AU664071B2 - Antibodies recognizing the fourth immunoglobulin-like domain of VCAM1 - Google Patents

Description

OPI DATE 03/08/93 APPLN. ID 34318/93 AOJP DATE 14/10/93 PCT NUMBER PCT/US93/00031 1111111 111111111 AU9334318 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5: (II) International Publication Number: WO 93/14220 C12P 21/08, C12N 5/20 Al C07K 3/18, COIN 33/577 (43) International Publication Date: 22 July 1993 (22.07.93) GOIN 33/68, A61K 39/395 (21) International Application Number: PCT/US93/00031 (72) Inventors; and Inventors/Applicants (for US only) OSBORN. Laurelee (22) International Filing Date: 12 January 1993 (12.01.93) [US/US]; 19 Harding St., Cambridge, MA 02141 BENJAMIN, Christopher, D. [US/US]; 2 Oak Hill Lane, Beverly, MA 01915 (US).

Priority data: 821,712 13 January 1992 (13.01.92) US (74) Agent: McNICHOLAS, Janet, Allegretti Witcoff, Ltd., Ten South Wacker Drive, Chicago, IL 60606 (US).

Parent Application or Grant (63) Related by Continuation (81) Designated States: AU, CA, JP, US, European patent (AT, US 821,712(CIP) BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, Filed on 13 January 1992 (13.01.92) NL, PT, SE).

(71) Applicant (for all designated States except US): BIOGEN, Published INC. [US/US]; 14 Cambridge Center, Cambridge, MA With international search report.

02142 Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

664 071 (54)Title: ANTIBODIES RECOGNIZING THE FOURTH IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM1 (57) Abstract Monoclonal antibodies recognizing immunoglobulin domain 4 of Vascular Cell Adhesion Molecule-I (VCAM I) are described. Such antibodies are useful, in the treatment of acute inflammation and disorders characterized by VCAM I-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.

kk i- t. 1 t 41~r~i^l i115PUi(ir_31 WO 93/14220 PCr/US93/00031 ANTIBODIES RECOGNIZING THE FOURTH IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM1 FIELD OF THE INVENTION This invention relates to monoclonal antibodies (MAbs), specifically monoclonal antibodies that bind to the domain 4 of Vascular Cell Adhesion Molecule-i (VCAM1).

BACKGROUND OF THE INVENTION VCAM1 (also known as INCAM-110) was first identified as an adhesion molecule induced on endothelial cells by inflammatory cytokines (TNF and IL-1) and LPS (Rice et al., 1989 Osborn et al., 1989 VCAM1 binds to cells exhibiting the integrin VLA-4 (a43i), including T and B lymphocytes, monocytes, and eosinophils, but not neutrophils, and is thought to participate in recruitment of these cells from the bloodstream to areas of infection and inflammation (Elices et al, 1990 Osborn, 1990 The VCAM1/VLA-4 adhesion pathway has been associated with a number of physiological and pathological processes. Although VLA-4 is normally restricted to hematopoietic lineages, it is found on melanoma cell lines, and thus it has been suggested that VCAM1 may participate in metastasis of such tumors (Rice et al., 1989 i- l i WO 93/14220 PCT/US93/00031 2 In vivo, VCAM1 is found on areas of arterial endothelium representing early atherosclerotic plaques in a rabbit model system (Cybulsky and Gimbrone, 1991 VCAM1 is also found on follicular dendritic cells in human lymph nodes (Freedman et al., 1990 It is also present on bone marrow stromal cells in the mouse (Miyake et al., 1991 thus VCAM1 appears to play a role in Bcell development.

VCAM1 belongs to the immunoglobulin (Ig) superfamily. The major form on endothelial cells, referred to herein as VCAM-7D, has seven Ig homology units or domains; domains 4, 5 and 6 are similar in amino acid sequence to domains 1, 2 and 3, respectively, suggesting an intergenic duplication event in the evolutionary history of the gene (Osborn et al., 1989 Polte et al.

1990 Hession et al., 1991 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 Hession et al.

1991 Cybulsky et al., 1991 The biological significance of this alternate splicing is not known, however as shown below VCAM-6D can bind VLA-4-expressing cells and thus clearly has potential functionality in vivo.

The apparent involvement of the VCAM1/VLA-4 adhesion pathway in infection, inflammation and possibly atherosclerosis has led to co ntinuing 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 Sdiseases, particularly inflammation (Osborn et al., 1989 I 7., pp- I WO 93/14220 PCT/US93/00031 3 tI Monoclonal antibodies that inhibit VCAM1 binding to VLA-4 are known. For example, anti-VLA-4 MAbs HP2/1 and HP1/3 have been shown to block attachment of VLA-4expressing Ramos cells to human umbilical vein cells and VCAM1-transfected COS cells (Elices et al., 1990 Also, anti-VCAMl antibodies such as the monoclonal antibody 4B9 (Carlos et al., 1990 have been shown to inhibit adhesion of Ramos (B-cell-like), Jurkat (T-celllike) and HL60 (granulocyte-like) cells to COS cells transfected to express VCAM-6D and VCAM-7D (Hession et al., 1991 Although blocking antibodies that might be potential therapeutics are known, new monoclonal antibodies recognizing epitopes on other, non-binding domains of VCAM1 would be important research tools to map the portion(s) of VCAM1 essential for binding to VLA-4.

Antibodies that bind to VCAM1 without competing for the epitope of a blocking antibody such as 4B9 would also have important diagnostic uses. For instance, a radiolabeled anti-VCAM1 antibody recognizing an epitope not essential to VLA-4 binding could be used to monitor the course and location of acute inflammatory events but would not interfere with the action of a later-administered therapeutic that interacts with the VLA-4-binding domain of VCAM1.

SUMMARY OF THE INVENTION We have now isolated a panel of monoclonal antibodies which bind to VCAM-7D but not to VCAM-6D, and therefore presumably recognize an epitope in domain 4 of VCAM1. We have surprisingly discovered that some of these domain 4 antibodies inhibit Ramos binding to VCAM-7Dexpressing cells, and through the use of these antibodies we have demonstrated that domain 4 of VCAM-7D contains a 1 I, i i WO 93/14220 PCT/US93/00031 $1

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4 site involved in VLA-4 binding, which is in addition to the site recognized and blocked by MAb 4B9 (domain 1).

Thus, VCAM1 is identified as the first known adhesion molecule to have two binding sites for a single receptor (VLA-4); and the domain 4 monoclonal antibodies according to this invention are useful not only in mapping the molecular functionality of VCAM1 but also in at least partially inhibiting VCAM1 recognition by VLA-4.

Accordingly, it is an object of this invention to provide monoclonal antibodies recognizing VCAM-7D but not VCAM-6D.

It is a further object of this invention to provide antibodies recognizing an epitope dependent on the fourth immunoglobulin-like domain ("domain of VCAM-7D ("domain 4 antibodies"), and particularly domain 4 antibodies that do not cross-block the anti-VCAM1 MAb 4B9.

It is a further object of the present invention to provide domain 4 antibodies capable of blocking adhesion of VIA-4-expressing cells to VCAM1.

These and other objects are accomplished by the monoclonal antibodies described and claimed herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of VCAM-7D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus. These are the signal peptide immunoglobulin-like domains 1 through 7, the transmembrane sequence and the cytoplasmic domain or "tail" Also indicated are the relative positions of several restriction enzyme recognition sites, which were used to prepare additional VCAM1 constructs having one or more altered domains (described infra). In the examples set forth below, cDNA t r :1 P -7 ~I WO 93/14220 PCr/US93/00031 5 encoding VCAM-7D having the diagrammed conformation was used to transfect COS7 cells to produce COS7 cells exhibiting VCAM-7D on their surfaces.

Figure 2 is a schematic diagram of VCAM-6D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus the signal peptide immunoglobulin-like domains 1, 2, 3, 5, 6 and 7, the transmembrane sequence and the cytoplasmic domain or "tail" 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 VCAM1, designated VCAM/ICAM-1, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except the cDNA encoding VCAM-7D was modified so that a segment encoding most of domain 1 and part of domain 2 was excised and replaced by cDNA encoding analogous portions of Intercellular Adhesion Molecule-1 (ICAM1). The modified portion of the VCAM-7D molecule is indicated by shading.

Figure 4 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-2, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.

Figure 5 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-3, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for part of domain 1, all

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'j p. WO 93/14220 PCT/US93/00031 6 of domains 2 and 3, and a small N-terminal part of domain 4 was excised and replaced with ICAM1 cDNA.

Figure 6 is a graph showing relative levels of expression in COS7 cells transfected with either cDNA encoding VCAM-6D or cDNA encoding VCAM-7D. Expression of either VCAM-6D or VCAM-7D was analyzed by flow cytofluorometry following indirect immunofluorescent staining with MAb 4B9. The number of cells with fluorescent intensity greater than that shown by 99% of control transfectants (pCDM8 vector only) was determined.

The data depicted combines the results of two experiments; standard deviation is indicated by error bars.

Figure 7 is a graph showing the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding VCAM-7D, VCAM-6D, VCAM/ICAM-1, VCAM/ICAM-2, and VCAM/ICAM-3. In addition, the effects on Ramos binding of preincubation of the transfected COS cells with 20 Ag/ml of MAb 4B9 (anti-VCAMl) and 10 g/ml of MAb HP1/2 (anti- VLA-4) are shown. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown. Binding is expressed as Ramos cells bound per square millimeter ("Ramos cells bound/mm" in the figures).

Figure 8 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-1, showing the Slinear arrangement of functional domains. This polypeptide has the general structure of VCAM-6D (no 3 domain however, as indicated by the shaded area, the 30 cDNA coding for most of domain 1 and a small part of domain 2 was excised and replaced with ICAM1 cDNA.

Figure 9 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-2, showing the ii

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i ii WO 93/14220 PCT/US93/00031 7 linear arrangement of functional domains. This polypeptide has the general structure of VCAM-6D (no domain however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.

Figure 10 is a graph depicting the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, VCAM/ICAM-2, and VCAM6D/ICAM-2. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown.

Figure 11 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/VCAM4-1, showing the linear arrangement of functional domains. This polypeptide has the general structure of VCAM-6D (no domain however, as indicated by the shaded area, the cDNA coding for domain 1 and a small part of domain 2 was excised and replaced with cDNA encoding domain 4 (and one 20 amino acid cf domain 5) of VCAM-7D.

Figure 12 is a graph depicting the extent of Ramos cell binding of COS cells separately tranfected with cDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, and VCAM6D/VCAM4-1. Negligible Ramos binding to ICAM1 and negative (pCDM8) controls is also shown.

Figure 13 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any interfering antibody or in the presence of MAb 4B9 or MAb GH12 (this invention).

Figure 14 is a graph depicting the extent of Ramos cell binding to transfectants expressini various chimeric VCAM1 constructs in the absence of any i_

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r r r, interfering antibody or in the presence of MAb 4B9 or MAb ED11 (this invention).

Figure 15 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any interfering antibody or in the presence of MAb 4B9 or MAb GE4 (this invention).

Figures 16, 17, 18 and 19 are graphs showing Ramos cell binding to cultured HUVECs treated with 10 ng/ml of recombinant human TNF for 4 hours (Fig. 16), 24 hours (Fig. 17), 48 hours (Fig. 18), and 72 hours (Fig.

19). Binding of Ramos cells to uninduced HUVECs (control), induced (TNF-treated) HUVECs alone, and in the presence of HP1/2 'anti-VLA-4 a 4 MAb), 4B9 (anti-VCAM1 15 MAb), ED11 (anti-VCAM1 MAb, this invention), and GH12 (anti-VCAM1 MAb, this invention) is compared.

DETAILED DESCRIPTION OF THE INVENTION The technology for producing monoclonal antibodies is well known. Briefly, an immortal cell line (typically murine myeloma cells) is fused to lymphocytes (typically splenocytes) from a mammal immunized with whole cells expressing a given antigen, VCAM1, and/or with purified antigen, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen. See, generally, Kohler and Milstein, 1975 [12].

Immunization may be accomplished using standard procedures. The unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the 30 body weight of the mammal, etc. Typically, the immunized mammals are bled and the serum from each blood sample is assayed for particulai antibodies using appropriate

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r WO 93/14220 PCT/US93/00031 9 screening assays. For example, anti-VCAM1 antibodies according to the present invention were identified by testinc 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-VCAMl antibodies using such screening assays.

Typically, the immortal cell line 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 tiiymidine ("HAT medium").

Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol PEG 3350). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants using assays which detect MAbs having the desired specificity. For example, hybridomas according to the invention were identified by their ability to bind to VCAM-7D-expressing cells but not to VCAM-6D-expressing cells. (See Example 1, supra.) To produce anti-VCAM1 antibodies, hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue i. r 6L

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i; s if WO 93/14220 PCT/US93/00031 10 culture techniques and culture media suitable for hybridoma cells are well known. The conditioned hybridoma culture supernatant may be collected and the anti-VCAM1 antibodies optionally further purified by well-known methods.

Alternatively, the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of a mouse primed with 2,6,10,14tetramethylpentadecane (PRISTANE; Sigma Chemical Co., St.

Louis MO). The hybridoma cells proliferate in the peritoneal cavity, secreting the antibody which accumulates as ascites fluid. The antibody may be harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe.

The monoclonal antibodies of the present invention recognize epitopes dependent on the fourth Ig domain of the seven-Ig-domain form of VCAM1, 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 eliminat-on of the fourth Ig domain eliminates their ability to recognize the VCAM1 structure. These MAbs are referred to as "domain 4 MAbs" herein.

The domain 4 MAbs bind to VCAM-7D but do not bind to VCAM-6D. All of the antibodies of this invention are distinct from known VCAM1/VLA-4 blocking antibodies such as 4B9. We show herein that 4B9 recognizes an epitope in domain 1 of VCAM1, and thus recognizes both VCAM-7D and VCAM-6D. The monoclonal antibodies of the present invention recognize VCAM-7D but do not cross-block MAb 4B9. Some of the monoclonal antibodies described herein are effective to inhibit adhesion of Ramos and ,2-rF p:t WO 93/14220 PCT/US93/00031 11 other VLA-4-expressing cells to VCAM1-expressing cells.

Some of the domain 4 monoclonal antibodies described herein bind to epitopes of VCAM1 which are not involved in the adhesion pathway between mononuclear leukocytes and VCAM1-expressing cells that is mediated by VCAM1. This latter type of antibody may be useful, for example, to detect the presence of VCAM1, in particular VCAM-7D, in vitro or in vivo without interference with the binding of VCAM1.

Using the monoclonal antibodies of the presert invention and other anti-VCAM1 MAbs obtained from other investigators, we have discovered that binding of VLA-4 to VCAM1 can occur via two separate sites, one requiring domain 1, and the other requiring domain 4. The fact that some of the anti-VCAMl domain 4 MAbs of this invention can inhibit domain 4-dependent cell binding and can inhibit cell binding to VCAM1 on cultured endothelial cells, indicates that binding through domain 4 is a significant functional component of the VCAM1/VLA-4 int,raction in vivo.

The monoclonal antibodies of the present invention may be produced naturally as outlined above or may be synthesized using recombinant DNA techniques.

Suitable recombinant antibodies include antibodies produced, by transforming a host cell with a suitable expression vector containing DNA encoding the light and heavy immunoglobulin chains of the desired antibody, and recombinant chimeric antibodies, wherein some or all of the hinge, constant and/or variable regions of the heavy and/or the light chains of the anti-VCAM1 antibody have been substituted with corresponding regions of an immunoglobulin light or heavy chain of a different species. This includes so-called "humanized" antibodies.

(2 WO 93/14220 PCT/US93/00031 -12- (See, Jones et al., 1986 Ward et al., 1989 and U.S. Patent 4,816,397 (Boss et al.) all incorporated herein by reference.) Furthermore, domain 4-binding fragments of anti- VCAM1 antibodies, such as Fab, Fab', 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, by cleaving an intact antibody with a protease, such as papain or pepsin, or via recombinant DNA techniques, by using host cells transformed with truncated heavy and/or light chain genes. Heavy and light chain monomers may similarly be produced by treating an intact antibody with a reducing agent such as dithiothreitol or 0-mercaptoethanol or by using host cells transformed with DNA encoding either the desired heavy chain or light chain or both. As an alternative to hybridoma technology, antibody fragments having similar specificities may be isolated by phage cloning methods.

(See, Clackson et al. (1991) Monoclonal antibodies of the present invention may be used in any application where antibody recognition of domain 4 of VCAM1 is advantageous, including applications where inhibiting binding between VCAM1 and its ligand, VLA-4, is desired. For example, monoclonal antibodies of the present invention may be used in the treatment of inflammation or diseases characterized by leukocyte binding to endothelium, such as post-reperfusion 30 injury, microbial infections or other cell migration metastasis) dependent on a VCAM1-mediated binding pathway, vasculitis, etc.

13 The monoclonal antibodies of the present invention may also be used in combination with other antibodies, bioactive agents or materials for various purposes. For example, the present monoclonal antibodies may be used in combination with 4B9 or other anti-VCAMI antibodies in the treatment of disorders characterized by VCAM1 expression in endothelium. Alternatively, the present monoclonal antibodies may be used in combination with antibodies recognizing other endothelial cell receptors identified in inflammatory events ELAM1, ICAM1, etc.) in a combination therapy designed to suppress a range of leukocyte-endothelial cell binding pathways.

Also, the antibodies of the present invention, or VCAM1recognizing fragments thereof, may be combined or linked to cytotoxic molecules such as TNF, ricin or the A chain of diphtheria toxin, in order to provide antibody/toxin conjugates capable of targeting cytotoxic elements to VCAM1-expressing cells. The antibodies of the present invention may also be immobilized on a chromatographic substrate Protein A-sepharose) to provide an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.

Detectably labeled antibodies according to the present invention may also be used in screening methods or diagnostic methods for detecting VCAM1 protein in a sample or VCAM1-expressing endothelial cells in vitro or in vivo.

k For example, samples may be screened for the presence of VCAM1 or VCAM1-expressing cells by contacting the sample with a labeled antibody according to the present invention and detecting whether a VCAM1/anti-VCAMI antibody complex is formed.

~Z fl~ Si I WO 93/14220 PCT/US93/00031 14 Suitable labels can be radioactive, enzymatic, fluorescent, magnetic or chemiluminescent. Radiolabeled antibodies are prepared in known ways by coupling a radioactive isotope such as 3 H, "Fe, 1I, which can then be detected by gamma counter, scintillation counter or by autoradiography. Antibodies of this invention may be suitably labeled with enzymes such as yeast alcohol dehydrogenase, horseradish peroxidase, alkaline phosphatase, and the like, then developed and detected spectrophotometrically or visually. Suitable fluorescent labels include fluorescein isothiocyanate, fluorescamine, rhodamine, and the like. Suitable chemiluminescent labels include luminol, imidazole, oxalate ester, luciferin, and the like.

For therapeutic uses, the monoclonal antibodies of the present invention may be formulated as a pharmaceutical composition comprising an effective amount of the antibody admixed with a pharmaceutically acceptable carrier. Typically, the antibodies of the present invention will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of the active ingredients or to prolong their presence in a patient's system. 1umerous suitable drug delivery systems are known and include, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like.

The pharmaceutical compositions contemplated herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally.

Ordinarily, intravenous or parenteral administration will be preferred.

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T, I WO 93/14220 PCT/US93/00031 15 Example 1 MAbs Recognizing VCAM-7D But Not VCAM-6D Monoclonal antibodies according to the invention that recognize domain 4 of VCAM-7D were prepared in the following manner. We injected four BALB/c mice with whole CHO cells that express VCAM-6D on their surfaces ("VCAM- 6D/CHO" cells). The CHO culture was a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA). Initially, each mouse received approximately 107 cells in PBS intraperitoneally We injected complete Freund's adjuvant i.p. at a different site 2-24 hours later. We boosted the mice twice i.p. with VCAM- 6D/CHO cells, without adjuvant. We then immunized each of the mice with 55 Ag of a recombinant soluble form of VCAM- 7D (rsVCAM-7D, consisting of the N-terminal 674 amino acids of mature VCAM-7D and lacking the transmembrane and cytoplasmic regions; obtained from Biogen, Inc., Cambridge, MA) injected intraperitoneally with incomplete Freund's adjuvant. We boosted the mice twice with rsVCAM- 7D i.p. without adjuvant. Four days after the rsVCAMI injections, we removed the spleen from one mouse and fused the splenocytes with P3/X63-Ag8.653 murine myeloma cells according to the method described by Lerner [17].

Supernatants of cultures of individual clones of fused cells were screened for their ability to bind to rsVCAM-7D-coated plates. ELISA plates (Corning) were coated with rsVCAM-7D (0.1 ig/ml, 50 Il/well) and incubated overnight at 40 C. The coating solution was aspirated and blocking buffer was added (phosphate buffered saline (PBS) with 5% fetal calf serum (FCS), pH 7.2) to block the remaining sites, and the plates were incubated for 2 hrs. at room temperature. The blocking iE pp.

I I- WO 93/14220 PCT/US93/00031 16 i i2Z buffer was removed, and 50 Al of hybridoma culture supernatants (or supernatant diluted into blocking buffer) were added to the plates and then incubated for 1 hr. at room temperature. Next, the plates were washed with PBS containing 0.05% detergent (Tween-20) to remove unbound antibody and incubated with a horseradish peroxidaseconjugated goat anti-mouse immunoglobulin for 1 hr. at room temperature. The plates were washed with the solution and developed with the chromogenic horseradish peroxidase substrate, TMB tetramethylbenzidine; ICN, Lisle IL) 42mM in 0.1M sodium acetate-citric acid, pH 4.9. The reaction was initiated by addition of hydrogen peroxide (15 Al, 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 VCAM-7D. In these experiments cDNA encoding VCAM-6D or VCAM-7D was inserted into an expression vector (pCDM8) and subsequently transfected into COS7 cells by electroporation, as described in Osborn et al. 1989 [2] and Hession et al. 1991 The COS7 cells, transfected with either VCAM-6D or VCAM-7D cDNA, were washed and suspended in suspension buffer (PBS/5mM EDTA/2% FCS/0.05% sodium azide) and incubated 45 min. at 40 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) r WO 93/14220 PCT/US93/00031 -17for 45 min. at 40 C. The cells were washed, fixed with 0.2% paraformaldehyde in PBS and evaluated using a FACStar (Becton Dickinson, Mountain View CA).

FACS analysis showed that the isolated anti- VCAM1 monoclonal antibodies bound to VCAM-7D but not to VCAM-6D. The binding data for these antibodies are summarized in Table I (infra p. 28).

Example 2 Ramos Cell Binding Assay Ramos is a B-lymphoblastoid cell line that expresses VLA-4 and thus binds to VCAM1 but does not bind to other induced endothelial cell molecules such as ICAM-1 or ELAM-1 (Osborn et al., 1989 Ramos is therefore a convenient model cell line with which to assay VLA-4dependent binding. Ramos cells bind to both recombinant VCAM-6D and recombinant VCAM-7D when these molecules are expressed on COS cells. We had previously noted that binding to VCAM-7D was somewhat (less than 2 fold) greater Sthan to VCAM-6D in transient transfection assays of a single preparation of each plasmid (Hession et al., 1991 To determine if this quantitative difference in binding activity was reproducible, several preparations of each plasmid were made and electroporated in parallel into COS cells. Repeated transfections revealed that although expression of the two plasmids is similar, VCAM-7D on average binds significantly more cells per square millimeter than does VCAM-6D. (Cf. Figure 6 and Figure Binding to both six- and seven-domain forms of VCAM1 was inhibited by the anti-VLA-4 MAb HP1/2 or by anti-VCAM 30 MAb 4B9 (Figure 7).

In all of our procedures, the binding of Ramos cells was measured as follows: h ;ii: ~1 WO 93/14220 PCT/US93/00031 18 Confluent monolayers of the target cells COS transfectants or induced HUVECs) in 48-well plastic tissue culture plates were washed with RPMI/10% fetal calf serum (FCS), and an amount 10 gg/ml, varying per MAb and per experiment) of the test MAb 4B9, HP1/2, GH12, etc.) in a volume of 0.15 ml was added. In the case of HUVECs, the cells were preincubated 4, 24, 48 or 72 hours at 370 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)- 6)-carboxy-fluorescein ("BCECF", Molecular Probes, Eugene OR) according to the manufacturer's instructions.

The cells were first centrifuged and then resuspended in fetal calf serum (FCS) to a concentration of 5 x 106 cells ml. The BCECF was added to 2 AM and the suspension incubated for 30 min. at 370 C. The cells were washed twice in RPMI/10% FCS and then resuspended in the same solution at 3 x 106 cells/ml.

x 10' 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 370 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 followed by incubation for 5 min. at 370 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

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WO 93/14220 PCT/US93/00031 19 i ii ii i I ::1I Antibody Staining Assay The antibodies were tested for their ability to bind to VCAM-7D, VCAM-6D and various VCAM/ICAM constructs exhibiting domain 4-dependent cell binding (see Figures 1- 13 and Example 3, infra). COS7 cells transfected with a particular VCAM, ICAM (control) or chimeric VCAM/ICAM (or VCAM/VCAM, see Fig. 11) cDNA construct were removed from 100 mm culture dishes by incubation in HBSS/5 mM EDTA for min. at 370 C, followed by vigorous pipetting.

Aliquots of 3-5 x 101 cells were incubated individually with 2 pg/ml of the MAb being tested, or with rabbit polyclonal antiserum raised against rsVCAM-7D (diluted 1:500), in PNF (PBS/0.1% sodium azide/2% FCS) for 20 min.

at room temperature. After incubation the cells were washed twice with PNF and incubated 20 min. at room temperature with 0.2 gg/ml of a secondary fluoresceinated antibody (rabbit anti-mouse immunoglobulin or goat antirabbit immunoglobulin) in PNF. Cells were then washed 3X with PBS, suspended in PBS/1% formaldehyde, stored dark at 20 40 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 histograms.

Fluorescence peaks for each transfectant and for negative controls (pCDM8 vector only) run in parallel were compared and the peaks marked to exclude 99% of the negative cells.

The number of positive cells (to the right of the marker) was determined for each transfectant. This method results in a slight underestimate of the number of positive cells in each case, since weakly positive cells that overlap the negative control fluorescence peak are excluded.

r 6k 1r

I

WO 93/14220 PCT/US93/00031 20 Example 3 Construction and Expression of Chimeric VCAM/ICAM Polypeptides In order to identify regions of VCAM1 involved in recognition of VLA-4-expressing cells, recombinant genes encoding VCAM/ICAM chimeras were constructed by excising portions of the VCAM-7D and VCAM-6D cDNAs and replacing them with analogous regions of ICAM-1, using unique restriction endonuclease sites in the VCAM1 cDNA (see Fig. 1) and polymerase chain reaction (PCR)-generated fragments of ICAM1. Substitution of the excised VCAM1 regions was done to eliminate as much as possible the structural distortions that would accompany deletion of Ione or more domains of the VCAM1 molecules. ICAM1 was selected as a source of substituent structures because it is also a member of the Ig superfamily and has the highest degree of amino acid homology to VCAM1 among Ig superfamily members (Osborn et al., 1989 Schematic diagrams of the VCAM1/ICAM1 chimeric polypeptides are shown in Figures 1-5, 8, 9 and 11. The cDNA constructs coding for each of these were prepared as follows: Aliquots of the parent plasmids VCAM1E11/CDM8 (encoding VCAM-7D; see, Hession et al., 1991 or VCAM41/CDM8 (encoding VCAM-6D; Osborn et al., 1989 were digested using restriction endonucleases at the appropriate sites indicated in Figure 1 and agarose gel purified. ICAM1 4. inserts were made by PCR from an ICAM1/CDM8 plasmid isolated from an endothelial cDNA library previously described (Osborn et al., 1989 using oligonucleotide probes based on the published sequence of ICAM1 (Simmons et al., 1988 Staunton et al., 1988 The PCR primers were designed as follows: IwL i 1 i

I

p.

2~ "rN 93/14220 PCF/US93/00031 21 Primer P-1 (SEQ ID NO:1) EcoRV TTCTGCCCCG GGAGGCTCCG TGCTG-3' The first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues 9-12; the following twenty-three nucleotides correspond to the coding region for ICAM1 amino acid residues 11-18.

Primer P-2 (SEQ ID NO:2) Bgl2 5'-GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA-3' The first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 86-89; the following twenty-one nucleotides correspond to the coding region for ICAM1 amino acid residues 84-90.

Primer P-3 (SEQ ID NO:3) Hind3 5'-TCTCAAGCTT TTACTGTTGA GATCTCCCCT GG-3' The first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues the following twenty nucleotides correspond to the coding region for ICAM1 amino acid residues 300-306.

Primer P-4 (SEQ ID NO:4) Bsu361 5'-CTGGATCCTT AGGAGTCCAG TACACGGTGA GGAAGG-3' 25 The first fourteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following twenty-two nucleotides correspond to the coding region for ICAM1 amino acid residues 79-85.

Primer P-5 (SEQ ID BspHl GCTTCTCCC CACGGAGCAG CACCAC-3' The first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 134-137; the

U

WO 93/14220 PCT/US93/00031 22 following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 121-128.

Primer P-6 (SEO ID NO:6) Bgl2 5'-GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT-3' The first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 302-305; the following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 279-286.

Primer P-7 (SEQ ID NO:7) Bsu361 AGGGAATGAG TAGAGCTCCA C-3' The first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following eighteen nucleotides correspond to the coding region for VCAM1 amino acid residues 385-394.

PCR synthesis for inserts for construct VCAM/ICAM-1 (Fig. 3) was primed with P-1 and P-4; for construct VCAM/ICAM-2 (Fig. P-1 and P-5; for construct VCAM/ICAM-3 (Fig. P-2 and P-6; for construct VCAM6D/VCAM4-1 (Fig. 11), P-3 and P-7. VCAM6D/ICAM-1 and VCAM6D/ICAM-2 (Figs. 8 and 9) were made using the same inserts as for VCAM/ICAM-1 and VCAM/ICAM-2, respectively, cloned into the VCAM-6D expression vector (VCAM41/CDM8) instead of the VCAM-7D expression vector (VCAM1E11/CDM8).

After PCR synthesis, performed as described in Hession et al. (1991) the insert fragments were digested with the appropriate restriction endonucleases and gel -purified. Expression vectors and insert fragments were S "30 ligated using NEB ligase and buffer, ethanol precipitated, then electroporated into bacterial host strain MC1061/p3 using a Biorad Gene Pulser.

-j .ii WO 93/14220 PCT/US93/00031 23 :-r

II

j;: xia :r Clones were screened by PCR and restriction digests, and at least one isolate of each clone was sequenced across cloning junctions and insert using a Sequenase kit (United States Biochemical Corp.). The complete cDNA sequences for VCAM-7D, VCAM-6D, and each of the chimeric constructs VCAM/ICAM-1, VCAM/ICAM-2, VCAM/ICAM-3, VCAM6D/ICAM-1, VCAM6D/ICAM-2, and VCAM6D/VCAM4-1 are set forth in the Sequence Listing below (in order, SEQ ID NOS: 8 through The diagrammed constructs were transfected into COS7 cells and found to be well expressed when tested by FACS analysis after staining with polyclonal antiserum to VCAM1.

In constructs VCAM/ICAM-1 (Fig. 3) and VCAM/ICAM-2 (Fig. most of VCAM domain 1, or most of domain 1 and the first half ot domain 2, were'repluced by ICAM1 sequence (see shaded areas, Figs. 3 and 4, respectively). In construct VCAM/ICAM-3 (Fig. VCAM domains 2 and 3, and small portions of domains 1 and 4, were replaced by ICAM1 sequence (see shaded area, Fig. Example 4 MAb 4B9 Binding to VCAM1 domain 1.

It has been previously shown that the N-terminal three domains of VCAM1 support adhesion to VLA-4 (Taichman et al., 1991 and that the anti-VCAM1 MAb 4B9 significantly inhibits binding of VLA-4-bearing cells to induced HUVECs or VCAM1 transfectants (Carlos et al., 1990 These results suggested that the binding site of VCAM1 recognized by VLA-4 and obstructed by 4B9 is in the N-terminal portion of the VCAM1 molecule.

Using the MAb binding assay described above (Example we confirmed that MAb 4B9 recognizes an "i~ r 1 ii WO 93/14220 PCT/US93/00031 24 epitope in VCAM1 domain 1. It was found that 4B9 bound readily to COS7 transfectants expressing both seven- and six-domain forms of VCAM1. Furthermore, chimeric constructs VCAM/ICAM-1 and VCAM/ICAM-2 (Figs. 3 and 4, respectively), wherein most of domain 1 was replaced by ICAM1 amino acid residues, did not bind to 4B9 (see, Table I, infra). These results indicated that the epitope to which 4B9 binds requires domain 1 for recognition.

Finally, construct VCAM/ICAM-3 (see, Fig. 5. domain 1 mostly intact) did bind 4B9, confirming that domain 1 contains the 4B9 epitope. The binding and Ramos blocking characteristics of 4B9 are summarized in Table I, infra.

Although constructs VCAM/ICAM-1 and VCAM/ICAM-2 did not bind MAb 4B9, both bound Ramos cells at about of the level seen with intact VCAM-7D (Figure This binding was mediated by VLA-4/VCAM1, since anti-VLA-4 mAb HP1/2 completely inhibited the interaction, while a blocking antibody to the ICAM-1 pathway (MAb 60.3) did not affect binding (data not shown). Construct VCAM/ICAM-3 bound Ramos cells weakly but measurably, at about 10% of the level seen with intact VCAM-7D (Figure 7).

Ramos Cells Bind At Two Regions On VCAM1 To determine whether Ramos cell binding to constructs VCAM/ICAM-1 and VCAM/ICAM-2 was due to residual portions of the domain 1-dependent binding site, or to domain 4 (which is structurally similar to domain 1), constructs analogous to VCAM/ICAM-1 and VCAM/ICAM-2 were made, except that cDNA coding for VCAM-6D (no domain 4) instead of VCAM-7D was used as the substrate or backbone 30 for the substituted ICAM1 inserts (see, Figs. 8 and 9).

These constructs, dc'ignated VCAM6D/ICAM-1 and VCAM6D/ICAM-2, respectively, were well expressed in COS7 cells as measured by anti-VCAMI polyclonal antiserum rr I SWO 93/14220 PCT/US93/00031 staining, but were unable to bind Ramos cells (Figure These results indicated that the binding of Ramos cells to constructs VCAM/ICAM-1 and VCAM/ICAM-2 appears to be due to the presence of domain 4.

We next prepared a construct designated VCAM6D/VCAM4-1, in which domain 4 of VCAM-7D was substituted for domain 1 of VCAM-6D (Fig. 11). Referring to the results depicted in Figures 7 and 12, Ramos binding of VCAM6D/VCAM4-1 was comparable to that of VCAM-6D (see Fig. 2, intact domain 1) and VCAM/ICAM-1 (see Fig. 3, intact domain indicating that domain 4 can indeed bind Icells. This result was somewhat surprising in light of i the fact that MAb 4B9, which binds to domain 1 but not to domain 4, can completely inhibit binding of Ramos cells to VCAM-7D. Stearic hindrance is one possible explanation; it is also possible that binding of 4B9 to domain 1 perturbs the structure of the molecule so that domain 4 is no longer in the proper conformation to bind cells.

Example Monoclonal Antibodies That Block Domain 4-dependent Cell Adhesion According to the procedure set forth in Example 1 1, three monoclonal antibodies were produced that recognize VCAM-7D but not VCAM-6D, indicating that they bind domain 4, or a domain 4-dependent epitope. These MAbs were designated GE4, ED11 and GH12. All three MAbs were of IgGi isotype. The binding and Ramos blocking characteristics of each antibody were determined using the antibody and Ramos binding assays described above (Example f 2).

SMAb GH12 bound to COS7 transfectants expressing VCAM-7D, VCAM/ICAM-1 and 2, and VCAM6D/VCAM4-1 but did not bind to transfectants expressing VCAM-6D or VCAM/ICAM-3.

0.

i r pi I il I"~ WO 93/14220 PCT/US93/00031 -26- Referring to Figure 13, Ramos binding experiments showed that MAb GH12 at 10 Ag/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1, VCAM/ICAM-2 and VCAM6D/VCAM4-1 constructs but did not bind to the VCAM/ICAM-3-expressing tranfectants, indicating that the small portion of domain 4 eliminated from the VCAM/ICAM-3 construct was important to GH12 binding. MAb GH12 did not block Ramos cell binding to VCAM-6D or VCAM-7D.

Binding experiments showed that MAb ED11 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-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 pg/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1 and VCAM/ICAM-2 constructs but did not inhibit VCAM-6D-expressing, VCAM/ICAM-3-expressing or VCAM6D/VCAM4-l-expressing tranfectants. Surprisingly, MAb ED11 did partially block Ramos cell binding to VCAM-7Dexpressing COS7 cells.

Binding experiments showed that MAb GE4 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-1, VCAM/ICAM-2 and VCAM/ICAM-3 but not to cells expressing VCAM-6D or VCAM6D/VCAM4-1. Referring to Figure 15, Ramos binding experiments showed that MAb GE4 at 10 gg/ml did not inhibit Ramos binding to COS7 transfectants expressing VCAM/ICAM-1 and VCAM/ICAM-2. MAb GE4 did not block Ramos adhesion to transfectants expressing VCAM6D/VCAM4-1, VCAM/ICAM-3 or VCAM-6D, and also did not inhibit Ramos binding to VCAM-7D-expressing COS7 cells at the single concentration tested.

The binding and blocking data are summarized below in Table I. From these results it can be concluded that each of the three domain 4 antibodies recognize t ii:- -x WO 93/14220 PCT/US93/00031 27 distinct epitopes, and two are capable of blocking domain 4-dependent adhesion of VLA-4 to VCAM1. These antibodies represent a new class of anti-VCAMl blocking antibody.

Table I constructs* A B C MAb GH12 binding blocking VCAM-7D VCAM-6D ED11 binding blocking GE4 binding blocking 4B9 (control) binding blocking A VCAM/ICAM-1 B VCAM/ICAM-2 C VCAM/ICAM-3 D VCAM6D/VCAM4-1 Example 6 Domain 4 MAb Inhibition of Ramos Binding to Cytokine-activated Endothelial Cells To determine whether the domain-4-dependent MAbs ED11 or GH12 were capable of blocking adhesion to VCAM1 on endothelial cells (which is predominantly VCAM-7D), human umbilical vein endothelial cells (HUVECs) were treated with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72 hours, and binding of Ramos cells and the ability of the antibodies to block binding was measured. All MAbs were added at a concentration of 10 ig/ml. The results are shown for each activation period in Figures 16-19.

I'

WO 93/14220 PCT/US93/00031 28 The results indicate overall that MAb GH12 did not block HUVEC/Ramos binding reliably at the single concentration tested (10 Ag/ml), but MAb ED11 blocked more than 80% of Ramos cell binding to induced HUVECs at all time points tested. MAb 4B9 blocked HUVEC/Ramos binding by greater than 90%. These results suggest that although the two cell binding sites of VCAM1 (domain 1- and domain- 4-dependent) can be separated and remain functional, antibodies to.each can interfere with Ramos cell binding to the other. This is so even though the antibodies do not cross-react with each other's binding sites (shown by the antibody binding data to constructs, above).

In contrast to stimulated endothelial cells, as in this Example, where VCAM-7D is the predominantly expressed (RNA and protein) form of VCAM-1, VCAM-6D (RNA) may be the predominant species of VCAM-1 in bone marrow stromal cells and tonsillar follicular dendritic cells.

This suggests a potential therapeutic advantage of domain 4 specific anti-VCAM1 antibodies over domain 1 specific anti-VCAM1 antibodies, in that domain 4 antibodies according to the present invention might potentially block the recruitment of leukocytes via stimulated endothelial cells into sites of inflammation, without blocking lymphopoiesis, which is the role that VCAM-1 plays in bone marrow (Miyake et al., 1977 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 Sillustration 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 WO 93/14220 PCT/US93/00031 29experienced in this art. All such modifications and additional embodiments are within the contemplation of this application and within the scope of the appended claims.

We deposited subclones of hybridomas producing ED11, GE4 and GH12 monoclonal antibodies under the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, MD (US) on January -941992. The cultures were identified as follows: designation ATCC accession no.

MAb CB.ED11.AG3 HB 10962 MAb CB.GE4.BG5 HB 10961 MAb CB.GH12.AA12 HB 10963 104 k j WO 93/14220 PCT/US93/00031 30 CITED PUBLICATIONS Rice, G.E. and Bevilacqua, M.P. (1989), "An inducible endothelial cell surface glycoprotein mediates melanoma adhesion," Science, 246, 1303-1306.

Osborn, Hession, Tizard, Vassallo, C., Luhowskyj, Chi-Rosso, and Lobb, R. (1989), "Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes," Cell, 59, 1203-1211.

Elices, Osborn, Takada, Crouse, C., Luhowskyj, Hemler, and Lobb, R.R. (1990), "VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site," Cell, 60, 577-584.

Osborn, L. (1990), "Leukocyte adhesion in inflammation," Cell, 62, 3-6.

Cybulsky, M.I. and Gimbrone, Jr. (1991), "Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis," Science, 251 788-791.

Freedman, Munro, Rice, Bevilacqua, Morimoto, McIntyre, Rhynhart, K., Pober, and Nadler, L.M. (1990), "Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110," Science, 249, 1030-1033.

Miyake, Medina, Ishihara, 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.

S[8] Polte, Newman, and Gopal, T.V. (1990), "Full length vascular cell adhesion molecule 1 (VCAM-1)," Nuc. Ac. Res., 18, 5901.

I WO 93/14220 PCT/US93/00031 31 Hession, Tizard, Vassallo, Schiffer, Goff, Moy, Chi-Rosso, Luhowskyj, Lobb, and Osborn, L. (1991), "Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1)", J. Biol. Chem., 266, 6682-6685.

Cybulsky, Fries, Williams, A.J., Sultan, Eddy, Byers, Shows, Gimbrone, Jr., and Collins, T. (1991), "Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene," Proc. Natl. Acad.

Sci. USA, 88, 7859-7863.

[11] Carlos, Schwartz, Kovach, Yee, E., Russo, Osborn, Chi-Rosso, Newman, Lobb, and Harlan, J.M. (1990), "Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokineactivated cultured human endothelial cells. Blood, 76, 965-970.

[12] Kohler, G. and Milstein, C. (1975), "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", Nature, 256, pp. 495-497.

[13] Jones, P. et al. (1986), "Replacing the Complementarity-Determining Regions in a Human Antibody with Those From a Mouse," Nature, 321, 522- I 25 525.

[14] Ward, E. et al. (1989), "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature, 341, 544- 546.

[15] U.S. Patent No. 4,816,397, Boss et al., "Multichain Polypeptides Or Proteins And Processes For Their Production", issued March 28, 1989.

[16] Clackson, Hoogenboom, H. 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.

IWO 93/14220 PCr/US93/00031 -32 [18] Gimbrone, Jr., Obin, Brock, Luis, Hass, Hebert, Yip, Leung, Lowe, Kohr, Darbonne, W.C., Bachtol, and Baker, J.B. (1989), "Endothelial interleukin-8: A novel inhibitor of leukocyteendothelial interactions," Science, 246 1601-1603.

[19] Simmons, Hakgoba, and Seed, B. (1988), "ICAM, an adhesion ligand of LFA-l, is homologous to the neural cell adhesion molecule NCAM,"1 Nature, 3311 624-627.

Staunton, Dustin, Erickson, and Springer, T.A. (1990), "The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-l and rhinovirus," Cell, 61, 243-254.

[21] Taichman, Cybulsky, Djaffar, I., Longenecker, Teixido, Rice, Aruffa, and Bevilacqua, M.P. (1991), "Tumor cell surface a fl integrin mediates adhesion to vascular endothelium: Demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-l,"1 Cell Regulation, 347-355.

[22] Carlos, Schwartz, Kovach, Yee, E., Rosso, Osborn, Chi-Rosso, Newman, B., Lobb, and Harlan, J.M. (1990), "Vascular cell adhesion molecule-i mediates lymphocyte adherence to cytokine-activated cultredhumn edothlia cels, Blood, 76(5), 965-970.

The foregoing publications are incorporated herein by reference.

-33- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: BIOGEN, Inc.

STREET: 14 Cambridge Center CITY: Cambridge STATE: Massachusetts COUNTRY: USA POSTAL CODE (ZIP): 02142 NAME: OSBORN, Laurelee STREET: 19 Harding Street, #2 CITY: Cambridge STATE: Massachusetts COUNTRY: USA POSTAL CODE (ZIP): 02141 NAME: BENJAMIN, Christopher D.

STREET: 2 Oak Hill Lane CITY: Beverly STATE: Massachusettes COUNTRY: USA POSTAL CODE (ZIP): 01915 (ii) TITLE OF INVENTION: ANTIBODIES RECOGNIZING THE FOURTH IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM-1 (iii) NUMBER OF SEQUENCES: 23 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Vers' a #1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO PCT/US93/00031 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/821,712 FILING DATE: 13-JAN-1992 INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE

SHEET

00S"^' 'r cr r r r r -34- SEQUENCE DESCRIPTION: SEQ ID NO:1: TCTAGATATC TTCTGCCCCG GGAGGCTCCG TGCTG INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA 34 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTCAAGCTT TTACTGTTGA GATCTCCCCT GG 32 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTGGATCCTT AGGAGTCCAG TACACGGTGA GGAAGG 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET st14 r

I

1 CP rCI-r i II I' SEQUENCE DESCRIPTION: SEQ ID CTCTTCATGA GCTTCTCCCC ACGGAGCAGC ACCAC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid S-RANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTGGATCCTT AGGGAATGAG TAGAGCTCCA C INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 2217 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2217 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

mat_peptide 73..2217 sig_peptide 1..72 SUBSTITUTE

SHEET

I .I-

F

-36- (xi) SEQUENCE DESCRIPTION: SEC ID NO:8: ATC CCT CCC AAC ATG GT! Met Pro Cly Lys Met Val -24 -20 CTC ATC CTT Val Ile Leu CCA CC Cly Ala -15 TCA AAT ATA Ser Asn Ile CTT TG Leu Trp ATA ATO TTT CCA CCT TCT CAA OCT TT!-T AAA ATC GAG ACC ACC CCA OAA Ile Met Phe Akla Ala Ser Gin Ala Phe Lys Ile Glu Thr Thr Pro Giu TCT AGA Ser Arg TAT OTT CCT CAC Tyr Leu Ala Gin COT CAC TCC GTC Oly Asp Ser Val TTO ACT TCC AC Leu Thr Cys Ser ACA 0CC TCT GAG Thr Gly Cys Ciu CCA TTT TTC TOT Pro Phe Phe Ser ACA ACC CAC ATA Arg Thr Gin Ile ACT CCA CTG AAT Ser Pro Leu Asn AAO GTO ACG AAT Lys Val Thr Asn CCC ACC ACA TCT Oly Thr Thr Ser ACC CTG Thr Leu ACA ATG AAT Thr Met Asn GTT ACT TTT CCC Val Ser Phe Oly GAA CAC TOT TAC Oiu His Ser Tyr OCA ACT TOT CAA TCT AGO AAA Ala Thr Cys Giu Ser Arg Lys GAA AAA GGA ATC Clii Lys Oly Ile CTG TGC ACA Leu Cys Thr OTO GAO ATC Val Oiu Ile CCT CTC GAO Pro Leu Giu TAO TCT Tyr Ser TTT CCT AAG CAT OCA GAC ATT CAT TTG Phe Pro Lys Asp Pro Oiu Ile His Leu ACT GC Ser Oly 100

CCT

Ala 105 CCC AAG CCC ATC Oly Lys Pro Ile CTC AAO TCT TCA CTT CCT CAT CTA TAO Val Lys Cys Ser Val Ala Asp Val Tyr 115

I:

TTT CAC AGO CTC CAG ATA CAC TTA CIG Phe Asp Arg Leu Oiu Ile Asp Leu Leu 125 ACT CAG GAA TTT CTG GAG CAT GCA GAC Ser Gin Glu Phe Leu Giu Asp Ala Asp CCA CAT CAT CTC Gly Asp His Leu ATC AAC Met Lys 135 AGG AAG TCC CTG Arg Lys Ser Leu GAA ACC AAO Giu Thr Lys 150 GGA AAA OTT Gly Lys Val ACT TTG GAA Ser Leu Glu 155 GTA ACC TTT Val Thr Phe ACT CCT Thr Pro 160 GTC ATT GAG CAT ATT Val Ile Giu Asp Ile 165 Ju800TITUTEISHEET -37- CTT ,GTT Leu Val 170 TG GGA OCT AAA Cys Arg Ala Lys CG ATT OAT OAA His Ile Asp Oiu OAT TOT GTG CCC Asp Ser Val Pro ACA GTA AGG GAG GCT Thr Val Arg Gin Ala

GTA

Val 190 AAA OAA TTO Lys Oiu Leu CAA OTO Gin Val 195 TAG ATA TGA 000 Tyr Ile Ser Pro AAT ACA OTT ATT Asn Thr Val Ile OTO AAT OGA TOG Val Asn Pro Ser AAO GTG CAA OAA Lys Leu Gin Giu GOT 000 Gly Gly 215 TOT OTG AGO Ser Val Thr ACC TOT TOO AGO Thr Oys Ser Ser GOT OTA OOA GGT Gly Leu Pro Ala OOA GAG ATT Pro Giu Ile 230 TTO TGO AOT AAG AAA Phe Trp Ser Lys Lys TTA OAT AAT Leu Asp Asn 240 000 AAT OTA CG CAC OTT TOT GC.

Oly Asn Leu Gin His Leu Ser Oly AAT OCA Asn Ala 250 ACT CTO AGO TTA Thr Leu Thr Leu GOT ATO AOO ATO Aia Met Arg Met OAT TOT OGA ATT Asp Ser Oly Ile

TAT

Tyr 265 OTO TOT OAA OGA Val Cys Oiu Gly AAT TTO ATT 000 Asn Leu Ile Oly AAO AGA AAA GAG Asn Arg Lys Oiu OAA TTA ATT OTT CAA GAO AAA OCA TTT Oiu Leu Ile Val Gin Oiu Lys Pro Phe OTT GAO ATO TOO COT OGA Val Oiu Ile Ser Pro Giy 295 000 000 ATT Pro Arg Ile OTO ATO 000 Vai Met Oly 315 GOT CG ATT OCA Ala Gin Ile Oly TCA OTC ATO TTO Ser Val Maht Leu ACA TOT AOT Thr Cys Ser 310 CAG ATA GAO Gin Ile Asp 1008 TOT OAA TOO GCA Cys Oiu Ser Pro TTO TCG TOO AGA Phe Ser Trp Arg 1056 AGO COT Ser Pro 330 OTO AGO 000 AAO Leu Ser Oly Lys AGO AOT GAG 000 Arg Ser Oiu Oly PAT TOO AOO OTO Asn Ser Thr Leu 1104

ACC

Thr 345 OTO AOC OCT OTO Leu Ser Pro Val TTT GAO PACGAOA CJAC TOT TAT OTO TOO Phe Oiu Asn Olu His Ser Tyr Leu Gys 1152 1200 OTO ACT TOT OOA OAT PAG AAA OTO OAA Val Thr Cys Oly His Lys Lys Leu Giu GOA ATO Oly Ile CA.O OTO GAO OTO Gin Val Oiu Leu 375 SUBSTiTUTE SHEET

I

-38- TAC TCA TTCG CCT AGA GAT CCA CAA ATC GAG ATG ACT GGT CCC CTC GTG Tyr Ser Phe Pro Arg Asp Pro Clu Ile Ciu Met Ser Cly Cly Leu Val 380 385 390 AAT CCC AC Asn Cly Ser 395 TCT GTC ACT GTPA Ser Val Thr Val

AC

S er 400 TCC AAG CTT Cys Lys Val CCT AC Pro Ser 405 GTC TAC CCC Val Tyr Pro 1248 1296 1344 CTT GAC Leu Asp 410 CCC CTC GAG ATT Arg Leu Giu Ile TTA CTT AAG CCC Leu Leu Lys Cly ACT ATT CTC GAG Thr Ile Leu Clu ATA GAG TTT TTC Ile Giu Phe Leu CAT ACC GAT ATG Asp Thr Asp Met TCT CTA GAC AAC Ser Leu Giu Asn 1392 1440 ACT TTC CAA ATC Ser Leu Clu Met TTC ATC CCT ACC Phe Ile Pro Thr CAA CAT ACT CCA Giu Asp Thr Cly AAA CCT Lys Ala 455 CTT CTT TCT Leu Val Cys AAA CAA ACC Lys Gin Arg 475 CCT AAC TTA CAT Ala Lys Leu His CAT CAC ATC GAA Asp Asp Met Glu TTC CAA CCC Phe Ciu Pro 470 1488 GAG ACT ACC C!kA (rin Ser Thr Gin

ACA

Thr 480 CTT TAT GTC AAT GTT CCC CCC AGA Leu Tyr Val Asn Val Ala Pro Arg 485 1536 CAT AGA Asp Thr 490 ACC CTC TTC GTC Thr Val Leu V CCT TCC TCC ATC Pro Ser Ser Ile CTC GAG CAA CCC ACT Leu Ciii Glu Cly Ser 500 OCT CCT CCG. AAA ATC Pro Ala Pro Lys Ile GTC AAT ATC AGA Val Asn Met Thr TTC AGO GAG GC Leu Ser Gin Cly 1584 1632 1680 CTG TCC AGO ACC GAG OTO COT AAC CCC Leu Trp Ser Arg Gin Leu Pro Asn Gly 525 CTA GAG OCT OTT Leu Gin Pro Leu TOT GAG Ser Giu 535 AAT GCA ACT Asn Ala Thr TAT TtA TCT Tyr Leu Cys 555 AGO TTA ATT TOT Thr Leu Ile Ser AAA ATC CAA CAT Lys Met Giu Asp TOT CCC GTT Ser Cly Val 550 AAC GAA CTC Lys Clu Val 1728 GAA GCA ATT AAC Ciii Clv Ile Asn CT CGA AGA AGO Ala Gly Arg Ser 1776 CAA TTA Ciu Leu 570 ATT ATC CAA CTT ACT OCA AAA GAG Ile Ile Gin Val Thr Pro Lys Asp ATA AAA OTT AGA CT TTT Tie -Lys Leu Thr Ala Phe 580 1824 SUSSTIaTUTE SHEET pp.~ h -39- TCT GAG AGT GTC Ser Giu Ser Val

AAA

Lys GAA GGA GAO ACT Glu Gly Asp Thr ATO ATO TCT TOT Ile Ile Ser Cys TOT 'A GTT Cys Gly .Xsn Val GAA ACA TOG ATA Glu Thr Trp Ile CTO AAG AAA AAA Leu Lys Lys Lys GOG GAO Ala Glu 615 1872 1920 1968 ACA GGA GAG Thr Gly Asp ACA GTA Thr Val 620 OTA AAA TOT Leu Lys Ser CAT 000 GC Asp Oly Ala TAT AGO ATO OGA Tyr Thr Ile Arg 630 AAO 000 CG TTG AAO OAT 000 OGA OTA TAT OAA TOT GAA TOT AAA AAG Lys Ala Gin Leu Lys Asp Ala Oly Val Tyr Giu Gys Oiu Ser Lys Asn 635 640 645 2016 AAA OTT 000 TGA CAA TTA AGA AOT TTA ACA OTT OAT Lys Val Oly Ser Gin Leu Arg Ser Leu Thr Leu Asp 650 655 660 OTT CAA OGA AGA Val Gin Oly Arg 2064 OAA AAO AAO AAA GAO Giu Asn Asn Lys Asp 665 GA TOG TGG TTA ATA Ala Ser Ser Leu Ile TTT TOT OCT GAO Phe Ser Pro Giu GTC OTG CTC TAT Leu Val Leu 141r 2112 ATA GOT 000 ATT Ile Pro Ala Ile ATO ATA ATT TAO TTT GA Met Ile Ile Tyr Phe Ala AGA AAA 000 AAO Arg Lys Ala Asn ATO AAO 000 TGA Met Lys Oly Ser PqT OTT OTA GAA Ser Leu Val Oiu GA CAG AAA Ala Gin Lys 710 2160 2208 2217 TOA AAA GTO Ser Lys Val 715 INFORMATION FOR SEQ ID NO:9: SEQUENCE CH-ARACTERISTICS: LENGTH: 739 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: SUSWITUTE SHIET Met Pro Gly Lys Met Val Val Ile Leu -24 -20 Ile S er Thr S er Thr Ala Tyr Ala 105 Phe S er Ser Leu Thr 185 Asn S er Phe Asn Phe Tyr Gly Leu Asn Gys Phe Lys Arg Giu Giu 155 Cys Arg Val Thar Ser 235 Thr Ala Leu Cys Asn Pro Glu Pro Pro Leu Phe 140 Val Arg Gin Ile Met 220 Lys Leu S er Gin S er 30 Lys S er Arg Asp Thr 110 Ile Giu Phe Lys Val 190 Val Cys Leu Leu Gin Ile Pro Val Phe Lys Pro Val Asp Asp Thr Leu 175 Lys Asn Ser Asp Ile 255 Phe 1 Asp Phe As n Asn 65 Giu Ile Cys Leu Asp 145 Val Ile Leu Ser Giu 225 Gly Met G ly -i5 Lys S er S er Giu Giu Lys His S er Lys 130 Arg Ile Asp Gin Thr 210 Gly Asn Arg Ala Ser Asn Ile Giu Thr Val Ser Leu Trp Arg Thr Gly Thr Thr His Ser Tyr Giy Ile Gin Leu Ser Gly 100 Val Ala Asp 115 Gly Asp His Lys Ser Leu Giu Asp Ile 165 Giu Met Asp 180 Val Tyr Ile 195 Lys Leu Gin Leu Pro Ala Leu Gin His 245 Met Glu Asp 260 Ile Thr Thr Gin S er Leu Val Pro Val Leu Giu 150 Gly Ser Ser Giu Pro 230 Leu Ser Leu Pro Cys Ile Thr Cys Giu Leu Tyr Met 135 Thr Lys Val Pro Gly 215 Giu S er Gly OuSUBSjTUTE

S~

-41- Tyr Val Cys Glu Gly Val Asn Leu Ile Gly lii

U

if 265 Giu Leu Pro Arg Val Met Ser Pro 330 Thr Leu 345 Val Thr Tyr Ser Asn Gly Leu Asp 410 Asn Ile 425 Ser Leu Leu Val Lys Gin Asp Thr 490 Ser Val 505 Ile Ile Gly 315 Leu Ser Cys Phe Ser 395 Arg Giu G iu Cys Arg 475 Thr Asn Val Ala 300 Cys S er Pro Gly Pro 380 S er Leu Phe Met Gin 460 Gin Val Met Gin 285 Ala Giu Gly Val His 365 Arg Val G iu Leu Thr 445 Ala Ser Leu Thr Lys TIle Pro Val 335 Phe Lys Pro Val G iu 415 Asp Ilie Leu Gin S er 495 Leu Thr 290 S er Ser Giu Glu Lys 370 G iu Lys Lys Met Ile 450 Asp Tyr Ser Gly Asn Arg Giu Ile Met Leu Arg Thr 325 Thr Asn 340 Ser Tyr Ile Gin Ser Gly Pro Ser 405 Giu Thr 420 Ser Leu Asp Thr Met Giu Asn Val 485 Leu Giu 500 Pro Ala SUBSTITUTE :1

A

'I

rr r r -42- Leu Trp Ser Arg Gin Leu Pro Asn Gly Glu Leu Gin Pro Leu Ser Glu 525 530 535 Asn Tyr Glu Pro 585 Cys Thr Lys Lys Glu 665 Ala Ala Leu Leu 570 Ser Gly Gly Ala Val 650 Asn Ser Thr Leu Thr 540 Cys Glu Gly 555 Ile lie Gin Glu Ser Val Asn Val Pro 605 Asp Thr Val 620 Gin Leu Lys 635 Gly Ser Gin Asn Lys Asp Ser Leu Ile 685 Leu lie Val Lys 590 Glu Leu Asp Leu Tyr 670 Ile Ile Asn Thr 575 Glu Thr Lys Ala Arg 655 Phe Pro Ser Gin 560 Pro Gly Trp Ser Gly 640 Ser Ser Ala Thr 545 Ala Lys Asp ile Ile 625 Val Leu Pro Ile Lys Gly Asp Thr Ile 610 Asp Tyr Thr Glu Gly 690 Met Arg Ile Val 595 Leu Gly Glu Leu SLeu 675 SMet Glu Asp Ser Arg 565 Lys Leu 580 Ile Ile Lys Lys Ala Tyr Cys Glu 645 Asp Val 660 Leu Val lie Ile Ser 550 Lys Thr Ser Lys Thr 630 Ser Gin Leu Tyr Gly Val Glu Val Ala Phe Cys Thr 600 Ala Glu 615 lie Arg Lys Asn Gly Arg Tyr Phe 680 Phe Ala 695 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu 700 705 Ser Lys Val 715 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1941 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 'ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1941 Ala Gin Lys 710 SUBSTITUTE

SHEET

I i hk -43- (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 73. .1941 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 72 (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

Met -24 CCT CCC AAG ATG Pro Gly Lys Met -20 GTC OTG ATC CTT Val Vai Ile Leu OGA GCC Oly Ala TCA AAT ATA CTT TOO Ser Asn Ile Leu Trp ATA ATG TTT OCA OCT TCT CAA GCT TTT AAA ATC GAO ACC ACC CCA OAA Ile Met Phe Ala Ala Ser Gin Ala Phe Lys Ile Giu Thr Thr Pro Olu TCT AGA Ser Arg TAT CTT OCT CAC Tyr Leu Ala Gin GOT GAC TCC GTC Gly Asp Ser Val TTG ACT TGC AGC Leu Thr Cys Ser ACA GGC TGT GAO Thr Giy Cys Oiu CCA TTT TTC TOT Pro Phe Phe Ser AGA AGO GAG ATA Arg Thr 0h Ile ACT CCA CTO AAT Ser Pro Leu Asn AAO OTO ACO AAT GAO 000 ACC ACA TCT ACO CTO Lys Val Thr Asn 0Th Giy Thr Thr Ser Thr Leu ACA ATO AAT Thr Met Asn OCA ACT TOT Ala Thr Cys OTT AOT TTT 000 Val Ser Phe Oly OAA CAC TOT TAG Olu His Ser Tyr CTG TG ACA Leu Cys Thr OAA TOT AGO AAA Giu Ser Arg Lys OAA AAA OGA ATC CG OTO GAO ATC Giu Lys Oly Ile Gin Val 0h Ile TAG TCT.

Tyr Ser TTT COT AAO OAT Phe Pro Lys Asp GAO ATT CAT TTO Giu Ile His Leu GOC CCT CG GAO Oly Pro Leu Giu 000 AAO 000 ATC Oly Lys Pro Ile

ACA

Thr 110 OTO AAO TOT TOA OTT OCT OAT OTA TAG CCA Val Lys Cys Ser Val Ala Asp Val Tyr Pro TTT GAG AGO CTO Phe Asp Arg Leu ATA GAO TTA CTO Ile Asp Leu Leu OGA OAT CAT OTC Oly Asp His Leu ATO AAO Met Lys 135 fw4~N<?\ I~A~/~1 SUBSTITUTE

SHEET

sl -44- *ACT 'GAG CAA TTT Ser Gin Clu Phe 140 AGT TTG GAA OTA Ser Leu Glu Val 155 CTT GTT TCC CGA Leu Val Cys Arg 170 QTG GAG GAT CCA Leu Ciu Asp Ala AGG AAG TCC CTG Arg Lys Ser Leu GAA ACC AAC Clii Thr Lys 150 GCA AAA GTT Cly Lys Vai ACC TTT ACT Thr Phe Thr CCT AAA TTA Ala Lys Leu 175 CTC ATT CAG CAT Val Ile Ciu Asp

ATT

Ile 165 GAG ATT CAT CAA His Ile Asp Giu CAT TOT CTC CCC Asp Ser Val Pro

ACA

Thr 185 CTA ACG GAG CT Val Arg Gin Ala AAA GAA TTC CAA Lys Ciu Leu Gin TAG ATA TCA CCC Tyr Ile Ser Pro PAT ACA CTT ATT Asn Thr Val Ile GTG AAT OGA TOG ACA AAG GTC CAA CPA Val Asn Pro Ser Thr Lys Leu Gin Giu 210 GGT GC Cly Gly 215 TOT GTG AGO Ser Val Thr TTG TGG ACT Phe Trp Ser 235 AGO TCT TOG AGO Thr Cys Ser Ser GGT OTA OGA GOT Cly Leu Pro Ala GGA GAG ATT Pro Giu Ile 230 OTT TOT CGA Leu Ser Cly PAG APA TTA CAT Lys Lys Leu Asp GCG PAT OTA GAG Cly Asn Leu Gin PAT GCA Asn Ala 250 ACT OTO AGO TTA Thr Leu Thr Leu CT ATG AGC ATG Ala Met Arg Met CAT TOT GGA ATT Asp Ser Cly Ile GTC TGT CPA CGA Val Cys Ciu Cly PAT TTG ATT CCC Asn Leu Ile Cly AAO AGA AAA GAG Asn Arg Lys Giu CPA TTA ATT GTT Clii Leu Ile Val CCA TTO OCT AGA Ala Phe Pro Arg OGA CPA ATO GAG Pro Clu Ile Glu ATC ACT Met Ser 295 GCT CCC OTO Cly Gly Leu AGO GTG TAO Ser Val Tyr 315 PAT CCC AGO TOT Asn Cly Ser Ser ACT CTA AGO TGO Thr Val Ser Cys AC GTT OCT Lys Val Pro 310 1008 4 COO OTT GAO CCC Pro Leu Asp Arg GAG ATT CPA TTA OTT AC CCC GAG Giu Ile Giu Leu Leu Lys Cly Giu 1056 1104 ACT ATT Thr Ile 330 OTG GAG PAT ATA Leu Glu Asn Ile TTT TTG GAG Phe Leu Ciu CAT AOG Asp -Thr 340 CAT ATG AAA TOT Asp Met Lys Ser DO I fi U I L, "T

CTA

Leu 345 GAG AAC AAA AGT Ciu Asn Lys Ser CAA ATC ACC TTC Giu Met Thr Phe CCT ACC ATT CAA Pro Thr Ile Ciiu 1.152 1200 ACT CGA AAA GCT Thr Gly Lys Ala GTT TGT CAG GCT Val Cys Gin Ala TTA CAT ATT CAT Leu His Ile Asp CAC ATG Asp Met 375 CAA TTC GAA Ciu Phe Ciu CTT GCC CCC Val Ala Pro 395 CCC AAA CAA AGC CAC Pro Lys Gin Arg Gin 3,20 AGA CAT ACA ACC CTC Arg Asp Thr Thr Val 400 ACG CAA ACA CTT Thr Gin Thr Leu TAT GTC AAT Tyr Val Asn 390 TCC ATC CTC Ser Ile Leu 1248 TTG CTC ACC CCT Leu Val Ser Pro GAG GAA Ciu Glu 410 CCC ACT TCT CTG Cly Ser Ser Val ATC ACA TCC TTC Met Thr Cys Leu GAG CCC TTT CCT Gin Gly Phe Pro CCG AAA ATC CTC TGG Pro Lys Ile Leu Trp 430 AGC ACG CAG CTC Ser Arg Gin Leu AAC CCC GAC CTA Asn Cly Ciu Leu

CAG

Cmn 440 CCT CIT TCT GAG Pro Leu Ser Ciu CCA ACT GIG ACC Ala Thr Leu Ihr AlT TCT ACA AAA Ile Ser Ihr Lys ATC CAA Met Ciu 455 1296 1344 1392 1440 1488 1536 1584 CAT TCT CCC Asp Ser Gly AGA AAG GAA Arg Lys Ciu 475 TAT TTA TCT GAA CCA ATT AAC CAG GC Tyr Leu Cys Giu Cly Ile Asn Gin Ala CCA AGA AC Cly Arg Ser 470 CAC ATA AAA Asp Ile Lys CTC CAA ITA ATT Val Giu Leu Ile CAA CTT ACT CCA Gin Val Thr Pro CTI ACA Leu Thr 490 GCT ITT CCI ICI Ala Phe Pro Ser

GAG

Clii 495 ACT GTC AAA CA Ser Val Lys Ciu GAG ACT GTC ATG Asp Ihr Val Ile TCT TCT ACA TCT Ser Gys Thr Gys PAT CTT GGA CA Asn Val Pro Glu GAG ACA CIA CIA Asp Thr Val Leu 530 ICC ATA ATG CG Trp Ile Ile Leu 1632 AAA AAA CC GAG ACA CGA Lys Lys Ala Clu Thr Cly 525 AAA ICT ATA CAT Lys Ser Ile Asp CCC CC Cly Ala 535 1680 TAT ACC AIC Tyr Thr Ile PAG CCC GAG TIC Lys Ala Gin Leu GAT CC CCA CIA Asp Ala -Cly Val TAT CPA TCT Tyr Ciu Cys 550 1728 SUBtTlk-fUTE SKEMt

A

it

I

p 1- 11 1 -46- GAA' -TGT AAA AAC AAA GTT CCC TCA CAA TTA A G1u Ser Lys Asn Lys Val Gly Ser Gin Leu A 555 560 GTT GAA GGA AGA GAA AAG AAC AAA GAG TAT TI Val Gin Cly Arg Ciu Asn Asn Lys Asp Tyr P 570 575 GTG GTC TAT TTT GGA TGC TCG TTA ATA ATA Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile 585 590 ATT TAG TTT CA AGA AAA CCC AAC ATC AAG Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys 605 610 GAA GGA GAG AAA TCA AAA GTG Glu Ala Gin Lys Ser Lys Val 620 INFORMATION FOR SEQ ID NO:ll: SEQUENGE GHARACTERISTIGS: LENGTH: 647 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLEGULE TYPE: protein (xi) SEQUENGE DESGRIPTION: SEQ ID Met Pro Gly Lys Met Val Val Ile Leu Cly -24 -20 -15 Ile Met Phe Ala Ala Ser Gin Ala Phe Lys 1 Ser Arg Tyr Leu Ala Gin Ile Cly Asp Ser 15 Thr Thr Gly Gys Giu Ser Pro Phe Phe Ser 30 Ser Pro Leu Asn Cly Lys Val Thr Asn Ciu 45 50 Thr Met Asn Pro Val Ser Phe Cly Asn Giu Ala Thr Gys Giu Ser Arg Lys Leu Ciu Lys 80

CA

~rg

TT

~he

CGT

?ro 595 3,GG ly

ACT

S er

TGT

Ser 580

CG

Al a

TGA

S er GTT CAT Leu Asp GTT GTG Leu Leu ATC ATA Met Ile 600 GTT CTA Leu Val 615 1776 1824 1372 1920 1941

N

NO: 11: Ala Ser Ile Clu Vai Ser Trp Arg Cly Thr His Ser Gly Ile Asn Thr Leu Thr Thr Tyr Gin Leu Pro Gys Ile Thr Gys Giu SUBu S'i is "iu* SH EET kk -47- Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu 95 Ala 105 Phe S er Ser Leu Thr 185 Asn Ser Phe Asn Tyr 265 Giu dly S er Thr Lys Pro Arg Leu Giu Phe 140 Glu Val 155 Cys Arg Arg Gin Val Ile Thr Met 220 Ser Lys 235 Thr Leu Cys Glu Ile Val Leu Val 300 Tyr Pro 315 Leu Giu Ile Glu 125 Leu Thr Al a Ala S er 205 Thr Lys Thr dly Gin 285 Asn Leu Asn Asp Thr Leu 175 Lys As n Ser Asp Ile 255 Asn Phe S er Arg Glu 335 Ala Pro 160 His G iu Pro S er Asn 240 Ala Leu Pro S er Leu 320 Phe Ser Lys 130 Arg Ile Asp Gin Thr 210 Gly Asn Arg G ly Asp 290 Thr Ile Giu Val 115 Gly Lys Glu Glu Val 195 Lys Leu Leu Met Lys 275 Pro Val Glu Asp Ser 100 Ala Asp Ser Asp Met 180 Tyr Leu Pro Gin G lu 260 Asn Glu S er Leu Thr 340 Gly Asp His Leu Ile 165 Asp Ile Gin Ala His 245 Asp Arg Ile Gys Leu 325 Asp Pro Val Leu Glu 150 Gly Ser S er Giu Pro 230 Leu Ser Lys Glu Lys 310 Lys Met Giu Pro 120 Lys Lys Val Pro Lys 200 Gly Ile G ly Ile Val 280 Ser Pro Giu S er Leu Giu Asn Lys Ser Leu Giu Met Thr Phe Ile Pro Thr Ile Giu 345 350 355- SUBS i !ii 6"-EET .Thr bly Lys Ala Leu Val Cys Gin Ala Clii Val Clii Ala 425 Pro Asp Arg Leu Ile 505 Lys Tyr Giu Val Val 585 Ile Giu ?he ka Giu 410 Pro Leu Ser Lys Thr 490 Ser Lys Thr Ser Cir 570 Lei.

Tyi Al~ Ciu Pro 395 Cly Lys S er Cly Giu 475 Ala Cys Ala Ile Lys 555 IGly Tyr Phe ~Gin Pro 380 Arg Ser Ile C iu Val 460 Val Phe Thr C iu Arg 540 Asn Arg Phe Ala Lys 6 365 Lys Gin Arg Asp Thr Thr Ser Vai Asn 415 Leu Trp Ser 430 Asn Ala Thr 445 Tyr Leu Cys Ciu Leu le Pro Ser Ciu 495 Cys Cly Asn 510 Thr Cly Asp 525 Lys Ala Gin Lys Val Cly Ciu Asn Asn 575 Ala Ser Ser 590 Arg Lys Ala 605 Ser Lys Val .,in Val 400 Me t Arg Leu Clii Ile 480 Ser Val Thr Leu Ser 560 Lys Leu Ser 385 Leu 1'hr Gln Thr Cly 465 Gin Val Pro Val Lys 545 Gin Asp Ile -48- Lys 370 Thr Val Cys Leu Leu 450 Ile Val Lys Ciu Leu 530 Asp Leu ITyr Ile iln Ser Leu Pro 435 Ile Asn Thr Ciu Thr 515 Lys Ala Arg Phe Pro 595 Leu His Ile Asp Thr Pro Ser 420 Asn S er C in Pro Cly 500 Trp Ser Cly Ser Set 580 Ala Leu Ser 405 Gin Cly Thr Ala Lys 485 Asp Ile Ile *Val *Leui 565 Pro Ile Tyr 390 S er Giy Giu Lys Cly 470 Asp Thr Ile Asp Tyr 550 Thr Clu Ci3 Asp Met 375 Val Asn Ile Leu The Pro Leu Cmn 440 Met Ciu 455 Arg Ser Ile Lys Val Ile Leu Lys 520 Cly Ala 535 Clu Cys Leu Asp Leu Leu Met Ile 600 Asn Met Lys Cly Ser Tyr Ser Leu Val S~as~TESHEET I i

I;$

t -49- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 2205 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2205 (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 73..2205 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ii

II

ATG

Met -24 CCT GGG AAG ATG Pro Gly Lys Met -20 GTC GTG ATC .CTT Val Val Ile Leu GCC TCA AAT ATA Ala Ser Asn lie CTT TGG Leu Trp ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA Ile Met Phe Ala Ala Ser Gin Ala Phe Lys Ile Glu Thr Thr Pro Glu TCT AGA Ser Arg 10 TAT CTT CTG Tyr Leu Leu CCC CGG Pro Arg GGA GGC TCC GTG Gly Gly Ser Val GTG ACA TGC AGC Val Thr Cys Ser TCC TGT GAC CAG Ser Cys Asp Gin AAG TTG TTG GGC ATA GAG ACC CCG TTG Lys Leu Leu Gly Ile Glu Thr Pro Leu AAA AAG GAG TTG Lys Lys Glu Leu CTG CCT GGG AAC Leu Pro Gly Asn CGG AAG GTG TAT Arg Lys Val Tyr GAA CTG Glu Leu AGC AAT GTG CAA GAA GAT AGC CAA Ser Asn Val Gin Glu Asp Ser Gin ATG TGC TAT TCA Met Cys Tyr Ser AAC TGC CCT Asn Cys Pro TGG ACT CCT Trp Thr Pro GAT GGG CAG Asp Gly Gin TCA ACA GCT AAA Ser Thr Ala Lys TTC CTC ACC GTG Phe Leu Thr Val SUBSTITIMrIT r'r j AAC 'GAT Tys7 Asp ATC ACA Ile Thr 105 CCA GAG ATT CAT TTG ACT GOC CCT CTG GAG OCT CG AAC CCC Pro G1u Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro OTC AAG TOT Val Lys Gys OTT GCT CAT GTA Val Ala Asp Val TAC OCA Tyr Pro 115 TTT GAO AGO OTO Phe Asp Arg Leu GAC ATA GAG TTA Ciu Ile Asp Leu AAA OCA CAT CAT Lys Gly Asp His ATO AAC ACT GAO Met Lys Ser Cmn CAA TTT Oiu Phe 135 OTO GAO OAT Leu Giu Asp AGO TTT ACT Thr Phe Thr 155 GAG AGO AAG TOG Asp Arg Lys Ser GAA AGO AAC ACT Ciii Thr Lys Ser TTC GAA OTA Leu Ciii Val 150 GOT OTC ATT GAO Pro Val Ile Giu ATT OGA AAA OTT OTT OTT TOO OGA Ile Cly Lys Val Leu Val Gys Arg CT AAA Ala Lys 170 TTA G.AO ATT OAT Leu His Ile Asp ATO OAT TOT OTO Met Asp Ser Val ACA OTA AGO CG Thr Val Arg Gin

I

I

1'

U.

N) OTA AAA CAA TTO CAA OTO TAO ATA TOA Val Lys Clii Leu Gin Val Tyr Ile Ser AAC AAT ACA OTT Lys Asn Thr Val TOT OTO AAT OGA Ser Val Asn Pro ACA AAG OTO CAA Thr Lys Leu Gin OCT CCC TOT OTO Oly Oly Ser Val AGO ATO Thr Met 215 AGO TOT TOG Thr Gys Ser AAA TTA CAT Lys Leu Asp 235 GAO GOT OTA OGA Olu Oly Leu Pro OCA GAO ATT TTO Pro Ciu Ile Phe TOG ACT AAG Trp Ser Lys 230 GA ACT OTO Ala Thr Leu AAT CCC PAT OTA CG GAO OTT TOT OGA Asn Cly Asn Leu Cmn His Leu Ser Gly

PAT

As n 245 AGO TTA ATT GOT ATO AGO Thr Leu Ile Ala Met Arg CPA OAT TOT OCA Glu Asp 5cr Gly TAT OTO TOT CA Tyr Val Gys Glu OTT PAT TTG ATT Vai Asn Leu Ile

COO

Gly 270 AAA PAC AGA AA Lys Asn Arg Lys OTO CPA TTA ATT Val Clii Leu Ile CPA GAG PAA OCA Gin Ciu Lys Pro ACT OTT GAO ATO Thr Val Clii Ile COT OCA CCC COO Pro-Giy Pro Arg ATT OCT Ile Ala 295 SUBSTITUTE SHIEE 1'~ I I- r r -51- GCT 'CAG ATT Ala Gin Ile GAA TCC CCA Giu Ser Pro 315 GAG TCA GTC Asp Ser Val ATG TTG Met Leu 305 ACA TGT ACT GTC Thr Cys Ser Val ATG 3GC TGT Met Gly Cys 310 1008 1056 TCT TTC TCC TGC Ser Phe Ser Trp ACC CAC ATA CAC ACCGCT CTC AGC Thr Gin Ile Asp Ser Pro Leu Ser GCG AAC Cly Lys 330 GTC AGC ACT GAG Val Arg Ser Ciu ACC AAT TCC ACC Thr Asn Ser Thr ACC CTG ACC CCT Thr Leu Ser Pro 1104 ACT TTT GAG AAG Ser Phe Ciu Asn GAG TCT TAT CTC His Ser Tyr Leu ACA CTC ACT TCT Thr Val Thr Cys 1152 CAT AAC AAA CTC His Lys Lys Leu AAC CCA ATC GAG Lys Cly Ile Gin GAG CTC TAG TCA Ciu Leu Tyr Ser TTC CCT Phe Pro 375 ACA GAT GCA Arg Asp Pro CTC ACT CTA Val Thr Val 395 ATG GAG ATC AGT Ile Ciu Met Ser GGC CTC GTC AAT Cly Leu Val Asn CCC ACC TCT Gly Ser Ser 390 GA6 CCC CTC Asp Arg Leu 1200 1248 1296 ACG TGC AAG GTT Ser Gys Lys Vai ACC GTG TAG CCC Ser Vai Tyr Pro

CTT

Leu 405 GAG ATT Ciu Ile 410 CAA TTA GTT AAC Ciu Leu Leu Lys (iukl ACT ATT CTC Ciu Thr Ile Leu AAT ATA GAG TTT Asn Ile Giu Phe 1344

TTC

Leu 425 GAG CAT AOC GAT Glu Asp Thr Asp ATC AAA TCT Met Lys Ser 430 -Z AAC AAA ACT TTG CAA G-Vr &sn Lys Ser Leu Giu 1392 ACC TTC ATG CCT Thr Phe Ile Pro ATT CAA CAT ACT Ile Ciu Asp Thr A, CCT CTT CTT Lys Ala Leu Val TCT CAC Cys Gin 455 1440 CCT AAC TTA Ala Lys Leu ACT ACC CAA Ser Thr Gin 475 ATT CAT GAG ATC Ile Asp Asp Met TTC CAA CCC AAA Phe Giu Pro Lys CAA ACC CAC Gin Arg Gin 470 ACA ACC CTC Thr Thr Val 1488 ACA GTT TAT GC Thr Leu Tyr Val AAT CTT CCC CCC AGA Asn Val Ala Pro Arg 480 CTG OAG GAA CCC ACT Leu Giu Giu Giy-Ser 500 1536 TTG GC Leu Val 490 AGG GGT TGG TOG Ser Pro Ser Ser TOT GTG AAT ATO Ser Val Asn Met 1584 SUBSTITUTE SffEET -52- ACA 'TGC Thr Cys 505 TTG AGO GAG Leu Ser Gin TTT OCT GCT Phe Pro Ala COG AAA Pro Lys 515 ATC OTG TOG AOC Ile Leu Trp Ser 1632 1680 CAG OTC OCT AAC Gin Leu Pro Asn GAG CTA CG COT 01u Leu Gin Pro TCT GAG AAT OCA Ser Oiu Asn Ala ACT OTO Thr Leu 535 ACC TTA ATT Thr Lou Ile OGA ATT AAC Gly Ile Asn 555 ACA AAA ATO OAA OAT TOT 000 OTT TAT Thr Lys Met Giu Asp Ser Oly Val Tyr 545 TTA TOT GAA Leu Cys 01u 550 TTA ATT ATO Lou Ia2 Ile 1729 1776 CAG OCT OGA AGA Gin Ala Oly Arg AGA AAO OAA OTO Arg Lys Oiu V~al CAA OTT Gin Val 570 ACT OCA AAA GAO Thr Pro Lys Asp AAA OTT ACA OCT Lys Lou Thr Ala OCT TOT GAO AOT Pro Ser Oiu Ser 1824 1872 AAA OAA OGA GAO Lys Giu Oiy Asp OTO ATO ATO TOT Vai Ile Ile Ser ACA TOT OGA AAT Thr Cys Oly Asn OCA GAA ACA TG Pro Giu Thr Trp ATO OTO AAO AAA Ile Lou Lys Lys 000 GAG ACA OGA Ala Giu Thr Gly GAO ACA Asp Thr 615 1920 OTA OTA AAA Vai Lou Lys ATA OAT 000 000 Ile Asp Oly Ala ACC ATO OGA AYAO Thr Ile Arg Lys 000 CAG TTO Ala Gin Lou 630 OTT 000 TOA Val Oly Ser 1968 AAG OAT 000 OGA OTA TAT OAA Lys Asp Ala Gly Val Tyr Oiu OAA TOT AAA AAO Olu Ser Lys Asn 2016 2064 CAA TTA Gin Leu 650 AGA AOT TTA ACA OTT OAT OTT CAA OGA Arg Ser Leu Thr Lou Asp Val Gin Gly 655 OAA AAO AAO AAA Oiu Asn Asn Lys TAT TTT TOT COT Tyr Phe Ser Pro OTT OTO OTO OTO Leu Lou Val Lei- TTT OCA TOO TOO Phe Ala Ser Ser 2112 ATA ATA COT CCC ATT OGA ATO ATA ATT Ile Ile Pro Ala Ile Gly Met Ile Ile TTT OCA AGA AAA Phe Ala Arg Lys 000 AAC Ala Asn 695 2160 2205 ATO AAO 000 Met Lys Gly TAT ACT OTT Tyr Ser Lou OTA GAA Val Glu 705 OCA GAG AAA TCA Ala GinLys Ser AAA OTO Lys Val 710 SUBSTirTT SHEET ;_i t sirr ,1 I rr~ -53- INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 735 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met -24 lie Ser Thr Lys Ser Asp Lys Ile 105 Glu Leu Thr Ala (xi) SEQUENCE Pro Gly Lys Met Met Phe Ala Ala Arg Tyr Leu Leu Ser Cys Asp Gin Lys Glu Leu Leu Asn Val Gln-Glu Gly Gin Ser Thr Asp Pro Glu Ile Thr Val Lys Cys Ile Asp Leu Leu 125 Glu Asp Ala Asp 140 Phe Thr Pro Val 155 Lys Leu His Ile 170 DESCRIPTION: SEQ ID NO:13: Val Val lie Leu Ala Ser Asn Ile Leu Trp Gin Arg Lys Pro Ser Lys Leu Val Gly Lys Glu Glu 175 Ala Gly Leu Gly Gin Thr Ser Ala Asp Ser Asp 160 Met Ile Val Ile 35 Arg Cys Thr Leu Tyr 115 Met Thr Lys Val Glu Ser Pro Leu Pro Pro Pro Leu 120 Phe Val Arg Gin SUBSTITUTE

SHEET

I

I'I

-54- 'Ala Val Lys Glxi Leu Gin Val Tyr Ile Ser Pro Lys Ar~ Thr Vai lie Ser Val Asn Pro Ser Thr Lys Thr Lys Thr Giy 265 Gin Ala G iu Giy Val 345 His Arg Val :ys Leu Leu 250 Val Giu Gin S er Lys 330 S er Lys Asp Thr Ser Asp 235 Ile Asn Lys Ile Pro 315 Vai Phe Lys Pro Val S er 220 Asn Aia Leu Pro Giy 300 S er Arg Giu Leu Aiu 380 Ser 205 Giu Giy Met Ile Phe 285 Asp Phe S er Asn Glxi 365 Ile Cys Asn Arg Giy 270 Thr S er Ser Glu Glxi 350 Lys G iu Lys Leu Leu Met 255 Lys Vai Vai Trp Gly 335 His G iy Met Val G13 415 Ly Cix Leu Pro Gin 240 Giu Asn.

G iu Met Arg 320 Thr Ser Ile Ser Pro 400 Giu Ser .x Asp 31n kla 225 His Asp Arg Ile Leu 305 Thr Asn Tyr Gin C iy 385 Ser Thi Lex Th Giu C 21i0 Pro G Leu S SerC Lys Ser 290 Thr Gin Ser Leu Vai 370 Giy Vai Ile .t Giu r Gly 450 iy Iiu er ;iu 275 Pro Cys Ile Thr Cys 355 Leu Tyr Le.

Asi 43

LY~

Gly S Ile I Giy Ile 2 Vai Giy Ser Asp Leu 340 Thr Leu Vai Pro Giu 420 1i Lys 52 s Ala er 'he sn 245 Giu Pro Val Ser 325 Thr Val Tyr Asn Leu 405 Asr Set Let Vai Trp 230 Ala Vai Leu Arg Met 310O Pro Leu Thr S er C ly 390 Asp Ile Leu xVal 2 Thr 1 215 SerI Thr Cys Ile Ile 295 G iy Lexi S er Cys Phe 375 S er Ar g Giu Giu ys 455 .00 e t .,ys eu Glu Val 280 Ala Cys S er Pro C iy 360 Pro Ser Leu Phe Met 440 Gin 395 Glu Leu 425 Thr Ile Gixi 410 Giu Asp Phe Ile Leu Thr Pro Leu Asp Thr 445 SUBSTITUTE

SHEET

K r C Ala Lys Leu Ser Thr Gin 475 Ile Leu Leu Thr 505 Gin i Thr Gly Gin Val 585 Pro Val Lys Gin Asp 665 Ile Tal Cys Leu Leu Ile Val 570 Lys Glu Leu Asp Leu 650 Tyr IlE Ser Leu Pro Ile Asn 555 Thr Glu Thr Lys Ala 635 Arg Phe Pro Pro Ser Ser Gin Asn Gly 525 Ser Thr 540 Gin Ala Pro Lys Gly Asp Trp Ile 605 Ser Ile 620 Gly Vat Ser LeL Ser Prc Ala il 68 Asp Asp Tyr Val I Ser Ile I 495 Gly Phe 510 Glu Leu Lys Met Gly Arg Asp Ile 575 Thr Val 590 Ile Leu Asp Gly Tyr Glu Thr'Leu 655 Glu Leu 670 a Gly Met 5 let G 4 sn 880 Leu C Pro I Gln Glu Ser 560 Lys Ile Lys Ala Gys 640 Asp Leu Ile lu ~65 Tal flu kla Pro Asp 545 Arg Leu Ile Lys Tyr 625 Gb.

Val Va.

Tb Gb Phe C Ala I Glu Pro I Leu 530 Ser Lys Thr Ser Lys 610 Thr 1 Ser Gin L Leu e Tyr 690 lu 'ro fly Lys 515 Ser Gly Glu Ala Cys 595 Ala Ile Lys Gly Tyr 675 Phe Pro Arg Ser 500 Ile Glu Val Val Phe 580 Thr Glu Arg Asn Arg 660 Phe Ala Lys G 4 Asp 1 485 Ser Leu Asn Tyr Glu 565 Pro Cys Thr Lys Lys 645 Glu Ala Arg 1n hr Jal Crp Ala Leu 550 Leu Ser Gly Gly Ala 630 Val Asn Ser Lys Arg Thr Asn Ser Thr 535 Cys Ile Glu Asn Asp 615 Gin Gly Asn Ser Ala 695 Gln Val Met Arg 520 Leu Glu ile Ser Val 600 Thr Leu Ser Lys Leu 680 Asn Kf~ Met Lys Gly Tyr Ser Leu Val 51Ala Gin Lys Ser Lys Val 710 SUBSTITUTE SHEET :1 C- ~ilrra~-~q i

II

r r' it i i~c >1 d; 11

A

I h:~

K

-56- INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 2208 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2208 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 73..2208 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATG CCT GGG AAG ATG GTC GTG ATC Met Pro Gly Lys Met Val Val Ile CTT GGA Leu Gly GCC TCA AAT ATA Ala Ser Asn Ile CTT TGG Leu Trp ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val GTG ACA TGC AGC Val Thr Cys Ser TCC TGT GAC CAG Ser Cys Asp Gin AAG TTG TTG GGC Lys Leu Leu Gly GAG ACC CCG TTG Glu Thr Pro Leu AAA AAG GAG TTG Lys Lys Glu Leu CTG CCT GGG Leu Pro Gly AAC AAC Asn Asn 50 CGG AAG GTG TAT Arg Lys Val Tyr GAA CTG Glu Leu AGC AAT GTG Ser Asn Val GAT GGG CAG Asp Gly Gln GAA GAT AGC CAA CCA ATG TGC TAT TCA Glu Asp Ser Gin Pro Met Cys Tyr Ser AAC TGC CCT Asn Cys Pro TGG ACT CCA Trp Thr Pro TCA ACA GCT AAA Ser Thr Ala Lys TTC CTC ACC GTG Phe Leu Thr Val SUBSTITUTE SHEET -57- G AA 'CGG GTG GAA Giu Arg Vai Giu CTG GCA CCC Leu Ala Pro CTC CCC TCT TGG GAG CCA GTG GGC AAG Leu Pro Ser Trp Gin Pro Val Cly Lys

AAC

Asn 105 CTT ACC CTA CGC TGC GAG GTG GAG GGT Leu Thr Leu Arg Cys Gin Vai Giu Gly GGG GCA Cly Ala 115 CCC CGG GCC Pro Arg Ala CTC ACC GTG GTG CTG CTC CGT GGG GAG Leu Thr Vai Val Leu Leu Arg Gly Giu CTC ATG AAG AGT Leu Met Lys Ser GAG GAA Gin Giu 135 TTT CTG GAG Phe Leu Giu GCA GAG AGG AAG Ala Asp Arg Lys CTG GAA ACC AAG Leu Giu Thr Lys AGT TTG GAA Ser Leu Giu 150 CTT GTT TGG Leu Val Cys GTA ACC TTT ACT CCT GTC ATT Vai Thr Phe Thr Pro Vai Ile CAT ATT CGA AAA Asp Ile Gly Lys CCA CCT Arg Ala 170 AAA TTA GAG ATT Lys Leu His Ile GAA ATG CAT TCT Giu Met Asp Ser CCC ACA GTA ACC Pro Thr Val Arg CCT GTA AAA GAA Ala Val Lys Ciu CAA CTC TAG ATA Gin Val Tyr Ile CCC AAG MAT AGA Pro Lys Asn Thr ATT TCT GTC AAT ile Ser Val Asn TCC ACA AAG CTC Ser Thr Lys Leu CAA CGT CCC TCT Giu Gly Cly Ser CTC ACC Val Thr 215 ATC ACC TCT Met Thr Gys AAC AAA TTA Lys Lys Leu 235 ACC GAG CCT CTA CCA GCT CCA GAG ATT Ser Ciu Gly Leu Pro Ala Pro Clii TIe TTC TCC ACT Phe Trp Ser 230 AAT CCA ACT Asn Ala Thr CAT AAT CCC AAT Asp Asn Gly Asn GAG GAG CTT TCT Gin His Leu Ser GTC ACC Leu Thr 250 TTA ATT GCT ATC Leu Ile Ala Met ATG CAA CAT TCT Met Clu Asp Ser ATT TAT CTG TCT Ile Tyr Vai Cys GAA GCA GTT AAT TTC Giu Cly Vai Asn Leu CCC MAA AAC AGA Gly Lys Asn Arg GAG GTC CMA TTA Giu Val Ciu Leu CTT GAA GAG AAA Vai Gin Giu Lys TTT ACT CTT GAG Phe Thr Val Giu TCG GGT OCA CCC Ser Pro Gly Pro CCC ATT Arg Ile 295

LEE

p.

I :ii j I -t .e -58- GCT' GCT GAG Ala Ala Gin TGT GAA TCC Cys Giu Ser 315 AGO GGG AAG Ser Gly Lys 330

ATT

Ile 300 GGA GAG TCA GTG Gly Asp Ser Val TTG ACA TGT Leu Thr Cys CCA TOT TTO TCC Pro Ser Phe Ser GTG AGO AGT GAG Val Arg Ser Glu 335 AGA ACC CAG ATA Arg Thr Gin Ile AGT GTG ATG GGC Ser Vai Met Gly 310 GAG AGC CCT OTG Asp Ser Pro Leu 325 OTG ACC CTG AGO Leu Thr Leu Ser 1008 1056 GGG ACC AAT TCC Gly Thr Asn Ser 1104 GTG AGT TTT GAG Vai Ser Phe Glu GAA GAC TOT TAT Glu His Ser Tyr TGC ACA GTG ACT Gys Thr Val Thr 1152 OGA OAT AAG AAA Gly His Lys Lys GAA AAG GGA ATO Glu Lys Gly Ile GTG GAG CTC TAG Val Giu Leu Tyr TCA TTG Ser Phe 375 CCT AGA OAT Pro Arg Asp

CCA

Pro 380 GAA ATO GAG ATO Giu Ile Giu Met GGT GGC OTC GTG Gly Giy Leu Vai AAT GGG AGO Asn Giy Ser 390 OTT GAC CGG Leu Asp Arg 1200 1248 1296 1344 TCT GTG ACT GTA Ser Vai Thr Val 395 AGO TGO AAG Ser Gys Lys CCT AGO GTG TAO Pro Ser Val Tyr GTG GAG Leu Glu 410 ATT GPA TTA OTT Ile Giu Leu Leu GGG GAG ACT ATT Gly Giu Thr Ile GAG PAT ATA GAG Glu Asn Ile Glu

TTT

Phe 425 TTG GAG GAT ACG Leu Giu Asp Thr ATG AAA TOT OTA Met Lys Ser Leu

GAG

Glu 435 AAC AAA AGT TTG Asn Lys Ser Leu 1392 ATG ACC TTO ATO Met Thr Phe Ile ACC ATT GPA OAT Thr Ile Giu Asp GGA AAA GOT GTT Gly Lys Ala Leu OTT TGT Val Gys 455 1440 GAG GOT PAG Gin Ala Lys

TTA

Leu 460 OAT ATT GAT GAG ATG GPA TTC GAA CCC His Ile Asp Asp Met Giu Phe Glu Pro 465 AAA CPA AGG Lys Gin Arg 470 OAT ACA ACC Asp Thr Thr 1488 r: r ic GAG AGT ACG CPA ACA OTT TAT Gin Ser Thr Gin Thr Leu Tyr PAT OTT GCC CCC Asn Val Ala Pro CTG GAG GAA GGC Leu Giu GluGly 500

AGA

Arg 485 1536 1584 GTC TTG Val Leu 490 GTC AGC CCT TCC TCC ATO Val Ser Pro Ser Ser Ile 495 AGT TOT GTG AAT Ser Ser Val Asn SUB3STIrUTE

SWEET

-59- AGA TGC TTG Thr Cys Leu AGC GAG GGC TTT OCT OCT GGG AAA ATC CTG TOG AGC Ser Gin Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser 510 515 520 AAC GGG GAG CTA CAG OCT OTT TOT GAG AAT GCA ACT Asn Gly Giu Leu Gin Pro Leu Ser Giu Asn Ala Thr 525 530 535 1632 1680 AGG GAG GTG GOT Arg Gin Leu Pro CTC AGC TTA Leu Thr Leu GAA GGA ATT Oiu Oly Ile 555 TGT AGA AAA ATG Ser Thr Lys Met GAT TGT GOG GTT Asp Ser Oly Val TAT TTA TGT Tyr Leu Gys 550 1728 AAG CG GCT OGA Asn Gin Ala Oly AGG AGA AAG GAA GTG GAA TTA ATT Ser Arg Lys Gilt Val Giu Leu Ile 565 1776 ATG CAA Ile Gin 570 GTT ACT OGA AAA Val Thr Pro Lys ATA AAA GTT AGA Ile Lys Leu Thr TTT COT TGT GAO Phe Pro Ser Oiu

I

A

IiA

A

GTO AAA GAA GGA Val Lys Giu Gly AGT OTG ATC ATG Thr Val Ile Ile TGT ACA TGT GGA Oys Thr Gys Gly

AAT

Asn 600 OTT GGA GAA ACA TOG ATA ATG OTO AAO Val Pro Oiu Thr Trp Ile Ile Leu Lys AAA OCO GAG ACA Lys Ala Oiu Thr OGA GAG Oly Asp 61i5 AGA OTA OTA Thr Vai Leu TTO AAO OAT Leu Lys Asp 635 TGT ATA OAT GC Ser Ile Asp Oly TAT AOG ATG OGA Tyr Thr Ile Arg AAO 0CC GAG Lys Ala Gin 630 OCO OGA OTA TAT Aia Oiy Vai Tyr TOT GAA TOT AAA AAO AAA OTT GG Gys Gilt Ser Lys Asn Lys Val Oly 1824 1872 1920 1968 2016 2064 2112 2160 2208 TOA CAA Ser Gin 650 TTA AGA ACT TTA Leu Arg Ser Leu CTT OAT OTT CAA Leu Asp Val Gin AGA GAA AAO AAG Arg Oiu Asn Asn GAC TAT TTT TOT Asp Tyr Phe Ser

GOT

Pro 670 GAG OTT CTC OTO Glu Leu Leu Val TAT TTT GA TGO Tyr Phe Ala Ser TTA ATA ATA OCT Leu Ile Ile Pro ATT OGA ATO ATA Ile Gly Met Ile TAG TTT OCA AGA Tyr Phe Ala Arg AAA 0CC Lys Ala 695 AAC ATO AAG Asn Met Lys TCA TAT AGT CTT Ser Tyr Ser Leu OTA OAA Val Glu 705 OCA GAG AAA Ala -Gl Lys TCA AAA OTG Ser Lys Val 710 SUBSTITUTEm

HEET

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 736 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp -24 -20 -15 Ile Met Phe Ala Ala Ser Gin Ala Phe Lys Ile Giu Thr Thr Pro Glu 1 Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser 15 Thr Ser Cys Asp Gin Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro 30 35 Lys Lys Clu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu 50 Ser Asn Val Gin Glu Asp Ser Gin Pro Met Cys Tyr Ser Asn Cys Pro 65 Asp Gly Gin Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro 80 Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gin Pro Val Gly Lys 95 100 Asn Leu Thr Leu Arg Cys.Gin Val Glu Gly Gly Ala Pro Arg Ala Asn 105 110 115 120 Leu Thr Val Val Leu Leu Arg Gly Giu Lys Leu Met Lys Ser Gin Glu 125 130 135 Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu 140 145 150 Val Thr Phe Thr Pro Val Ile Giu Asp Ile Gly Lys Val Leu Val Cys 155 160 165 Arg Ala Lys Leu His Ile Asp Giu Met Asp Ser Val Pro Thr Val Arg 170 175 180 Gin Ala Val Lys Glu Leu Gin Val Tyr Ile Ser Pro Lys Asn Thr Val 185 190 195 200 SU6841U( Ii E SHET 14

I,

H

ii It I 'I 11 -61- Iie'Ser Val Asn Pro Ser Thr Lys Leu 205 Met Lys Leu Glu 265 Val Ala Gys S er Pro 345 G ly Pro S er Leu Phe 425 Met Thr Lys Thr 250 Gly Gin Al a Giu Gly 330 Val His Ar g Val G iu 410 Leu Thr Gys Leu 235 Leu Val Giu Gin S er 315 Lys S er Lys Asp Thr 395 Sle *Giu *Phe Ser 220 Asp Ile Asn Lys Ile 300 Pro Val Phe Lys Pro 380 Val Giu Asp Ile Ser Asn Al a Leu Pro 285 Gly S er Arg Gi 1u Leu 365 Giu S er Leu Thr Pro 445 Giu Gly Met Ile 270 Phe Asp Phe Ser Asn 350 ,1i1u Ile Gys Leu Asp 430 Thr Gly Asn Arg 255 G ly Thr S er S er G iu 335 Giu Lys G li Lys TLys 415 Met Ile Leu Leu 240 Met Lys Val Val Trp 320 G ly His G ly Met Val 400 C ly Lys G iu Pro 225 Gin Glu Asn Giu Met 305 Arg Thr Ser Ile S er 385 Pro Giu Ser Asp Gin Glu Gly 210 Ala Pro Giu His Leu Ser Asp Ser Gly 260 Arg Lys Giu 275 Ile Ser Pro 290 Leu Thr Gys Thr Gin Ile Asn Ser Thr 340 Tyr Leu Gys 355 Gin Val Giu 370 Gly Gly Leu Ser Val Tyr Thr Ile Leu 420 Leu Glu Asn 435 Thr Gly Lys Gly Ile G ly 245 Ile Val G ly S er Asp 325 Leu Thr Leu Val Pro 405 Giu Lys Ala S er Phe 230 Asn Tyr Giu Pro Val 310 S er Thr Val Tyr Asn 390 Leu Asn S er Leu Val 215 Trp Ala Val Leu Ar g 295 Met Pro Leu Thr Ser 375 G ly Asp Ile Leu Val Thr S er Thr Gys Ile 280 Ile Gly Leu Ser Cys 360 Ph e Ser Arg Giu Giu 440 Gy s 450 Gin Ala Lys Leu 460 His Ile Asp Asp Glu Phe Glii Pro Lys Gin Arg 470 SUBSTf fiFu""PE 'SEET

I

-62- Gin Ser Thr Gin Thr Leu Tyr Val Asn Val Ala 475 480 4 ii ii

I

J

I

it

I

A

d Val Met 505 Arg Leu Giu Ile S er 585 Val Thr Leu S er Lys 665 Leu Leu 490 Thr Gin Thr Gly Gin 570 Val Pro Val Lys Gir 650 Asp IlE Val Cys Leu Leu Ile 555 Val Lys Giu Leu Asp 635 Leu Tyr Ile Ser Leu Pro Ile 540 As n Thr Giu Thr Lys 620 Ala Arg Phe Pro Gly 700 Pro Ser Asn 525 Ser Gin Pro G ly Trp 605 S er Gly Ser Ser Ala 685 Ser Gin 510 Gly Thr Ala Lys Asp 590 Ile Ile Val Leu Pro 670 Ile Ser 495 Giy Giu Lys Giy Asp 575 Thr Ile Asp Tyr Thr 655 Giu Gly Ile Leu Glu Phe Pro Ala Leu Gin Pro 530 Met Glu Asp 545 Arg Ser Arg 560 Ile Lys Leu .AVaL Ile Ile Leu Lys Lys 610 Gly Ala Tyr 625 Giu Gys Giu 640 Leu Asp Val Leu Leu Vai Met Ile Ile 690 J lu Pro 515 Leu S er Lys Thr Ser 595 Lys Thr S er Gin Leu 675 Tyr Pro Gly 500 Lys S er Giy Giu Ala 580 Gys Ala Ile Lys Giy 660 Tyr Phe Arg 485 Ser Ile G lu Val Vai 565 Phe Thr Giu Arg Asn 645 Arg Phe Ala Asp S er Leu Asn Tyr 550 Giu Pro Gys Thr Lys 630 Lys Glu Aila Arg Thr Thr Val Asn Trp Ser 520 Aia Thr 535 Leu Gys Leu Ile Ser Giu Gly Asn 600 Gly Asp 615 Ala Gin Val Gly Asn Asn Ser Ser 680 Lys Ala 695 Asn Met Lys Ser Tyr Ser Leu Glu Ala Gin Lys Ser Lys Val 710 SUBS"Irrur 13 i:il :I -63- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 2220 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2220 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 73..2220 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC Met Pro Gly Lys Met Val Val Ile Leu Gly Ala TCA AAT ATA CTT Ser Asn Ile Leu -24 -20 ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA CAA Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu TCT AGA TAT Ser Arg Tyr CTT GCT CAG Leu Ala Gln GGT GAC TCC GTC Gly Asp Ser Val TTG ACT TGC AGC Leu Thr Cys Ser ACC ACA GGC TGT GAG TCC CCA TTT TTC TCT Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG Ser Pro Leu Asn Gly Lys Val Thr Asn Glu AGA ACC CAG ATA Arg Thr Gln Ile GGG ACC ACA TCT Gly Thr Thr Ser ACG CTG Thr Leu ACA ATG AAT CCT GTT AGT TTT GGG AAC GAA CAC TCT TAC Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr CTG TGC ACA Leu Cys Thr GCA ACT TGT Ala Thr Cys GAA TCT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC Glu Ser Arg Lys Leu Glu Lys Gly Ile Gin Val Glu Ile SUBSTITUTE

SHEET

1 I- -64- TAC TG Tyr Trp ACT GCA GAA COO Thr Pro Glu Arg GAA OTG OCA COO Giu Leu Ala Pro

CTC

Leu 100 CCC TOT TOG GAG Pro Ser Trp Gin GTG GO AAG AAG Val Gly Lys Asn AGO CTA 000 TG GAG GTG GAO OGT CG Thr Leu Arg Gys Gin Val Glu Gly Gly 115 COO COG 000 AAC Pro Arg Ala Asn AGO GTO GTO OTO Thr Val Val Leu OTO GT Leu Arg 130 000 GAG AAG GAG OTO Oly Olu Lys Olu Leu 135 AAA 000 GAO Lys Arg Giu OTO GTG AGO Leu Val Arg 155

OGA

Pro 140 GGT OTO 000 GAO Ala Val Gly Oiu GOT GAO OTO AOO Ala Glu Val Thr AGO AG OTO Thr Thr Val 150 COO ACT OAA Arg Thr Giu AGA OAT GAG OAT Arg Asp His His

OGA

dly 160 000 AAT TTO TOO Ala Asn Phe Ser CTO GAO Leu Asp 170 OTO CG9 COG CAA Leu Arg Pro Gin OTO GAO OTO TTT Leu Glu Leu Phe AAO ACG TOO 000 Asn Thr Ser Ala

COO

Pro 185 TAG CG OTO GAG Tyr Gin L~u Gin

AGC

Thr 190 TTT OTO CTO OCA Phe Val Leu Pro ACT 000 GCA CAA Thr Pro Pro Gin OTO AGO 000 000 Val Ser Pro Arg OTA GAO OTO GAG Leu Olu Val Asp CG 000 AGO OTO Gin Oly Thr Val OTC TOT Val Gys 215 TOO OTO GAG Ser Leu Asp OTO 000 GAO Leu Gly Asp 235 OTO TTG OCA OTO TOG GAO 000 CAG OTO Leu Phe Pro Val Ser Oiu Ala Gin Val CAC OTO GA His Leu Ala 230 AAO GAO TOO Asn Asp Ser OAG AGO TTG AAO Gin Arg Leu Asn ACA OTO AGO TAT Thr Val Thr Tyr TTO TOO Phe Ser 250 000 AAO GO TGA Ala Lys Ala Ser AOT OTO AGO OCA Ser Val Thr Ala GAO GAO 000 ACC Asp Glu Oly Thr 000 CTO AG TOT Arg Leu Thr Gys OTA ATA OTO 000 Val Ile Leu Oly GAG AGO GAG GAG Gin Ser Gin Olu OTO CG AGA OTO Leu Gin Thr Val AGO ATO Thr Ile 285 TAG AGO ITT COO 000 000 GAG ATO Tyr Ser Phe Pro Ala'Pro Olu Ile TOO GOT Ser Pro 295 WWBII, u C C r ,it 'i GGA CCC CGG Gly Pro Arg

ATT

Ile 300 GCT GGT CAG ATT Ala Ala Gin Ile GAG TCA GTG ATG Asp Ser Val Met TTG AGA TGT Leu Thr Gys 310 ACC GAG ATA Thr Gin Ile AGT GTG ATG GGC TGT GAA TGC CCA TGT TTG TCC TGG Ser Val Met Gly Gys Glu Ser Pro Ser Phe Ser Trp GAG AGC Asp Ser 330 GCT CTG AGG GGG Pro Leu Ser Gly GTG AGG AGT GAG Val Arg Ser Glu ACC AAT TCC ACG Thr Asn Ser Thr ACC CTG AGC CCT Thr Leu Ser Pro AGT TTT GAG AAC GAA CAC TGT TAT GTG Ser Phe Giu Asn Giu His Ser Tyr Leu 1008 1056 1104 1152 1200 1248 1296 1344 ACA GTG AGT TGT Thr Val Thr Gys GAT AAG AAA GTG His Lys Lys Leu AAG GGA ATC GAG Lys Giy Ile Gin GTG GAG Val Glu 375 CTC TAG TCA Leu Tyr Ser GTG AAT GGG Vai Asn Gly 395 CCT AGA GAT CCA Pro Arg Asp Pro ATG GAG ATG AGT Ile Giu Met Ser GGT GGC CTC Gly Gly Leu 390 AGG GTG TAG Ser Vai Tyr AGG TGT GTG ACT Ser Ser Vai Thr AGG TGG AAG GTT Ser Gys Lys Vai CCC CTT Pro Leu 410 GAC CGG GTG GAG Asp Arg Leu Glu GAA TTA GTT AAG Glu Leu Leu Lys

GGG

Gly 420 GAG AGT ATT GTG Glu Thr Ile Leu

GAG

Glu 425 AAT ATA GAG TTT Asn Ile Giu Phe GAG GAT AGG GAT Glu Asp Thr Asp AAA TGT GTA GAG Lys Ser Leu Glu 1392 1440 AAA AGT TTG GAA Lys Ser Leu Glu ACC TTG ATC CCT Thr Phe Ile Pro

ACC

Thr 450 ATT GAA GAT ACT Ile Giu Asp Thr GGA AAA Gly Lys 455 GCT GTT GTT Ala Leu Val CCC AAA CAA Pro Lys Gin 475 GAG GGT AAG TTA Gin Ala Lys Leu ATT GAT GAG ATG Ile Asp Asp Met GAA TTG GAA Glu Phe Glu 470 GTT GCC CCC Val Ala Pro 1488 AGG CAG AGT AGG CAA AGA GTT TAT GTG Arg Gin Ser Thr Gin Thr Leu Tyr Val 1536 AGA GAT Arg Asp 490 ACA ACC GTG TTG Thr Thr Val Leu AGG CGT TCC Ser Pro Ser TCC ATC Ser- Ile 500 GTG GAG GAA GGG Leu Giu Giu Gly 1584 sULrj Si-IEET TCT GTG AAT ATG Ser Val Asn Met TGC TTG AGC GAG Gys Leu Ser Gin TTT CGT GGT CG Phe Pro Ala Pro ATC CTG TGG AGO Ile Leu Trp Ser

AGG

Arg 525 GAG CTC CCT AAC Gin Leu Pro Asn

GG

Gly 530 GAG CTA GAG CCT CTT TCT Giu Leu Gin Pro Leu Ser 535 1632 1680 1728 GAG AAT GCA Giu Asn Ala GTT TAT TTA Val Tyr Leu 555 CTO ACC TTA ATT Leu Thr Leu Ile ACA AAA ATG GAA Thr Lys Met Giu GAT TGT GGG Asp Ser Gly 550 TGT GAA GGA ATT Gys Glu Gly Ile AAO GAG GGT GGA AGA AGG AGA AAG GAA Asn Gin Ala Gly Arg Ser Arg Lys Giu 560 565 1776 GTG GAA Vai Giu 570 TTA ATT ATG CAA Leu Ile Ile Gin AGT GGA AAA GAG Thr Pro Lys Asp AAA GTT AGA GG* Lys Leu Thr Ala 1824 GGT TGT GAG AGT Pro Ser Giu Ser AAA GAA GGA GAG AGT GTG ATG ATG TGT Lys Giu Oiy Asp Thr Val Ile Ile Ser 595 il '~i AGA TOT GGA AAT Thr Gys Gly Asn GGA OAA AGA TGG Pro Giu Thr Trp ATG GTG AA. AAA Ile Leu Lys Lys AAA GG Lys Ala 615 GAG AGA GGA Giu Thr Gly OGA AAG 000 Arg Lys Ala 635 AGA OTA GTA AAA Thr Val Leu Lys ATA OAT 000 000 Ile Asp Giy Ala TAT AGO ATO Tyr Thr Ile 630 1872 1920 1968 16 2064 GAG TTG NAG CAT Gin Leu Lys Asp 000 66~A GTA TAT OAA TOT GNA TOT AAA Ala Oly Val Tyr Giu Gys Giu Ser Lys 640 645 AG AAA OTT GCG TOA CAA Asn Lys Val Oly Ser Gin AGA ACT TTA ACA Arg Ser Leu Thr OAT OTT CAA OGA Asp Val Gin Oly OAA AAG AG AAA Oiu Asn Asn Lys TAT TTT TOT GOT GAG OTT OTO OTO OTO Tyr Phe Ser Pro Giu Leu Leu Val Leu TTT GA TOG TOG Phe Ala Ser Ser ATA ATA GOT 000 Ile Ile Pro Ala OGA ATO ATA ATT Gly Met Ile Ile TAG TTT Tyr Phe 695 2112 2160 2208 OCA AGA AAA Ala Ar& Lys AG ATO NAG 000 Asn Met Lys Oly TAT AGT-GTT CTA Tyr Ser Leu Val GAA GA CAO Oiu Ala Gin 710 SUBSTITUTE

SHEET

-67- AAA TCA AAA GTG Lys Ser Lys Val 715 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 740 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 2220 Met -24 Ile Ser Thr S er Thr Al a Tyr Pro 105 Pro Lys (xi) Pro Gl) Met Phe Arg Tyi Thr G1 Pro Lex Met Asi Thr Cy~ Trp Th: Val Cl1 Arg Al Arg GlI SEQUENCE DESCRIPI Lys Met Val Val -20 -Ala Ala Ser Gin *Leu Ala Gin Ile 15 SCys Glu Ser Pro 30 i Asn Cly Lys Val i Pro Val Ser Phe C lii Ser Arg Lys r Pro Clii Arg Val 95 y Lys Asn Leu Thr 110 a Asn Leu Thr Val 125 ui Pro Ala Val Gly 140 ~ION: SEQ ID NO:17: Ile Leu Cly Ala Ser -15 Ala Phe Lys Ile Giu 1

G

1 y Asp Ser Val Ser Phe Phe Sur Trp Arg 35 Thr Asn Ciu Cly Thr 50 Cly Asn Ciu His Ser 65 Leu Giu Lys Gly Ile Clu Leu Ala Pro Leu 100 Leu Arg Cys Gin Val 115 Val Leu Leu Arg Cly 130 Ciu Pro Ala Ciu Val 145 Ile Thr Thr Gin Ser Leu Val Ser Gly Lys Thr 150 Leu Pro Cys Ile Thr Cys Clu Trp C ly Clu 135 Thr Trp Clu Ser Asp Leu Thr I le Gin Ala 120 Leu Val Leu Val Arg 155 Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Ciu SUBSTITUTE SHEET 1 I -68- Leu Pro 185 Val Ser Leu Phe Gin 265 Leu dly Ser Asp Leu 345 Thr Leu Val Pro Asp Leu 170 Tyr Gin Ser Pro Leu Asp Gly Asp 235 Ser Ala 250 Arg Leu Gin Thr Pro Arg Val Met 315 Ser Pro 330 Thr Leu Val Thr Tyr Ser Asn Gly 395 Leu Asp 410 Arg Pro Gin Gly 175 Leu Gin Thr Phe 190 Arg Val Leu Glu 205 Gly Leu Phe Pro 220 Gin Arg Leu Asn Lys Ala Ser Val 255 Thr Cys Ala Val 270 Val Thr Ile Tyr 285 Ile Ala Ala Gin 300 Gly Cys Giu Ser Leu Ser Gly Lys 335 Ser Pro Val Ser 350 Cys Gly His Lys 365 Phe Pro Arg Asp 380 Ser Ser Val Thr Arg Leu Glu Ile 415 Leu Glu Leu Phe Val Val Val Pro 240 S er Ile S er Ile Pro 320 Val Phe Lys Pro Val 400 Glu Leu Asp Ser 225 Thr Val Leu Phe G ly 305 Ser Arg Giu Leu Glu 385 Ser Leu Ala 195 Gin Ala Thr Ala Asn 275 Al a Ser Ser Glut Gilt 355 Lys Giu Lys Lys Glu Asn 180 Thr Pro Gly Thr Gin Val Tyr Gly 245 Glu Asp 260 Gin Ser Pro Giu Val Met Trp Arg 325 Gly Thr 340 His Ser Gly Ile Met Ser Val Pro 405 Gly Giu 420 Thr Pro Val1 His 230 Asn Glu Gin Ile Leu 310 Thr Asn Tyr Gin Gly 390 Ser Thr Glu Asn Ile Giu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu~ Glu Asn 425 430 435' 440 SVBSTITU1 E SHEE p..

I P CC C Ct~~ C -69- Lys Ser Leu Glu Met Thr Phe Ile Pro 445 Al a Pro Arg S er 505 Ile G iu Val Vai Phe 585 Thr Giu Arg Asn Arg 665 Phe Ala Val Gin 475 Thr Val Trp Aia Leu 555 Leu S er Gly G ly Al a 635 Vai Asn Ser Lys Gys 460 Arg Asn S er Thr 540 Cys Ile C iu Asn Asp 620 Gin G ly Asn Ser Ala 700 Gin Gin Val Met Arg 525 Leu Giu Ile Ser Vai 605 Thr Leu Ser Lys Leu 685 Asn Lys Thr Val 495 Gys Leu Leu Ile Val 575 Lys Giu Leu Asp Leu 655 Tyr Ile Lys Leu Gin 480 Ser Leu Pro Ile Asn 560 Thr Glu Thr Lys Al a 640 Arg Phe Pro G ly Thr Ile Giu 450 Ile Asp Asp Leu Tyr Vai Ser Ser Ile 500 Gin Giy Phe 515 Giy Glu Leu 530 Thr Lys Met Ala Gly Arg Lys Asp Ile 580 Asp Thr Val 595 Ile Ile Leu 610 Ile Asp Giy Val Tyr Giu Leu Thr Leu 660 Pro Giu Leu 675 Ile Gly Met 690 Tyr Ser Leu Asp Met Asn 485 Leu Pro Gin Glu Ser 565 Lys Ile Lys Ala Gys 645 Asp Leu Ile Val G iy 455 Phe Ala Glu Pro Leu 535 Ser Lys Thr Ser Lys 615 Thr S er Gin Leu Tyr 695 Ala Lys Giu Pro Gly Lys 520 Ser Gly Giu Al a Gys 600 Al a Ile Lys Gly Tyr 680 Phe Gin S UBS5T I T UTE SHENIET I it Lys Ser Lys Val 715 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 1929 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1929 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 73..1929 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

I

1 1( ATG CCT GGG AAG ATG GTC GTG ATC CTT Met Pro Gly Lys Met Val Val Ile Leu -24 -20 GCC TCA AAT ATA Ala Ser Asn Ile CTT TGG Leu Trp ATA ATG TTT Ile Met Phe GCA GCT TCT CAA GCT TTT Ala Ala Ser Gln Ala Phe 1 AAA ATC GAG ACC ACC CCA GAA Lys Ile Glu Thr Thr Pro Glu TCT AGA Ser Arg TAT CTT CTG CCC'CGG GGA GGC TCC GTG CTG GTG ACA TGC AC' Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser ACC TCC TGT GAC CAG Thr Ser Cys Asp Gln AAG TTG TTG Lys Leu Leu GGC ATA Gly Ile AAC CGG Asn Arg GAG ACC CCG TTG Glu Thr Pro Leu AAA AAG GAG TTG Lys Lys Glu Leu CTG CCT GGG AAC Leu Pro Gly Asn AAG GTG Lys Val TAT GAA CTG 240 Tyr Glu Leu AAC TGC CCT 288 Asn Cys Pro AGC AAT GTG CAA GAA GAT AGC Ser Asn Val Gin Glu Asp Ser CAA CCA Gin Pro ATG TGC TAT TCA Met Cys Tyr Ser suesu(s T I I I -71- CAT CCC CAG TCA ACA OCT AAA Asp Cly Gin Ser Thr Ala Lys TTC CTC ACC CTG Phe Leu Thr Val TCO ACT CCT Trp Thr Pro A-AC CAT Lys Asp CCA GAO ATT CAT Pro Ciu Ile His ACT CCC CCT CTG Ser Cly Pro Leu CCT CCC AAO CCC Ala Cly Lys Pro ATC ACA OTC AAC TOT TCA OTT OCT CAT CTA Ile Thr Val Lys Cys Ser Val Ala Asp Val 105 110 GAO ATA GAO TTA CTC AAA OCA CAT CAT CTC Clu Ile Asp Leu Leu Lys Gly Asp His Leu 125 130 CCA TTT CAC AGO Pro Phe Asp Arg ATO AAO ACT CAG Met Lys Ser Gln OAA TTT Clu Phe 135 480

A

CTC GAO OAT Leu 01u Asp ACC TTT ACT Thr Phe Thr 155 GAC ACO AAO TCC CTG CAA ACC AAC ACT Asp Arg Lys Ser Leu Oiu Thr Lys Ser TTO CAA GTA Leu Oiu Val 150 OTT TOO CCA Val Cys Arg CCT OTO ATT GAG Pro Val Ile Glu ATT OCA AAA OTT Ile Oly Lys Val GCT AAA Ala Lys 170 TTA CAC ATT CAT OAA ATO OAT TCT OTO Leu His Ile Asp Olu Met Asp Ser Val ACA GTA AOO CAC Thr Val Arg Cmn OTA AAA OAA TTC Val Lys Oiu Leu CTC TAO ATA TCA Val Tyr Ile Ser AAO AAT ACA OTT Lys Asn Thr Val TOT OTC AAT OCA Ser Val Asn Pro ACA AAO OTO CAA Thr Lys Leu Gin COT CCC TOT OTO Otmy Oly Ser Val ACC ATO Thr Met 215 ACC TOT TOO Thr Cys Ser AAA TTA OAT Lys Leu Asp 235 GAG OCT OTA OCA Oiu Oly Leu Pro OCA GAG ATT TTO Pro Glu Ile Phe TOG AOT AAC Trp Ser Lys 230 OCA ACT OTO Ala Thr Leu 768 816 AAT CCC AAT OTA Asn Oly Asn Leu CAC OTT TOT OGA His Leu Ser Oly ACC TTA Thr Leu 250 ATT OCT ATO AGO Ile Ala Met Arg OAA OAT TOT OCA Ciu Asp Ser Oly TAT OTO TOT OAA Tyr Val Cys Glu CPA TTA ATT OTT 912 Oiu Leu Ile Val 280 OTT AAT TTO ATT Val Asn Leu Ile MAA MAC AGA MAA GAG OTO Lys Asn Arg Lys Glu-Val SU B ST 1" U TE S H- T I -72- CAA GCA TTC CCT Gin Ala Phe Pro GAT CCA CAA ATG Asp Pro Giu Ile ATG ACT OCT CCC CTC CTC Met Ser Cly Cly Leu Val 295 AAT GCC AGC Asn Cly Ser CTT GAG CCC Leu Asp Arg 315 CTC ACT GTA AGC Val Thr Val Ser AAG GTT CGT AC Lys Val Pro Ser CTG TAG CCC Val Tyr Pro 310 ATT CTG GAG Ile Leu Oiu CTG GAG ATT GAA Leu Ciu Ile Clii CTT AAC CCC GAG Leu Lys Oly Ciu 1008 1056 1104 1152 AAT ATA Asn Ile 330 GAG TTT TTG GAG Giu Phe Leu Giu

CAT

Asp 335 ACC CAT ATO AAA Thr Asp Met LYS GTA GAG AAC AAA Leu Giu Asn Lys TTG GAA ATG ACC Leu Giu Met Thr ATG OCT ACC ATT Ile-'Pro Thr Ile CAT ACT OGA AAA Asp Thr Gly Lys GTT CIT TGT GAG Leu Val Cys Gin AAC TTA CAT ATT Lys Leu His Ile GAG ATG CAA TTG Asp Met Giu Phe GAA CCC Giu Pro 375 AAA CAA AGG Lys Gin Arg OAT ACA ACC Asp Thr Thr 395 ACT ACG CAA ACA Ser Thr Gin Thr TAT GTC AAT OTT Tyr Val Asn Val CCC CCC AGA Ala Pro Arg 390 CAA CCC ACT Ciu Gly Ser 1200 1248 1296 1344 GTC TTG GIG AGC Val Leu Val Ser

CCT

Pro 400 TGG TCC ATG CG Ser Ser Ile Leu TGT CTG Ser Val 410 AAT ATO ACA TGG Asn Met Thr Gys AGC GAG CCC TTT Ser Gin Cly Phe GGT CCC AAA ATG Ala Pro Lys Ile ICC AGC AGO CG Trp Ser Arg Gin

GTC

Leu 430 CCI AACGGCG GAO GTA GAG CCI CIT TGT Pro Asn Cly Oiu Leu Gin Pro Leu Ser 1392 Ii *1

K

AAT GGA ACT GTG Asn Ala Thr Leu TTA ATT TGT ACA Leu Ile Ser Thr ATO GAA CAT TGT Met Giu Asp Ser CCC OTT Cly Val 455 1440 TAT TTA TCT Tyr Leu Gys CAA TTA AT Ciu Leu Ile 475

CAA

Oiu 460 GCA ATT AAG CAC Oly Ile Asn Gin GCA AGA AC AGA AAG GAA G Oly Arg Ser Arg Lys Ciu Val 470 GAG ATA AAA GTT ACA GCT TTT Asp Ile-Lys Leu Thr Ala Phe 485 1488 1536 ATC CAA OTT ACT Ile Gin Val Thr GCA AAA Pro Lys 480 SUBSTITUTE SNEMT 3 -73- CCT TCT Pro Ser 490 GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr GGA AAT GTT CCA Gly Asn Val Pro ACA TGG ATA ATC Thr Trp Ile Ile AAG AAA AAA GCG Lys Lys Lys Ala ACA GGA GAG ACA Thr Gly Asp Thr CTA AAA TCT ATA Leu Lys Ser Ile GGC GCC TAT ACC Gly Ala Tyr Thr ATC CGA Ile Arg 535 AAG GCC CAG Lys Ala Gin AAG GAT GCG GGA Lys Asp Ala Gly CAA TTA AGA AGT Gin Leu Arg Ser TAT GAA TGT GAA Tyr Glu Cys Glu TCT AAA AAC Ser Lys Asn 550 1584 1632 1680 1728 1776 1824 1872 1920 AAA GTT GGC TCA Lys Val Gly Ser 555 TTA ACA CTT GAT GTT CAA GGA AGA Leu Thr Leu Asp Val Gin Gly Arg GAA AAC Glu Asn 570 AAC AAA GAC TAT Asn Lys Asp Tyr TCT CCT GAG CTT Ser Pro Glu Leu GTG CTC TAT TTT Val Leu Tyr Phe TCC TCC TTA ATA Ser Ser Leu Ile

ATA

Ile 590 CCT GCC ATT GGA ATG ATA ATT TAC TTT Pro Ala Ile Gly Met Ile Ile Tyr Phe AGA AAA GCC AAC Arg Lys Ala Asn AAG GGG TCA TAT Lys Gly Ser Tyr CTT GTA GAA GCA Leu Val Glu Ala CAG AAA Gin Lys 615 i i ;i 1 TCA AAA GTG Ser Lys Val INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 643 amino acids TYPE: amino'acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:19: 1929 Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp -24 -20 -15 SUBSTITUTE

SHEET

-74- Ile Met Phe Ala Ala Ser Gin Ala Phe Lys Ile S er Thr Lys S er Asp Lys Ile 105 Glu Leu Thr Ala Al a 185 S er Thr Lys Arg S er Lys Asn Gly Asp Thr Ile Glu Phe Lys 170 Val Val Cys Leu Tyr Cys Glu Val Gin Pro Val Asp Asp Thr 155 Leu Lys Asn Ser Asp 235 Leu Asp Leu Gin S er Glu Lys Leu Ala 140 Pro His Glu Pro Ser 220 Asn Leu Gin Leu Glu Thr Ile Gys Leu 125 Asp Val Ile Leu S e r 205 Glu Gly Pro Pro 30 Leu Asp Al a His Ser 110 Lys Ar g Ile Asp Gin 190 Thr Gly Asn Arg 15 Lys Pro Ser Lys Leu 95 Val Gly Lys G lu Giu 175 V %l Lys Leu Leu Gly Gly Leu Leu Gly Asn Gin Pro 65 Thr Phe 80 Ser Gly Ala Asp Asp 'His Ser Leu 145 Asp Ile 160 Met Asp Tyr Ile Leu Gin Pro Ala 225 Gin His 240 S er G ly Asn 50 Met Leu Pro Val Leu 130 Glu G ly Ser Ser Giu 210 Pro Leu Val Ile Arv Gys Thr Leu Tyr 115 Met Thr Lys Val Pro 195 Gly Giu S er Glu Leu G lu Lys Tyr Val G lu 100 Pro Lys Lys Val Pro 180 Lys C ly Ile Gly Thr Thr Val Thr Thr Pro Val Tyr Ser Asn Tyr Trp Ala Gly Phe Asp Ser Gin Ser Leu 150 Leu Val 165 Thr Val Asn Thr Ser Val Phe Trp 230 Asn Ala 245 Pro Gys Leu Glu Gys Thr Lys Arg Giu 135 Giu Gys Ar g Val Thr 215 S er Thr Thr Leu Ile Ala Met Arg Met Giu Asp Ser Gly Ile Tyr Val Gys Glu SUBSTITUTE So=T Gly 265 Gin Asn Leu Asn S er 345 Leu Lys Asp S er Leu 425 Asn Tyr Giu Pro Cys 505 Thr Vai Asn Leu Ile Ala Phe Pro Gly Ser Ser 300 Asp Arg Leu 315 Ile Giu Phe 330 Leu Giu Met Val Gys Gin Gin Arg Gin 380 Thr Thr Vai 395 Val Asn Met 410 Trp Ser Arg Aia Thr TLeu Leu Cys Giu 460 Leu Ile Ile 475 Ser Giu Ser 490 Gly Asn Vai Giy Asp Thr Arg 285 Vai Giu Leu Thr Aia 365 S er Leu Thr Gin Thr 445 G iy Gin Val Pro Vai 525 G iy 270 Asp Thr Ile Giu Phe 350 Lys Thr Vai Gys Leu 430 Leu Ile Vai Lys G iu 51i0 Leu Pro Vai G iu Asp 335 Ile Leu Gin Ser Leu 415 Pro Ile Asn Thr Giu 495 Thr Lys Lys Asn Arg Lys Giu Giu Ser Leu 320 Thr Pro His Thr Pro 400 Ser As11 Ser Gin Pro 480 Gly Trp Ser G iu 290 Lys Lys Met Ile Asp 370 Tyr Ser G iy Giu Lys 450 G iy Asp Thr Ile Asp 530 275 Met Vai C ly Lys Giu 355 Asp Val Ile Phe Leu 435 Met Arg Ile Vai Leu 51i5 Giy Vai Ser Pro Giu S er 340 Asp Met Asn Leu Pro 420 Gin Giu S er Lys Ile 500 Lys Al a Giu C iy Ser Thr 325 Leu Thr Giu Val Giu 405 Ala Pro Asp Arg Leu 485 Ile Lys Tyr Leu G iy Val 31i0 Ile G iu Gly Phe Ala 390 Giu Pro Leu S er Lys 470 Thr S er Lys Thr Ile Leu 295 Tyr Leu Asn Lys Clii 375 Pro C iy Lys Ser Ciy 455 Giu Aia Gys Ala Ile 535 Vai 280 Val Pro Clii Lys Aia 360 Pro Arg Ser Ilie Ciu 440 Val Val Phe Thr Giu 520 Arg SUBSTITUTES

S{T

-76- Lys Ala Gin Lys Val Gly 555 Lys Asp Ala Gly Val Tyr Glu Cys Glu 545 Ser Lys Asn 550 Gin Gly Arg Ser Gin Leu Arg Leu Thr Leu Asp Glu Asn 570 Asn Lys Asp Tyr Phe 575 Ser Pro Glu Leu Val Leu Tyr Phe Ser Ser Leu Ile Ile Pro Ala Ile Gly 590 Lys Gly Ser Tyr Ser 610 Ile Ile Tyr Phe Arg Lys Ala Asn Leu Val Glu Ala Gin Lys 615 Ser Lys Val INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1932 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1932 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 73..1932 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..72 (xi) SEQUENCE DESCRIPTION: SEQ ID CCT GGG AAG ATG GTC Pro Gly Lys Met Val GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 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 Ile Met Phe Ala Ala Ser Gin Ala Phe Lys Ile Glu Thr Thr Pro Glu TCT AGA Ser Arg TAT CTT CTG CCC Tyr Leu Leu Pro CGG GGA GGC TCC GTG CTG GTG Arg Gly Gly Ser Val Leu Val 15 ACA TGC AGC Thr Cys Ser SUBSTITUTE

SHEET

p,~ -77- AGC TC Thr Ser TOT GAG GAG Cys Asp Gin AAG TTG TTG GGC Lys Leu Leu Gly GAG ACC CCG TTG Guu Thr Pro Leu AAA AAG GAG TTG Lys Lys 0Th Leu OTG GOT GGG AAO Leu Pro Gly Asn CC AAG GTG TAT Arg Lys Val Tyr GAA OTG Giu Leu 240 AGO AAM GTG Ser Asn Val CAA GAT AGO CAA Giu Asp Ser Gin ATG TGG TAT TOA Met 0 ys Tyr Ser AAG TGO GOT Asn Cys Pro TGG ACT OGA Trp Thr Pro GAT CGG GAG TGA ACA GOT AAA Asp Gly Gin Ser Thr Ala Lys TTO OTO AGO CTG Phe Leu Thr Val GAA CC Giu Arg GTG CPA OTG GA Val Giu Leu Ala OTO 000 TOT TGC Leu Pro Ser Trp OGA GTG GO AAC Pro Val Gly Lys OTT AGO OTA 000 Leu Thr Leu Arg GAG GTG GAG GGT Gin Val 011. Cly GGA 000 CCC GOG Ala Pro Arg Ala CTO AGO GTC GTG Leu Thr Val Val OTO OGT GGG GAG Leu Arg Giy Clii OTC ATG AAG AGT Leu Met Lys Ser GAG GAA Gin Clii 135 TTT OTO GAG Phe Leu Ciu CTA AGO TTT Val Thr Phe 155 GGA GAG AGO AC Ala Asp Arg Lys CG CPA AGO AC ACT TTC CA Leu Giu Thr Lys Ser Leu Ciu 150 ACT GOT GTO ATT Thr Pro Val Ile CAT ATT GGA AAA Asp Ile Gly Lys OTT OTT TC Leu Vai Gys OGA GOT Arg Ala 170 AAA TTA GAG ATT Lys Leu His Ile CPA ATG CAT TOT Clu Met Asp Ser 000 ACA CTA AGC Pro Thr Vai Arg GOT GTA AAA CPA TTG CPA GTO TAG ATA Ala Val Lys Giu Leu Gin Vai Tyr Ile 190 000 PAG PAT ACA Pro Lys Asn Thr ATT TOT GTG PAT Ile Ser Val Asn TCO ACA PAG OTG Ser Thr Lys Leu

CA

Gin 210 CPA GGT GCC TOT GTG AGO Glu Gly Cly Ser Val Thr ATG AGO TOT Met Thr Cys AGO GAG GOT OTA OCA GOT OCA GAG ATT Ser Glu*Giy Leu Pro Ala Pro-Glu Ile 225 TTO TCG ACT Phe Trp Ser 230 SUBSTITUTE SHEET L- -I~

I-

-78- AAG AAA TTA GAT AAT CCC AAT CTA GAG GAG CTT TCT GCA AAT CCA ACT Lys Lys Leu Asp Asn Gly Asn Leu Gin Hius Leu Ser Cly Asn Ala Thr 235 240 245 CTC ACG Leu Thr 250 TTA ATT CT ATC Leu Ile Ala Met ATC CAA CAT TGT Met Clu Asp Ser ATT TAT CTC TCT Ile Tyr Val Cys

CAA

Giu 265 GCA GTT AAT TTC Gly Val Asn Leu CCC AAA AAC AGA Cly Lys Asn Arg

AAA

Lys 275 GAG CTC CAA TTA Glu Val Ciu Leu CTT CAA CCA TTC Val Gin Ala Phe GTC AAT GCG AC Val Asn Gly Ser AGA CAT CCA GAA Arg Asp Pro Giu GAG ATG ACT CCT Ciu Met Ser Cly CCC CTC Gly Leu 295 960 1008 TECT CTC ACT CTA Ser Val Thr Val TCC AAC CTT CCT Cys Lys Val Pro ACC CTG TAG Ser Val Tyr 310 ACT ATT CTG Thr Ile Leu CCC CTT GAG Pro Leu Asp 315 CCC CTC GAG ATT CAA TTA CTT AAG CCC Arg Leu Ciu Ile Ciu Leu Leu Lys Gly 10 56 1104 GAG AAT Giu Asn 330 ATA GAG TTT TTC Ile Glu Phe Leu GAT AG CAT ATG Asp Thr Asp Met TCT CTA GAG A-AC Ser Leu Ciu Asn

A

x

V

I'

ACT TTG CAA ATG Ser Leu Giu Met TTC ATG CGT ACC Phe Ile Pro Thr CAA CAT ACT CGA Ciu Asp Thr Cly 1152 GCT CTT GTT TCT Ala Leu Val Cys CCT AAG TTA CAT Ala Lys Leu His CAT GAG ATC CAA Asp Asp Met Ciu TTC CAA Phe Ciu 375 1200 CCC AAA CAA Pro Lys Gin GAG ACT AG CAA Gin Ser Thr Gin CTT TAT CTC AAT Leu Tyr Val Asn GTT CCC CCC Val Ala Pro 390 GAG GAA GC Glu Giu Cly 1248 1296 AGA CAT ACA ACC GC TTC GC AC Arg Asp Thr Thr Val Leu Val Ser 395 400 CCT TGG TGC ATG Pro Ser Ser Ile

CTC

Leu 405 ACT TCT Ser Ser 410 GTG AAT ATC ACA Val Asn Met Thr TTG AC CGAG GC Leu Ser Gin Gly CCT CCT CCC AAA Pro Ala Pro Lys 1344 ATC GTG TCC ACC AGG Ile Leu Trp Ser Arg 425 GAG GTC Gin Leu 430 CCT MG C C AG-CTA CAG GGT Pro Asn Gly Glu Leu Gin Pro 435 GTT TCT Leu Ser 440 1392 SUBSTITUT Ff "-rT'

I

1 4 GAG AAT GOA ACT Glu Asn Ala Thr ACC TTA ATT TOT Thr Leu Ile Ser

ACA

Thr 450 AAA ATG OAA CAT TOT COO Lys Met Glu Asp Ser Gly 1440 1488 OTT TAT TTA Val Tyr Leu OTO OAA TTA Val Olu Leu 475 OAA OCA ATT AAO CAC CT OCA ACA ACC Clu Cly Ile Asn Cmn Ala Cly Arg Ser 465 AGA AAC OAA Arg Lys Olu 470 OTT ACA OCT Leu Thr Ala ATT ATO CAA OTT Ile Ile Cmn Val OCA AAA GAO ATA Pro Lys Asp Ile 1536 TTT COT Phe Pro 490 TOT GAG ACT OTO 8cr Olu Ser Val OAA OCA CAC ACT Clu Oly Asp Thr ATO ATO TOT TOT Ile Ile Ser Cys

ACA

Thr 505

GAG

Glu TOT OCA AAT OTT Cys Cly Asn Val ACA OCA CAC ACA Thr Cly Asp Thr 525 CAA ACA TOO ATA 0Th Thr Trp Ile CTO AAC AAA AAA Leu Lys Lys Lys 1584 1632 1680 OTA OTA AAA TOT Val Leu Lys Ser

ATA

Ile 530 OAT CCC CCC TAT Asp Cly Ala Tyr ACC ATO Thr Ile 535 CA AAO GCC Arg Lys Ala AAC AAA OTT Asn Lys Val 555 AGA CPA AAC Arg Oiu Asn 570 040 Gin 540 TIC AAC CAT GC Leu Lys Asp Ala OTA TAT CPA TOT Val Tyr Oiu Cys CPA TOT AAA Clu Ser Lys 550 OTT CAA OCA Vlal Cmn Oly 1728 1776 CCC TICA CPA TTA Oly Ser Cmn Leu ACT TTA ACA OTT Ser LeU Thr Leu PAC AAA GC Asn Lys Asp TTT TCT OCT GAG Phe Ser Pro Oiu CTC 010 OTO TAT Leu Val Leu Tyr -1824 OCA TOO TCC TTA Ala Ser Ser Leu ATA COT CCC ATT Ile Pro Ala Ile ATO ATA ATT TAC Met Ile Ile Tyr 1872 ~2Z OCA AGA AAA GCC Ala Arg Lys Ala PAC ATO Asn Met 605 AC COO TCA Lys Oly Ser ACT OTT OTA CA Ser Leu Val Ciu OCA CAC Ala Gin 615 1920 AAA TCA AA Lys Ser Lys 1932 SUO, I~ INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 644 amino acids i TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein is Ii

K

Met -24 Ile Ser Thr Lys Ser Asp Glu Asn 105 Leu Phe Val Arg (xi) SEQUENCE Pro Gly Lys Met -20 Met Phe Ala Ala Arg Tyr Leu Leu Ser Cys Asp Gin Lys Glu Leu Leu Asn Val Gin Glu 60 Gly Gin Ser Thr Arg Val Glu Leu Leu Thr Leu Arg Thr Val Val Leu 125 Leu Glu Asp Ala 140 Thr Phe Thr Pro 155 Ala Lys Leu His 170 Val Val Ile Ser Pro Pro 30 Leu Asp Ala Ala Cys 110 Leu Asp Val Ile Gin Arg 15 Lys Pro Ser Lys Pro 95 Gin Arg Arg Ile Asp 175 DESCRIPTION: SEQ ID NO:21: Ala Gly Leu Gly Gin Thr 80 Leu Val Gly Lys Glu 160 Glu Leu Gly -15 Phe Lys 1 Gly Ser Leu Gly Asn Asn 50 Pro Met 65 Phe Leu Pro Ser Glu Gly Glu Lys 130 Ser Leu 145 Asp Ile Met Asp lie Glu Val Leu lie Glu 35 Arg Lys Cys Tyr Thr Val Trp Gin 100 Gly Ala 115 Leu Met Glu Thr Gly Lys Ser Val 180 Ser Pro 195 Thr Val Thr Val Ser Tyr Pro Pro Lys Lys Val 165 Pro Lys Ala Ser Asn lie Leu Trp Thr Pro Thr Cys Pro Leu Tyr Glu Asn Cys Trp Thr Val Gly Arg Ala Ser Gin 135 Ser Leu 150 Leu Val Thr Val Asn Thr Glu Ser Pro Leu Pro Pro Lys Asn 120 Glu Glu Cys Arg Val 200 Gin Ala Val Lys Glu Leu Gin Val Tyr Ile SUBWsITUTE SHEET I -81- Ile Ser Val Asn Pro Ser Thr Lys Leu Met Lys Leu Giu 265 Val Val Pro Giu Lys 345 Ala Pro Arg S er Ile 425 Giu Thr Lys Thr 250 Gly Gin Asn Leu Asn 330 S er Leu Lys Asp S er 410 Leu Asn Gys Ser Ser 220 Leu Asp As.

235 Leu Ile Ala Vai Asn Leu Ala Phe Pro 285 Gly Ser Ser 300 Asp Arg Leu 315 Ile Glu Phe Leu Giu Met Val Cys Gin 365 Gin Arg Gin 380.

Thr Thr Val 395 Val Asn Met Trp Ser Arg Ala Thr Leu 445 Giu Gly Met Ile 270 Arg Val Giu Leu Thr 350 Ala S er Leu Thr Gin 430 Thr Gly As n Arg 255 Gly Asp Thr Ile Giu 335 Phe Lys Thr Val Gys 415 Leu Leu Leu Leu 240 Met Lys Pro VJal Giu 320 Asp Ile Leu Gin S er 400 Leu Pro Ile Pro 225 Gin Giu Asn Glu S er 305 Leu Thr Pro His Thr 385 Pro S er Asn S er Gin Giu Gly 210 Ala Pro Giu His Leu Ser Asp Ser Giy 260 Arg Lys Giu 275 Ile Giu Met 290 Gys Lys Val Leu Lys Gly Asp Met Lys 340 Thr Ile Glu 355 Ile Asp Asp 370 Leu Tyr Vai Ser Ser Ile Gin Gly Phe 420 Giy Glu Leu 435 Thr Lys Met 450 Gly Ile Gly 245 Ilc Val S er Pro G lu 325 Ser Asp Met Asn Leu 405 Pro Gin Glu S er Phe 230 Asn Tyr Giu G ly S er 310 Thr Leu Thr Giu Val 390 Giu Ala Pro Asp Val 215 Trp Ala Val Leu Gly 295 Val Ile Giu G ly Phe 375 Ala Giu Pro Leu Ser 455 Thr S er Thr Gys Ile 280 Leu Tyr Leu Asn Lys 360 Giu Pro Val Tyr Leu Gys 460 Giu Gly Ile Asn Gin Ala Gly Arg Ser Arg Lys Giu 470 SUBSTITUTE

SWEET

f rrr pc rr rr -82- Val Glu Leu lie lie Gin 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 490 495 500 Thr Cys Gly Asn Val Pro Glu Thr Trp lie lie Leu Lys Lys Lys Ala 505 510 515 520 Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile 525 530 535 Arg Lys Ala Gin Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys 540 545 550 i Asn Lys Val Gly Ser Gin Leu Arg Ser Leu Thr Leu Asp Val Gin Gly 555 560 565 Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr 570 575 580 Phe Ala Ser Ser Leu Ile lie Pro Ala lie Gly Met lie lie Tyr Phe 585 590 595 600 Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gin 605 610 615 Lys Ser Lys Val 620 S(2) INFORMATION FOR SEQ ID NO:22: i SEQUENCE CHARACTERISTICS: LENGTH: 1941 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS 1(B) LOCATION: 1..1941 m (ix) FEATURE: 1' NAME/KEY: mat_peptide LOCATION: 73..1941 (ix) FEATURE: S(A) NAME/KEY: sig_peptide LOCATION: 1..72 SUBSTITUTE SHEET rr rnr I' I' r -83- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATC CCT GGG AAC ATG Met Pro Cly Lys Met -24 -20 CTC GTC ATC CTT Val Val Ile Leu GGA CCC TCA AAT ATA CTT TGG Cly Ala Ser Asn Ile Leu Trp -15 ATA ATC TTT Ile Met Phe CCC CGG ATT Pro Arg Ile CCA CCT Ala Ala TCT CAA CCT TTT ACT GTT CAG ATC TCC CCT GGA Ser Gin Ala Phe Thr Val Clu Ile Ser Pro Cly GCT CCT CAC ATT GGA CAC TCA CTC ATC TTC ACA TCT ACT Ala Ala Gn Ile Cly Asp Ser Val Met Leu Thr Cys Ser ATG GGC TCT CAA Met Cly Cys Glu CCA TCT TTC TCC Pro Ser Phe Ser AGA ACC CAC ATA Arg Thr Gin Ile AGC CCT CTC AGC Ser Pro Leu Ser AAG GTG AGG AGT Lys Val Arg Ser GGG ACC AAT TCC Cly Thr Asn Ser ACC CTC Thr Leu ACC CTC ACC Thr Leu Ser GTG ACT TGT Val Thr Cys GTG ACT TTT GAG Val Ser Phe Glu CAA CAC TCT TAT Glu His Ser Tyr CTC TCC ACA Leu Cys Thr GTG GAG CTC Val Giu Leu GGA CAT AAG AAA Cly His Lys Lys CAA AAG CGA ATC Glu Lys Cly Ile TAC TCA Tyr Ser TTC CCT AAG CAT Phe Pro Lys Asp GAG ATT CAT TTC Glu Ile His Leu GGC CCT CTC GAG Gly Pro Leu Glu GGG AAG CCC ATC Gly Lys Pro Ile CTC AAG TCT TCA CTT CCT CAT CTA TAC Val Lys Cys Ser Val Ala Asp Val Tyr 115

I~

ma i TTT GAC AGG CTG Phe Asp Arg Leu ATA GAG TTA CTG Ile Asp Leu Leu GGA CAT CAT CTC Cly Asp His Leu ATC AAC Met Lys 135 AGT GAG CAA Ser Gin Glu AGT TTC GAA Ser Leu Glu 155 CTG GAG CAT CCA Leu Clu Asp Ala AGG AAG TCC CTC Arg Lys Ser Leu CAA ACC AAC Glu Thr Lys 150 GTA ACC TTT ACT Val Thr Phe Thr CCT CTC Pro Val 160 ATT GAG CAT ATT GGA Ile Glu Asp Ile Gly 165 AAA CTT Lys Val SUBSITUTE S1 EET i j

I

-84- CTT GTT Leu Val 170 TG COA OCT AAA TTA GAC ATT OAT OAA ATG GAT TCT OTO CCC Gys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro

ACA

Thr 185 GTA AGG GAG OCT Val Arg Gin Ala AAA GAA TTG CAA Lys Glu Leu Gin TAG ATA TCA COG Tyr Ile Ser Pro AAG 672 Lys 200 AAT ACA GTT ATT Asn Thr Val Ile GTG AAT GCA TCC AGA AAO OTG CAA GAA Val Asn Pro Ser Thr Lys Leu Gin Giu GT GGG Gly Oly 215 TGT GTG ACC Ser Val Thr ACC TOT TCG AGG Thr Cys Ser Ser AAA TTA OAT AAT Lys Leu Asp Asn GOT CTA OCA OCT Oly Leu Pro Ala TTG TOO ACT AAO Phe Trp Ser Lys 235 000 MAT CTA CG Oly Asn Leu Gin OCA GAG ATT Pro Olu Ile 230 OTT TOT OGA Leu Ser Gly TOT OOA ATT Ser Oly Ile MAT OCA Asn Ala 250 ACT GTC ACC TTA Thr Leu Thr Leu OCT ATO AGO ATO Ala Met Arg M4et GMA OAT Oiu Asp 260 OTO TOT GMA GOA Vai Gys Giu Oly MAT TTG ATT 000 Asn Leu Ile Oly MAC AGA AAM GAG Asn Arg Lys Giu GMA TTA ATT OTT Giu Leu Ile Val GA TTG OCT AGA Ala Phe Pro Arg GCA OMA ATG GAG Pro Oiu Ile Glu ATO ACT Met Ser 295 GOT OG GTG Gly Gly Leu AGCGOTO TAG Ser Val Tyr 315 MAT 000 AGO TCT Asn Gly Ser Ser ACT OTA AOG TOG Thr Vai Ser Gys MOC GTT OCT Lys Val Pro 310 MOG 000 GAO Lys Oly Oiu 1008 CCC GTT GAG CG Pro Leu As ,p rg GAO ATT OMA TTA Glu Ile Giu Leu 1056 ACT ATT Thr Ile 330 CTO GAO MAT ATA Leu Glu Asn Ile TTT TTO GAO OAT Phe Leu Oiu Asp OAT ATO AAA TCT Asp Met Lys Ser 1104 GAO G AMAA AGT Giu Asn Lys Ser GMA ATO AGO TTG ATC OCT ACC ATT GMA Glu Met Thr Phe Ile Pro Thr Ile Oiu 1152 ACT OGA AAA OCT Thr Oly Lys Ala OTT TOT CG Val Cys Gin OCT MOG TTA CAT ATT OAT Ala Lys Leu-His Ile Asp 370 GAG ATO Asp Met 375 1200 SUBSmul".SET GAA TTC CA Ciu Phe Glu CCC AAA Pro Lys 380 CPA ACC GAG Gin Arg Gin ACG CAA ACA GTT Thr Gin Thr Leu TAT CTC PAT Tyr Val Asn 390 1248 1296 GTT CCC CCC AGA CAT ACA ACC CTC TTC CTC ACC CGT TCC TCC ATC CTG Vai Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu 395 400 405 GAG CA Ciu Giu 410 CCC ACT TCT GTG Gly Ser Ser Vai ATC ACA TCC TTC Met Thr Cys Leu GAG CCC TTT CCT Gin Gly Phe Pro 1344

CCT

Ala 425 CCC AAA ATC CTC Pro Lys Ile Leu ACC ACC GAG CTC Ser Arg Gin Leu PAG CCC GAC CTA Asn Cly Clu Leu 1392 CCT CTT TCT GAG Pro Leu Ser Ciu

PAT

Asn 445 GCA ACT CTC ACC Ala Thr Leu Thr ATT TCT ACA AAA Ile Ser Thr Lys ATG CA Met Glu 455 1440 CAT TCT CCC Asp Ser Cly TAT TTA TGT CA Tyr Leu Cys Giu

GCA

Gly 465 ATT PAC GAG CCT Ile Asn Cmn Ala GCA AGA AC Cly Arg Ser 470 GAG ATA AAA Asp Ile Lys AGA PAG CPA CTC Arg Lys Gilu Val 475 CPA TTA ATT Ciu Leu Ile CPA CTT ACT CCA Gin Val Thr Pro 1488 1536 1584 CTT ACA Leu Thr 490 CCT TTT CCT TCT Ala Phe Pro Ser

GAG

C iu 495 ACT CTC AAA CA Ser Val Lys Ciu GAG ACT CTC ATC Asp Thr Val Ile TCT TCT ACA TCT Ser Cys Thr Cys PAT CTT CCA CA Asn Vai Pro Giu TCG ATA ATC CTC Trp Ile Ile Leu 1632 AAA AAA CC GAG Lys Lys Ala Ciu CCA GAG ACA CTA Ciy Asp Thr Vai AAA TCT ATA CAT Lys Ser Ile Asp CCC CC Cly Ala 535 1680 TAT ACC ATC Tyr Thr Ile CPA TCT AAA Gilt Ser Lys 555 AAC CCC GAG TTC' Lys Aia Gin Leu CAT CC GGA CTA Asp Ala Cly Vai TAT CPA TGT Tyr Giu Cys 550 ACA CTT CAT Thr Leu Asp 1728 n PAC AAA GTT GC Asn Lys Vai Gly TCA CPA TTA AGA ACT TTA Ser Gin Leu Arg Ser Leu 560 565 1776 GTT CA Val Gin 570 CCA AGA CPA PAC Giy Arg Ciu Asn PAC AAA GAG TAT Asn Lys Asp Tyr 575 TTT TCT Phe-Ser 580 CCT GAG CTT CTC Pro Ciu Leu Leu 1824 SUR STITUTE CMIEET

I

ii

UI

i i-~ r I F~ C

GTG

Val 585

ATT

Ile

GAA

Glu (2) -86- CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly 590 595 TAC TTT GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser 605 610 GCA CAG AAA TCA AAA GTG Ala Gin Lys Ser Lys Val 620 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 647 amino acids TYPE: amino acid TOPOLOGY: linear ATG ATA Met Ile 600 CTT GTA Leu Val 615 1872 1920 1941 (ii) MOLECULE TYPE: protein ii

I

Met -24 lle Pro Val Ser Thr Val Tyr Ala 105 (xi) SEQUENCE Pro Gly Lys Met -20 Met Phe Ala Ala Arg Ile Ala Ala Met Gly Cys Glu Pro Leu Ser Gly Leu Ser Pro Val Thr Cys Gly His 75 Ser Phe Pro Lys Gly Lys Pro Ile DESCRIPTION: SEQ ID Val Val Ile Leu Gly -15 Ser Gin Ala Phe Thr 1 Gin Ile Gly Asp Ser 15 Ser Pro Ser Phe Ser 30 Lys Val Arg Ser Glu 50 Ser Phe Glu Asn Glu 65 Lys Lys Leu Glu Lys Asp Pro Glu Ile His 95 Thr Val Lys Cys Ser 110 NO:23: Ala Ser Val Glu Val Met Trp Arg Gly Thr His Ser Gly Ile Leu Ser 100 Val Ala 115 Ile Ser Thr Gln Ser Leu Val Pro Val Leu Pro Cys Ile Thr Cys Glu Leu Tyr SUBSTITUTE SHEET r 1 i -i, 1 -3 11; -87- Phe Asp Arg Leu Giu Ile Asp Leu Leu Ser S er Leu Thr 185 Asn S er Phe Asn Tyr 265 Giu G ly Ser Thr Leu 345 Thr Giu Giu 155 Cys Arg Val Thr Ser 235 Thr Cys Ile Leu Tyr 315 Leu Asn Lys Phe 140 Val Arg Gin Ile Met 220 Lys Leu Giu Val Val 300 Pro Giu Lys Ala Pro 380 Asp Thr Leu 175 Lys Asn Ser Asp Ile 255 Asn Phe S er Arg Giu 335 C iu Cys Lys Giy Asp His 130 Arg Lys Ser Leu Ile Glu Asp Ile 165 Asp Giu Met Asp 180 Gin Val Tyr Ile 195 Thr Lys Leu Gin 210 Gly Leu Pro Ala Asn Leu Gin His 245 Arg Met Giu Asp 260 Gly Lys Asn Arg 275 Asp Pro Giu Ile 290 Thr Val Ser Cys Ile Giu Leu Leu 325 Giu Asp Thr Asp 340 Phe Ile Pro Thr 355 Lys Leu His Ile 370 Giu Phe Giu Lys Gin Arg Gin Ser Thr Gin Thr Leu 385 Tyr Val Asn 390 SUBSTITUTE SHEET -88- Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser 395 400 Giu Ala 425 Pro Asp Arg Leu Ile 505 Lys Tyr Giu Val Val 585 Ile Glii Gly 410 Pro Lys Leu Ser Ser Gly Lys Clii 475 Thr Ala 490 Ser Cys Lys Ala Thr Ile Ser Lys 555 Gin Gly 570 Leu Tyr Tyr Phe Ser Ile Giu Vai 460 Val Phe Thr Giu Arg 540 Asn Arg Phe Ala Ser Val Leu Trp 430 Asn Ala 445 Tyr Leu Glii Leu Pro Ser Cys Gly 510 Thr Gly 525 Lys Ala Lys Val Giu Asn Ala Ser 590 Arg Lys 605 Asn Met Thr 415 Ser Arg Gin Thr Leu Thr Gys Clii Gly 465 Ile Ile Gin 480 Glu Ser Val 495 Asn Vai Pro Asp Thr Vai Gin Leu Lys 545 Gly Ser Gin 560 Asn Lys Asp 575 Ser Leu Ile Aia Asn Met Cys Leu Leu 450 Ile Val Lys Giu Leu 530 Asp Leu Tyr Ile Lys 610 Leu Pro 435 Ile Asn Thr Giu Thr 515 Lys Ala Arg Phe Pro 595 Pro Ser 420 Asn Ser Gin Pro C iy 500 T rp S er Gly S er S er 580 Ala Ser 405 Gin Gly Thr Aila Lys 485 Asp Ile Ile Val L~iu 565 Pro Ile S er C ly Giu Lys Gly 470 Asp Thr Ile Asp Tyr 550 Thr Giu Giy Ile Phe Leu Met~ 455 Arg 11 r Vai Leu C iy 535 Giu Leu Leu Met Leu Pro Gin.

440 Giu Ser Lys Ile Lys 520 Ala Gys Asp Leu Ile 600

K.

Gly Ser Tyr Ser Leu Val Giu Aia Gin Lys 620 Ser Lys Val SUBSTITUTE

SHEET

,1

Claims (12)

1. A monoclonal antibody recognizing an epitope dependent on the fourth immunoglobulin-like domain I of VCAM-7D.

2. A monoclonal antibody as defined in Claim 1, which blocks Ramos adhesion to VCAMl-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: ED11.AG6 (ATCC HB 10962), GE4.BG5 (ATCC HB 10961), and GH12.AA12 (ATCC HB 10963).

4. A monoclonal antibody having the characteristics of ED11, Fab, Fab', 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', 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 Scharacteristics of GH12, Fab, Fab', 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 r WO 93/14220 90 PCT/US93/00031 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 ED11.AG6 (ATCC HB 10962), GE4.BG5 (ATCC HB 10961), and GH12.AA12 (ATCC HB 10963). A method for purifying VCAM-7D from a solution also containing VCAM-6D comprising contacting the solution with a substrate on which is immobilized a monoclonal antibody according to Claim 1.

11. A method for separating cells expressing VCAM-7D from cells expressing VCAM-6D comprising contacting a solution including cells expressing VCAM-7D with a substrate on which is immobilized an antibody according to Claim 1.

12. An in vitro or in vivo method for detecting the presence, in a biological sample or a mammal, of VCAM- 7D or cells expressing VCAM-7D, comprising contacting said biological sample with a detectably labeled antibody according to Claim 1. 30 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, r -44 WO 93/14220 PCT/US93/00031

91- or Fab, Fab', 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 IlL;i trtially inhibit in vivo said VCAM-7D-mediated cell-cell adhesion. 14. A therapeutic reagent comprising a monoclonal antibody selected from ED11.AG6 (ATCC I 10 HB 10962), GE4.BG5 (ATCC HB 10961), or GH12.AA12 (ATCC HB 10963), or Fab, Fab', 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 neavy chain and one light chain formed therefrom, in a pharmaceutically acceptable carrier. A diagnostic kit comprising as a reagent, a detectably labeled monoclonal antibody selected from ED11, GE4'or GH12; Fab, Fab', 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 complete instructions for use of said reagent according to the method of claim 12. I h