WO1993014220A1 - Antibodies recognizing the fourth immunoglobulin-like domain of vcam1 - Google Patents

Antibodies recognizing the fourth immunoglobulin-like domain of vcam1 Download PDF

Info

Publication number
WO1993014220A1
WO1993014220A1 PCT/US1993/000031 US9300031W WO9314220A1 WO 1993014220 A1 WO1993014220 A1 WO 1993014220A1 US 9300031 W US9300031 W US 9300031W WO 9314220 A1 WO9314220 A1 WO 9314220A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
ser
glu
val
thr
Prior art date
Application number
PCT/US1993/000031
Other languages
French (fr)
Inventor
Laurelee Osborn
Christopher D. Benjamin
Original Assignee
Biogen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biogen, Inc. filed Critical Biogen, Inc.
Priority to JP5512513A priority Critical patent/JPH07506244A/en
Priority to AU34318/93A priority patent/AU664071B2/en
Priority to EP93902915A priority patent/EP0623173A1/en
Publication of WO1993014220A1 publication Critical patent/WO1993014220A1/en

Links

Classifications

    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to monoclonal antibodies
  • MAbs specifically monoclonal antibodies that bind to the domain 4 of Vascular Cell Adhesion Molecule-1 (VCAM1).
  • VCAM1 (also known as INCAM-110) was first identified as an adhesion molecule induced on endothelial cells by inflammatory cytokines (TNF and IL-1) and LPS (Rice et al., 1989 [1]; Osborn et al., 1989 [2]). VCAM1 binds to cells exhibiting the integrin VLA-4 ( ⁇ 4 ⁇ 1 ), including T and B lymphocytes, monocytes, and eosinophils, but not neutrophils, and is thought to participate in recruitment of these cells from the bloodstream to areas of infection and inflammation (Elices et al, 1990 [3];
  • VCAM1/VLA-4 adhesion pathway has been associated with a number of physiological and
  • VCAM1 may participate in metastasis of such tumors (Rice et al., 1989 [1]).
  • VCAM1 is found on areas of arterial endothelium representing early atherosclerotic plaques in a rabbit model system (Cybulsky and Gimbrone, 1991 [5]).
  • VCAM1 is also found on follicular dendritic cells in human lymph nodes (Freedman et al., 1990 [6]). It is also present on bone marrow stromal cells in the mouse (Miyake et al., 1991 [7]), thus VCAM1 appears to play a role in B- cell development.
  • VCAM1 belongs to the immunoglobulin (Ig)
  • VCAM-7D has seven Ig homology units or domains; domains 4, 5 and 6 are similar in amino acid sequence to domains 1, 2 and 3, respectively, suggesting an intergenic duplication event in the evolutionary history of the gene (Osborn et al., 1989 [2]; Polte et al. 1990 [8]; Hession et al., 1991 [9]). There is also a minor 6-domain form (referred to as VCAM-6D herein) generated by alternative splicing, in which the fourth domain is deleted (Osborn et al., 1989 [2]; Hession et al. 1991 [9], Cybulsky et al., 1991 [10]). The biological significance of this alternate splicing is not known, however as shown below VCAM-6D can bind VLA-4-expressing cells and thus clearly has potential functionality in vivo.
  • VCAM1/VLA-4 adhesion pathway The apparent involvement of the VCAM1/VLA-4 adhesion pathway in infection, inflammation and possibly atherosclerosis has led to continuing intensive research to understand the mechanisms of cell-cell adhesion on a molecular level and has led investigators to propose intervention in this adhesion pathway as a treatment for diseases, particularly inflammation (Osborn et al., 1989 [2]).
  • Monoclonal antibodies that inhibit VCAM1 binding to VLA-4 are known.
  • anti-VLA-4 MAbs HP2/1 and HP1/3 have been shown to block attachment of VLA-4- expressing Ramos cells to human umbilical vein cells and VCAM1-transfected COS cells (Elices et al., 1990 [3]).
  • anti-VCAM1 antibodies such as the monoclonal
  • antibody 4B9 (Carlos et al., 1990 [11]) have been shown to inhibit adhesion of Ramos (B-cell-like), Jurkat (T-cell- like) and HL60 (granulocyte-like) cells to COS cells transfected to express VCAM-6D and VCAM-7D (Hession et al., 1991 [9]).
  • VCAM1 antibodies recognizing epitopes on other, non-binding domains of VCAM1 would be important research tools to map the portion(s) of VCAM1 essential for binding to VLA-4.
  • domain 4 of VCAM-7D contains a site involved in VLA-4 binding, which is in addition to the site recognized and blocked by MAb 4B9 (domain 1).
  • VCAM1 is identified as the first known adhesion molecule to have two binding sites for a single receptor (VLA-4); and the domain 4 monoclonal antibodies according to this invention are useful not only in mapping the molecular functionality of VCAM1 but also in at least partially inhibiting VCAM1 recognition by VLA-4.
  • VLA-4-expressing cells adhesion of VLA-4-expressing cells to VCAM1.
  • FIG. 1 is a schematic diagram of VCAM-7D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus. These are the signal peptide ("sig”), immunoglobulin-like domains 1 through 7, the transmembrane sequence ("tm”), and the cytoplasmic domain or “tail” ("cyt"). Also indicated are the relative positions of several restriction enzyme recognition sites, which were used to prepare additional VCAM1 constructs having one or more altered domains
  • FIG. 2 is a schematic diagram of VCAM-6D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus --the signal peptide ("sig”), immunoglobulin-like domains 1, 2, 3, 5, 6 and 7, the transmembrane sequence ("tm”), and the
  • cDNA encoding VCAM-6D having the diagrammed conformation was used to transfect C0S7 cells to produce COS7 cells exhibiting VCAM-6D on their surfaces.
  • FIG. 3 is a schematic diagram of a chimeric VCAM1, designated VCAM/ICAM-1, showing the linear
  • This polypeptide has the general structure of VCAM-7D, except the cDNA encoding VCAM-7D was modified so that a segment encoding most of domain 1 and part of domain 2 was excised and replaced by cDNA encoding analogous portions of Intercellular Adhesion Molecule-1 (ICAM1).
  • IAM1 Intercellular Adhesion Molecule-1
  • FIG. 4 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-2, showing the linear arrangement of functional domains.
  • This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
  • FIG. 5 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-3, showing the linear arrangement of functional domains.
  • This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for part of domain 1, all of domains 2 and 3, and a small N-terminal part of domain 4 was excised and replaced with ICAM1 cDNA.
  • Figure 6 is a graph showing relative levels of expression in COS7 cells transfected with either cDNA encoding VCAM-6D or cDNA encoding VCAM-7D. Expression of either VCAM-6D or VCAM-7D was analyzed by flow
  • Figure 7 is a graph showing the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding VCAM-7D, VCAM-6D, VCAM/ICAM-1, VCAM/ICAM-2, and VCAM/ICAM-3.
  • the effects on Ramos binding of preincubation of the transfected COS cells with 20 ⁇ g/ml of MAb 4B9 (anti-VCAM1) and 10 ⁇ g/ml of MAb HP1/2 (anti- VLA-4) are shown.
  • the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown. Binding is expressed as Ramos cells bound per square millimeter ("Ramos cells bound/mm" in the figures).
  • FIG. 8 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-1, showing the linear arrangement of functional domains. This
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a small part of domain 2 was excised and replaced with ICAM1 cDNA.
  • FIG. 9 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-2, showing the linear arrangement of functional domains. This
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
  • Figure 10 is a graph depicting the extent of Ramos cell binding to COS cells separately tranfected with CDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, VCAM/ICAM-2, and VCAM6D/ICAM-2. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown.
  • FIG 11 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/VCAM4-1, showing the linear arrangement of functional domains.
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for domain 1 and a small part of domain 2 was excised and replaced with cDNA encoding domain 4 (and one amino acid of domain 5) of VCAM-7D.
  • Figure 12 is a graph depicting the extent of Ramos cell binding of COS cells separately tranfected with cDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, and
  • VCAM6D/VCAM4-1 Negligible Ramos binding to ICAM1 and negative (pCDM8) controls is also shown.
  • Figure 13 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any
  • Figure 14 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any interfering antibody or in the presence of MAb 4B9 or MAb
  • Figure 15 is a graph depicting the extent of
  • anti-VCAM1 MAb (anti-VCAM1 MAb, this invention) is compared.
  • an immortal cell line typically murine myeloma cells
  • lymphocytes typically murine myeloma cells
  • splenocytes from a mammal immunized with whole cells expressing a given antigen, e.g., VCAM1, and/or with purified antigen, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen.
  • a given antigen e.g., VCAM1
  • purified antigen e.g., VCAM1
  • the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen. See, generally, Kohler and Milstein,
  • Immunization may be accomplished using standard procedures.
  • the unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc.
  • the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays.
  • anti-VCAM1 antibodies according to the present invention were identified by testing the ability of the immune serum to block Ramos binding to plates coated with a recombinant soluble form of VCAM-7D (rsVCAM-7D).
  • the lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti-VCAM1 antibodies using such screening assays.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants using assays which detect MAbs having the desired
  • hybridomas according to the invention were identified by their ability to bind to VCAM-7D-expressing cells but not to VCAM-6D-expressing cells. (See Example 1, supra.)
  • hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue culture techniques and culture media suitable for
  • hybridoma cells are well known.
  • the conditioned hybridoma culture supernatant may be collected and the anti-VCAM1 antibodies optionally further purified by well-known methods.
  • the desired antibody may be produced by injecting the hybridoma cells into the desired antibody.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the invention recognize epitopes dependent on the fourth Ig domain of the seven-Ig-domain form of VCAM1, i.e., VCAM- 7D. That is, the MAbs of the present invention bind to epitopes that either are contained within the fourth Ig domain of VCAM-7D or are partly contained within the fourth Ig domain, such that elimination of the fourth Ig domain eliminates their ability to recognize the VCAM1 structure.
  • domain 4 MAbs are referred to as "domain 4 MAbs" herein.
  • the domain 4 MAbs bind to VCAM-7D but do not bind to VCAM-6D.
  • All of the antibodies of this invention are distinct from known VCAM1/VLA-4 blocking antibodies such as 4B9.
  • 4B9 recognizes an epitope in domain 1 of VCAM1, and thus recognizes both VCAM-7D and VCAM-6D.
  • the monoclonal antibodies of the present invention recognize VCAM-7D but do not cross-block MAb 4B9. Some of the monoclonal antibodies described herein are effective to inhibit adhesion of Ramos and other VLA-4-expressing cells to VCAM1-expressing cells.
  • Some of the domain 4 monoclonal antibodies described herein bind to epitopes of VCAM1 which are not involved in the adhesion pathway between mononuclear leukocytes and VCAM1-expressing cells that is mediated by VCAM1.
  • This latter type of antibody may be useful, for example, to detect the presence of VCAM1, in particular VCAM-7D, in vitro or in vivo without interference with the binding of VCAM1.
  • inventions may be produced naturally as outlined above or may be synthesized using recombinant DNA techniques.
  • Suitable recombinant antibodies include antibodies
  • suitable expression vector containing DNA encoding the light and heavy immunoglobulin chains of the desired antibody, and recombinant chimeric antibodies, wherein some or all of the hinge, constant and/or variable regions of the heavy and/or the light chains of the anti-VCAM1 antibody have been substituted with corresponding regions of an immunoglobulin light or heavy chain of a different species.
  • This includes so-called "humanized” antibodies. See, e.g., Jones et al., 1986 [13], Ward et al., 1989 [14], and U.S. Patent 4,816,397 (Boss et al.) [15], all incorporated herein by reference.)
  • domain 4-binding fragments of anti- VCAM1 antibodies such as Fab, Fab', F(ab) 2 , and F(v) fragments; heavy chain monomers or dimers; light chain monomers or dimers; and dimers consisting of one heavy chain and one light chain are also contemplated herein.
  • antibody fragments may be produced by chemical methods, e.g., by cleaving an intact antibody with a protease, such as papain or pepsin, or via recombinant DNA techniques, e.g., by using host cells transformed with truncated heavy and/or light chain genes.
  • Heavy and light chain monomers may similarly be produced by treating an intact antibody with a reducing agent such as
  • antibody fragments having similar specificities may be isolated by phage cloning methods. (See, e.g., Clackson et al. (1991) [16].)
  • Monoclonal antibodies of the present invention may be used in any application where antibody recognition of domain 4 of VCAM1 is advantageous, including
  • monoclonal antibodies of the present invention may be used in the treatment of inflammation or diseases characterized by leukocyte binding to endothelium, such as post-reperfusion injury, microbial infections or other cell migration
  • the present monoclonal antibodies may be used in combination with 4B9 or other anti-VCAM1 antibodies in the treatment of disorders characterized by VCAM1 expression in endothelium.
  • the present monoclonal antibodies may be used in combination with antibodies recognizing other endothelial cell
  • the antibodies of the present invention, or VCAM1- recognizing fragments thereof may be combined or linked to cytotoxic molecules such as TNF, ricin or the A chain of diphtheria toxin, in order to provide antibody/toxin conjugates capable of targeting cytotoxic elements to VCAM1-expressing cells.
  • the antibodies of the present invention may also be immobilized on a chromatographic substrate (e.g., Protein A-sepharose) to provide an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.
  • a chromatographic substrate e.g., Protein A-sepharose
  • an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.
  • Detectably labeled antibodies according to the present invention may also be used in screening methods or diagnostic methods for detecting VCAM1 protein in a sample or VCAM1-expressing endothelial cells in vitro or in vivo.
  • samples may be screened for the presence of VCAM1 or VCAM1-expressing cells by contacting the sample with a labeled antibody according to the present invention and detecting whether a VCAM1/anti-VCAM1 antibody complex is formed.
  • Suitable labels can be radioactive, enzymatic, fluorescent, magnetic or chemiluminescent.
  • Radiolabeled antibodies are prepared in known ways by coupling a radioactive isotope such as 3 H, 32 P, 35 S, 59 Fe, 125 I, which can then be detected by gamma counter, scintillation counter or by autoradiography.
  • Antibodies of this invention may be suitably labeled with enzymes such as yeast alcohol dehydrogenase, horseradish peroxidase, alkaline
  • Suitable fluorescent labels include fluorescein isothiocyanate, fluorescamine, rhodamine, and the like.
  • Suitable chemiluminescent labels include luminol, imidazole, oxalate ester, luciferin, and the like.
  • the monoclonal antibodies of the present invention may be formulated as a
  • composition comprising an effective amount of the antibody admixed with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier typically, the antibodies of the present
  • compositions may alternatively be formulated to control release of the active ingredients or to prolong their presence in a patient's system.
  • suitable drug delivery systems include, e.g., hydrogels,
  • microemulsions are microspheres, and the like.
  • compositions contemplated herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally.
  • VCAM-7D Monoclonal antibodies according to the invention that recognize domain 4 of VCAM-7D were prepared in the following manner.
  • VCAM- 6D/CHO whole CHO cells that express VCAM-6D on their surfaces
  • the CHO culture was a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA). Initially, each mouse received approximately 10 7 cells in PBS intraperitoneally (i.p.).
  • i.p. a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA).
  • each mouse received approximately 10 7 cells in PBS intraperitoneally (i.p.).
  • rsVCAM-7D a recombinant soluble form of VCAM- 7D (rsVCAM-7D, consisting of the N-terminal 674 amino acids of mature VCAM-7D and lacking the transmembrane and cytoplasmic regions; obtained from Biogen, Inc.,
  • PBS buffered saline
  • FCS fetal calf serum
  • the cells in positive culture wells were:
  • the COS7 cells transfected with either VCAM-6D or VCAM-7D cDNA, were washed and suspended in suspension buffer (PBS/5mM EDTA/2% FCS/0.05% sodium azide) and incubated 45 min. at 4° C with supernatant from hybridoma cultures. The transfected cells were then washed with suspension buffer and incubated with a fluoresceinated goat anti-mouse Ig (Jackson Immunoresearch, West Grove PA) for 45 min. at 4° C. The cells were washed, fixed with 0.2% paraformaldehyde in PBS and evaluated using a FACStar (Becton Dickinson, Mountain View CA).
  • FACStar Becton Dickinson, Mountain View CA.
  • Ramos is a B-lymphoblastoid cell line that expresses VLA-4 and thus binds to VCAM1 but does not bind to other induced endothelial cell molecules such as ICAM-1 or ELAM-1 (Osborn et al., 1989 [2]). Ramos is therefore a convenient model cell line with which to assay VLA-4- dependent binding. Ramos cells bind to both recombinant VCAM-6D and recombinant VCAM-7D when these molecules are expressed on COS cells.
  • VCAM-7D binding to VCAM-7D was somewhat (less than 2 fold) greater than to VCAM-6D in transient transfection assays of a single preparation of each plasmid (Hession et al., 1991 [9]). To determine if this quantitative difference in binding activity was reproducible, several preparations of each plasmid were made and electroporated in parallel into COS cells. Repeated transfections revealed that although expression of the two plasmids is similar, VCAM-7D on average binds significantly more cells per square
  • VCAM-6D millimeter than does VCAM-6D.
  • Binding to both six- and seven-domain forms of VCAM1 was inhibited by the anti-VLA-4 MAb HP1/2 or by anti-VCAM MAb 4B9 ( Figure 7).
  • the binding of Ramos cells was measured as follows: Confluent monolayers of the target cells (e.g., COS transfectants or induced HUVECs) in 48-well plastic tissue culture plates were washed with RPMI/10% fetal calf serum (FCS), and an amount (e.g., 10 ⁇ g/ml, varying per MAb and per experiment) of the test MAb (e.g., 4B9, HP1/2, GH12, etc.) in a volume of 0.15 ml was added.
  • FCS RPMI/10% fetal calf serum
  • the cells were preincubated 4, 24, 48 or 72 hours at 37° C with 20 ng/ml recombinant human TNF (Biogen, Inc.) to induce expression of VCAM1.
  • Ramos cells were fluorescently labeled by adding 2',7'-bis(2-carboxyethyl)- 5(and 6)-carboxy-fluorescein ("BCECF", Molecular Probes, Eugene OR) according to the manufacturer's instructions.
  • BCECF 2',7'-bis(2-carboxyethyl)- 5(and 6)-carboxy-fluorescein
  • the cells were first centrifuged and then resuspended in RPMI/10% fetal calf serum (FCS) to a concentration of 5 x 10 6 cells ml.
  • FCS fetal calf serum
  • VCAM-7D VCAM-7D
  • VCAM-6D various VCAM/ICAM constructs exhibiting domain 4-dependent cell binding
  • COS7 cells transfected with a particular VCAM, ICAM (control) or chimeric VCAM/ICAM (or VCAM/VCAM, see Fig. 11) cDNA construct were removed from 100 mm culture dishes by incubation in HBSS/5 mM EDTA for 15 min. at 37° C, followed by vigorous pipetting.
  • VCAM1/ICAM chimeras were constructed by excising portions of the VCAM-7D and VCAM-6D cDNAs and replacing them with analogous regions of ICAM-1, using unique restriction endonuclease sites in the VCAM1 cDNA (see Fig. 1) and polymerase chain reaction (PCR)-generated fragments of ICAM1. Substitution of the excised VCAM1 regions was done to eliminate as much as possible the structural distortions that would accompany deletion of one or more domains of the VCAM1 molecules. ICAM1 was selected as a source of substituent structures because it is also a member of the Ig superfamily and has the highest degree of amino acid homology to VCAM1 among Ig
  • VCAM1/ICAM1 chimeric polypeptides are shown in Figures 1-5, 8, 9 and 11.
  • the cDNA constructs coding for each of these were prepared as follows:
  • ICAM1 inserts were made by PCR from an ICAM1/CDM8 plasmid isolated from an endothelial cDNA library previously described (Osborn et al., 1989 [2]), using oligonucleotide probes based on the published sequence of ICAM1 (Simmons et al., 1988 [19]; Staunton et al., 1988 [20]).
  • the PCR primers were designed as follows: Primer P-1 (SEQ ID NO: 1)
  • the first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues 9-12; the following twenty-three nucleotides correspond to the coding region for ICAM1 amino acid residues 11-18.
  • Primer P-2 (SEQ ID NO: 2 )
  • the first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 86-89 ; the following twenty-one nucleotides correspond to the coding region for
  • Primer P-3 (SEQ ID NO: 3)
  • the first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues -3-1; the following twenty nucleotides correspond to the coding region for
  • Primer P-4 (SEO ID NO: 4)
  • the first fourteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following twenty-two nucleotides correspond to the coding region for
  • Primer P-5 (SEQ ID NO: 5)
  • the first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 134-137; the following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 121-128.
  • Primer P-6 (SEO ID NO: 6)
  • the first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 302-305; the
  • nucleotides correspond to the coding region for ICAM1 amino acid residues 279-286.
  • Primer P-7 (SEO ID NO: 7)
  • the first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following eighteen nucleotides correspond to the coding region for VCAM1 amino acid residues 385-394.
  • VCAM/ICAM-1 (Fig. 3) was primed with P-1 and P-4; for construct VCAM/ICAM-2 (Fig. 4), P-1 and P-5; for construct VCAM/ICAM-3 (Fig. 5), P-2 and P-6; for construct
  • VCAM6D/VCAM4-1 (Fig. 11), P-3 and P-7.
  • VCAM6D/ICAM-1 and VCAM6D/ICAM-2 (Figs. 8 and 9) were made using the same inserts as for VCAM/ICAM-1 and VCAM/ICAM-2, respectively, cloned into the VCAM-6D expression vector (VCAM41/CDM8) instead of the VCAM-7D expression vector (VCAM1E11/CDM8).
  • VCAM41/CDM8 VCAM-6D expression vector
  • VCAM7D expression vector VCAM1E11/CDM8
  • VCAM/ICAM-3 VCAM6D/ICAM-1, VCAM6D/ICAM-2, and
  • VCAM6D/VCAM4-1 are set forth in the Sequence Listing below (in order, SEQ ID NOS: 8 through 15).
  • COS7 cells found to be well expressed when tested by FACS analysis after staining with polyclonal antiserum to VCAM1.
  • VCAM/ICAM-1 (Fig. 3) and VCAM/ICAM-2 (Fig. 4)
  • ICAM1 sequence see shaded areas, Figs. 3 and 4,
  • VCAM/ICAM-3 (Fig. 5)
  • VCAM domains 2 and 3 and small portions of domains 1 and 4 were replaced by ICAM1 sequence (see shaded area, Fig. 5).
  • VCAM1 significantly inhibits binding of VLA-4-bearing cells to induced HUVECs or VCAM1 transfectants (Carlos et al., 1990 [22]). These results suggested that the binding site of VCAM1 recognized by VLA-4 and obstructed by 4B9 is in the N-terminal portion of the VCAM1 molecule.
  • VCAM/ICAM-1 and VCAM/ICAM-2 did not bind MAb 4B9, both bound Ramos cells at about 30% of the level seen with intact VCAM-7D ( Figure 7).
  • This binding was mediated by VLA-4/VCAM1, since anti-VLA-4 mAb HP1/2 completely inhibited the interaction, while a blocking antibody to the ICAM-1 pathway (MAb 60.3) did not affect binding (data not shown).
  • Construct VCAM/ICAM-3 bound Ramos cells weakly but measurably, at about 10% of the level seen with intact VCAM-7D ( Figure 7).
  • VCAM/ICAM-1 and VCAM/ICAM-2 were made, except that cDNA coding for VCAM-6D (no domain 4) instead of VCAM-7D was used as the substrate or backbone for the substituted ICAM1 inserts (see. Figs. 8 and 9). These constructs, designated VCAM6D/ICAM-1 and
  • VCAM6D/ICAM-2 were well expressed in COS7 cells as measured by anti-VCAM1 polyclonal antiserum staining, but were unable to bind Ramos cells (Figure 10). These results indicated that the binding of Ramos cells to constructs VCAM/ICAM-1 and VCAM/ICAM-2 appears to be due to the presence of domain 4.
  • VCAM6D/VCAM4-1 in which domain 4 of VCAM-7D was
  • MAb GH12 bound to COS7 transfectants expressing VCAM-7D, VCAM/ICAM-1 and 2, and VCAM6D/VCAM4-1 but did not bind to transfectants expressing VCAM-6D or VCAM/ICAM-3.
  • Ramos binding experiments showed that MAb GH12 at 10 ⁇ g/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1, VCAM/ICAM-2 and VCAM6D/VCAM4-1 constructs but did not bind to the
  • VCAM/ICAM-3-expressing tranfectants indicating that the small portion of domain 4 eliminated from the VCAM/ICAM-3 construct was important to GH12 binding.
  • MAb GH12 did not block Ramos cell binding to VCAM-6D or VCAM-7D.
  • Binding experiments showed that MAb ED11 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-l and
  • VCAM/ICAM-2 but not to cells expressing VCAM-6D
  • VCAM/ICAM-3 or VCAM6D/VCAM4-1 VCAM/ICAM-3 or VCAM6D/VCAM4-1.
  • MAb ED11 at 5 ⁇ g/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1 and VCAM/ICAM-2 constructs but did not inhibit VCAM-6D-expressing, VCAM/ICAM-3-expressing or VCAM6D/VCAM4-1-expressing tranfectants.
  • MAb ED11 did partially block Ramos cell binding to VCAM-7D- expressing COS7 cells.
  • VCAM/ICAM-1 and VCAM/ICAM-2 did not block Ramos adhesion to transfectants expressing VCAM6D/VCAM4-1
  • VCAM/ICAM-3 or VCAM-6D did not inhibit Ramos binding to VCAM-7D-expressing COS7 cells at the single concentration tested.
  • EDll or GH12 were capable of blocking adhesion to VCAM1 on endothelial cells (which is predominantly VCAM-7D), human umbilical vein endothelial cells (HUVECs) were treated with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72 hours, and binding of Ramos cells and the ability of the antibodies to block binding was measured. All MAbs were added at a concentration of 10 ⁇ g/ml. The results are shown for each activation period in Figures 16-19.
  • VCAM-7D is the predominantly expressed (RNA and protein) form of VCAM-1
  • VCAM-6D may be the predominant species of VCAM-1 in bone marrow stromal cells and tonsillar follicular dendritic cells. This suggests a potential therapeutic advantage of domain 4 specific anti-VCAM1 antibodies over domain 1 specific anti-VCAM1 antibodies, in that domain 4 antibodies
  • lymphopoiesis which is the role that VCAM-1 plays in bone marrow (Miyake et al., 1977 [7]). Such an inhibition of B-cell lymphopoiesis may be undesirable in longer term therapy with anti-VCAM1 antibodies.
  • VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site," Cell, 60, 577-584.
  • VCAM1 vascular cell adhesion molecule-1
  • Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine- activated cultured human endothelial cells. Blood, 76 , 965-970.
  • ICAM an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM," Nature, 331, 624-627.
  • AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
  • AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CIT GTA GAA GCA CAG AAA 2208 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
  • GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392 Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
  • AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC 1680 Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
  • Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
  • GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
  • GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT ATT 672 Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle
  • CAA GAG AAA CCA TTT ACT CTT GAG ATC TCC CCT GGA CCC CGG ATT GCT 960 Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle Ala
  • GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG CTG 1296 Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu
  • GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG TTG 1968 Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln Leu

Abstract

Monoclonal antibodies recognizing immunoglobulin domain 4 of Vascular Cell Adhesion Molecule-1 (VCAM1) are described. Such antibodies are useful, e.g., in the treatment of acute inflammation and disorders characterized by VCAM1-mediated adhesion of leukocytes and in purification methods, diagnostic methods and diagnostic kits, especially where distinguishing between the six-domain and seven-domain forms of VCAM1 is advantageous.

Description

ANTIBODIES RECOGNIZING THE FOURTH
IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM1
FIELD OF THE INVENTION
This invention relates to monoclonal antibodies
(MAbs), specifically monoclonal antibodies that bind to the domain 4 of Vascular Cell Adhesion Molecule-1 (VCAM1).
BACKGROUND OF THE INVENTION
VCAM1 (also known as INCAM-110) was first identified as an adhesion molecule induced on endothelial cells by inflammatory cytokines (TNF and IL-1) and LPS (Rice et al., 1989 [1]; Osborn et al., 1989 [2]). VCAM1 binds to cells exhibiting the integrin VLA-4 (α4β1), including T and B lymphocytes, monocytes, and eosinophils, but not neutrophils, and is thought to participate in recruitment of these cells from the bloodstream to areas of infection and inflammation (Elices et al, 1990 [3];
Osborn, 1990 [4]). The VCAM1/VLA-4 adhesion pathway has been associated with a number of physiological and
pathological processes. Although VLA-4 is normally restricted to hematopoietic lineages, it is found on melanoma cell lines, and thus it has been suggested that VCAM1 may participate in metastasis of such tumors (Rice et al., 1989 [1]). In vivo, VCAM1 is found on areas of arterial endothelium representing early atherosclerotic plaques in a rabbit model system (Cybulsky and Gimbrone, 1991 [5]). VCAM1 is also found on follicular dendritic cells in human lymph nodes (Freedman et al., 1990 [6]). It is also present on bone marrow stromal cells in the mouse (Miyake et al., 1991 [7]), thus VCAM1 appears to play a role in B- cell development.
VCAM1 belongs to the immunoglobulin (Ig)
superfamily. The major form on endothelial cells,
referred to herein as VCAM-7D, has seven Ig homology units or domains; domains 4, 5 and 6 are similar in amino acid sequence to domains 1, 2 and 3, respectively, suggesting an intergenic duplication event in the evolutionary history of the gene (Osborn et al., 1989 [2]; Polte et al. 1990 [8]; Hession et al., 1991 [9]). There is also a minor 6-domain form (referred to as VCAM-6D herein) generated by alternative splicing, in which the fourth domain is deleted (Osborn et al., 1989 [2]; Hession et al. 1991 [9], Cybulsky et al., 1991 [10]). The biological significance of this alternate splicing is not known, however as shown below VCAM-6D can bind VLA-4-expressing cells and thus clearly has potential functionality in vivo.
The apparent involvement of the VCAM1/VLA-4 adhesion pathway in infection, inflammation and possibly atherosclerosis has led to continuing intensive research to understand the mechanisms of cell-cell adhesion on a molecular level and has led investigators to propose intervention in this adhesion pathway as a treatment for diseases, particularly inflammation (Osborn et al., 1989 [2]). Monoclonal antibodies that inhibit VCAM1 binding to VLA-4 are known. For example, anti-VLA-4 MAbs HP2/1 and HP1/3 have been shown to block attachment of VLA-4- expressing Ramos cells to human umbilical vein cells and VCAM1-transfected COS cells (Elices et al., 1990 [3]). Also, anti-VCAM1 antibodies such as the monoclonal
antibody 4B9 (Carlos et al., 1990 [11]) have been shown to inhibit adhesion of Ramos (B-cell-like), Jurkat (T-cell- like) and HL60 (granulocyte-like) cells to COS cells transfected to express VCAM-6D and VCAM-7D (Hession et al., 1991 [9]).
Although blocking antibodies that might be potential therapeutics are known, new monoclonal
antibodies recognizing epitopes on other, non-binding domains of VCAM1 would be important research tools to map the portion(s) of VCAM1 essential for binding to VLA-4. Antibodies that bind to VCAM1 without competing for the epitope of a blocking antibody such as 4B9 would also have important diagnostic uses. For instance, a radiolabeled anti-VCAM1 antibody recognizing an epitope not essential to VLA-4 binding could be used to monitor the course and location of acute inflammatory events but would not interfere with the action of a later-administered
therapeutic that interacts with the VLA-4-binding domain of VCAM1.
SUMMARY OF THE INVENTION
We have now isolated a panel of monoclonal antibodies which bind to VCAM-7D but not to VCAM-6D, and therefore presumably recognize an epitope in domain 4 of VCAM1. We have surprisingly discovered that some of these domain 4 antibodies inhibit Ramos binding to VCAM-7D- expressing cells, and through the use of these antibodies we have demonstrated that domain 4 of VCAM-7D contains a site involved in VLA-4 binding, which is in addition to the site recognized and blocked by MAb 4B9 (domain 1). Thus, VCAM1 is identified as the first known adhesion molecule to have two binding sites for a single receptor (VLA-4); and the domain 4 monoclonal antibodies according to this invention are useful not only in mapping the molecular functionality of VCAM1 but also in at least partially inhibiting VCAM1 recognition by VLA-4.
Accordingly, it is an object of this invention to provide monoclonal antibodies recognizing VCAM-7D but not VCAM-6D.
It is a further object of this invention to provide antibodies recognizing an epitope dependent on the fourth immunoglobulin-like domain ("domain 4") of VCAM-7D ("domain 4 antibodies"), and particularly domain 4
antibodies that do not cross-block the anti-VCAM1 MAb 4B9.
It is a further object of the present invention to provide domain 4 antibodies capable of blocking
adhesion of VLA-4-expressing cells to VCAM1.
These and other objects are accomplished by the monoclonal antibodies described and claimed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram of VCAM-7D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus. These are the signal peptide ("sig"), immunoglobulin-like domains 1 through 7, the transmembrane sequence ("tm"), and the cytoplasmic domain or "tail" ("cyt"). Also indicated are the relative positions of several restriction enzyme recognition sites, which were used to prepare additional VCAM1 constructs having one or more altered domains
(described infra). In the examples set forth below, cDNA encoding VCAM-7D having the diagrammed conformation was used to transfect COS7 cells to produce COS7 cells
exhibiting VCAM-7D on their surfaces.
Figure 2 is a schematic diagram of VCAM-6D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus --the signal peptide ("sig"), immunoglobulin-like domains 1, 2, 3, 5, 6 and 7, the transmembrane sequence ("tm"), and the
cytoplasmic domain or "tail" ("cyt"). In the examples set forth below, cDNA encoding VCAM-6D having the diagrammed conformation was used to transfect C0S7 cells to produce COS7 cells exhibiting VCAM-6D on their surfaces.
Figure 3 is a schematic diagram of a chimeric VCAM1, designated VCAM/ICAM-1, showing the linear
arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except the cDNA encoding VCAM-7D was modified so that a segment encoding most of domain 1 and part of domain 2 was excised and replaced by cDNA encoding analogous portions of Intercellular Adhesion Molecule-1 (ICAM1). The modified portion of the VCAM-7D molecule is indicated by shading.
Figure 4 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-2, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
Figure 5 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-3, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for part of domain 1, all of domains 2 and 3, and a small N-terminal part of domain 4 was excised and replaced with ICAM1 cDNA.
Figure 6 is a graph showing relative levels of expression in COS7 cells transfected with either cDNA encoding VCAM-6D or cDNA encoding VCAM-7D. Expression of either VCAM-6D or VCAM-7D was analyzed by flow
cytofluorometry following indirect immunofluorescent staining with MAb 4B9. The number of cells with
fluorescent intensity greater than that shown by 99% of control transfectants (pCDM8 vector only) was determined. The data depicted combines the results of two experiments; standard deviation is indicated by error bars.
Figure 7 is a graph showing the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding VCAM-7D, VCAM-6D, VCAM/ICAM-1, VCAM/ICAM-2, and VCAM/ICAM-3. In addition, the effects on Ramos binding of preincubation of the transfected COS cells with 20 μg/ml of MAb 4B9 (anti-VCAM1) and 10 μg/ml of MAb HP1/2 (anti- VLA-4) are shown. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown. Binding is expressed as Ramos cells bound per square millimeter ("Ramos cells bound/mm" in the figures).
Figure 8 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-1, showing the linear arrangement of functional domains. This
polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a small part of domain 2 was excised and replaced with ICAM1 cDNA.
Figure 9 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-2, showing the linear arrangement of functional domains. This
polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
Figure 10 is a graph depicting the extent of Ramos cell binding to COS cells separately tranfected with CDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, VCAM/ICAM-2, and VCAM6D/ICAM-2. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown.
Figure 11 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/VCAM4-1, showing the linear arrangement of functional domains. This
polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for domain 1 and a small part of domain 2 was excised and replaced with cDNA encoding domain 4 (and one amino acid of domain 5) of VCAM-7D.
Figure 12 is a graph depicting the extent of Ramos cell binding of COS cells separately tranfected with cDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, and
VCAM6D/VCAM4-1. Negligible Ramos binding to ICAM1 and negative (pCDM8) controls is also shown.
Figure 13 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any
interfering antibody or in the presence of MAb 4B9 or MAb GH12 (this invention).
Figure 14 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any interfering antibody or in the presence of MAb 4B9 or MAb
ED11 (this invention).
Figure 15 is a graph depicting the extent of
Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any
interfering antibody or in the presence of MAb 4B9 or MAb
GE4 (this invention).
Figures 16, 17, 18 and 19 are graphs showing
Ramos cell binding to cultured HUVECs treated with 20 ng/ml of recombinant human TNF for 4 hours (Fig. 16), 24 hours (Fig. 17), 48 hours (Fig. 18), and 72 hours (Fig.
19). Binding of Ramos cells to uninduced HUVECs
(control), induced (TNF-treated) HUVECs alone, and in the presence of HP1/2 (anti-VLA-4 α4 MAb), 4B9 (anti-VCAM1 MAb), ED11 (anti-VCAM1 MAb, this invention), and GH12
(anti-VCAM1 MAb, this invention) is compared.
DETAILED DESCRIPTION OF THE INVENTION
The technology for producing monoclonal antibodies is well known. Briefly, an immortal cell line (typically murine myeloma cells) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with whole cells expressing a given antigen, e.g., VCAM1, and/or with purified antigen, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen. See, generally, Kohler and Milstein,
1975 [12].
Immunization may be accomplished using standard procedures. The unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc. Typically, the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays. For example, anti-VCAM1 antibodies according to the present invention were identified by testing the ability of the immune serum to block Ramos binding to plates coated with a recombinant soluble form of VCAM-7D (rsVCAM-7D). The lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti-VCAM1 antibodies using such screening assays.
Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol
(e.g., PEG 3350). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants using assays which detect MAbs having the desired
specificity. For example, hybridomas according to the invention were identified by their ability to bind to VCAM-7D-expressing cells but not to VCAM-6D-expressing cells. (See Example 1, supra.)
To produce anti-VCAM1 antibodies, hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue culture techniques and culture media suitable for
hybridoma cells are well known. The conditioned hybridoma culture supernatant may be collected and the anti-VCAM1 antibodies optionally further purified by well-known methods.
Alternatively, the desired antibody may be produced by injecting the hybridoma cells into the
peritoneal cavity of a mouse primed with 2,6,10,14- tetramethylpentadecane (PRISTANE; Sigma Chemical Co., St. Louis MO). The hybridoma cells proliferate in the
peritoneal cavity, secreting the antibody which
accumulates as ascites fluid. The antibody may be
harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe.
The monoclonal antibodies of the present
invention recognize epitopes dependent on the fourth Ig domain of the seven-Ig-domain form of VCAM1, i.e., VCAM- 7D. That is, the MAbs of the present invention bind to epitopes that either are contained within the fourth Ig domain of VCAM-7D or are partly contained within the fourth Ig domain, such that elimination of the fourth Ig domain eliminates their ability to recognize the VCAM1 structure. These MAbs are referred to as "domain 4 MAbs" herein.
The domain 4 MAbs bind to VCAM-7D but do not bind to VCAM-6D. All of the antibodies of this invention are distinct from known VCAM1/VLA-4 blocking antibodies such as 4B9. We show herein that 4B9 recognizes an epitope in domain 1 of VCAM1, and thus recognizes both VCAM-7D and VCAM-6D. The monoclonal antibodies of the present invention recognize VCAM-7D but do not cross-block MAb 4B9. Some of the monoclonal antibodies described herein are effective to inhibit adhesion of Ramos and other VLA-4-expressing cells to VCAM1-expressing cells. Some of the domain 4 monoclonal antibodies described herein bind to epitopes of VCAM1 which are not involved in the adhesion pathway between mononuclear leukocytes and VCAM1-expressing cells that is mediated by VCAM1. This latter type of antibody may be useful, for example, to detect the presence of VCAM1, in particular VCAM-7D, in vitro or in vivo without interference with the binding of VCAM1.
Using the monoclonal antibodies of the present invention and other anti-VCAM1 MAbs obtained from other investigators, we have discovered that binding of VLA-4 to VCAM1 can occur via two separate sites, one requiring domain 1, and the other requiring domain 4. The fact that some of the anti-VCAM1 domain 4 MAbs of this invention can inhibit domain 4-dependent cell binding and can inhibit cell binding to VCAM1 on cultured endothelial cells, indicates that binding through domain 4 is a significant functional component of the VCAM1/VLA-4 interaction in vivo.
The monoclonal antibodies of the present
invention may be produced naturally as outlined above or may be synthesized using recombinant DNA techniques.
Suitable recombinant antibodies include antibodies
produced, e.g., by transforming a host cell with a
suitable expression vector containing DNA encoding the light and heavy immunoglobulin chains of the desired antibody, and recombinant chimeric antibodies, wherein some or all of the hinge, constant and/or variable regions of the heavy and/or the light chains of the anti-VCAM1 antibody have been substituted with corresponding regions of an immunoglobulin light or heavy chain of a different species. This includes so-called "humanized" antibodies. (See, e.g., Jones et al., 1986 [13], Ward et al., 1989 [14], and U.S. Patent 4,816,397 (Boss et al.) [15], all incorporated herein by reference.)
Furthermore, domain 4-binding fragments of anti- VCAM1 antibodies, such as Fab, Fab', F(ab)2, and F(v) fragments; heavy chain monomers or dimers; light chain monomers or dimers; and dimers consisting of one heavy chain and one light chain are also contemplated herein. Such antibody fragments may be produced by chemical methods, e.g., by cleaving an intact antibody with a protease, such as papain or pepsin, or via recombinant DNA techniques, e.g., by using host cells transformed with truncated heavy and/or light chain genes. Heavy and light chain monomers may similarly be produced by treating an intact antibody with a reducing agent such as
dithiothreitol or β-mercaptoethanol or by using host cells transformed with DNA encoding either the desired heavy chain or light chain or both. As an alternative to hybridoma technology, antibody fragments having similar specificities may be isolated by phage cloning methods. (See, e.g., Clackson et al. (1991) [16].)
Monoclonal antibodies of the present invention may be used in any application where antibody recognition of domain 4 of VCAM1 is advantageous, including
applications where inhibiting binding between VCAM1 and its ligand, VLA-4, is desired. For example, monoclonal antibodies of the present invention may be used in the treatment of inflammation or diseases characterized by leukocyte binding to endothelium, such as post-reperfusion injury, microbial infections or other cell migration
(e.g., metastasis) dependent on a VCAM1-mediated binding pathway, vasculitis, etc. The monoclonal antibodies of the present
invention may also be used in combination with other antibodies, bioactive agents or materials for various purposes. For example, the present monoclonal antibodies may be used in combination with 4B9 or other anti-VCAM1 antibodies in the treatment of disorders characterized by VCAM1 expression in endothelium. Alternatively, the present monoclonal antibodies may be used in combination with antibodies recognizing other endothelial cell
receptors identified in inflammatory events (e.g., ELAM1, ICAM1, etc.) in a combination therapy designed to suppres a range of leukocyte-endothelial cell binding pathways. Also, the antibodies of the present invention, or VCAM1- recognizing fragments thereof, may be combined or linked to cytotoxic molecules such as TNF, ricin or the A chain of diphtheria toxin, in order to provide antibody/toxin conjugates capable of targeting cytotoxic elements to VCAM1-expressing cells. The antibodies of the present invention may also be immobilized on a chromatographic substrate (e.g., Protein A-sepharose) to provide an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.
Detectably labeled antibodies according to the present invention may also be used in screening methods or diagnostic methods for detecting VCAM1 protein in a sample or VCAM1-expressing endothelial cells in vitro or in vivo. For example, samples may be screened for the presence of VCAM1 or VCAM1-expressing cells by contacting the sample with a labeled antibody according to the present invention and detecting whether a VCAM1/anti-VCAM1 antibody complex is formed. Suitable labels can be radioactive, enzymatic, fluorescent, magnetic or chemiluminescent. Radiolabeled antibodies are prepared in known ways by coupling a radioactive isotope such as 3H, 32P, 35S, 59Fe, 125I, which can then be detected by gamma counter, scintillation counter or by autoradiography. Antibodies of this invention may be suitably labeled with enzymes such as yeast alcohol dehydrogenase, horseradish peroxidase, alkaline
phosphatase, and the like, then developed and detected spectrophotometrically or visually. Suitable fluorescent labels include fluorescein isothiocyanate, fluorescamine, rhodamine, and the like. Suitable chemiluminescent labels include luminol, imidazole, oxalate ester, luciferin, and the like.
For therapeutic uses, the monoclonal antibodies of the present invention may be formulated as a
pharmaceutical composition comprising an effective amount of the antibody admixed with a pharmaceutically acceptable carrier. Typically, the antibodies of the present
invention will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of the active ingredients or to prolong their presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., hydrogels,
hydroxymethylcellulose, microcapsules, liposomes,
microemulsions, microspheres, and the like.
The pharmaceutical compositions contemplated herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally.
Ordinarily, intravenous (i.v.) or parenteral
administration will be preferred. Example 1
MAbs Recognizing VCAM-7D But Not VCAM-6D
Monoclonal antibodies according to the invention that recognize domain 4 of VCAM-7D were prepared in the following manner. We injected four BALB/c mice with whole CHO cells that express VCAM-6D on their surfaces ("VCAM- 6D/CHO" cells). The CHO culture was a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA). Initially, each mouse received approximately 107 cells in PBS intraperitoneally (i.p.). We injected complete Freund's adjuvant i.p. at a different site 2-24 hours later. We boosted the mice twice i.p. with VCAM- 6D/CHO cells, without adjuvant. We then immunized each of the mice with 55 μg of a recombinant soluble form of VCAM- 7D (rsVCAM-7D, consisting of the N-terminal 674 amino acids of mature VCAM-7D and lacking the transmembrane and cytoplasmic regions; obtained from Biogen, Inc.,
Cambridge, MA) injected intraperitoneally with incomplete Freund's adjuvant. We boosted the mice twice with rsVCAM- 7D i.p. without adjuvant. Four days after the rsVCAM1 injections, we removed the spleen from one mouse and fused the splenocytes with P3/X63-Ag8.653 murine myeloma cells according to the method described by Lerner [17].
Supernatants of cultures of individual clones of fused cells were screened for their ability to bind to rsVCAM-7D-coated plates. ELISA plates (Corning) were coated with rsVCAM-7D (0.1 μg/ml, 50 μl/well) and
incubated overnight at 4° C. The coating solution was aspirated and blocking buffer was added (phosphate
buffered saline (PBS) with 5% fetal calf serum (FCS), pH 7.2) to block the remaining sites, and the plates were incubated for 2 hrs. at room temperature. The blocking buffer was removed, and 50 μl of hybridoma culture
supernatants (or supernatant diluted into blocking buffer) were added to the plates and then incubated for 1 hr. at room temperature. Next, the plates were washed with PBS containing 0.05% detergent (Tween-20) to remove unbound antibody and incubated with a horseradish peroxidase- conjugated goat anti-mouse immunoglobulin for 1 hr. at room temperature. The plates were washed with the
PBS/Tween-20 solution and developed with the chromogenic horseradish peroxidase substrate, TMB (3,3',5,5'- tetramethylbenzidine; ICN, Lisle IL) 42mM in 0.1M sodium acetate-citric acid, pH 4.9. The reaction was initiated by addition of hydrogen peroxide (15 μl, 30% solution) and quenched by addition of 2N sulfuric acid. The colored product was quantified at 450 nm using a Thermofax plate reader (Molecular Devices; Palo Alto CA).
The cells in positive culture wells were
subcloned by limiting dilution and re-evaluated using the screening assay described above. We then evaluated the hybridoma supernatants for their ability to stain, by FACS analysis, C0S7 cells transiently expressing VCAM-6D or VCAM-7D. In these experiments cDNA encoding VCAM-6D or VCAM-7D was inserted into an expression vector (pCDM8) and subsequently transfected into COS7 cells by
electroporation, as described in Osborn et al. 1989 [2] and Hession et al. 1991 [9],
The COS7 cells, transfected with either VCAM-6D or VCAM-7D cDNA, were washed and suspended in suspension buffer (PBS/5mM EDTA/2% FCS/0.05% sodium azide) and incubated 45 min. at 4° C with supernatant from hybridoma cultures. The transfected cells were then washed with suspension buffer and incubated with a fluoresceinated goat anti-mouse Ig (Jackson Immunoresearch, West Grove PA) for 45 min. at 4° C. The cells were washed, fixed with 0.2% paraformaldehyde in PBS and evaluated using a FACStar (Becton Dickinson, Mountain View CA).
FACS analysis showed that the isolated anti- VCAM1 monoclonal antibodies bound to VCAM-7D but not to VCAM-6D. The binding data for these antibodies are summarized in Table I (infra p. 28).
Example 2
Ramos Cell Binding Assay
Ramos is a B-lymphoblastoid cell line that expresses VLA-4 and thus binds to VCAM1 but does not bind to other induced endothelial cell molecules such as ICAM-1 or ELAM-1 (Osborn et al., 1989 [2]). Ramos is therefore a convenient model cell line with which to assay VLA-4- dependent binding. Ramos cells bind to both recombinant VCAM-6D and recombinant VCAM-7D when these molecules are expressed on COS cells. We had previously noted that binding to VCAM-7D was somewhat (less than 2 fold) greater than to VCAM-6D in transient transfection assays of a single preparation of each plasmid (Hession et al., 1991 [9]). To determine if this quantitative difference in binding activity was reproducible, several preparations of each plasmid were made and electroporated in parallel into COS cells. Repeated transfections revealed that although expression of the two plasmids is similar, VCAM-7D on average binds significantly more cells per square
millimeter than does VCAM-6D. (Cf. Figure 6 and Figure 7.) Binding to both six- and seven-domain forms of VCAM1 was inhibited by the anti-VLA-4 MAb HP1/2 or by anti-VCAM MAb 4B9 (Figure 7).
In all of our procedures, the binding of Ramos cells was measured as follows: Confluent monolayers of the target cells (e.g., COS transfectants or induced HUVECs) in 48-well plastic tissue culture plates were washed with RPMI/10% fetal calf serum (FCS), and an amount (e.g., 10 μg/ml, varying per MAb and per experiment) of the test MAb (e.g., 4B9, HP1/2, GH12, etc.) in a volume of 0.15 ml was added. In the case of HUVECs, the cells were preincubated 4, 24, 48 or 72 hours at 37° C with 20 ng/ml recombinant human TNF (Biogen, Inc.) to induce expression of VCAM1. Ramos cells were fluorescently labeled by adding 2',7'-bis(2-carboxyethyl)- 5(and 6)-carboxy-fluorescein ("BCECF", Molecular Probes, Eugene OR) according to the manufacturer's instructions. The cells were first centrifuged and then resuspended in RPMI/10% fetal calf serum (FCS) to a concentration of 5 x 106 cells ml. The BCECF was added to 2 μM and the
suspension incubated for 30 min. at 37° C. The cells were washed twice in RPMI/10% FCS and then resuspended in the same solution at 3 x 106 cells/ml.
4.5 X 105 labeled Ramos cells were added to each well of the 48-well tissue culture plates (final volume, 0.3 ml) and incubated 10 minutes at 37° C. The plates were then emptied by inversion with gentle shaking and washed 3X with HANKS Buffered Salt Solution (HBSS) with Ca++ and Mg++. Adherent cells were lysed by addition of 0.12 ml of a 1% detergent solution (Nonidet P-40),
followed by incubation for 5 min. at 37° C. The lysate was transferred to 96-well microtiter plates by
multichannel micropipette, and the number of adherent Ramos cells was calculated by reading the plate in a
Titertek Fluorskan II ELISA reader (Flow Laboratories). (See, also, Gimbrone et al., 1989 [18].) Antibody Staining Assay
The antibodies were tested for their ability to bind to VCAM-7D, VCAM-6D and various VCAM/ICAM constructs exhibiting domain 4-dependent cell binding (see Figures 1- 13 and Example 3, infra). COS7 cells transfected with a particular VCAM, ICAM (control) or chimeric VCAM/ICAM (or VCAM/VCAM, see Fig. 11) cDNA construct were removed from 100 mm culture dishes by incubation in HBSS/5 mM EDTA for 15 min. at 37° C, followed by vigorous pipetting.
Aliquots of 3-5 x 105 cells were incubated individually with 2 μg/ml of the MAb being tested, or with rabbit polyclonal antiserum raised against rsVCAM-7D (diluted 1:500), in PNF (PBS/0.1% sodium azide/2% FCS) for 20 min. at room temperature. After incubation the cells were washed twice with PNF and incubated 20 min. at room temperature with 0.2 μg/ml of a secondary fluoresceinated antibody (rabbit anti-mouse immunoglobulin or goat anti- rabbit immunoglobulin) in PNF. Cells were then washed 3X with PBS, suspended in PBS/1% formaldehyde, stored dark at 4° C, and analyzed using a FACStar (Becton Dickinson;
Mountain View CA). The number of cells versus fluorescen intensity was plotted, and the number of positive cells in each sample was determined from the FACS histograms.
Fluorescence peaks for each transfectant and for negative controls (pCDM8 vector only) run in parallel were compared and the peaks marked to exclude 99% of the negative cells. The number of positive cells (to the right of the marker) was determined for each transfectant. This method results in a slight underestimate of the number of positive cells in each case, since weakly positive cells that overlap the negative control fluorescence peak are excluded. Example 3
Construction and Expression of
Chimeric VCAM/ICAM Polypeptides
In order to identify regions of VCAM1 involved in recognition of VLA-4-expressing cells, recombinant genes encoding VCAM/ICAM chimeras were constructed by excising portions of the VCAM-7D and VCAM-6D cDNAs and replacing them with analogous regions of ICAM-1, using unique restriction endonuclease sites in the VCAM1 cDNA (see Fig. 1) and polymerase chain reaction (PCR)-generated fragments of ICAM1. Substitution of the excised VCAM1 regions was done to eliminate as much as possible the structural distortions that would accompany deletion of one or more domains of the VCAM1 molecules. ICAM1 was selected as a source of substituent structures because it is also a member of the Ig superfamily and has the highest degree of amino acid homology to VCAM1 among Ig
superfamily members (Osborn et al., 1989 [2]).
Schematic diagrams of the VCAM1/ICAM1 chimeric polypeptides are shown in Figures 1-5, 8, 9 and 11. The cDNA constructs coding for each of these were prepared as follows:
Aliquots of the parent plasmids VCAM1E11/CDM8 (encoding VCAM-7D; see, Hession et al., 1991 [9]) or VCAM41/CDM8 (encoding VCAM-6D; Osborn et al., 1989 [2]) were digested using restriction endonucleases at the appropriate sites indicated in Figure 1 and agarose gel purified. ICAM1 inserts were made by PCR from an ICAM1/CDM8 plasmid isolated from an endothelial cDNA library previously described (Osborn et al., 1989 [2]), using oligonucleotide probes based on the published sequence of ICAM1 (Simmons et al., 1988 [19]; Staunton et al., 1988 [20]). The PCR primers were designed as follows: Primer P-1 (SEQ ID NO: 1)
EcoRV
5'-TCTAGATATC TTCTGCCCCG GGAGGCTCCG TGCTG-3 '
The first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues 9-12; the following twenty-three nucleotides correspond to the coding region for ICAM1 amino acid residues 11-18.
Primer P-2 (SEQ ID NO: 2 )
Bgl2
5'-GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA-3 '
The first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 86-89 ; the following twenty-one nucleotides correspond to the coding region for
ICAM1 amino acid residues 84-90.
Primer P-3 (SEQ ID NO: 3)
Hind3
5'-TCTCAAGCTT TTACTGTTGA GATCTCCCCT GG-3'
The first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues -3-1; the following twenty nucleotides correspond to the coding region for
ICAM1 amino acid residues 300-306.
Primer P-4 (SEO ID NO: 4)
BSU361
5'-CTGGATCCTT AGGAGTCCAG TACACGGTGA GGAAGG-3 '
The first fourteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following twenty-two nucleotides correspond to the coding region for
ICAM1 amino acid residues 79-85.
Primer P-5 (SEQ ID NO: 5)
BspHl
5'-CTCTTCATGA GCTTCTCCC CACGGAGCAG CACCAC-3 '
The first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 134-137; the following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 121-128.
Primer P-6 (SEO ID NO: 6)
Bgl2
5'-GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT-3 '
The first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 302-305; the
following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 279-286.
Primer P-7 (SEO ID NO: 7)
Bsu361
5'-CTGGATCCTT AGGGAATGAG TAGAGCTCCA C-3'
The first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following eighteen nucleotides correspond to the coding region for VCAM1 amino acid residues 385-394.
PCR synthesis for inserts for construct VCAM/ICAM-1 (Fig. 3) was primed with P-1 and P-4; for construct VCAM/ICAM-2 (Fig. 4), P-1 and P-5; for construct VCAM/ICAM-3 (Fig. 5), P-2 and P-6; for construct
VCAM6D/VCAM4-1 (Fig. 11), P-3 and P-7. VCAM6D/ICAM-1 and VCAM6D/ICAM-2 (Figs. 8 and 9) were made using the same inserts as for VCAM/ICAM-1 and VCAM/ICAM-2, respectively, cloned into the VCAM-6D expression vector (VCAM41/CDM8) instead of the VCAM-7D expression vector (VCAM1E11/CDM8). After PCR synthesis, performed as described in Hession et al. (1991) [9], the insert fragments were digested with the appropriate restriction endonucleases and gel
purified. Expression vectors and insert fragments were ligated using NEB ligase and buffer, ethanol precipitated, then electroporated into bacterial host strain MC1061/p3 using a Biorad Gene Pulser. Clones were screened by PCR and restriction digests, and at least one isolate of each clone was sequenced across cloning junctions and insert using a Sequenase kit (United States Biochemical Corp.). The complete cDNA sequences for VCAM-7D, VCAM-6D, and each of the chimeric constructs VCAM/ICAM-1, VCAM/ICAM-2,
VCAM/ICAM-3, VCAM6D/ICAM-1, VCAM6D/ICAM-2, and
VCAM6D/VCAM4-1 are set forth in the Sequence Listing below (in order, SEQ ID NOS: 8 through 15).
The diagrammed constructs were transfected into
COS7 cells and found to be well expressed when tested by FACS analysis after staining with polyclonal antiserum to VCAM1.
In constructs VCAM/ICAM-1 (Fig. 3) and VCAM/ICAM-2 (Fig. 4), most of VCAM domain 1, or most of domain 1 and the first half of domain 2, were replaced by ICAM1 sequence (see shaded areas, Figs. 3 and 4,
respectively). In construct VCAM/ICAM-3 (Fig. 5), VCAM domains 2 and 3, and small portions of domains 1 and 4, were replaced by ICAM1 sequence (see shaded area, Fig. 5).
Example 4
MAb 4B9 Binding to VCAMl domain 1.
It has been previously shown that the N-terminal three domains of VCAM1 support adhesion to VLA-4 (Taichman et al., 1991 [21]) and that the anti-VCAM1 MAb 4B9
significantly inhibits binding of VLA-4-bearing cells to induced HUVECs or VCAM1 transfectants (Carlos et al., 1990 [22]). These results suggested that the binding site of VCAM1 recognized by VLA-4 and obstructed by 4B9 is in the N-terminal portion of the VCAM1 molecule.
Using the MAb binding assay described above (Example 2), we confirmed that MAb 4B9 recognizes an epitope in VCAM1 domain 1. It was found that 4B9 bound readily to COS7 transfectants expressing both seven- and six-domain forms of VCAM1. Furthermore, chimeric
constructs VCAM/ICAM-1 and VCAM/ICAM-2 (Figs. 3 and 4, respectively), wherein most of domain 1 was replaced by ICAM1 amino acid residues, did not bind to 4B9 (see. Table I, infra). These results indicated that the epitope to which 4B9 binds requires domain 1 for recognition.
Finally, construct VCAM/ICAM-3 (see, Fig. 5, domain 1 mostly intact) did bind 4B9, confirming that domain 1 contains the 4B9 epitope. The binding and Ramos blocking characteristics of 4B9 are summarized in Table I, infra.
Although constructs VCAM/ICAM-1 and VCAM/ICAM-2 did not bind MAb 4B9, both bound Ramos cells at about 30% of the level seen with intact VCAM-7D (Figure 7). This binding was mediated by VLA-4/VCAM1, since anti-VLA-4 mAb HP1/2 completely inhibited the interaction, while a blocking antibody to the ICAM-1 pathway (MAb 60.3) did not affect binding (data not shown). Construct VCAM/ICAM-3 bound Ramos cells weakly but measurably, at about 10% of the level seen with intact VCAM-7D (Figure 7).
Ramos Cells Bind At Two Regions On VCAM1
To determine whether Ramos cell binding to constructs VCAM/ICAM-1 and VCAM/ICAM-2 was due to residual portions of the domain 1-dependent binding site, or to domain 4 (which is structurally similar to domain 1), constructs analogous to VCAM/ICAM-1 and VCAM/ICAM-2 were made, except that cDNA coding for VCAM-6D (no domain 4) instead of VCAM-7D was used as the substrate or backbone for the substituted ICAM1 inserts (see. Figs. 8 and 9). These constructs, designated VCAM6D/ICAM-1 and
VCAM6D/ICAM-2, respectively, were well expressed in COS7 cells as measured by anti-VCAM1 polyclonal antiserum staining, but were unable to bind Ramos cells (Figure 10). These results indicated that the binding of Ramos cells to constructs VCAM/ICAM-1 and VCAM/ICAM-2 appears to be due to the presence of domain 4.
We next prepared a construct designated
VCAM6D/VCAM4-1, in which domain 4 of VCAM-7D was
substituted for domain 1 of VCAM-6D (Fig. 11). Referring to the results depicted in Figures 7 and 12, Ramos binding of VCAM6D/VCAM4-1 was comparable to that of VCAM-6D (see Fig. 2, intact domain 1) and VCAM/ICAM-l (see Fig. 3, intact domain 4), indicating that domain 4 can indeed bind cells. This result was somewhat surprising in light of the fact that MAb 4B9, which binds to domain 1 but not to domain 4, can completely inhibit binding of Ramos cells to VCAM-7D. Stearic hindrance is one possible explanation; it is also possible that binding of 4B9 to domain 1 perturbs the structure of the molecule so that domain 4 is no longer in the proper conformation to bind cells.
Example 5
Monoclonal Antibodies That Block
Domain 4-dependent Cell Adhesion
According to the procedure set forth in Example
1, three monoclonal antibodies were produced that
recognize VCAM-7D but not VCAM-6D, indicating that they bind domain 4, or a domain 4-dependent epitope. These MAbs were designated GE4, ED11 and GH12. All three MAbs were of IgGl isotype. The binding and Ramos blocking characteristics of each antibody were determined using the antibody and Ramos binding assays described above (Example 2).
MAb GH12 bound to COS7 transfectants expressing VCAM-7D, VCAM/ICAM-1 and 2, and VCAM6D/VCAM4-1 but did not bind to transfectants expressing VCAM-6D or VCAM/ICAM-3. Referring to Figure 13, Ramos binding experiments showed that MAb GH12 at 10 μg/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1, VCAM/ICAM-2 and VCAM6D/VCAM4-1 constructs but did not bind to the
VCAM/ICAM-3-expressing tranfectants, indicating that the small portion of domain 4 eliminated from the VCAM/ICAM-3 construct was important to GH12 binding. MAb GH12 did not block Ramos cell binding to VCAM-6D or VCAM-7D.
Binding experiments showed that MAb ED11 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-l and
VCAM/ICAM-2 but not to cells expressing VCAM-6D,
VCAM/ICAM-3 or VCAM6D/VCAM4-1. Referring to Figure 14, Ramos binding experiments showed that MAb ED11 at 5 μg/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1 and VCAM/ICAM-2 constructs but did not inhibit VCAM-6D-expressing, VCAM/ICAM-3-expressing or VCAM6D/VCAM4-1-expressing tranfectants. Surprisingly, MAb ED11 did partially block Ramos cell binding to VCAM-7D- expressing COS7 cells.
Binding experiments showed that MAb GE4 bound to
COS7 cells expressing VCAM-7D, VCAM/ICAM-1, VCAM/ICAM-2 and VCAM/ICAM-3 but not to cells expressing VCAM-6D or VCAM6D/VCAM4-1. Referring to Figure 15, Ramos binding experiments showed that MAb GE4 at 10 μg/ml did not inhibit Ramos binding to COS7 transfectants expressing
VCAM/ICAM-1 and VCAM/ICAM-2. MAb GE4 did not block Ramos adhesion to transfectants expressing VCAM6D/VCAM4-1,
VCAM/ICAM-3 or VCAM-6D, and also did not inhibit Ramos binding to VCAM-7D-expressing COS7 cells at the single concentration tested.
The binding and blocking data are summarized below in Table I. From these results it can be concluded that each of the three domain 4 antibodies recognize distinct epitopes, and two are capable of blocking domain 4-dependent adhesion of VLA-4 to VCAM1. These antibodies represent a new class of anti-VCAM1 blocking antibody.
Figure imgf000029_0001
Example 6
Domain 4 MAb Inhibition of Ramos Binding
to Cytokine-activated Endothelial Cells
To determine whether the domain-4-dependent MAbs
EDll or GH12 were capable of blocking adhesion to VCAM1 on endothelial cells (which is predominantly VCAM-7D), human umbilical vein endothelial cells (HUVECs) were treated with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72 hours, and binding of Ramos cells and the ability of the antibodies to block binding was measured. All MAbs were added at a concentration of 10 μg/ml. The results are shown for each activation period in Figures 16-19. The results indicate overall that MAb GH12 did not block HUVEC/Ramos binding reliably at the single concentration tested (10 μg/ml), but MAb ED11 blocked more than 80% of Ramos cell binding to induced HUVECs at all time points tested. MAb 4B9 blocked HUVEC/Ramos binding by greater than 90%. These results suggest that although the two cell binding sites of VCAM1 (domain 1- and domain- 4-dependent) can be separated and remain functional, antibodies to each can interfere with Ramos cell binding to the other. This is so even though the antibodies do not cross-react with each other's binding sites (shown by the antibody binding data to constructs, above).
In contrast to stimulated endothelial cells, as in this Example, where VCAM-7D is the predominantly expressed (RNA and protein) form of VCAM-1, VCAM-6D (RNA) may be the predominant species of VCAM-1 in bone marrow stromal cells and tonsillar follicular dendritic cells. This suggests a potential therapeutic advantage of domain 4 specific anti-VCAM1 antibodies over domain 1 specific anti-VCAM1 antibodies, in that domain 4 antibodies
according to the present invention might potentially block the recruitment of leukocytes via stimulated endothelial cells into sites of inflammation, without blocking
lymphopoiesis, which is the role that VCAM-1 plays in bone marrow (Miyake et al., 1977 [7]). Such an inhibition of B-cell lymphopoiesis may be undesirable in longer term therapy with anti-VCAM1 antibodies.
The foregoing examples are intended as an illustration of the present invention and are not
presented as a limitation of the invention as claimed hereinafter. From the foregoing disclosure, numerous modifications and additional embodiments of the invention will be apparent to and easily practiced by those experienced in this art. All such modifications and additional embodiments are within the contemplation of this application and within the scope of the appended claims.
We deposited subclones of hybridomas producing
EDll, GE4 and GH12 monoclonal antibodies under the
Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, MD (US) on January 9, 1992. The cultures were identified as follows:
designation ATCC accession no.
MAb CB.ED11.AG3 HB 10962
MAb CB.GE4.BG5 HB 10961
MAb CB.GH12.AA12 HB 10963
CITED PUBLICATIONS
[1] Rice, G.E. and Bevilacqua, M.P. (1989), "An inducible endothelial cell surface glycoprotein mediates melanoma adhesion," Science, 246, 1303-1306.
[2] Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso, G., and Lobb, R. (1989), "Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes," Cell, 59, 1203-1211.
[3] Elices, M.J., Osborn, L., Takada, Y., Crouse, C.,
Luhowskyj, S., Hemler, M.E., and Lobb, R.R. (1990), "VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site," Cell, 60, 577-584.
[4] Osborn, L. (1990), "Leukocyte adhesion in
inflammation," Cell, 62, 3-6.
[5] Cybulsky, M.I. and Gimbrone, M.A., Jr. (1991),
"Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis," Science,
251, 788-791.
[6] Freedman, A.S., Munro, J.M., Rice, G.E., Bevilacqua, M.P., Morimoto, C., Mclntyre, B.W., Rhynhart, K., Pober, J.S., and Nadler, L.M. (1990), "Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110," Science, 249, 1030-1033.
[7] Miyake, K., Medina, K., Ishihara, K., Kimoto, M.,
Auerbach, R. and Kincade, P.W. (1991), "A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in
culture," J. Cell Biol., 114, 557-565.
[8] Polte, T., Newman, W., and Gopal, T.V. (1990), "Full length vascular cell adhesion molecule 1 (VCAM-1)," Nuc. Ac. Res., 18, 5901. [9] Hession, C, Tizard, R., Vassallo, C, Schiffer,
S.B., Goff, D., Moy, P., Chi-Rosso, G., Luhowskyj, S., Lobb, R., and Osborn, L. (1991), "Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1)", J. Biol. Chem., 266, 6682-6685.
[10] Cybulsky, M.I., Fries, J.W.U., Williams, A.J.,
Sultan, P., Eddy, R., Byers, M., Shows, T., Gimbrone, M.A., Jr., and Collins, T. (1991), "Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene," Proc. Natl. Acad. Sci. USA, 88, 7859-7863.
[11] Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, E., Russo, M., Osborn, L., Chi-Rosso, G., Newman, Lobb, R., and Harlan, J.M. (1990), "Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine- activated cultured human endothelial cells. Blood, 76 , 965-970.
[12] Kohler, G. and Milstein, C. (1975), "Continuous
Cultures of Fused Cells Secreting Antibody of
Predefined Specificity", Nature, 256, pp. 495-497.
[13] Jones, P. T., et al. (1986), "Replacing the
Complementarity-Determining Regions in a Human
Antibody with Those From a Mouse," Nature, 321, 522- 525.
[14] Ward, E. S., et al. (1989), "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature, 341, 544- 546.
[15] U.S. Patent No. 4,816,397, Boss et al., "Multichain Polypeptides Or Proteins And Processes For Their Production", issued March 28, 1989.
[16] Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and Winter, G. (1991), "Making antibody fragments using phage display libraries," Nature, 352, 624-628.
[17] Lerner, L.A. (1981), "How to Make a Hybridoma," Yale J. Biol. Med., 54, 387-402. [18] Gimbrone, M.A., Jr., Obin, M.S., Brock, A.F., Luis, W.A., Hass, P.E., Hebert, C.A., Yip, Y.K., Leung, D.W., Lowe, D.G., Kohr, W.J., Darbonne, W.C.,
Bachtol, K.B., and Baker, J.B. (1989), "Endothelial interleukin-8: A novel inhibitor of leukocyte- endothelial interactions," Science, 246, 1601-1603.
[19] Simmons, D., Makgoba, M.W., and Seed, B. (1988),
"ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM," Nature, 331, 624-627.
[20] Staunton, D.E., Dustin, M.L., Erickson, H.P., and
Springer, T.A. (1990), "The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus," Cell, 61, 243-254.
[21] Taichman, D.B., Cybulsky, M.I., Djaffar, I.,
Longenecker, B.M., Teixido, J., Rice, G.E., Aruffo, A., and Bevilacqua, M.P. (1991), "Tumor cell surface α4β1 integrin mediates adhesion to vascular
endothelium: Demonstration of an interaction with the N-terminal domains of IΝCAM-110/VCAM-1," Cell Regulation, 2, 347-355.
[22] Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, E., Rosso, M., Osborn, L., Chi-Rosso, G., Newman, B., Lobb, R., and Harlan, J.M. (1990), "Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells," Blood, 76(5), 965-970.
The foregoing publications are incorporated herein by reference.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Osborn, Laurelee
Benjamin, Christopher D.
(ii) TITLE OF INVENTION: ANTIBODIES RECOGNIZING THE FOURTH
IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM1
(iii) NUMBER OF SEQUENCES: 23
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Allegretti & Witcoff, Ltd.
(B) STREET: 10 South Wacker Drive
(C) CITY: Chicago
(D) STATE: IL
(E) COUNTRY: US
(F) ZIP: 60606
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McNicholas, Janet M.
(B) REGISTRATION NUMBER: 32, 918
(C) REFERENCE/DOCKET NUMBER: 92, 306-A; D001 CIP PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (312) 715-1000
(B) TELEFAX: (312) 715-1234
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) SERANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCTAGATATC TTCTGCCCCG GGAGGCTCCG TGCTG 35 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA 34
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(XI) SEQUENCE DESCRIPNCN: SEQ ID NO: 3:
TCTCAAGCTT TTACTGTTGA GATCTCCCCT GG 32
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CTGGATCCTT AGGAGTCCAG TACACGGTGA GGAAGG 36
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CTCTTCATGA GCTTCTCCCC ACGGAGCAGC ACCAC 35
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT 35
(2) INFORMATION FOR SEQ ID NO: 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CTGGATCCTT AGGGAATGAG TAGAGCTCCA C 31
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2217 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .2217
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..2217 (ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1. .72
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Mat Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144 Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
ACC ACA GGC TGT GAG TGC CCA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192 Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
ACA ATG AAT CCT GTT AGT TTT GGG AAC GAA CAC TCT TAC CTG TGC ACA 288 Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GCA ACT TGT GAA TCT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC 336 Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85
TAC TCT TTT CCT AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384 Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG AAG CCG ATC ACA GTC AAG TGT TCA GTT GCT GAT GTA TAC CCA 432 Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG 480 Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
AGT CAG GAA TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG 528 Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150 ACT TTG GAA GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT 576 Ser Leu Glu Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165
CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624 Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro
170 175 180
ACA CTA. AGG CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG 672 Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys
185 190 195 200
AAT ACA GTT ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GCT GGC 720 Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GCT CTA CCA GCT CCA GAG ATT 768 Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
TTC TGG ACT AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA 816 Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864 Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle
250 255 260
TAT GTG TGT GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912 Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val
265 270 275 280
GAA TTA ATT GTT CAA GAG AAA CCA TTT ACT GTT GAG ATC TCC CCT GGA 960 Glu Leu lle Val Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly
285 290 295
CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT 1008 Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Mat Leu Thr Cys Ser
300 305 310
GTC ATG GGC TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC 1056 Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp
315 320 325
AGC CCT CTG AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG 1104 Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
330 335 340
ACC CTG AGC CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA 1152 Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
345 350 355 360 GTG ACT TGT GGA CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC 1200 Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu
365 370 375
TAC TCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG 1248 Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val
380 385 390
AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC 1296 Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
395 400 405
CTT GAC GGG CTG GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG GAG 1344 Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu
410 415 420
AAT ATA GAG TTT TTG GAG GAT AGG GAT ATG AAA TCT CTA GAG AAC AAA 1392 Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys
425 430 435 440
AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA GCT 1440 Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala
445 450 455
CTT GTT TGT CAG GCT AAG TEA CAT ATT GAT GAC ATG GAA TTC GAA CCC 1488 Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro
460 465 470
AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC AGA 1536 Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
475 480 485
GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT 1584 Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser
490 495 500
TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCS AAA ATC 1632 Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle
505 510 515 520
CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG 1680 Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
525 530 535
AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG GTT 1728 Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Mat Glu Asp Ser Gly Val
540 545 550
TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA GTG 1776 Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
560 565 GAA TEA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT 1824 Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe
570 575 580
CCT TCT GAG ACT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA 1872 Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr
585 590 595 600
TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG 1920 Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu
605 610 615
ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA 1968 Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg
620 625 630
AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TCT GAA TCT AAA AAC 2016 Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
635 640 645
AAA CTT GGC TCA CAA TTA AGA ACT TTA ACA CTT GAT GTT CAA GGA AGA 2064 Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
650 655 660
GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT 2112 Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
665 670 675 680
GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA 2160 Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala
685 690 695
AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CIT GTA GAA GCA CAG AAA 2208 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
700 705 710
TCA AAA GIG 2217
Ser Lys Val
715
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 739 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp -24 -20 -15 -10 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser 10 15 20
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp 25 30 35 40
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85
Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu 90 95 100
Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro 105 110 115 120 Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165
Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro 170 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys 185 190 195 200
Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245 Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle 250 255 260
Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val 265 270 275 280
Glu Leu lle Val Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly
285 290 295
Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser
300 305 310
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp
315 320 325
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu 330 335 340
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr 345 350 355 360
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu
365 370 375
Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val
380 385 390
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
395 400 405
Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu 410 415 420
Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys 425 430 435 440
Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala
445 450 455
Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro
460 465 470
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
475 480 485
Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser 490 495 500
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle 505 510 515 520 Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu 525 530 535
Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val
540 545 550
Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
555 560 565
Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe 570 575 580
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr 585 590 595 600
Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu
605 610 615
Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg
620 625 630
Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
635 640 645
Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg 650 655 660
Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe 665 670 675 680 Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala
685 690 695
Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
700 705 710
Ser Lys Val
715
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1941 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLLGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1941 (ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73. .1941
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1..72
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144 Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
ACC ACA GGC TGT GAG TCC CCA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
ACT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
ACA ATG AAT CCT GTT AGT TTT GGG AAC GAA CAC TCT TAC CTG TGC ACA 288 Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GCA ACT TGT GAA TCT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC 336 Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85
TAC TCT TTT CCT AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384 Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG AAG CCG ATC ACA GTC AAG TGT TCA CTT GCT GAT GTA TAC CCA 432 Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG 480 Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135 ACT CAG GAA TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG 528 Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
ACT TTG GAA CTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA CTT 576 Ser Leu Glu Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165
CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624 Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro
170 175 180
ACA GTA AGG CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG 672 Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys
185 190 195 200
AAT ACA CTT ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC 720 Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CIA CCA GCT CCA GAG ATT 768 Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
TTC TGG AGT AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA 816 Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864 Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle
250 255 2.60
TAT GTG TGT GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912 Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val
265 270 275 280
GAA TTA ATT GTT CAA GCA TTC CGT AGA GAT CCA GAA ATC GAG ATG AGT 960 Glu Leu lle Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser
285 290 295
GGT GGC CTC CTG AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT 1008 Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
AGC GTG TAC CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG 1056 Ser Val Tyr Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu
315 320 325
ACT ATT CTG GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT 1104 Thr lle Leu Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 340 CTA GAG AAC AAA ACT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT 1152 Leu Glu Asn Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp
345 350 355 360
ACT GGA AAA GCT CTT CTT TCT CAG GCT AAG TTA CAT ATT GAT GAC ATG 1200 Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met
365 370 375
GAA TTC GAA CCC AAA CAA AGG CAG AGT ACG CAA ACΑ CTT TAT GTC AAT 1248
Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
GTT GCC CCC AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG 1296 Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu
395 400 405
GAG GAA GGC AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT 1344 Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392 Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
425 430 435 440
CCT CTT TCT GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA 1440 Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu
445 450 455
GAT TCT GGG GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC 1488 Asp Ser Gly Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser
460 465 470
AGA AAG GAA GTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA 1536 Arg Lys Glu Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys
475 480 485
CTT ACA GCT TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC 1584 Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle
490 495 500
ATC TCT TGT ACA TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG 1632 lle Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys
505 510 515 520
AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC 1680 Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
525 530 535
TAT ACC ATC CGA AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TGT 1728 Tyr Thr lle Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550 GAA TCT AAA AAC AAA GTT GGC TCA CAA TTA AGA ACT TTA ACA CTT GAT 1776 Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
GTT CAA GGA AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC 1824 Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu
570 575 580
GTG CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA 1872 Val Leu Tyr Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle
585 590 595 600
ATT TAC TTT GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA 1920 lle Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
GAA GCA CAG AAA TCA AAA GTG 1941
Glu Ala Gln Lys Ser Lys Val
620
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 647 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85 Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu 90 95 100
Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro 105 110 115 120
Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165
Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro 170 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys 185 190 195 200
Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle 250 255 260
Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val 265 270 275 280
Glu Leu lle Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser
285 290 295
Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
Ser Val Tyr Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu
315 320 325
Thr lle Leu Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser 330 335 340
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp 345 350 355 360 Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met 365 370 375
Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu
395 400 405
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro 410 415 420
Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln 425 430 435 440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu
445 450 455
Asp Ser Gly Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser
460 465 470
Arg Lys Glu Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys
475 480 485
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle 490 495 500
lle Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys 505 510 515 520
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
525 530 535
Tyr Thr lle Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu 570 575 580
Val Leu Tyr Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle 585 590 595 600 lle Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
Glu Ala Gln Lys Ser Lys Val
620 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2205 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .2205
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73. .2205
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1. .72
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CIT CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC 144 Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240 Lyy Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AAC TGC CCT 288 Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC CTG TAC TGG ACT CCT 336 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85 AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG GCT GGG AAG CCG 384 Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro
90 95 100
ATC ACA GTC AAG TGT TCA GTE GCT GAT GTA TAC CCA TTT GAC AGG CTG 432 lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 1 10 1 15 120
GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG AGT CAG GAA TTT 480 Glu lle Asp Leu. Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG AGT TTG GAA GTA 528 Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC CGA 576 Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys Arg
155 160 165
GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 175 180
GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT ATT 672 Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle
185 190 195 200
TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC ATG 720 Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 215
ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT AAG 768 Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser Lys
220 225 230
AAA TTA GAT AAT GGG AAT CTA CAG CAC CIT TCT GGA AAT GCA ACT CTC 816 Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT GTG TGT GAA 864 Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu
250 255 260
GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT GTT 912 Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle Val
265 270 275 280
CAA GAG AAA CCA TTT ACT CTT GAG ATC TCC CCT GGA CCC CGG ATT GCT 960 Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle Ala
285 290 295 GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT ACT CTC ATG GGC TGT 1008 Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys
300 305 310
GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC AGC CCT CTG AGC 1056 Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp Ser Pro Leu Ser
315 320 325
GGG AAG CTG AGG ACT GAG GGG ACC AAT TCC ACG CTG ACC CTG AGC CCT 1104 Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro
330 335 340
CTG ACT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA GTG ACT TCT GGA 1152 Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly
345 350 355 360
CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC TAC TCA TTC CCT 1200 His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu Tyr Ser Phe Pro
365 370 375
AGA GAT CCA GAA ATC GAG ATC ACT GCT GGC CTC GTG AAT GGG AGC TCT 1248 Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser
380 385 390
GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG CTG 1296 Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu
395 400 405
GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG GAG AAT ATA GAG TTT 1344 Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu Asn lle Glu Phe
410 415 420
TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC AAA AGT TTG GAA ATG 1392 Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met
425 430 435 440
ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA GCT CTT GTT TCT CAG 1440 Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala Leu Val Cys Gln
445 450 455
GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC AAA CAA AGG CAG 1488 Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln
460 465 470
ACT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC AGA GAT ACA ACC GTC 1536 Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val
475 480 485
TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT TCT GTG AAT ATG 1584 Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser Ser Val Asn Met
490 495 500 ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC CTG TGG AGC AGG 1632 Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle Leu Trp Ser Arg
505 510 515 520
CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG AAT GCA ACT CTC 1680 Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu
525 530 535
ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG GTT TAT TTA TGT GAA 1728 Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu
540 545 550
GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA GTG GAA TTA ATT ATC 1776 Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu lle lle
555 560 565
CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT CCT TCT GAG AGT 1824 Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe Pro Ser Glu Ser
570 575 580
GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA TGT GGA AAT GTT 1872 Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr Cys Gly Asn Val
585 590 595 600
CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG ACA GGA GAC ACA 1920 Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr
605 610 615
GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG TTG 1968 Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln Leu
620 625 630
AAG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA AAC AAA GTT GGC TCA 2016 Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser
635 640 645
CAA TTA AGA AGT TEA ACA CTT GAT GTT CAA GGA AGA GAA AAC AAC AAA 2064 Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys
650 655 660
GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT GCA TCC TCC TTA 2112 Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu
665 670 675 680
ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA AGA AAA GCC AAC 2160 lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala Arg Lys Ala Asn
685 690 695
ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA TCA AAA GTG 2205 Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 735 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp -24 -20 -15 -10 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser 10 15 20
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro 25 30 35 40
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro 90 95 100
lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu 105 110 115 120
Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys Arg
155 160 165
Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln 170 175 180 Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle 185 190 195 200
Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 215
Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser Lys
220 225 230
Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu 250 255 260
Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle Val 265 270 275 280 Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle Ala
285 290 295 Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys
300 305 310
Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp Ser Pro Leu Ser
315 320 325
Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro 330 335 340
Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly 345 350 355 360
His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu Tyr Ser Phe Pro
365 370 375
Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser
380 385 390
Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu
395 400 405
Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu Asn lle Glu Phe 410 415 420
Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met 425 430 435 440
Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala Leu Val Cys Gln
445 450 455 Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln 460 465 470
Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val
475 480 485
Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser Ser Val Asn Met 490 495 500
Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle Leu Trp Ser Arg 505 510 515 520 Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu
525 530 535
Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu
540 545 550
Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu lle lle
555 560 565
Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe Pro Ser Glu Ser 570 575 580
Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr Cys Gly Asn Val 585 590 595 600
Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr
605 610 615
Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln Leu
620 625 630
Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser
635 640 645
Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys 650 655 660
Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu 665 670 675 680 lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala Arg Lys Ala Asn
685 690 695
Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2208 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..2208
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..2208
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1..72
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC 144 Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192 Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240 Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AAC TGC CCT 288 Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCA 336 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85 GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TGG CAG CCA GTG GGC AAG 384 Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
90 95 100
AAC CTT ACC CTA CGC TGC CAG GTG GAG GCT GGG GCA CCC CGG GCC AAC 432 Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn
105 110 115 120
CTC ACC GTG GTG CTG CTC CGT GGG GAG AAG CTC ATG AAG AGT CAG GAA 480 Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG AGT TTG GAA 528 Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT CTT TGC 576 Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Veil Leu Val Cys
155 160 165
CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG 624 Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 180
CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT 672 Gln Ala Val Lys Glu Leu Gln Veύ. Tyr lle Ser Pro Lys Asn Thr Val
185 190 195 200
ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC 720 lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT 768 Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser
220 225 230
AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CIT TCT GGA AAT GCA ACT 816 Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT GTG TGT 864 Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys
250 255 260
GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT 912 Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle
265 270 275 280
GTT CAA GAG AAA CCA TTT ACT GTT GAG ATC TCC CCT GGA CCC CGG ATT 960 Val Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle
285 290 295 GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT GTC ATG GGC 1008 Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly
300 305 310
TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC AGC CCT CTG 1056 Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp Ser Pro Leu
315 320 325
AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG ACC CTG AGC 1104 Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser
330 335 340
CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA GTG ACT TGT 1152 Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys
345 350 355 360
GGA CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC TAC TCA TTC 1200 Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu Tyr Ser Phe
365 370 375
CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG AAT GGG AGC 1248 Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val Asn Gly Ser
380 385 390
TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG 1296 Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg
395 400 405
CTG GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG GAG AAT ATA GAG 1344 Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu Asn lle Glu
410 415 420
TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC AAA AGT TTG GAA 1392 Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu
425 430 435 440
ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA GCT CTT GTT TGT 1440 Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala Leu Val Cys
445 450 455
CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC AAA CAA AGG 1488 Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro Lys Gln Arg
460 465 470
CAG AGT AGG CAA ACA CTT TAT GTC AAT GET GCC CCC AGA GAT ACA ACC 1536 Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr
475 480 485
GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT TCT GTG AAT 1584 Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser Ser Val Asn
490 495 500 ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC CTG TGG AGC 1632 Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle Leu Trp Ser
505 510 515 520
AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG AAT GCA ACT 1680 Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr
525 530 535
CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG GTT TAT TTA TCT 1728 Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys
540 545 550
GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA CTG GAA TTA ATT 1776 Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu lle
555 560 565
ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT CCT TCT GAG 1824 lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe Pro Ser Glu
570 575 580
ACT GTC AAA GAA GGA GAC ACT CTC ATC ATC TCT TGT ACA TGT GGA AAT 1872 Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr Cys Gly Asn
585 590 595 600
GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG ACA GGA GAC 1920 Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu Thr Gly Asp
605 610 615
ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG 1968 Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln
620 625 630
TTG AAG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA AAC AAA GTT GGC 2016 Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly
635 640 645
TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT CAA GGA AGA GAA AAC AAC 2064 Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn
650 655 660
AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT GCA TCC TCC 2112 Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser
665 670 675 680
TTA ATA ATA CCT GCC AIT GGA ATG ATA ATT TAC TTT GCA AGA AAA GCC 2160 Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala Arg Lys Ala
685 690 695
AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA TCA AAA GTG 2208 Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp -24 -20 -15 -10 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser 10 15 20
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro 25 30 35 40
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys 90 95 100
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn 105 110 115 120
Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135 Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys
155 160 165
Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg 170 175 180
Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val 185 190 195 200 lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr 205 210 215
Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser
220 225 230
Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys 250 255 260
Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle 265 270 275 280
Val Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle
285 290 295 Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly
300 305 310
Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp Ser Pro Leu
315 320 325
Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser 330 335 340
Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys 345 350 355 360
Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu Tyr Ser Phe
365 370 375
Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val Asn Gly Ser
380 385 390
Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg
395 400 405
Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu Asn lle Glu 410 415 420
Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu 425 430 435 440
Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala Leu Val Cys
445 450 455 Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro Lys Gln Arg
460 465 470 Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr 475 480 485
Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser Ser Val Asn 490 495 500
Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle Leu Trp Ser 505 510 515 520
Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr
525 530 535 Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys
540 545 550
Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu lle
555 560 565
lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe Pro Ser Glu 570 575 580
Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr Cys Gly Asn 585 590 595 600
Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu Thr Gly Asp
605 610 615
Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln
620 625 630
Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly
635 640 645
Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn 650 655 660
Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser 665 670 675 680
Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala Arg Lys Ala
685 690 695
Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2220 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..2220
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..2220
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1..72
(xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 16:
ATG CCT GGG AAG ATG GTC CTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144 Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
ACC ACA GGC TCT GAG TCC CCA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
ACA ATG AAT CCT GTT AGT TTT GGG AAC GAA CAC TCT TAC CTG TGC ACA 288 Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GCA ACT TGT GAA TGT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC 336 Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85 TAC TGG ACT CCA GAA CGG CTG GAA CTG GCA CCC CTC CCC TCT TGG CAG 384 Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln
90 95 100
CCA GTG GGC AAG AAC CTT ACC CTA OGC TGC CAG GTG GAG GGT GGG GCA 432 Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala
105 110 115 120
CCC CGG GCC AAC CTC ACC GTG CTG CTG CTC CCT GGG GAG AAG GAG CTG 480 Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu
125 130 135
AAA CGG GAG CCA GCT GTG GGG GAG CCC GCT GAG GTC ACG ACC ACG GTG 528 Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val
140 145 150
CTG GTG AGG AGA GAT CAC CAT GGA GCC AAT TTC TCG TGC CGC ACT GAA 576 Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu
155 160 165
CTG GAC CTG CGG CCC CAA GGG CTG GAG CTG TTT GAG AAC ACC TCG GCC 624 Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala
170 175 180
CCC TAC CAG CTC CAG ACC TTT GTC CTG CCA GCG ACT CCC CCA CAA CTT 672 Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu
185 190 195 200
GTC AGC CCC CGG GTC CTA GAG GTG GAC ACG CAG GGG ACC GTG GTC TGT 720 Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys
205 210 215
TCC CTG GAC GGG CTG TTC CCA GTC TCG GAG GCC CAG GTC CAC CTG GCA 768 Ser Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala
220 225 230
CTG GGG GAC CAG AGG TTG AAC CCC ACA GTC ACC TAT GGC AAC GAC TCC 816 Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser
235 240 245
TTC TCG GCC AAG GCC TCA GTC AGT GTG ACC GCA GAG GAC GAG GGC ACC 864 Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr
250 255 260
CAG CGG CTG ACG TGT GCA GTA ATA CTG GGG AAC CAG AGC CAG GAG ACA 912 Gln Arg Leu Thr Cys Ala Val lle Leu Gly Asn Gln Ser Gln Glu Thr
265 270 275 280
CTG CAG ACA GTG ACC ATC TAC AGC TTT CCG GCG CCC GAG ATC TCC CCT 960 Leu Gln Thr Val Thr lle Tyr Ser Phe Pro Ala Pro Glu lle Ser Pro
285 290 295 GGA CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT 1008 Gly Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys
300 305 310
AGT GTC ATG GGC TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA 1056 Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle
315 320 325
GAC AGC CCT CTG AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG 1104 Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr
330 335 340
CTG ACC CTG AGC CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC 1152 Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys
345 350 355 360
ACA GTG ACT TGT GGA CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG 1200 Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu
365 370 375
CTC TAC TCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC 1248 Leu Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu
380 385 390
GIG AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC 1296 Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
395 400 405
CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG 1344 Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu
410 415 420
GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC 1392 Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn
425 430 435 440
AAA ACT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA 1440 Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys
445 450 455
GCT CTT GTT TGT CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA 1488 Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu
460 465 470
CCC AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC 1536 Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
475 480 485
AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC 1584 Arg Asp Thr Thr Val leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly
490 495 500 AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CGG AAA 1632 Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
505 510 515 520
ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT 1680 lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
525 530 535
GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG 1728 Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly
540 545 550
GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA 1776 Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu
555 560 565
GTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT 1824 Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala
570 575 580
TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT 1872 Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys
585 590 595 600
ACA TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG 1920 Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala
605 610 615
GAG ACA GGA GAC ACA GTA CEA AAA TCT ATA GAT GGC GCC TAT ACC ATC 1968 Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle
620 625 630
CGA AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA 2016 Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
635 640 645
AAC AAA GTT GGC TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT CAA GGA 2064 Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
650 655 660
AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT 2112 Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
665 670 675 680
TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT 2160 Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe
685 690 695
GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG 2208 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
700 705 710 AAA TCA AAA GTG 2220
Lys Ser Lys Val
715
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 740 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Ala Gln lle Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly lle Gln Val Glu lle
75 80 85
Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln
90 95 100
Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala
105 110 115 120
Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu
125 130 135
Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val
140 145 150
Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu
155 160 165 Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala 170 175 180
Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu 185 190 195 200
Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys
205 210 215
Ser Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala
220 225 230
Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser
235 240 245
Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr 250 255 260
Gln Arg Leu Thr Cys Ala Val lle Leu Gly Asn Gln Ser Gln Glu Thr 265 270 275 280
Leu Gln Thr Val Thr lle Tyr Ser Phe Pro Ala Pro Glu lle Ser Pro
285 290 295
Gly Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys
300 305 310
Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle
315 320 325
Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr 330 335 340
Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys 345 350 355 360
Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu
365 370 375
Leu Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu
380 385 390
Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
395 400 405
Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu 410 415 420
Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn 425 430 435 440 Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys 445 450 455 Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu
460 465 470
Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
475 480 485
Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly 490 495 500
Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys 505 510 515 520 lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
525 530 535
Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly
540 545 550
Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu
555 560 565
Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala 570 575 580
Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys 585 590 595 600
Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala
605 610 615
Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle
620 625 630
Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
635 640 645
Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly 650 655 660
Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr 665 670 675 680 Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe
685 690 695 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser .Leu Val Glu Ala Gln
700 705 710 Lys Ser Lys Val
715
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1929 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .1929
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73. .1929
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1. .72
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTE CTG CCC CGG GGA GGC TCC GTG GTG GTG ACA TGC AGC 144 Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192 Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240 Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AAC TGC CCT 288 Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70 GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCT 336 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
AAG GAT CCA GAG ATT CAT TTG ACT GGC CCT CTG GAG GCT GGG AAG CCG 384 Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro
90 95 100
ATC ACA GTC AAG TGT TCA CTT GCT GAT GTA TAC CCA TTT GAC AGG CTG 432 lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 110 115 120
GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATC AAG AGT CAG GAA TTT 480 Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG ACT TTG GAA GTA 528 Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC GGA 576 Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys Arg
155 160 165
GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 175 180
GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT ATT 672 Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle
185 190 195 200
TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC ATG 720 Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 215
ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT AAG 768 Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser Lys
220 225 230
AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA AAT GCA ACT CTC 816 Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT GTG TGT GAA 864 Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu
250 255 260
GGA CTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT GTT 912 Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle Val
265 270 275 280 CAA GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG ACT GGT GGC CTC GTG 960 Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val
285 290 295
AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC 1008
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
300 305 310
CTT GAC CGG CTG GAG ATT GAA TTA C3T AAG GGG GAG ACT ATT CTG GAG 1056
Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu
315 320 325
AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC AAA 1 104
Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys
330 335 340
ACT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA GCT 1152
Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala
345 350 355 360
CTT CTT TCT CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC 1200 Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro
365 370 375
AAA CAA AGG CAG ACT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC AGA 1248
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
380 385 390
GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT 1296
Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly Ser
395 400 405
TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC 1344
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle
410 415 420
CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG 1392
Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
425 430 435 440
AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA.ATG GAA GAT TCT GGG GTT 1440
Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val
445 450 455
TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA GTG 1488
Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
460 465 470
GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT 1536
Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe
475 480 485 CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA 1584 Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr
490 495 500
TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG 1632 Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu
505 510 515 520
ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC GGA 1680 Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg
525 530 535
AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA AAC 1728 Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
540 545 550
AAA GTT GGC TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT CAA GGA AGA 1776 Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
555 560 565
GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT 1824 Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
570 575 580
GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA 1872 Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala
585 590 595 600
AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA 1920 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
605 610 615
TCA AAA GTG 1929
Ser Lys Val
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 643 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu -5 1 5
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser 10 15 20
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro 25 30 35 40
Lys Lys Glu Lsu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro 90 95 100
lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu 105 110 115 120
Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135 Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys Arg
155 160 165
Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln 170 175 180
Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle 185 190 195 200
Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 215
Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser Lys
220 225 230
Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu 250 255 260 Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle Val 265 270 275 280 Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val
285 290 295
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
300 305 310
Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu Glu
315 320 325
Asn lle Glu Phe leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys 330 335 340
Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys Ala 345 350 355 360
Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu Pro
365 370 375
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
380 385 390
Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle leu Glu Glu Gly Ser
395 400 405
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys lle 410 415 420
Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
425 430 435 440
Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly Val
445 450 455
Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
460 465 470
Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala Phe
475 480 485
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys Thr 490 495 500
Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala Glu 505 510 515 520
Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg
525 530 535 Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
540 545 550
Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
560 565
Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
570 575 580
Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe Ala
585 590 595 600
Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
605 610 615
Ser Lys Val
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1932 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .1932
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73. .1932
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) L OCATION: 1. .72
(xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 20:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC 144
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20 ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240 Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AAC TGC CCT 288 Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCA 336 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TGG CAG CCA GTG GGC AAG 384 Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
90 95 100
AAC CTT ACC CTA CGC TGC CAG GTC GAG GGTGGG GCA CCC CGG GCC AAC 432 Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn
105 110 115 120
CTC ACC GTG GTG CTG CTC CGT GGG GAG AAG CTC ATG AAG AGT CAG GAA 480 Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG AGT TTG GAA 528 Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC 576 Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys
155 160 165
CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG 624 Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 180
CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT 672 Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val
185 190 195 200
ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC 720 lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT 768 Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser
220 225 230 AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA AAT GCA ACT 816 Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT CTG TCT 864 Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys
250 255 260
GAA GGA GTT AAT TTG AIT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT 912 Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle
265 270 275 280
GTT CAA GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG ACT GCT GGC CTC 960 Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu
285 290 295
GTG AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG CTT CCT AGC GTG TAC 1008 Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
300 305 310
CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG 1056 Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu
315 320 325
GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC 1104 Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn
330 335 340
AAA ACT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA 1152 Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys
345 350 355 360
GCT CTT GTT TGT CAG GCT AAG TEA CAT ATT GAT GAC ATG GAA TTC GAA 1200 Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu
365 370 375
CCC AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC 1248 Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
380 385 390
AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC 1296 Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly
395 400 405
AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA 1344 Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
410 415 420
ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT 1392 lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
425 430 435 440 GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG 1440 Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly
445 450 455
CTT TAT TTA TCT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA 1488 Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu
460 465 470
GTG GAA TTA ATT ATC CAA CTT ACT CCA AAA GAC ATA AAA CTT ACA GCT 1536 Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala
475 480 485
TTT CCT TCT GAG ACT CTC AAA GAA GGA GAC ACT CTC ATC ATC TCT TCT 1584 Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys
490 495 500
ACA TCT GGA AAT CTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GGG 1632 Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala
505 510 515 520
GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC 1680 Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle
525 530 535 CGA AAG GCC CAG TTG AAG GAT GCG GGA CTA TAT GAA TGT GAA TCT AAA 1728 Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
540 545 550
AAC AAA GTT GGC TCA CAA TTA AGA ACT TTA ACA CTT GAT GTT CAA GGA 1776 Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
555 560 565
AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTC CTC TAT 1824 Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
570 575 580
TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT 1872 Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe
585 590 595 600
GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG 1920 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
605 610 615
AAA TCA AAA GTG 1932
Lys Ser Lys Val
620 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 644 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp -24 -20 -15 -10 lle Met Phe Ala Ala Ser Gln Ala Phe Lys lle Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser 10 15 20
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly lle Glu Thr Pro Leu Pro 25 30 35 40
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys 90 95 100
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn 105 110 115 120
Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val Leu Val Cys
155 160 165
Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg 170 175 180
Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val 185 190 195 200 lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr 205 210 215
Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle Phe Trp Ser
220 225 230
Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys 250 255 260
Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val Glu Leu lle 265 270 275 280
Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu
285 290 295
Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
300 305 310
Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu Thr lle Leu
315 320 325
Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn 330 335 340
Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp Thr Gly Lys 345 350 355 360 Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met Glu Phe Glu
365 370 375
Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
380 385 390
Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu Glu Glu Gly
395 400 405
Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys 410 415 420
lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser 425 430 435 440
Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu Asp Ser Gly
445 450 455
Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser Arg Lys Glu
460 465 470 Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys Leu Thr Ala 475 480 485
Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle lle Ser Cys 490 495 500
Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys Lys Lys Ala 505 510 515 520
Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle
525 530 535
Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
540 545 550
Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
555 560 565
Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr 570 575 580
Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle lle Tyr Phe 585 590 595 600
Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
605 610 615
Lys Ser Lys Val
620
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1941 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. .1941
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73. .1941
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1. .72 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT ACT CTT GAG ATC TCC CCT GGA 96 Ile Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu lle Ser Pro Gly
-5 1 5
CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TCT ACT 144
Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser
10 15 20
GTC ATG GGC TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC 192
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
AGC CCT CTG AGC GGG AAG GTC AGG ACT GAG GGG ACC AAT TCC ACG CTG 240
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
45 50 55
AOC CTG AGC CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA 288
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GTG ACT TGT GGA CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC 336
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu
75 80 85
TAC TCA TTC CCT AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384
Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG AAG COG ATC ACA GTC AAG TGT TCA GTT GCT GAT GTA TAC CCA 432 Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG 480
Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
AGT CAG GAA TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG 528
Ser Gln Glu Hie Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
AGT TTG GAA GTA ACC TTT ACT OCT GTC ATT GAG GAT ATT GGA AAA GTT 576
Ser Leu Glu Val Thr Hie Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165 CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624 Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro
170 175 180
ACA GEA AGG CAG GCT GTA AAA GAA TTG CAA CTC TAC ATA TCA CCC AAG 672 Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys
185 190 195 200
AAT ACA CTT ATT TCT CTG AAT CCA TCC ACA AAG CTG CftA GAA GCT GGC 720 Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GCT CTA CCA GCT CCA GAG ATT 768 Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
TTC TGG ACT AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA 816 Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864 Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle
250 255 260
TAT CTG TGT GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912 Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val
265 270 275 280
GAA TTA ATT CTT CAA GCA TIC CCT AGA GAT CCA GAA ATC GAG ATG ACT 960 Glu Leu lle Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser
285 290 295
GCT GGC CTC CTG AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTE CCT 1008 Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
AGC GTG TAC CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG 1056 Ser Val Tyr Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu
315 320 325
ACT AIT CTG GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT 1104 Thr lle Leu Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 340
CIA GAG AAC AAA AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT 1152 Leu Glu Asn Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp
345 350 355 360
ACT GGA AAA GCT CTT GTT TGT CAG GGT AAG TTA CAT ATT GAT GAC ATG 1200 Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met
365 370 375 GAA TTC GAA CCC AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT 1248
Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
GTT GCC CCC AGA GAT ACA ACC GTC TTG GIC AGC CCT TCC TCC ATC CTG 1296
Val Ala Pro Arg Asp Thr Thr Val leu Val Ser Pro Ser Ser lle Leu
395 400 405
GAG GAA GGC ACT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT 1344
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392 Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
425 430 435 440
CCT CTT TCT GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA 1440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu
445 450 455
GAT TCT GGG CTT TAT TTA TCT GAA GGA ATT AAC CAG GCT GGA AGA AGC 1488
Asp Ser Gly Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser
460 465 470
AGA AAG GAA CTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA 1536
Arg Lys Glu Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys
475 480 485
CTT ACA GCT TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC 1584 Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle
490 495 500
ATC TCT TGT ACA TCT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG 1632 lle Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys
505 510 515 520
AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC 1680
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
525 530 535
TAT ACC ATC CGA AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TGT 1728
Tyr Thr lle Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
GAA TGT AAA AAC AAA GTT GGC TCA CAA TTA AGA AGT TTA ACA CTT GAT 1776
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
GTT CAA GGA AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC 1824
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu
570 575 580 GTG CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA 1872 Val Leu Tyr Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle
585 590 595 600
ATT TAC TTT GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA 1920 lle Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
GAA GCA CAG AAA TCA AAA GTG 1941
Glu Ala Gln Lys Ser Lys Val
620
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 647 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Pro Gly Lys Met Val Val lle Leu Gly Ala Ser Asn lle Leu Trp
-24 -20 -15 -10
lle Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu lle Ser Pro Gly
-5 1 5
Pro Arg lle Ala Ala Gln lle Gly Asp Ser Val Met Leu Thr Cys Ser
10 15 20
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln lle Asp
25 30 35 40
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
45 50 55
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly lle Gln Val Glu Leu
75 80 85
Tyr Ser Phe Pro Lys Asp Pro Glu lle His Leu Ser Gly Pro Leu Glu
90 95 100
Ala Gly Lys Pro lle Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120 Phe Asp Arg Leu Glu lle Asp Leu Leu Lys Gly Asp His Leu Met Lys 125 130 135
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu Val Thr Phe Thr Pro Val lle Glu Asp lle Gly Lys Val
155 160 165
Leu Val Cys Arg Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro 170 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys 185 190 195 200
Asn Thr Val lle Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu lle
220 225 230
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
Asn Ala Thr Leu Thr Leu lle Ala Met Arg Met Glu Asp Ser Gly lle 250 255 260
Tyr Val Cys Glu Gly Val Asn Leu lle Gly Lys Asn Arg Lys Glu Val 265 270 275 280
Glu Leu lle Val Gln Ala Phe Pro Arg Asp Pro Glu lle Glu Met Ser
285 290 295
Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
Ser Val Tyr Pro Leu Asp Arg Leu Glu lle Glu Leu Leu Lys Gly Glu
315 320 325
Thr lle Leu Glu Asn lle Glu Phe Leu Glu Asp Thr Asp Met Lys Ser 330 335 340
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe lle Pro Thr lle Glu Asp 345 350 355 360
Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His lle Asp Asp Met
365 370 375
Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390 Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser lle Leu 395 400 405
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro 410 415 420
Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln 425 430 435 440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu lle Ser Thr Lys Met Glu
445 450 455
Asp Ser Gly Val Tyr Leu Cys Glu Gly lle Asn Gln Ala Gly Arg Ser
460 465 470
Arg Lys Glu Val Glu Leu lle lle Gln Val Thr Pro Lys Asp lle Lys
475 480 485
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val lle 490 495 500
lle Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp lle lle Leu Lys 505 510 515 520
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
525 530 535
Tyr Thr lle Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu 570 575 580
Val Leu Tyr Phe Ala Ser Ser Leu lle lle Pro Ala lle Gly Met lle 585 590 595 600 lle Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
Glu Ala Gln Lys Ser Lys Val
620

Claims

CLAIMS :
1. A monoclonal antibody recognizing an epitope dependent on the fourth immunoglobulin-like domain
Of VCAM-7D.
2. A monoclonal antibody as defined in Claim
1, which blocks Ramos adhesion to VCAM1-expressing cells and which does not cross-block monoclonal antibody 4B9.
3. A monoclonal antibody as defined in Claim 1, selected from the group produced by hybridomas:
(a) ED11.AG6 (ATCC HB 10962),
(b) GE4.BG5 (ATCC HB 10961),
and (c) GH12.AA12 (ATCC HB 10963).
4. A monoclonal antibody having the characteristics of ED11, Fab, Fab', F(ab)2, and F(v) fragments thereof, chimeric antibodies derived therefrom, heavy chain monomers or dimers formed therefrom, light chain monomers or dimers formed therefrom, or dimers consisting of one heavy chain and one light chain formed therefrom.
5. A monoclonal antibody having the characteristics of GE4, Fab, Fab', F(ab)2, and F(v) fragments thereof, chimeric antibodies derived therefrom, heavy chain monomers or dimers formed therefrom, light chain monomers or dimers formed therefrom, or dimers consisting of one heavy chain and one light chain formed therefrom.
6. A monoclonal antibody having the characteristics of GH12, Fab, Fab', F(ab)2, and F(v) fragments thereof, chimeric antibodies derived therefrom, heavy chain monomers or dimers formed therefrom, light chain monomers or dimers formed therefrom, or dimers consisting of one heavy chain and one light chain formed therefrom.
7. A monoclonal antibody that binds to VCAM- 7D, does not bind to VCAM-6D, at least partially inhibits binding between VLA-4-expressing cells and VCAM1- expressing cells, and does not cross-block anti-VCAM1 monoclonal antibody 4B9.
8. A monoclonal antibody according to
Claim 7, selected from monoclonal antibodies ED11, GE4, or GH12.
9. A hybridoma selected from the group
consisting of
(a) ED11.AG6 (ATCC HB 10962),
(b) GE4.BG5 (ATCC HB 10961),
and (C) GH12.AA12 (ATCC HB 10963).
10. A method for purifying VCAM-7D from a solution also containing VCAM-6D comprising contacting the solution with a substrate on which is immobilized a monoclonal antibody according to Claim 1.
11. A method for separating cells expressing
VCAM-7D from cells expressing VCAM-6D comprising
contacting a solution including cells expressing VCAM-7D with a substrate on which is immobilized an antibody according to Claim 1.
12. An in vitro or in vivo method for detecting the presence, in a biological sample or a mammal, of VCAM- 7D or cells expressing VCAM-7D, comprising contacting said biological sample with a detectably labeled antibody according to Claim 1.
13. A treatment for diseases in mammals
characterized by cell-cell adhesion mediated by VCAM-7D comprising administering to a mammal suffering from such a disease an amount of a VCAM1 domain 4 monoclonal antibody, or Fab, Fab', F(ab)2, or F(v) fragments thereof, chimeric antibodies derived therefrom, heavy chain monomers or dimers formed therefrom, light chain monomers or dimers formed therefrom, or dimers consisting of one heavy chain and one light chain formed therefrom, effective to at least partially inhibit in vivo said VCAM-7D-mediated cell-cell adhesion.
14. A therapeutic reagent comprising a monoclonal antibody selected from ED11.AG6 (ATCC
HB 10962), GE4.BG5 (ATCC HB 10961), or GH12.AA12 (ATCC HB 10963), or Fab, Fab', F(ab)2, and F(v) fragments thereof, chimeric antibodies derived therefrom, heavy chain monomers or dimers formed therefrom, light chain monomers or dimers formed therefrom, or dimers consisting of one heavy chain and one light chain formed therefrom, in a pharmaceutically acceptable carrier.
15. A diagnostic kit comprising (1) as a reagent, a detectably labeled monoclonal antibody selected from ED11, GE4 or GH12; Fab, Fab', F(ab)2, or F(v)
fragments thereof; chimeric antibodies derived therefrom; heavy chain monomers or dimers formed therefrom; light chain monomers or dimers formed therefrom; or dimers consisting of one heavy chain and one light chain formed therefrom; and (2) complete instructions for use of said reagent according to the method of claim 12.
PCT/US1993/000031 1992-01-13 1993-01-12 Antibodies recognizing the fourth immunoglobulin-like domain of vcam1 WO1993014220A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP5512513A JPH07506244A (en) 1992-01-13 1993-01-12 Antibody that recognizes the fourth immunoglobulin-like domain of VCAM1
AU34318/93A AU664071B2 (en) 1992-01-13 1993-01-12 Antibodies recognizing the fourth immunoglobulin-like domain of VCAM1
EP93902915A EP0623173A1 (en) 1992-01-13 1993-01-12 Antibodies recognizing the fourth immunoglobulin-like domain of vcam1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US82171292A 1992-01-13 1992-01-13
US821,712 1992-01-13

Publications (1)

Publication Number Publication Date
WO1993014220A1 true WO1993014220A1 (en) 1993-07-22

Family

ID=25234110

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/000031 WO1993014220A1 (en) 1992-01-13 1993-01-12 Antibodies recognizing the fourth immunoglobulin-like domain of vcam1

Country Status (5)

Country Link
EP (1) EP0623173A1 (en)
JP (1) JPH07506244A (en)
AU (1) AU664071B2 (en)
CA (1) CA2127532A1 (en)
WO (1) WO1993014220A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007139359A1 (en) 2006-05-31 2007-12-06 Hanwha Chemical Corporation Vcam-1 specific monoclonal antibody
WO2010114312A3 (en) * 2009-03-31 2011-03-31 Hanwha Chemical Corporation Human monoclonal antibody that specifically binds to vcam-1 and a composition for treating an inflammatory disease or a cancer comprising the same
WO2013160676A1 (en) * 2012-04-24 2013-10-31 Isis Innovation Limited Antibodies against cd106 (vcam-1)
WO2017205560A1 (en) * 2016-05-27 2017-11-30 Albert Einstein College Of Medicine, Inc. Methods for treating cancer by targeting vcam1 and maea
US10947311B2 (en) 2015-11-20 2021-03-16 The Board Of Trustees Of The Leland Stanford Junior University VCAM-1 mediated methods and compositions for treating aging-associated impairments
US11560433B2 (en) 2016-05-27 2023-01-24 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CELL REGULATION vol. 2, no. 5, May 1991, BETHESDA MD, US pages 347 - 355 D. TAICHMAN ET AL. 'Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: Demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.' *
CELLULAR AND MOLECULAR MECHANISMS OF INFLAMMATION vol. 2, 1991, SAN DIEGO CA, US pages 151 - 169 R. LOBB ET AL. 'Vascular cell adhesion molecule-1.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 173, no. 6, 1 June 1991, NEW YORK, US pages 1553 - 1556 B. BOCHNER ET AL. 'Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: Contributions of endothelial cell adhesion molecules.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 175, no. 6, 1 June 1992, NEW YORK, US pages 1433 - 1442 R. VONDERHEIDE ET AL. 'Lymphocyte adhesion through very late antigen 4: Evidence for a novel binding site in the alternatively spliced domain of vascular cell adhesion molecule 1 and an additional alpha 4 integrin counter-receptor on stimulated endothelium.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 176, no. 1, 1 July 1992, NEW YORK, US pages 99 - 107 L. OSBORN ET AL. 'Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.' *
THE FASEB JOURNAL vol. 5, no. 6, 19 March 1991, BETHESDA MD, US page A1602 P. DICORLETO ET AL. 'Monoclonal antibodies (Mab) to ELAM-1 and VCAM-1 that inhibit monocytic cell adhesion to activated and sparse endothelial cells (EC).' *
THE JOURNAL OF IMMUNOLOGY vol. 146, no. 2, 15 January 1991, BALTIMORE MD, US pages 592 - 598 M. THORNHILL ET AL. 'Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.' *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7655417B2 (en) * 2006-05-31 2010-02-02 Hanwha Chemical Corporation VCAM-1 specific monoclonal antibody
WO2007139359A1 (en) 2006-05-31 2007-12-06 Hanwha Chemical Corporation Vcam-1 specific monoclonal antibody
US8715670B2 (en) 2009-03-31 2014-05-06 Hanwha Chemical Corporation Human monoclonal antibody that specifically binds to VCAM-1 and a composition for treating an inflammatory disease or a cancer comprising the same
WO2010114312A3 (en) * 2009-03-31 2011-03-31 Hanwha Chemical Corporation Human monoclonal antibody that specifically binds to vcam-1 and a composition for treating an inflammatory disease or a cancer comprising the same
EP2414392A2 (en) * 2009-03-31 2012-02-08 Hanwha Chemical Corporation Human monoclonal antibody that specifically binds to vcam-1 and a composition for treating an inflammatory disease or a cancer comprising the same
CN102369216A (en) * 2009-03-31 2012-03-07 韩华石油化学株式会社 Human monoclonal antibody that specifically binds to vcam-1 and a composition for treating an inflammatory disease or a cancer comprising the same
EP2414392A4 (en) * 2009-03-31 2013-03-13 Hanwha Chemical Corp Human monoclonal antibody that specifically binds to vcam-1 and a composition for treating an inflammatory disease or a cancer comprising the same
WO2013160676A1 (en) * 2012-04-24 2013-10-31 Isis Innovation Limited Antibodies against cd106 (vcam-1)
US10040854B2 (en) 2012-04-24 2018-08-07 Oxford University Innovation Limited Antibodies against CD106 (VCAM-1)
US10947311B2 (en) 2015-11-20 2021-03-16 The Board Of Trustees Of The Leland Stanford Junior University VCAM-1 mediated methods and compositions for treating aging-associated impairments
WO2017205560A1 (en) * 2016-05-27 2017-11-30 Albert Einstein College Of Medicine, Inc. Methods for treating cancer by targeting vcam1 and maea
US11560433B2 (en) 2016-05-27 2023-01-24 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA
US11732053B2 (en) 2016-05-27 2023-08-22 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA

Also Published As

Publication number Publication date
CA2127532A1 (en) 1993-07-22
AU664071B2 (en) 1995-11-02
AU3431893A (en) 1993-08-03
EP0623173A1 (en) 1994-11-09
JPH07506244A (en) 1995-07-13

Similar Documents

Publication Publication Date Title
CA2132091C (en) Antibodies to alphavbeta3 integrin
CA2140538C (en) Monoclonal antibodies that block ligand binding to the cd22 receptor in mature b cells
AU688751B2 (en) Recombinant anti-VLA4 antibody molecules
JP3684228B2 (en) Endothelial cell-leukocyte adhesion molecule (ELAM) and molecule involved in leukocyte adhesion (MILA)
AU674302B2 (en) Treatment for inflammatory bowel disease
US5766947A (en) Monoclonal antibodies reactive with an epitope of a Vβ3.1 variable region of a T cell receptor
US5217870A (en) Monoclonal antibodies against CDX
US7666417B2 (en) Methods and compositions for treating autoimmune diseases or conditions
US5445940A (en) Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease
EP0555880A2 (en) The CD40CR receptor and ligands therefor
US20130059337A1 (en) Therapeutic Uses of Humanized Antibodies Against ALPHA-4 Integrin
JPH09511644A (en) Protein tyrosine kinase agonist antibody
US6482925B1 (en) Mutants of the LAG-3 proteins and nucleotides encoding LAG-3 mutants
JPH0638789A (en) Hybridoma and monoclonal antibody for inhibiting growth of anti-cd 3 stimulating t-cell
US5599676A (en) Method for isolating a novel receptor for α4 integrins
AU664071B2 (en) Antibodies recognizing the fourth immunoglobulin-like domain of VCAM1
WO1996040942A1 (en) Humanized antibodies to e-selectin
WO1995020604A1 (en) Antibodies to mammalian natural killer antigens and uses
WO1992016563A1 (en) Monoclonal antibodies recognizing lymphocyte function associated antigen-3
WO1990007321A2 (en) Lymphocyte receptor homing sequences and their uses
US5747036A (en) Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease
US6361939B1 (en) Isolated mammalian dendritic cell genes; related reagents
US5679346A (en) Methods of blocking adhesion with anti-lami-3 antibody
US7037496B2 (en) Chimeric immunoglobulin for CD4 receptors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
LE32 Later election for international application filed prior to expiration of 19th month from priority date or according to rule 32.2 (b)
EX32 Extension under rule 32 effected after completion of technical preparation for international publication
WWE Wipo information: entry into national phase

Ref document number: 2127532

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1993902915

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1993902915

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1993902915

Country of ref document: EP