CA2127532A1 - Antibodies recognizing the fourth immunoglobulin-like domain of vcam1 - Google Patents

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

WO 93/14220 PCr~US93/00031

2~7 ~32 ANTIBODIES RECOGNI ZING THE FOURTH
I~Ot:;LOBULIN-LIKE_DOMAIN OF VCAMl FIELD OF THE INVENTIOl~
This invention relates to monoclonal antibodies (MAbs), specifically monoclonal antibodies that bind to the domain 4 cf Vascular Cell Adhesion Molecule-1 (VCAM1~.

BACKGROUND OF THE TNV~NTION
VCA~l (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., 1~89 [2]). VCAM1 binds tQ cells sxhîbiting the integrin VLA~4 (~4~
including T ànd B l~mphocytes, monocytes/ and-eosinophils, but not neutrophils, and is thought to participate in recruitment of these cells from the bloodstream to a~eas of infection and infla~mation (Elices et al, 1990 [3];
Osborn, lg90 [4~. The VCAMl/~LA-4 adhesion pathway has been associated with a number of physiolQgic.al and pathological processes. Although VL~-4 is normally restricted to hematopoietic lineages, it is found cn melanoma cell lines, and thus it has been suggested that VCAM1 may participate in metastasis of such tumors (Rice st al., 1989 ~1]~.

, 2 In vivo, VCAMl 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 fo~llicular dendritic cells in human 5 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 VCAMl appears to play a role in B-cell development.
VCAMl 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 ~ 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.
lg90 [83; 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 ViVQ .
The apparent involvement of the VCAMlIVLA-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 inflàmmation (Os~orn et al., 1989 [2]).

2~7 ~

- 3 - .

Monoclonal antibodies that inhibit VCAMl binding to VLA-4 are known. For example, anti-YLA-4 MAbs HP2/1 and HPlt3 have been shown to block attachment of VLA-4-expressing Ramos cells to human umbilical vein cells and VCAM~-transfected COS cells (Elices et al., 1990 [3]).
Also, anti-VCAN1 antibodies such as the monoclonal a~tibody 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 portionts) of VCAM1 a sential for binding to VLA-4.
Antibodies that bind to VCAM1 without competing for the epitope of a blocking antibody such as 4Bs would also have import~nt diagnostic uses. For instance, a radiolabeled anti-V~AM1 antibody recognizing an epitopa not essential to VLA-4 binding could be used to monitor the course and location of acute inflammatory events but would not ~nterfere with the action of a 1 ter-administëred therapeutic that interacts with the VLA-4-binding domain of ~CA~l.
SUM~ARY 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 VC~M-7D-expressing cells, and through the use of these antibodies we have demonstrated that domain 4 of VCAN-7D contains a site involved in VLA-4 binding, which is in addition to the site recognized and blocked by MAb 4B9 (domain l).
Thus, VCAMl is identified as the first known adhesion molecule to have two binding sites for a single receptor ;~
5 (VLA-4); and the domain 4 monoclonal antibodies according ~-to this invention are useful not only in mapping the -molecular functionality of VCAMl but al o in at least partially inhibiting VCAMl recognition by VLA-4.
Accordingly, it is an object of this invention to provide monoclonal antibodies recogni2ing VCAM-7D but not VCAM-6~. ;
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 ~articularly domain 4 antibodies that do not cross-block the anti-VCAMl 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 VCAMl.
These and other objects are accomplished by the monoclonal antibodies described and claimed herein.

RIEF DESCRIPTION OF THE DRAWIN~S
Figure 1 is a schematic diagram of VCAM-7D, showing the linear arrangemPnt of functional domains from the amino terminus to the carboxyl terminus. These are the signal peptide ('Isig")~ 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 VCAMl constructs having one or more altered domains (described infra~. In the examples set forth below, cDNA

~4~ ~f itif2 encoding VCAM-7D having the diagrammed conformation was used to transfect CoS7 cells to produce CoS7 cells exhibiting VC~M-7D on their surfaces.
Figure 2 is a schematic diagram of VCAM-6D, 5 showing the linear arrangement of functional domains from :~
the amino terminus to the carboxyl terminus --the signal peptide ("sig"), immunoglobulin-like domains l, 2, 3, 5, 6 and 7, the transmembrane sequence ("tm")y 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 COS7 cells to produce COS7 cells exhibiting VCAM-6D on their surfaces.
Figure 3 is a schematic diagram of a chimeric VCANl, designated VCAM/ICAM-l, 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 l and part of domain 2 was excised and replaced by cDNA encoding analogous portions of Intercellular Adhesion Molecule-l (I~AMl). The modified portion of the VCAM-7D
molecule is indicated by shading.
Figura 4 is a schematic diagram of another chimeric VCAMl, 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 l and a large part of domain 2 was excised and replaced with ICAM1 cDNA .
Figure 5 is a schematic diagram of another 30 chimeric VCAMl, designated VCAM/ICAM-3, ~howing 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 l, all ~1 2~r'J32 of domains 2 and 3, and a small N-terminal part of domain

4 was excised and replaced with ICAMl cDNA.
Fîgure 6 is a graph showing relative le~els 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 indireot immunofluorescent staining with MAb 4B9. The number of cells with fluorescent intensity greater than that shown by g9% of control transfectants (pCDM8 vector only) waæ det:ermined.
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 VC~M-7D, VCAM-6D, VCAM/ICAM-l, VCAM/ICAM-2, and VCAM/ICAM-3. In addition, the effects on Xamos binding of preincubation of the transfected COS cells with 20 ~g/ml of M~b 4B~ (anti-VCAMl) and lO ~g/ml of MAb HPl/2 (anti- :~
~LA-4) are shown. In addition, the negli~ible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAMl is shown. Binding :is expressed as Ramos cells bound per square millimeter ("Ramos cel~s bound/mm"
in the figures).
Figure 8 is a schematic diagram of another chimeric VCAMl, designated VCAM6D/IC~M-l, showing the linear arrangement of functional domains. This polypeptide has the general structure of VC~M-6D (no`
domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain l and a small part of domain 2 was excised and replaced with ICAMl cDNA.
Figure 9 is a schematic d~agram of another chimeric VCAMl, designated VCAM6D/ICAM-2, showing the :

W093/l4220 PCT/US93/00031 3 ~

linear arrangement of functional domains. This polypeptide has the general structure of VCAM-6D ~no domain 4), however, as indicated by the sh~ed area, the cDNA coding for most of domain 1 and a large part of S domain 2 was excised and replaced with ICAMl cDNA.
Figure 10 is a graph depicting the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding ~CAM/ICAM-l, VCAM~D/ICAM-l, 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 ICAMl is shown.
Figure 11 is a schematic diagram of another chimeric VC~M1, designated VCAM6D/V~M4-1, showing the ;
linear arrangement of functional dom.~ins. This polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, ths 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 i5 a graph depicting the extent of Ramos cell binding of COS cells separately tranfected with cDNA encoding VCAM/ICAM-l, VC~M~D/ICAM-l, an~- .
VC~M6D/VCAM4-1. Negligible Ramos binding to ICAM1 and negative (pCDM83 controls is also shown.
Figure 13 is a graph depicting the ~xtent of Ramos cell binding to transfectants expressing various chimeric VCAMl 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 VCAMl constructs in the absence of any WO 93/14220 PCrtUS93/00031 '~ 1'"~ ~ r~"~ .z interfering antibody or in the presence of MAb 4B9 or MAb ED11 (this invention).
Figure 15 is a ~raph 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 4~9 or MAb GE4 (this invention).
Figures 16, 17, 18 and 19 are graphs showing Ramos cell binding to cultured H W ECs treated with 20 ng/ml of recombinant human TNF for 4 hours tFig. 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 HPl/2 (anti-VLA-4 ~4 MAb), 4B9 (anti-VCAM1 --MAb), EDll (anti-VCAMl NAb, this invention), and GH12 (anti-VCAMl MAb, this invention) is compared.
DE~AILED DESCRIP~ L~OF THE INVENTION
Th~ 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 ma~mal immunized with whole `~
cells expressing a given antigen, e.g., VCAMl, and/or with purified antigen, and the culture supernatants of ~he resulting hybridoma cells are screened for antibodiPs against the antigen. See, generally, Kohler and Milstein, lg75 [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, et~. Typically, the immunized mammals are bled and the serum from each blood cample is assayed for particular antibodies using appropriate 212 ~ 2 g screening assays. For example, anti-VCAMl 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.
10Typically, 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 ~350). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenoGytes die after several days because they are not transformed). Hybxidomas producing a desired antibody are detected by screening the hybridoma culture s~pernatants 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 VCA~-6D-expressing cells~ (See Example 1, suPra.) To produce anti-VCAMl antibodies, hybridoma cells that tested positive in ~uch screening assays were cultured in a nutrient medium under conditions and for a time suffici~nt to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue WO93/14220 PCT~US93/00031 532 ' 10-culture techniques and culture media suitable for ~ybridoma 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 accu~ulates 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 pr~sent -învention recognize epitopes dependent on the fourth Ig domain of the seven-Ig-domain form of VCAMl, 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 ~o recognize the VCAMl structure. These M~bs 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 VCAMl/VL~-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 2 i . ~

other VLA-4-expressing cells to VCAM1-expressing cells.
Some of the domain 4 monoclonal antibodies described herein bind to epitopes of VCAMl 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-VCAMl 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 YCAM1 on cultured endothelial cells, indicates that binding through domain 4 is a ~ignificant functional component of the VCAMl/VLA-4 interaction n 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 ~NA 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 andlor 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.

WOg3/a4220 PCT/US93/00031 2~i 2~S~3~ ~

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(See, e.g., ~ones et al., ~986 [13], Ward et al., 1989 ~14], and U.S. Patent 4,816,397 (Boss et al.~ tl5], all incorporated herein by reference.) `~
Furthermore, domain 4-binding fragments of anti-VCAMl 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 monemers may similarly be produced by treating an intact antibody with a reducing agent such as dithiothreitol or ~-mercaptoethanol or by using host cells transformed with DNA encoding either the desired heavy ~-chain or light chain or both. As an alternative to hybxidoma technology, antibody fragments having similar specificities may be isolated by phage cloning methods.
~See, e.g., Clackson et al. (ls9l) ~16].) Monoclonal antibodies of the present invention may be used in any application where antibody~recognition of domain 4 of VCAMl 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 VCAMl-mediated binding pathway, vasculitis, etc.

WO93/14220 PCT/VS93/~31 The monoclonal antibodies of the present invention may also be u~ed in combination with other antibodies, bioactive agents or materials for various purposes. For example, the present monoclonal antibodies S may be used in combination with 4B9 or other anti-VCAM1 antibodies in the treatment of disorder~ characterized by VCAMl 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., ELAMl, ICAM1, etc.) in a combination therapy designed to suppress a range of leukocyte-endothelial cell binding pathways.
Alæo, 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 VCAMl-expressing cells. The antibodies of the present invention may also be i~mobi~i2ed on a chromatographic ~ubstrate (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 ~abeled anti~odies according to the present invention may also be used in screening methods or diagnostic methods for detecting VCAMl protein in a sample or VCAMl-expre~sing endothelial cells ln_yi~Q or in vivo.
For example, samples may be screened for the presence of VCAN1 or VCAM1-expressing cells by contacting the sample with a labeled antibody according to the present invention and detecting whether a VCAMl/anti-VCAM1 antibody complex iæ formed.

W093/14220 PCT/US93/~31 2~ 32 - 14 - :.

Suitable labels can be radioactive, enzymatic, fluorescent, maqnetic or chemiluminescent. Radiolabeled antibodies are prepared in known ways by coupling a ~
radioactive isotope such as ~, 32p~ ~S, ~Fe, '~I~ which can ~:

5 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 aster, 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 ~he 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 actiYe ingredients or to prolong their prese~ce 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, intrana~ally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally.
Ordinarily, intravenous (i.v.) or parenteral administration will be preferred.

WO 93/14220 PCI`~US93/00031 ~ ~ ~ 7 -, ~ 9 ' J

~ pl~ 1 MAbs Recoan zinq 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 Chin~se 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 P8S intraperitoneally (i.p.). We in3ected 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 immu~ zed each of the mice with 55 ~g of a recombinant soluble ~rm 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 m~loma cells according to the method described by Lerner t17].
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 ~g/ml, 50 ~l/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 ~erum (FCS), pH
7.2) to block the remaining sites, and the plates were incubated for 2 hrs. at room temperature. The blocking W093/14220 P~T/US93/00031 2 ~ 2 7 .1 3 2 buffer was removed, and 50 ~1 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 r2move 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.lM sodium acetate-citric acid, pH 4.9. The reaction was initiated by addition of hydrogen peroxide (15 ~1, 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, COS7 cells transiently expressing VCAM-6D or ~CAM-7D. In these experiments cDNA encoding VCAM-6D or VCAM-7D was inserted into an expression vector (pCDM8) and subsequently transfected into C~S7 cells by electroporation, as described in Osborn et al. 19S9 [2 and Hession et al. 1991 [9~.
The COS7 cells, transfected with either VCAM-6D
or VCAM-7D cDNA, were washed and suspendad in suspension buffer (PBS/5mM EDTA/2% ~CS/o.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) W093~14220 PCT/US~3~31 ~ ~ 7~
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for 45 min. at 4O C. The cells weire washed, fixed with o.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 ~ummarized in Table I (infra p. 28).
E~mpl~ 2 Ramos Cell Bindinq_Assay Ramos is a B-lymphoblastoid cell line t.hat expre~ses VLA-4 and thus binds to VCAM1 but does not bind to other induced endothelial cell molecules such as ICAM-l or ELAM-l (Osborn et al., 1989 [2]). Ramos is therefore a convenient model cell line with which to assay VLA-4- ~:
15 dependent bindinq. Ramos cells bind to both recombinant ~
VCAM-6D and recombinant VCAM-7~ 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 ~uantitative difference in binding activity was reproducible, several preparations of ~.
each plasmid were made and electroporated in parallel into COS cells. Reipeated 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 VC~Ml was inhibited by the anti-VLA-4 M~b HP1~2 or by anti-VCAM
MA~ 4B9 (Figurei 7).
In all of our procedures, the binding of Ramos cells was measured as follows:
: `

~2~ ~t~32 Confluent monolayers of the target cells (e.g., COS
transfectants or induced H W ECs) in 48-well plastic tissue culture plates were washed with RPMI/10% fetal calf serum (FCS), and an amount (e.g~ g/ml, varying per MAb and 5 per experiment) of the test MAb (e.g., 4B9, HPl/2, GH12, ~;
etc.) in a volume of 0.15 ml was added. In the case of H W ECs, 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 S x 106 cells ml. The BCECF was added to 2 ~M 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 10~ 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].) W093~14220 PCT/US93/~031 ~ 1~7 ` 32 Antibody staininq 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 aparticular 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 indiviclually with 2 ~g/ml of the MAb being tested, or with rabbit polyclonal antiserum raised against rsVCAM-7D (diluted 1:500), in PNF tPBS/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 ~g/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 fluorescent intensity was plotted, and the number of positive cells in each sample was determined from the FACS hist~grams.
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 ~ase, since weakly positive cells that overlap the negative control fluorescence peak are excluded.

WO93/14~20 PCT/US93/~031 ,~ ~7 1 ~2 ~ pl~ 3 Construction and Expression of Chimeric VCAM/ICAM PolyDeptides In order to identify regions of VCAMl involved in recognition of VLA-4-expressing cells, recombinant genes encoding VCAM/ICAM chimeras were constru~ted by excising portions of the VCAM-7D and VCAM-6D cDNAs and replacing them with analogous regions of ICAM-l, using unique restriction endonuclease sites in the VCAMl cDNA
tsee Fig. l) and polymerase chain reaction tPCR)-generated fragments of ICAMl. Substitution of the excised VCAMl regions was done to eliminate as much as possible the ~tructural distortions that would accompany deletion of one or more domains of the VCAMl molecules. ICAMl was `~
selected as a source of substituant structures because it is also a member of the Ig superfamily and has the highest degree of amino acid homology to VCAMl among Ig superfamily members (Osborn et al., 1989 ~2]).
- Schematic diagrams of the VCAMl/ICAMl chimeric polypeptides are shown in Figures 1-5, 8,~ 9 and ll. The cDNA constructs coding for each of these were prepared as follows: ~
Aliquots of the parent plasmids VCAMlEll/CDM8 (encoding VCAM-7D; see, Hession et al., l99l [~]~ or VCAM4ltCDM8 (encod~ng VCAM-6D; Osborn et al., 1989 [2]) were diqested using restriction endonucleases at the appropriate sites indicated in Figure 1 and agarose gel purified. ICAMl inserts were made by PCR from an ICAMl/CDN8 plasmid isolated from an endothelial cDNA library previously described (Osborn et al., l989 [2]), using oligonucleotide probes based on the published sequence of ICAMl (Simmons et al~, 1988 [l9~; Staunton et al., 1988 [20]). The PCR
primers were designed as follows:

WO~3/14220 PCT/US93/~031 21~7 vJ~ `

Primer P-1 (SE0 ID N0:1) ;.
EcoRV
5'-TCTAGATATC TTCTGCCCCG GGAGGCTCCG TGCTG-3' The first twelve nucleotides correspond to the coding 5 region for VCAMl amino acid rssidues 9-12; the following .
twenty-three nucleotides correspond to the coding region for ICAMl amino acid residues 11-18.
Primer P-2 ISE0 ID N0:2) Bgl2 5'-GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA-3' The first thirteen nucleotides correspond to the coding region for VCAMl amino acid residues 86-89; the following twenty-one nucleotides correspond to the coding region for ICAMl amino acid residues 84-90.
Primer P-3 (SE0 ID N0:3) Hind3 5'-TCTCAA CTT TTACTGTTGA GATCTCCCCT GG-3' The first twelve nucleotides correspond to the coding region for VCAMl amino acid residues -3-1; the following twenty nucleotides correspond to the coding region for ICAM1 amino acid residues 300-30~.
PrinLer P-4 (SEQ ID N0:4) Bsu3 6 1 5 '--CTGGAT CT~ AGGAGTCCAG TACACGGTGA GGAAGG-3' The first fourteen nucleotides correspond to the coding region for VCAMl amino a~id resi~ues 92-9~; the following twenty-two nucleotides correspond to the coding region for ICAMl amino acid residues 79-85.
Primer P-5 fSEQ ID N0:5) BspHl 5'-CTCTTCATGA GCTTCTCCC C~CGGAGCAG CACCAC-3' The first eleven nucleotides correspond to the coding region for VCAMl amino acid residues 134-137; the ` -,`, WO93~14220 PCT/US93/~31 ~

5 J ~ ;~

following twenty-four nucleotides correspond to the coding region for ICAMl amino acid residues 121-128.
Primer P-6 ~SE0 ID NO:6~ -Bgl2 5'-GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT-3' The first eleven nucleotides correspond to the coding region for VCAMl amino acid residues 302-305; the following twenty-four nuclsotides correspond to the coding region for ICAM1 amino acid residues 279-2~6.
Primer P-7 (SE0 ID N0:7 Bsu361 5'-CTGGATCCTT AGGGAATGAG TAGAGCTCCA C-3' The first thirteen nucleotides correspond to the coding region ~or VCAMl 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 primad with P-l and P~4; for construct V~A~/ICAM-2 (Fig. 4), P-l and P-5; for construct VCAM/ICAM-3 (Fig. 5), P-2 and P-6; for construct VCAM6D/V~AM4-l (Fig. 11), P-3 and P-7. VCAM6D/ICAM-l and ~CAM6D/ICAM-2 ~Figs. 8 and 9~ were made using the same inserts as for VCAM/I~AM-1 and VC~M/ICAM-2, respectively, clonad into the VCAM-6D expressic)n vector (VCAM41/CDM8) 25 instead of the VCAM-7D expression vector (VCAM~Ell/CDM8).
~fter PCR synthesis, performed as described in Hession at al. (1991) [9], the insert fragments were digested with the appropriate r~striction endonuclaases and gel purified. Expression vectors and insert fragments were ligated using NEB ligase and buffer, ethanol precipitated, then electroporatad into bacterial host strain MCl061/p3 using a Biorad Gene Pulser.

WO93/14220 PCT/US93/00031 ~
2 ~ 3~ J
- 23 - ~.

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 Stàtes Biochemical Corp~). The complete cDNA sequences for VCAM-7D, VCAM-6D, and each of the chimeric constructs VCAN/ICAM-l, VCAM/ICAM-2, VCAMtICAM-3, VCAM6DtICAM-1, VCAM6D/ICAM-2, and VCAM6D/VCAM4-l 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 expresæed when tested by FACS analysis after staining with polyclonal anticerum to VCAMl.
In constructs VCAM/ICAM-l (Fig. 3) and 15 VCAM/ICAM-2 (Fig. 4), most of VCAM domain l, or most of `:
domain l and the first half of domain 2, were replaced by ICAMl 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 l and 4, 20 were replaced by ICAMl sequence (see shaded area, Fig. 5). :~:

Ex~mple 4 MAb 4B9 Bindinq to VCAMl domain l.
It has been previously shown that the N-terminal three domains of VCAMl support adhesion to VLA-4 (Taichman et al., l99l [2l]) and that the anti-VCA`Ml MAb 4B9 ::
significantly inhibits binding of VLA-4-bearing cells to in~uced HUVECs or VCAMl transfectants (Carlos et al., l990 [22]). These results suggested that the binding site of VCAMl recognized by VLA-4 and obstructed by 4B9 is i~ the N-terminal portion of the VCAMl molecule.
Using the MAb binding assay described above (Example 2), we confirmed that MAb 4B9 recognizes an `~:

' '' 2127~2 - 24 -epitope in VCAMl domain l. It was found that 4B9 bound -readily to COS7 transfectants expressing both seven~ and six-domain forms of VCAMl. Furthermore, chimeric constructs VCAM/ICAM-l and VCAM/ICAM-2 ~Figs. 3 and 4, respectively), wherein most of domain l was replaced by ICAMl amino acid residues, did not bind to 4B9 (see, Table I, nf~a). Th~se results indicated that the epitope to which 4Bg binds requires domain l for recognition.
Finally, construct VCAM/ICAM-3 (see, Fig. 5, domain 1 mostly intact) did bind 4B9, confirming that domain l contains the 4B9 epitope. The binding and ~amos blocking ~
characteristics of 4B~ 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/VCAMl, since anti-VLA-4 mAb HPl/2 completely inhibited the interaction, while a blocking antibody to the ICAM-l 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 Reions On VCA~l To determine whether Ramos cell binding to constructs VCAM/ICAM-l and VC~M/ICAM-2 was due to residual 2S portions of the domain l-dependent binding site, or to domain 4 (which is structurally similar to domain l~, constructs analogous to VCAM/ICAM-l 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 ICAMl inserts (see, Figs. 8 and 9).
These constructs, designated VCAM6D/ICAM-l and VCAM6~/ICAM-2, respectively, were wèll expressed in COS7 cells as measured by anti-VCAMl polyclonal antiserum g .~

staining, but were unable to bind ~amos cells (Figure lO).
These results indicated that the binding of Ramos cells to constructs VCAM~ICAM-l and VCAM/ICAM-~ appears to be due to the presence of domain 4.
~e 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 10 Fig. 2, intact domain 1) and VCAM/ICAM-l ~see Fig. 3, :~
intact domain 4), indicating that domain 4 can incleed 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.
~x~mple S -Nonoclonal Antibodies That Block Domain 4-deendent Cell Adhçsion According to the procedure set forth in Ex~mple l, 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 IgG1 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-l and 2, and VCAM6D/VCAM4-1 but did not bind to transfectants expressing VCAM-6D or VCAM/ICAM-3.
`:

WO93/14220 PCT/US93/~03~

2 i27i.i32 Referring to Figure 13, Ramos binding experiments showed that MAb GH~2 at 10 ~g/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-l, VCAM/ICAM-2 and VCAM6D/VCAM4-1 constructs but did not bind to the S VCAM/ICAM-3-expressing tranfectants, indicating that the ~mall 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 EDll bound to COS7 cells expressing VCAM-7~, VCAM/ICAM-1 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 ~g/ml inhibited Ramos binding to COS7 transfectants expressing th~ VCA~'/ICAM-1 and VCAM/ICAM-2 constructs but did not inhibit VCAM-6D-expressing, VCA~/ICAM-3-expressing or VCAM6D/VCAM4-1-expressing tranfectants. Surprisingly, MAb EDll did partially block Ramos cell binding to VCAM-7D-expressing COS7 cells.
Bi~ding experiments showed that MAb GE4 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-l, VC~M/ICAM-2 and VC~M/ICAM-3 but not to cells expressing VCAM-6D or VCAM6D/VCAM4-1. Referring to Figure 15, Ramos binding experiments showed that MAk GE4 at 10 ~glml did not inhibit Ramos binding to COS7 transfectants expressing VCAM/ICAM-1 and VC~M/ICAM-2. MAb GE4 did not block Ramos adhesion to transfectants expressing VCAM6~/VCAM4-1, VCAMlICAM-3 or VCAM-6D, and also did not inhibit Ramos binding to VCAM-7D-expressing COS7 ~ells at the single concentration testad.
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 WO93/14220 PCT/US93/~031 2~27.i.~

distinct epitopes, and two are capable of blocking domain 4-dependent adhesion of VL~-4 to VCAM1. These antibodies represent a new rlass of anti-VCAMl blocking antibody.

Table I
constructs* .
_~a~ VCAM-7D VCAM-6D A B C D

binding + - ~ +
blocking - - + ~ - +
10 EDll .. :
binding + - + ~ - -blocking ~ +

binding + - + ~ + - -::
15 blocking 489 ~control) --~
binding ~ + - ::
blockîng + +
20 * A - VCAM/ICAM-l C = VCAM/ICAM-3 B = VCAM/ICAM-2 D - VCAM6D/VCAM4-1 .

E~ample 6 Domain 4 MAb Inhibition of Ramos Binding to Cvtokine-activated EndQthelial Cells To determine whether the domain-4-dependent MAbs ED11 or GH12 were capable of blockin~ adhesion to VCAMl on e~dothelial cells (which is predominantly VCAM-7D), human `~
umbilical vein endothelial cells (H W ECs) were treated with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72 hours, and binding of Ramos cells and t~e 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.

WO93/14220 pcT/us93/noo3l ~1275~

The results indicate overall that MAb GH12 did not block HUVEC/Ramos binding reliably at the cingle concentration tested (10 ~glml), but MAb EDll blocked more than 80% of Ramos cell binding to induced H W ECs 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 VCAMl (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-l, VCAM-6D tRNA) may be the predominant species of VCAM-l in bone marrow stromal cells and tonsillar follicular dendritic cells.
This suggests a potential therapeutic advantage of domain 4 spe~ific anti-VCAMl antibodies over domain 1 specific anti-VCAMl 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 infla~mation, without blocking lymphopoiesis, which is the role that VCAM-l 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 WO93/14220 PCT/US93~31 2127 -;3 ~ ~

experienced in this art. All such modifications and .`
additional embodiments are within the contemplation of - :
t~is 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: .
desiq__tion ATCC_accession no. `:
MAb CB.EDll.AG3 HB 10962 MAb CB.GE4.BG5 HB 10961 ~ -MAb CB.GH12.AAl2 HB 10963 ~

W093/14220 PCT/U~93/~031 3 ~

CIT~D P~BLICATION8 tl] Rice, G.E. and Bevilacqua, M.P. (1989), "An inducible endothelial cell surface glycoprotein mediates melanoma adhesion," Science, 246, 1303-1306.
~2] Osborn, L., He~sion, C., Tizard, R., Vassallo, C., Lu~owskyj, 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 lymphocyte~," Cell, 59, 1203-1211.
t3~ Elices, M.J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M.E., and Lobb, R.R. ~1990), "VCAM-l on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct ~rom the VLA-4/fibronectin binding site," Cell, 60, 577-584.
15 ~4~ Osborn, L. (1990), "Leukocyte adhesion in inflammation," Cell, 62, 3-6~
~5] Cybulsky, M.I. and Gimbrone, M.A., 3r. (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., McIntyre, B.W., Rhynhart, K., Pober, J.S., and Nadler, L.M. (1990), "Adhesion of human B cells to germinal centers in ~itro invol~es VLA-4 and INCAM-110t" 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. tl990), "Full length vascular cell adhesion molecule 1 (VCAM-l),"
Nuc. Ac. Res., 18, 5901.

WO93/14220 PCT/US93/~31 ~ 27 ., 3 ~

~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-l (VCAMl~", J. Biol Chem., 266, 6682-6685.
[10] ~ybulsky, 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), NGene structure, chromosomal location, and basis for alternative mRNA
splicing of the human VCAM1 gene," Proc. Natl. Acad.
Sci. USA, 88, 7859-7863.
C11~ Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, E., Russo, M., Osborn, L., Chi-Rosso, G., Newman, Lo~b, ~
R., and Harlan, J.M. (1990), "Vascular cell adhesion .
molecule-1 mediates lymphocyte adherence to cyt~kine- :
activated cultured human endothelial cells. Blood, 7~, 965-970~ :
[12~ Kohler, G. and Milstein, C. (1975), "Continuous Culture of Fused Cells Secreting Antibody of ~
Predefined Specificity", Nature, 256, pp. 4g5-497. -;
~13] Jones, P. T., et al. (1986), "Replacing the Complementarity-D~termining 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 Reper~oire of Single Immunoglobulin Variable Domains Secrete~ Fr~m Escherichia coli," Nature, 341, 54~-546.
~153 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.

WQ93/14220 PCT/US93/0003l ~ ~ h~ ~ 3 2 ~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, ~.B~ (1989), "Endothelial interleukin-8: A novel inhibitor of leukocyte-endothelial interactions," Science, 246, l601-1603.
Cl9] Simmons, D., Makgoba, M.W., and Seed, B. (1988), "I~AM, an adhesion ligand of LFA-l, 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-l and the binding sites for LFA-l and rhinovirus," Cell, 61, ~43-254.
[21] Taichman, D.B., Cybulsky, M.I., Djaffar, I., Longenecker, B.M., Teixido, J., Rice, ~.E., Aruffo, A., and ~evilacqua, M.P. (l991~, "Tumor cell surface ~4~1 integrin media'es adhesion to vascular endothelium: Demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1," Cell Req~lation, 2, 347-355.
[22~ Carlos, T.M., Schwartz, B.R., Kovach, N.L., Yee, ~., Rosso, M., Osborn, L., Chi-Rosso, G., Newman, B., Lobb, R., and Harlan, ~.M. (1990), "Vascular cell adhesion molecule-l mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells,"
Blood, 76(5), 965-970.

The foregoing publications are incorporated herein by reference.

r 3 7 . ~ ;) ~ .J ~
SEQUENCE LISTING

(1) GENERAL INFO~ATION:
(i) APPLICANT:
(A) NAME: BIOGEN, Inc.
(B) STREET: 14 Cambridge Center (C) CITY: Cambridge (D) STATE: Massachusetts (E~ COUNTRY: USA
(F) POSTAL CODE (ZIP): 02142 (A) NAME: OSBORN, Laurelee (B) STREET: 19 Harding Street, ~2 (C) CITY: Cambridge (D) STATE: Massachusetts (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02141 (A) NAME: BENJAMIN, Christopher D.
(B) STREET: 2 Oak Hill Lane (C) CITY: Beverly (D) STATE: Massachu;ettes (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 01915 (ii) TITLE OF INVENTION: ANTIBODIES RECOGNIZING THE FOURTH
IMMVNOGLOBULIN-LIKE DOMAIN OF VCAM-l (iii) NUMBER OF SEQUENCES: 23 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATINC SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: WQ PCT/US93/00031 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/821,712 (B) FILING DATE: 13-JAN-1992 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :
(D) TOPOLOGY: linear ~UeSTl~ E S~IEET

212~ ~ 2~ 2 ' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

(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:

(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 DESCRIPTION: SEQ ID NO:3:
TCTC M GCTT TTACTGTTGA GATCTCCCCT GG 32 . ~.
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: ~ucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - -(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 ~;116S~ IJ~E S~EE~T

~ 1 2 ~ ;3 3 ~

-35- :~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(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:

(2) INFORMATION FOR SEQ ID NO:7:
(i) ~EQUENCE 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:
tA) NAME/KEY: CDS
(B) LOCATION: 1..2217 (ix) FEATURE:
(A) NAME/KEY: mat peptide (B) LOCATION: 73..2217 (ix) FEATURE:
tA) NAME/KEY: sig_peptide (B) LOCATION: 1..72 81~3S~3~E SffE~ ~

2 1 ~ ~ . i ~ 2 .... .

(~i) SEQUENCE DESCRIPTION: SEQ ID No:8:

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 .
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile 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 Ile 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 Ile 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 .
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 Lyc Gly Ile Gln Val Glu Ile -~;
75 80 85 , Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Gys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val elJBSTlTllTE SHE~ ` ` `
.' `,``' ~ 1 27'~ ~2 Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pr~ Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile TTC TGG AGT AAG AAA TTA GAT MT 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 `~

Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val I:lu Leu Ile Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly .

Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser GTC ATG GGC TGT GM TCC CCA TCT TTC TCC TGG AGA AC,C CAG ATA GAC 1055 Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr LPU

Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu ~U13ST~ E S~

2~ 7'.3f32 '~

Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val 380 385 39~ -Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro :~

Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys 425 430 435 440 .

Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala -Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile L,eu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe ~:

2 127 t~ 32 Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile 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 Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg ~ :
620 ~25 630 Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg 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 Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val (2) INFORMATION FOR SEQ ID NO:9:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 739 amino acids (B) l~PE: amino acid -~;
(~) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

SU~S~ T

212'7`132 ' `

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn ~le Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe Phe Se. Trp Ar~ Thr Gln Ile Asp er Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr la Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile ~5 yr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu la Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro he Asp Arg Leu Glu Ile~ Asp Leu Leu Lys Gly Asp His Leu Met Lys er Gln Glu Phie Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg A~a Lys Leu His Ile Asp Glu Met Asp Ser Val Pro ~.70 175 180 Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly er Val Thr Met ~r Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile 220 225 ~30 Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln Hi.q Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile T~ E ~

21 27~-j32 Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Va~

Glu Leu Ile Val Gln Glu Lys Pro Pha Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu -~:
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr ,~:
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro 395 400 4~5 Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser 51n Gly Phe Pro Ala Pro Lys Ile SllBST~7 UTE S~

.

` 2~7~3~ -eu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu sn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe 570 575 580 - `
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr 585 590 595 600 : ~:
ys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg ~ .
620 625 630 .
Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn ~:

ys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg 65~ 655 660 lu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe 665 670 675 680 `
Ala Ser Ser Leu Ile Ile Pro Ala Il~ Gly Met Ile Ile Tyr Phe Ala ~ .-685 690 ~5 ~:
rg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val -(2) INFO~ATION FOR SEQ ID NQ:10:
( i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 1941 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: 1inear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..19 Sll~3ST~T~ $~

( i~c) FEATURE:
(A) NAME/KEY: mat peptide (B) LOCATION: 73. .1941 ( i:c ) FEATURE:
(A) NAME/KEY: sig peptide (B) LOCATION: 1. .72 : -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: .

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu --5 1 5 : .

Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys SUBSTI~V~E S~ET

2 I 2~

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 AGT 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 Ile Glu Asp Ile Gly Lys Val -~ ~
155 160 165 .- .

Leu Val Cys Ar~ Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro 170 175 180 .

Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly .

TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT 768 ~:
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile 220 225 230 :~.
TTC TGG AGT ~AG AAA TTA GAT AAT ÇGG MT CTA CAG CAC CTT TCT GGA 816 :
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile .
250 255 260 - ~-TAT GTG TGT GM. GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912 Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val GM TTA ATT GTT CM GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT . 960 Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro AGC GTG TAC (::CC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG 1056 Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser SIJ~ T

2.-i 2 rl ~ 3 ~

Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Ar~s Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu `~' 445 450 455 Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys ~er Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys SU~

~ ~ 2i2~5r~2 r ' , Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp ~:

Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu GTG CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT ~CC ATT GGA ATG ATA 1872 Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile -~

Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val (2) INFORMATION FOR SEQ ID NO~
~i~ SEQUENCE CHARACTERISTICS: .
(A) LENGT~: 647 amino acids ~
(B) TYPE: amino acid - .
(D) TOPOLOGY: linear -~
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: :~
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp -24 -20 -15 -10 ;
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu -5 l 5 Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr

6~ 65 70 Ala Thr Cys ~lu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile 8UB~ T

212~7 ~232 -4~-Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro he Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys er Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu V~l Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val 155 160 1~5 Leu~Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly er Val Thr Met Thr Cys Ser Ser Gl~ Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val lu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser 285 290 2~5 ly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr lle Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser 330 335 3~0 Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp SUE~ EET ~;

' ' '. ?J'~ J~J~
-48- :
Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Mee ~: .

lu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu ~:

Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile 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 Ile Ser Thr Lys Met Glu sp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys : -Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile -490 4g5 500 le Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys 505 510 515 520 .
ys Lys Ala S:lu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala 525 530 535 .
yr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu 570 575 5~0 Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile le Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val su~ s~

2127 t~32 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2205 base pairs ~B~ TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: 1 inear ( 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:

Me~ Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 :.
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu .

Ser ArE~ 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 Ile Glu Thr Pro Leu Pro 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 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro ~VB~T~

` 2ri 2 7 ~ 3 2 -so~

Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG MG AGT CAG GAA TTT 4 8 0Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC CGA 5 7 6Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Lèu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln GCT. GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC MG AAT ACA GTT ATT 672 Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile l~S 190 l9S 200 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 GGT CCA GAG ATT TTC TGG AGT AAG 7 6 8 .
Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys AAA TTA GAT MT GGG AAT CTA CAG CAC CTT TCT GGA AAT GCA ACT CTC . 816Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu ACC TTA ATT GCT ATG AGG ATG GM GAT TCT GGA ATT TAT GTG TGT GM 864 -~
Thr Leu Ile Ala Met Arg Me~ Glu Asp Ser Gly Ile Tyr Val Cys Glu :
250 255 260 ~ :
GGA GTT A.AT TTG ATT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT GTT912 Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Gl.u Leu Ile Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala 81JE~ E ~;H~Fr ~ ~ ~7~ ~
~J ' ~

GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGï AGT GTC ATG GGC TGT 1008 Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Sar Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu S~r Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC TAC TCA TTC CCT :L200 His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG AAT ~GG AGC TCT 1248 Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser :;

Val Thr ~al Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe 410 415 4~0 `:
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 . ~ :`

Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met SIIE~STITUTE ~gtÆET

52- ~127~.?~

Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Giy Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu 525 53~ 535 Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA TGT GGA AAT GTT1872 ~
Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cy.g Gly Asn Val `
585 590 595 600 -~ ~-Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG TTG1968 : :
Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu 620 625 630 ,:

Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser :: :

Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys 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 Ile I1Q PrO AIa Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn ATG AAG GGG TCA TAT AGT CTl' GTA GAA GCA CAG AM TCA AAA GTG 2205 Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln~Lys Ser Lys Val S~ E Sff~E~

-53- , (2) INF0RMATION 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 Ile Leu Gly Ala Ser Asn Ile Leu ~rp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu ~;
_5 1 5 `:-.
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser l0 15 20 ~
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile 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 Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu lu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe eu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Ar~

Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln SU~STIT~IESlLi!~

3~ 5 3 ~

ds D N0:13:
.y Ala Ser Asn Ile Leu Trp .5 -10 s Ile Glu Thr Thr Pro Glu s r Val Leu Val Thr Cys Ser .y Ile Glu Thr Pro Leu Pro ~;n Arg Lys Val Tyr Glu Leu ~t Cys Tyr Ser Asn Cys Pro ~u Thr Val Tyr Trp Thr Pro ~-o Leu Glu Ala Gly Lys Pro Tyr Pro Phe Asp Arg Leu llS 120 ~u Met Lys Ser Cln Glu Phe u Thr Lys Ser Leu Glu Val y Lys Val Leu Val Cys Arg ~r Val Pro Thr Val Arg Gln T~
:

~ r r r ~ ~ r 2~2~ 2 la Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg ln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu hr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Ar~s Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val ro Glu ll~r Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr al Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser 6~5 640 645 Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu le Ile ~ro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn et Lys Gly Ser Tyr Ser Leu Val Gl.u Ala Gln Lys Ser Lys Val SUI~STITU~ EEr 2 ~ ` 3 ~

(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 (i~) 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:

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile GIu Thr Thr Pro Glu -5 l 5 Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu l'hr Pro Leu Pro L~s Lys Glu Leu Leu Le~ Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro SU135~Tl~TE ~

c ' '2127 ,,'3?,' --~7-- -GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TG(' CAG CCA GTG GGC AAG 384 Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser AAG AAA TTA GAT MT GGG MT CTA CAG CAC CTT TCT GGA AAT GCA ACT . 816 T~y5 Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr CTC ACC TTA ATT GCT ATG AGG AT& GM GAT TCT GGA ATT TAT GTG TGT 864 Leu lhr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile 265 270 27S ~0 Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Ar~g Ile SU~ EE~ ~

~127~33~

GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT G,TC ATG GGC 1008 Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu ~::

Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys L~u Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg : ~-395 . 400 405 Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met l,ys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Yal Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr ~75 480 485 Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn su~ r~ ET

2 ~ 2 7 ~ 3 2 _59_ Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gl~ Pro Leu Ser Glu Asn Ala Thr ~25 530 535 Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA TGT GGA AAT 1872 ~-Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp 605 610 615 .~.

Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln 620 625 630 ~-Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly `

TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT C M GGA AGA GAA .~AC AAC- 2064Ser Gln Leu Arg Ser Leu Thx Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser -TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA AGA AAA GCC ~160 Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val 81JBST~T~TE ~EEl' 2 } 27~3 ~

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 a~ino 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 Ila Leu Gly Ala Ser Asn Ile Leu Trp le Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu _5 1 5 er Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cy5 Ser 10 15 20 :
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro ys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu ' 45 50 55 er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn 105 11.0 115 12 eu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu he Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val SU~ ET

; 2~7S~- 2 Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr et Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser ys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Ar~ Lys Glu Val Glu Leu Ile :~

Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile 285 290 295 :
la Ala Gln I18 Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly ys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu 315 320 325 ;
Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser 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 Ile Gln Val Glu Leu Tyr Ser Phe `
365 370 375 `:
ro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser er Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg 3g5 400 ~OS
Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys ln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg SlJB~ S~

~ r ~ r ~

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 Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser rg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr eu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys lu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val I.ys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn 585 5~0 595 600 al Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp 605 610 61 s hr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Ç20 625 630 eu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser eu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala sn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val -~JBS~ TF~

~1"7~2 (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 -~ .
(i~) 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 ID NO:16:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TG~ 48 Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser lQ 15 20 Thr Thr Gly Cys Clu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile SlJB~T~TU~E ~

2127 ~32 Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln ~0 95 100 Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val 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 Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys Ser Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala Leu Gly Asp Gln Axg Leu~Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser ~35 240 245 Phe Sex Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu Thr I.eu Gln Thr Val Thr Ile Tyr Ser Phe Pro Ala Pro Glu Ile Ser Pro ~;~.IB5~ ; t ~ T

2 7 ~ 3 2 Gly Pro Arg Ile Ala Ala Gln Ile 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 Ile ::~

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 GM~CAC TCT TAT CTG TGC 1152 :
Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu CTC TAC TCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC 1~48 Leu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val-Thr Val Ser Cys Lys Val Pro Ser Val Tyr 3gS 400 405 CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT A.9G G(;G GAG ACT ATT CTG 1344 Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys 4~5 450 455 Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu CCC AAA CM AGG C:AG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC 1536 Pro Lys Gln Ar~s Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu ~lal Ser Pro Ser Ser Ile Leu Glu Glu Gly r ~ ~
J~

AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA 1632Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys 505 510 515 5~0 ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT 1680Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser GAG M T GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG 1728Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA M G GAA 1776Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu GTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT 1824Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT 1872Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys ACA TGT GGA AAT GTT CCA G M ACA TGG ATA ATC CTG AAG AAA AAA GCG 1920Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC 1968Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile 620 625 630 ~ :-CGA AAG GCC CAG TTG ~AG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA 2016Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys M C AAA GTT GGC TCA CM TTA AGA AGT TTA ACA CTT GAT GT~ C~A GGA 2064Asn Lys ~al Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly 650 655 6~0 AGA GAA M C AAC AAA GAC TAT TTT TCT CCT GAG CTT CTG GTG CTC TAT 2112Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro ~la Ile Gly Met Ile Ile Tyr Phe 685 690 6~5 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln `, :.
8UE~STIT~E ~

: 212~2 -:

Lys S e r Lys Val (2) INFORMATION FOR SEQ ID NO:17:
( i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 740 amino acids (B) TYPE: amino acid -( D ) TOPOLOGY: 1 ine ar (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO :17:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp --24 --20 --15 --lO
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp er Pro Leu Asn Gly ~ys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu 5~
hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala ro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu ys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu ~;U8~;1 IT~)TE S~EE~

2 1 ~ 7 ~5 3 ~

Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala 170 175 lB0 Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys er Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu Thr eu Gln Thr Val Thr Ile Tyr Ser Phe Pro Ala Pro Glu Ile Ser Pro ly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys 30~ 305 310 Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys hr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu eu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser- Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met. Lys Ser Leu Glu Asn 811BST~Tl~E S~

Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly As~ Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile S2(~ 625 630 Arg Lys Ala Gln Leu l.ys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu lhr l,eu Asp Val Gln Gly Ar~ Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln 700 705 ~ 710 ~;UB~;T~Tl~E ~Ef 2 ~ 2 7 !J 3 ~

Lys Ser Lys Val (2) INFORMATION FOR SE~ ID NO:18:
(i) SEQUENCE CHAXACTERISTICS:
(A) LENGTH: 1929 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ix) FEATURE:
(A~ NAME/KEY: GDS
(B) LOCATION: 1..1929 ( ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 73..1929 ( ix) FEATURE:
(A) NAME/KEY: sig_peptide (B~ LOCATION: l . . 72 (xi) SEQUENCE DESCRIPTION: SE~ ID N0:18:
ATG CCT GGG AAG ~TG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 :~
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu TCT AGA TAT CTT GTG 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 Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro 60 65 ` 70 SUE~ U~ LE~

~; ~ ?, 7 ~ 3 2 Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro 7S 80 ~5 Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val 140 145 150 ~:~

Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 : .
Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln 170 175 1~0 Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile 185 190 l9S 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 ACC TGT TCC AGC GAG GGT CTA CCA (:CT CCA GAG ATT TIC TGG AGT MG 7 6 8 Thr Cys Ser Ser Gl~ Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu -~35 240 245 Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val I;U~ 7a~E ~3~.r~r 2 ~ ~ 7 ~ 3 2 Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys ~ .

Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala 345 350 355 360 :

Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro :
365 370 375 ;-AAA CAA AGG CAG AGT ACG CM 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 -:

Asp Thr Thr Val Leu Val Ser Pro Sar Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GM GTG 14~8 Tyr Leu Cys &lu Gly Ile Asn Gln Ala Gly Ar~ Ser Arg Lys Glu Val GM TTA ATT ATC CM GTT.ACT CCA MA GAC ATA AM CTT ACA GCT TTT 1536 Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile~Lys Leu Thr Ala Phe SUE~S~lTtlTE ~fflEf - 21~75~
--73-- .

Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu ACA GGA GAC ACA GTA CTA AAA TCT ATA :;AT GGC GCC TAT ACC ATC CGA 1680 Thr Gly Asp Thr ~.'al Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile 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 555 560 565 ~
GAA AAC MC 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 Lell Val Leu Tyr Phe 570 575 580 .

Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala ;

Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys TCA AAA GTG 1~29 Ser Lys Val (2) INFORMATION FOR SEQ ID NO: 19:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 643 anlino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECUI,E TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp 8UBSTIT~T~

lle Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser ~-Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu - ~ -- 45 50 55 ~.
er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro .

Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly I~s Pro g5 100 Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile 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 Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met 205 210 ~15 Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys :~

Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu 250 255 ~ 260 SUE3ST~T~E S~

~ 1 2 7 i~ 3 ~

Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val 265 270 275 280 :~
ln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val 285 2gO 295 sn Gly Ser Ser Val Thr Val Ser Cy5 Lys Val Pro Ser Val Tyr Pro eu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala eu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro 365 ~370 375 ys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg sp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu 425 ~30 435 440 sn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val yr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu 505 510 515 s2n hr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg ~u~ r ~ ~ 12 rl ~ 3 ~ ;:

ys Ala Gln Leu Lys Asp Ala Gly Val Tyr Gl~l Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val (2) I~FORMATION FOR SEQ ID NO:20:
(i) SEQUENCE C~ ACTERISTICS:
(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) FEAT~RE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 73..1932 (ix) FEATURE:
(A~ NAME/KEY: sig_peptide (B) LOCATION: 1.. 72 ~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: :
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA .~Ar ATA CTT TGG 48 Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp -24 -20 -15 -lO
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA G.~ 96 Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile 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 lO 15 20 `

81JB~TIT~T~

-b~ 2~32 Thr Ser Cys Asp Gln Pr~ Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe l.eu Thr Val Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys 90 95 lC0 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 Val lrhr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG ('CC ACA GTA AGG 6 24 Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg 170 175 lB0 Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr ATG ACC TGT TCC AGC GAG .GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT 7 6 8 Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Sll~S~3Tt~TE S~~

2~ 2~3~

Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu :
285 290 2~5 GTG MT GGG RGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC 1008 :~
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 Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu 3~5 320 325 Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser I.eu Glu Asn 33~ 335 340 :

Lys Ser Leu Glu Met Thr Phe lle Pro Thr Ile Glu Asp Thr Gly Lys.
345 350 355 ~ 360 Ala Le.u Val Cys Gln Ala Lys Leu His Ile 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 AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GA& G~ GGC 1296 Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Çlu Gly AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT C:CG AAA 1344 Ser Sex Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser ~3U!13STIT~

7 ~ <J h Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala TTT CCl` TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT 1584 Phe Prc Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys 490 495 500 :

Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp,Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gin Leu Lys A.~p Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe 585 590 59~ 603 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln . 605 610 615 Lys Ser Lys ~al S~

: 2 ~ 2 7 ~ `, 2 (2) I~FORMATION 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 Ile Leu Gly Ala Ser Asn Ile Leu Trp :.

Ile Me~ Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu -5 1 5 ~:
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro ys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro 60 ~5 70 ::
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro -Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala E'ro Arg Ala Asn eu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu he Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly I.ys V~l Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val 185 190 195 ~00 S~ TlTl~

. -81- 2127~32 Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr et Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser 220 ~25 230 Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile al Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly l.eu al Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys la Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu ro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser lu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly al Tyr Leu Cys Glu &ly Ile Asn Gln Ala Gly Ar~ Ser Arg Lys Glu 460 465 ~ 470 SU~3T~

r ~ ~ .
--8 2-- 2 1 2 7 J r3 Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala 475 480 485 .:
Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys ~hr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu ~ys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile ..

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 Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr 570 575 580 .
Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln :~605 610 615 -~.
Lys Ser Lys Val (2) INFORMATION FOR SEQ ID NO:22: ~ `
(i) SEQUENCE C~ARACTERISTICS:
(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 SUB~ F~

2127~32 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp Ile Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu Ile Ser Pro Gly _5 1 5 Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro 105 110 115 12~

Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val SUE~S~UTE~

r r r 2 1 2 7 5 ~3 2 -84- :

Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro :

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 Ile Ser Pro Lys Asn Thr Val Ile Sar Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT 768 :~
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile 220 225 230 :~

Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864 ` :
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile ~:
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 Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser 285 290 295 :
GGT GGC CTC GTG AAT GGC: AGC TCT GTC ACT GTA AGC TGC MG GTT CCT 1008 Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro -Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu-His Ile Asp Asp Mec SU~

2 1 2 7 ~ 3 2 Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys CTT ACA GCT TTT CCT TCT GAG AGT GTC AAA GM GGA GAC ACT. GTC ATC 1584 Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GC~ 1680 Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu SU~

~ 1 2 7 '.~

Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val (2) INFO~MATION FOR SE~ 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 Ile Leu Gly Ala Ser Asn Ile Leu Trp :
-24 -2~ -15 -10 Ile Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp er Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu hr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro iO5 110 llS 120 SUI3!~T~9~E ~tEES

2 7 ~

Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met l.ys er Gln Glu Phe Leu Glu Asp Ala Asp Ar~ Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Ar~ Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly er Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp SPr Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Rsn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val ~65 270 275 280 lu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser ly 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 Ile Glu Leu Leu Lys Gly Glu-Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp hr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met 3~5 370 375 lu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn 380 385 ` 390

8)Jg3ST~TJ~ ~

~ ~ 2 7 ~ .~ 2 :~

Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg 51n Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser ~ly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser . 460 465 470 Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile 490 4~5 500 Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala 525 530 535 ~:
Tyr Thr Ile Arg Lys Ala Gln Leu Lyc Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp 555 56Q S65 .
Val Gln Gly Arg Glu Asn Asn Lys Asp-Tyr Phe Ser Pro Glu Leu Leu ~;-Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile 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 ... . ~

8UBÇT3TU~E~ S9~EE~

Claims (15)

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 ceils 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.