WO1991019511A1 - CONTROLLING CELLULAR IMMUNE/INFLAMMATORY RESPONSES WITH β2 INTEGRINS - Google Patents

CONTROLLING CELLULAR IMMUNE/INFLAMMATORY RESPONSES WITH β2 INTEGRINS Download PDF

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WO1991019511A1
WO1991019511A1 PCT/US1991/004338 US9104338W WO9119511A1 WO 1991019511 A1 WO1991019511 A1 WO 1991019511A1 US 9104338 W US9104338 W US 9104338W WO 9119511 A1 WO9119511 A1 WO 9119511A1
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PCT/US1991/004338
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French (fr)
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M. Amin Arnaout
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • This invention relates to controlling cellular immune/inflammatory responses, particularly phagocyte-mediated tissue injury and inflammation.
  • Circulating phagocytic white blood cells are an important component of the cellular acute inflammatory response. It is believed that a number of important biological functions such as chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endothelium, phagocytosis, antibody-dependent cellular cytotoxicity, superoxide, and lysosomal enzyme release are mediated by a family of leukocyte surface glycoprotein adhesion receptors known as ⁇ 2 integrins or the CD11/CD18 complex. Arnaout et al., Blood 75:1037 (1990) .
  • the CD11/CD18 family consists of three heterodimeric surface glycoproteins, each with a distinct subunit (CDlla, CDllb or CDllc) non-covalently associated with a common ⁇ subunit (CD18) .
  • the divalent cations Ca + and Mg are essential in the stabilization and function of the ⁇ (CD11/CD18) complex.
  • the ⁇ 2 integrins are expressed only on leukocytes.
  • CDlla/CD18 also known as LFA-1, TA- 1
  • CDllb/CDl ⁇ and CD11C/CD18 are expressed primarily on monocytes, poly orphonuclear leukocytes, macrophages and natural killer cells CDllc/CD18 is also expressed on certain lymphocytes.
  • CDlla/CD18 and not CDllb/CD18 or CDllc/CD18, is expressed on B- and T-lymphocytes; accordingly CDlla/CD18 plays a role in itogen-, antigen-, and alloantigen- induced proliferation, T-cell-mediated cytotoxicity, lymphocyte aggregation, and Ig production.
  • CD11/CD18 molecules are important for monocyte/macrophage and granulocyte adhesion-dependent functions.
  • CDllb/CD18 and CDllc/CD18 mediate enhanced adhesiveness of activated phagocytes through quantitative and qualitative changes in these proteins on the surface of activated cells. For example, in granulocytes, these proteins are translocated from intracellular storage pools present in secondary and tertiary granules. Arnaout et al., J . Clin . Invest . 74:1291 (1984); Arnaout et al., New Eng . J. Med . 312:457 (1985); Todd et al., J . Clin . Invest . 74:1280 (1984). CDllb/CD18 is also known as complement receptor type 3 (CR3) , Mol, Mac-1 or MAM.
  • CR3 complement receptor type 3
  • CDllb/CD18 consists of two non-covalently associated subunits.
  • the ⁇ subunit of CDllb/CD18 has an apparent molecular mass of 155-165 kD and associates non- covalently with a ⁇ subunit, CD18, of apparent molecular mass 95 kD. Todd et al., Hybridoma 1:329 (1982).
  • Monoclonal antibodies have been used to identify at least two distinct functional domains of CDllb/CD18, one mediating homotypic and heterotypic adhesion and the other mediating binding to the complement C3 fragment (iC3b) , the major C3 opsonin in vivo. Dana et al., J. Immunol . 137:3259 (1986).
  • the peptides and heterodimeric proteins of the invention are capable of antagonizing CD11/CD18 ( ⁇ 2 integrin) mediated immune response.
  • CD11/CD18 mediated immune responses which it may be desirable to block include acute inflammatory functions mediated by neutrophils.
  • the molecules of the invention are useful for treatment of ischemia reperfusion injury (e.g., in the heart, brain, skin, liver or gastrointestinal tract) , burns, frostbite, acute arthritis, asthema, and adult respiratory distress syndrome.
  • Peptides and heterodimeric proteins of the invention may also be useful for blocking intra-islet infiltration of macrophages associated with-insulin-dependent diabetes mellitus.
  • the invention features a purified peptide which includes at least one extracellular region of a ⁇ 2 integrin subunit capable of inhibiting a CD11/CD18 mediated immune response, the peptide lacks the transmembrane and cytoplasmic portions of the ⁇ 2 integrin subunit.
  • the ⁇ 2 integrin subunit is a human ⁇ 2 integrin subunit; more preferably the ⁇ 2 integrin subunit is CDlla, CDllb, CDllc or CD18; most preferably the ⁇ 2 integrin subunit is CDllb.
  • the peptide includes all or part of the A domain of CDllb.
  • the peptide includes one of the following sequences: DIAFLIDGS (SEQ ID NO: 32); FRRMKEFVS (SEQ ID NO: 33); FKILWITDGE (SEQ ID NO: 34); VIRYVIGVGDA (SEQ ID NO: 35); DGEKFGDPLG (SEQ ID NO: 36); YEDVIPEADR (SEQ ID NO: 37); DGEKFGDPLGYEDVIPEADR
  • the peptide includes the amino acid sequence YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 38); the peptide includes the amino acid sequence KSTRDRLR (SEQ ID NO: 15) .
  • the peptide includes one of the following amino acid sequences: AYFGASLCSVDVDSNGSTDLVLIGAP (SEQ ID NO: 1) ; GRFGAALTVLGDVNGDKLTDVAIGAP (SEQ ID NO: 2); QYFGQSLSGGQDLTMDGLVDLTVGAQ (SEQ ID NO: 3); YEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 4); DIAFLIDGSGSIIPHDFRRMK (SEQ ID NO: 5) ; RRMKEFVSTVMEQLKKSKTLF (SEQ ID NO: 6) ; SLMQYSEEFRIHFTFKEFQNN (SEQ ID NO: 7) ; PNPRSLVKPITQLLGRTHTATGIRK (SEQ ID NO: 8); RKWRELFNITNGARKNAFK (SEQ ID NO: 9); FKILWITDGEKFGDPLGYEDVIPEADR (SEQ ID NO: 10); REGVIRYVIGVGDAFR
  • RQNTGMWESNANVKGT SEQ ID NO: 21
  • TSGSGISPSHSQRIA SEQ ID NO: 22
  • NQRGSLYQCDYSTGSCEPIR SEQ ID NO: 23
  • PRGRARWQC SEQ ID NO: 24
  • KLSPRLQYFGQSLSGGQDLT SEQ ID NO: 25
  • QKSTRDRLREGQ SEQ ID NO: 26
  • SGRPHSRAVFNETKNSTRRQTQ SEQ ID NO: 27
  • CETLKLQLPNCIEDPV SEQ ID NO: 28
  • FEKNCGNDNICQDDL (SEQ ID NO: 29) ; VRNDGEDSYRTQ (SEQ ID NO: 30) ; SYRKVSTLQNQRSQRS (SEQ ID NO: 31) .
  • the peptide includes one or more metal binding domains of CDllb. More preferably, the metal binding domains encompass amino acids 358-412,
  • the peptide includes one of the following sequences: DVDSNGSTD (SEQ ID NO: 46); DVNGDKLTD (SEQ ID NO: 47); DLTMDGLVD (SEQ ID NO: 48); DSDMNDAYL (SEQ ID NO: 49).
  • the peptides are soluble under physiological conditions.
  • the invention features a heterodimer which includes a first peptide and a second peptide; the first peptide includes at least one extracellular region of a CDll subunit and lacks the transmembrane and cytoplasmic portions of the CDll subunit; the second peptide comprising at least one extracellular region of a CD18 subunit and lacks the transmembrane and cytoplasmic portions of the CD18 subunit; the first and second peptides are associated to form the heterodimer; and the heterodimer is capable of inhibiting a CD11/CD18 mediated immune response.
  • the CDll subunit is: CDlla; CDllb; CDllc.
  • the heterodimer is CDllb 1089 /CD18 699 .
  • the invention features a method of controlling phagocyte-mediated tissue damage to a human patient.
  • the method includes administering a therapeutic composition to a patient; the therapeutic composition includes a physiologically acceptable carrier and a peptide or a heterodimer of the invention. More preferably, the method is used to control phagocyte- mediated tissue damage due to ischemia-reperfussion.
  • the method is used to control phagocyte- mediated tissue damage to the heart muscle associated with reduced perfusion of heart tissue during acute cardiac insufficiency.
  • the invention features a method of producing a recombinant ⁇ 2 integrin heterodimer.
  • the method includes the steps of: (a) providing a recombinant cell encoding a CDll peptide lacking both the transmembrane domain and the cytoplasmic domain and a CD18 peptide lacking both the transmembrane domain and the cytoplasmic domain; (b) culturing the recombinant cell; and (c) isolating the heterodimer from the culture supernatant.
  • the method is used to produce a soluble recombinant ⁇ 2 integrin heterodimer.
  • the CDll peptide of the heterodimer is a CDlla peptide; is a CDllb peptide; is a CDllc peptide.
  • the invention features a monoclonal antibody which is raised to a peptide or a heterodimer of the invention and which is capable of inhibiting a CD11/CD18 mediated immune response.
  • ⁇ 2 integrins include all leukocyte adhesion molecules which include a CD18 subunit.
  • a domain of CDllb is meant the amino acid sequence corresponding to the sequence of CDllb from Cys 128 to Glu321 or an am o acid sequence produced by introducing one or more conservative amino acid substitutions in an amino acid sequence corresponding to the sequence of CDllb from Cys 128 to Glu 321 .
  • CDll/CD18-mediated immune response includes those CDll/CD18-related functions mentioned above: chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endotheliu , phagocytosis, antibody-dependent or -independent cellular cytotoxicity, and superoxide and lysosomal enzyme release. Inhibition of these immune functions can be determined by one or more of the following inhibition assays as described in greater detail below: iC3b binding, cell-cell aggregation, phagocytosis, adhesion to endothelium, and chemotaxis.
  • a human CDllb recombinant peptide is a chain of amino acids derived from recombinant CDllb-encoding cDNA, or the corresponding synthetic DNA.
  • CD11 1089 /CD 18699 is a heterodimer which comprises amino acids 1-1089 of human CDll and amino acids 1-699 of CD18.
  • Figure 1 is the cDNA sequence and deduced amino acid sequence of the open reading frame of human CDllb from Arnaout et al., J. Cell . Biol . 106:2153 (1988).
  • Figure 2 is a representation of the results of an immunoprecipitation assay.
  • Figure 3 is a representation of the results of an immunoprecipitation assay.
  • Figure 4 is a representation of the results of an immunoprecipitation assay.
  • Figure 5 is a graph of the effect of various proteins and antibodies on neutrophil adhesion to endothelium.
  • Figure 6 is the cDNA sequence and deduced amino acid sequence of human CDlla from Larson et al., J. Cell . Biol . 108:703 (1989).
  • Figure 7 is the cDNA sequence and deduced amino acid sequence of human CDllc from Corbi et al., EMBO J. 6:4023 (1987).
  • Figure 8 is the cDNA sequence of human CD18 from Law et al., EMBO J. 6:915 (1987).
  • Peptides As described in greater detail elsewhere, each member of the ⁇ 2 integrin family is a heterodimer consisting of two subunits: a CDll subunit (with at least three variants designated CDlla, CDllb, and CDllc) and a CD18 subunit. Each subunit includes a transmembrane anchor which connects a cytoplasmic segment to an extracellular segment. The two subunits interact to form a functional heterodimer. As described in greater detail below, the extracellular segments of the ⁇ 2 integrin subunits contain various functional domains which are the focus of the invention.
  • the peptides of the invention antagonize CDll/CD18-mediated immune responses by competitively inhibiting binding of leukocytes bearing a member of the ⁇ 2 integrin family to the respective binding partners of that family.
  • the peptides of the invention include an immune-response inhibiting extracellular segment of any one of the ⁇ 2 integrin subunits —CDlla, CDllb, CDllc, CD18— or a heterodimer composed of a portion of an ⁇ (CDlla, CDllb, or CDllc) subunit together with a portion of a ⁇ subunit (CD18) .
  • Candidate ⁇ 2 integrin subunits can be evaluated for their ability to antagonize CDll/CD18-mediated immune responses by any of several techniques. For example, subunits may be tested for their ability to interfere with neutrophil adhesion to endothelial cells using an assay described in detail below. Specific regions of the ,92 integrin subunits can be evaluated in a similar manner. Any extracellular region of a ⁇ 2 integrin subunit may be screened for its ability to interfere with CD11/CD18 mediated immune response. Regions of CDll whose sequences are conserved between two or more subunits are preferred candidates for antagonizing
  • CD11/CD18 - mediated immune response For example, the A domain (corresponding to Cys 128 to Glu 321 of CDllb) is conserved between CDlla, CDllb, and CDllc. The A domain is 64% identical in CDllb and CDllc and 36% homologous between these two subunits and CDlla. This domain is also homologous to a conserved domain in other proteins involved in adhesive interactions including von Willebrand's factor, cartilage matrix protein, VLA2, and - li ⁇
  • CDlla, CDllb and CDllc include seven homologous tandem repeats of approximately 60 amino acids. These repeats are also conserved in the ⁇ subunits of other integrin subfamilies (e.g., fibronectin receptor). Arnaout et al.. Blood 75:1037 (1990) .
  • Regions of CD18 which are conserved among ⁇ intergrin subunits are also good candidates for regions capable of interfering with CD11/CD18 - mediated immune response.
  • CD18 has four tandem repeats of an eight- cysteine motif. This cysteine-rich region is conserved among ⁇ subunits. Just amino terminal to this cysteine rich region is another conserved region, 247 amino acids long, which is conserved in several integrin ⁇ subunits.
  • Fig. 6 depicts the cDNA sequence of human CDlla (SEQ ID NO: 39) ;
  • Fig. 7 depicts the cDNA sequence of human CDllc (SEQ ID NO: ) ;
  • Fig. 8 depicts the cDNA sequence of CD18 (SEQ ID NO: 41) .
  • CDllb, CDllc or CD18 can be obtained by means of polymerase chain reaction amplification.
  • two short DNA primers are used to generate multiple copies of a DNA fragment of interest from cells known to harbor the RNA of produced by the gene of interest.
  • Polymerase chain reaction methods are generally described by Mullis et al. (U.S. Patent Nos. 4,683,195 and 4,683,202) .
  • CDlla For example, to clone a portion of CDlla, the known sequence of CDlla is used to design two DNA primers which will hybridize to opposite strands outside (or just within) the region of interest. The primers must be oriented so that when they are extended by DNA polymerase, extension proceeds into the region of interest.
  • poyA RNA is isolated from cells expressing CDlla. A first primer and reverse transcriptase are used to generate a cDNA form the mRNA. A seond primer is added; and Tag DNA polymerase is used to amplify the cDNA genrated in the previous step.
  • CDlla, CDllb, CDllc and CD18 can be used to design highly specific probes for identifying cDNA clones harboring the DNA of interest.
  • a cDNA library suitable for isolation of CDlla, CDllb, and CDllc DNA can be generated using phorbol ester-induced HL-60 cells (ATCC Accession No. CCL 240) as described by Corbi et al. (EMBO J. 6:4023, 1987) and Arnaout et al., Proc . Nat ' l Acad Sci . USA 85:2776, 1988) ; CD18 DNA can be isolated from a library generated using U937 cells (ATCC Accession No. CRL 1593) as described by Law et al. (EMBO J. 6:915, 1987). These cell lines are also suitable for generating cDNA by polymerase chain reaction amplification of mRNA as sescribed above.
  • Heterodimers comprised of part of CDllc and CD18 can be produced as described below for CDllb/CD18 by changing a codon amino terminal to the transmembrane region (e.g. Pro 1086 ) to a stop codon.
  • Heterodimers comprised of part of CDlla can be produced by changing a codon amino terminal to the transmembrane region (e.g., - 13
  • DNA encodi •ng the truncated CDll subunit is then introduced into cells along with DNA encoding a similarly truncated CD18 molecule (described below) . These cells are then used as a source of heterodimer.
  • a 378 base pair (bp) cDNA clone encoding guinea pig CDllb was used as a probe to isolate three additional cDNA clones from a human monocyte/lymphocyte cDNA library as described in Arnaout et al., Proc . Nat ' l . Acad . Sci . USA 85:2776 (1988); together these three clones contain the 3,048 nucleotide sequence encoding the CDllb gene shown in Fig. 1 (SEQ ID NO: 40). Arnaout et al., J. Cell . Biol . 106:2153 (1988).
  • a mammalian expression vector was constructed by assembling the above-described three cDNA clones. Appropriate restriction enzyme sites within the CDllb gene can be chosen to assemble the cDNA inserts so that they are in the same translation reading frame. Arnaout et al., J. Clin . Invest . 85:977 (1990).
  • a suitable basic expression vector can be used as a vehicle for the 3,048 bp complete cDNA fragment encoding the human CDllb peptide; the recombinant cDNA can be expressed by transfection into, e.g., COS-l cells, according to conventional techniques, e.g., the techniques generally described by Aruffo et al., Proc.
  • the CDllb protein can be purified from the lysate of transfected COS-1 cells, " sing affinity chromatography and lentil-lectin Sepharose and available anti-CDllb monoclonal antibody as described by Pierce et al. (1986) supra and Arnaout et al., Meth . Enzymol . 150:602 (1987).
  • DNA encoding the desired peptide can be expressed in the same mammalian expression vector described above using the selected DNA fragment and the appropriate restriction enzyme site, as outlined above.
  • the selected DNA fragment may be isolated according to conventional techniques from one of the CDllb cDNA clones or may be synthesized by standard polymerase chain reaction amplification, as described above. See also Saiki et al. , (Science 239:487, 1988). Characterization of the CDllb Polypeptide The coding sequence of the complete CDllb protein is preceded by a single translation initiation methionine.
  • the translation product of the single open reading frame begins with a 16-amino acid hydrophobic peptide representing a leader sequence, followed by the NH 2 -terminal phenylalanine residue.
  • the translation product also contained all eight tryptic peptides isolated from the purified antigen, the amino-terminal peptide, and an amino acid hydrophobic domain representing a potential transmembrane region, and a short 19-amino acid carboxy-terminal cytoplasmic domain
  • Fig. 1 illustrates the amino acid sequence of CDllb; SEQ ID NO: 43) .
  • the coding region of the 155-165 kD CDllb (1,136 amino acids) is eight amino acids shorter than the 130-150 kD alpha subunit of CDllc/CD18 (1,144 amino acids) .
  • the cytoplasmic region of CDllb contains one serine residue that could serve as a potential phosphorylation site.
  • the cytoplasmic region is also relatively rich in acidic residues and in proline (Fig. 1) . Since CDllb/CD18 is involved in the process of phagocytosis and is also targeted to intracellular storage pools, these residues are candidates for mediating these functions.
  • the long extracytoplasmic amino-terminal region contains three or four metal-binding domains (outlined by broken lines in Fig. 1) that are similar to Ca 2+ -binding sites found in other integrins.
  • Each metal binding site may be composed of two noncontiguous peptide segments and may be found in the four internal tandem repeats formed by amino acid residues 358-412, 426-483, 487-553, and 554-614.
  • the portion of the extracytoplasmic domain between Tyr 465 and Val is homologous to the fibronectin-like collagen binding domain and IL-2-receptor.
  • the extracytoplasmic region also contains an additional unique 187-200 amino acid domai •n, the A domain, between Cys128 to Glu321, which is not present in the homologous ( ⁇ ) subunits of fibronectin, vitronectin, or platelet Ilb/IIIa receptors.
  • This sequence is present in the highly homologous CDllc protein ( ⁇ of pl50,95) with 64% of the amino acids identical and 34% representing conserved substitutions.
  • CDllb/CD18 and CDllc/CD18 have a binding site for complement fragment C3 and this unique region may be involved in C3 binding.
  • This region of CDllb also has significant homology (17.1% identity and 52.9% conserved substitutions) to the collagen/heparin/platelet Gpl binding regions of the mature von Willebrand factor (domains A1-A3) .
  • the A domain is also homologous to a region in CDlla. Larson et al., J. Cell Biol . 108:703 (1989) .
  • the A domain is also referred to as the L domain or the I domain. Larson et al., supra (1988); Corbi et - 16 -
  • peptides can be used to inhibit CDllb/CD18 activity: a) peptides identical to the above-described A domain of CDllb, or a portion thereof, e.g., DIAFLIDGS (SEQ ID NO:32), FRRMKEFVS (SEQ ID N0:33), FKILWITDGE (SEQ ID NO:34), DGEKFGDPLGYEDVIPEADR (SEQ ID NO:17), or VIRYVIGVGDA SEQ ID NO:35); b) peptides identical to the above-described fibronectin-like collagen binding domain, or a portion thereof, e.g.,
  • YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO:38); c) peptides identical to one or more of the four metal binding regions of CDllb, or a portion thereof, e.g., DVDSNGSTD (SEQ ID NO:46), DVNGDKLTD (SEQ ID NO:47), DLTMDGLVD (SEQ ID NO:48), DSDMNDAYL (SEQ ID NO:49); d) peptides substantially identical to the complete CDllb; or e) other CDllb domains, e.g. KSTRDRLR (SEQ ID NO:15).
  • a recombinant peptide which includes part of the A domain, e.g, NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO: 50) .
  • the A domain binds iC3b, gelatin, and fibrinogen and binding is disrupted by EDTA.
  • the A domain also binds both Ca 2+ and Mg 2+ . This result unexpected since the A doamin lies outside of the region of CDllb previously predicted (Arnaout et al., J. Cell Biol . 106:2153, 1988; Corbi et al., J. Biol . Chem . 25:12403, 1988) to contain metal binding sites.
  • heterodimer formed by the CDllb and CD18 proteins It is advantageous to administer the heterodimer formed by the CDllb and CD18 proteins. Expression of CDllb is described elsewhere in this application. Expression of CD18 has been ' reported by others. Law et al. Embo, J. 6:915 (1987); Kishimoto et al. Cell 48:681 (1987) . The strategies described above or in those reports can be used to obtain CD18 to make such a heterodimer. Preferred heterodimers are soluble under physiological conditions. The heterodimer described below is generated by changing the codon for Leu 1 in CDllb (SEQ ID NO: 40) to a stop codon and the codon for Asn 700 of CD18 (SEQ ID NO: 41) to a stop codon. Other potentially soluble heterodimers can be generated by introducing a stop codon at positions amino terminal to those described below.
  • CDllb/CD18 heterodimer A soluble form of a CDllb/CD18 heterodimer was produced in COS cells. To produce this molecule the codons for Leu 1090 and Asn 700 located at the predicted extracellular boundaries of CDllb and CD18 respectively, were replaced with in-frame translational stop codons using oligonucleotide-directed gapped-duplex mutagenesis of the wild-type cDNAs (described below) .
  • COS cells were co-transfected with cDNA encoding the truncated forms of CDllb (CDllb 1089 ) and CD18 (CDll 699) .
  • Secreted proteins were analyzed by immunoprecipitation and SDS-PAGE. The results of this analysis are presented in Fig. 2. Briefly, COS cells were transfected as previously described (Arnaout et al., J . Clin . Invest . 85:977, 1990) .
  • both CDllb 1089 and CD18 699 were immunoprecipitated from supernatants of cells transfected with DNA encoding the truncated subunits.
  • the secreted CDllb 1089 had an apparent molecular weight of gqg
  • CDllb 1089/CD18699 complex and that neither the cytoplasmic nor the transmembrane region of the subunits are necessary for heterodimer formation.
  • These mAbs did not precipitate receptor subunits from the supernatants of mock-transfected cells.
  • Arrowheads at left indicate the positions of molecular weight size markers: myosin (200kD) , phosphorylase b (92.5 kD) , bovine serum albumin (69 kD) , and ovalbumin (46 kD) .
  • Arrows at right indicate the expected positions of CDllb 1089 and CD18 699 .
  • CDllb 1089 /CD18 699 was next tested for its ability to bind iC3b (the receptor bound by wild-type CDllb/CD18) .
  • COS cells were transfected CDllb 1089 and CD18 699 cDNA as described above. Cells were labeled with 35 S-methionine as described by Dana et al., J. Clin . Invest . 79:1010 (1987).
  • Supernatants from both co-transfected COS cells (7 _x 10 cells) and mock- transfected COS cells (7 x 10 6 cells) were concentrated to one ml using collodion bags (10,000 MW cut off).
  • Fig. 3 illustrates the results of SDS-PAGE analysis of neutrophil-derived 125 I-surface-labeled glycoproteins eluted from C3b-sepharose and iC3b- sepharose.
  • Eluants from C3b-sepharose (lane a) contained complement receptor type 1 (250kD) and the C3-binding regulatory protein gp45/70 (45-70 kD) .
  • Eluants from iC3b-sepharose (lane b) contained two additional proteins at 155 kD, 94 kD, representing wild-type CDllb and CD18.
  • CDllb/CD18 was immunoprecipitated with 44a mAb (an anti- CDllb mAb) from material eluted from iC3b-sepharose (lane d) , but not from material eluted from C3b-sepharose (lane c) , confirming previous results. Malhorta et al., Eur. J. Immunol . 16:177, (1986).
  • the arrowheads at right indicate the positions of molecular weight standards: yosin (200 kD) , phosphorylase b (92.5 kD) , and bovine serum albumin (69 kD) .
  • the arrows at left indicate the expected position of CR1, CDllb, CD18 and gp45/70.
  • Fig. 4 shows.the results of SDS-PAGE analysis of CDllb 1089 /CD18 699 heterodimer binding to iC3b.
  • CDllb mAb (44a) was used to immunoprecipitate proteins from culture supernatants of mock-transfected COS cells (lane a) , and from COS cells co-transfected with CDllb - 20 -
  • CDllb 1089 /CD18 699 The ability of CDllb 1089 /CD18 699 to inhibit binding of human neutrophils to inflamed endothelium was examined and compared to the inhibition induced by anti- CDllb mAb and anti-CD18 mAb.
  • Adherence of purified human neutrophils to confluent monolayers of human umbilical vein endothelial cells (HUVE) pre-treated with recombinant IL-1 (10 units/ml for 4 hours at 37°C) was measured as described by Arnaout et al.,( «7. Cell . Physiol . 137:305, 1988) with the following modifications.
  • Neutrophils were labeled with carboxyfluorescein (CF, Molecular Probes, Eugene, OR) by incubating 4 x 10 6 cells with 30 ⁇ g of CF in one ml of Tris-buffered saline for 10 minutes on ice, followed by three washes.
  • HUVE were pre- incubated for 10 minutes at 37°C with supernatants of COS cells co-transfected with CDllb 1089 and CD18 699 cDNA supernatants, or for 5 minutes at room temperature with the non-reactive monoclonal antibody NS1, 44a (anti- CDllb) or TS18 (anti-CD18) ascites (1:100 dilution). Labeled neutrophils were then added and incubation was continued for an additional 10 minutes. The plates HUVE were washed twice, and adherent neutrophils were harvested by washing with 0.1% SDS and 0.1N NaOH. - 21 -
  • Relative numbers of neutrophils were measured (at Exc. , 490 nm; Em, 300nm) using a Fluorometer (SLM 8000, SLM Aminco, Urbana, IL) . All assays were done in triplicate. Labels along the horizontal axis indicate the molecule added to HUVE. 'Buffer 1 indicates that no antibodies were added. 'Sham 1 indicates that supernatant from mock transfected cells was added.
  • culture supernatants containing CDllb 1089 /CDl8 699 were found to be at least as effective in blocking neutrophil adhesion to rIL-1-induced endothelium as monoclonal antibodies directed against CDllb or CD18.
  • CDllb 1089 /CD18 699 was more effective than 44a mAb (an anti-CDllb mAb) in inhibiting adhesion to rIL-1-activated endothelium and comparable to inhibition seen using TS18 mAb (an anti-CD18 mAb) , suggesting the presence of multi •ple functi•onal si•tes on CDllb1089 and/or the possibility that CD18 (like other ⁇ integrins) contains a recognition site(s) for interacting with ligand(s) expressed on endothelium.
  • Truncated CDllb and CD18 PAT-X plasmid containing the partial CD18 cDNA clone J19 was linearized with Hindlll or digested with Ncol (to generate a 1331 bp gap) . These two plasmids were mixed with an excess of the synthetic and 5'-end phosphorylated 18-mer (5'-aggccccTaGatcgccgc) containing desired nucleotide mutations (caps) . The mixture was denatured by boiling and renatured by stepwise cooling.
  • Reannealed DNA (containing single-stranded region to which the mutant 18-mer is hybridized) was primer extended to fill the gap, and used to transform E. coli strain BMH 71-18 mutL. Arnaout et al., J. Clin . Invest . 85:977 (1990). Plasmids containing the mutation were 22 -
  • Monoclonal antibodies directed against CDll or CD18 can be used to antagonize CDll/CD18-mediated immune response.
  • Useful monoclonal antibodies can be generated by using a peptide of the invention as an immunogen.
  • monoclonal antibodies can be raised against the A domain of CDllb, CDlla or CDllc.
  • Anti-CDllb monoclonal antibodies which inhibit iC3b binding mAb 903 , neutrophil adhesive interactions, e.g., aggregation and chemotaxis, (mAb 904), or both activities (mAb44a) have been identified.
  • Other monoclonal antibodies OKM-1, which inhibits fibrinogen binding, and OKM9 have also been mapped to this region. Dana et al., J. Immunol . 137:3259 (1986). These monoclonal antibodies recognize epitopes in the A domain of CDllb. Dana et al., JASON 1:549 (1990).
  • human monoclonal antibodies can be produced.
  • Human monoclonal antibodies can be isolated from a combinatorial library produced by the method of Huse et al. (Science , 246:1275, 1988) .
  • the library can be generated in vivo by immunizing nude or SCID mice whose immune system has been reconstituted with human peripheral blood lymphocytes or spleen cells or in vitro by immunizing human peripheral blood lymphocytes or spleen cells.
  • the immunogen can be any CDllb or CD18 peptide. Similar techniques are described by Duchosal et al., J. Exp. Med. 92:985 (1990) and Mullinax et al., Proc. Nat'l. Acad. USA 87:8095 (1990) .
  • Peptides derived from the A domain of CDlla, CDllb, or CDllc are preferred immunogens. These peptides can be produced in E. coli transformed by a plasmid encoding all or part of the A domain.
  • a CD18 peptide can also be used as an immunogen.
  • Three anti-CD18 mAbs with anti-inflammatory properties (TS18, 10F12, 60.3) have been identified. Binding each of these antibodies to CD18 can be abrogated by a specific point mutation within a particular region of CD18 (Asp 128 to Asn 361 of Fig. 8) (SEQ ID No.: 45). Peptide corresponding to this region can be produced in E. coli using a plasmid encoding the A domain.
  • CDllb (or CDllc) peptides, heterodimers, and monoclonal antibodies such as those described above can be tested in vitro for inhibition in one of the following five assays: iC3b binding, inhibition of phagocytosis, inhibition of monocyte/granulocyte adhesion to endothelium, inhibition of chemotaxis, or inhibition of cell-cell aggregation. Alternatively, they may be tested 24 -
  • neutrophils possess a number of specific cell surface receptors that promote this interaction, such as a receptor which binds to complement C3 (iC3b) , e.g. the CDllb/CD18 receptor.
  • a receptor which binds to complement C3 (iC3b) e.g. the CDllb/CD18 receptor.
  • Human neutrophilic polymorphonuclear granulocytes can be isolated from EDTA-anticoagulated blood on Ficoll-Hypaque gradients. Boyu , Scand. J. Clin . Invest . (Suppl.) 21:77 (1968) modified as described by Dana et al., J. Clin . Invest .
  • Phagocytes can be prepared by incubating the mononuclear cell fraction (obtained from Ficoll-Hypaque centrifugation) on plastic petri dishes. Todd et al., J . Immunol . 126:1435 (1981). Peptides of the invention can be tested for their ability to inhibit iC3b mediated binding of granulocytes to sheep erythrocytes as described in Dana et al. supra , 1984; and Arnaout et al., supra, 1985. Inhibition of Phagocytosis Phagocytosis is an important biological function resulting in clearing of damaged tissue from the body, and in elimination of foreign particles (bacteria, fungi) . An in vitro test for inhibition of phagocytosis - 25 -
  • Granulocytes/monocytes must cross vascular endothelium during their egress from blood to extravascular tissues.
  • Studies of leukocyte kinetics in animals indicate that acute inflammatory reactions may be marked by a massive increase in transendothelial monocyte/granulocyte traffic.
  • perivascular monocytes accumulate in skin windows more slowly than neutrophils, but later become the predominant cell type.
  • monocytes leaving the circulation can rapidly acquire the morphology of resident tissue macrophages—in some cases within a few hours of their departure from plasma.
  • vascular endothelium may be considered an important substrate with which monocytes/granulocytes must interact during adherence, diapedesis, and differentiation.
  • An in vitro assay for monocyte/granulocyte interaction with the vessel wall consists of binding radiolabeled or fluorescein monocyte/granulocyte preparations to cultured vascular endothelium, as described in Arnaout et al., J. Cell Physiol . 137:305 (1988). Mentzer et al., J . Cell Physiol . 125:285 (1986) describes a lymphocyte adhesion assay. These endothelial adhesion assays are appropriate for CDlla, CDllb or CDllc peptides, heterodimers and monoclonal antibodies when the endothelial cells are pre-activated. When the granulocytes/monocytes (or leukocytes) are pre-activated, these assays are suitable for CDllb peptides, heterodimers or monoclonal antibodies. Inhibition of Chemotaxis.
  • a peptide of the invention can be tested for its ability to inhibit chemotaxis, as described in Dana et al., (1986), supra.
  • a granulocyte aggregation assay can be performed as described by. Arnaout et al., New Engl . J. Med. 306:693 (1982). Aggregation can be induced by zymosan-activated autologous serum or with chemotactic peptides, e.g. FMLP. Aggregation can then be recorded as incremental change in light transmission [ ⁇ T] using a platelet aggregometer. The results can be confirmed by phase microscopy. Assays for CDlla peptides, heterodimers and monoclonal antibodies
  • CDlla peptides, heterodimers and monoclonal antibodies can be tested using the inhibition of endothelial adhesion assay (described above) or a lymphocyte proliferation assay.
  • endothelial adhesion assay described above
  • a lymphocyte proliferation assay Arnaout et al., J. Clin . Invest . 74:1291 (1984) describes an assay for inhibition of antigen/mitogen induced lymphocyte proliferation.
  • Damage to tissues injured by ischemia- reperfussion can be minimized by administering to an animal an inhibitor of CD11/CD18 mediated immune response.
  • a peptide of the invention may be tested for in vivo effectiveness using animals, e.g., dogs, which have been induced to undergo myocardial infarction. See, e.g. Simpson et al. supra .
  • the peptide or monoclonal antibody can be administered intravenously in saline solution generally - 27 -
  • the peptide can be administered in combination with other drugs, for example, in combination with, or within six hours to three days after a clot dissolving agent, e.g., tissue plasminogen activator (TPA) , Activase, or Streptokinase.
  • a clot dissolving agent e.g., tissue plasminogen activator (TPA) , Activase, or Streptokinase.
  • GGT GCC CCA CTG TTC TAT GGG GAG CAG AGA GGA GGC CGG GTG TTT 1579 ACT CTG GAG CTG GTG GGA GAG ATC GAG GCC TCT TCC ATG TTC AGC 324
  • AGC GGC ATC GGG GGG CTG CTG CTG CTG CTG CTC ATT TNC ATA GTG 342
  • CACACTTCCA CCACCCTGCA CTACTCCCTC AAAGCACACG TCATGTTTCT 4404
  • CTGCTAACTT GGAGCCCCAG TGCCAAGCAC AGTGCCTGCA TGTATTTATC 51
  • aat aac ttt gag get ctg aag ace att eag aac eag ctt egg gag 1064
  • ctg act gta gga gcc eag ggg cac gtg ctg ctg etc agg tec eag 1919
  • gac tct aag get tec ctt gga aac aaa ctg etc etc aag gcc aat 2774
  • CAC AAT GGG GGC CAG AAG CAG CTG TCC CCA CAA AAA GTG ACG CTT 3
  • ATC CAG CCC ATC TTC GCG GTG ACC AGT AGG ATG GTG AAG ACC TAC 990
  • GAG AAA CTC ACC GAG ATC ATC CCC AAG TCA GCC GTG GGG GAG CTG 1035

Abstract

The invention features human CD11 recombinant or synthetic peptide capable of inhibiting a CD11/CD18-mediated immune response, a purified DNA encoding a human CD11b peptide, soluble heterodimeric molecules composed of a CD11 peptide and a CD18 peptide, and a method of controlling any phagocyte-mediated tissue damage such as that associated with reduced perfusion of heart tissue during acute cardiac insufficiency.

Description

CONTROLLING CELLULAR IMMUNE/INFLAMMATORY RESPONSES WITH β2 INTEGRINS
Background of the Invention This invention, at least in part, was funded by a grant from the United States Government and the Government has certain rights in the invention.
This application is a continuation-in-part of my earlier, co-pending application USSN 539,842, filed June 18, 1990, which is in turn a continuation-in-part of my earlier application USSN 212,573, filed June 28, 1988, now abandoned, both of which are hereby incorporated by reference.
This invention relates to controlling cellular immune/inflammatory responses, particularly phagocyte-mediated tissue injury and inflammation.
Circulating phagocytic white blood cells are an important component of the cellular acute inflammatory response. It is believed that a number of important biological functions such as chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endothelium, phagocytosis, antibody-dependent cellular cytotoxicity, superoxide, and lysosomal enzyme release are mediated by a family of leukocyte surface glycoprotein adhesion receptors known as β2 integrins or the CD11/CD18 complex. Arnaout et al., Blood 75:1037 (1990) . Inherited deficiency of CD11/CD18 impairs leukocyte adhesion-dependent inflammatory functions and predisposes to life-threatening bacterial infections. Dana et al., J. Clin . Invest . 73:153 (1983); Arnaout et al., J. Clin . Invest . 74:1291 (1984).
The CD11/CD18 family consists of three heterodimeric surface glycoproteins, each with a distinct subunit (CDlla, CDllb or CDllc) non-covalently associated with a common β subunit (CD18) . The divalent cations Ca+ and Mg are essential in the stabilization and function of the β (CD11/CD18) complex. The β2 integrins are expressed only on leukocytes. While CDlla/CD18 (also known as LFA-1, TA- 1) is expressed on all leukocytes, CDllb/CDlδ and CD11C/CD18 (also known as LeuM5 or pl50,95) are expressed primarily on monocytes, poly orphonuclear leukocytes, macrophages and natural killer cells CDllc/CD18 is also expressed on certain lymphocytes. Arnaout, Blood 75:1037 (1990) .
CDlla/CD18, and not CDllb/CD18 or CDllc/CD18, is expressed on B- and T-lymphocytes; accordingly CDlla/CD18 plays a role in itogen-, antigen-, and alloantigen- induced proliferation, T-cell-mediated cytotoxicity, lymphocyte aggregation, and Ig production. In contrast, all three CD11/CD18 molecules are important for monocyte/macrophage and granulocyte adhesion-dependent functions.
It is believed that CDllb/CD18 and CDllc/CD18 mediate enhanced adhesiveness of activated phagocytes through quantitative and qualitative changes in these proteins on the surface of activated cells. For example, in granulocytes, these proteins are translocated from intracellular storage pools present in secondary and tertiary granules. Arnaout et al., J . Clin . Invest . 74:1291 (1984); Arnaout et al., New Eng . J. Med . 312:457 (1985); Todd et al., J . Clin . Invest . 74:1280 (1984). CDllb/CD18 is also known as complement receptor type 3 (CR3) , Mol, Mac-1 or MAM. See, Arnaout et al., J. Clin . Invest . 72:171 (1983) ,- and references cited therein; Dana et al., J. Immunol . 137:3259 (1986); Wallis et al., J. Immunol . 135:2323 (1985); Arnaout et al., New Eng . J. Med . 312:457 (1985); Dana et al., J. Clin . Invest . 73:153 (1984); and Beatty et al., J. Immunol . 131:2913 (1983). Like all β2 integrins, CDllb/CD18 consists of two non-covalently associated subunits.
Kishimoto et al., Cell 48:681 (1987); Law et al., EMBO J. 6:915 (1987); Arnaout et al. J. Clin . Invest . 72:171 (1983) . The α subunit of CDllb/CD18 has an apparent molecular mass of 155-165 kD and associates non- covalently with a β subunit, CD18, of apparent molecular mass 95 kD. Todd et al., Hybridoma 1:329 (1982).
Monoclonal antibodies have been used to identify at least two distinct functional domains of CDllb/CD18, one mediating homotypic and heterotypic adhesion and the other mediating binding to the complement C3 fragment (iC3b) , the major C3 opsonin in vivo. Dana et al., J. Immunol . 137:3259 (1986).
Law et al., EMBO J. 6:915 (1987) and Kishimoto et al., Cell 48:681 (1987) disclose the nucleotide sequence of human CD18. Arnaout et al., J. Cell Biol . 106:2153 (1988); Corbi et al., J. Biol . Chem . 263:12403 (1988); and Hickstein et al., Proc . Nat ' l . Acad . Sci . USA 86:275 (1989) disclose the nucleotide sequence of human CDllb. Larson et al., J. Cell . Biol . 108:703 (1989) disclose the nucleotide sequence of CDlla. Corbi et al., EMBO J.
6:4023 (1987) disclose the nucleotide sequence of CDllc.
Cosgrove et al. (Proc . Nat ' l . Acad . Sci . USA 83:752, 1986) report a human genomic clone which produces "a molecule(s) " reactive with monoclonal antibodies to CDllb.
Sastre et al. (Proc . Nat ' l . Acad . Sci . USA 83:5644, 1986) report a mouse genomic clone coding for an amino-terminal partial exon of murine CDllb. Pytela et al. , EMBO J. 7:1371 (1988) report a cDNA sequence of murine CDllb.
Simpson et al., J. Clin . Invest . 81:624 (1988) disclose that a monoclonal antibody (904) directed to an adhesion-promoting domain of CDllb (Dana et al., J.
Immunol . 137:3259, 1986) reduces the extent of cardiac damage in dogs associated with myocardial infarction, presumably by limiting reperfusion injury. Vedder et al. (J". Clin . Invest . 81:939, 1988) similarly found that a monoclonal antibody directed against CD18 subunit of CDllb/CD18 reduced organ injury and improved survival from hemorrhagic shock in rabbits. In animal models, anti-CDll/CD18 antibodies have been shown to have protective effects in shock, frostbite, burns, cerebral edema, onset of diabetes mellitus (Hutchings et al..
Nature 348:639, 1990) and transplant rejection. Reviewed in Carlos et al., Immunol . Rev. 114:5 (1990).
Summary of the Invention The peptides and heterodimeric proteins of the invention are capable of antagonizing CD11/CD18 (β2 integrin) mediated immune response. CD11/CD18 mediated immune responses which it may be desirable to block include acute inflammatory functions mediated by neutrophils. The molecules of the invention are useful for treatment of ischemia reperfusion injury (e.g., in the heart, brain, skin, liver or gastrointestinal tract) , burns, frostbite, acute arthritis, asthema, and adult respiratory distress syndrome. Peptides and heterodimeric proteins of the invention may also be useful for blocking intra-islet infiltration of macrophages associated with-insulin-dependent diabetes mellitus. The invention features a purified peptide which includes at least one extracellular region of a β2 integrin subunit capable of inhibiting a CD11/CD18 mediated immune response, the peptide lacks the transmembrane and cytoplasmic portions of the β2 integrin subunit. In a preferred embodiment the β2 integrin subunit is a human β2 integrin subunit; more preferably the β2 integrin subunit is CDlla, CDllb, CDllc or CD18; most preferably the β2 integrin subunit is CDllb. Preferably, the peptide includes all or part of the A domain of CDllb. More preferably the peptide includes one of the following sequences: DIAFLIDGS (SEQ ID NO: 32); FRRMKEFVS (SEQ ID NO: 33); FKILWITDGE (SEQ ID NO: 34); VIRYVIGVGDA (SEQ ID NO: 35); DGEKFGDPLG (SEQ ID NO: 36); YEDVIPEADR (SEQ ID NO: 37); DGEKFGDPLGYEDVIPEADR
(SEQ ID NO: 17); NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO: 50); DGEKF (SEQ ID NO: 51). In preferred embodiments, the peptide includes the amino acid sequence YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 38); the peptide includes the amino acid sequence KSTRDRLR (SEQ ID NO: 15) . Preferably, the peptide includes one of the following amino acid sequences: AYFGASLCSVDVDSNGSTDLVLIGAP (SEQ ID NO: 1) ; GRFGAALTVLGDVNGDKLTDVAIGAP (SEQ ID NO: 2); QYFGQSLSGGQDLTMDGLVDLTVGAQ (SEQ ID NO: 3); YEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 4); DIAFLIDGSGSIIPHDFRRMK (SEQ ID NO: 5) ; RRMKEFVSTVMEQLKKSKTLF (SEQ ID NO: 6) ; SLMQYSEEFRIHFTFKEFQNN (SEQ ID NO: 7) ; PNPRSLVKPITQLLGRTHTATGIRK (SEQ ID NO: 8); RKWRELFNITNGARKNAFK (SEQ ID NO: 9); FKILWITDGEKFGDPLGYEDVIPEADR (SEQ ID NO: 10); REGVIRYVIGVGDAFRSEKSR (SEQ ID NO: 11); QELNTIASKPPRDHVFQVNNFE (SEQ ID NO: 12);
ALKTIQNQLREKIFAIEGT (SEQ ID NO: 13); QTGSSSSFEHEMSQE (SEQ ID NO: 14); FRSEKSRQELNTIASKPPRDHV (SEQ ID NO: 16); KEFQNNPNPRSL (SEQ ID NO: 18); GTQTGSSSSFEHEMSQEG (SEQ ID NO: 19); SNLRQQPQKFPEALRGCPQEDSD (SEQ ID NO: 20);
RQNTGMWESNANVKGT (SEQ ID NO: 21) ; TSGSGISPSHSQRIA (SEQ ID NO: 22); NQRGSLYQCDYSTGSCEPIR (SEQ ID NO: 23); PRGRARWQC (SEQ ID NO: 24); KLSPRLQYFGQSLSGGQDLT (SEQ ID NO: 25); QKSTRDRLREGQ (SEQ ID NO: 26) ; SGRPHSRAVFNETKNSTRRQTQ (SEQ ID NO: 27); CETLKLQLPNCIEDPV (SEQ ID NO: 28);
FEKNCGNDNICQDDL (SEQ ID NO: 29) ; VRNDGEDSYRTQ (SEQ ID NO: 30) ; SYRKVSTLQNQRSQRS (SEQ ID NO: 31) .
Preferably, the peptide includes one or more metal binding domains of CDllb. More preferably, the metal binding domains encompass amino acids 358-412,
426-483, 487-553, and 554-614 of CDllb. Most preferably, the peptide includes one of the following sequences: DVDSNGSTD (SEQ ID NO: 46); DVNGDKLTD (SEQ ID NO: 47); DLTMDGLVD (SEQ ID NO: 48); DSDMNDAYL (SEQ ID NO: 49). In a preferred embodiment, the peptides are soluble under physiological conditions.
In a related aspect, the invention features a heterodimer which includes a first peptide and a second peptide; the first peptide includes at least one extracellular region of a CDll subunit and lacks the transmembrane and cytoplasmic portions of the CDll subunit; the second peptide comprising at least one extracellular region of a CD18 subunit and lacks the transmembrane and cytoplasmic portions of the CD18 subunit; the first and second peptides are associated to form the heterodimer; and the heterodimer is capable of inhibiting a CD11/CD18 mediated immune response. In preferred embodiments, the CDll subunit is: CDlla; CDllb; CDllc. In a more preferred embodiment, the heterodimer is CDllb1089/CD18699.
In another aspect, the invention features a method of controlling phagocyte-mediated tissue damage to a human patient. The method includes administering a therapeutic composition to a patient; the therapeutic composition includes a physiologically acceptable carrier and a peptide or a heterodimer of the invention. More preferably, the method is used to control phagocyte- mediated tissue damage due to ischemia-reperfussion.
Most preferably, the method is used to control phagocyte- mediated tissue damage to the heart muscle associated with reduced perfusion of heart tissue during acute cardiac insufficiency. In another aspect, the invention features a method of producing a recombinant β2 integrin heterodimer. The method includes the steps of: (a) providing a recombinant cell encoding a CDll peptide lacking both the transmembrane domain and the cytoplasmic domain and a CD18 peptide lacking both the transmembrane domain and the cytoplasmic domain; (b) culturing the recombinant cell; and (c) isolating the heterodimer from the culture supernatant. More preferably, the method is used to produce a soluble recombinant β2 integrin heterodimer. In preferred embodiments, the CDll peptide of the heterodimer is a CDlla peptide; is a CDllb peptide; is a CDllc peptide.
In another aspect, the invention features a monoclonal antibody which is raised to a peptide or a heterodimer of the invention and which is capable of inhibiting a CD11/CD18 mediated immune response.
In another aspect, the features a human CDllb recombinant peptide. "β2 integrins" include all leukocyte adhesion molecules which include a CD18 subunit. By the "A domain of CDllb" is meant the amino acid sequence corresponding to the sequence of CDllb from Cys 128 to Glu321 or an am o acid sequence produced by introducing one or more conservative amino acid substitutions in an amino acid sequence corresponding to the sequence of CDllb from Cys128 to Glu321. "CDll/CD18-mediated immune response" includes those CDll/CD18-related functions mentioned above: chemotaxis, immune adherence (homotypic cell adhesion or aggregation) , adhesion to endotheliu , phagocytosis, antibody-dependent or -independent cellular cytotoxicity, and superoxide and lysosomal enzyme release. Inhibition of these immune functions can be determined by one or more of the following inhibition assays as described in greater detail below: iC3b binding, cell-cell aggregation, phagocytosis, adhesion to endothelium, and chemotaxis. As used herein, a human CDllb recombinant peptide is a chain of amino acids derived from recombinant CDllb-encoding cDNA, or the corresponding synthetic DNA. "CD111089/CD18699" is a heterodimer which comprises amino acids 1-1089 of human CDll and amino acids 1-699 of CD18.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawings will first briefly be described. Drawings
Figure 1 is the cDNA sequence and deduced amino acid sequence of the open reading frame of human CDllb from Arnaout et al., J. Cell . Biol . 106:2153 (1988).
Figure 2 is a representation of the results of an immunoprecipitation assay.
Figure 3 is a representation of the results of an immunoprecipitation assay.
Figure 4 is a representation of the results of an immunoprecipitation assay.
Figure 5 is a graph of the effect of various proteins and antibodies on neutrophil adhesion to endothelium.
Figure 6 is the cDNA sequence and deduced amino acid sequence of human CDlla from Larson et al., J. Cell . Biol . 108:703 (1989).
Figure 7 is the cDNA sequence and deduced amino acid sequence of human CDllc from Corbi et al., EMBO J. 6:4023 (1987).
Figure 8 is the cDNA sequence of human CD18 from Law et al., EMBO J. 6:915 (1987). Peptides As described in greater detail elsewhere, each member of the β2 integrin family is a heterodimer consisting of two subunits: a CDll subunit (with at least three variants designated CDlla, CDllb, and CDllc) and a CD18 subunit. Each subunit includes a transmembrane anchor which connects a cytoplasmic segment to an extracellular segment. The two subunits interact to form a functional heterodimer. As described in greater detail below, the extracellular segments of the β2 integrin subunits contain various functional domains which are the focus of the invention.
Without wishing to bind myself to a particular theory, it appears that the peptides of the invention antagonize CDll/CD18-mediated immune responses by competitively inhibiting binding of leukocytes bearing a member of the β2 integrin family to the respective binding partners of that family. Specifically, the peptides of the invention include an immune-response inhibiting extracellular segment of any one of the β2 integrin subunits —CDlla, CDllb, CDllc, CD18— or a heterodimer composed of a portion of an α (CDlla, CDllb, or CDllc) subunit together with a portion of a β subunit (CD18) . Candidate β2 integrin subunits can be evaluated for their ability to antagonize CDll/CD18-mediated immune responses by any of several techniques. For example, subunits may be tested for their ability to interfere with neutrophil adhesion to endothelial cells using an assay described in detail below. Specific regions of the ,92 integrin subunits can be evaluated in a similar manner. Any extracellular region of a β2 integrin subunit may be screened for its ability to interfere with CD11/CD18 mediated immune response. Regions of CDll whose sequences are conserved between two or more subunits are preferred candidates for antagonizing
CD11/CD18 - mediated immune response. For example, the A domain (corresponding to Cys128 to Glu321 of CDllb) is conserved between CDlla, CDllb, and CDllc. The A domain is 64% identical in CDllb and CDllc and 36% homologous between these two subunits and CDlla. This domain is also homologous to a conserved domain in other proteins involved in adhesive interactions including von Willebrand's factor, cartilage matrix protein, VLA2, and - li ¬
the complement C3b/C4b - binding proteins C2 and factor B. The extracellular portions of CDlla, CDllb and CDllc include seven homologous tandem repeats of approximately 60 amino acids. These repeats are also conserved in the α subunits of other integrin subfamilies (e.g., fibronectin receptor). Arnaout et al.. Blood 75:1037 (1990) .
Regions of CD18 which are conserved among β intergrin subunits (i.e., the β subunits of βl , β2 and β3 integrins) are also good candidates for regions capable of interfering with CD11/CD18 - mediated immune response. For example, CD18 has four tandem repeats of an eight- cysteine motif. This cysteine-rich region is conserved among β subunits. Just amino terminal to this cysteine rich region is another conserved region, 247 amino acids long, which is conserved in several integrin β subunits.
Described in detail below are techniques for generating CDllb peptides and heterodimers. The same techniques may be used to generate CDlla, CDllc, and CD18 peptides as well as CDlla/CD18 and CDllc/CD18 heterodimers. Fig. 6 depicts the cDNA sequence of human CDlla (SEQ ID NO: 39) ; Fig. 7 depicts the cDNA sequence of human CDllc (SEQ ID NO: ) ; Fig. 8 depicts the cDNA sequence of CD18 (SEQ ID NO: 41) . DNA molecules encoding all or part of CDlla,
CDllb, CDllc or CD18 can be obtained by means of polymerase chain reaction amplification. In this technique two short DNA primers are used to generate multiple copies of a DNA fragment of interest from cells known to harbor the RNA of produced by the gene of interest. This technique is described in detail by Frohman et al., Proc. Nat ' l Acad Sci . USA 85:8998 (1988). Polymerase chain reaction methods are generally described by Mullis et al. (U.S. Patent Nos. 4,683,195 and 4,683,202) .
For example, to clone a portion of CDlla, the known sequence of CDlla is used to design two DNA primers which will hybridize to opposite strands outside (or just within) the region of interest. The primers must be oriented so that when they are extended by DNA polymerase, extension proceeds into the region of interest. To generate the CDlla DNA, poyA RNA is isolated from cells expressing CDlla. A first primer and reverse transcriptase are used to generate a cDNA form the mRNA. A seond primer is added; and Tag DNA polymerase is used to amplify the cDNA genrated in the previous step. Alternatively, the known sequences of CDlla, CDllb, CDllc and CD18 can be used to design highly specific probes for identifying cDNA clones harboring the DNA of interest. A cDNA library suitable for isolation of CDlla, CDllb, and CDllc DNA can be generated using phorbol ester-induced HL-60 cells (ATCC Accession No. CCL 240) as described by Corbi et al. (EMBO J. 6:4023, 1987) and Arnaout et al., Proc . Nat ' l Acad Sci . USA 85:2776, 1988) ; CD18 DNA can be isolated from a library generated using U937 cells (ATCC Accession No. CRL 1593) as described by Law et al. (EMBO J. 6:915, 1987). These cell lines are also suitable for generating cDNA by polymerase chain reaction amplification of mRNA as sescribed above.
Heterodimers comprised of part of CDllc and CD18 can be produced as described below for CDllb/CD18 by changing a codon amino terminal to the transmembrane region (e.g. Pro1086) to a stop codon. Heterodimers comprised of part of CDlla can be produced by changing a codon amino terminal to the transmembrane region (e.g., - 13
Lys 10S7) to a stop codon. DNA encodi •ng the truncated CDll subunit is then introduced into cells along with DNA encoding a similarly truncated CD18 molecule (described below) . These cells are then used as a source of heterodimer.
Isolation of a Human CDllb cDNA clone.
A 378 base pair (bp) cDNA clone encoding guinea pig CDllb was used as a probe to isolate three additional cDNA clones from a human monocyte/lymphocyte cDNA library as described in Arnaout et al., Proc . Nat ' l . Acad . Sci . USA 85:2776 (1988); together these three clones contain the 3,048 nucleotide sequence encoding the CDllb gene shown in Fig. 1 (SEQ ID NO: 40). Arnaout et al., J. Cell . Biol . 106:2153 (1988). In order to express CDllb, a mammalian expression vector was constructed by assembling the above-described three cDNA clones. Appropriate restriction enzyme sites within the CDllb gene can be chosen to assemble the cDNA inserts so that they are in the same translation reading frame. Arnaout et al., J. Clin . Invest . 85:977 (1990). A suitable basic expression vector can be used as a vehicle for the 3,048 bp complete cDNA fragment encoding the human CDllb peptide; the recombinant cDNA can be expressed by transfection into, e.g., COS-l cells, according to conventional techniques, e.g., the techniques generally described by Aruffo et al., Proc. Nat ' l . Acad . Sci . USA 84:8573 (1987) or expressed in E. coli using standard techniques. Smith et al., Gene 67:31 (1988) . Isolation of CDllb Peptide from Mammalian Cells
The CDllb protein can be purified from the lysate of transfected COS-1 cells, " sing affinity chromatography and lentil-lectin Sepharose and available anti-CDllb monoclonal antibody as described by Pierce et al. (1986) supra and Arnaout et al., Meth . Enzymol . 150:602 (1987).
If the desired CDllb peptide is shorter than the entire protein, DNA encoding the desired peptide can be expressed in the same mammalian expression vector described above using the selected DNA fragment and the appropriate restriction enzyme site, as outlined above. The selected DNA fragment may be isolated according to conventional techniques from one of the CDllb cDNA clones or may be synthesized by standard polymerase chain reaction amplification, as described above. See also Saiki et al. , (Science 239:487, 1988). Characterization of the CDllb Polypeptide The coding sequence of the complete CDllb protein is preceded by a single translation initiation methionine. The translation product of the single open reading frame begins with a 16-amino acid hydrophobic peptide representing a leader sequence, followed by the NH2-terminal phenylalanine residue. The translation product also contained all eight tryptic peptides isolated from the purified antigen, the amino-terminal peptide, and an amino acid hydrophobic domain representing a potential transmembrane region, and a short 19-amino acid carboxy-terminal cytoplasmic domain
(Fig. 1 illustrates the amino acid sequence of CDllb; SEQ ID NO: 43) . The coding region of the 155-165 kD CDllb (1,136 amino acids) is eight amino acids shorter than the 130-150 kD alpha subunit of CDllc/CD18 (1,144 amino acids) . The cytoplasmic region of CDllb contains one serine residue that could serve as a potential phosphorylation site. The cytoplasmic region is also relatively rich in acidic residues and in proline (Fig. 1) . Since CDllb/CD18 is involved in the process of phagocytosis and is also targeted to intracellular storage pools, these residues are candidates for mediating these functions. The long extracytoplasmic amino-terminal region contains three or four metal-binding domains (outlined by broken lines in Fig. 1) that are similar to Ca2+-binding sites found in other integrins. Each metal binding site may be composed of two noncontiguous peptide segments and may be found in the four internal tandem repeats formed by amino acid residues 358-412, 426-483, 487-553, and 554-614. The portion of the extracytoplasmic domain between Tyr465 and Val is homologous to the fibronectin-like collagen binding domain and IL-2-receptor. The extracytoplasmic region also contains an additional unique 187-200 amino acid domai •n, the A domain, between Cys128 to Glu321, which is not present in the homologous (α) subunits of fibronectin, vitronectin, or platelet Ilb/IIIa receptors. This sequence is present in the highly homologous CDllc protein (α of pl50,95) with 64% of the amino acids identical and 34% representing conserved substitutions. Arnaout et al., J. Cell Biol . 106:2153, 1988; Arnaout et al. Blood 75:1037 (1990). It is known that both CDllb/CD18 and CDllc/CD18 have a binding site for complement fragment C3 and this unique region may be involved in C3 binding. This region of CDllb also has significant homology (17.1% identity and 52.9% conserved substitutions) to the collagen/heparin/platelet Gpl binding regions of the mature von Willebrand factor (domains A1-A3) . The A domain is also homologous to a region in CDlla. Larson et al., J. Cell Biol . 108:703 (1989) . The A domain is also referred to as the L domain or the I domain. Larson et al., supra (1988); Corbi et - 16 -
al., J . Biol . Chem . 263:12,403 (1988). CDllb Peptides
The following peptides can be used to inhibit CDllb/CD18 activity: a) peptides identical to the above-described A domain of CDllb, or a portion thereof, e.g., DIAFLIDGS (SEQ ID NO:32), FRRMKEFVS (SEQ ID N0:33), FKILWITDGE (SEQ ID NO:34), DGEKFGDPLGYEDVIPEADR (SEQ ID NO:17), or VIRYVIGVGDA SEQ ID NO:35); b) peptides identical to the above-described fibronectin-like collagen binding domain, or a portion thereof, e.g.,
YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO:38); c) peptides identical to one or more of the four metal binding regions of CDllb, or a portion thereof, e.g., DVDSNGSTD (SEQ ID NO:46), DVNGDKLTD (SEQ ID NO:47), DLTMDGLVD (SEQ ID NO:48), DSDMNDAYL (SEQ ID NO:49); d) peptides substantially identical to the complete CDllb; or e) other CDllb domains, e.g. KSTRDRLR (SEQ ID NO:15).
Also of interest is a recombinant peptide which includes part of the A domain, e.g, NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO: 50) . The A domain binds iC3b, gelatin, and fibrinogen and binding is disrupted by EDTA. The A domain also binds both Ca2+ and Mg2+. This result unexpected since the A doamin lies outside of the region of CDllb previously predicted (Arnaout et al., J. Cell Biol . 106:2153, 1988; Corbi et al., J. Biol . Chem . 25:12403, 1988) to contain metal binding sites. Heterodimers
It is advantageous to administer the heterodimer formed by the CDllb and CD18 proteins. Expression of CDllb is described elsewhere in this application. Expression of CD18 has been'reported by others. Law et al. Embo, J. 6:915 (1987); Kishimoto et al. Cell 48:681 (1987) . The strategies described above or in those reports can be used to obtain CD18 to make such a heterodimer. Preferred heterodimers are soluble under physiological conditions. The heterodimer described below is generated by changing the codon for Leu1 in CDllb (SEQ ID NO: 40) to a stop codon and the codon for Asn700 of CD18 (SEQ ID NO: 41) to a stop codon. Other potentially soluble heterodimers can be generated by introducing a stop codon at positions amino terminal to those described below.
Generation of Soluble Heterodimers
A soluble form of a CDllb/CD18 heterodimer was produced in COS cells. To produce this molecule the codons for Leu1090 and Asn700 located at the predicted extracellular boundaries of CDllb and CD18 respectively, were replaced with in-frame translational stop codons using oligonucleotide-directed gapped-duplex mutagenesis of the wild-type cDNAs (described below) .
To determine if COS cells can express a soluble form of CDllb/CD18, COS cells were co-transfected with cDNA encoding the truncated forms of CDllb (CDllb1089) and CD18 (CDll 699) . Secreted proteins were analyzed by immunoprecipitation and SDS-PAGE. The results of this analysis are presented in Fig. 2. Briefly, COS cells were transfected as previously described (Arnaout et al., J . Clin . Invest . 85:977, 1990) . 7 x 106 transfected cells were labeled overnight with 0.1 mCi of 35S methionine, and the harvested supernatants were used for immunoprecipitation with NS1, a non-reactive monoclonal antibody (mAb) (lane 1) ; 44a, an anti-CDllb mAb (lane 2);_or TS18, an anti-CD18 mAb (lane 3) . Immunoprecipitation and antibodies as described by Arnaout et al., J . Cell . Physiol . 137:305 (1988); Trowbridge et al., J. Exp . Med. 154:1517 (1981); and Sanchez-Madrid et al., J. Exp. Med. 158:1785 (1983).
As shown in Fig. 2, both CDllb1089 and CD18699 were immunoprecipitated from supernatants of cells transfected with DNA encoding the truncated subunits.
The secreted CDllb 1089 had an apparent molecular weight of gqg
149 kD; the secreted CD18 had an apparent molecular weight of 84 kD (compared to 155 kD and 94 kD respectively for the wild-type subunits) . Arnaout et al.. New Engl . J. Med . 312:457 (1985); Dierner et al., J. Immunol . 135:537 (1985); Arnaout et al., J. Clin . Invest . 72:171 (1983); Klebanoff et al., J. Immunol . 134:1153 (1985) . That mAbs directed against either the CDllb or CD18 immunoprecipitated both truncated forms, indicates that the secreted subunits are expressed as an
CDllb 1089/CD18699 complex and that neither the cytoplasmic nor the transmembrane region of the subunits are necessary for heterodimer formation. These mAbs did not precipitate receptor subunits from the supernatants of mock-transfected cells. Arrowheads at left indicate the positions of molecular weight size markers: myosin (200kD) , phosphorylase b (92.5 kD) , bovine serum albumin (69 kD) , and ovalbumin (46 kD) . Arrows at right indicate the expected positions of CDllb1089 and CD18699. CDllb1089/CD18699 was next tested for its ability to bind iC3b (the receptor bound by wild-type CDllb/CD18) . Briefly, COS cells were transfected CDllb1089 and CD18699 cDNA as described above. Cells were labeled with 35S-methionine as described by Dana et al., J. Clin . Invest . 79:1010 (1987). Supernatants from both co-transfected COS cells (7 _x 10 cells) and mock- transfected COS cells (7 x 106 cells) were concentrated to one ml using collodion bags (10,000 MW cut off). 100 μl of the concentrated supernatant were used for immunoprecipitation, and the rest of the supernatant was incubated with C3b-sepharose or iC3b-sepharose. C3b- sepharose and iC3b-sepharose was washed, eluted with 0.4 M NaCl and the eluted proteins were analyzed by SDS-PAGE and autoradiography. Binding of wild-type, membrane- bound CDllb/CD18 to iC3b-sepharose or C3b-sepharose was performed as described by Arnaout et al., (In Methods in Enzymology, DiSabato, Ed., Acad. Press Inc., Fl., 1987) using the detergent soluble fracti •on from 1 x 108 1 ~-2"5j_- surface-labelled neutrophils.
Fig. 3 illustrates the results of SDS-PAGE analysis of neutrophil-derived 125I-surface-labeled glycoproteins eluted from C3b-sepharose and iC3b- sepharose. Eluants from C3b-sepharose (lane a) contained complement receptor type 1 (250kD) and the C3-binding regulatory protein gp45/70 (45-70 kD) . Eluants from iC3b-sepharose (lane b) contained two additional proteins at 155 kD, 94 kD, representing wild-type CDllb and CD18. CDllb/CD18 was immunoprecipitated with 44a mAb (an anti- CDllb mAb) from material eluted from iC3b-sepharose (lane d) , but not from material eluted from C3b-sepharose (lane c) , confirming previous results. Malhorta et al., Eur. J. Immunol . 16:177, (1986). The arrowheads at right indicate the positions of molecular weight standards: yosin (200 kD) , phosphorylase b (92.5 kD) , and bovine serum albumin (69 kD) . The arrows at left indicate the expected position of CR1, CDllb, CD18 and gp45/70.
Fig. 4 shows.the results of SDS-PAGE analysis of CDllb1089/CD18699 heterodimer binding to iC3b. An anti-
CDllb mAb (44a) was used to immunoprecipitate proteins from culture supernatants of mock-transfected COS cells (lane a) , and from COS cells co-transfected with CDllb - 20 -
and CD18 cDNAs (lane b) . No specific radiolabeled material was present in eluant of iC3b-sepharose exposed to culture supernatant of mock-transfected COS cells (lane c) . CDllb1089/CD18699 was eluted from iC3b- sepharose (lane d) , but not from C3b-sepharose (lane e) exposed to culture supernatant of co-transfected cells. Arrowheads at right indicate the positions of molecular weight standard standards (as in Fig. 2) . Arrows at left indicate the expected positions of CDllb1089 and CD18699. Similar results were seen with supernatants from two other transfections.
The ability of CDllb1089/CD18699 to inhibit binding of human neutrophils to inflamed endothelium was examined and compared to the inhibition induced by anti- CDllb mAb and anti-CD18 mAb. Adherence of purified human neutrophils to confluent monolayers of human umbilical vein endothelial cells (HUVE) pre-treated with recombinant IL-1 (10 units/ml for 4 hours at 37°C) was measured as described by Arnaout et al.,(«7. Cell . Physiol . 137:305, 1988) with the following modifications. Neutrophils were labeled with carboxyfluorescein (CF, Molecular Probes, Eugene, OR) by incubating 4 x 106 cells with 30 μg of CF in one ml of Tris-buffered saline for 10 minutes on ice, followed by three washes. HUVE were pre- incubated for 10 minutes at 37°C with supernatants of COS cells co-transfected with CDllb1089 and CD18699 cDNA supernatants, or for 5 minutes at room temperature with the non-reactive monoclonal antibody NS1, 44a (anti- CDllb) or TS18 (anti-CD18) ascites (1:100 dilution). Labeled neutrophils were then added and incubation was continued for an additional 10 minutes. The plates HUVE were washed twice, and adherent neutrophils were harvested by washing with 0.1% SDS and 0.1N NaOH. - 21 -
Relative numbers of neutrophils were measured (at Exc. , 490 nm; Em, 300nm) using a Fluorometer (SLM 8000, SLM Aminco, Urbana, IL) . All assays were done in triplicate. Labels along the horizontal axis indicate the molecule added to HUVE. 'Buffer1 indicates that no antibodies were added. 'Sham1 indicates that supernatant from mock transfected cells was added.
As shown in Fig. 5, culture supernatants containing CDllb1089/CDl8699 (approximately 10-50 ng/ml) were found to be at least as effective in blocking neutrophil adhesion to rIL-1-induced endothelium as monoclonal antibodies directed against CDllb or CD18. CDllb1089/CD18699 was more effective than 44a mAb (an anti-CDllb mAb) in inhibiting adhesion to rIL-1-activated endothelium and comparable to inhibition seen using TS18 mAb (an anti-CD18 mAb) , suggesting the presence of multi •ple functi•onal si•tes on CDllb1089 and/or the possibility that CD18 (like other β integrins) contains a recognition site(s) for interacting with ligand(s) expressed on endothelium.
Generation of Truncated CDllb and CD18 PAT-X plasmid containing the partial CD18 cDNA clone J19 (Law et al. supra , 1987) was linearized with Hindlll or digested with Ncol (to generate a 1331 bp gap) . These two plasmids were mixed with an excess of the synthetic and 5'-end phosphorylated 18-mer (5'-aggccccTaGatcgccgc) containing desired nucleotide mutations (caps) . The mixture was denatured by boiling and renatured by stepwise cooling. Reannealed DNA (containing single-stranded region to which the mutant 18-mer is hybridized) was primer extended to fill the gap, and used to transform E. coli strain BMH 71-18 mutL. Arnaout et al., J. Clin . Invest . 85:977 (1990). Plasmids containing the mutation were 22 -
identified by differential hybridization with 32P-labeled wild-type- or mutant 18-mers and DNA used to transform E. coli JM109. Positive colonies were identified following rehybridization, sequenced to verify the mutation, then used to replace the corresponding fragment in wild-type full length CD18 cDNA cloned in 7TH3M expression vector. Arnaout et al., J. Clin . Invest . 85:977 (1990). A stop codon was similarly introduced in CDllb. Blue Script (Stratagene, La Jolla, CA) plasmid vector containing the full coding region of membrane-bound CDllb was used. A mixture of Kpnl-linearized and gapped (by removing a Smal fragment, 1048 bp long) CDllb cDNAs were mixed with an excess of the synthetic mutant 18-mer (51- caaccccTAgccgctcat) . Mutant plasmid was produced and isolated as detailed above. Monoclonal Antibodies
Monoclonal antibodies directed against CDll or CD18 can be used to antagonize CDll/CD18-mediated immune response. Useful monoclonal antibodies can be generated by using a peptide of the invention as an immunogen. For example, monoclonal antibodies can be raised against the A domain of CDllb, CDlla or CDllc.
Anti-CDllb monoclonal antibodies which inhibit iC3b binding (mAb 903) , neutrophil adhesive interactions, e.g., aggregation and chemotaxis, (mAb 904), or both activities (mAb44a) have been identified. Other monoclonal antibodies (OKM-1, which inhibits fibrinogen binding, and OKM9) have also been mapped to this region. Dana et al., J. Immunol . 137:3259 (1986). These monoclonal antibodies recognize epitopes in the A domain of CDllb. Dana et al., JASON 1:549 (1990).
Additional useful monoclonal antibodies can be generated by standard techniques. Preferably, human monoclonal antibodies can be produced. Human monoclonal antibodies can be isolated from a combinatorial library produced by the method of Huse et al. (Science , 246:1275, 1988) . The library can be generated in vivo by immunizing nude or SCID mice whose immune system has been reconstituted with human peripheral blood lymphocytes or spleen cells or in vitro by immunizing human peripheral blood lymphocytes or spleen cells. The immunogen can be any CDllb or CD18 peptide. Similar techniques are described by Duchosal et al., J. Exp. Med. 92:985 (1990) and Mullinax et al., Proc. Nat'l. Acad. USA 87:8095 (1990) .
Peptides derived from the A domain of CDlla, CDllb, or CDllc are preferred immunogens. These peptides can be produced in E. coli transformed by a plasmid encoding all or part of the A domain.
A CD18 peptide can also be used as an immunogen. Three anti-CD18 mAbs with anti-inflammatory properties (TS18, 10F12, 60.3) have been identified. Binding each of these antibodies to CD18 can be abrogated by a specific point mutation within a particular region of CD18 (Asp128 to Asn361 of Fig. 8) (SEQ ID No.: 45). Peptide corresponding to this region can be produced in E. coli using a plasmid encoding the A domain. Assays for CDllb (or CDllc) peptides, heterodimers and monoclonal antibodies
CDllb (or CDllc) peptides, heterodimers, and monoclonal antibodies such as those described above, can be tested in vitro for inhibition in one of the following five assays: iC3b binding, inhibition of phagocytosis, inhibition of monocyte/granulocyte adhesion to endothelium, inhibition of chemotaxis, or inhibition of cell-cell aggregation. Alternatively, they may be tested 24 -
in vivo for controlling damage associated with reduced perfusion or immune injury of tissues, as a result of myocardial infarction, burns, frost bite, glomerulonephritis, asthma, adult respiratory distress syndrome, transplant rejection, onset of diabetes mellitus, ischemia, colitis, shock liver syndrome, and resuscitation from hemorrhagic shock. Inhibition of Granulocyte or Phagocyte Adhesion to iC3b-Coated Erythrocytes or Bacteria The antimicrobial activity of the neutrophil depends to a significant degree on the ability of this cell to establish a firm attachment to its target. For this purpose, neutrophils possess a number of specific cell surface receptors that promote this interaction, such as a receptor which binds to complement C3 (iC3b) , e.g. the CDllb/CD18 receptor. Human neutrophilic polymorphonuclear granulocytes can be isolated from EDTA-anticoagulated blood on Ficoll-Hypaque gradients. Boyu , Scand. J. Clin . Invest . (Suppl.) 21:77 (1968) modified as described by Dana et al., J. Clin . Invest .
73:153 (1984). Phagocytes can be prepared by incubating the mononuclear cell fraction (obtained from Ficoll-Hypaque centrifugation) on plastic petri dishes. Todd et al., J . Immunol . 126:1435 (1981). Peptides of the invention can be tested for their ability to inhibit iC3b mediated binding of granulocytes to sheep erythrocytes as described in Dana et al. supra , 1984; and Arnaout et al., supra, 1985. Inhibition of Phagocytosis Phagocytosis is an important biological function resulting in clearing of damaged tissue from the body, and in elimination of foreign particles (bacteria, fungi) . An in vitro test for inhibition of phagocytosis - 25 -
is described in Arnaout et al., New Eng . J . Med. 306:693
(1982).
Inhibition Adhesion to Endothelium.
Granulocytes/monocytes must cross vascular endothelium during their egress from blood to extravascular tissues. Studies of leukocyte kinetics in animals indicate that acute inflammatory reactions may be marked by a massive increase in transendothelial monocyte/granulocyte traffic. In many chronic inflammatory lesions, perivascular monocytes accumulate in skin windows more slowly than neutrophils, but later become the predominant cell type. In addition, monocytes leaving the circulation can rapidly acquire the morphology of resident tissue macrophages—in some cases within a few hours of their departure from plasma. Thus, vascular endothelium may be considered an important substrate with which monocytes/granulocytes must interact during adherence, diapedesis, and differentiation. An in vitro assay for monocyte/granulocyte interaction with the vessel wall consists of binding radiolabeled or fluorescein monocyte/granulocyte preparations to cultured vascular endothelium, as described in Arnaout et al., J. Cell Physiol . 137:305 (1988). Mentzer et al., J . Cell Physiol . 125:285 (1986) describes a lymphocyte adhesion assay. These endothelial adhesion assays are appropriate for CDlla, CDllb or CDllc peptides, heterodimers and monoclonal antibodies when the endothelial cells are pre-activated. When the granulocytes/monocytes (or leukocytes) are pre-activated, these assays are suitable for CDllb peptides, heterodimers or monoclonal antibodies. Inhibition of Chemotaxis.
The ability of cells of the immune system to - 26 -
migrate is essential to the cellular immune response that results in tissue inflammation. Therefore, a peptide of the invention can be tested for its ability to inhibit chemotaxis, as described in Dana et al., (1986), supra. Cell-Cell Aggregation
A granulocyte aggregation assay can be performed as described by. Arnaout et al., New Engl . J. Med. 306:693 (1982). Aggregation can be induced by zymosan-activated autologous serum or with chemotactic peptides, e.g. FMLP. Aggregation can then be recorded as incremental change in light transmission [ΔT] using a platelet aggregometer. The results can be confirmed by phase microscopy. Assays for CDlla peptides, heterodimers and monoclonal antibodies
CDlla peptides, heterodimers and monoclonal antibodies can be tested using the inhibition of endothelial adhesion assay (described above) or a lymphocyte proliferation assay. Arnaout et al., J. Clin . Invest . 74:1291 (1984) describes an assay for inhibition of antigen/mitogen induced lymphocyte proliferation. In Vivo Model for Testing Peptide
Damage to tissues injured by ischemia- reperfussion (e.g., heart tissue during myocardial infarction) can be minimized by administering to an animal an inhibitor of CD11/CD18 mediated immune response. A peptide of the invention may be tested for in vivo effectiveness using animals, e.g., dogs, which have been induced to undergo myocardial infarction. See, e.g. Simpson et al. supra . Use
The peptide or monoclonal antibody can be administered intravenously in saline solution generally - 27 -
on the order of mg quantities per 10 kilograms of body weight. The peptide can be administered in combination with other drugs, for example, in combination with, or within six hours to three days after a clot dissolving agent, e.g., tissue plasminogen activator (TPA) , Activase, or Streptokinase.
SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Arnaout, M. Amin (ϋ) TITLE OF INVENTION: Controlling Cellular Immune/Infla atory Responses with B2 Integrins
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(Vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/637,830
(B) FILING DATE: 01/04/91
(C) CLASSIFICATION: •
(Vii) PRIOR APPLICATION DATA:
Prior applications total, including application described below: 2
(A) APPLICATION NUMBER: 07/212,573
(B) FILING DATE : 28-06-88
(A) APPLICATION NUMBER: 07/539,842
(B) FILING DATE: 18-06-90
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: John W. Freeman
(B) REGISTRATION NUMBER: 29,066
(C) REFERENCE/DOCKET NUMBER: 108/33003
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE:
(B) TELEFAX:
(C) TELEX:
Figure imgf000031_0001
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Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp Val Asp Ser Asn
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Gly Arg Phe Gly Ala Ala Leu Thr Val Leu Gly Asp Val Asn Gly
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in Tyr Phe Gly Gin Ser Leu Ser Gly Gly Gin Asp Leu Thr Met
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Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro Leu Pro
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Asp lie Ala Phe Leu lie Asp Gly Ser Gly Ser lie lie Pro His
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Ser Leu Met Gin Tyr Ser Glu Glu Phe Arg lie His Phe Thr Phe
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Pro Asn Pro Arg Ser Leu Val Lys Pro lie Thr Gin Leu Leu Gly
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Arg Lys Val Val Arg Glu Leu Phe Asn He Thr Asn Gly Ala Arg
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Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys Phe Gly Asp
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Arg Glu Gly Val He Arg Tyr Val He Gly Val Gly Asp Ala Phe
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Gin Glu Leu Asn Thr He Ala Ser Lys Pro Pro Arg Asp His Val
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Gin Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gin Glu
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Lys Ser Thr Arg Asp Arg Leu Arg
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Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu Asn Thr He Ala Ser
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Lys Glu Phe Gin Asn Asn Pro Asn Pro Arg Ser Leu
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(D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Gly Thr Gin Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser
5 10 15
Gin Glu Gly
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Ser Asn Leu Arg Gin Gin Pro Gin Lys Phe Pro Glu Ala Leu Arg
5 10 15
Gly Cys Pro Gin Glu Asp Ser Asp
20
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Arg Gin Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val Lys Gly
5 10 15
Thr
(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15
(B) TYPE: ~ amino acid (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Thr Ser Gly Ser Gly He Ser Pro Ser His Ser Gin Arg He Ala
5 10 15
(2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Asn Gin Arg Gly Ser Leu Tyr Gin Cys Asp Tyr Ser Thr Gly Ser
5 10 15
Cys Glu Pro He Arg
20
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Pro Arg Gly Arg Ala Arg Trp Gin Cys
5
(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Lys Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly
5 10 15 3 7
Gly Gin Asp Leu Thr
20
(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Gin Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gin
5 10
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Ser Gly Arg Pro His Ser Arg Ala Val Phe Asn Glu Thr Lys Asn
5 10 15
Ser Thr Arg Arg Gin Thr Gin
20
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Cys Glu Thr Leu Lys Leu Gin Leu Pro Asn Cys He Glu Asp Pro
5 10 15
Val 3 8
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Phe Glu Lys Asn Cys Gly Asn Asp Asn He Cys Gin Asp Asp Leu
5 10 15
(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg Thr Gin
5 10
( 2 ) INFORMATION FOR SEQ ID NO : 31 :
( i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 16
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Ser Tyr Arg Lys Val Ser Thr Leu Gin Asn Gin Arg Ser Gin Arg
5 10 15
Ser
(2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear 3 9
( ii) SEQUENCE DESCRIPTION : SEQ ID NO : 32 :
Asp He Ala Phe Leu He Asp Gly Ser
5
(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Phe Arg Arg Met Lys Glu Phe Val Ser
5
(2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Phe Lys He Leu Val Val He Thr Asp Gly Glu
5 10
(2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Val He Arg Tyr Val He Gly Val Gly Asp Ala
5 10
(2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly
5 10
(2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Tyr Glu Asp Val He Pro Glu Ala Asp Arg
5 10
(2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Ser Val Cys
5 10 15
Pro Arg Gly Arg Ala Arg Trp Gin Cys Asp Ala Tyr
20 25
(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5138
(B) TYPE: nucleic acid 4 1
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GAATTCCCTC TTTCACCCTG TCTAGGTTGC CAGCAAATCC CACGGGCCTC 50
CTGACGCTGC CCCTGgGGCC ACAgGTCCCT CGAGTGCTGG AAGG 94
ATG AAG GAT TCC TGC ATC ACT GTG ATG GCC ATG GCG CTG CTG TCT 139
GGG TTC TTT TTC TTC GCG CCG GCC TCG AGC TAC AAC CTG GAC GTG 184
CGG GGC GCG CGG AGC TTC TCC CCA CCG CGC GCC GGG AGG CAC TTT 229
GGA TAC CGC GTC CTG CAG GTC GGA AAC GGG GTC ATC GTG GGA GCT 274
CCA GGG GAG GGG AAC AGC ACA GGA AGC CTC TAT CAG TGC CAG TCG 319
GGC ACA GGA CAC TGC CTG CCA GTC ACC CTG AGA GGT TCC AAC TAT 364
ACC TCC AAG TAC TTG GGC ATG ACC TTG GCA ACA GAC CCC ACA GAT 409
GGA AGC ATT TTG GCC TGT GAC CCT GGG CTG TCT CGA ACG TGT GAC 454
CAG AAC ACC TAT CTG AGT GGC CTG TGT TAC CTC TTC CGC CAG AAT 499
CTG CAG GGT CCC ATG CTG CAG GGG CGC CCT GGT TTT CAG GAA TGT 544
ATC AAG GGC AAC GTA GAC CTG GTA TTT CTG TTT GAT GGT TCG ATG 589
AGC TTG CAG CCA GAT GAA TTT CAG AAA ATT CTG GAC TTC ATG AAG 634
GAT GTG ATG AAG AAA CTC AGC AAC ACT TCG TAC CAG TTT GCT GCT 679
GTT CAG TTT TCC ACA AGC TAC JAAA ACA GAA TTT GAT TTC TCA GAT 724
TAT GTT AAA TGG AAG GAC CCT GAT GCT CTG CTG AAG CAT GTA AAG 769 4 2
CAC ATG TTG CTG TTG ACA AAT ACC TTT GGT GCC ATC AAT TAT GTC 814
GCG ACA GAG GTG TTC CGG GAG GAG CTG GGG GCC CGG CCA GAT GCC 859
ACC AAA GTG CTT ATC ATC ATC ACG GAT GGG GAG GCC ACT GAC AGT 904
GGC AAC ATC GAT GCG GCC AAA GAC ATC ATC CGC TAC ATC ATC GGG 949
ATT GGA AAG CAT TTT CAG ACC AAG GAG AGT CAG GAG ACC CTC CAC 994
AAA TTT GCA TCA AAA CCC GCG AGC GAG TTT GTG AAA ATT CTG GAC 1039
ACA TTT GAG AAG CTG AAA GAT CTA TTC ATC GAG CGG CAG AAG AAG 1084
ATC TAT GTC ATT GAG GGC ACA AGC AAA CAG GAC CTG ACT TCC TTC 1129
AAC ATG GAG CTG TCC TCC AGC GGC ATC AGT GCT GAC CTC AGC AGG 1174
GGC CAT GCA GTC GTG GGG GCA GTA GGA GCC AAG GAC TGG GCT GGG 1219
GGC TTT CTT GAC CTG AAG GCA GAC CTG CAG GAT GAC ACA TTT ATT 1264
GGG AAT GAA CCA TTG ACA CCA GAA GTG AGA GCA GGC TAT TTG GGT 1309
TAC ACC GTG ACC TGG CTG CCC TCC CGG CAA AAG ACT TCG TTG CTG 1354
GCC TCG GGA GCC CCT CG TAC CAG CAC ATG GGC CGA GTG CTG CTG 1399
TTC CAA GAG CCA CAG GGC GGA GGA CAC TGG AGC CAG GTC CAG ACA 1444
ATC CAT GGG ACC CAG ATT GGC TCT TAT TTC GGT GGG GAG CTG TGT 1489
GGC GTC GAC GTG GAC CAA GAT GGG GAG ACA GAG CTG CTG CTG ATT 1534
GGT GCC CCA CTG TTC TAT GGG GAG CAG AGA GGA GGC CGG GTG TTT 1579 ACT CTG GAG CTG GTG GGA GAG ATC GAG GCC TCT TCC ATG TTC AGC 324
CTC TGC AGC TCC CTC TCC ATC TCC TTC AAC AGC AGC AAG CAT TTC 328
CAC CTC TAT GGC AGC AAC GCC TCC CTG GCC CAG GTT GTC ATG AAG 333
GTT GAC GTG GTG TAT GAG AAG CAG ATG CTC TAC CTC TAC GTG CTG 337
AGC GGC ATC GGG GGG CTG CTG CTG CTG CTG CTC ATT TNC ATA GTG 342
CTG TAC AAG GTT GGT TTC TTC AAA CGG AAC CTG AAG GAG AAG ATG 346
GAG GCT GGC AGA GGT GTC CCG AAT GGA ATC CCT GCA GAA GAC TCT 351
GAG CAG CTG GCA TCT GGG CAA GAG GCT GGG GAT CCC GGC TGC CTG 355
AAG CCC CTC CAT GAG AAG GAC TCT GAG AGT GGT GGT GGC AAG GAC 360
TGAGTCCAGC CTGTGAGGTG CAGAGTGCCC AGAACTGGAC TCAGGATGCC 365
CAGGGCCACT TCGCCTCTGC CTGCATTCTG CCGTGTGCCC TCGGGCGAGT 370
CACTGCCTCT CCCTGGCCCT CAGTTTCCCT ATCTCGAACA TGGAACTCAT 375
TCCTGAATGT CTCCTTTGCA GGCTCATAGG GAAGACCTGC TGAGGGACCA 3804
GCCAAGAGGG CTGCAAAAGT GAGGGCTTGT CATTACCAGA CGGTTCACCA 3854
GCCTCTCTTG GTTCCTTCCT TGGAAGAGAA TGTCTGATCT AAATGTGGAG 3904
J ACTGTAGT CTCAGGACCT AGGGATGTTC TGGCCCTCAC CCCTGCCCTG 3954
GGATGTCCAC AGATGCCTCC ACCCCCCAGA ACCTGTCCTT GCACACTCCC 4004
CTGCACTGGA GTCCAGTCTC TTCTGTTGGC AGAAAGCAAA TGTGACCTGT 4054
GTCACTACGT GACTGTGGCA CACGCCTTGT TCTTGGCCAA AGACCAAATT 4104
CCTTGGCATG CCTTCCAGCA CCCTGCAAAA TGAGACCCTC GTGGCCTTCC 4154
CCAGCCTCTT CTAGAGCCGT GATGCCTCCC TGTTGAAGCT CTGGTGACAC 4204
CAGCCTTTCT CCCAGGCCAG GCTCCTTCCT GTCTTCCTGC ATTCACCCAG 4254
ACAGCTCCCT CTGCCTG AC CTTCCATCTC GCCCACCCCT CCTTCCTTGA 4304
CCAGCAGATC CCAGCTCACG TCACACACTT GGTTGGGTCC TCACATCTTT 4354
CACACTTCCA CCACCCTGCA CTACTCCCTC AAAGCACACG TCATGTTTCT 4404
TCATCCGGCA GCCTGGATGT TTTTTCCCTG TTTAATGATT GACGTACTTA 4454
GCAGCTATCT CTCAGTGAAC TGTGAGGGTA AAGGCTATAC TTGTCTTGTT 4504
CACCTTGGGA TGACGCCGCA TGATATGTCA GGGCGTGGGA CATCTAGTAG 4554
GTGCTTGACA TAATTTCACT GAATTAATGA CAGAGCCAGT GGGAAGATAC 4604
AGAAAAAGAG GGCCGGGGCT GGGCGCGGTG GTTCACGCCT GTAATCCCAG 4654
CACTTTGGGA GGCCAAGGAG GGTGGATCAC CTGAGGTCAG GAGTTAGAGG 4704
CCAGCCTGGC GAAACCCCAT CTCTACTAAA AATACAAAAT CCAGGCGTGG 4754
TGGCACACAC CTGTAGTCCC AGCTACTCAG GAGGTTGAGG TAGGAGAATT 4804
GCTTGAACCT GGGAGGTGGA GGTTGCAGTG AGCCAAGATT GCGCCATTGC 4854
ACTCCAGCCT GGGCAACACA GCGAGACTCC GTCTCAAGGA AAAAATAAAA 4904 CCT TTT GAG AAG AAC TGT GGG GAG GAC AAG AAG TGT GAG GCA AAC 2434
TTG AGA GTG TCC TTC TCT CCT GCA ACA TCC AGA GCC CTG CGT CTA 2479
ACT GCT TTT GCC AGC CTC TCT GTG GAG CTG AGC CTG AGT AAC TTG 2524
GAA GAA GAT GCT TAC TGG GTC CAG CTG GAC CTG CAC TTC CCC CCG 2569
GGA CTC TCC TTC CGC AAG GTG GAG ATG CTG AAG CCC CAT AGC CAG 2614
ATA CCT GTG AGC TGC GAG GAG CTT CCT GAA GAG TCC AGG CTT CTG 2659
TCC AGG GCA TTA TCT TGC AAT GTG AGC TCT CCC ATC TTC AAA GCA 2704
GGC CAC TCG GTT GCT CTG CAG ATG ATG TTT AAT ACA CTG GTA AAC 2749
AGC TCC TGG GGG GAC TCG GTT GAA TTG CAC GCC AAT GTG ACC TGT 2794
AAC AAT GAG GAC TCA GAC CTC CTG GAG GAC AAC TCA GCC ACT ACC 2839
ATC ATC CCC ATC CTG TAC CCC ATC AAC ATC CTC ATC CAG GAC CAA 2884
GAA GAC TCC ACA CTC TAT GTC AGT TTC ACC CCC AAA GGC CCC AAG 2929
ATC CAC CAA GTC AAG CAC ATG TAC CAG GTG AGG ATC CAG CCT TCC 2974
ATC CAC GAC CAC AAC ATA CCC ACC CTG GAG GCT GTG GTT GGG GTG 3019
CCA CAG CCT CCC AGC GAG GGG CCC ATC ACA CAC CAG TGG AGC GTG 3064
CAG ATG GAG CCT CCC GTG CCC TGC CAC TAT GAG GAT CTG GAG AGG 3109
CTC CCG GAT GCA GCT GAG CCT TGT CTC CCC GGA CCC CTG TTC CGC 3154
TGC CCT GTT GTC TTC AGG CAG GAG ATC CTC GTC CAA GTG ATC GGG 3199 ATAAAAAGCG GGCACGGGCC CGGACATCCC CACCCTTGGA GGCTGTCTTC 49
TCAGGCTCTG CCCTGCCCTA GCTCCACACC CTCTCCCAGG ACCCATCACG 50
CCTGTGCAGT GGCCCCCACA GAAAGACTGA GCTCAAGGTG GGAACCACGT 50
CTGCTAACTT GGAGCCCCAG TGCCAAGCAC AGTGCCTGCA TGTATTTATC 51
CAATAAATGT GAAATTCTGT CCAAAAAAAA AAAA 51
( 2 ) INFORMATION FOR SEQ ID NO : 40 :
( i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 3533
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 40: tggcttcctt gtggttcctc agtggtgcct gcaacccctg gttcacctcc 5 ttccaggttc tggcccttcc agcc 7 atg get etc aga gtc ctt ctg tta aca gcc ttg ace tta tgt cat 11 ggg ttc aac ttg gac act gaa aac gca atg ace ttc caa gag aac 16 gca agg ggc ttc ggg eag age gtg gtc eag ctt eag gga tec agg 20 gtg gtg gtt gga gcc ccc eag gag ata gtg get gcc aac caa agg 25
ggc age etc tac eag tgc gac tac age aca ggc tea tgc gag ccc 29
ate cgc ctg eag gtc ccc gtg gag gcc gtg aac atg tec ctg ggc 34
ctg tec ctg gca gcc ace ace age ccc cct eag ctg ctg gcc tgt 38
ggt ccc ace gtg cac eag act tgc agt gag aac acg tat gtg aaa 43
ggg etc tgc ttc ctg ttt gga tec aac eta egg eag eag ccc eag 47
aag ttc cca gag gcc etc cga ggg tgt cct caa gag gat agt gac 524
att gcc ttc ttg att gat ggc tct ggt age ate ate cca cat gac 569
ttt egg egg atg aag gag ttt gtc tea act gtg atg gag caa tta 614
aaa aag tec aaa ace ttg ttc tct ttg atg eag tac tct gaa gaa 659 ATC TAC CAG AGA AGA CAG TTG GGG TTT GAA GAA GTC TCA GAG CTG 162
CAG GGG GAC CCC GGC TAC CCA CTC GGG CGG TTT GGA GAA GCC ATC 166
ACT GCT CTG ACA GAC ATC AAC GGC GAT GGG CTG GTA GAC GTG GCT 171
GTG GGG GCC CCT CTG GAG GAG CAG GGG GCT GTG TAC ATC TTC AAT 175
GGG AGG CAC GGG GGG CTT AGT CCC CAG CCA AGT CAG CGG ATA GAA 180
GGG ACC CAA GTG CTC TCA GGA ATT CAG TGG TTT GGA CGC TCC ATC 184
CAT GGG GTG AAG GAC CTT GAA GGG GAT GGC CTG GCA GAT GTG GCT 189
GTG GGG GCT GAG AGC CAG ATG ATC GTG CTG AGC TCC CGG CCC GTG 193
GTG GAT ATG GTC ACC CTG ATG TCC TTC TCT CCA GCT GAG ATC CCA 198
GTG CAT GAA GTG GAG TCG TCC TAT TCA ACC AGT AAC AAG ATG AAA 202
GAA GGA GTT AAT ATC ACA ATC TGT TTC CAG ATC AAG TCT CTC TAC 207
CCC CAG TTC CAA GGC CGC CTG GTT GCC AAT CTC ACT TAC ACT CTG 211
CAG CTG GAT GGC CAC CGG ACC AGA AGA CGG GGG TTG TTC CCA GGA 216
GGG AGA CAT GAA CTC AGA AGG AAT ATA GCT GTC ACC ACC AGC ATG 220
TCA TGC ACT GAC TTC TCA TTT CAT TTC CCG GTA TGT GTT CAA GAC 225
CTC ATC TCC CCC ATC AAT GTT TCC CTG AAT TTC TCT CTT TGG GAG 229
GAG GAA GGG ACA CCG AGG GAC CAA AGG GCG CAG GGC AAG GAC ATA 234
CCG CCC ATC CTG AGA CCC TCC CTG CAC TCG GAA ACC TGG GAG ATC 238 47
ttc egg att cac ttt ace ttc aaa gag ttc eag aac aac cct aac 70
cca aga tea ctg gtg aag cca ata acg eag ctg ctt ggg egg aca 74
cac acg gcc acg ggc ate cgc aaa gtg gta cga gag ctg ttt aac 79
ate ace aac gga gcc cga aag aat gcc ttt aag ate eta gtt gtc 839
ate acg gat gga gaa aag ttt ggc gat ccc ttg gga tat gag gat 884
gtc ate cct gag gca gac aga gag gga gtc att cgc tac gtc att 929
ggg gtg gga gat gcc ttc cgc agt gag aaa tec cgc caa gag ctt 974
aat ace ate gca tec aag ccg cct cgt gat cac gtg ttc eag gtg 1019
aat aac ttt gag get ctg aag ace att eag aac eag ctt egg gag 1064
aag ate ttt gcg ate gag ggt act eag aca gga agt age age tec 1109
ttt gag cat gag atg tct eag gaa ggc ttc age get gcc ate ace 1154
tct aat ggc ccc ttg ctg age act gtg ggg age tat gac tgg get 1199
ggt gga gtc ttt eta tat aca tea aag gag aaa age ace ttc ate 1244
aac atg ace aga gtg gat tea gac atg aat gat get tac ttg ggt 1289
tat get gcc gcc ate ate tta egg aac egg gtg caa age ctg gtt 1334
ctg ggg gca cct cga tat eag cac ate ggc ctg gta gcg atg ttc 1379
agg eag aac act ggc atg tgg gag tec aac get aat gtc aag ggc 1424
ace eag ate ggc gcc tac ttc_ggg gcc tec etc tgc tec gtg gac 1469 4 8
gtg gac age aac ggc age ace gac ctg gtc etc ate ggg gcc ccc 1514
cat tac tac gag eag ace cga ggg ggc eag gtg tec gtg tgc ccc 1559
ttg ccc agg ggg agg get egg tgg eag tgt gat get gtt etc tac 1604
ggg gag eag ggc caa ccc tgg ggc cgc ttt ggg gca gcc eta aca 1649
gtg ctg ggg gac gta aat ggg gac aag ctg acg gac gtg gcc att 1694
ggg gcc cca gga gag gag gac aac egg ggt get gtt tac ctg ttt 1739
cac gga ace tea gga tct ggc ate age ccc tec cat age eag egg 1784
ata gca ggc tec aag etc tct ccc agg etc eag tat ttt ggt eag 1829
tea ctg agt ggg ggc eag gac etc aca atg gat gga ctg gta gac 1874
ctg act gta gga gcc eag ggg cac gtg ctg ctg etc agg tec eag 1919
cca gta ctg aga gtc aag gca ate atg gag ttc aat ccc agg gaa 1964
gtg gca agg aat gta ttt gag tgt aat gat caa gtg gtg aaa ggc 2002
aag gaa gcc gga gag gtc aga gtc tgc etc cat gtc eag aag age 2054
aca egg gat egg eta aga gaa gga eag ate eag agt gtt gtg act 2099
tat gac ctg get ctg gac tec ggc cgc cca cat tec cgc gcc gtc 2144
ttc aat gag aca aag aac age aca cgc aga eag aca eag gtc ttg 2189
ggg ctg ace eag act tgt gag ace ctg aaa eta eag ttg ccg aat 2234
tgc ate gag gac cca gtg age"ccc att gtg ctg cgc ctg aac ttc 2279 49
tct ctg gtg gga acg cca ttg tct get ttc ggg aac etc egg cca 2324
gtg ctg gcg gag gat get eag aga etc ttc aca gcc ttg ttt ccc 2369
ttt gag aag aat tgt ggc aat gac aac ate tgc eag gat gac etc 2414
age ate ace ttc agt ttc atg age ctg gac tgc etc gtg gtg ggt 2459
ggg ccc egg gag tct aac gtg aca gtg act gtg aga aat gat ggt 2504
gag gac tec tac agg aca eag gtc ace ttc ttc ttc ccg ctt gac 2549
ctg tec tac egg aag gtg tec aca etc eag aac eag cgc tea eag 2594
cga tec tgg cgc ctg gcc tgt gag tct gcc tec tec ace gaa gtg 2639
tct ggg gcc ttg aag age ace age tgc age ata aac cac ccc ate 2684
ttc ccg gaa aac tea gag gtc ace ttt aat ate acg ttt gat gta 2729
gac tct aag get tec ctt gga aac aaa ctg etc etc aag gcc aat 2774
gtg ace agt gag aac aac atg ccc aga ace aac aaa ace gaa ttc 2819
caa ctg gag ctg ccg gtg aaa tat get gtc tac atg gtg gtc ace 2864
age cat ggg gtc tec act aaa tat etc aac ttc acg gcc tea gag 2909
aat ace agt egg gtc atg eag cat caa tat eag gtc age aac ctg 2954
ggg eag agg age ccc ccc ate age ctg gtg ttc ttg gtg ccc gtc 2999
egg ctg aac eag act gtc ata tgg gac cgc ccc eag gtc ace ttc 3044
tec gag aac etc teg agt acg "tgc cac ace aag gag cgc ttg ccc 3089 tct cac tec gac ttt ctg get gag ctt egg aag gcc ccc gtg gtg 313
aac tgc tec ate get gtc tgc eag aga ate eag tgt gac ate ccg 317
ttc ttt ggc ate eag gaa gaa ttc aat get ace etc aaa ggc aac 322
etc teg ttt gac tgg tac ate aag ace teg cat aac cac etc ctg 326
ate gtg age aca get gag ate ttg ttt aac gat tec gtg ttc ace 331
ctg ctg ccg gga eag ggg gcg ttt gtg agg tec eag acg gag ace 335
aaa gtg gag ccg ttc gag gtc ccc aac ccc ctg ccg etc ate gtg 340
ggc age tct gtc ggg gga ctg ctg etc ctg gcc etc ate ace gcc 344
gcg ctg tac aag etc ggc ttc ttc aag egg caa tac aag gac atg 349
atg agt gaa ggg ggt ccc ccg ggg gcc gaa ccc eag tag 353
(2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2310
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
ATG CTG GGC CTG CGC CCC CCA CTT CTC GCC CTG GTG GGG CTG CTC 4
TCC CTC GGG TGC GTC CTC TCT CAG GAG TGC ACG AAG TTC AAG GTC 9
AGC AGC TGC CGG GAA TGC ATC GAG TCG GGG CCC GGC TGC ACC TGG 13
TGC CAG AAG CTG AAC TTC ACA GGG CCG GGG GAT CCT GAC TCC ATT 18
CGC TGC GAC ACC CGG CCA CAG CTG CTC ATG AGG GGC TGT GCG GCT 22
GAC GAC ATC ATG GAC CCC ACA AGC CTC GCT GAA ACC CAG GAA GAC 27 5 1
CAC AAT GGG GGC CAG AAG CAG CTG TCC CCA CAA AAA GTG ACG CTT 3
TAC CTG CGA CCA GGC CAG GCA GCA GCG TTC AAC GTG ACC TTC CGG 36
CGG GCC AAG GGC TAC CCC ATC GAC CTG TAC TAT CTG ATG GAC CTC 40
TCC TAC TCC ATG CTT GAT GAC CTC AGG AAT GTC AAG AAG CTA GGT 45
GGC GAC CTG CTC CGG GCC CTC AAC GAG ATC ACC GAG TCC GGC CGC 49
ATT GGC TTC GGG TCC TTC GTG GAC AAG ACC GTG CTG CCG TTC GTG 54
AAC ACG CAC CCT GAT AAG CTG CGA AAC CCA TGC CCC AAC AAG GAG 58
AAA GAG TGC CAG CCC CCG TTT GCC TTC AGG CAC GTG CTG AAG CTG 63
ACC AAC AAC TCC AAC CAG TTT CAG ACC GAG GTC GGG AAG CAG CTG 67
ATT TCC GGA AAC CTG GAT GCA CCC GAG GGT GGG CTG GAC GCC ATG 72
ATG CAG GTC GCC GCC TGC CCG GAG GAA ATC GGC TGG CGC AAC GTC 76
ACG CGG CTG CTG GTG TTT GCC ACT GAT GAC GGC TTC CAT TTC GCG 810
GGC GAC GGA AAG CTG GGC GCC ATC CTG ACC CCC AAC GAC GGC CGC 855
TGT CAC CTG GAG GAC AAC TTG TAC AAG AGG AGC AAC GAA TTC GAC 900
TAC CCA TCG GTG GGC CAG CTG GCG CAC AAG CTG GCT GAA AAC AAC 945
ATC CAG CCC ATC TTC GCG GTG ACC AGT AGG ATG GTG AAG ACC TAC 990
GAG AAA CTC ACC GAG ATC ATC CCC AAG TCA GCC GTG GGG GAG CTG 1035
TCT GAG GAC TCC AGC AAT GTG GTC CAT CTC ATT AAG AAT GCT TAC 1080 AAT AAA CTC TCC TCC AGG GTC TTC CTG GAT CAC AAC GCC CTC CCC 1125
GAC ACC CTG AAA GTC ACC TAC GAC TCC TTC TGC AGC AAT GGA GTG 1170
ACG CAC AGG AAC CAG CCC AGA GGT GAC TGT GAT GGC GTG CAG ATC 1215
AAT GTC CCG ATC ACC TTC CAG GTG AAG GTC ACG GCC ACA GAG TGC 1260
ATC CAG GAG CAG TCG TTT GTC ATC CGG GCG CTG GGC TTC ACG GAC 1305
ATA GTG ACC GTG CAG GTT CTT CCC CAG TGT GAG TGC CGG TGC CGG 1350
GAC CAG AGC AGA GAC CGC AGC CTC TGC CAT GGC AAG GGC TTC TTG 1395
GAG TGC GGC ATC TGC AGG TGT GAC ACT GGC TAC ATT GGG AAA AAC 1440
TGT GAG TGC CAG ACA CAG GGC CGG AGC AGC CAG GAG CTG GAA GGA 1485
AGC TGC CGG AAG GAC AAC AAC TCC ATC ATC TGC TCA GGG CTG GGG 1530
GAC TGT GTC TGC GGG CAG TGC CTG TGC CAC ACC AGC GAC GTC CCC 1575
GGC AAG CTG ATA TAC GGG CAG TAC TGC GAG TGT GAC ACC ATC AAC 1620
TGT GAG CGC TAC AAC GGC CAG GTC TGC GGC GGC CCG GGG AGG GGG 1665
CTC TGC TTC TGC GGG AAG TGC CGC TGC CAC CCG GGC TTT GAG GGC 1710
TCA GCG TGC CAG TGC GAG AGG ACC ACT GAG GGC TGC CTG AAC CCG 1755
CGG CGT GTT GAG TGT AGT GGT CGT GGC CGG TGC CGC TGC AAC GTA 1800
TGC GAG TGC CAT TCA GGC TAC CAG CTG CCT CTG TGC CAG GAG TGC 1845
CCC GGC TGC CCC TCA CCC TGT GGC AAG TAC ATC TCC TGC GCC GAG 1890 TGC CTG AAG TTC GAA AAG GGC CCC TTT GGG AAG AAC TGC AGC GCG 193
GCG TGT CCG GGC CTG CAG CTG TCG AAC AAC CCC GTG AAG GGC AGG 198
ACC TGC AAG GAG AGG GAC TCA GAG GGC TGC TGG GTG GCC TAC ACG 202
CTG GAG CAG CAG GAC GGG ATG GAC CGC TAC CTC ATC TAT GTG GAT 207
GAG AGC CGA GAG TGT GTG GCA GGC CCC AAC ATC GCC GCC ATC GTC 211
GGG GGC ACC GTG GCA GGC ATC GTG CTG ATC GGC ATT CTC CTG CTG 216
GTC ATC TGG AAG GCT CTG ATC CAC CTG AGC GAC CTC CGG GAG TAC 220
AGG CGC TTT GAG AAG GAG AAG CTC AAG TCC CAG TGG AAC AAT GAT 2250
AAT CCC CTT TTC AAG AGC GCC ACC ACG ACG GTC ATG AAC CCC AAG 2295
TTT GCT GAG AGT TAG 2310
(2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1170
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Met Lys Asp Ser Cys He Thr Val Met Ala Met Ala Leu Leu Ser
5 10 15
Gly Phe Phe Phe Phe Ala Pro Ala Ser Ser Tyr Asn Leu Asp Val
20 25 30
Arg Gly Ala Arg Ser Phe Ser Pro Pro Arg Ala Gly Arg His Phe
35 40 50
Gly Tyr Arg Val Leu Gin Val Gly Asn Gly Val He Val Gly Ala
55 ~ 60 65
Pro Gly Glu Gly Asn Ser Thr Gly Ser Leu Tyr Gin Cys Gin Ser 70 75 80
Gly Thr Gly His Cys Leu Pro Val Thr Leu Arg Gly Ser Asn Tyr
85 90 95
Thr Ser Lys Tyr Leu Gly Met Thr Leu Ala Thr Asp Pro Thr Asp
100 105 115
Gly Ser He Leu Ala Cys Asp Pro Gly Leu Ser Arg Thr Cys Asp
120 125 130
Gin Asn Thr Tyr Leu Ser Gly Leu Cys Tyr Leu Phe Arg Gin Asn
135 140 145
Leu Gin Gly Pro Met Leu Gin Gly Arg Pro Gly Phe Gin Glu Cys
150 155 160
He Lys Gly Asn Val Asp Leu Val Phe Leu Phe Asp Gly Ser Met
165 170 175
Ser Leu Gin Pro Asp Glu Phe Gin Lys He Leu Asp Phe Met Lys
180 185 190
Asp Val Met Lys Lys Leu Ser Asn Thr Ser Tyr Gin Phe Ala Ala
195 200 205
Val Gin Phe Ser Thr Ser Tyr Lys Thr Glu Phe Asp Phe Ser Asp
215 220 225
Tyr Val Lys Trp Lys Asp Pro Asp Ala Leu Leu Lys His Val Lys
230 235 240
His Met Leu Leu Leu Thr Asn Thr Phe Gly Ala He Asn Tyr Val
245 250 255
Ala Thr Glu Val Phe Arg Glu Glu Leu Gly Ala Arg Pro Asp Ala
260 265 270
Thr Lys Val Leu He He He Thr Asp Gly Glu Ala Thr Asp Ser
275 280 285
Gly Asn He Asp Ala Ala Lys Asp He He Arg Tyr He He Gly
290 295 300
He Gly Lys His Phe Gin Thr Lys Glu Ser Gin Glu Thr Leu His
305 310 315
Lys Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Lys He Leu Asp
320 325 330
Thr Phe Glu Lys Leu Lys Asp-Leu Phe He Glu Arg Gin Lys Lys
335 340 345
He Tyr Val He Glu Gly Thr Ser Lys Gin Asp Leu Thr Ser Phe 350 355 360
Asn Met Glu Leu Ser Ser Ser Gly He Ser Ala Asp Leu Ser Arg
365 370 375
Gly His Ala Val Val Gly Ala Val Gly Ala Lys Asp Trp Ala Gly
380 385 390
Gly Phe Leu Asp Leu Lys Ala Asp Leu Gin Asp Asp Thr Phe He
395 400 405
Gly Asn Glu Pro Leu Thr Pro Glu Val Arg Ala Gly Tyr Leu Gly
415 420 425
Tyr Thr Val Thr Trp Leu Pro Ser Arg Gin Lys Thr Ser Leu Leu
430 435 440
Ala Ser Gly Ala Pro Arg Tyr Gin His Met Gly Arg Val Leu Leu
445 450 455
Phe Gin Glu Pro Gin Gly Gly Gly His Trp Ser Gin Val Gin Thr
460 465 470
He His Gly Thr Gin He Gly Ser Tyr Phe Gly Gly Glu Leu Cys
475 480 485
Gly Val Asp Val Asp Gin Asp Gly Glu Thr Glu Leu Leu Leu He
490 495 500
Gly Ala Pro Leu Phe Tyr Gly Glu Gin Arg Gly Gly Arg Val Phe
505 510 515
He Tyr Gin Arg Arg Gin Leu Gly Phe Glu Glu Val Ser Glu Leu
520 525 530
Gin Gly Asp Pro Gly Tyr Pro Leu Gly Arg Phe Gly Glu Ala He
535 540 545
Thr Ala Leu Thr Asp He Asn Gly Asp Gly Leu Val Asp Val Ala
550 555 560
Val Gly Ala Pro Leu Glu Glu Gin Gly Ala Val Tyr He Phe Asn
565 570 575
Gly Arg His Gly Gly Leu Ser Pro Gin Pro Ser Gin Arg He Glu
580 585 590
Gly Thr Gin Val Leu Ser Gly He Gin Trp Phe Gly Arg Ser He
595 600 605
His Gly Val Lys Asp Leu Glu Gly Asp Gly Leu Ala Asp Val Ala
610 615 620
Val Gly Ala Glu Ser Gin Met He Val Leu Ser Ser Arg Pro Val 625 630 635
Figure imgf000058_0001
875 880 885
Ser Ser Trp Gly Asp Ser Val Glu Leu His Ala Asn Val Thr Cys
890 895 900
Asn Asn Glu Asp Ser Asp Leu Leu Glu Asp Asn Ser Ala Thr Thr
905 910 915
He He Pro He Leu Tyr Pro He Asn He Leu He Gin Asp Gin
920 925 930
Glu Asp Ser Thr Leu Tyr Val Ser Phe Thr Pro Lys Gly Pro Lys
935 940 945
He His Gin Val Lys His Met Tyr Gin Val Arg He Gin Pro Ser
950 955 960
He His Asp His Asn He Pro Thr Leu Glu Ala Val Val Gly Val
965 970 975
Pro Gin Pro Pro Ser Glu Gly Pro He Thr His Gin Trp Ser Val
980 985 990
Gin Met Glu Pro Pro Val Pro Cys His Tyr Glu Asp Leu Glu Arg
995 1000 1005
Leu Pro Asp Ala Ala Glu Pro Cys Leu Pro Gly Pro Leu Phe Arg
1010 1015 1020
Cys Pro Val Val Phe Arg Gin Glu He Leu Val Gin Val He Gly
1025 1030 1035
Thr Leu Glu Leu Val Gly Glu He Glu Ala Ser Ser Met Phe Ser
1040 1045 1050
Leu Cys Ser Ser Leu Ser He Ser Phe Asn Ser Ser Lys His Phe
1055 1060 1065
His Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gin Val Val Met Lys
1070 1075 1080
Val Asp Val Val Tyr Glu Lys Gin Met Leu Tyr Leu Tyr Val Leu
1085 1090 1095
Ser Gly He Gly Gly Leu Leu Leu Leu Leu Leu He Xaa He Val
1100 1105 1110
Leu Tyr Lys Val Gly Phe Phe Lys Arg Asn Leu Lys Glu Lys Met
1115 1120 1125
Glu Ala Gly Arg Gly Val Pro Asn Gly He Pro Ala Glu Asp Ser
1130 _ 1135 1140
Glu Gin Leu Ala Ser Gly Gin Glu Ala Gly Asp Pro Gly Cys Leu 1145 1150 1155
Lys Pro Leu His Glu Lys Asp Ser Glu Ser Gly Gly Gly Lys Asp
1160 1165 1170
(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1152
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His
5 10 15
Gly Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gin Glu Asn
20 25 30
Ala Arg Gly Phe Gly Gin Ser Val Val Gin Leu Gin Gly Ser Arg
35 40 50
Val Val Val Gly Ala Pro Gin Glu He Val Ala Ala Asn Gin Arg
55 60 65
Gly Ser Leu Tyr Gin Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro
70 75 80
He Arg Leu Gin Val Pro Val Glu Ala Val Asn Met Ser Leu Gly
85 90 95
Leu Ser Leu Ala Ala Thr Thr Ser Pro Pro Gin Leu Leu Ala Cys
100 105 115
Gly Pro Thr Val His Gin Thr Cys Ser Glu Asn Thr Tyr Val Lys
120 125 130
Gly Leu Cys Phe Leu Phe Gly Ser Asn Leu Arg Gin Gin Pro Gin
135 140 145
Lys Phe Pro Glu Ala Leu Arg Gly Cys Pro Gin Glu Asp Ser Asp
150 155 160
He Ala Phe Leu He Asp Gly Ser Gly Ser He He Pro His Asp
165 170 175
Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val Met Glu Gin Leu
180 185 190 Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gin Tyr Ser Glu Glu
195 200 205
Phe Arg He His Phe Thr Phe Lys Glu Phe Gin Asn Asn Pro Asn
215 220 225
Pro Arg Ser Leu Val Lys Pro He Thr Gin Leu Leu Gly Arg Thr
230 235 240
His Thr Ala Thr Gly He Arg Lys Val Val Arg Glu Leu Phe Asn
245 250 255
He Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys He Leu Val Val
260 265 270
He Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp
275 280 285
Val He Pro Glu Ala Asp Arg Glu Gly Val He Arg Tyr Val He
290 295 300
Gly Val Gly Asp Ala Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu
305 310 315
Asn Thr He Ala Ser Lys Pro Pro Arg Asp His Val Phe Gin Val
320 325 330
Asn Asn Phe Glu Ala Leu Lys Thr He Gin Asn Gin Leu Arg Glu
335 340 345
Lys He Phe Ala He Glu Gly Thr Gin Thr Gly Ser Ser Ser Ser
350 355 360
Phe Glu His Glu Met Ser Gin Glu Gly Phe Ser Ala Ala He Thr
365 370 375
Ser Asn Gly Pro Leu Leu Ser Thr Val Gly Ser Tyr Asp Trp Ala
380 385 390
Gly Gly Val Phe Leu Tyr Thr Ser Lys Glu Lys Ser Thr Phe He
395 400 405
Asn Met Thr Arg Val Asp Ser Asp Met Asn Asp Ala Tyr Leu Gly
415 420 425
Tyr Ala Ala Ala He He Leu Arg Asn Arg Val Gin Ser Leu Val
430 435 440
Leu Gly Ala Pro Arg Tyr Gin His He Gly Leu Val Ala Met Phe
445 450 455
Arg Gin Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val Lys Gly
460 _ 465 470 Thr Gin He Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp 475 480 485 Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu He Gly Ala Pro 490 495 500 His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro 505 510 515 Leu Pro Arg Gly Arg Ala Arg Trp Gin Cys Asp Ala Val Leu Tyr 520 525 530 Gly Glu Gin Gly Gin Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr 535 540 545 Val Leu Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala He 550 555 560 Gly Ala Pro Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe 565 570 575 His Gly Thr Ser Gly Ser Gly He Ser Pro Ser His Ser Gin Arg 580 585 590 He Ala Gly Ser Lys Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin 595 600 605 Ser Leu Ser Gly Gly Gin Asp Leu Thr Met Asp Gly Leu Val Asp 610 615 620 Leu Thr Val Gly Ala Gin Gly His Val Leu Leu Leu Arg Ser Gin 625 630 635 Pro Val Leu Arg Val Lys Ala He Met Glu Phe Asn Pro Arg Glu 640 645 650 Val Ala Arg Asn Val Phe Glu Cys Asn Asp Gin Val Val Lys Gly 655 670 675 Lys Glu Ala Gly Glu Val Arg Val Cys Leu His Val Gin Lys Ser 680 685 690 Thr Arg Asp Arg Leu Arg Glu Gly Gin He Gin Ser Val Val Thr 695 670 675 Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser Arg Ala Val 680 685 690 Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gin Thr Gin Val Leu 695 700 705 Gly Leu Thr Gin Thr Cys Glu Thr Leu Lys Leu Gin Leu Pro Asn 710 715 720 Cys He Glu Asp Pro Val Ser Pro He Val Leu Arg Leu Asn Phe
725 730 735
Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro
740 745 750
Val Leu Ala Glu Asp Ala Gin Arg Leu Phe Thr Ala Leu Phe Pro
755 760 765
Phe Glu Lys Asn Cys Gly Asn Asp Asn He Cys Gin Asp Asp Leu
770 775 780
Ser He Thr Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly
785 790 795
Gly Pro Arg Glu Ser Asn Val Thr Val Thr Val Arg Asn Asp Gly
800 805 810
Glu Asp Ser Tyr Arg Thr Gin Val Thr Phe Phe Phe Pro Leu Asp
815 820 825
Leu Ser Tyr Arg Lys Val Ser Thr Leu Gin Asn Gin Arg Ser Gin
830 835 840
Arg Ser Trp Arg Leu Ala Cys Glu Ser Ala Ser Ser Thr Glu Val
845 850 855
Ser Gly Ala Leu Lys Ser Thr Ser Cys Ser He Asn His Pro He
860 865 870
Phe Pro Glu Asn Ser Glu Val Thr Phe Asn He Thr Phe Asp Val
875 880 885
Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu Leu Leu Lys Ala Asn
890 895 900
Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn Lys Thr Glu Phe
905 910 915
Gin Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met Val Val Thr
920 925 930
Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala Ser Glu
935 940 945
Asn Thr Ser Arg Val Met Gin His Gin Tyr Gin Val Ser Asn Leu
950 955 960
Gly Gin Arg Ser Pro Pro He Ser Leu Val Phe Leu Val Pro Val
965 970 975
Arg Leu Asn Gin Thr Val He Trp Asp Arg Pro Gin Val Thr Phe
980 985 990 Ser Glu Asn Leu Ser Ser Thr Cys His Thr Lys Glu Arg Leu Pro
995 1000 1005
Ser His Ser Asp Phe Leu Ala Glu Leu Arg Lys Ala Pro Val Val
1010 1015 1020
Asn Cys Ser He Ala Val Cys Gin Arg He Gin Cys Asp He Pro
1025 1030 1035
Phe Phe Gly He Gin Glu Glu Phe Asn Ala Thr Leu Lys Gly Asn
1040 1045 1050
Leu Ser Phe Asp Trp Tyr He Lys Thr Ser His Asn His Leu Leu
1055 1060 1065
He Val Ser Thr Ala Glu He Leu Phe Asn Asp Ser Val Phe Thr
1070 1075 1080
Leu Leu Pro Gly Gin Gly Ala Phe Val Arg Ser Gin Thr Glu Thr
1085 1090 1095
Lys Val Glu Pro Phe Glu Val Pro Asn Pro Leu Pro Leu He Val
1100 1105 1110
Gly Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu He Thr Ala
1115 1120 1125
Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gin Tyr Lys Asp Met
1130 1135 1140
Met Ser Glu Gly Gly Pro Pro Gly Ala Glu Pro Gin
1145 1150
(2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1163
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 44
Met Thr Arg Thr Arg Ala Ala Leu Leu Leu Phe Thr Ala Leu Ala
5 10 15
Thr Ser Leu Gly Phe Asn Leu Asp Thr Glu Glu Leu Thr Ala Phe
20 25 30
Arg Val Asp Ser Ala Gly Phe Gly Asp Ser Val Val Gin Tyr Ala
35 - 40 50 Asn Ser Trp Val Val Val Gly Ala Pro Gin Lys He Thr Ala Ala
55 60 65
Asn Gin Thr Gly Gly Leu Tyr Gin Cys Gly Tyr Ser Thr Gly Ala
70 75 80
Cys Glu Pro He Gly Leu Gin Val Pro Pro Glu Ala Val Asn Met
85 90 95
Ser Leu Gly Leu Ser Leu Ala Ser Thr Thr Ser Pro Ser Gin Leu
100 105 115
Leu Ala Cys Gly Pro Thr Val His His Glu Cys Gly Arg Asn Met
120 125 130
Tyr Leu Thr Gly Leu Cys Phe Leu Leu Gly Pro Thr Gin Leu Thr
135 140 145
Gin Arg Leu Pro Val Ser Arg Gin Glu Cys Pro Arg Gin Glu Gin
150 155 160
Asp He Val Phe Leu He Asp Gly Ser Gly Ser He Ser Ser Arg
165 170 175
Asn Phe Ala Thr Met Met Asn Phe Val Arg Ala Val He Ser Gin
180 185 190
Phe Gin Arg Pro Ser Thr Gin Phe Ser Leu Met Gin Phe Ser Asn
195 200 205
Lys Phe Gin Thr His Phe Thr Phe Glu Glu Phe Arg Arg Thr Ser
215 220 225
Asn Pro Leu Ser Leu Leu Ala Ser Val His Gin Leu Gin Gly Phe
230 235 240
Thr Tyr Thr Ala Thr Ala He Gin Asn Val Val His Arg Leu Phe
245 250 255
His Ala Ser Tyr Gly Ala Arg Arg Asp Ala Thr Lys He Leu He
260 265 270
Val He Thr Asp Gly Lys Lys Glu Gly Asp Ser Leu Asp Tyr Lys
275 280 285
Asp Val He Pro Met Ala Asp Ala Ala Gly He He Arg Tyr Ala
290 295 300
He Gly Val Gly Leu Ala Phe Gin Asn Arg Asn Ser Trp Lys Glu
305 310 315
Leu Asn Asp He Ala Ser Lys Pro Ser Gin Glu His He Phe Lys
320 325 330 Val Glu Asp Phe Asp Ala Leu Lys Asp He Gin Asn Gin Leu Lys
335 340 345
Glu Lys He Phe Ala He Glu Gly Thr Glu Thr Thr Ser Ser Ser
350 355 360
Ser Phe Glu Leu Glu Met Ala Gin Glu Gly Phe Ser Ala Val Phe
365 370 375
Thr Pro Asp Gly Pro Val Leu Gly Ala Val Gly Ser Phe Thr Trp
380 385 390
Ser Gly Gly Ala Phe Leu Tyr Pro Pro Asn Met Ser Pro Thr Phe
395 400 405
He Asn Met Ser Gin Glu Asn Val Asp Met Arg Asp Ser Tyr Leu
415 420 425
Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly Val Gin Ser Leu
430 435 440
Val Leu Gly Ala Pro Arg Tyr Gin His Thr Gly Lys Ala Val He
445 450 455
Phe Thr Gin Val Ser Arg Gin Trp Arg Met Lys Ala Glu Val Thr
460 465 470
Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val
475 480 485
Asp Val Asp Thr Asp Gly Ser Thr Asp Leu Val Leu He Gly Ala
490 495 500
Pro His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys
505 510 515
Pro Leu Pro Arg Gly Trp Arg Arg Trp Trp Cys Asp Ala Val Leu
520 525 530
Tyr Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu
535 540 545
Thr Val Leu Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Val
550 555 560
He Gly Ala Pro Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr Leu
565 570 575
Phe His Gly Val Leu Gly Pro Ser He Ser Pro Ser His Ser Gin
580 585 590
Arg He Ala Gly Ser Gin Leu Ser Ser Arg Leu Gin Tyr Phe Gly
595 600 605 Gin Ala Leu Ser Gly Gly Gin Asp Leu Thr Gin Asp Gly Leu Val
610 615 620
Asp Leu Ala Val Gly Ala Arg Gly Gin Val Leu Leu Leu Arg Thr
625 630 635
Arg Pro Val Leu Trp Val Gly Val Ser Met Gin Phe He Pro Ala
640 645 650
Glu He Pro Arg Ser Ala Phe Glu Cys Arg Glu Gin Val Val Ser
655 670 675
Glu Gin Thr Leu Val Gin Ser Asn He Cys Leu Tyr He Asp Lys
680 685 690
Arg Ser Lys Asn Leu Leu Gly Ser Arg Asp Leu Gin Ser Ser Val
695 670 675
Thr Leu Asp Leu Ala Leu Asp Pro Gly Arg Leu Ser Pro Arg Ala
680 685 690
Thr Phe Gin Glu Thr Lys Asn Arg Ser Leu Ser Arg Val Arg Val
695 700 705
Leu Gly Leu Lys Ala His Cys Glu Asn Phe Asn Leu Leu Leu Pro
710 715 720
Ser Cys Val Glu Asp Ser Val Thr Pro He Thr Leu Arg Leu Asn
725 730 735
Phe Thr Leu Val Gly Lys Pro Leu Leu Ala Phe Arg Asn Leu Arg
740 745 750
Pro Met Leu Ala Ala Leu Ala Gin Arg Tyr Phe Thr Ala Ser Leu
755 760 765
Pro Phe Glu Lys Asn Cys Gly Ala Asp His He Cys Gin Asp Asn
770 775 780
Leu Gly He Ser Phe Ser Phe Pro Gly Leu Lys Ser Leu Leu Val
785 790 795
Gly Ser Asn Leu Glu Leu Asn Ala Glu Val Met Val Trp Asn Asp
800 805 810
Gly Glu Asp Ser Tyr Gly Thr Thr He Thr Phe Ser His Pro Ala
815 820 825
Gly Leu Ser Tyr Arg Tyr Val Ala Glu Gly Gin Lys Gin Gly Gin
830 835 840
Leu Arg Ser Leu His Leu Thr Cys Asp Ser Ala Pro Val Gly Ser
845 ~ 850 855 Gin Gly Thr Trp Ser Thr Ser Cys Arg He Asn His Leu He Phe
860 865 870
Arg Gly Gly Ala Gin He Thr Phe Leu Ala Thr Phe Asp Val Ser
875 880 885
Pro Lys Ala Val Leu Gly Asp Arg Leu Leu Leu Thr Ala Asn Val
890 895 900
Ser Ser Glu Asn Asn Thr Pro Arg Thr Ser Lys Thr Thr Phe Gin
905 910 915
Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Thr Val Val Ser Ser
920 925 930
His Glu Gin Phe Thr Lys Tyr Leu Asn Phe Ser Glu Ser Glu Glu
935 940 945
Lys Glu Ser His Val Ala Met His Arg Tyr Gin Val Asn Asn Leu
950 955 960
Gly Gin Arg Asp Leu Pro Val Ser He Asn Phe Trp Val Pro Val
965 970 975
Glu Leu Asn Gin Glu Ala Val Trp Met Asp Val Glu Val Ser His
980 985 990
Pro Gin Asn Pro Ser Leu Arg Cys Ser Ser Glu Lys He Ala Pro
995 1000 1005
Pro Ala Ser Asp Phe Leu Ala His He Gin Lys Asn Pro Val Leu
1010 1015 1020
Asp Cys Ser He Ala Gly Cys Leu Arg Phe Arg Cys Asp Val Pro
1025 1030 1035
Ser Phe Ser Val Gin Glu Glu Leu Asp Phe Thr Leu Lys Gly Asn
1040 1045 1050
Leu Ser Phe Gly Trp Val Arg Gin He Leu Gin Lys Lys Val Ser
1055 1060 1065
Val Val Ser Val Ala Glu He Thr Phe Asp Thr Ser Val Tyr Ser
1070 1075 1080
Gin Leu Pro Gly Gin Glu Ala Phe Met Arg Ala Gin Thr Thr Thr
1085 1090 1095
Val Leu Glu Lys Tyr Lys Val His Asn Pro Thr Pro Leu He Val
1100 1105 1110
Gly Ser Ser He Gly Gly Leu Leu Leu Leu Ala Leu He Thr Ala
1115 1120 1125 Val Leu Tyr Lys Val Gly Phe Phe Lys Arg Gin Tyr Lys Glu Met
1130 1135 1140
Met Glu Glu Ala Asn Gly Gin He Ala Pro Glu Asn Gly Thr Gin
1145 1150 1155
Thr Pro Ser Pro Pro Ser Glu Lys
1160
( 2 ) INFORMATION FOR SEQ ID NO : 45 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 769
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Met Leu Gly Leu Arg Pro Pro Leu Leu Ala Leu Val Gly Leu Leu
5 10 15
Ser Leu Gly Cys Val Leu Ser Gin Glu Cys Thr Lys Phe Lys Val
20 25 30
Ser Ser Cys Arg Glu Cys He Glu Ser Gly Pro Gly Cys Thr Trp
35 40 50
Cys Gin Lys Leu Asn Phe Thr Gly Pro Gly Asp Pro Asp Ser He
55 60 65
Arg Cys Asp Thr Arg Pro Gin Leu Leu Met Arg Gly Cys Ala Ala
70 75 80
Asp Asp He Met Asp Pro Thr Ser Leu Ala Glu Thr Gin Glu Asp
85 90 95
His Asn Gly Gly Gin Lys Gin Leu Ser Pro Gin Lys Val Thr Leu
100 105 115
Tyr Leu Arg Pro Gly Gin Ala Ala Ala Phe Asn Val Thr Phe Arg
120 125 130
Arg Ala Lys Gly Tyr Pro He Asp Leu Tyr Tyr Leu Met Asp Leu
135 140 145
Ser Tyr Ser Met Leu Asp Asp Leu Arg Asn Val Lys Lys Leu Gly
150 155 160
Gly Asp Leu Leu Arg Ala Leu Asn Glu He Thr Glu Ser Gly Arg
165 170 175 He Gly Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val
180 185 190
Asn Thr His Pro Asp Lys Leu Arg Asn Pro Cys Pro Asn Lys Glu
195 200 205
Lys Glu Cys Gin Pro Pro Phe Ala Phe Arg His Val Leu Lys Leu
215 220 225
Thr Asn Asn Ser Asn Gin Phe Gin Thr Glu Val Gly Lys Gin Leu
230 235 240
He Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Leu Asp Ala Met
245 250 255
Met Gin Val Ala Ala Cys Pro Glu Glu He Gly Trp Arg Asn Val
260 265 270
Thr Arg Leu Leu Val Phe Ala Thr Asp Asp Gly Phe His Phe Ala
275 280 285
Gly Asp Gly Lys Leu Gly Ala He Leu Thr Pro Asn Asp Gly Arg
290 295 300
Cys His Leu Glu Asp Asn Leu Tyr Lys Arg Ser Asn Glu Phe Asp
305 310 315
Tyr Pro Ser Val Gly Gin Leu Ala His Lys Leu Ala Glu Asn Asn
320 325 330
He Gin Pro He Phe Ala Val Thr Ser Arg Met Val Lys Thr Tyr
335 340 345
Glu Lys Leu Thr Glu He He Pro Lys Ser Ala Val Gly Glu Leu
350 355 360
Ser Glu Asp Ser Ser Asn Val Val His Leu He Lys Asn Ala Tyr
365 370 375
Asn Lys Leu Ser Ser Arg Val Phe Leu Asp His Asn Ala Leu Pro
380 385 390
Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Val
395 400 405
Thr His Arg Asn Gin Pro Arg Gly Asp Cys Asp Gly Val Gin He
415 420 425
Asn Val Pro He Thr Phe Gin Val Lys Val Thr Ala Thr Glu Cys
430 435 440
He Gin Glu Gin Ser Phe Val He Arg Ala Leu Gly Phe Thr Asp
445 ~ 450 455 He Val Thr Val Gin Val Leu Pro Gin Cys Glu Cys Arg Cys Arg
460 465 470
Asp Gin Ser Arg Asp Arg Ser Leu Cys His Gly Lys Gly Phe Leu
475 480 485
Glu Cys Gly He Cys Arg Cys Asp Thr Gly Tyr He Gly Lys Asn
490 495 500
Cys Glu Cys Gin Thr Gin Gly Arg Ser Ser Gin Glu Leu Glu Gly
505 510 515
Ser Cys Arg Lys Asp Asn Asn Ser He He Cys Ser Gly Leu Gly
520 525 530
Asp Cys Val Cys Gly Gin Cys Leu Cys His Thr Ser Asp Val Pro
535 540 545
Gly Lys Leu He Tyr Gly Gin Tyr Cys Glu Cys Asp Thr He Asn
550 555 560
Cys Glu Arg Tyr Asn Gly Gin Val Cys Gly Gly Pro Gly Arg Gly
565 570 575
Leu Cys Phe Cys Gly Lys Cys Arg Cys His Pro Gly Phe Glu Gly
580 585 590
Ser Ala Cys Gin Cys Glu Arg Thr Thr Glu Gly Cys Leu Asn Pro
595 600 605
Arg Arg Val Glu Cys Ser Gly Arg Gly Arg Cys Arg Cys Asn Val
610 615 620
Cys Glu Cys His Ser Gly Tyr Gin Leu Pro Leu Cys Gin Glu Cys
625 630 635
Pro Gly Cys Pro Ser Pro Cys Gly Lys Tyr He Ser Cys Ala Glu
640 645 650
Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly Lys Asn Cys Ser Ala
655 670 675
Ala Cys Pro Gly Leu Gin Leu Ser Asn Asn Pro Val Lys Gly Arg
680 685 690
Thr Cys Lys Glu Arg Asp Ser Glu Gly Cys Trp Val Ala Tyr Thr
695 670 675
Leu Glu Gin Gin Asp Gly Met Asp Arg Tyr Leu He Tyr Val Asp
680 685 690
Glu Ser Arg Glu Cys Val Ala Gly Pro Asn He Ala Ala He Val
695 ~ 700 705 Gly Gly Thr Val Ala Gly He Val Leu He Gly He Leu Leu Leu
710 715 720
Val He Trp Lys Ala Leu He His Leu Ser Asp Leu Arg Glu Tyr
725 730 735
Arg Arg Phe Glu Lys Glu Lys Leu Lys Ser Gin Trp Asn Asn Asp
740 745 750
Asn Pro Leu Phe Lys Ser Ala Thr Thr Thr Val Met Asn Pro Lys
755 760 765
Phe Ala Glu Ser
(2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Asp Val Asp Ser Asn Gly Ser Thr Asp
5
(2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Asp Val Asn Gly Asp Lys Leu Thr Asp
5
(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 48: Asp Leu Thr Met Asp Gly Leu Val Asp
5
( 2 ) INFORMATION FOR SEQ ID NO : 49 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 9
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Asp Ser Asp Met Asn Asp Ala Tyr Leu
5
( 2 ) INFORMATION FOR SEQ ID NO : 50 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Asn Ala Phe Lys He Leu Val Val He Thr Asp Gly Glu Lys Phe
5 10 15
Gly Asp Pro Leu Gly Tyr Glu Asp Val He Pro Glu Ala Asp Arg
20 25 30
Glu Gly Val
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 51: sp Gly Glu Lys Phe
5

Claims

Claims
1. A purified peptide comprising at least one extracellular region of a β2 integrin subunit capable of inhibiting a CD11/CD18 mediated immune response, said peptide lacking the transmembrane and cytoplasmic portions of said β2 integrin subunit, wherein said subunit is CDllb, CDllc or CD18.
2. The purified peptide of claim 1 wherein said jS2 integrin subunit is CDllb.
3. The peptide of claim 3, said peptide comprising all or part of the A domain of CDllb.
4. The peptide of claim 3, said peptide comprising one of the following amino acid sequences: a. DIAFLIDGS (SEQ ID NO: 32) , b. FRRMKEFVS (SEQ ID NO: 33), c. FKILWITDGE (SEQ ID NO: 34) , d. VIRYVIGVGDA (SEQ ID NO: 35),
5. The peptide of claim 3, said peptide comprising one of the following amino acid sequences: a. DGEKFGDPLG (SEQ ID NO: 36) , b. YEDVIPEADR (SEQ ID NO: 37), c. DGEKFGDPLGYEDVIPEADR (SEQ ID NO: 17) or d. NAFKILWITDGEKFGDPLGYEDVIPEADREGV (SEQ ID NO:50) e. DGEKF (SEQ ID NO: 51)
6. The peptide of claim 2 wherein said peptide comprises the f ollowing amino acid sequence : YYEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 38) .
73 7. The peptide of claim 2 wherein said peptide comprises the following amino acid sequence: KSTRDRLR (SEQ ID NO: 15) .
8. The peptide of claim 2, said peptide comprising one of the following amino acid sequences: a. AYFGASLCSVDVDSNGSTDLVLIGAP (SEQ ID NO: 1), b. GRFGAALTVLGDVNGDKLTDVAIGAP (SEQ ID NO: 2), C. QYFGQSLSGGQDLTMDGLVDLTVGAQ (SEQ ID NO: 3), d. YEQTRGGQVSVCPLPRGRARWQCDAV (SEQ ID NO: 4), e. DIAFLIDGSGSIIPHDFRRMK (SEQ ID NO: 5) , f. RRMKEFVSTVMEQLKKSKTLF (SEQ ID NO: 6), g. SLMQYSEEFRIHFTFKEFQNN (SEQ ID NO: 7), h. PNPRSLVKPITQLLGRTHTATGIRK (SEQ ID NO: 8), i. RKWRELFNITNGARKNAFK (SEQ ID NO: 9), j. FKILWITDGEKFGDPLGYEDVIPEADR (SEQ ID NO: 10), k. REGVIRYVIGVGDAFRSEKSR (SEQ ID NO: 11) , 1. QELNTIASKPPRDHVFQVNNFE (SEQ ID NO: 12), m. ALKTIQNQLREKIFAIEGT (SEQ ID NO: 13), n. QTGSSSSFEHEMSQE (SEQ ID NO: 14), o. FRSEKSRQELNTIASKPPRDHV (SEQ ID NO: 16), p. KEFQNNPNPRSL (SEQ ID NO: 18), q. GTQTGSSSSFEHEMSQEG (SEQ ID NO: 19), r. SNLRQQPQKFPEALRGCPQEDSD (SEQ ID NO: 20) , S. RQNTGMWESNANVKGT (SEQ ID NO: 21), t. TSGSGISPSHSQRIA (SEQ ID NO: 22), U. NQRGSLYQCDYSTGSCEPIR (SEQ ID NO: 23), v. PRGRARWQC (SEQ ID NO: 24), W. KLSPRLQYFGQSLSGGQDLT (SEQ ID NO: 25) , X. QKSTRDRLREGQ (SEQ ID NO: 26), y. SGRPHSRAVFNETKNSTRRQTQ (SEQ ID NO: 27), z. CETLKLQLPNCIEDPV (SEQ ID NO: 28), a'. FEKNCGNDNICQDDL (SEQ ID NO: 29), b1. VRNDGEDSYRTQ (SEQ I-D NO: 30), C1. SYRKVSTLQNQRSQRS (SEQ ID NO: 31).
9. The peptide of claim 2, said peptide comprising one or more metal binding domains of CDllb.
10. The peptide of claim 9, said metal binding domains encompassing amino acids 358-412, 426-483, 487-553, and 554-614 of CDllb.
11. The peptide of claim 10, said peptide comprising one of the following sequences: a. DVDSNGSTD (SEQ ID NO: 46) , b. DVNGDKLTD (SEQ ID NO: 47) , C. DLTMDGLVD (SEQ ID NO: 48) , or d. DSDMNDAYL (SEQ ID NO: 49)
12. The peptide of claim 1 or 2 wherein said peptide is soluble under physiological conditions.
13. A heterodimer comprising a first peptide and a second peptide, said first peptide comprising at least one extracellular region of a CDll subunit and lacking the transmembrane and cytoplasmic portions of said CDll subunit, said second peptide comprising at least one extracellular region of CD18 and lacking the transmembrane and cytoplasmic portions of CD18, said peptides being associated to form said heterodimer, said heterodimer being capable of inhibiting a CD11/CD18 mediated immune response.
14. The heterodimer of claim 13 wherein said CDll subunit is CDllb.
15. The heterodimer of claim 13 wherein said CDll subunit is CDllc.
16. The heterodimer of claim 14 wherein said heterodimer is CDllb1089/CD18699
17. A method of controlling phagocyte-mediated tissue damage to a human patient, said method comprising administering a therapeutic composition to a patient said therapeutic composition comprising a physiologically acceptable carrier and either a peptide according to claim 1 or 2 or a heterodimer according to claim 13.
18. The method of claim 17 wherein said therapeutic composition is administered to control phagocyte-mediated tissue damage associated with ischemia-reperfussion.
19. The method of claim 17 wherein said therapeutic composition is administered to control phagocyte-mediated tissue damage to the heart muscle associated with reduced perfusion of heart tissue during acute cardiac insufficiency.
20. A method of producing a recombinant β2 integrin heterodimer, said method comprising: (a) providing a recombinant cell encoding a CDll peptide lacking both the transmembrane domain and the cytoplasmic domain and a CD18 peptide lacking both the transmembrane domain and the cytoplasmic domain; (b) culturing said recombinant cell; and (c) isolating said heterodimer from the culture supernatant.
21. The method of claim 20 wherein said recombinant β2 integrin heterodimer is soluble under physiological conditions.
22. The method of claim 20 wherein said CDll peptide is a CDllb peptide.
23. The method of claim 20 wherein said soluble CDll peptide is a recombinant CDllc peptide.
24. A monoclonal antibody which is raised to the peptide of claim 1 or claim 2 or the heterodimer of claim 13, said monoclonal antibody being capable of inhibiting a CD11/CD18 mediated immune response.
PCT/US1991/004338 1990-06-18 1991-06-18 CONTROLLING CELLULAR IMMUNE/INFLAMMATORY RESPONSES WITH β2 INTEGRINS WO1991019511A1 (en)

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US539,842 1990-06-18
US63783091A 1991-01-04 1991-01-04
US637,830 1991-01-04

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