et al. , Cell, 69: 11-25 (1992)). The leukocyte integrin subfamily has three members. LFA-1 (CD11a / CD18), Mac-1 (CD11b / CD1 8) and p150,95 (CD11c / CD18), which are unique but dense. It has a common β subunit that is non-covalently linked to the α chain associated with the junction (Kishimot o et al., Adv. Immunol. 46: 149-182 (1989); Springer et al., Nature, 346: 425-433. (1990)). These glycoproteins share the common CD18 β subunit (95,000 M_r), But structurally homologous Individual unique CD11α-subunits (175,000, 160,000, 150,000M_r) Respectively (Larson et al., J. Cell Biol. 108: 703-7). 12 (1989)). All three members have two distinct features in the extracellular region of the molecule. Or putative divalent cation binding region consisting of three tandem repeats of the EF hand motif Region with an inserted 200 amino acid or "I" domain (Arnaout Et al., J. Cell Biol. 106: 2153-2158 (1988); Corbi et al., J. Am. Biol. Chem. 263: 1 2403-12411 (1988); Corbi et al., EMBO J. 6: 4023-4028 (1987); Kaufman et al., J. ． Immunol. 147: 369-371 (1991); Larson et al. Cell Biol. 108: 703-712 (19 89); Pytela, EMBO J. 7: 1371-1378 (1988)). I domain is the integrin V Α of the LA subfamily₁And α₂-All other known subunits except subunits Not present in the tegrin α-subunit (Hemler, Annu. Rev. Immunol. 8: 3 65-400 (1990); Hynes, Cell, 69: 11-25 (1992)).   Leukocyte integrins mediate several adhesion events that are important for immune system function It They are T lymphocyte targeted cytolysis (Davignon et al., Proc. Natl. Acad. Sci. U. SA, 78: 4535-4539 (1981)), T lymphocyte proliferation (Davignon et al., Proc. Natl. Acad. ． Sci. USA, 78: 4535-4539 (1981)), natural extinction (Krensky et al., J. Immun. ol. 131: 611-616 (1983)), leukocyte adhesion to and migration from endothelial cells (Dust in et al. Cell Biol. 107: 321-331 (1988); Harlan et al., Blood, 66: 167-178 (1985); H. askard et al. Immunol. 137: 2901-2906 (1986); Lo et al. Exp. Med. 169: 17 79-1793 (1989); Lo et al. Immunol. 143 (10): 3325-3329 (1989); Smith et al. , J. Clin. Invest. 83: 2008-2017 (1989); Smith et al. Clin. Invest. 82: 1746-1756 (1988)), neutrophil homomorphic aggregation and neutrophil chemotaxis (Anderson et al., J. Immu). nol. 137: 15-27 (1986)) promotes the necessary adhesion.   Mac-1 migrates to the cell surface by inflammatory mediators in circulating neutrophils and monocytes Expressed in certain intracellular vesicle compartments (Todd et al., J. Clin. Invest. 74: 1280-1290 ( 1984); Springer et al., In: Biochemistry of Macrophages (CIBA Symposium 118). ), Pitman, London, pp. 102-126 (1986); Lanier et al., Eur. J. Immunol. 15: 713-718 (1985); Yancey et al. Immunol. 135: 465-470 (1985)). This move Correlates with increased adhesion (Anderson et al. Ann. Rev. Med. 38: 175-194 (1987)) . Mac-1 α subunit message is present in most cells of the T or B lineage , Mac-1 protein detected in blood monocytes and PMA-induced myeloid cell lines Correlates with quality surface expression.   Some cytotoxic T lymphocyte clones have similar amounts It is known to express p150,95 and LFA-. LFA- and p1 Monoclonal antibodies against the 50,95 α subunit are similar to this What It inhibits killing by CTL clones and their inhibitory effects are additive. (Keizer et al., J. Immunol. 138: 3130-3136 (1987)). Furthermore, p150, 95 Antibodies to the α subunit were shown to inhibit monocyte adhesion to endothelial cells (Keizer et al., Eur. J. Immunol. 17: 1317-1322 (1987)).   Monoclonal antibody against Mac-1 or p150,95 Inhibits neutrophil aggregation and adhesion to the surface of puffy coat, bacteria, protozoan parasites and fungi. Harm et al., Blood, 66: 167-178 (1985); Springer et al., In: Biochemist. ry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 (19 86); Dana et al. Immunol. 137: 3259 (1986); Bullock et al. Exper. Med. 16 5: 195-210 (1987); Mosser et al., J. Am. Immunol. 135: 2785-2789 (1985)).   Mac-1 (CD11b / CD18) is a leukocyte adhesion glycoprotein In addition to its role in cell-cell and cell-substrate adhesion interactions, iC3b (Be ller et al. Exper. Med. 156: 1000-1009 (1982)), Fibrinogen (Altieri Et al., J. Cell. Biol. 107: 1893-1900 (1988); Wright et al., Proc. Natl. Acad. S ci. USA, 85: 7734-7738 (1988)) and Factor X (Altieri et al., J. Biol. Chem. 863). : 7007-7015 (1988)). World Surface-active agent soluble Mac-1 and p150,95 binds to iC3b-sepharose (Micklem et al., Biochem. J. 231: 233-236 (1985)).   The α-1 subunit of Mac-1 has a long extracellular domain (residues 1092) and 1 It is a transmembrane protein of 1137 residues containing a 9 amino acid cytoplasmic tail. Extracellular matrix Mainly 3 putative divalent cation binding sequences and 19 potential N-glycosylation sites Is included. The amino acid sequence of Mac-1 α is integrin super It is shown to be a member of the family; Mac-1 α is a leukocyte adhesion sugar 63% α subunit of protein p150,95, extracellular matrix receptor Putter platelet glycoprotein IIb / IIIa) fibrinogen receptor and It shows a 25% identity with the α subunit of the vitronectin receptor. Mac- 1 α subunit Putative divalent cation binding site and its adjacent region are p150,95   α subunit (87% identical at the amino acid level) and the rest of the integrins It shows a high degree of identity with both α subunits (38%). Mac-1 α sub unit Is Not present in other integrins, like the p150,95 α subunit 18 It contains a 7 amino acid domain in the extracellular region. This inserted or "I" Mainly homologous to the A domain of van Willebrand factor And it is homologous to the C3 binding protein factor B and C2 regions . These discoveries Drew attention to this region of Mac-1 as a potential binding site for iC3b.   The functional role of Mac-1 is primarily iC3 in macrophages and polymorphonuclear leukocytes. Anti-Mac-1 alpha subunit monoc blocks rosette formation of b-coated erythrocytes Proven by the ability of local antibodies (MAbs) (Beller et al., J. Exper. Med. 156 : 1000-1009 (1982)), Mac-1 is distinguished from complement receptor type 3 (CR3) He showed that he couldn't. The involvement of Mac-1 in the inflammatory process is then followed by anti-M Ac-1 α-subunit and anti-β-subunit specific MAb for endothelial cells (Anderson et al., J. Immunol. 137: 1). 5-27 (1986); Dana et al. Immunol. 137: 3259-3263 (1986); Vedder et al. C lin. Invest. 81: 672-682 (1988)). In recent epitope mapping studies, iC The site involved in 3b binding is associated with neutrophil aggregation and protein-coated plastic. Suggesting that they are different from those involved in dressing (Anderson et al., J. Immunol. 137: 1. 5-27 (1986); Dana et al. Immunol. 137: 3259-3263 (1986); Rosen et al., J. Am. Ex per. Med. 166: 1685-1701 (1987)). Therefore, Mac-1 is It appears to be a mature receptor in at least two parts of its function.   Expression of Mac-1 functional activity is regulated during leukocyte differentiation and activation. Myeloid Differentiation and maturation of monocyte cell lines Increase Mac-1 expression (Miller et al., J. Immunol. 137: 2891-2900 (1986)), Blood monocyte differentiation into tissue macrophages depends on the amount of Mac-1 on the whole cell surface. With a marked decrease (Hogg et al., Eur. J. Immunol. 16: 240-248 (1986)). Circulation Expression of Mac-1 on the surface of spheres and monocytes is upregulated by inflammatory stimuli Mac-1 is intracellularly rapidly moved to the cell surface by a chemotactic inducing substance Stored in the vesicle compartment (Todd et al., J. Clin. Invest. 74: 1280-1290 (1984 ); Miller et al. Clin. Invest. 80: 535-544 (1987)). Enhanced expression of Mac-1 Can increase adhesion, but qualitative changes after cell activation can also bind ligand Is important for the regulation of D. (Detmers et al., J. Cell Biol. 105: 1137-1145 (1987)). . Both qualitative and quantitative changes in leukocyte binding to the postcapillary endothelium at sites of inflammation Important for regulation.   In essence, the ability of white blood cells to maintain the health and growth of animals depends on their other factors. Ability to attach to cells (such as endothelial cells) and proteins (such as iC3b) Requires. This adhesion is mediated by specific receptor molecules on the surface of leukocytes. It was found that contact was required. Some of these cell surface receptor molecules are mutually It was also found to be highly relevant. White blood cells are these cell surface receptors Humans lacking the molecule exhibit chronic and recurrent infections and other clinical manifestations.   Experiments by several groups showed that leukocyte integrin adhesion was structural. It is not due to intracellular signals introduced from other surface receptors Proved to be controlled. For example, LFA-1 on resting T lymphocytes Until its signal is delivered from the antigen receptor, its counter-receptor-ICAM-1 Inability to bind (Duslin et al., Nature, 341: 619-624 (1989); van Kooyk et al., Nature , 342: 811-813 (1989)). The signal to attach is T-phosphorus within 30 minutes of stimulation. It is temporary because paw balls lose their ability to bind to ICAM-1. same Similarly, Mac-1 on neutrophils has specific signals (eg, chemotactic factors, sites ICAM- (Diamond et al., C until delivery of cain or phorbol ester) ell, 65: 961-971 (1991)), fibrinogen (Altieri et al., J. Cell. Biol. 10). 7: 1893-1900 (1988); Wright et al., Proc. Natl. Acad. Sci. USA, 85: 7734-773 8 (1988)), Factor X (Altieri et al., J. Biol. Chem. 263: 7007-7015 (1988)) or Its cellularity, including iC3b (Wright et al., J. Immunol. 136: 1759-1764 (1986)) Or does not bind to a soluble ligand; stimulation within seconds of Ma to that ligand Increases the binding activity of c-1 (Anderson et al., J. Immunol. 137: 15-27 (1986) Buyon et al., J .; Immunol. 140: 3156-3160 (1988); Detmers et al., J. Am. Exp. Med. 1 71: 1155-1162 (1990); Wright et al. Immunol. 136: 1759-1764 (1986)).   It is clear that activation promotes leukocyte integrin interactions with ligands However, little is known about the molecular mechanisms involved in altered binding activity. Up to. Of LFA-1 expression after antigen stimulation or phorbol ester treatment on T cells Since there is no increase, the change in the binding activity of LFA-1 to ICAM-1 is (Dustin et al., Nature, 341: 619-624 (1). 989)). On the other hand, the change in binding activity is a signal that concentrates adhesion receptors on the cell surface. (Detmers et al., J. Cell. Biol. 105: 1137-1145 (1987)). . Mechanisms involved in regulating the binding activity of Mac-1 on neutrophils are similar Apparently, the assay is complicated by a 10-fold or greater quantitative increase in expression following stimulation ( Berger et al., J. Clin. Invest. 74: 1566-1571 (1984); Detmers et al. Exp. Med ． 171: 1155-1162 (1990); Lo et al., J. Chem. Exp. Med. 169: 1779-1793 (1989); Mil ler et al. Clin. Invest. 80: 535-544 (1987); Todd et al. Clin. Invest. 74 : 1280-1290 (1984)). However, the change in surface expression that occurs after stimulation is Not parallel to dynamics or size (Buyon et al., J. Immunol. 140: 3156-3160 (1 988); Lo et al. Exp. Med. 169: 1779-1793 (1989)), the reason is Mac-1 An ion channel antagonist that inhibits the current quantitative increase (Vedder et al., J. Clin. Invest) ． 81: 676-682 (1988)) or temperature conditions (Schleiflenbaum et al., J. Immunol. 142: 3537-3545 (1989)). Does not block Mac-1-dependent adhesion with endothelial cells or in vitro substrates. It This causes Mac-1 to undergo an additional conformational change that promotes adhesion. (Buyon et al., J. Immunol. 140: 3156-3160 (1988); Lo et al., J. Ex. p. Med. 169: 1779-1793 (1989); Philips, J. et al. Clin. Invest. 82: 495-501 (1 988); Vedder et al. Clin. Invest. 81: 676-682 (1988)). Divalent cation (Alt ieri, J. Immunol. 147: 1891-1898 (1991); Dransfield et al. Cell Biol. 11 6: 219-226 (1992); Dransfield et al., EMBO J. 8: 3759-3765 (1989); van Kooy K et al. Cell Biol. 112: 345-354 (1991)) or Mac-1 or LFA-1 MAb against (Dransfield et al., J. Cell Biol. 116: 1527-1535 (1992); Hibbs et al., Science, 251: 1611-1613 (1991); Keizer et al. Immunol. 140: 1393-1400 (1988); Ro binson et al. Immunol. 148: 1080-1085 (1992)) increase adhesion in the absence of activation Studies showing that direct structural changes to the extracellular domain show leukocyte integrins Supports the model of modulating binding activity.   Despite the cDNA cloning and confirmation of the ligand interaction sequence, Regions for the α and β subunits of leukocyte integrins that mediate Gand binding Has not been characterized. Platelet adhesion molecule α_IIb-Β₃(CD41 / CD61) is The best characterized in subunit domain involved in ligand recognition Tegrinf It is an Amily member. Chemical crosslinking experiments (D'Souza et al., J. Biol. Chem. 265: 3440-3446 (1990)) and functional studies (D'Souza et al., Nature, 350: 66-68 (1991)) , Fibrinogen is α_IIb11-residue pepti within the divalent cation-binding repeats above RG peptide binds to β between amino acid residues 61-203₃Sabyou It is suggested that it binds to a site on the knit (D'Souza, Science, 242: 91-93 (1988); Smith et al. Biol. Chem. 263: 18726-18731 (1988)). β₃sub Naturally Occurring in a Patient with Grant's Thromboasthenia on Residue 119 of the Unit Point mutations disable ligand recognition and prevent divalent cation binding (Loftus et al. , Science, 249: 915-918 (1990)). However, the result is that leukocyte integrins It is not clear if it can be generalized to. Mac-1 is part of its ligand Was reported to bind to the RGD-like sequences in (Ross et al., J. Leukoc. ． Biol. 51: 109: 117 (1992); Wright et al. Exp. Med. 169: 175-183 (1989 ); Wright et al., Proc. Natl. Acad. Sci. USA, 84: 1965-1968 (1987); Wright Et al., Proc. Natl. Acad. Sci. USA, 85: 7734-7738 (1988)), recent research Do not utilize RGD-like sequences in binding to iC3b and fibrinogen (Altieri et al., J. Biol. Chem. 265: 12119-12122 (1990); Al tieri et al. Cell. Biol. 107: 1893-1900 (1988); Taniguchi-Sidle et al. Bi ol. Chem. 267: 635-643 (1992)). Similarly, LFA-1 (Staunton et al., Cell, 61: 243-254 (1990)) and Mac-1. (Diamond et al., Cell, 65: 961-971 (1991)) the binding site on ICAM-1 is It does not contain R-G-D residues. To date, any inte No data have been reported on the positioning of the ligand-binding site for gulin . I domain is ligand-binding to van Willebrand factor and complement factor B It is structurally homologous to the module (Larson et al., J. Cell Biol. 108: 703-712. (1989); Pytela, EMBO J. 7: 1371-1378 (1988)), this region became an integrin. It was speculated that it is involved in the recognition of the ligand by (Hynes et al., Cell, 69: 11- 25 (1992); Larson et al. Cell Biol. 108: 703-712 (1989); Pytela, EMBO J ． 7: 1371-1378 (1988)).   Mac-1 indiscriminately binds to several soluble and cell surface ligands Therefore, it is one of the biggest concerns of integrins containing I domain. . Previous studies have shown that the characteristic Mac-1-dependent adhesive function is MA-1α. Proving that it can be blocked by another group of b (Anderson et al., J. I. mmunol. 137: 15-27 (1986); Dana et al. Immunol. 137: 3259-3263 (1986); Diamond et al., "Differential Effects on Leukocyte Functions of CD11a, CD11b , And CD18 Monoclonal Antibodies "(CD11a, CD11b and CD18 models Different effects of noclonal antibodies on leukocyte function), Leukocyte Typing IV, Knapp et al., Eds., Oxford University, London, 570-574 ( 1989); Wright et al., Proc. Natl. Acad. Sci. USA, 80: 5699-5703 (1983); Wri ght et al., Proc. Natl. Acad. Sci. USA, 85: 7734-7738 (1988)), we are different It was speculated that the structural domain of P. Mac-1 and p1 High degree of structural identity between 50 and 95, antigenic difference and apparent functional phase Because of the difference, we used Mac-1 / p15 to locate the ligand binding site. A mutual set of 0,95 chimeras was designed. In this report, four functionally different Ligands are tested. We have found that the I domain on Mac-1 is It was found to be important for Gand's perception.                               Summary of the invention   The present invention provides for the presence of Mac on the cell surface when resting myeloid cells are stimulated. -Based on the new observation that not all 1 molecules are activated . Only selective subpopulations of Mac-1 molecules are activated and have the ability to bind ligand. To earn. The present invention shows that upon activation, an activation-specific epitope appears on the Mac-1 molecule. It also discloses the manifestation. These activation-specific epitopes are Activated Mac-1 Molecules and Stimulated Myeloid-Like Cells with Inactive Mac-1 Molecules and Rest It can be used to distinguish from myeloid cells.   Based on these activation-specific epitopes, the present invention is directed to stimulated myeloid cells. Class of monoclonal antibodies M that bind to activated Mac-1 molecules present in It discloses the creation of Abs. These antibodies are purified Mac-1 substrate and interface It was also found to have a novel ability to bind active agent solubilized cell lysates.   Members of this class of antibodies, CBRM1 / 5 below, have been described. CBR M1 / 5 is on stimulated neutrophils (which can be activated and bind ligand) It binds to a subset of the existing Mac-1 molecules (10-30%). CBRM1 / 5 is a good activating agent for Mac-1 ligand, fibrinogen and ICAM-1 It was further found that the binding of spheres could be blocked.   Another member of this class of antibodies, CBRM1 / 19, was further isolated. It was This antibody binds to an activation specific epitope, but by CBRM1 / 5 Recognize subpopulations that differ from those that combine.   The invention further provides methods for isolating other members of this class of antibodies. Special React with similar stimulated bone marrow-like cell subpopulations or similar activated Mac-1 subpopulations Antibodies to similar cells, such as in the case of CBRM1 / 5 or CBRM1 / 19 Confirmed by their ability to bind to subpopulations or similar activated Mac-1 subpopulations. Can be recognized.   The present invention selectively inhibits the binding of stimulated myeloid cells to the ligand of Mac-1. A method is also provided. In particular, stimulated myeloid cells are (1) on stimulated myeloid cells (2) non-activated Mac-1 and resting bone marrow capable of binding to existing activated Mac-1 (3) Targeted ligand / Mac-1 interaction By providing an antibody, antibody fragment or antibody derivative capable of inhibiting the action to bone marrow-like cells Can block binding to the Mac-1 ligand. For example, CBRM1 / 5 inhibits ICAM-1 and fibrinogen binding when subjected to myeloid cells. Stop.   The present invention provides a variety of mediated by bone marrow-like cell adhesion, migration and ligand interactions. Further provided are methods of treating a medical condition. In particular, flames mediated by non-specific defense systems Symptomatic response is the administration of an effective amount of antibody-based agent to patients in need of such treatment. Can be treated. The antibody-based agent used in the method is on stimulated myeloid cells. Capable of binding to existing activated Mac-1, but non-activated Mac-1 and resting bone marrow-like An antibody, an antibody fragment, or an antibody derivative that cannot substantially bind to cells.   The present invention further provides a method of locating the presence of stimulated bone marrow-like cells within the body to be treated. To serve. In particular, the presence and location of cells that express the active form of Mac-1 are Can bind to activated Mac-1 present on cell-like cells but is inactive Detectably labeled, substantially incapable of binding to modified Mac-1 and resting myeloid cells Can be determined in the treated body by administering the antibody or antibody fragment to the treated body. It The location of the labeled antibody or antibody fragment can then be detected using known methods.   The present invention identifies agents that can activate Mac-1 present on resting myeloid cells A method is also provided. In particular, resting myeloid cells or inactivated Mac-1 molecules were tested. Contacted with an agent. The cells are then activated M present on stimulated myeloid cells. Can bind ac-1, but substantially binds to non-activated Mac-1 and resting myeloid cells Contact with an antibody that cannot. The cells or purified Mac-1 molecule are then The body is tested to determine if it binds to cells.   The present invention provides an agent capable of inactivating activated Mac-1 present on stimulated myeloid cells. It also provides a method of recognition. In particular, stimulated myeloid cells or purified activation The Mac-1 molecule is contacted with the agent to be tested. The cells are then stimulated with bone marrow-like cells. Can bind to activated Mac-1 present on cells, but not activated Mac-1 and rest Contacted with an antibody that is substantially unable to bind to myeloid cells. Then the cells or purified The isolated Mac-1 molecule is used to determine whether cells bind to the antibody. Be tested.   The present invention selectively selects cells expressing activated Mac-1. It also provides a way to kill. In particular, having activated Mac-1 present on the cell surface Cells bind to the toxin-derived antibody containing the toxin moiety or the surface of activated Mac-1. And an antibody or antibody fragment that is capable of binding, but is substantially unable to bind to non-activated Mac-1. Such cells can be selectively killed by contacting them.   The present invention selects cells expressing activated Mac-1 from a solution or liquid such as blood. A method of selectively removing is also provided. In particular, containing activated Mac-1 on the cell surface Stimulated bone marrow-like cells can bind to activated Mac- but not activated Mac-1. From the liquid by passing the liquid through a fixed antibody or antibody fragment that cannot substantially bind to It can be selectively removed. Such methods affect unstimulated myeloid cells. Stimulated myeloid cells or myeloma expressing activated Mac-1 from patient blood without feeding Used in standard leukophoresis type procedure to remove ulcer cells Can be                         Brief description of the drawings   Figure 1. A. Treated or stimulated with PMA (100 ng / ml) for 10 minutes at 37 ° C Immunofluorescence flow cytometry of untreated peripheral blood neutrophils. Neutrophil negative control Roll (X63), MAb to Mac-1 α subunit (LM2 / 1) Alternatively, it was stained with CBRM1 / 5. B.¹²⁵Triton X-100 interface of I-labeled peripheral blood neutrophils Immunoprecipitation of Mac-1 from activator lysates. Immunoprecipitation with the following MAb reduced SD Subjected to S-5% PAGE and autoradiography: Row 1, CBRIC2 / 2 (ICAM-2); Row 2, LM2 / 1 (Mac-1α); Row 3, CBRM1 / 5 (Mac-1α). Molecular weight standards are shown on the left.   Figure 2. Saturation binding of CBRM1 / 5 IgG and Fab. A, untreated neutrophils; B , Neutrophils stimulated with PMA (100 ng / ml) for 10 minutes at 37 ° C. Excess neutrophils Unlabeled CBRM1 / 5 in the absence (open circles) or in the presence (open triangles) on ice for 4 hours¹²⁵ Incubated with I-labeled CBRM1 / 5. The specific binding amount (black circle) is It was measured by subtracting the non-specific binding amount from the total binding amount. Panel B, supplementary drawing Scatchard plot of specific binding from curves in A, panel B. Calculation K_dIs 15 nM. x-intercept (1010 pM) is 608 per neutrophil It corresponds to the 00 part. C, by CBRM1 / 5 Fab fragment¹²⁵I-CBRM 1/5 IgG competition. Paraformaldehyde solids stimulated with PMA (100 ng / ml) for 10 minutes at 37 ° C. Neutrophils, subsaturated¹²⁵I-CBRM1 / 5 IgG (1 nM) and concentration Ink with increasing unlabeled CBRM1 / 5 Fab fragments for 60 minutes at 37 ° C. Was added. Bars indicate standard error of the mean of triplicates. Panel C, supplementary drawing. CBR M1 / 5 SDS-6% PAGE of IgG and Fab fragments. CBRM1 / 5 IgG (lanes 1, 4), CBRM1 / 5 Fab '₂(Rows 2, 5) Alternatively, a sample (3-5 μg) of CBRM1 / 5 Fab (lanes 3, 5) was non-reduced ( 50 mM iodoacetamide, rows 1 to 3) or reduced (5% β-mercaptoethanol SDS-8% PAGE and silver staining under conditions of columns 4-6). Non-return and return Molecular weight standards under original conditions are shown.   Figure 3. Kinetics of Mac-1 epitope expression. No stimulation (white triangle) or fMLP ( 10^-7, Black square), IL-8 (25ng / ml, black circle) or PMA (100ng / ml, white) Circles) for neutrophils activated at 37 ° C for a predetermined time with A, CBRM1 / 5 or B, immunostained with LM2 / 1 and subjected to immunofluorescence flow cytometry. data Is expressed as relative linear fluorescence units and represents the average of 3 experiments.   Figure 4. A, Effect of temperature on Mac-1 expression in peripheral blood monocytes and B) neutrophils. Cells without stimulation or in the presence (fMLP, 10^-7PMA, 100ng / ml) Incubate at 4 ° C, 25 ° C and 37 ° C for 10 minutes, then negative control (X63), Mac-1 α-subunit MAb (LM2 / 1) or C Immunostaining was performed with BRM1 / 5. The display histogram shows log fluorescence intensity vs. cell number. are doing.   Figure 5. Outline of Mac-1 / p150,95 chimeric molecule Figure and flow cytometric analysis. A, MA used for flow cytometry b is X63 (unbound control), LM2 / 1 (anti-Mac-1α), OKM1 (Anti-Mac-1α), BLY6 (anti-p150,95α) and CBRM1 / 5 It was. The displayed histogram shows Iog fluorescence intensity versus cell number. B, chimera Molecules assembled by ligation of fragments from p150,95 or Mac-1 cDNA , Stably expressed in CHO cells. The part of the chimera from Mac-1 is shown as a white bar. Then The case from p150, 95 is represented by a shaded bar. The region of the EcoRV-BglII restriction site constitutes the I domain.   Figure 6. Divalent cations affect the expression of Mac-1 MAb epitope. A, Neutrophils in HBSS supplemented with the indicated divalent cation or chelating agent 4 ° C (Pause) and 37 ° C (fMLP, 10^-7M) was incubated for 10 minutes. Fine Cells negative control (X63), MAb against Mac-1 α subunit Immunostaining was performed with (LM2 / 1) or CBRM1 / 5. All subsequent cleaning is displayed Was carried out in the presence of divalent cations. In the displayed histogram, log fluorescence intensity vs. The number of cells is shown. B, non-specific by adsorbing purified Mac-1 on plastic Sequential with HSA and Tween 20 in the presence of site-marked divalent cations Blocked after washing (See materials and methods section). Solid-phase Mac-1 was replaced with MAb (CBRIC1 / 11, Anti-ICAM-1; LM2 / 1, Anti-Mac-1α; CBRM1 / 5, Anti-Mac-1 α) incubated with horseradish peroxidase coupled goat anti- Developed by ELISA with mouse IgG. This representative experiment was performed 3 times and the bar was flat. Shows the standard error of the mean.   Figure 7. SDS-5% PAGE of immunoprecipitates from neutrophil lysates. Iodination Dissolve the spheres in 1% Triton-X-100 and aliquot (60 μl, 1: 4 dilution) Sepharose coupled directly (lane 1, LM2 / 1; lane 4, CBRM1 / 5; rows 7 and 10) incubated with mouse IgG) for 2 hours at 4 ° C. B Pellets and collect the pre-clarified supernatant, which is then directly coupled. Sepharose (lane 2, LM2 / 1; lane 5, CBRM1 / 5; lanes 8 and 11, ma Re-precipitation for 2 hours at 4 ° C. After pelletizing, resuspend the supernatant Collected, but the LM2 / 1 pre-clarified lysate was sedimented with CBRM1 / 5 (lane 3), C BRM1 / 5 pre-cleared lysate was pelleted with LM2 / 1 (lane 6), mouse IgG pre Clarified lysate was LM2 / 1 (lane 9) or CBRMI / 5 Settled in (row 12). Run the material eluted from the beads under reducing conditions and It was submitted for tradography. Molecular weight standards are shown on the left.   Figure 8. Effect of CBRM1 / 5 on neutrophil adhesion to ligands. A, neutrophils MAb below: TS1 / 22 (anti-LFA-1α, ascites diluted 1/250), LPM 19c (anti-Mac-1α, 1/250 diluted ascites), CBRM1 / 5 (anti-Mac -1α, IgG and Fab '₂25 μg_/Fab; 25 μg / ml), CBRM1 / FM after preincubation with 23 (anti-Mac-1α, 25 μg / ml) for 10 minutes LP (10^-7M) purified sICAM-in a 6 cm Petri dish for 4 minutes at room temperature in the presence of Combined with 1. B, ICAM-1 to purified Mac-1⁺C for L cell adhesion The effect of BRM1 / 5. ICAM-1⁺The following MAb: TS1 / 22 (anti-L FA-1α), LPM19c (anti-Mac-1α), CBRM1 / 5 (anti-Mac- 1α) and CBRM1 / 23 (anti-Mac-1α) and preincubation for 25 minutes Afterwards, it was combined with purified Mac-1 in a 6 cm Petri dish for 60 minutes at 37 ° C. Data are expressed as percent cell binding compared to media controls. Data point G is the average of 5 microscopic fields, and the bar shows the standard error of the mean. C, CBR Dose-dependent inhibition of neutrophil binding to fibrinogen by M1 / 5. Neutrophil is C Preincubation for 10 minutes with increasing concentration of BRM1 / 5 IgG or Fab FMLP (10^-7M) in a 6 cm Petri dish for 4 minutes at room temperature in the presence of Made fibrinogen Combined with Unbound cells were removed by continuous washing with a Pasteur pipette. After leaving, it was examined with an optical microscope (40X). Data compared to media control It is displayed as the inhibition rate of cell binding. Data points are for 5 microscope fields Mean, bars represent standard error of the mean. Graph is a representative experiment repeated several times is there.   Figure 9. Outline of leukocyte integrin α chain and Mac-1 / p150,95 chimera FIG. Restriction sites that facilitate mutual exchange (e, EcoRV; b, BglII; a, Af II) is indicated by an arrow or the name of each chimera.   Figure 10.¹²⁵Mac-1 immunoprecipitated from I-COS cell detergent lysates , P150,95 or Mac-1 / p150,95 chimera SDS-PAGE. COS cells with β subunit and (A) Mac-1, (B) p150, 95, (C) M-e-X-b-M or (D) X-e-M-b- Co-transfected with X cDNA,¹²⁵Labeling the surface with I X63 (lane 1, negative control), TS 1/18 (lane 2) as described elsewhere. , Anti-CD18), LM2 / 1 (lane 3, anti-CD11b), Mn41 (lane 4, anti-CD). 11b), VIM12 (lane 5, anti-CD11b), OKM1 (lane 6, anti-CD11b). ), OKM10_old(Lane 7, anti-CD11b), SHCL3 (lane 8, anti-CD11c) ), BLY6 (row 9, anti-CD11c), L29 (row 10, anti-CD11c) Epidemically settled. SDS sample buffer containing 5% β-mercaptoethanol Boil in and electrophorese on a 7% polyacrylamide gel, autoradiograph I attached it. Molecular weights of protein standards are shown in the middle.   FIG. 11. Mac-1, p150,95 and Mac-1 / p150,95 chimeras Flow cytometry profile of CHO cells expressing A. CHO cell trans A negative control (X63), Mac-1 α M to the I domain (LM2 / 1) or C-terminal (OKM1) region of the subunit MAb to C-terminal region of Ab, p150,95 α subunit MA for (CBRp150 / 4G1) or CD18 β subunit Immunostaining was carried out with b (TS1 / 18). Cells subjected to immunofluorescence flow cytometry did. Negative control (X63, bright line) and display above each column in each histogram MAb (dark line).   Figure 12.¹²⁵Mac-1 immunoprecipitated from I-CHO cell detergent lysate , P150,95 or Mac-1 / p150,95 chimera SDS-PAGE. CHO cell transfectant (A) Mac-1, (B) Xe-M, (C) M-e-X, (D) X-a-M, (E) X-b-M, (F) X-e-M-b-X , (G) MbXaM, (H) p150,95, (I) MbX, (J ) XbMaX, (K) M-a-X and (L) M-e-X-b-M¹²⁵Surface labeling with I, materials and methods Negative control MAb (X63, row 1), Mac- MAbs for 1 I domain (LM2 / 1, columns A2, B2, F2, 12 and K 2), a MAb to the C-terminal region of Mac-1 (OKM1, rows D2, E2, G2 And L2), a MAb (CBRp150 / 4) to the C-terminal region of p150,95. MAbs for G1, lanes C2, H2, J2) and CD18 β-subunit (T S1 / 18, lane 3) was immunoprecipitated. Contains 5% β-mercaptoethanol Boiled in SDS sample buffer and electrophoresed on 5% polyacrylamide gel And subjected to autoradiography. The molecular weights of protein standards are shown on the left There is.   Figure 13. Wild-type or chimeric Mac-1 and p Immunofluorescence flow for CHO cells transfected with 150,95 molecules Summary of MAb reactivity examined by cytometry. (+++) is 100% of cells Shows that the MAb was stained with a pattern similar to that shown in FIG. (+ ) Shows that 100% of the cells were positively stained with MAb, but the fluorescence intensity was significantly lower. Indicates that (+/-) indicates that MAb positively stained a subpopulation of cells . (-) MAb staining did not differ significantly from the negative control (X63) Indicates that.   FIG. 14. Summary of functional effects of MAbs on Mac-1 interaction with ligands Me. Assays for interactions with four ligands are described in the methods section . OKM10_oldInhibition data from transfected neutrophils Obtained from iC3b binding to COS cells. Data are shown as inhibition rate by each MAb Shown is the average of at least 3 independent experiments. The standard error of the mean is shown after the ± symbol. Has been done. ND indicates that the value was not determined.   Figure 15. Aggregation profile of PMA-activated neutrophils. Neutrophils at room temperature Pre-incubate with the MAb for 25 minutes, equilibrate at 37 ° C, Activated at 100 ng / ml, t = Omin). Individual display tracing is at least 3 It shows the experiments that were performed once. I / I. Represents relative light transmittance.   Figure 16. CHO cell trans to ICAM-1 (A) and iC3b-E (B) Fectant coupling. Panel A. Mac-1, p150, 95, Mac-1 / p1 Desorption of 50,95 chimera and ICAM-1 transfected CHO cells to 1 m resuspend in 1 (8 x 10^FiveCells), PMA (100 ng / ml), and plast Immunoaffinity-purified ICAM-1 adsorbed on the screen for 90 minutes at room temperature Combined. Unbound cells were removed by transfer pipette with 5 washes . 5 connecting cells The cells were quantified by visually counting the number of cells in the microscope field (40 × magnification). Background transfection rates for plates lacking ICAM-1 for each transfectant I looked it up and subtracted it. Data are the average of 3 experiments and are for wild type Mac-1 Convert to the degree of coupling. Bars represent standard error of the mean. Panel B. Transfer Detached CHO cells and re-plated in a tissue culture medium treated 6-well plate. Swing (4 × 10^FiveCells / ml, 0.5 ml / well), 10% CO₂3 at 37 ° C I glued it over the time. Red blood cells (iC3b-E, 2 × 10⁸50 cells / ml μl) and incubate with the transfectant for 60 minutes at 37 ° C. I started. Non-adherent red blood cells were removed after eight washes with a transfer pipette and Optical Zet (> 10 red blood cells / CHO cells,> 100 test cells) at 100x magnification Counted by microscopy. Data are the average of 3 experiments. The bar is the standard error of the mean Is shown.                         Description of the preferred embodiment   The present invention provides for the presence of Mac on the cell surface when resting myeloid cells are stimulated. -1 Not all molecules are activated and can bind ligand Based on new observations. Selective subpopulations of Mac-1 molecules and myeloid-like cells Only the subpopulation is activated and becomes able to bind the ligand of Mac-1. Activate and live An activation specific epitope appears on the Mac-1 molecule, which results in activation. Activated Mac-1 Molecules and Stimulated Myeloid-Like Cells with Inactive Mac-1 Molecules and Rest It can be distinguished from myeloid cells.   Based on this disclosure, the present invention provides activated Ma present on stimulated myeloid cells. Non-activated Mac- that binds to the c-1 molecule but is on stimulated resting myeloid cells Preparation of a new class of monoclonal antibody MAb that cannot bind to a single molecule Is disclosed. These antibodies are soluble in purified Mac-1 substrate and detergent It has also been found to have a novel ability to bind to denatured cell lysates.   Anti-Mac-1 MAb is purified Mac-1 or activated Mac-1 as immunogen The expressed cells are made using routine manipulations known in the art (Harlow Et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)). Antibodies that selectively bind to activated Mac-1 when anti-Mac-1 antibodies are generated Yes It can be confirmed in several ways.   First, antibodies that selectively bind to stimulated myeloid-like cells are stimulated by neutrophil-like stimuli. Ability to bind to acute myeloid cells and ability to bind resting myeloid cells This can be confirmed by screening the antibody.   On the other hand, the antibody that binds to stimulated myeloid cells is purified purified activated Mac-1. And the ability to bind To screen antibodies for their inability to bind to inactivated Mac-1 But you can check.   As used herein, a monoclonal antibody is one in which the antibody stimulates myeloid cells. Or with activated Mac-1 or stimulated myeloid cells when bound to activated Mac-1 "Selectively binds" but does not substantially associate with non-activated Mac-1 or resting myeloid cells. It is said that they cannot fit. Antibodies have a level of binding to resting myeloid cells and to stimulator cells. If it is below 10% of the binding level, binding to resting myeloid cells is "parenchymal. I can't make it. "   As used herein, a bone marrow-like cell is one in which the cell has a ligand for Mac-1. Stimulated when it can bind or participate in Mac-1-dependent biological functions It is said that Therefore, stimulated myeloid cells are ICAM-1, iC3b, Mac-1-dependent fraction capable of binding to fibrinogen and / or homotypic aggregation Cells that can be involved in. FMLP and PMA were used to stimulate myeloid cells. Such substances include, but are not limited to.   As used herein, a bone marrow-like cell is one in which the cell has a ligand for Mac-1. Pause when unable to bind or participate in Mac-1-dependent biological functions It is said that Therefore, quiescent myeloid cells are ICAM-1, iC3b, Inability to bind fibrinogen and / or Mac-1 dependent homotypic aggregation Cells.   Specifically, the differential screening is performed on bone marrow-like cells or two populations of purified Mac-1 molecules. Is performed using. Myeloid cells such as neutrophils and Mac-1 molecule (active and inactive The isoform) is isolated using known methods. When using purified cells, Divide into groups. Two populations rest on Mac-1 on these cells in a resting conformation. Pre-incubate for about 5 minutes in HHMC at about 37 ° C. Be done. One population of cells may have, for example, 10 ng / ml under conditions that activate Mac-1.   Incubate in PMA-containing medium, while maintaining the other population in resting medium.   The antibody to be screened is then a sample of two populations of cells, ie, purified activation. And bone marrow of an activated population incubated with a sample of inactivated Mac-1 Antibodies that selectively bind to like cells or purified activated Mac-1 molecules are selected. Fine Any method of screening for antibodies that bind to cells or molecules can be used. These include flow cytometry, ELISA and RIA, but they are limited. Not limited. When a large number of antibodies are screened, for example 2% paraform Incubate with antibodies by incubating cells in mualdehyde Prior to fixing the bone marrow-like cells is preferred.   On the other hand, the above operation may be carried out on two populations of cells expressing recombinant Mac-1. Any cell capable of expressing Mac-1 can be used. These include yeast, E. coli like There are, but are not limited to, bacteria, mammalian cells and insect cells.   Using such an operation, it was named CBRM1 / 5 and CBRM1 / 19 Two antibodies were made and confirmed.   As a marker for stimulated myeloid cells and a characteristic subpopulation of activated Mac-1 molecules With CBRM1 / 5 or CBRM1 / 19, other members of this class The antibody can also be confirmed.   When bound by CBRM1 / 5 or by CBRM1 / 19 Similar stericity to the activation specific epitope or activated Mac-1 molecule Antibodies that bind to activation specific epitopes overlapping in This can be confirmed using a competitive binding assay. Such antibodies can be CBRM1 / 5 or C Recognizes a similar subpopulation of Mac-1 molecules as bound by BRM1 / 19 Or bind to different subpopulations of activated Mac-1 molecules.   As used herein, "activation specific epitope" means activated Ma. Epitope present on c-1 molecule but not on non-activated Mac-1 molecule Regarding Such epitopes are sometimes referred to as neoepitope.   As used herein, two epitopes are The ability of antibodies against the two epitopes to compete with each other and bind to the antigen. If they eliminate forces, they are said to be "like" or "sterically overlapping."   Antibodies that bind to activation-specific epitopes that are different or non-sterically overlapping , Bound by CBRM1 / 5 or bound by CBRM1 / 19 Similar to the case of their ability to bind non-competitively with stimulated myeloid cells in a subpopulation, Confirmed by its ability to non-competitively bind to activated Mac-1 of a ruby or similar subpopulation it can.   To test if two antibodies bind to similar cell populations or similar molecules The offset or known method is when combined by CBRM1 / 5 or CBRM1 / 1 Cells competitively or non-competitively with cells or molecules of the same population as those bound by 9. It can be modified by those skilled in the art to identify matching antibodies. Like this Assays include but are not limited to flow cytometry and ELISA analysis. Not.   The present invention further provides antibodies CBRM1 / 5 and CBRM1 / 19 in substantially purified form. provide. As used herein, the antibody is effectively administered to the subject. Or if it has been purified to the level required to be used for the desired purpose , "Substantially purified". In some cases antibodies are all cellular components Is substantially free, but in other cases the antibody may be It just does not contain epidemics.   The present invention further provides a hybridoma capable of producing the CBRM1 / 5 antibody. This The hybridoma producing the antibody of has been deposited with the ATCC, Was named.   The present invention further provides a hybridoma capable of producing the CBRM1 / 19 antibody. The hybridoma producing this antibody has been deposited with the ATCC, Was named.   The present invention provides a book that retains the ability to bind stimulated myeloid cells or activated Mac-1 Further included are fragments of the antibodies of the invention. Such fragments include Fv, Fab and Fab₂Disconnection There are pieces, but not limited to them. Those skilled in the art will use routine methods , An antibody fragment that retains the binding activity can be easily prepared.   The present invention also includes a derivative of the antibody of the present invention (antibody derivative). Used herein Thus, an “antibody derivative” is a combination of the antibody of the present invention or a fragment of the antibody and the antibody usually It contains additional parts that are not parts. Such a part is the solubility, adsorption, Improve the physical half-life, etc., or reduce the toxicity of the antibody, No side effects are eliminated or diminished. The part that can mediate such actions is Reming disclosed in ton's Pharmaceutical Sciences (1980).   "Toxin-derived" antibodies are a special class of antibody derivatives. What is a "toxin-inducing" antibody? An antibody or antibody fragment containing an additional toxin moiety. Bind these agents to the cells It then carries the toxin moiety very close to the cell, thereby promoting cell death. I Any suitable toxin moiety may also be used; however, for example, ricin toxin, difte Use toxins such as rear toxins, radioisotope toxins, membrane channel forming toxins, etc. Is preferred. Procedures for making such toxin derivatives are well known in the art.   “Detectably labeled” antibodies are also another special class of antibody derivatives of the invention. It is. Antibodies can be identified, visualized or located using known methods, including methods known It is said to be detectably labeled if it is bound to a determinable molecule . The detectable label of the present invention includes a radioisotope label and an affinity label (biotin label). , Avidin, etc.), fluorescent labels, paramagnetic atoms, etc., but are not limited to them. Not. Operations for making such labels are well known in the art.   The invention further includes humanized forms of the antibodies of the invention. The humanized form of the antibody of the invention is , By replacing the immunogenic portion of the antibody with the corresponding but non-immunogenic portion, (Ie, chimeric antibody) (Cabilly et al., European Patent Application No. 125,02). 3; Better et al., Science, 240: 1041-1043 (1988); Liu et al., Proc. Natl ． Acad. Sci. USA, 84; 3439-3443 (1987) Liu et al. Immunol. 139: 3521-3526 (1987); Wood et al., Natu. re, 314: 446-449 (1985); all references incorporated herein by reference. Is).   On the other hand, suitable "humanized" antibodies are produced by CDR or CEA grafting / substitution Yes (Jones et al., Nature, 321: 552-525 (1986); Verhoeyan et al., Science, 239) : 1534 (1988); Beidler, J. et al. Immunol. 141: 4053-4060 (1988); all references The references are incorporated herein by reference).   The present invention selectively inhibits the binding of stimulated myeloid cells to the ligand of Mac-1. A method is also provided.   As used herein, a ligand of Mac-1 is a ligand that binds to Mac-1. Is defined as a non-antibody molecule. IC3b and ICA are the ligands for Mac-1 There is, but is not limited to, M-1 and fibrinogen.   Specifically, stimulated myeloid cells are activated Mac-1 present on stimulated myeloid cells. Can be bound to, but deactivated Bone marrow-like cells with antibodies, antibody fragments or antibody derivatives that are substantially unable to bind to Mac-1 By providing, the binding to the Mac-1 ligand can be blocked. For example, CBR M1 / 5 is activated Mac- present on cells when subjected to stimulated myeloid cells. Binds to one molecule and blocks cell binding to ICAM-1 and fibrinogen It However, other Mac-1 / ligans such as Mac-1 / iC3b binding Activation-specific anti-Mac that inhibits the interaction -1 antibody can be isolated.   Adhesion, migration and biological activity of circulating myeloid cells such as neutrophils and monocytes are It is due to the interaction between ac-1 and its ligand. Cell adhesion favors inflammation On neutrophil and monocyte migration and various neutrophil and monocyte effector functions involved in inflammation I found it necessary.   A non-activating specific anti-Mac-1 antibody that inhibits such cell adhesion is used in human It has been proved to be effective in preventing an inflammatory reaction when administered to a medical treatment body. In addition, anti-Mac-1 antibody was shown to inhibit monocyte phagocytosis of foreign substances Was done. Furthermore, antibodies that bind Mac-1 have been found to be both in vitro and in vivo. And mediate other monocyte and neutrophil activities such as chemotaxis and chemotaxis. It was proved to be effective.   Due to their specificity and selectivity, the antibodies, antibody fragments and antibody derivatives of the invention Previously disclosed for use as anti-inflammatory, antimalarial, anti-HIV and anti-asthma agents The anti-Mac-1 antibody was improved. For example, Fisher et al., Lancet, 2: 1058 (1986); Perez et al., Bone Marrow Transp. 4,379 (1 989); Patarroyo et al., Scan. J. Immunol. 30: 129-164 (1989); Lindbom et al., Cl in. Immun. Immuno. Patholo. 57: 105-119 (1990); Butcher, Amer. J. Patho l. 136: 3-11 (1990); Falanga et al., Eur. J. Immunol. 21: 2259-2263 (1991) Dreyer et al., Circulation, 84: 400-411 (1991); Simpson et al., Circulation, 81: 226-237 (1990); Wegner et al., Chest. 101: 345-3 95 (1992); all of which are incorporated herein by reference. Already proved Anti-Mac-1 antibody treats inflammation caused by non-specific defense system response It is effective in doing.   The term “inflammation” as used herein includes only non-specific defense system reactions. It means the meaning. "Non-specific defense system reaction" is a bone marrow-like cell that does not have an immunological memory. Is a response mediated by. Such cells include neutrophils and macrophages is there. As used herein, inflammation is the response of inflammation to non-specific defense system responses. A nonspecific defense system, if more evoked, mediated, or associated with it Is said to be due to the response. At least in part due to the response of the non-specific defense system Examples of such inflammation include adult respiratory distress syndrome (ARDS) or sepsis or trauma. Secondary multiple organ injury syndrome; reperfusion injury of myocardium or other tissues; acute glomerulonephritis Reactive arthritis; skin disease due to acute inflammatory components; acute purulent meningitis or other central nervous system Inflammatory diseases; heat injury; hemodialysis; leukocyte export; ulcerative colitis; Crohn's disease; necrosis Enteritis; granulocyte transfusion-related syndrome; associated with symptoms such as cytokine-induced toxicity There is inflammation.   The activation-specific anti-Mac-1 antibody of the present invention, the antibody fragment and its derivative are For the above symptoms and non-specific defense system It is administered to patients as an anti-inflammatory agent to treat other more mediated conditions. This Agents such as Mac- because they can selectively target stimulated bone marrow-like cell populations. 1 / Different from common anti-inflammatory drugs in that it inhibits specific ligand interactions . Such specificity only affects the specific biological function of myeloid cells, By-products such as nephrotoxicity and bone marrow-like cell depletion seen with conventional less specific anti-inflammatory agents It doesn't work. Moreover, such agents have the effect that they influence the non-activated myeloid cell population. It differs from other conventionally known anti-Mac-1 antibody agents in that it does not give a sound. did Therefore, the antibodies, antibody fragments, and antibody derivatives of the present invention are liable to undergo non-specific defense system reactions. Improved over all conventional cognitive usage of anti-Mac-1 antibodies such as treatment of inflammation caused by Has been done.   The ability of activation-specific anti-Mac-1 antibody to inhibit the interactions required for inflammatory response , Chronic inflammatory diseases and autoimmune diseases such as erythematous lupus, autoimmune thyroiditis, fruit Experimental allergic encephalomyelitis (EAE), multiple sclerosis, some types of diabetic Provides the basis for their therapeutic use in the treatment of Raynaud's syndrome, rheumatoid arthritis, etc. . Such antibodies may be used as therapeutic agents in the treatment of psoriasis. Generally activated Monoclonal antibodies capable of binding Mac-1 are currently treatable with steroid therapy In addition, for the diseases for which previously known non-activating specific anti-Mac-1 antibody was suggested. It may be used in therapy.   Of particular interest in the present invention is the same as when bound by antibody CBRM1 / 5. Activated Macs Recognizing Stimulated Myeloid-Like Cells or Activated Mac-1 in a Subpopulation of Likes It is an antibody against -1. Such antibodies can be myeloid cells / ICAM-1 and bone marrow Used to block cell-like cell / fibrinogen binding, thus Without affecting the reaction and without the potential side effects of depleting non-activated neutrophils , Can be used in vivo as a means of treating inflammatory reactions of the non-specific defense system It   The present invention determines the location and distribution of activated Mac-1 cells expressed in the treated body Method is further provided. Of a detectably labeled activation-specific anti-Mac-1 antibody Administration to patients is performed by activating stimulated Mac-1 like stimulated myeloid and myeloid tumor cells. Provide a means for imaging or visualizing the location, migration and aggregation of cells expressing the To do. The clinical application of antibodies in diagnostic imaging is Grossman, Urol. Clin. North Amer ． 13: 465-474 (1986); Unger et al., Invest. Radiol. 20: 693-700 (1985); Kha W et al., Science, 209: 295-297 (1980).   In such applications, the antibody or fragment thereof of the present invention is detectably labeled. Administered to the patient in a form. As described previously for antibody derivatives, the antibody Radioisotopes, using procedures known in the art for use in Ming Affinity labels (such as biotin, avidin, etc.), fluorescent labels, paramagnetic atoms, etc. Can be detectably labeled.   The invention further provides methods for selectively killing activated Mac-1 expressing cells. Detailed In fact, cells expressing activated Mac-1 can bind to activated Mac-1, Contacting them with "toxin-derived antibodies" that are substantially incapable of binding to non-activated Mac-1. You can kill them selectively. As mentioned above, the antibody derivative of the present invention includes There are antibodies conjugated to toxic molecules such as ricin A chain. This operation is To selectively kill myeloid cells that mediate Mac-1-dependent biological processes More effective means of treating the process and selection of tumor cells expressing activated Mac-1 Providing alternative killing means.   Providing a patient with an antibody, antibody fragment or antibody derivative capable of binding to activated Mac-1 In the above, the dose of the agent to be administered is such that the antibody agent is administered as a diagnostic or therapeutic agent. It depends on such factors as whether or not. Patient's age, weight, height, sex, Factors such as general health status, past medical history, etc. will also affect the dose administered. Get   Dose determination techniques are well known in the art for diagnostic and therapeutic antibody agents. Generally, a dose of antibody that is within the range of about 1 pg / kg to 10 mg / kg (patient body weight) It is desirable to give it to the recipient. However, the following Therefore, the therapeutically effective dose is such that the anti-Mac-1 antibody agent is the anti-ICAM-1 antibody or its equivalent. It can be reduced if it is additionally administered with the substance.   As used herein, both compounds can be detected simultaneously in patient serum When two compounds are administered in close proximity, one compound can be combined with the second compound. It is said to be additionally administered with the product.   The activation-specific anti-Mac-1 antibody or fragment thereof of the present invention is used as a diagnostic agent in use. Or even as a therapeutic agent, intravenously, intranasally, intramuscularly, subcutaneously, enterally or parenterally Is administered. If such a molecule is administered by injection, the administration may be continuous infusion. May be a single or multiple bolus.   The anti-inflammatory agents of the present invention target Mac-1 ligands that mediate biological functions that are inhibited. Give recipient recipients an amount sufficient to inhibit binding of activated Mac-1. available. For example, antibodies used to treat inflammation are ICAM-1 / M It is administered to the patient at a concentration sufficient to inhibit ac-1 binding.   The amount of the drug depends on the dose, route of administration, etc. of Mac-1 / ligand interaction. To “suppress” Mac-1 / ligand interactions if sufficient to stop Is said to be sufficient. Such attenuation or prevention is mediated by target interactions. Biological function is occurring in vivo Whether to test for neutrophil entry into the site of inflammation, for example. , Or by correlating in vitro inhibition studies with expected in vivo efficacy. It   The activation-specific anti-Mac-1 antibody of the present invention may be used alone or in combination with one or more booster immunizations. It is administered in combination with a suppressive agent, especially to recipients of organ or tissue transplants. this Administration of such compounds is for either "prevention" or "treatment" purposes. Prevention When given above, the compound is given prior to the inflammatory response or condition. Compound Prophylactic administration helps to prevent or reduce the subsequent inflammatory response.   When given therapeutically, the compound is given at the beginning (or shortly after) of the actual inflammatory condition. Can be Therapeutic administration of compounds helps to reduce actual inflammation. For this reason, the book The anti-inflammatory agent of the invention may be used before the onset of inflammation or to prevent inflammation (to suppress expected inflammation). It will be given either after the start.   A composition is "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such compositions are such that the amount administered is physiologically significant. If so, it is said to be administered in a "therapeutically effective amount". The agent is a recipe whose existence It is physiologically significant if it causes a detectable change in the physiology of the ent patient.   The antibody-based agent of the present invention is formulated according to known methods for producing pharmaceutically useful compositions. This allows these substances or their functional derivatives to be pharmaceutically acceptable caps. Riabi Mixed with vehicle. Including other human proteins, such as human serum albumin Suitable vehicles and their formulations are described, for example, in Remington's Pharmaceutical Scie nces (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such composition Thing is an effective amount of Contains Mac-1 antibody.   Additional pharmaceutical methods may also be used to control the duration of action. Controlled release Formulations are obtained by using polymers that complex or adsorb the therapeutic agents of the invention. Controlled delivery refers to macromolecules suitable for controlling release (eg, polyesters). Tell, poly amino acid, polyvinyl, pyrrolidone, ethylene vinyl acetate, Chill cellulose, carboxymethyl cellulose or protamine sulfate) This can be done by selecting the concentration and the method of blending. Controlled release formulation Another possible way to control the duration is polyester, polyamid Acid, hydrogel, poly (lactic acid) or ethylene vinyl acetate copolymer Incorporating anti-Mac-1 antibody into particles of such polymeric material. On the other hand, this Instead of incorporating these agents into the polymer particles, for example coacervation or Microcapsules produced by interfacial polymerization, eg hydroxymethyl cells each Loin or gelatin microcapsules and poly (methyl) Methacrylate) in microcapsules or in colloidal drug delivery systems, eg Examples include liposomes, albumin microspheres, microemulsions, nanoparticles and nanoparticles. These substances can be placed in a capsule or in a macroemulsion. This Techniques such as are disclosed in Remington's Pharmaceutical Sciences (1980). It   The present invention identifies agents that can activate Mac-1 present on resting myeloid cells A method is also provided. The agent was treated with the agent due to the appearance of activation specific epitopes. Resting myeloid cells by testing the treated cells or purified Mac-1 molecules. Can be investigated for their ability to stimulate or activate Mac-1 It   Specifically, isolated myeloid cells such as neutrophils or isolated inactivated Mac-1 molecules are the most It is first contacted with the agent to be tested. The treated cells or Mac-1 molecules are then activated. Activated Mac-1 that can bind to non-activated Mac-1 or resting myeloid cells It is contacted with an antibody that is substantially unable to bind. The cell or Mac-1 molecule is then To see if the cell or molecule binds to the activation specific antibody To be tested. If cells or Mac-1 molecules can bind to such antibodies, cells or Is said to be a "stimulant" when contacted with the Mac-1 molecule.   The present invention further provides methods for identifying agents that can inactivate Mac-1. In detail Like PMA-treated neutrophils The stimulated bone marrow-like cells or isolated activated Mac-1 molecules are first contacted with the agent to be tested. Can be The treated cell or Mac-1 molecule can then bind to activated Mac-1 Are contacted with non-activated Mac-1 or antibodies that are substantially unable to bind resting myeloid cells. Can be done. The cell or Mac-1 molecule then becomes Tested to determine if it binds to the body. Cells or molecules with antibodies If it can no longer bind, the agent first contacted with the cell or Mac-1 molecule is It is said to be an "inactivating agent".   The present invention provides agents that can block activation of Mac-1 present on resting myeloid cells. It also provides a method of recognition. Specifically, resting myeloid cells such as neutrophils or isolation The inert Mac-1 molecule is first contacted with the agent to be tested. Then contact Cells or Mac-1 molecules stimulate myeloid cells or activate Mac-1 molecules. It is treated with a solubilizing agent such as PMA. Then these cells or Mac-1 Molecules can bind to activated Mac-1, but not activated Mac-1 or resting bone marrow-like Incubated with an antibody that is substantially unable to bind cells. Then cells or M The ac-1 molecule determines whether the cell or Mac-1 molecule binds to the antibody. Will be tested to decide. If the cell or Mac-1 molecule cannot bind to the antibody For example, an agent first contacted with a cell or Mac-1 molecule is "active." It is said to be an "antioxidant".   The present invention selects cells containing activated Mac-1 on the cell surface from a liquid or a tissue. A method of removing it selectively is also provided. Specifically, stimulated bone marrow-like cells containing activated Mac-1. Vesicles are able to bind to activated Mac-1 present on stimulated myeloid-like cells, but deactivate Liquid is passed over immobilized antibody that is substantially incapable of binding to Mac-1 or resting myeloid cells. Can be selectively removed from a liquid or tissue such as the patient's blood by It For example, stimulated bone marrow-like cells or tumor cells expressing activated Mac-1 are White blood in which patient blood is contacted with immobilized activation-specific anti-Mac-1 antibody by electrophoresis By subjecting the patient to electrophoresis, it can be removed from the patient's blood.   The present invention provides that most of the antibodies that block the Mac-1 / ligand interaction are Mac- Further based on the new observation that it binds to a region within the I domain of one molecule (See Example 2). Furthermore, it blocks Mac-1 / ligand interactions in specific subsets It was found that an antibody that can be produced can be prepared, for example, CBRM1 / 1 is Mac-1 / IC Completely blocks AM-1 interaction but has no effect on Mac-1 / neutrophil binding Yes. Based on this observation, the present invention can block specific Mac-1 / ligand interactions. To provide a method to identify possible agents.   Specifically, the antibody or phase described in FIGS. 13 and 14 Equivalent antibody is a peptide in the Mac-1 molecule that binds to a specific Mac-1 ligand It can be used to position residues. A similar approach to epitope localization is Filed Nov. 28, 1990, incorporated herein by reference Co-pending U.S. application Ser. No. 07 / 618,286.   In the present invention, Mac-1 and various Mac-1 ligands (iC3b, ICAM- 1 and the site of interaction with fibrinogen) are located within the I domain of Mac-1. However, it discloses that they are characteristic and have almost no overlap. This disclosure Based on, can substantially block a specific subset of Mac-1 / ligand interactions Short peptide fragments of Mac-1 sequence can be made.   As used herein, "specific subset of Mac-1 / ligand interactions" Relates to the ability of an agent to block one or more Mac-1 / ligand interactions. For example, Peptides block Mac-1 binding to single ligands such as ICAM-1 Inhibits a specific subset of Mac-1 / ligand interactions with.   Some preferred short peptide fragments of the present invention are derived from the amino acid sequence of Mac-1. A Mac-1 contact site on the Mac-1 ligand containing the derived amino acid sequence Contains a binding peptide and an amino acid sequence derived from a Mac-1 ligand , Ligand binding on Mac-1 There are peptides that bind to the site.   The monoclonal antibody blocking data presented here is for Mac-1 I domain. To further locate the MAb epitope in a characteristic region within Can be combined with the mutagenesis studies of. In addition, the amino acid position of Mac-1 Substitution and Domain Deletion Mutagenesis Bind Specific Mac-1 Ligands or Antibodies To create inefficient Mac-1 deletion and substitution mutants, for example, Kunkel (Pr oc. Natl. Acad. Sci. USA, 82: 488-492 (1985)), Peterson et al. (N. ature, 329: 842-846 (1987)). This can be done by specific mutagenesis. By using such an operation, Mac-1 The short peptide sequence that serves as the overlying ligand contact site can be determined.   The invention further provides chimeric molecules fused between Mac-1 and p150,95. It See example 2. Such a chimeric molecule is contained within the Mac-1 and p150,95 molecules. It is useful in locating certain epitope domains and ligand contact regions. Furthermore, it maintains the ability to associate with the β-subunit (CD11) of protein dimers Create a chimeric molecule involved in a selected set of α-subunit ligand binding it can. For example, by forming a dimer with CD18, ICAM-1 and fibrino Retains the ability to bind gen, but does not resemble neutrophil aggregation or Mac-1 dependent A chimeric Mac-1 / p150,95α-subunit, which cannot form type aggregates, was created. Wear. The method for creating such chimeras and suggestions for their use are described in this document for reference. Co-pending US application filed November 28, 1990 incorporated into the detailed text No. 07 / 618,286.   Having described the invention in general, it is referred to the following examples provided for illustration. It will be understood more easily by way of example, and unless otherwise indicated, examples are provided as limitations of the invention. Not the first.                                  Example 1             Confirmation of a new class of anti-Mac-1 antibody                            Materials and methods MAb   The following murine MAbs against human antigens were used: TSI / 22 (anti-CD11a, IgG1, ascites) (Sanchez-Madrid et al., Proc. Natl. Acad. Sci. USA, 79: 7489. -7493 (1982)); LM2 / 1 (anti-CD11b, IgG1, protein A-purified) (Miller et al., J. Immunol. 137: 28910-2900 (1986)); CBRM1 / 23 (anti-CD 11b, IgG2a, protein A-purified); OKMI (anti-CD11b, IgG 2b, protein A-purified) (Wright et al., Proc. Natl. Acad. Sci. USA, 80:56. 99-5703 (1983)); LPM19c (anti-CD11b) IgG2a, Dr. K. Pulford , Oxford, UK gifts) (Uciechowski et al., In Leukocyte Typing IV: White Cell Differentiation Antig ens, Knapp editor, Oxford University Press, Oxford, pages 543-551 (1989) ); BLY6 (anti-CD11c, IgG1, ascites) (Uciechowski et al., In Leukocyte Ty ping IV: White Cell Differentiation Antigens, Knapp editor, Oxford Unive rsity Press, Oxford, pages 543-551 (1989));% 6.5 (anti-CD54, IgG 2a, Dr. R. A kind gift from Rothlein, Boehringer-Ingelheim, Ridgefield, CT ) (Smith et al., J. Clin. Invest. 82: 1746-1756 (1988)); CBRIC1 / 11 (anti-CD54, IgG1, protein A-purified); My4 ( Anti-CD14, IgG2b, purified) (Griffin et al., J. Clin. Invest. 69: 932-941 ( 1981)) (Coulter Immunology, Hialeah, FL). X63 (non-binding antibody, IgG1 ) And 3G8 (anti-CD16, IgG1) (Fleit et al., In Leukocyte Typing IV: W hite Cell DifferentiationAntigens, Knapp et al., editors, Oxford University P ress, Oxford, pages 579-581 (1989)) was used as the culture supernatant.Protein purification   Mac-1 was immunoaffinity chromatographed after detergent solubilization as described. Purified from leukocyte lysates by chromatography (Diamond et al., Cell, 65: 961-971 (1 991)). Soluble ICAM-1 (sICAM-1, Dr. S. Marlin, Boehringer-I ngelheim Pharmaceuticals, Ridgefield, CT Kindly gifted) by immunoaffinity chromatography as described. Purified from the supernatant of CAM-1 transfected CHO cells (Marlin et al., Nature. , 344: 70-72 (1990)).Tissue culture, transfection and cell production   Neutrophils were dexmeated by Ficoll gradient centrifugation at room temperature as described. Isolated from whole blood of healthy volunteers by Tran sedimentation and hypotonic lysis at 4 ° C (English et al., J. Immunol. Methods, 5: 249 (1974); Miller et al., J. Clin. Inv. est. 80: 535-544 (1987)).¹²⁵Before the I-MAb binding experiment, neutrophils (2 x 10⁷ Cells / ml) in polypropylene tube [Falcoln 2097, vector HB at Becton Dickinson, Lincoln Park, NJ] SS, 10 mM HEPES pH 7.3, 1 mM MgCl₂1 mM CaCl₂(HHM Stored in C) at 4 ° C. Sample leukocyte-rich supernatant for time and temperature studies. It was used after the sedimentation of the tran. Immediately place the cell suspension on ice to remove HBSS) 10 mMH Wash 4 times with EPES pH 7.3 to remove platelets and 5x1 on HHMC 0⁶Resuspended at cells / ml. Flow neutrophils by forward and 90 ° scatter Identified during cytometry and confirmed by immunoprecipitation with MAbs against CD16 . Mononuclear cells were isolated from the interface of a Ficoll gradient, RPMI1640, 5 mM EDT A, 2.5% FCS [heat inactivation low Endotoxin, Hyclone, Utah] and washed 5 times with L1 at 4 ° C. 5, 2.5% FCS) 1 mg / ml human γ-globulin [ICN immunobiologic Luz (ICN ImmunoBiologicals), Costa Mesa, CA] 5 x 10⁶Cells / ml again Suspended. Mononuclear cells were immunostained according to the neutrophil protocol (see below). reference). Monocyte subpopulations are flow cytometric forward and 90 ° scatter patterns Immunoassay with MAb against monocyte-specific CD14 antigen. I accepted.   Of CHO cells expressing wild-type and chimeric forms of Mac-1 and p150,95 Creation and selection are described. They are α-MEM, 10% dialysis FCS, 16 μM thymidine, 0.05 μM methotrexate, 2 mM glutamine and 50 μg / ml Maintained in gentamicin.Preparation and labeling of CBRMI / 5 IgG and Fab fragments   The generation of MAbs for Mac-1 is described in Example 2. Simply put, ha BALB / c mice immunized with ibridoma with immunoaffinity purified Mac-1 Produced from Us. Screen the hybridoma (500) as follows: It was Purified neutrophils were isolated, divided into two groups, and preincubated in HHMC at 37 ° C for 5 minutes. I got it. One population was phorbol ester (PMA, 100 ng / ml) 10 Treated for minutes, the other remained untreated. Place cell suspension on ice for 5 minutes and With ibridoma supernatant Incubated and processed by flow cytometry. In some cases, good Neutrophils were fixed with 2% paraformaldehyde before incubation with MAbs. C Regarding its ability to bind BRM1 / 5 to PMA-stimulated resting neutrophils And confirmed. Clone it twice with limiting dilution,_FourSO_FourProfessional after sedimentation Purified from culture supernatant by Tin A-Sepharose affinity chromatography (Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor: C old Spring Harbor Laboratory (1988)). CBRM1 / 5 is an IgG1 subclass [ImmunoPure Monoclonal Antibody Isotyping Kit, Pierce, Rockford, IL].   Fab fragmentation of CBRM1 / 5 was achieved by pepsin cleavage followed by cysteine reduction. (Parham, P., In Immunological Methods in Biomedical Sciences, Weir Et al, editors, Blackwell, Oxford (1983)). Fab '1.5 x 75 cm S-20 0 Sephacryl [Pharmacia, Piscataway, NJ] size Separated by exclusion chromatography. Fab ’is apparent M_r  40,000 The column was eluted as a single uniform peak. Fab ’₂And Fab 'fragment purity Reduced (5% β-mercaptoethanol) and non-reduced (50 mM iodoacetamide) D) Confirmed by SDS-8% PAGE. Tampa The cytoplasm was visualized by silver staining (Morrissey, JH Anal Biochem. 117: 307-31. 0 (1981)). Unlabeled [Bio-Rad, Richmond, CA] or¹⁴C Labeled [GIBCO BRL, Gaithersburg, MD] protein standards are Oshin (M_r200,000), β-galactosidase (M_r116,000), Phosphorylase B (M_r97,000), serum albumin (M_r66,000), Ovalbumin (M_r43,000), carbonic anhydrase (M_r31, 000), trypsin inhibitor (M_r21,500) and lysozyme (M_r1 4,000).   MAb (100 μg) was added to 1,3,4,6-tetrachloro-3α, 6α-dife Nylglycouril (Pierce Chemical Company, Rockford, IL)¹²⁵ Labeled with I (1 mCi) (Fraker et al., Biochem. Biophys. Res. Communi. 80: 8. 49-857 (1978)). The specific activity of iodinated MAbs was determined (protein recovery was average 83 μg, specific activity 6-7 × 10⁶CPM / μg). ¹²⁵ I-MAb binding assay   Purified neutrophils (2 x 10 in HHMC⁷Cells / ml) on ice or at 37 ° C for 5 minutes Warm up and stimulate for 10 minutes (PMA, 100 ng / ml; fMLP, 10^-7M). Activated neutrophils were immediately placed on ice and 500,000 cells were added to 20-10. In the presence or absence of 0-fold excess cold MAb U-bottom plate containing iodinated MAb (0.109-112 nM) with increasing concentration under [96-well non-tissue culture solution treated microtitration plate, Limbro Titertech ( Linbro-Titertek), Flow Laboratories, McCree , VA]. Binding medium is L15, 2.5% FCS) 1 mg / ml human It contained γ-globulin. Keep neutrophils on ice for 4 hours with iodinated MAbs. Incubate and wash 5 times with L15, 2.5% FCS by 21 gauge needle suction. , L15, 2.5% FCS, 0.2N NaOH to dissolve the γ-radiation I don't know. Specific binding rate subtracted CPM in the presence of excess cold MAb Decided by. Create a Scatchard plot (Scatchard, G. Ann. NY. cad. Sci. 51: 660-672 (1949)), the specific binding of labeled MAbs is no longer increased. The data points were used to determine the site density.Surface labeling, immunoprecipitation and gel electrophoresis   Purified neutrophils (2 x 10⁷Cells) in PBS, 1 mM MgCl₂, 0.5 mM CaCl₂ Washed twice with and resuspended in 2 ml; fMLP (10^-7M) to some neutrophils It was Cells were iodinated, lysed and precleared as described (Diamond et al., Cell, 6 5: 961-971 (1991)).   Immunoprecipitation was performed in one of two ways: (i) 1 mg / ml purified rabbit anti-mouse Ig G [Zymed, Sanfra Cisco, CA] MAb with 1 μl (neat supernatant or 20 μg / ml purified IgG) This was done indirectly by incubating 250 μl at 4 ° C. for 4 hours. Professional Thein A-Sepharose (20 μl of 1: 1 slurry) was incubated overnight I added it. Centrifuge the Eppendorf tube, aspirate the supernatant, and iodinated neutrophil lysate 50 μl was added and the mixture was incubated for 2 hours at 4 ° C. with vigorous agitation (Ii) directly coupled MAb-Sepharose (CNBr activated Sepharose 60 μl of 1: 4 diluted lysate was added to purified MAb 2-3 mg in sucrose CL-4B ml). , Directly by incubating for 2 hours at 4 ° C with vigorous stirring . Washing and elution of immunoprecipitates has been described (Diamond et al., Cell, 65: 961-971. (1991)). Prepare the sample and perform SDS-in the presence of β-mercaptoethanol. 7% PAGE (Laemmli, UK Nature, 227: 680-685 (1970)) Clean and autoradiographed (Laskey et al., FEBS Letters, 82:31. 4-316 (1977)). The pre-cleaning experiment was performed with a directly coupled cephalo as follows. Control (mouse IgG) or specific sepharose (LM2). / 1, CBRM1 / 5), the beads were pelleted and the supernatant ( 60 μl) were transferred to a second portion of the same MAb-Sepharose and loaded as above. I had a cube. The beads are then pelleted and the supernatant (60 μl) Transferred to a portion of LM2 / 1 or CBRM1 / 5-Sepharose and incubated did. After each round of immunoprecipitation, wash the beads and elute the bound proteins as described. (Diamond et al., Cell, 65: 961-971 (1991)). Prepare the sample and SDS-5% PAGE in the presence of lucaptoethanol (Laemmli, UK ature, 227: 680-685 (1970)) and subjected to autoradiography (Laskey et al., F. EBS Letters, 82: 314-316 (1977)).Purified Mac-1 ELISA   Immunoaffinity purified human Mac-1 with 25 mM Tris HCl, 150 mM aCl, 2 mM MgCl₂Microtitration plate diluted with 1/20 with non-tissue culture solution (Limbro Titer Tech, Flow Laboratories, McLean, VA) The individual wells were allowed to adsorb at 37 ° C. for 1 hour. Wash plate 5 times and wash with 1% Blocked in HHMC supplemented with serum albumin for 1 hour at 37 ° C. Plates were then plated with HBSS, 10 mM HEPES pH 7.3, 10 mM EDTA, Twice with 0.05% Tween 20 twice, HBSS, 10 mM HEPES pH 7.3, 0 ． Washed twice with 05% Tween 20. 1 mM MgCl₂1 mM CaCl₂1 mM nCl₂Or HBSS, 10 mMH supplemented with 5 mM EDTA (HHT cation) EPES pH 7.3, 0.05% Tween MAb solution in 20 (100 μl of 20 μg / ml purified MAb) Was added to the wells and incubated at 37 ° C for 1 hour. Then plate for 5 minutes It was ice-cooled and washed 4 times with HHT cation at 4 ° C. Enzyme-linked secondary antibody (1 / 400 μl horse radish peroxidase goat anti-mouse IgG 50 μl, Zym Laboratories, San Francisco, CA) in HHT cation at 4 ° C Incubate for 1 hour at 4 ° C and wash plate 5 times with HHT cation at 4 ° C. did. Substrate (2,2-azinodi (3-ethylbenzothiazoline) sulfonic acid, the Id Laboratories, San Francisco, CA) 0.05% H₂O₂Contains 0. In addition to 1M citric acid, the results were measured using an ELISA plate reader (Titer Tech Ma Quantification at 414 nm in a Luciscan MCC 340).Flow cytometry research   A time course and temperature study was performed as follows: Purification after dextran precipitation Supernatant rich in spheres or leukocytes (5.0 x 10 in HHMC⁶Cells / ml) as a stimulant [IL -8, 25ng / ml (72 amino acid form), PeproTech, Rockyhi Le, NJ; fMLP, 10^-7M; PMA, 100 ng / ml] before being added Pre-incubate for 5 minutes at 0.25 or 37 ° C in a Schottendorf tube (1.5 ml) did. When the time comes, place the sample on ice immediately and a portion on ice containing MAb. U-bottom microtiter plates were added to individual wells. In some cases it was time Immediately fix the cells with paraformaldehyde (2%) and incubate on ice for 30 minutes. I had a cube. Immunofluorescence and flow cytometry were performed as described (Di amond et al. Cell. Biol. 111: 3129-3139 (1990)). Linear fluorescence from log fluorescence Protocols for conversion to values are described (Schmid et al., Cytometry, 9: 533-538 (1988)).Adhesion assay   Purified fibrinogen [2.0 mg / ml in PBS, Sigma Chemical Co. (Sigma C hemical Co. ), St. Louis, MO] or purified sICAM-1 (20 in PBS) 0 μg / ml) at room temperature for 90 minutes in a 6 cm bacterial petri dish [Fisher (Fisher) 1007] and spotted (25 μl). Plate as stated Blocked with surfactant Tween 20 (Diamond et al., Cell, 65: 961-971 (1991). ). Neutrophils (4 x 10 in 1 ml)⁶Cells) in HBSS, 10 mM HEPES pH7 ． 3, 1 mM MgCl₂Resuspend in a preincubator for 10 minutes at room temperature with MAb. I got it. The cells were then transferred to fMLP (10^-7M, final volume 3 ml) or not It was added to the dish in the presence and allowed to adhere for 4 minutes. Non-adherent cells are 0.5% BSA Gently agitate with the same buffer supplemented and then 12 times with a Pasteur pipette Was removed by washing. Coupling is performed with a magnification of 100X using an eyepiece grid. It was quantified by counting the number of adherent cells in at least 4 different fields. MAb Inhibition by Rates were determined by comparison with medium controls.   ICAM-1⁺The L cell binding method is a modification of the previously described protocol. (Diamond et al., J. Cell Biol. 111: 3129-3139 (1990)). Simply put, purified M ac-1 was diluted and adsorbed on a 6 cm Petri dish (30 μl). 90 minutes at room temperature After incubation, non-specific binding sites were blocked with 0.5% heat treated BSA. ICAM-1⁺Trypsin-EDTA (Gibco, Long Island, N Y), removed from the tissue culture plate and washed twice, PBS, 2 mM MgCl 2₂, 0 ． 5% heat treatment BSA (0.5 to 1.0 x 10⁶Cells / ml). 30 minutes before binding assay , Petri dishes or cells in PBS, 2 mM MgCl 2₂, 0.5% heat treated BSA (2ml) Medium MAb (1/200 dilution of ascites or 20 μg / ml purified MAb) at room temperature. Reincubated. Add cells (1 ml) to a Petri dish and incubate at 37 ° C for 60 minutes. I had a cube. Then stir the Petri dish and allow the non-adherent cells to plate at an angle. Tilt and remove the binding buffer with a Pasteur pipette and add fresh medium (3 ml) to the edge of the dish. Removed by adding to a portion. This operation was repeated 5 times. Then the number of adherent cells Were examined by light microscopy at 100X using an eyepiece grid. The binding rate is Converted to troll.                                 result MAb against neoepitope on Mac-1   If the conformational change of Mac-1 is involved in the regulation of its adhesiveness , A specific epitope should be expressed in activated and non-quiescent cells . In order to generate MAbs that recognize these activation-dependent epitopes, Immunoaffinity purification judged functional by its ability to bind to some ligands Mac-1 (Diamond et al., In Leukocyte Typing IV, Knapp et al., Editors, Oxford University, London, pages 570-574 (1989); Diamond et al. Cell Biol. 111 : 3129-3139 (1990)) was used to immunize BALB / c mice. Hybridoma To their differential ability to bind quiescent and phorbol ester-activated neutrophils I screened it. One MAb, CBRM1 / 5, rests with neutrophils However, it did not bind to neutrophils stimulated with phorbol ester. (FIG. 1a). CBRM1 / 5 is a previously characterized M for Mac-1 It bound to activated cells at significantly lower levels compared to Ab. Surface labeled neutrophils (Fig. 1b) or a detergent lysate of transfected COS cells (data not shown) It was confirmed by immunoprecipitation that CBRM1 / 5 recognizes Mac-1.   CBRM1 / 5 epitome on PMA-stimulated neutrophils Low-level expression of MAP was not due to insufficient amounts of MAb, but flow cytometry Studies suggested that saturation was reached at 20 μg / ml (data not shown). ). this is¹²⁵I-CBRM1 / 5 and¹²⁵Confirmed by the binding experiment with I-LM2 / 1 I accepted. CBRM1 / 5 favors activation (fMLP or PMA) but not resting neutrophils Note the difference in scale specifically bound to neutrophils (Figs. 2a and 2b), Table 1). Conclusion Despite the small absolute number of conjugation sites, the binding properties of CBRM1 / 5 are saturated , LM2 / 1 (K_d= 2 nM) Affinity (K)_d= 15_nM)Met. Neutrophils stimulated with fMLP or PMA are 139,000 to 197,000 sites / Total Mac-1 site density of cells (determined by LM2 / 1), 13,000-57 Each had a CBRMI / 5 neoepitope site density of 1,000 sites / cell (Table 1). The bivalent binding of CBRM1 / 5 to Mac-1 is not strictly necessary , The reason is that CBRM1 / 5 monovalent Fab 'fragment specifically binds to activated neutrophils. And replaced with bivalent CBRM1 / 5 IgG (Fab's K_i= 200 nM) (Fig. 2c, data not shown).Dynamics and temperature studies   IL-8, fMLP or PMA is time dependent for expression of CBRM1 / 5 epitope And increased to a stable level after 10 minutes (Fig. 3). Smell in all cases hand, CBRM1 / 5 is a subset of LM2 / 1-reactive Mac-1 molecules on the cell surface Reacted.   MAb binding studies showed that CBRM1 / 5 epitope expression was Mac-1 surface expression or To determine if the correlation with the increase in activation It was performed once (FIG. 4). When the temperature is raised from 4 ℃ to 37 ℃, Mac-1 Increases surface expression but does not necessarily promote Mac-1-dependent adhesion (Buyon Et al., J. Immunol. 140: 3156-3160 (1988); Lo et al., J, Exp. Med. 169: 1779-179 3 (1989); Schleiffenbaum et al., J. Immunol. 142: 3537-3545 (1989)). this The temperature dependence increase is prepared by a preliminary operation. We have 4 ° C to 37 ° C Although increasing the temperature at 50% significantly increased the total amount of surface Mac-1, Pitope expression remains virtually absent in both monocytes (Figure 4a) and neutrophils (Figure 4b) It was discovered that. Conversely, fMLP induces CBR in both monocytes and neutrophils. Expression of the M1 / 5 epitope was induced. These experiments show that expression of CBRM1 / 5 It is suggested that it is associated with activation of Mac-1.Epitope localization of CBRM1 / 5 on transfected cells   CHO cells transfected with wild-type and chimeric Mac-1 molecules Were tested for expression of CBRM1 / 5 (FIG. 5). Transfected C HO thin A subset of the Mac-1 molecules on the vesicle structurally expresses the CBRM1 / 5 epitope. Made it appear. This is because the activated MAb NKI-L16 was transfected with CHO cells. Our past view that it structurally reacted with a subset of LFA-1 molecules on the cell (Larson et al., Cell Reg. 1: 359-367 (1990): Keizer et al., J. Immun. ol. 140: 1393-1400 (1988)). CHO cell trans expressing wild-type Mac-1 Fectants are functional and they iC3b-coated erythrocytes and rosette formation, Leishmania promastigote And bound to purified ICAM-1 (data not shown). CBRM1 / 5 was specifically localized in the I domain of Mac-1 α chain, because LM2 Chimera X containing the Mac-1 I domain on the p150,95 frame, such as / 1 Because it bound to CHO cells expressing -eMbX and wild type Mac-1. is there. Conversely, CBRM1 / 5 has a p150,951 domain on the Mac-1 backbone. Does not bind to the included reciprocal chimera MexBx or wild-type p150,95 (Fig. 5).Effect of divalent cations on CBRM1 / 5 expression   In the case of integrins that bind ligands, divalent cations are required. Contact Adhesion disappears uniformly in the presence of EDTA and is quantitative in the type of divalent cations present. (Gailit et al., J. Biol. Chem. 263: 12927-12932 (1988); Diamond et al., In Leukocyte Typing IV, Knapp et al., Editors, London, Oxford Univ. ersity, pp. 570-574 (1989): Altieri, D.M. C. J. Immunol. 147: 1891-1898 ( 1991); Dransfield et al. Cell. Biol. 116: 219-226 (1992)). We are different CBRM1 / 5 and LM2 / 1 epitotes for sensitivity to divalent cations Was tested. Binding to cell surface (Fig. 6a) or purified Mac-1 (Fig. 6b) In order for the presence of divalent cations to be present in the CBRM1 / 5 epitope, not in LM2 / 1 Required for expression. EDTA (5 mM) abolished the expression of CBRM1 / 5 , LM2 / 1 expression was only slightly reduced. stimulated with fMLP Neutrophils are Mg²⁺Ca than only²⁺In the presence of However, in other studies, CBRM1 / 5 was Mg²⁺Stimulated with PMA in the presence of only Abundant expression in neutrophils (data not shown). In vitro Mac-1 adhesive machine Mn reported to enhance performance²⁺(Altieri, D.C.J.Immunol. 147: 1891-1. 898 (1991)) enhanced the expression of CBRM1 / 5 in purified Mac-1 and induced quiescent neutrophils. The expression of the epitope was promoted. Unlike Mac-1 on neutrophils, Mac-1 purified by affinity chromatography is a divalent cation It expressed comparable levels of CBRM1 / 5 and LM2 / 1 epitopes in the presence. Reverse IC3b-Sepharose chromatography (Hermanowski-Vosatka et al., Cel l, 68: 341-352 (1992)) purified Mac-1 and expressed the LM2 / 1 epitope. However, it lacks CBRM1 / 5 epitope and cannot sustain ligand binding (MS Diamond, KPM van Kessel, SD Wright and TA Springer, unpublished observations) .Structural subset of Mac-1   Control whether CBRM1 / 5 recognizes a subset of Mac-1 molecules. For this purpose, a continuous immunoprecipitation experiment was performed (Fig. 7). A negligible amount of CBRM1 / CBRM 1/5 sepharose is preferred until only 5-reactive Mac-1 remains. After preclearing the neutrophil lysate twice, LM2 / 1-Sepharose was -1 was immunoprecipitated. However, the lysate was first treated with LM2 / 1-Cephalo CBRM1 / 5-Sepharose immunoprecipitates Mac-1 when precleared with I couldn't get it down. These experiments show that CBRM1 / 5 It is suggested to recognize a subset of LM2 / 1 reactive Mac-1.Functional effects of CBRM1 / 5   CBRM 1/5 bivalent IgG, Fab₂Or, the monovalent Fab fragment is ICAM-1 It inhibited more than 80% of the Mac-1-dependent binding of fMLP-stimulated neutrophils to. CB RM1 / 5 binds to as many sites / cells as LM2 / 1 and binds to Mac-1-ICA Completely prevent M-1 interaction The MAb against Mac-1 and LPM19c, which are stopped, had comparable efficacy (Fig. 8a (Diamond et al., J. Cell Biol. 111: 3129-3139 (1990)), data not shown). As a control, the third MAb against Mac-1, CBRM1 / 23, is I It had no significant inhibitory effect on neutrophil adhesion to CAM-1. CBRM1 / 5 Blocking MAb (TS1 / 222) against LFA-1 as in LPM19c (Diamond et al., Cell, 65: 961-971 (1991)), the MAb combination is I Virtually abolished neutrophil binding to CAM-1. Mac-1 dependence with ICAM-1 The ability of CBRM1 / 5 to inhibit sex neutrophil interaction is associated with binding to purified Mac-1. Confirmed by assay (Fig. 8b). Playing purified Mac-1 with CBRM1 / 5 Incubate is ICAM-1⁺Completely eliminated L adhesion.   CBRM1 for fMLP-stimulated neutrophil adhesion to purified fibrinogen Almost complete loss of binding was also observed when testing the effect of / 5. Saturation concentration C BRM1 / 5 IgG, Fab '₂Or, Fab binds to fibrinogen about 9 times. 0% inhibition and half maximal inhibition was observed at IgG concentrations of approximately 1 μg / ml (Fig. 8c, data. Not shown). In addition, other MAbs (C-1) against Mac-1 used at similar concentrations BRM1 / 23) showed almost no inhibitory effect (data not shown). others Therefore, CBRM1 / 5 Confirmed 10% of the Mac-1 molecules present on neutrophils after fMLP stimulation Subset mediates at least 90% of the adhesive activity of Mac-1.                                 Consideration   Of the Mac-1 molecule on activated and non-resting peripheral blood neutrophils and monocytes Characterization of a novel MAb, CBRM1 / 5, that associates with subpopulations is disclosed herein. There is. This MAb recognizes an activation-dependent neoepitope and activates Mac Acts as a reporter for -1. Our result is CBRM1 / 5 Neoe The pitope-expressing subset is at least two characterized ligand ICs Structurally active Mac-1 min that retains adhesion to AM-1 and fibrinogen Prove that it contains children. This evidence includes: (i) CBRM 1/5 reacts with Mac-1 on the surface of resting neutrophils or monocytes by immunofluorescence Absent. However, chemotaxis-inducing agent fMLP, chemotaxis-inducing agent site Stimulation with kine IL-8 or phorbol ester is a sub-sequence of the Mac-1 molecule. Promptly promotes the expression of epitopes on the cell. Expression of neoepitope and Time course of Mac-1 adhesive activity (Anderson et al., J. Immunol. 137: 15-27 (1986 ); Detmers et al. Exp. Med. 171: 1155-1162 (1990)), both within 2 minutes It's parallel because it happens. (Ii) for Mac-1¹²⁵I labeled MAb Saturated bond of Experimental quantification of site density revealed that resting neutrophils had approximately 12000 LM2 / 1 binding sites. / Cells but not identified for CBRM1 / 5 site / cell Have proved. Treatment with fMLP and PMA is 10 times more than described ( Increasing the total site density of Mac-1 (as reported in LM2 / 1) (Berger et al. , J. Clin. Invest. 74: 1566-1571 (1984): Lo et al. Exp. Med. 169: 1779-1 793 (1989); Miller et al. Clin. Invest. 80: 535-544 (1987): Todd et al. Clin. Invest. 74: 1280-1290 (1984)), 10 to 30% of these molecules have CBR Induce expression of M1 / 5 neoepitope (13000-57000 sites after stimulation) /cell). (Iii) In continuous immunoprecipitation, CBRM1 / 5 was Mac- with neutrophils It was confirmed to recognize a structural subset of one molecule. CBRM 1/5 Epito The lysate pre-cleared for the immunoprecipitate was immunoprecipitated by LM2 / 1 -1 molecule but pre-cleared for LM2 / 1 epitope containing Mac-1 The clarified lysate contains no residual amount of CBRM1 / 5 sedimentable material. (Iv ) Functional studies showed that less than 15% of Mac-1 molecules were stimulated in fMLP-stimulated neutrophils. Despite binding, CBRM1 / 5 (IgG or Fab fragment) Inhibits Mac-1-dependent adhesion to Gen and ICAM-1 almost completely (> 85%) It shows that you do. Even in additional experiments, ICAM-1⁺Since the adhesion of L cells is eliminated, the blocking ability of CBRM1 / 5 is confirmed. I accepted.   Inhibits Mac-1-dependent adhesion to ICAM-1 and fibrinogen Several MAbs have been characterized (Altieri et al., J. Biol. Chem. 265: 12119-121. 22 (1990); Diamond et al. Cell. Biol. 111: 3129-3139 (1990); Smith et al., J. ． Clin. Invest. 83: 2008-2017 (1989): Wright et al., Proc. Natl. Acad. Sci. USA, 85: 7734-7738 (1988)), CBRM1 / 5 mediates ligand interaction. It is unique because it recognizes a subset of the Mac-1 molecules that do. CBRM1 / 5 is located in the I domain on Mac-1, where ICAM-1 and fibrino It seems to contain the main recognition site of the gene (see below). However, Mac-1 Not all MAbs that bind to a subset of molecules recognize functional epitopes is not. We have two more MAbs (anti-CD18, CBRM1 / 19; anti-CD1 1b, CBRM1 / 28), but these are activated and not resting It binds to neutrophils and does not block function (MS Diamond and TA Springer, unpublished data T). With the discovery of a structurally characteristic subset of the Mac-1 molecule, neutrophils are f Why does it show no significant change in redistribution of surface Mac-1 after activation with MLP? (Detmers et al., J. Cell Biol. 105: 1137-1145 (1987); Smith et al. , J. Clin. Invest. 65: 804-812 (1980)).   Neutrophils with chemotactic factor cytokines, nucleotides or phorbol esters And monocyte activation increase the binding activity of Mac-1 to some ligands Saru (Altieri et al., J. Cell. Biol. 107: 1893-1900 (1988); Altieri et al. Biol. Chem. 26: 7007-7015 (1988); Diamond et al. Cell. Biol. 111: 3129-3 139 (1990); Philips et al. Clin. Invest. 82: 495-501 (1988); Smith et al., J. ． Clin. Invest. 83: 2008-2017 (1989): Wright et al. Immunol. 136: 1759-1 764 (1986); Wright et al., Proc. Natl. Acad. Sci. USA, 85: 7734-7738 (1988) ), The molecular basis for this change remained unclear. Ma on neutrophils A quantitative increase in c-1 expression does not directly correlate with adhesive function (Buyon et al., J. Immunol. 140: 3156-3160 (1988); Lo et al. Exp. Med. 169: 1779-1793 (1989); Vedder et al., J. Clin. Invest. 81: 676-682 (1988)), the direct evidence so far is about structural changes. I couldn't get it. Global Mac-1 expression in 4 ° C, 25 ° C and 37 ° C studies Shows that the increase in TNF does not induce the CBRM1 / 5 epitope on neutrophils or monocytes Shows. Similarly, increased Mac-1 surface expression was associated with ICAM-1, fibrinose. Insufficient to promote Mac-1-dependent adhesion to gen or other ligands ( Buyon et al. Immunol. 140: 3156-3160 (1988); Vedder et al. Clin. Invest. 81: 676-682 (1988): MS Diamnd unpublished observation). Fibrinogen and ICA Adhesion to M-1 and CBR Expression of the M1 / 5 epitope both requires cell stimulation. We have a functional relationship Acquire continuous CBRM1 / 5 epitopes and combine with ICAM-1 and fibrinogen Activation activates a subset of Mac-1 molecules so that they are structurally qualified to bind. I think to encourage.   Pairs with activation-dependent neoepitope of integrin superfamily members Several other MAbs that have been described recently. Platelet integrin gpIIb Of MAb to -IIIa is induced by cell activation or ligand binding Reported to monitor changes (Du et al., Cell, 65: 409 (1991)). actually The first evidence for structural changes in integrins is ADP, epinephrine or With thrombin-treated resting platelets It came from the development of a PAC-1 MAb that recognizes gpIIb-IIIa (O'Toole Et al., Cell Reg. 1: 883-893 (1990); Shattil et al., J. Am. Biol. Chem. 260: 11107-1 1114 (1985)). PAC-1 is a subset of gpIIb-IIIa molecules (20 ˜50%) and inhibits fibrinogen-mediated platelet aggregation (Shattil et al. , J. Biol. Chem. 260: 11107-11114 (1985)). Furthermore, it expresses gpIIb-IIIa CHO cells are soluble in fibrinogen or PAC until structural changes occur. -1 Cannot bind to MAb (O'Toole et al., Cell Reg. 1: 883-893 (1990)). Structure Additional evidence for structural alterations is the ligand on gpIIb-IIIa Came from the characterization of a class of MAbs that recognize inducible binding sites (LIBS) (Frelinger et al., J. Biol. Chem. 265: Inpress (1990)). Many of gpIIb-IIIa The formation of MAbs for several conformations is linked to cell activation and ligand binding. Suggests that it causes characteristic structural changes that promote attachment and mediate function (Frelinger et al., J. Biol. Chem. 266: 17106-17111 (1991)). PAC-1 and C Since BRM1 / 5 has similar binding properties, we have determined that the platelet integrin -Like leukocyte integrins require similar structural changes to promote adhesion I believe.   Several activation-dependent epitopes on leukocyte integrins have also been described . MAb7E3 originally made against gpIIb-IIIa (Coller et al., J. Clin. Invest. 76: 101-108 (1985)) are monocytes or monocytes that have not been rested after ADP stimulation. Binds to all Mac-1 molecules on the globular cell line (Altieri et al., J. Immunol ． 141: 2656-2660 (1988)). Unlike PAC-1 or CBRM1 / 5, it is It has no declared functional effect. NKI-L16 MAb is on resting T lymphocytes It only weakly recognizes the leukocyte integrin LFA-1 in (Larson et al. Cell Reg. 1: 359-367 (1990); van Kooyk et al., J. Am. Cell. Biol. 112: 345-354 (1 991)), activated with MAbs that crosslink phorbol esters, IL-2, or CD3 T lymphocytes Combine with. NKI-L16 induces homotypic adhesion of B cell lines and T cell clones Transfects with wild-type LFA-1 chain and defective CD18 β-subunit Restores partial binding of purified CCAM cells to purified ICAM-1 (Hibbs et al., S cience, 251: 1611-1613 (1991)). However, the NKI-L16 epitope , Whether the cloned T cells are structurally expressed at a high level but do not self-aggregate Et al. Are insufficient for cell binding (van Kooyk et al., J. Cell. Blol. 112: 345-354. (1991)). MAb KIM127 recognizes the common CD18 β-subunit of leukocyte integrins Promotes LAF-1 and Mac-1 dependent adhesion phenomena (Robinson et al., J. Imm. unol. 148: 1080-1085 (1992)). MAb binding mimics activation and interacts with ligand Induces a conformational change that increases binding activity. To leukocyte integrin Compared to these activation-dependent MAbs against CBRM1 / 5, Binds to a subset of molecules that block, rather than promote, adhesion Without induction of the highly active (avid) form of Mac-1 on neutrophils and monocytes It seems to be unique because it has correlated epitopes.   Recent studies have begun to address structural phenomena that increase binding activity. One model Cells stimulate or alter the binding of divalent cations by integrins. ,this The phenomenon is that it induces a conformation with high binding activity (Allieri et al., J. Im. munol. 147: 1891-1898 (1991); Dransfield et al. Cell. Biol. 116: 219-226 (1 992); Dransfield et al., EMBO J. 8: 3759-3765 (1989)). Integrin a chain is Ca²⁺ Contains a region of extensive homology with divalent cation binding sites on regulatory proteins (Ar graves et al. Biol. Chem. 261: 12922-12924 (1986); Corbi et al., EMBO J. 6: 4 023-4028 (1987); Poncz et al. Biol. Chem. 262: 8476-8482 (1987); Suzuki Et al., J. Biol. Chem. 262: 14080-14085 (1987)), many studies are integrin machines. Noh has demonstrated the need for a divalent cation (Altieri et al., J. Immunol. 147: 1891). -1898 (1991); Bajt et al. Biol. Chem. 267: 3789-3794 (1992); Dransfield Et al., J. Cell, Biol. 116: 219-226 (1992); Elices et al. Cell. Biol. 112: 1 69-181 (1991); Gailit et al. Biol. Chem. 263: 12927-12932 (1988); Hynes , R. O., Cell, 48: 549-554 (1987); Kirchhofer et al. Biol. Chem. 266: 44 71-4477 (1991); Loftus et al., Science, 249: 915-918 (1990); Martz, Human I. mmunol. 18: 3-37 (1987); Rothlein et al. Exp. Med. 163: 1132-1149 (1986 ); Sonnenberg et al., Nature, 336: 487-489 (1988)). On white blood cell integrin Some activation-dependent neoepitope may require the presence of divalent cations. Was shown. Recognizes a common determinant on the a chain of three leukocyte integrins MAb24 (Dransfield et al., J. Cell. Biol. 116: 219-226 (1992); Dransfiel. d et al. EMBO J. 8: 3759-3765 (1989)) is a Mg²⁺Or Mn²⁺, But Ca²⁺ Disappear in the presence of. Conversely, expression of the NKI-L16 epitope requires Ca²⁺Request In the presence of EGTA or EDTA (van Kooyk et al., J. Cell. Biol. 112: 345-354 (1991)). CBRM1 / 5 requires divalent cation for expression A first example of an activation specific MAb for the integrin a subunit, It is known to bind to regions outside the divalent cation binding repeat. Mg²⁺Or Ca²⁺ Is sufficient and EDTA abolishes expression of the CBRM1 / 5 epitope; neutrophils At Mn²⁺Increases CBRM1 / 5 epitope expression in the absence of stimulation , Therefore, it may be possible to make structural changes that mimic cell activation (Al tieri, J. Immunol. 147: 1891-1898 (1991); Dransfield et al. Cell. Biol. 116: 219-226 (1992)). In these studies, divalent cations regulate integrin function. Suggesting that they are permissive for ligand binding , Or whether divalent cations are actively involved in adhesion regulation It remains.   Leukocyte trafficking is a protective response of the host defense system to foreign pathogens or tissue repair However, in some circumstances, white blood cells can mediate a significant degree of vascular and tissue injury. is there. Neutrophils are associated with adult respiratory distress syndrome, ischemia-reperfusion injury after myocardial infarction, vasculitis and Several clinical disorders including shock (For a review, Carlos et al., Immunol. Rev. 114: 1-24 (199 See 0)). Already, some groups have leukocyte-mediated inflammatory damage. Antibodies to leukocyte integrins have been shown to be attenuated (Mileski et al., S urgery, 108: 206-212 (1990); Rosen et al. Exp. Med. 169: 535-548 (1989) Simpson et al., J .; Clin. Invest. 81: 624-629 (1988); Vedder et al., J. Am. Clin. In vest. 81: 939-944 (1988)). C recognizing activated and non-resting white blood cells MAbs such as BRM1 / 5 target highly adherent cells that cause inflammatory injury, and It is clinically useful for nitrification or depletion.                                   Example 2           Confirmation of the ligand binding domain of Mac-1                             Materials and methods MAb   The following murine MAbs against human antigens were used from ascites: TS1 / 22 (anti-CD 11a, IgG1) (Sanchez-Madrid et al., Proc. Natl. Acad. Sci. USA, 79:74. 89-7493 (1982)): LPM19c (anti-CD11b, IgG2a, Dr. K. Pulford. , Oxford, UK gift) (Uciechowski and schmidt, "Cluster Report: CD11", in Leukocyte Typing IV: White Cell Differentiation Antigens, Knapp et al., eds ., Oxford University Press, Oxford, 543-551 (1989)); TMG-65 (anti-C D11b, IgG1, Dr. I. Gift of Ando, Szeged, Hungary) (Uciechowski and And Schmidt, "Cluster Report: CD11", in Leukocyte Typing IV: White Cell Differentiation Antigens, Knapp et al., Eds., Oxford University Press, Oxford , 543-551 (1989)); Mn41 (anti-CD11b, gift of Dr. J. Buyon, New York. ) (Eddy et al., Clin. Immunol. Immunopathol. 31: 371-389 (1984)); 0 (anti-CD11b, IgG2a, Dr. P. Rao, Ortho Pharmaceuticals, Raritan , NJ) (Wright et al., Proc. Natl. Acad. Sci. USA, 80: 5699-5703 (1983). ); VIM12 (anti-CD11b, IgG1) (Bernstein and Self, "joint R eport of the Myeloid Section of the Second International Workshop on Human Leukocyte Different iation Antigens ″, in HumanMyeloid and Hematopoietic Cells, Reinherz et al. eds., Springer-Verlag, New York, 1-25 (1986)); 14B6E. 2 (anti-CD11b, IgM) and 5A4. C5 (anti-CD11b, IgG1, Dr. L. Ashman, Adelaid e, a gift from Australia) (Uciechowski and Schmidt, "Cluster Report: CD11 ″, In Leukocyte Typing IV: White Cell Differentiation Antigens, Knapp et al. , Eds., Oxford University Press, Oxford, 543-551 (1989)); CBRp150. / 4G1 (anti-CD11c, IgG2a) (Stacker et al., J. Immunol. 146: 648-655. (1991)); L29 (anti-CD11c, IgG1) (Uciechowski et al., "Cluster Rep. ort: CD11 ″, in Leukocyte Typing IV: White Cell Differentiation Anti gens, Knapp et al., eds., Oxford University Press, Oxford, 543-551 (1989)); BLY6 (anti-CD11c, IgG1) (Uciechowski et al., "Cluster Report: C D11 ″, in Leukocyte Typing IV: White Cell Differentiation Antigens, K napp et al., eds., Oxford University Press, Oxford, 543-551 (1989)); TS1 / 18 (anti-CD18, IgG1) (Sanchez-Madrid et al., Proc. Natl. Acad. Sci. U) SA, 79: 7489-7493 (1982)). The following MAb was used as purified IgG: R15.7. (Anti-CD18, Dr. R. Rothlein, Boehringer-Ingelheim, Gift of Ridgefield, CT) (Entman et al., J. Clin. Invest. 85: 1497-1506 (1990)) OKM1 (anti-CD11b, IgG2a) (Wright et al., Proc. Natl. Acad. Sci. USA, 80: 5699-5703 (1983). ): OKM9 (anti-CD11b, IgG1, Dr. P. Rao, Ortho Pharmaceuticals , Raritan, NJ) (Wright et al., Proc. Natl. Acad. Sci. USA, 80: 5699-57. 03 (1983)); LM2 / 1 (anti-CD11b, IgG1, (Miller et al., J. Immunol. 137: 2891-2900 (1986)); SHCL3 (anti-CD11c, IgG2b) (Schwarti ng et al., Blood, 65: 974-983 (1985)). M1 / 87 (anti-Forsman antigen, IgM ) (Springer et al., Eur. J. Immunol. 8: 539-551 (1978)) at 60 ° C. for 1 hour It was later used as a culture supernatant. X63 (unbound MAb, IgG1) was also used as a culture supernatant. Used.   Preparation of MAb (CBRM1 series) for human Mac-1 has already been published Protocol (Stacker et al., J. Immunol. 146: 648-655 (1991). ). Briefly, male BALB / c mice were treated with non-secreting murine myeloma cells P3X6. 31 days before fusion with 3Ag8.653 (ATCC CRL1580) (intraperitoneal) And purified 3 days before (intraperitoneally and intravenously) (2 μg / immunization) Mac-1 (Diamond Et al., J. Cell. Biol. 111: 3129-3139 (1990)). Mac-1 is a manufacturer 10 μg of purified Mac-1 as described in the instruction manual of Mycobacter ium phlei) Trehalose dimycolate from Salmonella minneso ta) Monophosphoryl lipid A from R595, PBS, 0.2% Tween 80 and Prepared for initial immunization by mixing with squalene (RIBI Immunochemical Research, Hamillon, MT). The second immunization was performed with PBS. Fusion from mouse 8 days before the test, blood was collected and the antiserum by ELISA against purified Mac-1 The titer was 1/5000. Protocols for fusion and hybridoma maintenance As described (Springer et al., Eur. J. Immunol. 9: 301-306 (1979)). ).   MA binding to peripheral blood neutrophils using indirect immunofluorescence flow cytometry Confirm hybridoma producing b (Diamond et al., J. Cell Biol. 111: 3129). -3139 (1990)). Of the 500 hybridoma supernatants tested, about 20% are preferred. It contained MAbs that bound to neutrophils. Then these are those whose MAb is Mac-1 To determine whether it recognizes α or β chains, peripheral blood lymphocytes and Mac- 1 or p150.95 transfected COS or CHO cells that bind to it. Tested for their ability. Subcloning the MAb to produce Mac-1α It was investigated to be specific to Buunit. MAb is Immunopure MAb Isotai Isotyping was performed using a sorting kit (Pierce, Rockford, IL).Construction of Mac-1 / p150.95 chimera   Oligonucleotides for the production of chimeric Mac-1 and p150.95 α chains Site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA, 82: 488-492 (1 985); Peterson et al., Nature, 329: 842-846 (1987)) using α in pCDM8.^M (Mac-1) (Corbi et al., J. Biol. Chem. 263: 12403-12411 (1988) and α.^X (P150.95) (Corbi et al., EMBO J. 6: 4023-4028 (1987)) coding sequence The restriction sites were introduced unchanged in the homologous region of the cDNA. Nucleotide sequence Numbering followed published cDNA clones (Corbi et al., J. Biol. Chem. 263: 12403-12411 (1988); Corbi et al., EMBO J. 6: 4023-4028 (1987)). Change is Introduced with the following nucleotide sequence to facilitate reciprocal exchange: EcoRV (α^Mb p520-525, GACATT is changed to GATATC; α^Xbp515-5 20, GACATT is changed to GATATC), BglII (α^Mbp1064- 1069, AGATCT is unchanged; α^Xbp1059-1064, AGA TCT remains unchanged) and AflII (α^Mbp 1908-1913, GCTC Change AG to CTTAAG; α^Xbp 1900-1905, GCTCAG is C Change to TTAAG). These sites are amino acids in the leukocyte integrin family. Frame I-domains and divalent cation binding regions based on the acid sequence homology pattern Do (Larson et al., J. Cell Biol. 108: 703-712 (1989)). p150. Genome cloning of 95 (Corbi et al., J. Biol. Chem. 265: 2782-2788 (1990)). Confirms the exon-intron boundaries that distinguish these regions from RcoRV And BglII framed the I domain: the RcoRV site Located at the 23rd nucleotide in the first exon of the I domain (exon 7), Bg The lII site is the 15th nucleotide before the end of the last exon of the I domain (exon 10). It's Leotide. BglII and AflII restriction sites include exons 11-13 and three Defining Regions Containing Divalent Cation Bond Repeats (Exons 14-16): Simplified For this reason it is called the divalent cation binding region: the BglII site is at the end of exon 10. It is 10 nucleotides before the end, and the AflII site is exactly a divalent cation bond. At the end of the final exon of the repeat (exon 16) (Corbi et al., J. Biol. Chem. 265: 2782-2788 (1990)). Mac-1 α subunit containing silent mutation Showed that iC3b-E and the appropriate α chain after they were transfected into COS cells. It was antigenically and functionally complete as it bound to the specific MAb (data not shown). Sazu).   In all chimeras, M represents the region of the Mac-1 α subunit, X Represents the region of the p150.95 α subunit, and e, b and a represent the restriction site Rc. oRV, BglII and AflII. M-e-X and X-e-M are pCDM8-α^M (4.7 kb) or pCDM8-α^X(4.1 kb) RcoRV / NotI fragment pCDM8-α^XOr pCDM8-α^MNotI / RcoRV fragment (3.5 or 4.7 kb) and assembled. MbX and XbM, or Ma- X and X-a-M are also the corresponding plasmids BglII / NotI or AflII / It was assembled using the NotI fragment. MbXaM and XbMaX Is α^MOr α^x0.8 kb BglII / AflII of pCDM8-α^X(6.7kb) Is pCDM8-α^M(7.2 kb) AflII / BglII fragment ligated and assembled I was M-e-X-b-M is pCDM8-α^MSalI / RcoRV (4.5k b) Fragment pCDM8-α^MBglII / SalI (3.0 kb) fragment of α and α^XR It was assembled by ligation with coRV // BglII (0.5 kb). X-e-M-b- X is α^MRcoRV / BglII fragment (0.5 kb) of pCDM8-α^XBglII / RcoRV fragment (7.0 kb) and assembled. Assembly of each chimera is controlled Cleavage analysis, hybridization with appropriate segment of cDNA, and junk Confirmed by sequencing (Maniatis et al., Molecular Cloning: A Laborat ory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) ).Tissue culture, transfection and cell production   Neutrophils were treated with dextran sedimentation, Ficoll gradient centrifugation and hypotonic lysis as described. The solution isolated from whole blood of healthy volunteers (English et al., J. Immunol. Met. hods, 5: 249 (1974); Miller et al. Clin. Invest. 80: 535-544 (1987)). Real Prior to testing, cells were placed in polypropylene tubes (Falcon 2097, Becton Di Kinson, Lincoln Park, NJ) HBSS, 10 mM HEPESpH 7.3, 1 mM MgCl₂Stored at medium room temperature. COS cells treated with 15 cm tissue culture solution RPM 11640 on the processing plate, 10% FCS (JRH Bioscience, Nexa, KS), grown in 2 mM glutamine and 50 μg / ml gentamicin, Purification of CsCl in pCDM8 by DEAE-dextran method as described thereafter α^M  , Α^xOr chimera α^{M / X}And common β (Kishimoto et al., Cell, 48: 681-690 (1987) ) CDNA co-transfected (Staunton et al., Cell, 61: 243-254 (1990 )). Surface expression was determined by flow cytometry for transfection as described. Monitored after 3 days (Diamond et al., J. Cell. Biol. 111: 3129-3139 (1990)).   CHO DG44 cells with a deletion of the DHFR gene were identified as Dr. L. Chasin (Colum bia University, New York, NY) and maintained as described (Qu et al., JE). xp. Med. 167: 1195-1210 (1988)). The cells were either wild type or kissed in pCDM8. 25 μg of Mela α, 25 μg of wild type β in pCDM8 and CHO DHFR mini inheritance 5 μg of pDCHIP vector containing offspring at 380 V and 960 μF Co-transfected by tropolation (Chan et al., Mol. Biol. Cell, 3 : 157-166 (1992)). Cells initially lack hypoxanthine but 16 μM thymidine Α-MEM supplemented with 2 mM glutamine and 50 μg / ml gentamicin, 10 % Dialysis FCS (JRH Biosciences, Renexa, KS). 1 After 0 days, selection was reinforced with the addition of methotrexate (0.02 μM). Methotre After two weeks of growth in medium containing xetate, cells were detached with HBSS, 5 mM EDTA, 4 ° C with MAb against Mac-1 or p150.95 (chimera dependent) And incubated for 30 minutes. Cells are α-MEM, 2.5% dialysis FCS 3 times Wash and wash with 50 μg / ml rabbit anti-mouse IgG (Zymed Laboratories, Sanf 10 cm non-tissue coated with Lancisco, CA) and blocked with 10% FCS Culture solution treatment plate [Lab-Tek 4026, Nunc, Inc. (Nunc, Inc. ), Naperville, IL] at room temperature for 5 minutes. Non-contact Adherent cells were removed by eight medium changes and adherent cells were treated with trypsin-EDTA. It was recovered after the reason. The cells were cultured and the panning operation was repeated after a few days. High expression Cells increase the concentration of methotrexate in the medium (0.02 to 0.20 μM). As a control CHO cells expressing a deletion form of ICAM-1 (domain 3 deletion, F185) ( Staunton, D.D. E. Et al., Cell, 56: 849-853 (1989)) together with pDCHIP into pCD. Co-transfection of F185 cDNA in M8, followed by selection and selection as described. Made by Muno Panning. Positive table for all transfectants Surface expression was monitored by immunofluorescence flow cytometry.   ICAM-1⁺15 cm tissue culture plates as described previously for L cell stables. DME) 10% heat inactivated FCS [low endotoxin serum (normative), high claw Hyclone Laboratories, Logan, UT], 2 mM gluta Maintained in min and 50 μg / ml gentamicin (Diamond et al., J. Cell. Biol. 111: 3129-3139 (1990)). Cells were trypsin-EDTA paired prior to adhesion assay. Remove from woven culture plate, PBS, 2mM MgCl₂, 0.5% heat treated with BSA It was washed twice (Diamond et al., J. Cell. Biol. 111: 3129-3139 (1990)).Surface labeling, immunoprecipitation and gel electrophoresis   Transfected COS or CHO cells (1-2 x 10⁶Cells) to HB It was desorbed with SS, 5 mM DTA, washed 4 times with PBS, and resuspended in 2 ml. Fine The cells were iodinated, lysed and pre-clarified as described (Diamond et al., Cell, 65: 961-971 (1991)). Immunoprecipitation was performed as follows: Immune complex was purified. Rabbit anti-mouse antibody (5 μl of Img / ml) Zym Laboratories, Sanfra MAb (neat supernatant 250 μl volume, 1/100 dilution) Formed by incubating ascites, 20 μg / ml purified MAb) at 4 ° C. for 4 hours. I made it. Protein A-Sepharose (Pharmacia KLB, Piscataway , NJ) (20 μl of 1: 1 slurry) was added for overnight incubation. Centrifuge the Eppendorf tube, aspirate the supernatant, add iodinated lysate (50 μl) Incubated for 2 hours at 4 ° C. with vigorous stirring. Washing of immunoprecipitates and Elution is described (Diamond et al., Cell, 65: 961-971 (1991)). Sample Prepare for SDS-PAGE in the presence of 5% β-mercaptoethanol (La emmli, Nature, 227: 680-685 (1970)) and subjected to autoradiography (Las key et al., FEBS Letters, 82: 314-316 (1977)).Protein purification   Mac-1 is LM2 / 1-Sepharose immunoaffinity chromatography as described. Purification from leukocyte lysates after detergent solubilization by chromatography (Diamond et al. , J. CellBiol. 111: 3129-3139 (1990)). ICAM-1 is RR1 as described Erythroleukemia cells by 1 / 1-sepharose immunoaffinity chromatography Surface activity of system K562 Purified from sex agent lysate (Marlin et al., Cell, 51: 813-819 (1987)).iC3b-E Generation and Binding Assay   Modified IgM and iC3b coated red blood cells (IgM-E) iC3b-E And made as described previously (Rothlein et al., J. Immunol. 135: 2668- 2672 (1985)). In some experiments, sheep red blood cells [Colora Searum (Colora do Serum Company), Denver, CO] once with PBS and FITC 0. 22 μm filtered solution (0.8 mg / ml in PBS after being dissolved in 30 μl DMSO) Labeling at 37 ° C for 2 hours. Red blood cells in HBSS, 10 mM HEPES p H7.3, 2 mM MgCl₂Wash 3 times with and resuspend (6 x 10 in 10 ml).⁸cell), Heat-inactivated anti-Forsmann IgM MAb (1/128 dilution with gentle stirring) M1 / 87) and incubated for 60 minutes at room temperature. IgM-coated red blood cells ( IgM-E) was washed 3 times, and C5-deficient human serum (100 μl) [Sigma Chemica Replenishment (Sigma Chemical Co.), St. Louis, MO], HBSS, 1 0 mM HEPES, 1 mM MgCl₂1 mM CaCl₂Resuspend in 1 ml, and spin Incubated at 37 ° C for 60 minutes. iC3b coated red blood cells (iC3b-E) And IgM-E were washed 3 times, HBSS, 1 mM MgCl 2₂1 mM CaCl₂10, Resuspend to mMHEPES pH 7.3 Cloudy (4 x 10⁷Cells / ml).   Neutrophils and transfected CHO cells were tested for binding to iC3b-E. I looked it up. Peripheral blood neutrophils (1.25 x 10 in 50 μl^FiveCells / well) to 5% C O₂HBSS, 10 mM HEPES pH 7.3, 1 m in the incubator MMgCl₂1 mM CaCl₂96-well tissue culture medium treatment in medium 37 ° C for 20 minutes Plate [Corning Glass Works, Corni , NY]; non-adherent cells were removed after two washes with the same buffer. MAb (PBS, 1 mM MgCl 2)₂, Diluted ascites in 0.5% BSA or Pre-incubation with 50 μl of purified MAb) for 20 min; I went 5 times. Then iC3b-E or IgM-E (2 x 10 in 50 μl⁶)as well as PMA (final concentration 65 ng / ml) was added; red blood cells were centrifuged on neutrophils (300 RPM, 5 minutes, 4 ° C) and the plates were incubated for 15 minutes in a 37 ° C water bath. Non Adherent red blood cells in PBS, 1 mM MgCl₂, 0.5% BSA (175 μl / well) Was removed by flicking the plate 6 times after the addition of. Union Pandex fluorescence concentration analyzer [Baxter Healthcare (Ba xter Healthcare Corp.), Mandeline, IL] using a 96-well plate Was quantified by. The inhibition rate was converted as follows: Transfected CHO cells (2 x 10 in 250 μl^FiveCells / ml) to L15 6-well tissue culture medium treatment in 10 mM HEPES pH 7.3, 5% heat inactivated FCS The seeds were seeded on a processing plate and adhered at 37 ° C. for 3 hours or more. Then iC3b -E or IgM-E (2 x 10⁸/ ml, 50 μl) and PMA (final concentration 100 ng) / ml) and the cells were allowed to bind for 60 minutes at 37 ° C. Non-adherent red blood cells 6 to 8 times Remove after washing and remove rosettes (> 10 red blood cells / CHO cells,> 100 test cells) Counted by light microscopy at 200x magnification.Neutrophil homotypic adhesion assay   Previously, two methods were used to quantify the effects of MAbs on neutrophil aggregation Has been done: microscopic examination of the number of free neutrophils (not found in cell zygotes) (Di amond et al., "Differential Effects on Leukocyte Functions of CD11a, CD11b, and CD18 Monoclonal Antibodies ″, in Leukocyte Typing IV, Knapp et al., eds. , Oxford University, London, 570-574 (1989); Patarroyo et al., Scand. J. Immun ol. 22: 171-182 (1985)) or when the cells become a cell aggregate from a single cell dispersion. Aggregometry, which measures the relative increase in light transmission that occurs (Anderson et al., J. Immunol. 137: 15-27 (1986); Buyon et al. Immunol. 140: 3156-3160 (1988); Hammersch Midt et al., Blood, 55: 898-902 (1980)). Both methods are reproducible, but the former is laborious. High activity, the latter only indirectly measuring the number and size of cell aggregates. We have devised another way to use the Coulter counter; Automated, reproducible, mechanically counting the total number of cell particles in a fixed volume It The Coulter Counter handles cell aggregates and is therefore a single unit regardless of size. As particles, a few 2 or 3 cell aggregates have a dramatic effect on particle counts. Therefore, this assay distinguishes weakly blocking MAbs from strongly blocking ones. Sensitive above. Neutrophil (2 x 10⁷Cells / ml) in HBSS, 10 mM HEPES, 1 mM MgCl₂1 mM CaCl₂, And resuspended in 0.5% HSA (HHMCH). Part (0.325 ml) of MAb (1/200 diluted ascites, 20 μg / ml purified MAb) ) For 25 minutes at room temperature. Then these cells (0.3 pre-wet polypropylene Eppendorf tube (1. ml) containing 0.1 ml of HHMCH. 5 ml). Add PMA (final concentration 100 ng / ml) and pipe the tube to Berco Rocka -Bellco Rocker Platform (Belco, Vinland, N J) Gently rocked on both sides of them at 37 ° C. for 15 minutes. Then part (0.2m l) is HBSS, 10 mM HEPES, 2% para Conical tube containing formaldehyde (0.1 ml) [3 ml, Sarstedt , Germany]. The number of particles (single cells or cell aggregates) in a fixed volume Model S-Plus Coulter Counter (Coulter, Hiales, F L). The average of two readings was used as the individual data point. Each condition Repeated at least 3 times with different donors. The% aggregation was determined according to the following formula: The inhibition rate was determined according to the following formula:   Neutrophil aggregation measurements were performed as described (Hammerschmidt et al., Blood, 55: 898-90. 2 (1980)). Briefly, purified neutrophils were washed twice and HHMCH (phenol Resuspended (without red) (107 cells / ml). M of some cells (0.5 ml) Preincubated with Ab at room temperature for at least 25 minutes. Neutrophil (0.4 ml ) Is a standard platelet aggregometer (model DP-247F), Sienco, Stirring at 37 ° C in a siliconized cuvette (700 RP) M). Record the baseline for 3 minutes, then add PMA (100 ng / ml) and let Change of excessive rate Monitored with a strip chart recorder.Adhesion of Neutrophils to Purified Fibrinogen   Purified fibrinogen was resuspended in PBS (2.0 mg / ml) and 6 cm bacterial petri Plate (Falcon 1007, Becton Dickinson, Lincoln Park, NJ) Spotted over 90 minutes at room temperature. Replace the plate with the detergent Blocked with inen 20 (Diamond et al., Cell, 65: 961-971 (1991)). Neutrophil ( 4 x 10 in 1 ml⁶Cells) in HBSS, 10 mM HEPES pH 7.3, 1 mM Mg Cl₂Resuspended in and preincubated with MAb for 25 minutes at room temperature. Next When the cells are fMLP (10^-7M, final volume 3 ml) in the presence or absence of No, it was adhered for 3 minutes and 45 seconds. After gently stirring non-adherent cells, 0.5% BSA Removed by washing 12 times with a Pasteur pipette with the same buffer supplemented with . Join at 100x magnification with at least 4 different fields of view using the eyepiece grid. It was quantified by counting the number of adherent cells. Inhibition rate by MAb is medium control Decided in comparison with Le.Binding of ICAM-1 ⁺ L cells to purified Mac-1   ICAM-1 to Mac-1⁺The procedure for binding L cells has already been described. (Diamond et al., J. Cell. Biol. 111: 3129-3139 (1990)). simply In short, dilute the purified Mac-1 and let it adsorb on a 6 cm Petri dish. (30 μl), blocked with 0.5% heat treated BSA as described (Diamond et al. , J. Cell. Biol. 111: 3129-3139 (1990)). 30 minutes before binding assay, Rate is PBS, 2mM MgCl₂, 0.5% heat treated BSA (PMBSA, 2 ml) Pre-treatment with medium MAb (1/200 diluted ascites or purified MAb 20 μg / ml) at room temperature Incubated. ICAM-1⁺Trypsin-EDTA (Gibco, Long Island, NY) Remove from tissue culture plate, wash twice, PMB SA (0.5 to 1.0 x 10⁶Cells / ml). Control part or Pre-incubated with anti-ICAM-1 MAb for 25 minutes at room temperature. Combined The essay was performed as described (Micklem et al., J. Immunol. 144: 2295-2303 (1990). ). The inhibition rate by MAb was determined by comparison with the medium control.Binding of CHO cell chimera to purified ICAM-1   Method for binding transfected CHO cells to ICAM-1 It is a modification of the protocol previously described for transfectants (Di amond et al. Cell. Biol. 111: 3129-3139 (1990)). Simply put, purified ICA M-1 was diluted and adsorbed on a 6 cm Petri dish (25 μl). 90 minutes of incubation After beating, non-specific binding sites were blocked with 0.5% heat treated BSA. CHO thin Cell transfectants in HBSS, 10 mM After removal from tissue culture plates with HEPES pH 7.3, 5 mM EDTA, H BSS, 10 mM HEPES pH 7.3, 1 mM MgCl₂, 0.5% heat treated BSA Wash twice with and resuspend (8 x 10^FiveCells / ml), ICAM-1 coated Petri dish To room temperature for 90 minutes at room temperature. Use non-adherent cells 5 times with a Pasteur pipette After washing, remove and count the number of adherent cells under a light microscope at 100x using an eyepiece grid. I investigated more.                                   result Construction and expression of Mac-1 / p150,95 chimera   Using the natural restriction site and the site introduced by silent mutation, Mac-1 And the p150.95 α subunit were divided into four structurally distinct regions (Fig. 9). The chimeric α subunit and the common CD18 β subunit temporarily transfer The transfected COS cells were treated with Mac-1 and p150.95 α-subunit. Immunofluorescence flow cytometry with previously characterized MAb populations And immunoprecipitation. Most chimeras are expressed on the cell surface, including Mac-1 and It stained with a MAb to p150.95 in a unique pattern (Table 2). Immune firefly Data obtained with light were confirmed by immunoprecipitation and most chimeric α subunits Showed stable association with the β subunit (FIG. 10, data not shown). Unfortunately, all chimeras were expressed in COS cells Not two: two (XbM and XbMAX) are β subunits Synthesized to remain in the cell without meeting (data not shown), moderate cell surface The third (Xe-M) showing surface expression has an abnormal pattern of immunoprecipitation, The MAb for the .beta. Subunit precipitated only with the .alpha. Chain, and the MAb for the .beta. Subunit was the .alpha. Chain. Not only did β-chain not precipitate (data not shown).   Since we were unable to express the chimeric subset in COS cells Stable cell line for limited functional localization of b structural epitopes for functional analysis I tried to express the molecule. Chimeric α and wild-type β subunits are functional CHO DG44 cells lacking soluble dihydrofolate reductase (Qu et al., J. Exp. Med. ． 167: 1195-1210 (1988)) with the pDCHIP plasmid. I got it. After drug selection, positive cells were confirmed and recovered by immunopanning (Aruf fo et al., Proc. Natl. Acad. Sci. USA, 84: 8573-8577 (1987)), cultured, high level Re-panning until a CHO cell line expressing each individual chimeric molecule was obtained. In flow cytometric analysis, all chimeric molecules were well detected on the surface of CHO cells. It was expressed (FIG. 11) and is equivalent for each chimeric level of the α and β chains (FIG. 1). 1) proved that. In one case (MbXaM), on the surface The level of β subunit was higher than that of α subunit. Cell table However, immunoprecipitation studies have shown that three chimeras There was an incorrect association between the α and β subunits. β subunit MAb (TS 1/18) surface labeled XeM, XbM and XbMaX chimeras Immunoprecipitation was weak or absent (FIG. 12, groups B, E and J; lane 3). This These three chimeras showed TS1 / 18 epi on the β subunit after detergent solubilization. Apparently it was lost. Previously, we found that TS1 / 18 was not associated β subuni LFA-1, Mac-1 and p150.95 from the αβ complex but not from (Springer et al., J. Exp. Med. 160: 1901-1918 (1984). )). Despite the change in antigen reactivity, the β subunit is associated with the α subunit. Furthermore, in these chimeras, co-precipitation by To the extent associated with the α subunit (FIG. 12, groups B, E and J; lane 2). . Weak associations between subunits are also suggested by stoichiometric changes in the sedimentation complex It was XeM, XbM and XbM with MAb to α subunit -A-X immunoprecipitation results in significantly higher amounts of α subunit compared to β subunit. It was different from other chimeras because of the presence ofEpitope localization of MAb to Mac-1   Between α and β subunits in a subset of chimeras Since improper association of A will affect functional assays and our subsequent analysis, we To create a structure-function map of the ligand-binding domain on Mac-1 MAb was used for. That binds a MAb epitope to a particular subset of chimeras Their ability to position on the Mac-1 α subunit and the results to the native Mac -1 was correlated with the ability of the MAb to block the characteristic adhesive function of the molecule. Already featured A new MAb2 to the α-1 chain of Mac-1 to complement the excised MAb10 species Immunization of 4 species with purified Mac-1 and neutrophils, lymphocytes and transfected Cells produced by immunoprecipitation flow cytometry screening (Fig. 13).   Locating structural epitopes of 34 MAbs against Mac-1 α subunit To express wild-type and chimeric Mac-1 and p150.95 O cells were tested for their differential ability to bind MAbs (Figure 13). Of the 34 MAb species tested, 19 were in the I domain and 11 were in the C-terminal region. One (CBRM1 / 20) was located in the divalent cation bond repeat-containing region. Two kinds (CBRM1 / 32 and OKM10_old) Is the N-terminal and divalent cation binding region One is located in a discontinuous epitope that requires the presence of the other. Orto Pharmacutica OKM10 received from Ruz_newIs OKM10_oldDifferent from The new OKM10 As above We are OKM10_oldIC3b-E binding with Mac-1 The two MAbs are not located in the same region of Mac-1 (Fig. 13) . We have two MAbs (OKM10_newAnd CBRM 1/28) Could not be positioned. p150.95 Three Ms for α subunit Abs were also located in CHO cell chimeras: CBRp150 / 4G1 is C-terminal region BLY6 is in the I domain, SHCL3 is in the N-terminal and divalent cation binding domain. It was located in one area.MAb inhibitory activity IC3b-E binding with Mac-1   The ability to inhibit the characteristic Mac-1-dependent adhesive function has been studied in a broad MAb population. I investigated. To investigate that the MAb blocks iC3b binding to Mac-1, Adhesion of FITC-labeled iC3b erythrocytes in the presence of MAb to Mac-1 A quantitative assay was developed that allowed incubation with spheres (Figure 14). I domain MAbs against iC3b binding to neutrophils with an average of 72.6 ± 24.9% , MAbs to the C-terminus blocked with an average of 33.6 ± 16.6%. To the I domain To position Eleven of the 19 MAbs are strongly blocked (> 85%) and M located in the C-terminal region Ab did not show more than 60% inhibition. Located directly in the divalent cation binding region One MAb (CBRM1 / 20) had little inhibitory effect. N-terminal and two Two types of MAbs (OKM10_oldAnd C BRM1 / 32) blocked 81% and 65% of binding (Figure 14). These conclusions Fruits use a limited number of these MAbs to inhibit iC3b-Mac-1 interaction (Anderson et al., J. Immunol. 137: 15-27 (1986); Di amond et al., "Differential Effects on Leukocyte Functions of CD11a, CD11b, and CD18 Monoclonal Antibodies ″, in Leukocyte Typing IV, Knapp et al., eds. , Oxford University, London, 570-574 (1989); Mosser et al. Cell. Biol. 1 16: 511-520 (1992)).Neutrophil isomorphic adhesion   In some groups, activated neutrophils bind isomorphically and this aggregation response is It was shown to be blocked by MAbs against the α-1 or β chains of -1 (Anderson et al. , J. Immunol. 137: 15-27 (1986); Buyon et al. Immunol. 140: 3156-3160 (1 988); Patarroyo et al., Scand. J. Immunol. 22: 171-182 (1985)). Isomorphic media The neutrophil ligand for mediated Mac-1 has not yet been characterized. Good luck We have newly developed to determine the region of Mac-1 that is important for sphere homotypic adhesion. MA for the ability to inhibit this interaction in a modified Coulter counter assay A population of b was tested (see method section). Count the number of particles Assay is more rigorous than the classical aggregometry assay; MAbs like CBRM1 / 2 and Mn41 (FIG. 14) that inhibit 30% are light scattering. Aggregometry assay shows 56 and 67% inhibition (FIG. 15). For I domain MAbs blocked the agglutination assay with an average of 25.9 ± 16.1% and paired with the C-terminal region. MAbs that ran blocked on average 1.8 ± 1.1%. Two MAs for I domain b (LPM19c and CBRM1 / 29) showed 81% and 51% neutrophil homotypic adhesion I strongly discouraged. CBRM1 / 32M for N-terminal and divalent cation binding region Ab blocked by 21% and CBRM1 / 20MAb against divalent cation binding region 7% inhibition.Neutrophil adhesion to purified fibrinogen   Neutrophils are fMLP (10^-7When stimulated with M), they are almost completely Mac- It binds to fibrinogen in a 1-dependent manner. MA for I-domain of Mac-1 b strongly inhibited the binding to fibrinogen with an average of 77.6 ± 13.2% (Fig. 1). 4), MAbs to the C-terminal region weakly inhibited the binding with an average of 5.4 ± 22.1%. It was One located outside the I domain at the site with both N-terminal and divalent cation regions MAb (CBRM1 / 32) blocked binding to fibrinogen (85% ). All MAbs against p150,95 tested were stimulated with fMLP Inhibit the binding of neutrophils to fibrinogen (FIG. 14, data not shown).ICAM-1 ⁺ L cell adhesion with purified Mac-1   Neutrophils stimulated by fMLP bind Mac-1 and L on binding to ICAM-1 FA-1 is used cooperatively (Diamond et al., Cell, 65: 961-971 (1991); Smith et al. , J. Clin. Invest. 83: 2008-2017 (1989)). Check blocking MAbs more easily In order to test the Mac-1 interaction with ICAM-1 exclusively, SEI: ICAM-1 with purified Mac-1⁺Switched to L cell adhesion (Diamond et al. J. Cell. Biol. 111: 3129-3139 (1990)). Average MAb for I domain is 8 The binding was inhibited by 2.4 ± 26.1%, and the average MAb to the C-terminal region was 24.1. Inhibition was ± 26.8% (Fig. 14). 12 out of 19 I-domain MAbs are 9 The inhibition rate was 0% or more, and the maximum inhibition rate by MAb for the C-terminal region was 52%. It was One MAb located at the site with both N-terminal and divalent cation regions binds Was strongly inhibited (CBRM1 / 32, 97% decrease). Directly into the divalent cation binding region One MAb (CBRM1 / 20) that binds contiguously binds ICAM-1 to Mac-1 It didn't stop.Functional studies on Mac-1 / p150,95 chimera   To determine whether the I domain of Mac-1 is essential for ligand recognition , We have determined the ability of CHO cells to bind to ICAM-1 and iC3b-E. La Was tested. Control transfected or untransfected CHO cells Binds fibrinogen structurally, so this ligand was not tested.   Transfected CHO cells bind phorbol bound to purified ICAM-1 Of a functional form for stimulation by a luester-excited Mac-1 transfectant It contained Mac-1 (FIG. 16A). Into transfected COS cells Contrary to previous research by (Diamond et al., J. Cell. Biol. 111: 3129-3139 (1990)) , Binding to ICAM-1 was hardly observed in the absence of stimulation (data shown). Sazu). Two chimeras containing the I domain of Mac-1 (Max, Mb- X) bound to ICAM-1 but was weaker than expected. Surprisingly, wild type p15 CHO cells expressing 0.95 also bound to ICAM-1. For low level binding Nevertheless, it is reproducible and is inhibited by the MAb against p150,95. And was observed in control CHO cells transfected with ICAM-1 No data (Figure 16A) data not shown). We p150,95-ICAM- Not sure about the physiological relevance of one interaction, but p150,95 was stimulated Acts as a counter-receptor for uncharacterized adhesion molecules on endothelial cells (Stacker et al., J. Immunol. 146: 648-655 (1991) c), L cells expressing ICAM-1. Transfectants are purified Weakly binds to p150,95 (Stacker and Springer, unpublished data). Ma Chimeras in which the I domains of c-1 and p150,95 are exchanged (MeeXb- M, X-e-M-b-X) showed no significant difference in adhesion with ICAM-1 . Both wild-type Mac-1 and p150,95 transfectants were ICAM -1 was attached, but three chimeras (XeM, XbM and XbMa -X) did not bind to ICAM-1; however, these three chimeras Inappropriately expressed in COS and CHO cells (see above). Therefore, CHO ICAM-1 despite relatively high levels of expression of all chimeras in cells No readily analyzable pattern of binding to was observed.   CHO cell transfection for its ability to bind iC3b-E-coated red blood cells. The fectant was tested. Almost all CHO cells expressing wild-type Mac-1 Formed a rosette with iC3b-E (FIG. 16B). Binding occurs in the absence of stimulus However, the average number of iC3b coated particles per cell was Increased (data not shown, Diamond, Mosser and Springer, unpublished data). M The binding pattern of iC3b-E to the ac-1 / p150,95 chimera There was no limited positioning (Figure 16B). Appropriately expressed in COS and CHO cells Without binding to ICAM-1 in CHO cells None of the three chimeras (XeM, XbM and XbMaX) are iC No rosette formation with 3b-E. Four types of keys containing the I domain of Mac-1 Mela (MbX, MaX, XeMbX and MbXAM) Formed rosettes with iC3b-E, but wild type despite similar expression levels The level was significantly lower than that of Mac-1. Surprisingly, I dome of p150, 95 One chimera containing the inn (MeXbM) consistently bound iC3b. It was In an independent study in transfected COS cells, we found that It was observed that the set rosettes with iC3b-E (data not shown).                                   Consideration   In this report, using the chimera of Mac-1 and p150,95, we Epitope locating for the MAb 34 species against Mac-1 This was correlated with their ability to block the characteristic adhesive function of -1 molecules. we The 200 amino acid I domain on the α-1 subunit of Mac-1 is iC3b, I On Uncertain Paired Receptors for CAM-1, Fibrinogen and Neutrophil Aggregation It was found to be the main site of ligand recognition in the. N-terminal and divalent cation bond One MAb (CBRM) located outside the I domain in a region-shared epitope 1/32) also includes iC3b, ICAM-1 and F Blocked binding to fibrinogen. Because of this, the N-terminus and probably the divalent cation The binding domain may be involved in the recognition of some ligands. MAb Epito Analysis of results from functional assay with Mac-1 / p150,95 chimera Since it is not simple, it was used to position the ligand recognition site.   The I domain seems to be important for recognition of all four ligands by Mac-1, The reason is that in each of the experimental systems tested, there are several differences located in the I domain. MAb strongly blocked Mac-1 dependent binding (> 80%). Change In addition, in all cases, a separate MAb that almost completely eliminates binding is in the I domain. It was located. 2/3 of MAbs located in I domain are iC3b) fibrinogen And Mac-1 interaction with ICAM-1 was blocked by 70% or more, and neutrophil isomorphism All of the MAbs that strongly block attachment were located in the I domain.   The majority of MAbs against the I domain are strongly inhibitory in at least one assay. However, not all MAbs were able to block some ligand interactions Absent. There are 5 kinds of MAbs (LPM19c, TMG-65, Mn41, CBR). M1 / 29, CBRM1 / 34) only all Mac-1-ligand interactions It just strongly stopped him. With fibrinogen, ICAM-1 and iC3b Many of the MAbs that block binding are neutrophil isotypes It only partially blocked the adhesion. However, there were some exceptions . Two MAbs (OKM9 and CBRM1 / 27) enhance fibrinogen binding. However, the interaction with the other three ligands was only weakly inhibited, and Three MAbs (CBRM1 / that block adhesion to Brinogen and ICAM-1 1, CBRM1 / 2, CBRM1 / 32) partially inhibit the binding to iC3b. It was only Difference in inhibition pattern among MAbs located in I domain Yes It suggests the presence of some immune responses and functional subdomains. Each individual rig , Some or all of these subdomains contribute to the construction of the binding site. It may be Because of this, we have the same ligand recognition site in the I domain. I'm guessing they are duplicates, not one.   A novel MAb mask against Mac-1 solely by its ability to bind to peripheral blood neutrophils. Despite learning, most of the novel MAbs were located in the I domain . This is because the α subunit of human and murine Mac-1 is 74% at the amino acid level. Unexpected because they are identical (Arnaout et al., J. Cell. Biol. 106: 2153-2158. (1988); Corbi et al. Biol. Chem. 263: 12403-12411 (1988); Pytela, EMBO  J. 7: 1371-1378 (1988)), the divalent cation region is maximally identical in the extracellular domain. Rate (84%), and the C-terminal region showed the minimum identity rate (74%). Why I Domain Immunodominant It is unknown if there is; it is more homologous (81% identical) and smaller in size than the C-terminal region (200 for 500 amino acids). Not surprisingly, murine and human Ma The strong identity between the I domains of c-1 indicates that both Mac-1 species are human and murine i. C3b coated particles (Beller et al., J. Exp. Med. 156: 1000-1009 (1982)) and COS cells transfected with Zumi ICAM-1 (Diamond et al., Unpublished) Since it binds to, it retains the site for ligand recognition.   The homology between human Mac-1 and p150,95 is greater than that of murine and human Mac-1. The residues that confer ligand specificity appear to be conserved, although significantly lower than this. Human Mac-1 and p150,95 have 63% overall protein sequence identity, The identity rate between mains is 55% (Corbi et al., J. Biol. Chem. 263: 12403-12411 ( 1988); Corbi et al., EMBO J. 6: 4023-4028 (1987)). Which MAb was studied Also does not cross react between the α-1 subunits of Mac-1 and p150,95. However However, increasing experimental evidence shows that p150,95 is fibrinogen (Loike et al., P. roc. Natl. Acad. Sci. USA, 88: 1044-1048 (1991); Postigo et al., J. Chem. Exp. Med ． 174: 1313-1322 (1991)) and iC3b (Micklem et al., Biochem. J. 231: 233-23). 6 (1985); Myones et al. Clin. Invest. 82: 640-651 (1988); Ress et al., J. Am. Le ukoc. Biol. 51: 109-117 (1992)), including Mac-1 This conservation is associated with ligand recognition because it binds to a ligand repertoire similar to Suggesting that it is sufficient to preserve the site. 36% at the amino acid level Identical LFA-1 and Mac-1 share ICAM-1 as a ligand However, LFA-1 binds to the first Ig-like domain (Staunton et al., Cell, 61: 243-254 (1990)), Mac-1 binds to the third Ig-like domain in ICAM-1 Therefore (Diamond et al., Cell, 65: 961-971 (1991)) the binding appears to be different. A more sophisticated mutagenesis study showed that Mac-1, p150,95 and LFA-1 Whether to utilize related recognition sites that bind to their common ligand Required to find out.   MAb located in the C-terminal region of 493 amino acids interacts with Mac-1 as a ligand It showed little ability to block the effects. The average inhibition rate is related to neutrophil homotypic adhesion. From as low as 1.8% to as high as 33.6% for binding to iC3b. This For this reason, we have found that the C-terminal region of Mac-1 directly interacts with four ligands tested. We believe that it does not contain a recognition site that is important for interaction.   We have 294 amino acid divalent cation bond repeats and 68 amino acids immediately N-terminal to them. Only made one MAb located at 362 amino acids containing the mino acid Thus, there is little conclusive reference to the role of this part of the molecule in ligand recognition. I can't. Apparent lack of immunogenicity It has an 84% amino acid conservation ratio between the corresponding regions of human and murine Mac-1. It may have contributed. One MAb, C positioned exactly in this area BRM1 / 20 did not block adhesion above 30% in either assay. However, we exclude the presence of recognition sites that were not blocked by CBRM1 / 20. Cannot be removed. In fact, the experiment (D'Souza, J. Biol. Chem. 265: 3440-3446 (1990 ); D'Souza, Nature, 350: 66-68 (1991)), related platelet integrin α_{II b} β₃Uses a site within the divalent cation binding repeat to bind fibrinogen Suggesting that Future work with additional MAbs in this area will , Strong sequence conservation in the divalent cation-binding module is associated with receptor conformation. Whether it maintains the protein or sustains its direct interaction with the ligand. It is required to clarify this.   We have successfully positioned both MAbs exclusively to the N-terminal region Although it could not be done, two kinds of MAb (OKM10_oldAnd CBRM 1/32) and one MAb (SHCL3) for p150,95 is Mac-1 or Is a discontinuity requiring the presence of both the N-terminus and the divalent cation binding region of p150,95. Bound to subsequent epitopes. SHCL3 has only the N-terminal of p150,95 Different from CBRM1 / 32 and CBRM1 / 32 because it binds to the chimera containing But; Et al., That both the N-terminus of p150,95 and the divalent cation binding region are present. It was more strongly bound. Importantly, only these MAbs are located outside the I domain. It was placed so that it strongly blocked ligand interaction. CBR M1 / 32 blocks the binding of iC3b) to fibrinogen and ICAM-1 ( 70-95%), OKM10_oldBlock binding to iC3b (Fig. 14 and Ander son et al. Immunol. 137: 15-27 (1986); Wright et al., Proc. Natl. Acad. Sci. USA, 80: 5699-5703 (1983)), inhibiting the binding to fibrinogen (Wright et al. , Proc. Natl. Acad. Sci. USA, 85: 7734-7738 (1988)). SHCL3 is stimulated Endothelial cells (Stacker et al., J. Immunol. 146: 648-655 (1991)) and iC3b (B p150,95 binding with ilsland, Daimond, Mosser and Springer (unpublished observations) But not fibrinogen (Postigo et al., J. Exp. Med. 174: 1313-1322 (1991)). The positioning of the three MAbs with respect to the discontinuous region of the α chain is N-terminal and divalent cation binding regions separated by 200 amino acids of I domain Giving immunological evidence of an association between. Related integrin α_IIb-Β₃as well as α_Five-Β₁In previous electron microscopy studies on α and β subunits, membrane spanning Form a globular head at the N-terminus of the molecule distal to the spanning region (Carrell et al., J. Biol. Chem. 260: 1743-1749 (1985); Hynes , Cell, 69: 11-25 (1992); Nermut et al., EMBO J. 7: 4093-4099 (1988)). Mac -1 and the association from different regions of p150,95 show that the I domain is a series of physically It suggests that it does not get into the spherical head in forming a standing module. It Therefore, the structure of leukocyte integrins is based on the adhesion molecules immunoglobulin and selenium. Different from the linear sequence of domains presented for members of the Kuching family (Bevilacqua et al., Science, 243: 1160-1165 (1989); Driscoll et al., Nature, 3 53: 762-765 (1991); Kansas et al. Cell. Biol. 114: 351-358 (1991); Will iams et al., Annu. Rev. Immunol. 6: 381-405 (1988)).   Experiments with MAbs indicate that the I domain is the major ligand recognition site However, the distinguishable pattern is that the Mac-1 / p150,95 chimera is used in the functional assay. It was not observed when tested. Some factors are iC3b and To directly locate the ligand binding site on Mac-1 for ICAM-1 It may have been involved in the failure of these experiments. As mentioned above, others are Mac-1 And recently demonstrated that p150,95 appears to bind some similar ligands. It was Created a Mac-1 / LFA-1 chimera with little overlap in the ligand repertoire Attempts have been made to synthesize these molecules in their precursor form after transfection into COS cells. However, it was unsuccessful because it was not expressed on the surface. Others are p1 We reported complement receptor activity for 50 and 95 (Micklem et al., Bioch. em. J. 231: 233-236 (1985); Myones et al. Clin. Invest. 82: 640-651 (198 8); Ress et al. Leukoc. Biol. 51: 109-117 (1992)), in our experiments, iC 3b-coated erythrocytes bind to transfected CHO cells expressing p150,95. Not matched (Figure 16B) data not shown). Probably transfected In cells, the I domain of p150,95 contains structural information that binds to iC3b. However, it lacks proper interaction with other parts of the α or β subunits. right. In line with this, human α^XSubunit and chicken β₂Is it a subunit? And a human-chicken p150,95 hybrid expressed in COS cells. The rod molecule strongly rosettes with iC3b-E, but₂With subunits Paired human α^XDid not form (Bilsland and Springer, unpublished observations). I In various experiments, at least in vitro, p150,95 was identified as ICAM-1. It also suggests weak binding (FIG. 16A). I domain of p150,95 is Almost no binding to ICAM-1 due to wild-type p150,95 frame, ICAM-1 in relation to Mac-1 frame (Me-X-b-M) where the next site exists Coupling with was enhanced.   The analysis of the functional assay is based on three chimeric α subunits in COS and CHO cells. (X-E-M, X-b-M, X -B-M-a-X) associated with β subunit or incorrect expression It was complicated. Transfection of surface-labeled CHO cells expressing these three chimeras Immunoprecipitation of ctant with β subunit MAb (TS1 / 18) is weak or absent Absent, but the β subunit coprecipitates with an α subunit-specific MAb Therefore, it is still associated with the α subunit. From this point of view, Xe-M, X -BM and XbMaX are iC when expressed in CHO or COS cells It is not surprising that it did not bind to 3b or ICAM-1. Mac-1 I-do Two other textures, including main, that bind poorly to ICAM-1 and iC3b La (M-X and M-B-X) are subtle structural differences based on changes in antigen reactivity. It shows the usual evidence. M-X and M-B-X are located in the I domain Two defined MAbs, CBRM1 / 2 (Fig. 13) and CBRM1 / 5 (Diam Decrease or loss of binding with ond and Springer, supra). CBRM1 / 5 recognizes a subpopulation of functionally active Mac-1 molecules on neutrophils and monocytes (Diamond and Springer, supra), including the I domain of Mac-1 as a ligand Expressed in other chimeras that bind (XeMbX, MbXAM) It is worth noting that. Therefore, despite high levels of expression in CHO cells, M-X and M-B-X retained ligand binding It may not be a conformation; the molecular packing of the I domain is Is sufficiently corrupted by the improper content of the surrounding domains that the domain cannot be recognized. . To recognize ligands on leukocytes, Mac-1 and LFA-1 are low The binding activity must be converted to a high binding activity state (Buyon et al., J. Immunol. ． 140: 3156-3160 (1988); Dustin et al., Nature, 341: 619-624 (1989); Lo et al. J. Exp. Med. 169: 1779-1793 (1989); van Kooyk et al., Nature, 342: 811-813 (1 989)). Requires integrin to physically “open or close” the molecule to which it binds the ligand Moreover, the juxtaposition of homologous non-natural domains in some chimeras causes this change. It is tempting to speculate that it is preventing the occurrence of. Furthermore, the β subunit is the α subunit Regulates structural changes. According to this, in the cytoplasmic domain of β subunit Why the Deletion or Substitution of Residues in Abolish LFA-1 Binding to ICAM-1? (Hibbs et al., J. Exp. Med. 174: 1227-1238 (1991); Hibbs et al., Science, 251: 1. 611-1613 (1991)), why Xe-M, X-b-M and X-b-M-a-X are iC 3b and not bound to ICAM-1 or β in chicken but not in human₂sub Why Units Rosette p150,95 with iC3b Coated Particles Can be explained.   In summary, the studies presented here have shown that the I domain on Mac-1 is Both cells and soluble ligands It has been proved that it is a major recognition site for nigiri. This is the integrin alpha subunit This is the first study to show the limiting role of the I domain on the knit. Previously, And Willebrand factor, cartilage matrix protein and complement protein Because of its homology with the ligand binding region, It was speculated to be important for ligand recognition (Larson et al., J. Cell. Biol. 108: 703-712 (1989); Pytela, EMBO J. 7: 1371-1378 (1988) ).                 Translations of pages 74 and 75 of the text   Name of Depositary Authority; American Type Culture Collection   Deposit Authorities Address: 12301, Park Lawn, Drive, Rockville,                   Maryland, 20852 United States   Additional display: Hybridoma CBRM 1/5   Name of Depositary Authority; American Type Culture Collection   Deposit Authorities Address: 12301, Park Lawn, Drive, Rockville,                   Maryland, 20852 United States   Additional display: Hybridoma CBRM 1/19

[Procedure Amendment] Patent Act Article 184-8 [Submission date] October 28, 1994 [Correction content]                             The scope of the claims   1. To specifically bind to activated Mac-1 present on stimulated myeloid cells More blocking Mac-1 / ligand interaction, deactivating Mac-1 and resting bone marrow , Antigen-binding antibody fragments and antigen-binding antibody derivatives that do not substantially bind to like cells.   2. Antibodies, antigen-binding antibody fragments and antigen-binding antibody derivatives are selected as activated Mac-1. Selectively bind and block the binding of stimulated myeloid cells to ICAM-1. The described antibodies, antigen-binding antibody fragments and antigen-binding antibody derivatives.   7. Contacting activated Mac-1 with an antibody, antibody fragment or antibody derivative Activation consisting of an antibody, antibody fragment or antibody derivative present on stimulated bone marrow-like cells Can bind to Mac-1 and substantially bind to non-activated Mac-1 and resting myeloid cells No, a method of blocking the binding of activated Mac-1 to a ligand of Mac-1.   8. Activated Mac-1 and stimulated myeloid cells The method according to claim 7, which can bind to ICAM-1 or fibrinogen.   13. Comprising administering an effective amount of an anti-inflammatory agent to a patient in need of treatment, The inflammatory agent is an antibody or fragment thereof, and the antibody or antibody fragment is present on stimulated myeloid cells Can be bound to activated Mac-1 but not activated Mac-1 Mediated by a non-specific defense system that is substantially incapable of binding to resting myeloid cells How to treat illness reactions.   14. Activated Mac-1 and stimulated myeloid cells 14. The method of claim 13, which is capable of binding ICAM-1.   19. The mode of administration of the agent is enteral means, parenteral means, topical means, inhalation means and intranasal hand. 14. The method of claim 13, selected from the group consisting of steps.   20. 14. The method of claim 13, wherein the agent is administered prior to the onset of inflammation.   21. 14. The method of claim 13, wherein the agent is administered after initiation of inflammation.   22. The parenteral means is selected from the group consisting of intramuscular, intravenous and subcutaneous means, The method according to claim 19.   23. Inflammation secondary to adult respiratory distress syndrome (ARDS), sepsis, trauma or bleeding Multiple organ injury syndrome, myocardial or other tissue reperfusion injury, acute glomerulonephritis, Reactive arthritis, skin diseases caused by acute inflammatory components, acute purulent meningitis or other central nervous system Inflammatory disease (eg, seizures), heat injury, hemodialysis, leukocyte export, ulcerative colitis, liver It consists of Lawn's disease, necrotizing enteritis, granulocyte transfusion-related syndrome and cytokine-induced toxicity. 14. The method of claim 13, which is responsive to a condition selected from the group:   24. Detectable by administering a detectably labeled antibody or antibody fragment to the patient The antibody labeled to Detecting the site located inside the antibody or antibody fragment is activated Ma can bind to c-1, but binds substantially to non-activated Mac-1 and resting myeloid cells To determine the presence of cells expressing the active form of Mac-1 in the untreated subject Law.   25. 25. The method of claim 24, wherein activated Mac-1 is capable of binding ICAM-1. Law.   30. a) isolated resting bone marrow-like cells or isolated inactive Mac-1 molecule Contact the agent   b) Contacting the cells or Mac-1 molecule of step (a) with an antibody (the above antibody) Can bind to activated Mac-1 but to non-activated Mac-1 and resting myeloid cells Practically cannot be combined),   c) Whether the cell or the Mac-1 molecule binds to the antibody decide A method for confirming an agent capable of activating Mac-1 comprising steps.   31. Activated Mac-1 and stimulated myeloid cells 31. The method of claim 30, which is capable of binding ICAM-1.   36. a) isolated stimulated bone marrow-like cells or isolated activated Mac-1 molecules Contact the agent   b) Contacting the cells or Mac-1 molecule of step (a) with an antibody (the above antibody) Can bind to activated Mac-1 present on stimulated myeloid cells, but not to activated Ma c-1 And substantially unable to bind resting myeloid cells),   c) Whether the cell or the Mac-1 molecule binds to the antibody decide A method for confirming an agent capable of inactivating Mac-1 comprising steps.   37. Activated Mac-1 and stimulated myeloid cells 37. The method of claim 36, which is capable of binding ICAM-1.   42. Administration of an effective amount of antibody conjugate to the treated subject, The conjugate can bind to activated Mac-1, but substantially not to non-activated Mac-1. A method for selectively killing cells containing an active form of Mac-1 which cannot be activated.   43. 43. The method of claim 42, wherein activated Mac-1 is capable of binding ICAM-1. Law.   48. The step of subjecting the patient to leukophoresis is performed, wherein the leukocyte migration is performed on the patient's blood. The liquid can be contacted with the immobilized antibody and the antibody can bind to activated Mac-1. From the patient's blood, which is substantially unable to bind to non-activated Mac-1 and resting myeloid cells A method for selectively removing cells containing activated Mac-1.   49. 49. The method of claim 48, wherein activated Mac-1 is capable of binding ICAM-1. Law.   54. a) Isolated resting myeloid cells or isolated inactive Mac-1 molecule Contact the agent with   b) Activate the cell or Mac-1 molecule of step (a) Contact with an agent (the activator can activate the Mac-1 molecule),   c) Contacting the cells of step (b) with an antibody (the antibody binds to activated Mac-1). But not substantially bound to non-activated Mac-1 and resting myeloid cells),   d) Whether the cell or Mac-1 molecule of step (c) binds to the above antibody Decide about A method for identifying an agent capable of blocking activation of Mac-1 comprising steps.   55. Activated Mac-1 and stimulated myeloid cells 55. The method of claim 54, which is capable of binding ICAM-1.   56. 55. In claim 54, wherein the active agent is selected from the group consisting of fMLP and PMA. The method described.   61. Amino acids from two or more members of the CDII family of glycoproteins A chimeric CD11 molecule comprising a sequence,   The chimeric CD11 molecule can form a dimer with CD18, and the dimer is a glycoprotein. Glycoprotein capable of binding one or more ligands of the CD11 / CD18 family of proteins Quality The above members of the CD11 family are CD11a, A chimeric CD11 molecule selected from the group consisting of CD11b and CD11c.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), AU, CA, JP, KR

Claims (1)

[Claims]   1. Can bind to activated Mac-1 present on stimulated myeloid cells, but deactivate it Antibodies, antibody fragments and antibodies that are substantially incapable of binding to Mac-1 and resting myeloid cells Derivative.   2. Antibodies bind to activated Mac-1 and stimulate myeloid cells bind to ICAM-1 The antibody according to claim 1, which is capable.   3. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 3. The antibody of claim 2, which binds to 1 or stimulated myeloid cells.   4. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The antibody according to claim 3.   5. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Epitope that sterically overlaps with the epitope bound by the antibody produced by the antibody. 4. The antibody of claim 3, which binds to the taupe.   6. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC The antibody according to claim 1, which is produced by a mouse.   7. Contacting activated Mac-1 with an antibody, antibody fragment or antibody derivative Activation consisting of an antibody, antibody fragment or antibody derivative present on stimulated bone marrow-like cells Can bind to Mac-1 and substantially bind to non-activated Mac-1 and resting myeloid cells No, a method of blocking the binding of activated Mac-1 to a ligand of Mac-1.   8. Activated Mac-1 and stimulated bone marrow-like cells are ICAM-1 or fibrinogen The method of claim 7, wherein the method is capable of binding to a protein.   9. Antibody is hybridoma ATCC Activated Mac-1 or subpopulations similar to those bound by antibodies produced in 9. The method of claim 8, wherein the binds with stimulated myeloid cells.   10. Antibody is hybridoma ATCC Binding to an epitope similar to that bound by an antibody produced in 9. The method according to 9.   11. The antibody is the hybridoma CBRM1 / 5, Steric and epitope bound by antibodies produced by ATCC 10. The method of claim 9, wherein the method binds to an epitope that overlaps.   12. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC The method of claim 7, wherein the method is produced by an armor.   13. Comprising administering an effective amount of an anti-inflammatory agent to a patient in need of treatment, The inflammatory agent is an antibody or fragment thereof, and the antibody or antibody fragment is present on stimulated myeloid cells Non-activated Mac-1 and resting bone marrow-like cells capable of binding to activated Mac-1 A method of treating an inflammatory response mediated by a non-specific defense system that is substantially unable to bind to.   14. Activated Mac-1 and stimulated myeloid cells can bind to ICAM-1 The method according to claim 13.   15. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRMI / 19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 15. The method of claim 14, which binds to 1 or stimulated myeloid cells.   16. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The method according to claim 15.   17． Antibody is CBRM1 / 5, ATCC And a group consisting of CBRM 1/19, ATCC The epitopes bound by the antibodies produced by the selected hybridoma. The method according to claim 15, wherein the method binds to a physically overlapping epitope.   18. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC 14. The method according to claim 13, which is produced by an armor.   19. The mode of administration of the agent is enteral means, parenteral means, topical means, inhalation means and intranasal hand. 14. The method of claim 13, selected from the group consisting of steps.   20. 14. The method of claim 13, wherein the agent is administered prior to the onset of inflammation.   21. 14. The method of claim 13, wherein the agent is administered after initiation of inflammation.   22. The parenteral means is selected from the group consisting of intramuscular, intravenous and subcutaneous means, The method according to claim 19.   23. Inflammation secondary to adult respiratory distress syndrome (ARDS), sepsis, trauma or bleeding Multiple organ injury syndrome, myocardial or other tissue reperfusion injury, acute glomerulonephritis, Reactive arthritis, skin diseases caused by acute inflammatory components, acute purulent meningitis or other central nervous system Inflammatory disease (eg, seizures), heat injury, hemodialysis, leukocyte export, ulcerative colitis, liver It consists of Lawn's disease, necrotizing enteritis, granulocyte transfusion-related syndrome and cytokine-induced toxicity. Contractor responding to symptoms selected from the group The method according to claim 13.   24. Detectable by administering a detectably labeled antibody or antibody fragment to the patient The step of detecting the location where the antibody labeled on the The body or antibody fragment can bind to activated Mac-1, but not to inactivated Mac-1 and rest. Express the active form of Mac-1 in the recipient, which is virtually incapable of binding to myeloblast-like cells A method of locating the presence of cells.   25. 25. The method of claim 24, wherein activated Mac-1 is capable of binding ICAM-1. Law.   26. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 26. The method of claim 25, which binds to one or a similar stimulated bone marrow-like cell subpopulation.   27. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The method according to claim 26.   28. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Antibody produced by 27. Binding to an epitope that is sterically overlapping with the combined epitope. The method described in.   29. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC 25. The method of claim 24, produced by an armor.   30. a) isolated resting bone marrow-like cells or isolated inactive Mac-1 molecule Contact the agent   b) Contacting the cells or Mac-1 molecule of step (a) with an antibody (the above antibody) Can bind to activated Mac-1 but to non-activated Mac-1 and resting myeloid cells Practically cannot be combined),   c) Whether the cell or the Mac-1 molecule binds to the antibody decide A method for confirming an agent capable of activating Mac-1 comprising steps.   31. Activated Mac-1 and stimulated myeloid cells can bind to ICAM-1 The method according to claim 30.   32. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 32. The method of claim 31, which binds to 1 or stimulated myeloid cells.   33. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The method according to claim 31.   34. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Epitope that sterically overlaps with the epitope bound by the antibody produced by the antibody. 32. The method of claim 31, which is associated with a taupe.   35. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC 31. The method according to claim 30, produced by an armor.   36. a) isolated stimulated bone marrow-like cells or isolated activated Mac-1 molecules Contact the agent   b) Contacting the cells or Mac-1 molecule of step (a) with an antibody (the above antibody) Can bind to activated Mac-1 present on stimulated myeloid cells, but not to activated Ma c-1 warp and resting bone marrow-like cells cannot be bound substantially),   c) Whether the cell or the Mac-1 molecule binds to the antibody decide A method for confirming an agent capable of inactivating Mac-1 comprising steps.   37. Activated Mac-1 and stimulated myeloid cells are IC 37. The method of claim 36, which is capable of binding AM-1.   38. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 38. The method of claim 37, which binds to 1 or stimulated myeloid cells.   39. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The method according to claim 38.   40. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Epitope that sterically overlaps with the epitope bound by the antibody produced by the antibody. 39. The method of claim 38, which is associated with a taupe.   41. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC 38. The method of claim 37, produced by an armor.   42. Administration of an effective amount of antibody conjugate to the treated subject, The conjugate can bind to activated Mac-1, but substantially not to non-activated Mac-1. A method for selectively killing cells containing an active form of Mac-1 which cannot be activated.   43. 43. The method of claim 42, wherein activated Mac-1 is capable of binding ICAM-1. Law.   44. Antibody conjugate is CBRM1 / 5, ATCC       And CBRM 1/19, ATCC The activity of the subpopulation is similar to that bound by the antibody produced by the hybridoma. 44. The method of claim 43, which binds to cells of a sexualized Mac-1 or similar subpopulation. .   45. Antibody conjugate is CBRM1 / 5, ATCC       And CBRM 1/19, ATCC The same epitope as when bound by the antibody produced by the hybridoma 45. The method of claim 44, which binds.   46. Antibody conjugate is CBRM1 / 5, ATCC       And CBRM 1/19, ATCC Sterically overlaps with the epitope bound by the antibody produced by the hybridoma 45. The method of claim 44, wherein the method binds to an epitope that is   47. Antibody conjugate is CBRM1 / 5, ATCC       And CBRM 1/19, ATCC 43. The method according to claim 42, which is a conjugate of an antibody produced by the hybridoma. Law.   48. The step of subjecting the patient to leukophoresis is performed, wherein the leukocyte migration is performed on the patient's blood. Contact the liquid with the immobilized antibody The above antibody can bind to activated Mac-1, but not to inactivate Mac-1 and rest. Cells containing activated Mac-1 from the blood of a patient that are substantially incapable of binding to myeloid cells How to remove selectively.   49. 49. The method of claim 48, wherein activated Mac-1 is capable of binding ICAM-1. Law.   50. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 50. The method of claim 49, which binds to 1 or stimulated myeloid cells.   51. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Binding to an epitope similar to that bound by the antibody produced by the The method according to claim 50.   52. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Epitope that sterically overlaps with the epitope bound by the antibody produced by the antibody. 51. The method of claim 50, which is associated with a taupe.   53. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Claims produced by a license The method according to 48.   54. a) isolated resting bone marrow-like cells or isolated inactive Mac-1 molecule Contact the agent   b) Contacting the cells or Mac-1 molecule of step (a) with an activator (see above activity). Sexual agents can activate the Mac-1 molecule),   c) Contacting the cells of step (b) with an antibody (the antibody binds to activated Mac-1). But not substantially bound to non-activated Mac-1 and resting myeloid cells),   d) Whether the cell or Mac-1 molecule of step (c) binds to the above antibody Decide about A method for identifying an agent capable of blocking activation of Mac-1 comprising steps.   55. Activated Mac-1 and stimulated myeloid cells 55. The method of claim 54, which is capable of binding ICAM-1.   56. 55. In claim 54, wherein the active agent is selected from the group consisting of fMLP and PMA. The method described.   57. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Of activated subpopulations similar to those bound by antibody-produced antibody 56. The method of claim 55, which binds to 1 or stimulated bone marrow-like cells.   58. Antibody is CBRM1 / 5, ATCC And a group consisting of CBRM 1/19, ATCC The same type of antibody as that bound by the antibody produced by the selected hybridoma. 58. The method of claim 57, which is associated with a pitope.   59. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC Epitope that sterically overlaps with the epitope bound by the antibody produced by the antibody. 58. The method of claim 57, wherein the method is associated with a taupe.   60. Antibody is CBRM1 / 5, ATCC And a hybrid selected from the group consisting of CBRM 1/19 and ATCC 55. The method of claim 54, produced by an armor.   61. Amino acids from two or more members of the CD11 family of glycoproteins A chimeric CD11 molecule comprising a sequence,   The chimeric CD11 molecule can form a dimer with CD18, and the dimer is a glycoprotein. Glycoprotein capable of binding one or more ligands of the CD11 / CD18 family of proteins The above members of the quality CD11 family are CD11a, A chimeric CD11 molecule selected from the group consisting of CD11b and CD11c.   62. Dimer can bind to ICAM-1, but iC3b and fibrinogen 62. Substantially incapable of binding one or more ligands selected from the group consisting of: In The listed chimeric CD11 molecule.   63. The dimer can bind fibrinogen, but ICAM-1 and iC3b 62. Substantially incapable of binding one or more ligands selected from the group consisting of: The chimeric CD11 molecule described in 1.   64. Dimers can bind iC3b, but ICAM-1 and fibrinogen 62. Substantially incapable of binding one or more ligands selected from the group consisting of: The chimeric CD11 molecule described in 1.