Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
  • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70525—ICAM molecules, e.g. CD50, CD54, CD102
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
  • G01N2333/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18

Definitions

• LFA-1 The leukocyte function-associated antigen (LFA-1, CD 1 la/CD 18) is a leukocyte-specific ⁇ 2 integrin that participates in cell/cell adhesion. Binding activity of LFA-1 is essential to leukocyte extravasation from circulation to a site of injury in an inflammatory response. Three principle ligands are known to bind LFA-1 , ICAM-
• ICAM-1 intercellular adhesion molecules that play an important role in localizing leukocyte adhesion to endothelial cells at a site of injury.
• ICAM-4 and ICAM-5 have also been reported to bind LFA-1.
• Most leukocytes constitutively express LFA-1 but ligand binding requires activation believed to induce a conformational change and to increase avidity ligand binding. For example,
• ICAM-1 is normally expressed at low levels on the endothelium. However, injury- induced inflammatory mediators promote enhanced surface expression in cells at the site of the injury which, in turn, promotes localized leukocyte adhesion through binding to activated LFA-1.
• the structure of LFA-1 includes distinct intracellular and extracellular domains that are believed to participate in and/or regulate ICAM binding. Of particular interest is a region in the ⁇ L chain of approximately 200 amino acids, designated the I domain, that is found in all ⁇ 2 integrins, as well as many other proteins. Evidence suggests that the I domain is essential to LFA-1 binding to ICAM- 1 and 3. For example, anti-LFA-1 blocking monoclonal antibodies have been mapped to epitopes within the I domain.
• I domain polypeptide fragments have been shown to inhibit integrin-mediated adhesion and bind ICAM-1.
• ICAM-1 Within the I domain of LFA-1 (and other proteins) is a single metal ion dependent adhesion site (MIDAS) that preferentially binds manganese or magnesium ions. Binding of either cation is required for ligand interaction and is believed to induce conformational changes in LFA-1 necessary for binding. Cation binding may therefore be a regulatory mechanism that responds to changes in the extracellular leukocyte environment. This hypothesis is supported by the observation that calcium ion binding actually inhibits LFA-1 interaction with ICAM-1.
• MIDAS metal ion dependent adhesion site
• LFA-1 /ICAM binding sites provides targets to modulate leukocyte inflammatory responses.
• Numerous antibodies have been isolated that are capable of inducing LFA-1 activation [see, for example, Landis, et al, J. Cell Biol. 720:1519-1527 (1993)] or that are capable of preventing ICAM-1 interaction [see for example, Randi and Hogg, J. Biol Chem. 269:12395-
• the present invention provides methods for identifying a negative regulator of LFA-1 binding to a natural ligand that competes for binding to LFA-1 with ICAM-1 or ICAM-3 comprising the steps of (i) contacting LFA-1. or a ligand binding fragment thereof, and a ligand that binds LFA-1, or an LFA-1 -binding fragment thereof, in the presence and absence of a test compound under conditions that allow binding of LFA-1 to the ligand (ii) identifying as a negative regulator the compound which decreases LFA-1 binding to the ligand and which binds LFA-1 ⁇ L polypeptide at a site presenting a diaryl sulfide binding conformation defined by He 259 , Leu 298 , He 235 , Val' 57 , Leu 161 ' and He 306 of human LFA-1 as set out in SEQ ID NO: 2, which provides the amino acid sequence for mature (i.e., without the leader sequence) LFA-1.
• Natural ligand refers to any biological compound that binds LFA-1.
• the term “negative regulator” refers to a compound that decreases ICAM binding to LFA- 1, but does not directly compete with the ICAM for LFA-1 binding.
• a negative regulator may be an allosteric inhibitor or a compound that modulates the activation state of LFA-1.
• the negative regulator is a diaryl sulfide.
• the natural ligand is an ICAM. Most preferably, the ICAM is ICAM-1 or ICAM-3.
• the invention provides methods for identifying a negative regulator of LFA-1 binding to a natural ligand that binds LFA-1 comprising the steps of (i) contacting LFA-1, or a ligand binding fragment thereof, and a natural ligand that binds LFA-1, or an LFA-1 -binding fragment thereof, in the presence and absence of a test compound under conditions that allow binding of LFA-1 to the ligand, (ii) identifying as a negative regulator the compound which decreases LFA-1 binding to the ligand and which competes with (2-isopropyl-phenyl)[2-nitro-4-(E-((4- acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl] sulfide for binding to LFA-1 cc L polypeptide.
• the negative regulator is a diaryl sulfide.
• the ligand is an ICAM.
• the ICAM is ICAM-1 or ICAM- 3.
• the invention also provides screening methods for identifying a negative regulator of LFA-1 binding to a natural ligand that binds LFA-1 comprising the steps of (i) contacting LFA-1 , or a ligand binding fragment thereof, with (2- isopropyl-phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l -yl)carbonyl)ethenyl)phenyl]- sulfide in the presence and absence of a compound, and (ii) identifying the compound as a putative negative regulator wherein the compound competes with (2-isopropyl- phenyl)[2-nitro-4-(E-((4-acetylpiperazin- 1 -yl)carbonyl)ethenyl)phenyl]-sulf
• the invention also provides pharmaceutical compositions comprising a negative regulator of LFA-1 binding to a natural ligand that binds LFA-1 identified by a method of the invention.
• the invention further provides use of a negative regulator identified by a method of the invention in the production of a medicament to ameliorate pathologies arising from LFA-1 binding to an ICAM that binds LFA-1.
• the invention further provides methods for inhibiting LFA-1 binding to a natural ligand that binds LFA-1 comprising the step of contacting LFA-1, or a ligand binding fragment thereof, with a negative regulator compound; said negative regulator binding the LFA-1 L polypeptide.
• the negative regulator is a diaryl sulfide.
• methods of the invention include use of cells expressing either LFA-1 or the ligand.
• the other binding partner is either purified and isolated, in a fluid sample (purified, partially purified, or crude) taken from an individual, or in a cell lysate.
• the invention also comprehends methods wherein both LFA-1 and the
• ICAM are expressed in cells.
• the LFA-1 and ligand binding partners may be expressed on the same cell type or different cell types.
• the invention also provides methods to inhibit leukocyte adhesion to endothelial cells comprising the step of contacting said leukocyte with a negative regulator of LFA-1 binding to a natural ligand that binds LFA-1 , said negative regulator binding an LFA-1 regulatory site selected from the group consisting of a site that binds a diaryl sulfide, a site defined by He 2 ' 9 , Leu 29 ⁇ Ile 23 ⁇ Val l s , Leu 161 , and He 106 of human LFA-1 L polypeptide, and an LFA-1 domain that binds (2- ⁇ sopropyl- phenyl)[2-n ⁇ tro-4-(E-((4-acetylp ⁇ peraz ⁇ n-l - ⁇ l)carbonyl)ethenyl)phenyl]sulf ⁇ de.
• the negative regulator of the methods is a diaryl sulfide
• the invention also provides methods to ameliorate a pathology arising from LFA-1 binding to a natural ligand that binds LFA-1 comprising the step of administering to an individual in need thereof a negative regulator of LFA-1 binding to the ligand m an amount effective to inhibit LFA-1 binding to the ligand, said negative regulator binding to an LFA-1 regulatory site selected from the group consisting of a site that binds a diaryl sulfide, a site defined by He 259 , Leu 298 , He 235 , Val' 57 , Leu 161 , and He 306 of human LFA-1 and an LFA-1 domain that binds compound (2-isopropyl-phenyl)[2-nitro-4-(E-((4-acetylp ⁇ perazm-l-yl)carbonyl)ethenyl)phenyl]- sulfide.
• the invention also provides LFA-1 L polypeptides and fragments thereof comprising a regulatory binding site presenting a diaryl sulfide binding conformation.
• the LFA-1 polypeptide fragment comp ⁇ ses the ⁇ L polypeptide I domain sequence.
• the LFA-1 polypeptide contains less than all amino acids in the ⁇ polypeptide I domain.
• LFA-1 polypeptides wherein amino acid residues in the wild type ⁇ L polypeptide regulatory site are substituted with non-naturally occurring (i e , residues not found in the same position in the wild type molecule) ammo acid residues.
• Preferred mutant regulatory sites exhibit modified affinity and/or avidity for an ICAM, both in the presence and absence of an inducing agent (e.g., the monoclonal antibody 240Q described below which induces LFA-1 into an activated state required for ICAM binding).
• Presently preferred mutants include (i) those demonstrating wild type levels of ICAM-1 binding with or without monoclonal antibody 240Q induction, exemplified mutations having one or more of the single amino acid changes Val 157 - Ala, Glu 218 ⁇ Ala, Thr ⁇ ' ⁇ Ala, Lys 280 -Ala, and Lys 294 ⁇ Ala, (ii) mutants that support greater than wild type levels of binding without induction and wild type levels with induction, exemplified by mutations having one or more of the single amino acid changes He 235 - Ala, He 255 - Ala, Ser 83 -Ala, Glu 284 - Ala, Glu 30, ⁇ Ala, and Ile 306 ⁇ Ala, (iii) mutants with decreased levels of ICAM-1 binding relative to wild type binding in the absence of induction, but wild-type levels with antibody 240Q induction, exemplified by mutants having one or more of the substitutions Lys 160 ⁇ Ala,
• the invention also provides an LFA-1 -activating monoclonal antibody secreted by a hybridoma designated 240Q, mailed on March 30, 1999 to, and received on March 31, 1999 by the American Type Culture Collection, 10861 University Boulevard., Manassas, NA 20010-2209, and assigned Accession No: HB-12692.
• the present invention provides novel in vivo and in vitro methods for negatively, and preferably reversibly, regulating LFA-1 binding to a natural ligand that binds LFA-1 involving use of compounds which bind LFA-1 at a regulatory domain located remote from the ligand binding site.
• the LFA-1 regulatory site presents a conformation that binds a substituted diaryl sulfide.
• the binding site is defined by human LFA-1 amino acid residues He 259 , Leu 298 , He 235 , Val 157 , Leu 161 and He 306 .
• the site is defined by amino acid residues He 259, Leu 298 , He 235 , Val 157 , Leu 161 He 306 , Leu 302 , Tyr 257 , Leu 132 , Val 233 , Val 130 , and Tyr 166 .
• the binding site is defined by amino acid residues Lys 287 , Leu 298 , He 259 , Leu 302 , He 235 , Val 157 , Tyr 257 , Lys 305 , Leu 161 , Leu 132 , Val 233 , He 255 , Val 130 , Tyr 166 , He 306 , Phe 134 , Phe 168 , Phe 153 , Tyr 307 , Val 308 , He 309 , Thr 231 , Glu 284 , Phe 285 , Glu 301 , Met 154 , He 237 ,
• the ligand is an ICAM.
• the ICAM is ICAM-1 or ICAM-3.
• reversible negative regulation i.e., reversible inhibition
• LFA-1 binding to ligand ICAM is provided by substituted diaryl sulfide compounds which bind LFA-1 at the aforementioned regulatory domain and/or compounds that competitively inhibit diaryl sulfide binding to said domain.
• methods of the invention are carried out using LFA-1 and a binding partner protein, such as ICAM-1 , which are recombinant, purified from natural sources, or synthetic.
• a binding partner protein such as ICAM-1
• the LFA-1 and ICAM-1 are recombinant, purified from natural sources, or synthetic.
• the binding partner proteins are recombinant.
• the binding partner proteins may be holoproteins (e.g., including both and ⁇ chains of LFA-1), protein subunits (e.g., the isolated LFA-1 polypeptide chain), or fragments thereof, including, for example, extracellular domains of either LFA-1 or the ICAM, I domain fragments of LFA-1, less than complete I domain fragments of LFA-1 , and/or less than a complete extracellular domain of the ICAM.
• the invention provides methods wherein either LFA- 1 , the ligand, or both are expressed in a cell.
• the cell can be one that expresses an endogenous polynucleotide encoding LFA-1 or the ligand, or a host cell transformed and transfected with a heterologous polynucleotide encoding LFA-1 or the ligand and grown under conditions appropriate to permit expression of LFA-1 or the ligand on the cell surface.
• transcription of the polynucleotide can be directed by either endogenous or heterologous transcriptional control elements.
• endogenous control elements can be purified from a desired host cell and ligated in an operative position relative to the LFA-1 or the ligand-encoding polynucleotide.
• a cell expressing endogenous LFA-1 or the ligand can be modified, for example through homologous recombination, to provide the LFA-1 or ligand polynucleotide with one or more transcriptional control elements that modify wild type levels of proteins expression.
• leukocytes i.e., lymphocytes, monocytes, and granulocytes (e.g., neutrophils), and endothelial cells.
• the invention embraces methods to inhibit leukocyte adhesion to endothelial cells associated with LFA-1, expressed on leukocytes, binding to an ICAM that binds LFA-1, expressed on endothelial cells.
• Leukocyte adhesion to endothehum is characteristic of an inflammatory response arising from release of cell mediators at an injury site
• the invention also comprehends methods to inhibit an inflammatory response associated with LFA-1 binding to a natural ligand that binds LFA-1
• the invention provides methods to ameliorate pathologies associated with accumulation of leukocytes resulting from LFA-1 binding to an ICAM that binds LFA-1 , comprising the step of administering to an individual in need thereof an amount of an inhibitor of LFA-1 binding to the ICAM effective to inhibit LFA-1 binding to the ICAM, said inhibitor binding to LFA-1 at a site presented by ammo acid residues He 259 , Leu 298 , Ile 23 ⁇ Val 157 , Leu 161 and He 306 .
• Exemplary medical conditions include, without limitation, inflammatory diseases, autoimmune diseases, reperfusion injury, myocardial infarction, stroke, hemorrhagic shock, organ transplant, and the like.
• Methods of the invention provide for amelioration of a variety of pathologies, including, for example, but not limited to adult respiratory distress syndrome, multiple organ injury syndrome secondary to septicemia, multiple organ injury secondary to trauma, reperfusion injury of tissue, acute glomeruloneph ⁇ tis, reactive arthritis, dermatosis with acute inflammatory components, stroke, thermal injury, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion associated syndrome, cytokine induced toxicity, and T cell mediated diseases.
• Inflammatory cell activation and excessive or unregulated cytokine (e.g., TNF ⁇ and IL-1 ⁇ ) production are also implicated in disorders such as rheumatoid arthritis, osteoarthritis, gouty arthritis, spondylitis, thyroid associated ophthalmopathy,
• Behcet disease sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shock syndrome, asthma, chronic bronchitis, allergic respiratory distress syndrome, chronic pulmonary inflammatory disease, such as chronic obstructive pulmonary disease, sihcosis, pulmonary sarcoidosis, reperfusion injury of the myocardium, bram, and extremities, fibrosis, cystic fibrosis, keloid formation, scar formation, atherosclerosis, transplant rejection disorders, such as graft vs.
• chronic pulmonary inflammatory disease such as chronic obstructive pulmonary disease, sihcosis, pulmonary sarcoidosis, reperfusion injury of the myocardium, bram, and extremities, fibrosis, cystic fibrosis, keloid formation, scar formation, atherosclerosis, transplant rejection disorders, such as graft vs.
• inflammatory bowel disease such as ulcerative colitis
• proliferative lymphocyte diseases such as leukemia
• inflammatory dermatoses such as atopic dermatitis, psoriasis, urticaria, and uveitis.
• Other conditions characterized by elevated cytokine levels include brain injury due to moderate trauma (see J. Neurotrauma, 12, pp. 1035-1043 (1995); J Clin. Invest., 91, pp. 1421-1428 (1993)).
• cardiomyopathies such as congestive heart failure (see Circulation, 97, pp.
• cachexia cachexia secondary to infection or malignancy
• cachexia secondary to acquired immune deficiency syndrome AIDS
• ARC AIDS related complex
• fever myalgias due to infection cerebral malaria
• osteoporosis and bone resorption diseases keloid formation
• scar tissue formation scar tissue formation
• pyrexia pyrexia
• the ability of the negative regulators of the invention to treat arthritis can be demonstrated in a murine collagen-induced arthritis model [Kakimoto, et al. Immunol. 142:326-337 (1992)], in a rat collagen-induced arthritis model [Knoerzer, et al, Toxical Pathol. 25:13-19 (1997)], in a rat adjuvant arthritis model [Halloran, et al, Arthritis Rheum 39: 810-819 (1996)], in a rat streptococcal cell wall-induced arthritis model [Schimmer, et al, J. Immunol. 160:1466-1477 (1998)], or in a SCID- ouse human rheumatoid arthritis model [Oppenheimer-Marks, et al, J. Clin. Invest 707:1261-1272 (1998)].
• the ability of the negative regulators to treat asthma can be demonstrated in a murine allergic asthma model according to the method of Wegner, et al, Science, 247:456-459, (1990), or in a murine non-allergic asthma model according to the method of Bloemen, et al, Am. J. Respir. Crit. Care Med. 753:521- 529 (1996).
• the ability of the negative regulators to treat inflammatory lung injury can be demonstrated in a murine oxygen-induced lung injury model according to the method of Wegner, et al, Lung, 170:267-279, (1992), in a murine immune complex- induced lung injury model according to the method of Mulligan, et al, J. Immunol, 754: 1350-1363. ( 1995), or in a murine acid-induced lung injury model according to the method of Nagase, et al, Am. J. Respir. Crit. Care Med., 154:504-5X 0, (1996).
• the ability of the negative regulators to treat autoimmune diabetes can be demonstrated in an NOD mouse model according to the method of Hasagawa, et al, Int. Immunol. 6:831-838 (1994), or in a murine streptozotocin-induced diabetes model according to the method of Herrold. et al, Cell Immunol 757:489-500, (1994).
• the ability of the negative regulators to treat inflammatory liver injury can be demonstrated in a murine liver injury model according to the method of Tanaka, et al, J. Immunol, 757:5088-5095, (1993).
• the ability of the negative regulators to treat pulmonary reperfusion injury can be demonstrated in a rat lung allograft reperfusion injury model according to the method of DeMeester, et al, Transplantation, (52:1477-1485 (1996), or in a rabbit pulmonary edema model according to the method of Horgan, et al, Am. J. Physiol. 2 ⁇ 57:H1578-H1584 (1991).
• the ability of the negative regulators to treat stroke can be demonstrated in a rabbit cerebral embolism stroke model according to the method of Bowes, et al, Exp.
• the ability of the negative regulators to treat peripheral artery occlusion can be demonstrated in a rat skeletal muscle ischemia/reperfusion model according to the method of Gute, et al, Mol. Cell Biochem. , 779:169-187 (1998).
• the ability of the negative regulators to treat graft rejection can be demonstrated in a murine cardiac allograft rejection model according to the method of Isobe, et al, Science, 255: X 125-1127 (1992), in a murine thyroid gland kidney capsule model according to the method of Talento, et al, Transplantation, 55:418-422 (1993), in a cynomolgus monkey renal allograft model according to the method of Cosimi, et al, J.
• GVHD GVHD
• GVHD murine lethal GNHD model according to the method of Harning, et al, Transplantation, 52:842-845 (1991).
• the invention also provides an LFA-1 regulatory binding site.
• the regulatory binding site is displayed on the ⁇ L chain of LFA-1 in its wild type, or native, conformation. Fragments of the L chain that display the regulatory site are also contemplated, and preferred fragments of the invention include ⁇ L chain I domain sequences, as well as fragments consisting of less than a complete L chain I domain.
• the invention provides LFA-1 regulatory binding sites as part of a polypeptide comprising a human LFA-1 amino acid sequence, the amino acid sequence of a species homolog of human LFA-1 , the amino acid sequence of analogs of human LFA-1 , or the amino acid sequence of a synthetic polypeptide with homology to human LFA-1. Regulatory binding sites displayed on synthetic polypeptide-like mimetics are also contemplated.
• the regulatory binding site of the invention binds a diaryl sulfide (alternatively referred to as a diaryl thioether compound) comprising a first aryl ring and second aryl ring linked to one another through a sulfur atom.
• the site is defined by human LFA-1 amino acid residues He 259 , Leu 298 , He 235 , Nal 157 , Leu 161 and He 306 .
• the binding site is defined by other amino acid residues (i.e., conservative substitutions) or compounds that mimic the binding ability of a site defined by LFA-1 He 259 , Leu 298 , He 235 , Val 157 , Leu 161 and He 306 .
• the regulatory site is also defined by LFA-1 ⁇ L polypeptide amino acid residues that present a domain that binds (2-isopropyl-phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l-yl)carbonyl)ethenyl)- phenyljsulfide.
• the regulatory site of the invention reversibly binds a negative regulator compound.
• the invention also provides LFA-1 regulatory binding site mutants wherein one or more amino acid residues defining the site (i.e., presenting the (2- isopropyl-phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl]- sulfide binding site) is substituted with an alternative amino acid residue, wherein substitution of the wild type amino acid residues results in modified capacity for the mutant to bind (2-isopropyl-phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l-yl)carbonyl)- ethenyl)phenyl]sulfide compared to a wild type regulatory site.
• Preferred mutant regulatory sites exhibit modified affinity and/or avidity for ICAM-1, both in the presence and absence of an agent that induces ICAM-1 binding (e.g., the monoclonal antibody 240Q which induces LFA-1 into an activated state required for ICAM binding).
• an agent that induces ICAM-1 binding e.g., the monoclonal antibody 240Q which induces LFA-1 into an activated state required for ICAM binding.
• Presently preferred mutants include (i) those demonstrating wild type levels of ICAM-1 binding with or without monoclonal antibody 240Q induction, exemplified by mutants having one or more of the amino acid changes Val' 57 - Ala,
• mutants that support greater than wild type levels of binding without induction and wild type levels with induction exemplified by mutants having one or more of the amino acid changes He 235 - Ala, He 255 - Ala, Ser ⁇ Ala, Glu 284 - Ala, Glu 0 '-Ala, and He 306 - Ala
• mutants with decreased levels of ICAM-1 binding relative to wild type in the absence of induction, but wild-type levels with antibody 240Q induction exemplified by mutants having one or more of the amino acid substitutions Lys 160 -* Ala, Lys 232 ⁇ Ala, Asp 253 - Ala, Lys 287 - Ala, Gin 303 - Ala, Lys 304 - Ala, and Lys 305 - Ala
• mutants exemplified by mutants having one or more of the amino acid substitutions Lys 160 -* Ala, Lys 232 ⁇ Ala, Asp 253 - Ala, Lys 287
• Mutants of the LFA-1 regulatory site are useful in production of antibodies that more precisely define LFA-1 epitopes that can serve as targets for therapeutic intervention.
• soluble regulatory sites or LFA-1 regulatory sites as part of chimeric compounds with an increased ability to bind an ICAM that binds LFA-1 can modulate LFA-1 binding to the ICAM through competitive inhibition.
• the invention further provides methods for identifying a negative regulator of LFA-1 binding to an ICAM that binds LFA-1 comprising the steps of (i) contacting LFA-1 and the ICAM in the presence and absence of a test compound under conditions that allow binding of LFA-1 to the ICAM, (ii) identifying as a negative regulator the compound which decreases LFA-1 binding to the ICAM and which binds LFA-1 ⁇ L polypeptide at a site presenting a diaryl sulfide binding conformation defined by He 259 , Leu 298 , He 235 , Val' 57 , Leu 16 ', and He 306 of human LFA-1.
• IC 50 value for a compound is defined as the concentration of the compound required to produce 50% inhibition of a biological activity of interest.
• a negative regulator is defined as a compound characterized by an IC 50 for inhibition of LFA-1 binding to a natural ligand.
• Negative regulators of LFA-1 binding are defined to have an IC 50 of less than about 200 ⁇ M, less than about 100 ⁇ M, less than about 50 ⁇ M, and preferably from about 0.05 ⁇ M to 40 ⁇ M.
• the invention provides methods for identifying a negative regulator of LFA-1 binding to an ICAM that binds LFA-1 comprising the steps of (i) contacting LFA-1 and the ICAM under conditions that allow binding of LFA-1 to the ICAM in the presence and absence of a test compound, (ii) identifying as a negative regulator the compound which decreases LFA-1 binding to the ICAM and which competes with (2-isopropyl- phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl] sulfide for binding to LFA-1 ⁇ L polypeptide.
• the negative regulator competes with 4-amino-2-chlorophenyl-(l '-chloro-2-naphthylphenyl)-sulfide for binding to LFA-1 ⁇ L polypeptide.
• the regulatory site is defined as the site binding site for a negative regulatory that competes for binding to LFA-1 with one of 3-chloro-4-(l- chloro-naphthalen-2-ylsulfanyl)-phenylamine, 2-iso-propylphenyl)[2-nitro-4-(E-((4- acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl]sulfide, (4-methylphenyl)[2-nitro-4-(E- ((4-acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl] sulfide, 3-chloro-4-(2-chloro-4- (N,N-dimethylamino
• the invention also provides methods to identify candidate compounds particularly useful as negative regulators of LFA-1 binding to an ICAM that binds LFA-1 comprising the steps of (i) contacting LFA-1 with (2-isopropyl-phenyl)[2- nitro-4-(E-((4-acetylpiperazin-l-yl)carbonyl)ethenyl)phenyl] sulfide in the presence and absence of a compound, and (ii) identifying the compound as a putative negative regulator wherein the compound competes with (2-isopropyl-phenyl)[2-nitro-4-(E-((4- acetylp ⁇ perazin-l -yl)carbonyl)ethenyl)phenyl]sulf ⁇ de for binding to the LFA-1 L polypeptide.
• the invention therefore provides a method to screen for candidate negative regulators and/or to confirm the mode of action of compounds that decrease LFA-1 binding to an ICAM.
• the methods of the invention to identify negative regulators are particularly amenable to the various high throughput screening techniques known in the art.
• libraries used for the identification of small molecule modulators in these screening techniques of the invention, including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
• Chemical libraries consist of structural analogs of known compounds or compounds that are identified as "hits" or "leads" via natural product screening.
• Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 252:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.
• Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
• combinatorial chemistry and libraries created therefrom see Myers, Curr. Opin. Biotechnol 5:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit” (or “lead") to optimize the capacity of the "hit” to modulate activity.
• Negative regulators of the invention are compounds that decrease LFA-1 binding to an ICAM (as compared to binding in the absence of the compound) and compete with (2-isopropyl-phenyl)[2-nitro-4-(E-((4- acetylpiperazin- 1 -yl)carbonyl)ethenyl)phenyl]sulfide for binding to the ⁇ L polypeptide of LFA-1.
• Presently preferred inhibitors are substituted diaryl sulfides.
• Exemplary compounds include those as described in co-pending U.S. patent applications entitled “Cell Adhesion-Inhibiting Antiinflammatory and Immune Suppressive Compounds” filed April 2, 1999, attorney docket number 6446.US.Z3, Serial Number 09/286,645, incorporated herein by reference in its entirety, and
• the invention also provides compositions comprising negative regulators of the invention, and preferably pharmaceutical compositions further comprising a pharmaceutically acceptable diluent or carrier.
• Pharmaceutical compositions are particularly useful for treatment of a variety of pathological disorders in humans or other animals, e.g., disorders amenable to animal models as described above.
• the invention further provides use of a negative regulator identified by a method of the invention in the production of a medicament to ameliorate pathologies arising from LFA-1 binding to an ICAM that binds LFA-1.
• kits to identify inhibitors of LFA-1 binding to an ICAM that binds LFA-1 comprising one or more of a purified and isolated LFA-1 polypeptide, a purified and isolated ICAM polypeptide that binds
• LFA-1 LFA-1
• cells expressing LFA-1 cells expressing the ICAM.
• Appropriate control reagents and buffers are contemplated in kits of the invention.
• Example 1 describes a high throughput assay to identify inhibitors of LFA-1 binding to full length ICAM-1.
• Example 2 relates to identification of LFA-1 residues that participate in antagonist binding.
• Example 3 describes production of an antibody that activates LFA-1.
• Example 4 describes identification of an ICAM-1 binding site on LFA-1.
• HTS high throughput screening
• the extracellular domain of ICAM-1 was subcloned into plasmid pDCl by standard methods to generate an expression construct encoding a chimeric protein containing the ICAM-1 extracellular domain fused to the Fc region of the heavy chain of human IgGl just upstream of the hinge.
• the protein was expressed in CHO cells
• the fusion protein was biotinylated using a kit obtained from Pierce Chemical (Rockford, IL). Biotinylated protein (BioIgICAM-1) concentration was determined by measuring absorbance at 280 nm, and serial dilutions were prepared to give a final concentration range of 50 ⁇ g/ml to
• BioIgICAM-1 concentration selected as described above.
• the capture antibody i.e., a non-blocking anti-LFA-1 monoclonal antibody (TS2/4.1 ; ATCC #HB244)
• plate coating buffer 50 mM sodium carbonate/bicarbonate, 0.05%
• BioIgICAM-1 A 2X stock solution of BioIgICAM-1 was prepared containing 0.1 ⁇ g/ml BioIgICAM-1 and 4 ⁇ M crystal violet (an activator of LFA-1 /ICAM-1 binding) in Assay Buffer (EG&G Wallac, Gaithersburg, MD). Diluted aliquots (50 ⁇ l) of pooled chemicals (22 compounds/pool) from the chemical library were added to the wells, followed by addition of 50 ⁇ l of the 2X stock of BioIgICAM-1 to provide a final assay volume of 100 ⁇ l (containing 2% DMSO). The plates were incubated for one hour at room temperature and washed once with wash buffer.
• Europium-labeled streptavidin (Eu-SA; #1244-360, EG&G Wallac) was diluted 1 :500 in Assay Buffer, 100 ⁇ l of the diluted Eu-SA was added to each well, and the plates were incubated at room temperature for one hour.
• Controls included both positive and negative wells and 50% binding wells established using blocking antibodies, i.e., anti-LFA-1 monoclonal antibody (TS 1/22.1, ATCC #HB202) or an anti-ICAM-1 monoclonal antibody. Chemical pools in wells showing 50%) or greater inhibition of LFA-1 binding to ICAM-1 were identified and the experiment was repeated using individual chemicals from those pools. Inhibitors of LFA-1 /ICAM-1 binding were identified, and a further screen was performed to determine dose dependence of the inhibitory activity. Further study of selected compounds was carried out using biochemical and cellular assay techniques.
• the HTS assay identified more than 40 compounds as hits demonstrating potency in inhibiting LFA-1 ICAM-1 interaction. Of these many, compounds exhibited a diaryl sulfide structure, thereby identifying these compounds as a class of LFA-1 /ICAM-1 binding inhibitors.
• Nuclear magnetic resonance (NMR) spectroscopy has proven to be a useful technique to detect small molecule binding to proteins.
• This technique for screening, or establishing the structure activity relationship (SAR) by NMR [Shuker, et al, Science 274:1531-1534 (1996), incorporated herein by reference], has been successful to identify drug leads against several proteins [WO 97/18471, published May 22, 1997 and WO 97/18469, published May 22, 1997, both of which are incorporated herein by reference].
• This technique relies on detecting chemical shifts of amide proton and nitrogen atoms resulting from changes in the chemical environment of the peptide backbone, such as those that occur upon ligand binding.
• ICAM-1 binding, and recombinant I domain polypeptides can compete with intact LFA-1 for ICAM-1 binding.
• the approximately 200 amino acid I domain region was therefore subcloned, and the recombinant polypeptide was used in NMR experiments to assess whether antagonists of LFA -mediated adhesion interact with the I domain.
• the I domain polypeptide corresponding to residues 127-309 in SEQ ID NO:
• ID NO: 1 was isotopically labeled in E. coli and purified.
• the pET15b plasmids encoding residues 127-310, 127-309, or 127-305 of SEQ ID NO: 2 were prepared by PCR amplification of the respective sequences using the human LFA-1 gene as a template and cloned using standard techniques. Each expression plasmid was checked by sequencing.
• Models for the bound compounds were generated with a simulated annealing protocol using the program XPLOR.
• the docking calculations were performed using the NMR derived distance constraints.
• the starting protein coordinates in these calculations were derived from the x-ray crystal structure [Qu and Leahy, Proc. Natl. Acad. Sci.(USA) 92:10277-10281 (1995)].
• Starting structures for the compound were generated randomly.
• the backbone atoms of the protein were fixed in the docking calculations.
• the two dimensional heteronuclear single quantum correlation (HSQC) spectra of the 15 N-labeled I domain was indicative of a folded structure.
• Addition of (2-isopropyl-phenyl)[2-nitro-4-(E-((4-acetylpiperazin-l- yl)carbonyl)ethenyl)phenyl]sulfide induced multiple chemical shift changes in the LFA-1 domain spectrum thereby confirming that the I domain of LFA-1 binds to this antagonist.
• Negative regulators were docked using the NOE constraints and the program XPLOR. In the docking calculations, the protein backbone was kept rigid, but amino acid sidechains of the protein were allowed to relax to accommodate the ligand. Only minor changes in protein conformation were necessary to dock the regulator. In all of the docking calculations, the negative regulator was found to lie in the cleft between beta sheet 5 and the carboxy terminal helix, alpha 7 in the I domain, in agreement with the model based on chemical shift changes. The top of the binding pocket is formed from the loop connecting alpha helix 7 to beta sheet 5.
• Negative regulator ring A is positioned to the top part of the cleft by NOE constraints to He 259 and Leu 298 while ring B makes contact to the middle of the cleft with NOEs to He 235 , Val' 57 , Leu 161 and He 306 .
• the contacts Val 15 " and Leu 161 on the helix-5 indicate how deep into the protein pocket ring B sits in the complex.
• Residue He 235 is positioned near the center of the negative regulator and shows large chemical shifts upon regulator binding.
• these hydrophilic groups shield the hydrophobic binding pocket from solvent, possibly by forming salt bridges.
• these hydrophilic side chains move to accommodate the negative regulator.
• mice Female BALB/c mice were immunized with purified recombinant cc d /CD18 (described in U.S. Patent 5,837,478, issued November 17, 1998, and incorporated herein by reference).
• the protein was captured from CHO cell
• Hybridomas were screened by ELIS A for production of IgG by standard protocols.
• a secondary ELISA screen was performed to identify hybridoma supernatant reactive with either the integrin ⁇ or ⁇ chain. Briefly, plates were coated in standard buffer with 100 ng/ml of the F(ab') 2 fragment of the CD18-specific antibody, 195N.
• CHO cells supematants containing either soluble ⁇ d /CD18 or CD 1 la/CD 18 were added to the wells and capture of integrins was allowed to continue for four hours at 37 °C.
• Hybridoma supematants were incubated with bound integrin, after which bound mouse IgG was detected with a horseradish peroxidase-conjugated anti-mouse Fc-specific polyclonal antibody.
• Hybridomas that reacted with both ⁇ d /CD18 and CD 1 la/CD 18 were presumed to recognize either the common ⁇ chain or the leucine zipper region of the recombinant molecule.
• Supematants were tested by flow cytometry for recognition of native ⁇ d on ⁇ d -transfected JY cells and HL60 cells. Hybridomas that reacted with neither were presumed to be reactive with the leucine zipper peptides.
• PMA phorbol myristate acetate
• hybridomas Six hybridomas were identified that produced an anti-CD18 monoclonal antibody capable of enhancing PBL binding to ICAM-1 at the same level as the PMA control (three- to four- fold over unstimulated cells). The hybridomas were cloned in successive rounds using a modified limiting dilution method. Five clones survived the cloning process and were retested in the PBL assay and with B and T cells. The antibody 240Q was developed further since it appeared to be more effective at cell stimulation.
• Antibody/antigen complexes were isolated with an anti-mouse Ig conjugated to
• Antibody 240Q precipitated the identical series of proteins as the known CD18 antibodies.
• the bands represented known molecular weight proteins for all of the leukointegrin ⁇ chains and the CD 18 ⁇ chain. Extensive immunocytochemical analyses comparing 240Q staining with that of the other anti-CD 18 antibodies indicated that 240Q recognized the ⁇ chain.
• Biotinylated antibodies were incubated with both cell types at 0.3, 1.0, 3.0, and 10 ⁇ g/ml. Biotinylated antibody was detected with both streptavidin-FITC (to determine whether biotinylation was successful) and anti-mouse Ig/FITC (to determine whether biotinylated antibodies were still functional and binding at equivalent levels). Staining with 240Q with the streptavidin-FITC detection method was only 25% that of the CD18-specific antibody 2311 IB at any given concentration. The difference was more dramatic with the anti-mouse FITC detection. Affinity differences would not be expected to account for these results, since transformants stained equally well with both antibodies.
• the integrin-activation function of 240Q was further characterized in binding experiments using the TACO cell line. These cells were isolated from a patient diagnosed with a subtype of leukocyte adhesion deficiency (LAD). The subtype is characterized by normal surface expression of LFA-1 on lymphocytes, but the inability of LFA-1 to bind ICAM- 1. The functional phenotype is not recognized by phorbol myristate acetate (PMA). Treatment of the cells with the antibody 240Q rescued homotypic aggregation, which was determined to be ICAM-1 -dependent using an ICAM-1 -specific antibody.
• LAD leukocyte adhesion deficiency
• PMA phorbol myristate acetate
• the amplification product was digested with Hmdlll and Xbal and gel purified.
• the purified fragment was used in a three-way ligation including ICAM-1 domains 1 and 2 (the H/ndlll/Nb ⁇ l fragment), pDEFl 7 previously digested with Xbal and Notl, and pDEF17 previously digested with Notl and H. ' ndlll, and the resulting plasmid, pDEF17/ICAM-l domains 1 and 2, was sequenced.
• the plasmid was transformed into C ⁇ O cells by standard methods.
• a 70 ml immunoaffinity column was created by coupling 2 mg of a non-blocking anti-ICAM-1 18E3D monoclonal antibody per ml of activated
• the column was eluted with 2 M KSC ⁇ , p ⁇ 8.0, and fractions were analyzed by
• amino acid substitution mutants were generated and tested for the ability to bind ICAM-1.
• Amino acids most affected in NMR by compound binding and whose side chains are directed toward the surface of the I domain were substituted with alanine.
• Asp' 3 a residue located within and essential to a functional MIDAS and ICAM-1 binding site, was substituted with alanine.
• the various I domain mutants were expressed in COS cells and cell adhesion to ICAM-1 was determined in the presence of a CD18 monoclonal antibody, 240Q, that induces high avidity binding.
• CD1 la Twenty-five individual mutations in the LFA-1 ⁇ polypeptide (CD1 la) were generated. Each mutation was prepared using Stratagene's QuikChangeTM Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Briefly, two primers were synthesized that introduced a specific mutation in the amplification product. Primers utilized are set out below, with only the sense primer shown.
• D137A/S SEQ ID NO: 5 CTGGTATTTCTGTTTGCGGGTTCGATGAGCTTG
• V157A/S SEQ D NO: 6
• GCCCGGCCAGATGCCGCGAAAGTGCTTATCATC K2327 SEQ ID NO: 10 CGGCCAGATGCCACCGCGGTGCTTATCATCATC I235A S: SEQ ID NO: 1 1
• Control mutants included the following, wherein amino acid changes were introduced in regions reported by others to be involved in ICAM-1 binding.
• T243A/S SEQ ID NO: 25
• the primers were used in two PCR reactions, one with full-length CD1 la (residues 1- 1170) in plasmid pDCl as template and the other with CD1 la I domain (residues 152- 334) in plasmid pET15b as template.
• PCR reaction conditions varied depending on the melting temperature (T M ) of the primers. Details of the reaction for each mutation are described below.
• T M melting temperature
• the general format was as follows: one cycle at 95 °C for 30 seconds followed by 16 cycles of: 95 °C for 30 seconds, 55 °C for one minute, and 68 °C for 20 minutes.
• template DNA was digested with Dpnl at 37 °C for one hour and the remaining amplified DNA was used to transform supercompetent E. coli XL1 Blue (Stratagene) according to the manufacturer's suggested protocol.
• Mutations V157A, ⁇ 218A, T231A, 1235 A, I255A, E284A, K287A, K294A, K305A were generated in PCR including a 45 °C annealing step and a 58 °C extension step.
• extension times for the full length sequence in pDCl was 20 minutes and 15 minutes for I domain in pET15b.
• mutations D137A, K160A, K232A, K280A, S283A, E301A, Q303A, K304A and I306A the same temperatures as described above were used, but with both templates, the extension time for both templates was 20 minutes.
• mutants Y307A and D253A an extension step of 25 minutes was used.
• the annealing step was carried out at 45 °C, and extension was carried out at 60°C for 20 minutes.
• mutant S245 A PCR included 18 cycles rather than 16 cycles.
• COS Cell Transfections On day 1, COS cells were seeded at 1.6 x 10 6 cells per 10 cm plate in
• Cells were grown ovemight and media was removed and replaced. After overnight growth, cells were split 1 :2 and grown overnight again. Cells were removed from the plate with Versene, collected by centrifugation, resuspended in adhesion buffer containing (RPMI containing 5%> inactivated FBS), and counted. Cells were then used for adhesion assays and for fluorescence activated cell sorting (FACS) staining and analysis.
• FACS fluorescence activated cell sorting
• Adhesion assays were performed in 96-well Easy Wash plates (Coming, Coming NY) using a modification of a previously reported procedure [Sadhu, et al, Cell. Adhes. Commun. 2:429-440 (1994)]. Each well was coated overnight at 4°C with (i) 50 ⁇ l of ICAM-1/Fc (5 ⁇ g/ml), (ii) anti-CD18 monoclonal antibody TS1/18 [Sanchez-Madrid, et al, Proc. Natl. Acad. Sci. (USA) 79:7489-7493
• Adhesion buffer 100 ⁇ l
• COS transfectants 100 ⁇ l, approximately 0.75 x 10 6 cells/ml
• expressing the heterodimer with or without a mutation
• adhesion buffer with or without activating antibody 240Q (10 ⁇ g/ml) was added to each well and the plates incubated at 37°C for 15 to 20 min.
• Adherent cells were fixed by addition of 50 ⁇ l/well 14% glutaraldehyde in D-PBS and incubated at room temperature for 1.5 hr.
• the plates were washed with dH 2 O and stained with 100 ⁇ l/well 0.5%> crystal violet in 10%> ethanol for five minutes at room temperature. Plates were washed in several changes of dH 2 O. After washing, 70%> ethanol was added, and adherent cells were quantitated by determining absorbance at 570 nm and
• a 570 -A 410 (binding to CDl 8 + CDl la antibody)
• % wildtype binding (% mutant cell binding X 100
• FACS Stainin FACs staining was carried out in a 96 well plate. Each transfectant was stained with an antibody to CDl 8 (TS1/18), an antibody to CDl la (TS1/22), and an activating antibody to CDl 8 (240Q). Controls included unstained cells, cells stained with secondary antibody only, and cells stained with an isotype matched control antibody (1B7).
• 240Q induction (Val 157 - Ala, Glu 218 ⁇ Ala, Thr ⁇ ' ⁇ Ala, Lys 280 - Ala, and Lys 294 - Ala), 2) mutants that supported greater than wild type levels of binding without 240Q induction and wild type levels with induction (Ile 235 ⁇ Ala, Ue 255 ⁇ Ala, Ser ⁇ Ala, Glu 284 - Ala, Glu 301 -* Ala, and He 306 — Ala), 3) mutants that possessed decreased levels of binding relative to wild type binding in the absence of induction, but wild-type levels with 240Q induction (Lys' 60 ⁇ Ala, Lys 232 - Ala, Asp 253 ⁇ Ala, Lys 287 - Ala, Gin 303 - Ala, Lys 304 - Ala, and Lys 305 ⁇ Ala ), and 4) mutants that demonstrate severely decreased levels or no binding with or without 240Q (Tyr 307 -
• the residues Lys 232 , Lys 287 , Lys 304 , Lys "" ' " , and Tyr 307 are all hydrophilic residues that surround, but do not directly form, the small molecule ligand binding site. Residues Val 157 , He 235 , He 255 , and He 306 form the hydrophobic pocket of the small molecule binding site.
• LFA-1 binding activity is regulated through two different mechanisms which are not mutually exclusive: 1) control of individual receptor affinity (the strength of binding between two molecules), and 2) control of overall avidity (the affinity multiplied by the number of interactions which are occurring at one time) by the regulated aggregation of individual receptors through interactions with the cellular cytoskeleton. If the LFA-1 regulatory binding site, as defined above, is responsible for regulating individual receptor affinity, then the activating mutants, typified by I235A (described above), should possess higher binding affinity in cellular adhesion. These methods, however, are imprecise and do not accurately separate affinity from avidity. Therefore, in order to accurately measure the relative binding affinity of wildtype and mutant 1235 A for ICAM-1, the following assay was carried out.
• Recombinant I235A was produced in CHO cells in secreted form using the same method as that used for production of recombinant LFA-1 in Example 1.
• the soluble forms of recombinant LFA-1 (used here and in Example 1) and I235A (used here) contain deletions of the transmembrane and cytoplasmic domains of CDl la and
• CD18 (SEQ ID NO: 30 [full length polynucleotide] and 31 [full length amino acid], and substitution of these regions for acidic and basic leucine zipper cassettes, respectively, which promote and stabilize specific heterodimerization as described for the production of soluble T-cell receptor [Hsiu-Ching et al Proc. Natl. Acad. Sci. (USA) 91: 11408-11412 (1994)].
• Both wildtype and mutant 1235 A CDl la were truncated after position Q1063 in the mature polypeptide, and the 47 amino acid acidic leucine zipper cassette (SEQ ID NO: 32) was added in-frame, using standard methods.
• CDl 8 was truncated after position N678 in the mature polypeptide, and the 47 amino acid basic leucine zipper cassette (SEQ ID NO: 33) was added in-frame.
• Both recombinant LFA-1 and I235A were expressed in CHO cells and purified from the supematants using separate 8 ml immunoaffmity columns created by coupling 2 mg of anti-CD18 2311 IB monoclonal antibody per ml of activated CNBr-SepharoseTM according to the manufacturer's suggested protocol, and eluted using a 20 mM Tris
• Recombinant LFA-1 and 1235 A were then purified a second time by gel filtration chromatography over a Pharmacia HiLD SuperDex 200TM column in PBS buffer using standard methods, in order to remove any single chain, aggregated and/or degraded material.
• the resulting suspensions of purified heterodimers were concentrated using Millipore Ultrafree-4 Centrifugal Filter UnitsTM with Biomax-30TM membranes, then dialyzed in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM MgCl 2 ) at 4 oC, and quantitated using a BioRad Protein AssayTM and the manufacturer's protocol.
• LFA-1 or I235A was then injected at different concentrations, using a flow rate of 10 ml/min, and the surface plasmon resonance was recorded. After each concentration of LFA-1 or 1235 A was allowed to bind and dissociate, the chip was stripped of ICAM-1 /LFA-1 complexes with 0.1N HC1 and regenerated with fresh ICAM-1 /IgGl before the next concentration of LFA-1 or 1235 A was analyzed.
• the association and dissociation rate constants (k a and k d , respectively) for LFA-1 and 1235 A were calculated using the BIA evaluation 2.0 program and its 1 :1 Langmuir binding kinetics model (Pharmacia Biosensor AB).
• the k a for LFA-1 and 1235 A were identical and equaled 2.2 x 105 M “1 s "1 . However, the k d for LFA-1 and 1235 A were significantly different and equaled 1.2 x 10 "2 s " ' and 1.9 x 10 "3 s "1 , respectively. These values corresponded to an affinity dissociation rate (K D ) of 547 nM for LFA-1 , which is in close agreement with the value of 500 nM calculated by Tominaga using a similar method [Tominaga, et al J. Immunol, 161: 4016-4022 (1998)].
• K D affinity dissociation rate
• diaryl sulfide compounds which bind to the LFA-1 regulatory binding site are predicted to inhibit adhesion to ICAM-1 by lowering the affinity of LFA-1 for ICAM through an increase in the k d of the receptor or through stabilizing the low affinity state of LFA-1.

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