AU9340601A - A method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulins in therapy and in vivo diagnosis - Google Patents

Description

I

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Bristol-Myers Squibb Company and Mae Joanne Rosok Actual Inventor(s): Mae Joanne Rosok, Dale E Yelton Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: A METHOD FOR INHIBITING IMMUNOGLOBULIN-INDUCED TOXICITY RESULTING FROM THE USE OF IMMUNOGLOBULINS IN THERAPY AND IN VIVO DIAGNOSIS Our Ref: 657417 POF Code: 140109/140109, 261014 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): la A METHOD FOR INHIBITING IMMUNOGLOBULIN-INDUCED TOXICITY RESULTING FROM THE USE OF IMMUNOGLOBULINS IN THERAPY AND IN VIVO DIAGNOSIS The present application is a divisional application from Australian patent application number 39688/97, the entire disclosure of which is incorporated herein by reference.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

TECHNICAL FIELD OF THE INVENTION The present invention relates to methods for inhibiting or reducing immunoglobulin-induced toxicity resulting from therapy or in vivo diagnosis.

Specifically, in lieu of using unmodified antibodies or recombinant binding proteins for in vivo use. the invention provides the use of modified antibodies or recombinant binding proteins which have been structurally altered in the constant domain so that upon administration immunoglobulin-induced toxicity is reduced or inhibited.

BACKGROUND OF THE INVENTION Over the years investigators have attempted to harness the immune system for therapeutic use. Irmnunoglobulin (Ig) molecules which constitute an important part of the immune system are of great interest because they react with a diverse family of ligands, possess different effector functions and are of great biological importance. Despite its potential, a persistent problem with W. ancIetsped3968-dirvsionI doc immunoglobulin immunotherapy has been, among other problems, the toxic effect to normal cells of using antibodies which recognize both normal and diseased cells.

This problem is far-reaching because the majority of antibodies presently available recognize a target located on both normal and diseased cells (Slavin-Chiorini, et al., Int. J. Cancer 53: 97-103 (1993)).

The constant region can promote cell death through antibody dependent cell mediated cytotoxicity (ADCC) or by complement dependent cytotoxicirv (CDC).

Despite the deletion of portions of the constant region. particularly the CH- domain.

the antigen binding function can be retained Yelton. M. Scharf. Mutant monoclonal antibody with alterations in biological functions, J. Exp. Methods 156:1131-1148 (1982)).

Others have generated a CH:-deleted antibody (Mueller et al., Proc. Natl.. Acad. Sci.

USA 87: 5702-5705 (1990)). Their findings provide that the CH:-deleted antibody was cleared from the blood of tumor-bearing mice much faster than the corresponding intact antibody. Other in vivo findings also confirmed that a CH:deleted antibody, designated chl4.18DCH2, is a potentially useful reagent for radioinmmunodetection of human tumors because of its reduced immunogeniciv, increased targe: specificirv. and rapid clearance from circulation (Mueiler et al., Proc. Natl. Acad. Sci. USA 87: 5702-5705 (1990). G.J. Schreiber et al.. -'An Unmrcdified Anticarcinoma Antibody. BR96. Localizes to and Inhibits the Outgrowth of Human Tumors in Nude Mice," Cancer Res. 52: 3262-3266 (1992), reports the use of an IgGI class-switched variant of BR96 and fragments.

S.D. Gillies J.S. Wesolowski, ".Antigen Binding and Biological Activities of Engineered Mutant Chimeric Antibodies with Human Tumor Specificities," Hum.

Antibod. Hvbridomas 1: -17-54 (1990). report the use of fragments of chimeric antibodies to reduce toxicity. G.J. Weiner et al.. -The Role of T Cell Activation in Anti-CD3 X Antitumor Bispecific Antibody Therapy." J. mrmunol.

152: 2385-2392 (1994), report bispecific anti-CD3 x antitumor F(ab') 2 antibody fragment used for immune therapy.

Generally, whole antibody molecules are composed of two heavy and two light chains which are held together by covalent bonds (disulfide) and non-covalent interactions. Each chain contains a variable region and a constant region The variable regions at the amino termini of the two chains form the antigen binding region. The constant region of the H chain has three components or domains.

Occasionally, the first constant region domain (CHI) interacts with the C region of the L chain through hydrophobic interactions and generally a disulfide bond, depending on isotype. The next C region stretch is the hinge-acting disulfide bond stably introduced between two H chains. The second constant region domain (CH 2 is adjacent to the hinge region. CH 2 contains sequences important for effector functions of the antibody, such as the sequences responsible for complement fixation, and Fc receptor binding The third constant region domain (CH 3 is located at the carboxyl terminus of the H chain, and is considered to play an important role in H chain assembly as well as some C region finctions.

Today many antibodies in clinical trials are directed against tumor associated antigens. Most tumor associated antigens are not tumor specific but are also generally found on the cell surface of some normal, non-tumorigenic cells. The clinical use of some antibodies directed against tumor associated antigens are limited 20 because of the toxicity associated with their use. Therefore, there is a need for methods for inhibiting toxicity associated with immunoglobulin use in the field of disease therapy therapy for tumors, kidney disease, and the likeyand in vivo diagnosis.

We addressed this need by discovering methods for inhibiting or reducing toxicity to normal cells generally associated with immunoglobulin immunotherapy *or in vivo diagnosis, wherein the immunoglobulin recognizes both diseased and normal cells.

Our discovery involves generating immunoglobulin molecules or Ig fusion proteins having structurally altered constant regions which inhibit or reduce immunoglobulin-induced toxicity.

SUMMARY OF THE INVENTION The present invention provides methods for inhibiting immunoglobulin-induced toxicity by using known imnmunoglobulin or Ig fusion protein molecules which are structurally altered in their constant regions so that the resulting structurally altered immunoglobulin or Ig fusion protein molecules exhibit reduced or inhibited toxicity in vivo compared to their original unmodified counterparts.

Structural alteration of the constant region may be effected in a number of ways as long as it results in reducing or inhibiting immunoglobulin-induced toxicitv.

In accordance with the practice of one embodiment of the invention, structural alteration of the constant region is effected by deletion of the entire constant region.

In another embodiment, only the CH: domain is deleted. In another embodiment, only that portion of the CH: domain that binds the Fc receotor is deleted. In vet another embodiment, only that portion of the CH: domain that binds the complement component CIq is deleted. Alternatively, in another embodiment, multiple deletions in discrete Fc receptor and complement component bining domains are effected.

2 -Alternatively, strucurali alteration is effected by sing!e or multiple mutations in the CH domain such as amino acid insentions and substitutions. The mutation or mutations must result in inhibiting imnmrrnogiobuiin-induced toxiciy. By w-a of exampie. the amino acids in multiple toxicity associated domains in the constant region can be altered so as to render the constant region unable to mediate a DCC response or activate complement thereby inhibiting immunog!obuiin induced toxicity resulting from immunotherapy. Alternatively, multiple amino acids in a single toxicity associated domain in the constant region can be altered.

S. *S Further alternatively, structurai alteration can be effected by isotype switcring resulting in an altered immunoglobulin molecule that either does not induce toxicity or induces some limited toxicity but does not cause a harmful effect. For example.

isotype switching can result in the constant region being unable to mediate a CDC or ADCC response or some other activity which mediates toxicity.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a line graph showing plasma clearance in high Ley expressing dogs using chimeric BR96 versus constant region mutant ofcBR96-2.

Figure 2 is a schematic diagram of a plasmid designated pTWD-cJVK.Ll including the chimeric (c)BR96-light chain (SEQ ID NO. 11).

Figure 3 is a schematic diagram of a plasmid designated pD 16hJl.L1 including the human (h)BR96-light chain (SEQ ID NO. 13).

Figure 4 is a schematic diagram of a piasmid, designated pDl7-hJml4-dCH2.HI, of hBR96-2A human mutant BR96 having the HI, H2, and H3 mutations and the CH 2 deletion (PCT Application No. 95/305444, published March 6, 1996)).

Figure 5 is a schematic diagram of a plasmid, designated pDi7-cJ-dCH2.HL. of cBR96-A (SEQ ID NO. 10) chimeric BR96 having the CH 2 deletion (PCT 20 Application No. 95/305444, published March 6, 1996)).

Figure 6 is a schematic diagram of a plasmid, designated pD 7-cJ.H of cBR96.

Figure 7 is a line graph showing the results of an ELISA assay of hBR96-2A- Dox to Le y (closed diamond), hBR96-2A to Le y (96:0006A2 R/A)(closed square), hBR96-2A to Le y (96:0006B R/A)(closed triangle), and BR96-Dox to Le S Figure 8 is a line graph showing the results of an ELISA assay of(1) BR96-A-Dox 30 to Le y (closed diamond), chiBR96 to Le y (closed square), cBR96-A to Le y (96:0003 R/A)(closed triangle), and cBR96-Dox to Le y Figures 9a-c are schematic diagrams showing the steps for deleting a CH 2 domain.

Figures 10a-c are schematic diagrams showing the construction of BR96 IgGI CH, Uomain point mutations.

Figure 11 is a schematic diagram showing the construction of the pNg 1/14 vector.

Figure 12 is a schematic diagram showing the construction of pD7-hBR96-2.

Figure 13 is a schematic diagram showing the construction of oDl7-hJmi4dCH2.Hl.

Figures I4A-J are the nucleic acid sequence of pDl7-cJ-dCH-2.HI, the plasmid shown in Figure 5. chimeric BR96 having the CH. deletion.

Fiure 15 is a line graph showing the results of an ELISA assay comparing whole chiBR96 and deleted CH, chiBR96 on Ley.

Figure 16 is a description of the seven structural alterations.

Figure 17 is a schematic diagram of a plasmid designated pD I 7-hG i b.

Figures 18 A-F are the nucleic acid sequence of pDI7-himi4.HI.

Figures 19 A-N are the nucleic acid sequence of pD 17-hGlb.

Figure 20 is a line graph showing complement dependent cytotoxicit. In :he legend, the closed square is hBR96-1; closed diamond is hBR96-2B: closed circie is hBR96-2C; closed triangle is hBR96-2D; open square is hBR96-2H: open circle is hBR96-2A and open triangle is 2B8, anti-Pseudonomas aeruginosa flagella type b mAb, negative control.

Figure 21 is a line graph showing antibody dependent cell-mediated cvtotoxitv. In the legend, the closed square is hBR96-1; closed diamond is hBR96-2B; closed circle is hBR96-2C; closed triangle is hBR96-2D; open square is hBR96-2H; open circle is hBR96-2A and open triangle is 2B8, anti-Pseudonomas aeruginosa flagella type b monoclonal antibody (mAb), negative control.

Figure 22 is a line graph showing binding activity of hBR96-2 constant region mutants on LeY-HSA. In the legend, the solid diamond is hBR96-1; solid square is hBR96-2A (CH2 deletion); solid triangle is hBR96-2B (235, 237 mutations): open square is hBR96-2C (318, 320, 322 mutations); open circle is hBR96-2D (331 mutation); and open triangle is hBR96-2H (235, 237, 318, 320, 322, 331 mutations).

Figure 23 is a line graph showing binding activity of hBR96-2 constant region mutants on LNFPIII-BSA. LNFPIII is a lacto-N-fucopentasose, a Lewis X trisaccharide with an additional lactose spacer (V Labs, Covington, LA). In the legend, the solid diamond is hBR96-1; solid square is hBR96-2A (CH2 deletion); solid triangle is hBR96-2B (235, 237 mutations); open square is hBR96-2C (318, 320, 322 mutations); open circle is hBR96-2D (331 mutation); and open triangle is 20 hBR96-2H (235. 237, 318, 320, 322, 331 mutations).

Figures 24A and 24B provide a strategy for introducing multiple mutations by RPCR. Diagram of he 1.4 kpb IgG heavy chain region showing the hinge CH 2 and CH 3 domains as boxed regions. Site-specific mutations to be introduced into

CH

2 positions Ll, L2, and L3 are encoded by complementary sets of mutant PCR primers (Al and A2; B1 and B2; and Cl and C2). The asterisks indicate the number of amino acid changes introduced at each L position. The two PCR primers, Rs (Recombination -sense) and Ra (Recombination-antisense), flank the Eco-47- III restriction sites and mediate homologous recombination with vector ends. The 3' ends of the oligonucleotides are represented by arrowheads. A three-way homologous recombination event between fragments RsA2, AIRa and the linearized vector produces the L1 mutant IgG. Two distally located sets of mutations (LI and L2) are simultaneously introduced by increasing the number of recombining PCR produces as is shown in the four-way recombination of RsA2, ALB2,. BIRa with vector.

Figure 25 is a gel showing Eco-47-HI restriction endonuclease analysis of DNAs prepared from colonies generated by multiple PCR fragment RPCR. Lane ikb ladder DNA marker (GIBCO/BRL Life Science Technology). Lanes 1-12: Twe!ve randomly selected colonies resulting from quadruple homologous recombination events were used to prepare plasmid and digested with Eco47-1I1. Clones 1. 2. 6 and 9 contain the fully assembled 1.4 kob insert.

Figure 26 provides the amino acid sequence for hBR96-2 heavy-chain variable region and the human IgGI constant region.

Figure '7 provides the amino acid sequence for hBR96-'A heavy-chain variable reion and the human g12G1 constant rezion from which the CH2 reaion has been deleted.

Figure 28 provides the arnino acid sequence for chi BR96 heavy-chain varabie re2ion and the humazn itG1 constant regicn without the CH: domain.

DETAILED DESCRIPTION OF THE INVENTION *ooS DEFINITIONS As used herein the term "inhibiting immunoglobulin-induced toxicity"' means to reduce or alleviate symptoms generally associated with toxicity caused by S, irmmunoglobulin or Ig fusion protein therapy, toxicity mediated by efector functions of the Fc receptor. For example. BR96 antibody recognizes and binds BR96 antigen which is found at some levels in the gastrointestinal tract and at elevated levels in tumors (as compared to the gastrointestinal tract of normal tissues). The binding of BR96 antibody to BR96 antigen in vivo causes symptoms associated with gastrointestinal toxicity. These symptoms include rapid onset of vomiting, often with blood, and nausea. In humans the bleeding is limited to the fundus of the stomach, causing erosion of the superficial mucosa of the stomach.

The pathology of the wound is limited and resolves. However, the extreme nature of the nausea and vomiting, unrelieved by anti-emetics, defines it as the dose-limiting toxicity. For highly elevated levels of other antigens found in the central nervous system (CNS), liver, and other locations, the toxicity will be characterized by symptoms other than those described above.

As used herein the term "immunoglobulin molecule" can be produced by B cells or be generated through recombinant engineering or chemical synthetic means.

Examples of immunoglobulin molecules include antibodies, polyclonal and monoclonal antibodies, chimeric or humanized, and recombinant Ig. containing binding proteins, Ig fusion proteins. Recombinant Ig containing binding proteins include cell surface proteins, CD antigens (in one embodiment, CTLA4), to which an Ig tail is joined.

As used herein the terms "structurally altered" or "structural alteration" means 20 manipulating the constant region so that the resulting molecule or protein exhibits a diminished ability to induce toxicity. Structural alteration can be by chemical modification. proteolytic alteration, or by recombinant genetic means. Recombinant genetic means may include, but is not limited to, the deletion, insertion and substitution of amino acid moieties.

As used herein the terms "multiple toxicity associated domains" means more than one discrete toxicity associated domain. As there appear to be at least two toxicity associated domains in the immunoglobulin molecule, one roughly localized to amino acids 231-238 and another roughly localized to amino acids 310-331, an example of the structural alteration of multiple toxicity associated domains comprises the insertion, substitution or deletion of amino acid residues in both of these domains.

This definition excludes structural alterations targeting a single toxicity associated domain.

Merely by way of example, the constant region of the immunoglobulin molecule can be structurally altered so that the molecule no longer mediates a CDC or ADCC response. However, the methods of the invention encompasses the use of structurally altered immunoglobulin molecules regardless of whether it mediates a CDC or ADCC response. The underlying requirement is that the altered molecule must inhibit immunoglobulin-induced toxicity.

Structural alteration can be effected in a number of ways. For example, structural alteration can be effected by deletion of the entire constant region.

Alternatively, structural alteration can be effected by deletion of the entire CH 2 domain of the constant region. In this instance, deletion of the entire CH 2 domain may render the molecule unable to bind an Fc receptor thereby eliminating the molecule's possibility of mediating antibody-dependent cellular cytotoxicity (ADCC), bind Clq, or activate complement.

Alternatively, structural alteration can be effected by deletion of only that portion of 20 the CH 2 domain that binds the Fc receptor or complement.

Further alternatively, a single mutation or multiple mutations such as substitutions and insertions in the CH 2 domain can be made. The underlying requirement of any mutation is that it must inhibit, diminish, or block immunoglobulin-induced toxicity. For example, this can be achieved by mutating the constant region such 0 that the altered molecule is rendered unable to mediate a CDC response or an ADCC response, or to activate complement.

Alternatively, structural alteration can be effected by isotype switching (also known as class switching) so that the altered molecule does not induce toxicity in the subject. In one embodiment, the constant region of the immunoglobulin is structurally altered so that it no longer binds the Fc receptor or a complement component, switching a molecule's original IgG isotype from IgGI to IgG4.

Isotype switching can be effected regardless of species, an isotype from a nonhuman being can be switched with an isotype from a human being Finkelman et al. (1990) Annu. Rev. Immunol. 8:303-333; T. Honjo et al. (1979) Cell 18: 559- 568; T. Honjo et al. In "Immunoglobulin Genes" pp. 124-149 Academic Press, London)).

As used herein the term "Ig fusion protein" means any recombinantly produced antigen or ligand binding domain having a constant region which can be structurally altered.

As used herein "cytotoxic agent" includes antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, and chemotherapeutic agents.

Specific examples within these groups include but are not limited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide.

tenoposide. vincristine, vinblastine, colchicine, supporin, gelonin, PE40, brvodin, dihydroxy anthracin dione, actinomycin D, and 1-dehvdrotestosterone.

*0*g As used herein the term "BR96" refers to the whole BR96 monocional antibody 20 disclosed in PCT No. 95/305444, published March 6. 1996, chimeric BR96 monoclonal antibody disclosed in PCT No. 95/305444, published March 6, 1996, or BR96 mutant molecules disclosed in PCT No. 95/305444, published March 6, 1996.

*00* As used herein, "treating" means to provide tumor regression so that the tumor is S"not palpable for a period of time (standard tumor measurement procedures may be followed Miller et al. "Reporting results of cancer treatment" Cancer 47:207- 214 (1981)); stabilize the disease; or provide any clinically beneficial effects.

As used herein, an "effective amount" is an amount of the antibody, immunoconjugate, or recombinant molecule which kills cells or inhibits the proliferation thereof.

As used herein, "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular or subcutaneous administration, or the implantation of a slow-release device such as a miniosmotic pump, to the subject.

As used herein, "pharmaceutically acceptable carrier" includes any material which when combined with the antibody retains the antibody's specificity or efficacy and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carrii-s such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets ard capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions .o comprising such carriers are formulated by well known conventional methods.

As used herein, "mutation" means a single amino acid or nucleic acid mutation or multiple mutations by.whatever means, homologous recombination, error prone PCR, or site directed mutagenesis.

In order that the invention herein described may be more fully understood, the following description is set forth.

METHODS OF THE PRESENT INVENTION The present invention provides a method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulin during therapy or in vivo diagnosis. For example, the methods of the invention would be useful to minimize the toxicity associated with prolonged clinical exposure to immunoglobulin use during or after tumor imaging with radiolabeled antibodies.

In accordance with the practice of this invention, the subject includes, but is not limited to, human, equine, porcine, bovine, murine, canine, feline, and avian subjects. Other warm blooded animals are also included in this invention.

This method comprises administering an immunoglobulin molecule to the subject.

The immunoglobulin can be IgG, IgM, or IgA. IgG is preferred.

In one embodiment of the invention, the immunoglobulin molecule recognizes and binds Le y In another embodiment, the immunoglobulin recognizes and binds Le".

In a further embodiment, the immunoglobulin is a monoclonal antibody BR96 produced by the hybridoma deposited on February 22, 1989 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 and accorded ATCC Accession No.: HB 10036. in vet another embodiment, the immunoglobulin is a chimeric antibody ChiBR96 produced by the hybridoma deposited on May 23, 1990, with the ATCC, 12301 Parklawn Drive, Rockville, MD 20852 and accorded ATCC Accession No.: IHB 10460.

In accordance with the practice of the invention, the immunoglobulin can be a bispecific antibody with a binding specificity for two different antigens, one of the antigens being that with which the monoclonal antibody BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC binds. Also, in accordance with the practice of the invention, the immunoglobulin can be an anti-idiotypic antibody.

As required by the invention, at least a portion of the constant region of the immunoglobulin molecule is structurally altered. Structural alteration can be effected by a number of means. In one embodiment, the entire constant region, i.e., CHI, CH 2 and CH 3 domains, can be deleted.

In another embodiment, only the CH 2 domain is deleted from the immunoglobulin molecule cBR96-A (Figure hBR96-2A (Figure In this embodiment, the CH 2 deletion may result in a molecule unable to bind the Fc receptor or a complement component.

In another embodiment, only that portion of the CH 2 domain which binds the complement component Clq is deleted. In yet another embodiment, mutations in specific portions of the CH 2 domain are made. For example, the immunoglobulin molecule may be modified by structurally altering multiple toxicity associated domains in the constant region so that immunoglobulin-induced toxicity is inhibited. A discussion of such mutations are further found hereinafter.

Regardless of the means, the underlying requirement for any structural alteration of the constant region is that immunoglobulin-induced toxicity is substantially reduced or inhibited. In one embodiment, immunoglobulin-induced toxicity is inhibited by structurally altering the constant region such that the molecule's ability to mediate a CDC response or ADCC response and/or activate the complement cascade is prevented or inhibited. Methods for determining whether the molecule is able to inhibit a CDC response are well known, one method involves a 20 test Garrigues et al. Int. J. Cancer 29:511 (1982); I. Hellstrom et al. PNAS 82:1499 (1985)). Methods for determining whether the molecule is able to inhibit an ADCC response are well known Hellstrom et al. PNAS 82:1499 (1985)).

Methods for determining whether the molecule is able to activate a complement cascade are well known.

In another embodiment of the invention, the method comprises administering to the subject an Ig fusion protein having a structurally altered constant region. Structural alteration of the constant region may include deletion of the entire C region or o*.o portions thereof, alteration of the CH 2 domain so that the altered molecule no longer binds the Fc receptor or a complement component.

The invention further provides a method for inhibiting immunoglobulin-induced toxicity resulting from immunotherapy in a subject. The method comprises administering to the subject an antibody which has been modified so that at least a portion of the constant region has been structurally altered as discussed supra. In one embodiment, the antibody recognizes and binds Le y In another embodiment, the antibody recognizes and binds to Le.

In accordance with the practice of this invention, the antibody can be monoclonal antibody BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC. Alternatively, the antibody can be chimeric antibody ChiBR96 produced by the hybridoma having the identifying characteristics of HB 10460 as deposited with the ATCC. Further, the antibody can be a bispecific antibody with a binding specificity for two different antigens, one of the antigens being that with which the monoclonal antibody BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with-the ATCC binds.

Additionally, the present invention provides a method for inhibiting immunoglobulin-induced toxicity resulting from immunotherapy for a disease in a subject. The disease will vary with the antigen sought to be bound. Examples of 20 diseases include but are not limited to immunological diseases, cancer, S cardiovascular diseases, neurological diseases, dermatological diseases or kidney disease.

This method comprises the following steps. Step one provides selecting an antibody for a target. Generally, the target is associated with the disease and the antibody directed to the target is known. For example, the target can be the BR96 antigen and the antibody selected is BR96.

Step two of this method provides structurally altering the constant region of the antibody so selected so that immunoglobulin induced toxicity is inhibited.

Inactivation can include any of the means discussed above. For example, inactivation can be effected by structurally altering multiple toxicity associated domains in the CH 2 domain of the constant region of the Ig protein so selected.

Step three of this method provides administering the structurally altered antibody of step two to the subject under conditions that the structurally altered antibody recognizes and binds the target and that such binding directly or indirectly alleviates symptoms associated with the disease.

In accordance with the invention, in one embodiment step one provides selecting an Ig fusion protein for a target. Further, the method provides mutating the Ig fusion protein so selected by structurally altering the CH 2 domain of the constant region of the Ig protein by the same means discussed above.

The invention further provides methods to treat human carcinoma. -For example, the immunoglobulin, antibody, or Ig fusion protein discussed above can be used in combination with standard or conventional treatment methods such as chemotherapy, radiation therapy or can be conjugated or linked to a therapeutic drug, or toxin, as well as to a lymphokine or a tumor-inhibitory growth factor, for delivery of the therapeutic agent to the site of the carcinoma.

20 Techniques for conjugating therapeutic agents to irmmunoglobulins are well known o (see, Arnon et al.. "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.

pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstr6m et al., "Antibodies For Drug

O

Delivery", in Controlled Drug Delivery (2nd Robinson et al. pp. 623-53 25 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In o Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.

Rev., 62:119-58 (1982)).

Alternatively, the structurally altered antibody or Ig fusion protein can be coupled to high-energy radiative agents, a radioisotope such as 1311; which, when localized at the tumor site, results in a killing of several cell diameters (see, Order, "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. pp. 303-16 (Academic Press 1985)).

According to yet another embodiment, the structurally altered BR96 antibody can be conjugated to a second antibody to form an antibody heteroconjugate for the treatment of tumor cells as described by Segal in United States Patent 4,676,980.

Still other therapeutic applications for the structurally altered antibody or Ig fusion protein of the invention include conjugation or linkage, by recombinant DNA techniques or protein chemical techniques, to an enzyme capable of converting a prodrug into a cytotoxic drag and the use of that antibody-enzyme conjugate in combination with the prodrug to convert the prodrug to a cytotoxic agent at the tumor site (see, Senter et "Anti-Tumor Effects Of Antibody-alkaline Phosphatase", Proc. Natl. Acad. Sci. USA, 85:4842-46 (1988); "Enhancement of the in vitro and in vivo Antitumor Activities of Phosphorylated Mitomvcin C and Etoposide Derivatives by Monoclonai Antibody-Alkaline Phosphatase Conjugates", Cancer Research 49:5789-5792 (1989); and Senter. "Activation of Prodrugs by Antibody-Enzyme Conjugates: A New Approach to Cancer Therapy," FASEB J.

20 4:188-193(1990)).

It is apparent therefore that the present invention encompasses pharmaceutical compositions including immunoglobulin molecules, antibodies, and Ig fusion proteins all having structurally altered CH 2 domains, and their use in methods for treating human carcinomas. For example, the invention includes pharmaceutical compositions for use in the treatment of human carcinomas comprising a pharmaceutically effective amount of a structurally altered BR96 and a pharmaceutically acceptable carrier.

30 The compositions may contain the structurally altered antibody or Ig fusion protein or antibody fragments, either unmodified, conjugated to a therapeutic agent drug, toxin, enzyme or second antibody). The compositions may additionally include other antibodies or conjugates for treating carcinomas an antibody cocktail).

The compositions of the invention can be administered using conventional modes of administration including, but not limited to, intrathecal, intravenous, intraperitoneal, oral. intralymphatic or administration directly into the tumor. Intravenous administration is preferred.

The composition of the invention can be in a variety of dosage forms which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends upon the mode of administration and the therapeutic application.

The compositions of the invention also preferably include conventional pharmaceutically acceptable carriers and adjuvants known in the art such as human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate.

in accordance with the practice of the invention, the pharmaceutical carrier can be a lipid carrier. The lipid carrier can be a phospholipid. Further, the lipid carrier can be a fatty acid. Also, the lipid carrier can be a detergent. As used herein, a detergent is any substance that alters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent can be a nonionic detergent.

Examples of nonionic detergents include, but are not limited to, polysorbate 80 (also known as Tween® 80 or (polyoxyethylenesorbitan monooleate), Brij®, and Triton® (for example Triton® WR-1339 and Triton® 30 Alternatively, the detergent can be an ionic detergent. An example of an ionic detergent includes, but is not limited to, alkyltrimethylammonium bromide.

Additionally, in accordance with the invention, the lipid carrier can be a liposome.

As used in this application, a "liposome" is any membrane bound vesicle which contains any molecules of the invention or combinations thereof.

The most effective mode of administration and dosage regimen for the compositions of this invention depends upon the severity and course of the disease, the patient's health and response to treatment and the judgment of the treating physician.

The interrelationship of dosages for animals of various sizes and species and humans based on mg/m 2 of surface area is described by Freireich, et al. Cancer Chemother., Rep. 50 219-244 (1966). Adjustments in the dosage regimen can be made to optimize the tumor cell growth inhibiting and killing response,. e.g., doses can be divided and administered on a daily basis or the dose reduced proportionally depending upon the situation several divided doses can be administered daily or proportionally reduced depending on the specific therapeutic situation).

THE MOLECULES OF THE INVENTION 20 The present invention provides structurally altered BR96 or BR96 Ig fusion proteins.

Structurally altered BR96 antibodies or Ig fusion proteins have the variable region of BR96 and a modified constant region. This modification provides structurally altered BR96 antibodies or Ig fusion proteins with the ability to inhibit immunoglobulin-induced toxicity.

Various embodiments of structurally altered BR96 or BR96 Ig fusion proteins have S"been made.

In one embodiment, designated cBR96-A, the entire CH 2 domain of cBR96 was 30 deleted. CBR96-A is expressed by the plasmid having the sequence shown in SEQ.

ID. NO. 10. cBR96 is expressed by a plasmid having the sequence in SEQ ID NO.

9.

In another embodiment, designated hBR96-2A, the entire CH 2 domain of hBR96 was deleted. hBR96-A is expressed by the plasmid having the sequence shown in SEQ. ID. NO. 12. hBR96 is a mutant BR96 having the HI, H2, and H3 mutations described in PCT Application No. 95/305444, published March 6, 1996.

In yet another embodiment, designated hBR96-2B, the leucine residue located at amino acid position 235 is mutated to alanine. Additionally, the glycine residue located at amino acid position 237 is mutated to alanine. The amino acid position numbering used is described in Kabat et al. Sequences of Proteins of Immunological Interest 5th Edition (1991) United States Department of Health and Human Services.

In a further embodiment, designated hBR96-2C, the glutamic acid residue at position 318 is mutated to serine; the lysine residue located at position 320 is mutated to serine; and the lysine residue located at position 322 is mutated to serine using standard protocols (Alexander R. Duncan and Greg Winter "The binding site for Clq on IgG" Nature 332:738 (1988)).

In another embodiment, designated hBR96-2D, the proline residue at position 331 is 0 mutated to alanine Tao et al., "Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation" J. Exp.

Ii Med. 178:661-667 (1993); Y. Xu et al., "Residue at position 331 in the IgGI and IgG4 domains contributes to their differential ability to bind and activate complement" J. Biol. Chem. 269:3469-3474 (1994)).

In an additional embodiment, designated hBR96-2E, the leucine residue at position 235 is mutated to alanine; the glycine residue located at position 237 is mutated to alanine; the glutamic acid residue located at position 318 is mutated to serine; the lysine residue located at position 320 is mutated to serine; and the Ivsine residue 30 located at position 322 is mutated to serine Morgan et al., "The N-terminal end of the CH 2 domain of chimeric human IgGI anti-HLA-DR is necessary for Clq, Fc(gamma)RI and Fc(gamma)RIII binding" Immunol. 86:319-324 (1995)).

In yet a further embodiment, designated hBR96-2F, the leucine residue located at position 235 is mutated to alanine; the glycine residue located at position 237 is mutated to alanine; and the proline residue located at position 331 is mutated to alanine.

In yet another embodiment, designated hBR96-2G, the giutamic acid residue located at position 318 is mutated to serine; the lysine residue located at position 320 is mutated to serine; the lysine residue located at position 322 is mutated to serine; and the proline residue located at position 331 is mutated to alanine.

In another embodiment, designated hBR96-2H, the leucine residue located at position 235 is mutated to alanine; the glycine residue located at position 237 is mutated to alanine; the glutamic acid residue at position 318 is mutated to serine; the lysine residue located at position 320 is mutated to serine; the lysine residue located at position 322 is mutated to serine; and the proline residue located at position 331 is mutated to alanine.

Depending on its form. a structurally altered BR96 antibody or fusion protein can be a monofunctional antibody, such as a monoclonal antibody, or bifunctional antibody, such as a bispecific antibody or a heteroantibody. The uses of structurally altered BR96, as a therapeuti or diagnostic agent. will determine the different forms of structurally altered BR96 which is made.

S 25 Several options exists for antibody expression. Immunoexpression libraries can be combined with transfectoma technology, the genes for the Fab molecules 0 derived from the immunoglobulin gene expression library can be connected to the desired constant-domain exons. These recombinant genes can then be transfected and expressed in a transfectoma that would secrete an antibody molecule.

Once produced, the polypeptides of the invention can be modified, by amino acid modifications within the molecule, so as to produce derivative molecules. Such derivative molecules would retain the functional property of the polypeptide, namely, the molecule having such substitutions will still permit the binding of the polypeptide to the BR96 antigen or portions thereof.

It is a well-established principle of protein chemistry that certain amino acid substitutions, entitled "conservative amino acid substitutions," can frequently be made in a protein without altering either the conformation or the function of the protein.

Amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative".

Such changes include substituting any of isoleucine valine and leucine (L) for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa; giutamine for asparagine and vice versa: and serine (S) for threonine and vice versa.

Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine and alanine can frequently be interchangeable, as can alanine and valine .e Methionine which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in "particular environments.

30 In one embodiment of the present invention, the polypeptide is substantially pure, free of other amino acid residues which would inhibit or diminish binding of the polypeptide to its target and would inhibit or reduce gastrointestinal toxicity which are normally exhibited during or after antibody therapy.

NUCLEIC ACID MOLECULES ENCODING THE PRESENT INVENTION The nucleotide sequences and the amino acid sequences of the variable and constant regions of BR96 are known. The sequence for the immunoglobulin constant region is known and provided in Figure 18. Specific mutations in the constant region of the BR96 antibody were made. Nucleic acid molecules encoding the seven mutants described above (hBR96-2B through hBR96-2H) are as follows.

In hBR96-2B, alanine at amino acid positions 235 and 237 is encoded by codons GCU, GCC, GCA, or GCG.

In hBR96-2C, serine at positions 318, 320, and 322 is encoded by UCU, UCC, UCA. orUGG.

In hBR96-2D, alanine at position 331 is encoded by codons GCU, GCC, GCA, or

GCG.

in hBR96-2E, alanine at positions 235 and 237 is encoded by codons GCU, GCC, GCA, or GCG. Serine at positions 318, 320. and 322 is encoded by UCU, UCC.

UCA. or UGG.

In hBR96-2F, alanine at positions 235, 237, and 331 is encoded by codons GCU, GCC, GCA, or GCG.

In hBR96-2G, serine at positions 318, 320, 322 is encoded by UCU, UC UCUCA, or UGG. Further, the alanine at position 331 is encoded by codons GCU, GCC, GCA, or GCG.

In hBR96-2H, alanine at positions 235, 237, and 331 is encoded by codons GCU, GCC, GCA, or GCG. Additionally, serine at positions 318, 320, 322 is encoded by UCU, UCC, UCA, or UGG.

Any of the above can be deoxyribonucleic acid (DNA), complementary

DNA

(cDNA), or ribonucleic acid (RNA).

IMMUNOCONJUGATES

[mmunoconjugates (having whole antibody or Ig fusion proteins) may be constructed using a wide variety of chemotherapeutic agents such as folic acid and anthracyclines (Peterson et al., "Transport And Storage Of Anthracyclines In Experimental Systems And Human Leukemia", in Anthracycline Antibiotics In Cancer Therapy, Muggia et al. p. 132 (Martinus Nijhoff Publishers (1982); Smyth et al., "Specific Targeting of Chlorambucil to Tumors With the Use of Monoclonal Antibodies", J. Natl. Cancer Inst., 76:503-510 (1986)). including doxorubicin (DOX) (Yang and Reisfeld "Doxorubicin Conjugated with a Monoclonal Antibody Directed to a Human Melanoma-Associated Proteoglycan Suppresses Growth of Established Tumor xenografts in Nude Mice PNAS (USA)" 85:1189-1193 (1988)), Daunomycin (Arnon and Sela "In Vitro and in vivo Efficacy of Conjugates of Daunomycin With Anti-Tumor Antibodies" Inmunol. Rev., 65:5- 20 27 (1982)), and morpholinodoxorubicin (Mueller et al., "Antibody Conjugates With Morpholinodoxorubicin and Acid-Cleavable Linkers", Bioconjugate Chem., 1:325- 330 (1990)).

BR96 has been conjugated to doxorubicin and has been shown to be effective in therapy of certain cancers or carcinomas (Trail, Willner, Lasch, S.J., Henderson, Casazza, Firestone, Hellstr6m, and Hellstrom, K.E.

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Science, 261:212-215, 1993).

30 In accordance with the practice of the invention, structurally altered BR96 can be used in forms including unreduced IgG, reduced structurally altered IgG, and fusion proteins (PCT Application No. 95/305444, published March 6, 1996).

Suitable therapeutic agents for use in making the immunoconjugate includes Pseudomonas exotoxin A (PE) in either the native PE or LysPE40 form. LysPE40 is a truncated form containing a genetically modified amino terminus that includes a lysine residue for conjugation purposes. Doxorubicin is also a suitable therapeutic agent.

Additional examples of therapeutic agents include, but are not limited to.

antimetabolites, alkylating agents, anthracyclines, antibiotics, and anti-mitotic agents.

Antimetabolites include methotrexate, 6 -mercaptopurine, 6 -thioguanine, cytarabine, decarbazine.

Alklating agents include mechlorethamine, thiotepa chiorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cvclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichorodiamine platinum (II) (DDP) cisplatin.

Anthracyciines include daunorubicin (formerly daunomycin) and doxorubicin (also referred to herein as adria-iycin). Additional examples include mitozantrone and bisantrene.

Antibiotics include dactinomycin (formerly actinomycin), bleomycin. mithrarnvcin, and anthramycin

(AMC).

Antimitotic agents include vincristine and vinblastine (which are commonly referred to as vinca alkaloids).

30 Other cytotoxic agents include procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane interferons.

Further examples of cytotoxic agents include, but are not limited to, ricin, bryodin, gelonin, supporin, doxorubicin, taxol, cytochalasin B, gramicidin D, ethidium bromide, etoposide, tenoposide, colchicine, dihydroxy anthracin dione, 1dehydrotestosterone, and glucocorticoid.

Clearly analogs and homologs of such therapeutic and cytotoxic agents are encompassed by the present invention. For example, the chemotherapuetic agent aminopterin has a correlative improved analog namely methotrexate.

Further, the improved analog of doxorubicin is an Fe-chelate. Also, the improved analog for 1-methylnitrosourea is lomustine. Further, the improved analog of vinblastine is vincristine. Also, the improved analog ofmechlorethamine is cyclophosphamide.

METHODS FOR MAKING MOLECULES OF THE INVENTION There are multiple approaches to making site specific mutations in the CH 2 domain of an immunoglobulin molecule. One approach entails PCR amplification of the

CH

2 domain with the mutations followed by homologous recombination of the mutated CH 2 into the vector containing the desired immunoglobulin, hBR96-2.

For examnie, hBR96-2B and hBR96-2D have been made by this method.

Another approach would be to introduce mutations by site-directed mutagenesis of single-stranded DNA. For example, vector pD17-hGlb, which contains only the 25 constant region of IgG1 and not the V domain of hBR96, has the fl origin of replication. This gives the vector the properties of a phagemid and site-directed mutagenesis experiments can be performed according to the methods of Kunkel, et al. (Kunkel, J.D. Roberts, and R.A. Zakour, 1987 Methods Enzymol. 154:367- 383) as provided in the Bio-Rad Muta-Gene® phagemid in vitro mutagenesis kit, 30 version 2. For example, hBR96-2B, and -H were made by this method.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and arc not to be construed as limiting the scope of this invention in any manner.

EXAMPLE 1 The following standard ELISA protocol was used.

Materials: Immulon2 96 well plates and Genetic Systems Specimen Diluent Concentrate (1Ox); antibody conjugate was Goat Anti Human Kappa-HRP Mouse Adsorbed, Southern Biotech. at 1:10,000 in Genetic Systems Conjugate Diluent Genetic Systems EIA Chromogen Reagent (TMB) (1:100); Genetic Systems EIA Buffered Substrate primary antibody or antigen were AffiniPure F(ab')z Fragment Goat Anti Human IgG Fc Fragment specific (Jackson Irnuno Research), Goat Anti Human Kappa-UNLB (Southern Biotechnology Associates), Le-HSA (Alberta Research Council).

Methods: Dilute primary antibody or antigen to 1.0 Aig/ml in 0.05M Carb/Bicarb 20 buffer. Add 100 l of the diluted solution per well in immulon 2 plates. Seal plates and incubate O.N. at 4 0

C.

Block plates by flicking them and blotting on paper towels. Add 200ul/well of Genetic Systems, Specimen Diluent Concentrate Incubate at least 1 hour at room temperature and then dump the contents of the plates. Wash the plates 3x in saline/Tween. Blot to dry. Allow the plates to dry at R.T. (45 min. to 1 hour). Seal and store the plates at 4 0

C.

Test samples as follows. Dilute samples and standards in Specimen Diluent at 1:10.

30 Perform serial dilutions in separate round bottom plates. Transfer 100ul/well of final dilutions to antigen coated assay plates; then incubate O.N. at 4 0 C. Wash plates 3x with saline/Tween.

For conjugation add 100 l/well of antibody-HRP conjugate in Genetic Systems Conjugate Diluent Incubate plates at Rcom Temp. for 60 min. Wash plates 3x in saline/Tween.

Add 100 .l/well of Genetic Systems EIA Chromogen Reagent (TMB) 1:100 in EIA Buffered Substrate Incubate at R.T. for 15 min. and stop with IN H2SO 4 100 al/well. Read plate at 4 5 0 /630nm in EIA plate reader.

EXAMPLE 2 Construction of CH 2 deleted BR96 molecules Strategy for Deleting CH- Domains: To construct CH; deleted BR96 molecules, the hinge, CH 2 and CH 3 domains were removed from chimeric BR96 and humanized BR9696-2 IgG1 molecules by an Eco47-III restriction digestion in non-coding regions. The hinge and CH 3 domains were amplified by polymerase chain reaction (PCR) from a human IgG1 (pNyl.i4) molecule lacking the CH2 domain. Two oligonucleotides (Sense 49mer, Antisense 50mer) homologous to the sequences of 20 IgGI constant region at both sides preserving E.co47-III sites were synthesized.

The amplified hinge and CH 3 domain PCR fragments were added into Eco47-III sites on BR96 IgGI molecules by in vivo homologous recombination Bubeck et al., Nucleic Acid Research (1993) 21:3601-3602). The new BR96 IgG1 molecules t* were verified by restriction mapping and sequencing.

A sewing PCR strategy was used for the construction of CH 2 deleted human igG1 (pN /1.14) (Robert M. Horton, et al. (1990) Biotech 8 528).

The CHi domain was amplified as a 580 bp fragment with a sense oligonucleotide 30 TGG CAC CGA AAG CTT TCT GGG GCA GGC CAG GCC TGA (primer A) and an antisense oligonucleotide TCC GAG CAT GTT GGT ACC CAC GTG GTG GTC GAC GCT GAG CCT GGC TTC GAG CAG ACA (primer B) from a linearized human IgG 1 constant region vector (pNy The PCR fragment extends from the 5' end of the Hind-IIl site (in bold) through the Cel-II, Sal-I, Dra- III, Kpn-I, 6 bp nucleotide spacer and Mro-I sites (in bold) at the 3' end of the CHI domain.

The CH 3 domain was then partially amplified (to the Xba-I site) with a sense primer GTC GAC CAC CAC GTG GGT ACC AAC ATG TCC GGA GCC ACA TGG ACA GAG GCC GGC T (primer C) and an antisense primer CTG GTT CTT GTT CAT CTC CTC TCT AGA TGG (primer D) from a linearized human IgGI constant region vector (pNy A PCR fragment (about 150 bp) with Sal-I, Dra-III, Kpn-I, 6 nucleotide spacer and Mro-I sites (in bold) on its 5' end, extends only through the Xba-1 site (in bold) within the CH 3 domain.

The CHI and CH3 partial PCR fragments were combined in a PCR without any primer. The reaction was run through two full cycles of denaturation and reannealing to allow the fragments to combine at the homologous region at the 3' ends. Primers A and D (described above) were added to the reaction and the PCR cycle was completed. The polymerase extends the DNA with primer A and primer D, yielding a full- length (660 bp) PCR fragment. The newly extended PCR fragment is arranged from the 5' end to the 3' end in the following order: Hind-III C H Cei-II Sal-I Dra-III Kpn-I 6 bp spacer Mro-I ClH partial Xba-1.

The combined PCR fragment, with the CHI and partial CH3 domains, was then 2 5 cloned by a biunt end ligation into a Sma-I site on a pEMBL 18 vector and the sequence was confirmed by dideoxy sequencing (Sanger et al. (1977) PNAS (USA) 74:5463-5466).

To transfer the CHI and partial CH3 into a mammalian expression vector, both the 30 pEMBL18 and pNy 1.7 vectors were digested with Hind-III and Xba-I. The Hind- III and Xba-I fragment was ligated into the same sites on a linearized pNy1.7 vector.

The new construct, with CH, and a full CH3 domain, was designated the vector.

The hinge fragment was amplified from a Hind-III digested pNyl.7 vec:or with the primers designed to flank the hinge exon with a Sal-I and a Dra-lI cloning site at each end. These sites also exist between the CHI and CH 3 domains of the pNy 1.10 construct. The sense oligonucleotide ACC ATG GTC GAC CTC AGA CCT GCC AAG AGC CAT ATC with a 6 bp spacer and a Sal-I cloning site (in bold) and the antisense oligonucleotide CAT GGT CAC GTG GTG TGT CCC TGG ATG CAG GCT ACT CTA G with a 6 bp spacer and a Dra-III cloning site (in bold) were used for the amplication of the hinge fragment (250 bp).

The hinge region PCR fragment was cloned into a Sma-I site on pEMBL 8 by blunt end ligation. Both the pEMBL 18 with the hinge domain and the pNy 1.10 with the

CH

2 and CH 3 domains were digested with Sal-1 and Dra-III. The digested hinge fragment was cloned into the Sal-1 and Dra-III linearized sites on the pNy1.10 vector. The new construct, now carrying the CHi, hinge and CH 3 domains, was designated pNy1.11.

To make the final CH 2 deleted human igG1 construct, both the pN-i.11 construct 20 and pNy 1.11 vector were digested with BamH1 and HindIII. A fragment containing the CHI, hinge and CIH3 domains was cloned into the linearized pNy/l.11 vector.

The new constant region IgG1 construct lacks the CH 2 domain and is designated pNyl.14 (Figure 11).

25 For digestion of BR96 IgG1 with Eco47-III, a restriction fragment with hinge, Cl-h and CH 3 domains was identified on the constant region sequence of BR96 IgG1 vector in both chimeric and humanized molecules. The 5' end of this fragment lies inside the intron between CHI and hinge and the 3' end is located inside the CH 3 intron of the BR96 IgGI molecule. The hinge, CH 2 and CH 3 domains (1.368 kb 30 fragment) were removed from BR96 IgG1 molecules by Eco47-III restriction digestion. The Eco47-III is a blunt end cutter. The BR96 IgG1 DNA digested with this enzyme does not require any pretreatment before cloning. Figure 12 is a diagrammatic representation of the pD 7-hBR96-2 vector showing the Eco47-III sites used in cloning.

The CH 2 deleted BR96 IgGI was then constructed as follows. The hinge and CH 3 Sdomains were amplified from a CH, deleted L6 IgG1 (pNy1.14) construct with a sense oligonucleotide

CAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTGACCTCAG

A homologous to the constant region sequence of IgGI at the 5' end of the Eco47-III site (in bold) and an antisense oligonucleotide CCCAGGGCAGCGCTGGGTGCTT homologous to the constant region sequence of IgG1 at the 3' end of the Eco47-III site (in bold). The Eco47-III site at the 3' end of the pNyl.14 construct is modified in the cloning process. The Eco47- III site is thus introduced into an antisense primer and used in amplification of the hinge and CH 3 domains.

The pD17-BR96 IgG vector was digested with Eco47-III and the hinge, CHt and

CH

3 domains were removed. The linearized pD17-BR96 IgGI vector was mixed with equimolar amounts of hinge and CH 3 PCR fragments. Cotransformation of the 20 PCR fragment with linearized DNA into E.coli DH5a competent ceils resulted in a recombinant molecule. mediated by homologous recombination in bacteria. This construct lacks the CH 2 domain of BR96 IgG1 molecules, and is designated pD17- BR96-dCH2 (Figure 13).

C

25 1.9 grams of CH 2 -deleted chimeric BR96 was obtained as raw material from 89L of culture supernatant.

o C EXAMPLE 3 C 30 Toxicity, localization and clearance of CH 2 -deleted chimeric BR96 was tested in vivo as follows.

Three dogs received 400 mg/m 2 ofcBR96-A, the CH 2 deletion mutant of chimeric BR96, and two received chimeric BR96. Both molecules had been mildly reduced and alkylated. This is required to prevent dirnerization of the deletion mutant into a tetravalent form. Both control dogs experienced the typical GI toxicity and none of the three receiving the mutant displayed any toxicity. The control dogs and two of the test dogs were sacrificed at 1 hr to obtain duodenal tissue to measure antibody localization. Both control dogs had grossly visible GI pathology, and the test dogs had normal appearing GI tissue. The third dog has continued to show no signs of toxicity.

Results: A significant amount of localization of the CH 2 deleted cBR96 (cBR96-A) occurred to the GI tract in dogs treated with 400 mg/m 2 although the intact chiBR96 localized slightly better. The levels of localization indicate that roughly equivalent amounts of intact and CI-2 deleted cBR96 was delivered to the GI tract in these dogs.

Table 5. Localization of cBR96 to GI tissue.

Group Animal Specific mean Localization #271 155 a..

*o a a •no• .o a. a.

a a.

cBR96 135 #272 114 #273 126 cBR96-A 89 #274 52 Using the mean level of specific localization, an amount of cBR96-A equivalent to at least 66% of the amount of cBR96 was delivered to the target organ of toxicity, the duodenum. Based on the dose ranging done with cBR96 in dogs (some clinical signs of toxicity seen at doses of 10 mg/m 2 even if this difference is real, it could not explain the difference between significant toxicity and no toxicity, evaluation to date indicated that dogs treated with cBR96-A had no toxicity, pending microscopic histopathologic examination. This evaluation was based on analysis of 2 frozen blocks per dog and 2 sections per block. Replicates were quite good. We also ran historical frozen tissues from dogs treated with native cBR96 or F(ab)2/BR96 and the levels of localization for those tissues were 110 and 0. respectively, consistent with our previous data.

Assuming that there is no toxicity at marginally higher (2X) doses of cBR96-A, these data indicate that the CH 2 domain is associated with the induction of acute gastroenteropathy, and that the removal of this domain prevents the induction of gastroenteropathy mediated by BR96.

This study confirms the results showing that F(ab')2 is not toxic in the dog model and that the toxicity is mediated by the constant region. The CH 2 deletion mutant is a candidate for targeting agents clinically. Because of the very long half-life of chimeric BR96, some decrease in the mutant's half-life should be acceptable.

20 Figure 1 shows the measurement of the clearance of the cBR96-A in high Ley expressing dogs. The study used chimeric versus constant region mutant of cBR96- 2.

2 CBR96-2 did clear faster than the chimeric BR96. The localization ofcBR96-A to 25 the gastrointestinal epithelium is not significantly affected by this more rapid clearance. More than enough of the cBR96-A localized to have caused toxicity.

*Discussion: The constant region of chimeric IgG is responsible for the GI toxicity seen in clinical trials, e.g. with chiBR96-dox. The GI toxicity seen in the dog model 30 is very similar to the clinical toxicity. Both in man and dog, administration of the unconjugated antibody mediates an acute GI toxicity characterized by rapid onset of vomiting, often with blood.

In man the bleeding is limited to the fundus of the stomach, causing erosion of the superficial mucosa of the stomach. Although the pathology of the wound is limited and resolves, the extreme nature of the nausea and vomiting, unrelieved by antiemetics, defines it as the dose-limiting toxicity.

This toxicity is mediated in man and dog by the antibody molecule alone. At higher doses of the antibody-dox conjugate, additional toxicity is seen in the dog model, probably due to doxorubicin. Although the intact IgG of BR96 causes toxicity in dog and man, the F(ab')2 molecule (divalent and lacking only in the constant region) is not toxic in dogs. This finding has motivated our attempts at high levels, and improves the affinity and specificity of BR96 for tumor antigen.

The CH 2 domain is known to mediate complement and FcR binding. It was not known that structural alteration of the CH 2 domain would result in immunoglobuiininduced toxicity inhibition.

Toxicology study of hBR96-2B 20 The toxicology study of hBR96-2B in high Lewis Y expressor dogs showed that a dose of 400 mgi/m 2 did not cause hematemesis nor bloody stools, in contrast to BR96 which consistently causes one or both signs. A dog sacrificed at 24 hrs had normal gross appearance of the GI tract, again in marked contrast to chimeric BR96 which causes hemorrhagic lesions and mucosal erosions.

EXAMPLE 4 The polymerase chain reaction (PCR) is a widely used and versatile technique for the amplification and subsequent modification of immunoglobulin genes. The rapidity 30 and accuracy with which antibody genes can be modified in vitro has produced an Sassortment of novel antibody genes can be modified in vitro has produced an assortment of novel antibodies. For example, PCR methods have been used for engineering antibodies with increased affinity to antigen, for "humanizing" antibodies, and for modulating effector function (Marks, A.D. Griffiths, M.

Malmqvist, T. Clackson, J.M. Bye and G. Winter. 1992. Bypassing immunization: high affinity human antibodies by chain shuffling. Bio/Technology 10:779-783; Rosok, D.E. Yelton, L.J. Harris. J. Bajorath, Hellstrom. I. Hellstrom, G.A. Cruz, K. Kristensson, H. Lin, W.D. Huse and S.M. Glaser. 1996. A combinatorial library strategy for the rapid humanization of anticarcinoma BR96 Fab. J. Biol. Chem. 271:22611-22618; Morgan, D. Jones, A.M. Nesbitt, L.

Chaplin, M.W. Bodmer and S. Emtage. 1995. The N-terminal end of the CH2 domain of chimeric human IgGI anti-HLA-DR is necessary for Clq, FcyRI and FcyRIII binding. Immunology. 86:319-324).

As part of a more comprehensive study, we desired to introduce various site specific mutations in the CH 2 constant domain of human IgGi. Six specific amino acid residues distributed throughout the CH2 domain previously identified to play a role in immune effector function were marked as targets for mutagenesis (Morgan, A.N., D. Jones, A.M. Nesbitt, L. Chaplin, M.W. Bodmer and S. Emtage. 1995. The Nterminal end of the CH2 domain of chimeric human IgG 1 anti-HLA-DR is necessary for Clq, Fc-yRI and FcRIII binding. Immunology. 86:319-324; Duncan, 20 A.R. and G. Winter. 1988. The binding site for Clq on IgG. Nature 332:738-740; o. Tao, R.I.F. Smith and S.L. Morrison. 1993. Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation. J.Exp.Med. 178:661-667). five of the six residues were grouped into two clusters-one cluster consisting of two residues, two amino acids apart (Location 25 1, or L1); and a second cluster consisting of three residues spanning a sequence of five amino acids The remaining amino acid position (L3) made for the total of six residues. We were interested in constructing a panel of mutant CH 2 domain IgGs consisting of each L mutation by itself as well as in combination with other L mutants L1; L1; and L2; L1, L2 and L3; etc.).

S 30 SVarious in vitro methods have been described where PCR is used to simultaneously introduce distally located site-specific mutations within a gene sequence (Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen and L.R. Pease. 1989. Site-directed mutagenesis by overlap extension. Gene 77:51-59; Ge, L. and P. Rudolpf. 1996.

Simultaneous introduction of multiple mutations using overlap extention PCR.

BioTechniques 22:28-30). Alternatively. an in vivo procedure termed recombination PCR (RPCR) has also successfully been used for rapidly and efficiently generating distally located site-specific mutations (Jones, D.H. and S.C.

Winistorfer. 1993. Use of polymerase chain reaction for making recombinant constructs, p.241-250. In B.A. White Methods in Molecular Biology, Vol.

Humana Press Inc., Totowa, NJ, Jones, D.H. And B.H. Howard. 1991. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. BioTechniques 10:62-66).

RPCR

uses E. Coli's recombination machinery to generate intact circular recombinant plasmids from a transfected mixture of linear PCR-generated product and linearized vector. In vivo recombination is mediated through the joining of nucleotide sequences designed into the 5' ends of both PCR primers that are homologous to DNA sequences encoded by the vector. In this report we describe an extension of the RPCR procedure for simultaneously introducing complex combinations of mutations into an antibody CH2 domain.

20 Humanized BR96 variable region hcavv and light chain genes, previously cloned and co-expressed as an assembled active Fab fragment in an MI13 phage expression vector, provided the starting material (Rosok, D.E. Yelton, L.J. Harris, J.

Bajorath, Hellstrom, I. Hellstrom, G.A. Cruz, K. Kristensson, H. Lin, W.D.

Huse and S.M. Glaser. 1996. A combinatorial library strategy for the rapid 25 humanization of anticarcinoma BR96 Fab. J. Biol. Chem. 271:22611-22618). The heavy and light chain V genes were amplified by PCR from a single-stranded M13 DNA template and subcloned by in vivo recombination (Jones, D.H. And B.H.

Howard. 1991. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. BioTechniques 30 10:62-66) into vectors pD17-hGla and pD16-hCK, to form pBR96-hGla and pBR96-hCK respectively. pD17-hGla and pD16-hCK are eukaryotic immunoglobulin expression vectors derived from pcDNA3 (Invitrogen. San Diego, CA). The plasmid pBR96-hGla was further modified by site-directed mutagenesis to introduce two Eco47-III restriction sites flanking the immunoglobulin hinge-

CH

2

-CH

3 domains using standard procedures. The recipient vector was then prepared by digesting pBR96-hGla with Eco47-III, isolating the vector backbone by agarose gel electrophoresis followed by extracting the vector DNA from the excised gel slice using the Qiagen Gel Extraction kit (Qiagen, Chatsworth,

CA).

The strategy for introducing multiple mutations within the immunoglobulin CH2 gene, shown in Figure 24, relies on the in vivo homologous recombination of several independently amplified PCR products with each other as well as with the pBR96hGla vector DNA. For introducing mutations at two distal locations two PCR products are synthesized (Figure 24B). One end of each PCR product is for recombining with an homologous end of the linear vector, and the other end, encoding the mutation(s) of interest, is for recombining with the neighboring

PCR

product. As shown in Figure 24B, additional distally-located mutations can be introduced into a target sequence by increasing the number of PCR products proportionately. The recombination of neighboring PCR products always occurs across the regions containing the desired mutations, therefore the oligonucleotide primers encoding these ends Al, A2) contain complementary mutant residues.

20 The mutagenic PCR primers contain at least 15 nucleotides of wild-type sequence flanking each side of the mutant residues for either priming the polymerization reaction or mediating recombination. Two 49-nucleotide long PCR sense and antio sense primers (Rs and Ra) contain sequences for recombining with the end regions of the Eco47-III digested pBR96-hGla vector.

Each L mutation was amplified in a separate PCR reaction. The reaction conditions were 250 ng intact pBR96-hGla DNA template, 10 ul of IX Pfu buffer (Stratagene, Inc. San Diego, CA), 10 nmol dNTPs, 200ng each of the appropriate PCR primers, dimethysulfoxide (ATCC, Rockville, MD) and 2.5 units cloned Pfu DNA 30 polymerase in a 100ul reaction volume. Samples were first denatured at 950 C for S* min, cooled to 45 0 C for 5 min, and extended at 72 0 C for 1 min followed by cycles of denaturation at 94 0 C for 45 sec, annealing at 45 0 C for 45 sec, extension at 72 0 C for 1 min/kb, followed by a final extension at 72 0 C for 7 min in a Perkin- Elmer DNA Thermal Cycler (Norwalk, CT). The amplified products were purified from a 1% agarose gel, extracted with Qiagen Gel Extraction kit and the recovered DNA quantitated. 50 ng of each PCR product was mixed with 25 ng of the Eco47- III digested pBR96-hGla vector, transfected into Max competent E. coli according to the manufacturer's procedure (GIBCO BRL/Life Technologies, Gaithersburg, MD), and the entire transfection reaction plated onto selective LB agar plates containing 100 ug/ml ampicillin.

The results of several cloning experiments are summarized in the Table that follows.

Typically the transformations produced from 80 to 200 bacterial colonies.

Individual colonies were selected and grown overnight in 2 ml liquid cultures for isolation of miniprep plasmid DNA (Qiagen) and analysis by Eco47-III restriction endonuclease mapping. Among 24 independent transformants analyzed from triple homologous recombination events (two PCR products plus vector) 11 clones contained the predicted 1.4 kpb DNA insert.

Figure 25 shows a sample diagnostic restriction analysis of DNA prepared from clones derived from quadruple homologous recombination events (three PCR 20 products plus vector). Additional sampling of clones resulting from quadruple recombination yielded a cloning efficiency of 29% (7 clones containing inserts/24 clones sampled). At this point, due to the small sampling sizes, we do not know whether the differences in the cloning efficiencies observed between the triple and quadruple recombination events are meaningful.

C

To evaluate the expression of Lev -binding activity of the CH 2 mutant IgGs, miniprep DNAs from 6 clones derived from the triple recombination reaction and 6 •clones derived from the quadruple recombination reaction exhibiting the predicted diagnostic Eco47-III restriction patterns were isolated, mixed with pBR96- hCK 30 DNA and used to co-transfect COS7 cells. 48 hour spent supernatants from 3 ml cultures were assayed for total IgG production and for Le' binding activity by enzyme-linked immunosorbent assay (EIA) as described (Yelton, M.J. Rosok, G.A. Cruz, W.L. Cosand, J. Bajorath, I. Hellstom, Hellstorm, W.D. Huse and S.M. Glaser. 1995. Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis. J.Immunol. 155:1994-2004). All twelve cultures were found to secrete approximately 2-3 ug/ml Ley -reactive IgG. The spectrum of Ley binding activities were all similar to that of native humanized BR96 IgG indicating that the homologously recombined antibodies did not acquire any gross mutations that could affect antigen binding. To confirm that the desired CH 2 mutations had been incorporated, and to evaluate the recombined genes for misincorporated nucleotides, four of the clones producing functional antibody were sequenced using Sequenase Version 2 DNA Sequencing Kit (United States Biochemical). One clone was found to contain a single nucleotide change within the forward PCR primer used for mediating recombination with vector DNA. We are uncertain whether this error occurred during chemical synthesis of the oligonucleotide primer or is a result of misincorporation during the PCR reaction, despite the fact that we used a thermostable polymerase with proofreading activity.

A RPCR procedure for homologously recombining up to three separate PCRgenerated mutated antibody sequence products into a eukaryotic expression vector for the rapid construction of engineered IgG molecules is described herein. The 20 advantage of this approach is the ability to simultaneously introduce multiple distally-located mutations with PCR products synthesized by a single round of PCR.

Recombinant DNAs are produced with a reasonably high cloning efficiency and fidelity of correct nucleotide sequences. The ability to efficiently rejoin several distinct PCR products should permit combinatorial strategies for constructing 25 complexly mutated protein domains as well as broadening the number and location of desired mutations.

Analysis of transformants generated by multiple-fragment

RPCR.

S SS o g*o Mutant IgGs PCR HR' events Colonies Cloning Constructed Fragments in Analyzed Efficiencyb reaction 2 2 triple 24 S3 quadruple 24 33% aHR-homologous recombination bCloning efficiency (number of clones containing 1.4kbp insert/total number of colonies EXAMPLE This example provides two methods for introducing site specific mutations into the CH2 domain of human IgGI constant region containing vectors.

One method involves PCR amplification of a segment or segments of the constant region, wherein mutations are introduced using appropriately constructed 10 oligonucleotides. The vector receiving the fragment(s) is digested with a restriction enzyme to linearize the vector. PCR amplification primers are designed so that the 5' ends of the PCR fragments can hybridize to the DNA sequence of the vectors. If more than one PCR fragment is amplified, then common sequences to the two fragments are introduced by oligonucleotides. Bacteria are transfected with the PCR fragments and with the digested vector. The fragments and vector can recombine by homologous recombination using the bacteria's recombination machinery. Bacterial colonies are selected and the DNA is analyzed by size and restriction map as a preliminary determination that the vector and fragment(s) recombined correctly.

Correct insertion of fragments with the mutations is confirmed by dideoxynucleotide sequence analysis. DNA is then introduced into mammalian cells as described for the CH2 deleted antibody, and the expressed antibody analyzed for binding and functional activity.

By way of example, mutations Leu to Ala at residue 235 in CH2 and Gly to Ala at residue 237 were introduced by the procedure disclosed in Example 4. The heavy chain vector used for this procedure was pD 17-hG 1 a, similar to pD 17-BR96 vector described herein except that humanized V regions (Rosok, D.E. Yelton.

L.J.

Harris, J. Bajorath, K-E. Hellstrom, I, Hellstrom, G.A. Cruz, K. Kristensson, H. Lin, W.D. Huse, and S.M. Glaser, 1996. J. Biol. Chem 271 37:22611-22618) with three affinity mutations (HI, H2, and H3 mutations) were substituted.

pBR96-hGla contains two Eco47-III restriction sites flanking the Ig hinge-CH2- CH3 domains. The recipient vector was prepared by digesting pBR96-hGla with Eco47-III, isolating the vector by agarose gel electrophoresis, and (3) extracting the vector DNA from the excised gel slice using the Qiagen Gel Extraction kit (Qiagen, Chatsworth, CA). To introduce mutations at a single location, such as for positions 235 and 237, two PCR products were synthesized.

To introduce two distally located mutations, such as for mutant F (also referred to herein as hBR96-2F) with mutations at 235, 237, 331, requires 3 PCR products.

The recombination of neighboring PCR products occurs across the regions containing the desired mutations, therefore the oligonucleotide primers encoding 20 these ends contain complementary mutant residues. The mutagenic PCR primers contain at least 15 nucleotides of wild-type sequence flanking each side of the mutant residues for either priming the polymerization reaction or mediating recombination. Two 4 9 -nucieotide long PCR sense and anti-sense primers containing sequences for recombining with the end regions of the Ecc47-III digested 25 pBR96-hGla vector.

PCR amplification used 250 ng intact pBR96-hGla DNA template, 10 ul of Pfu buffer (Stratagene, Inc., San Diego, CA), 10 nmol dNTPs, 200 ng each of the appropriate PCR primers. 10% dimethylsulfoxide (ATCC, Rockville, MD) and 30 units cloned Pfu DNA polymerase (Stratagen, Inc. San Diego, CA) in 100 ul reaction. Samples were denatured at 95°C for 5 min, annealed at 45 0 C for 5 min, and extended at 72 0 C for 1 min followed by 25 cycles of denaturation at 94 0 C for sec, annealing at 45 0 C for 45 sec, extension at 72 0 C for 1 min/kb, and a final extension at 72°C for 7 min. The amplified products were purified from a 1% agarose gei, extracted with the Qiagen Gel Extraction kit and quantitated. 50 mg of each PCR product was mixed with 25 ng of the Eco47-III digested pBR96-hGla vector and transfected in E.coli MAX Efficiency DH5aTM according to the manufacturer's instructions (GIBCO BRL/Life Technologies, Gaithersburg,

MD).

The entire transfection reaction was plated onto LB agar plated containing 100 gg/ml ampicillin.

Bacterial colonies were selected and grown overnight at 370 C in 2 ml liquid cultures. DNA was isolated and analyzed by Eco47-III restriction endonuclease mapping. Clones with the correct size insert were sequenced (Sequenase Version 2, U.S. Biochemical Corp., Cleveland, OH).

The second method for introducing site specific mutations into the CH 2 domain of human IgG1 involved the method of Kunkel (1987 Methods Enzymology, supra).

For this procedure pD17-hGlb DNA with the Fl origin of replication was introduced into electrocompetent E. coli CJ236 dut-ung- (Bio-Rad Laboratories, Hercules, CA) by electroporation according to manufacturer's instructions. PD17hGIb is a vector having a constant region but no variable region. The Fl ori site allows treatment of this vector as a phagemid.

Bacteria containing the plasmid were selected by ampicillin resistance. Single stranded uridinylated DNA was prepared using the Muta-Gene Phagemid In Vitro 25 Mutagenesis Version 2 protocol (Bio-Rad). Mutations were introduced by sitedirected mutagenesis with the appropriate antisense oligonucleotide. For molecules with mutations at more than one location, mutations were introduced by either of the two methods discussed above. One method would be to prepare one mutant, for example, mutant 2C (also referred to herein as BR96-2C) with the mutations at 30 residues 318, 320, 322, isolate ssDNA, and introduce a second mutation set with the appropriate anti-sense oligonucleotide. The second method would be to anneal two antisense oligonucleotides with the same uridinylated ssDNA and screen for mutants with both sets of changes. Mutant 2H (hBR96-2H) was also prepared by a combination of thse methods.

The V region of humanized BR96-2 heavy chain was introduced by the homologous recombination method described above in pD17-hJml4.H1. The pD17-hJml4.H1 plasmid contains the BR96 humanized variable region with the H1/H2/H3 mutations and the plasmid was used to transfect mutant sequences into mammalian cells. The pD17Glb vector containing the Fc mutation(s) was digested with NheI for 3 hr at 370 C and the DNA isolated by methods described above. Insertion of the V region into the vector was determined by size and restriction enzyme mapping and confirmed by sequence analysis.

Transient expression of whole antibodies was performed by transfection of COS cells. For production of antibody, stable transfections of CHO cells were performed (see description of deleted CH2 mutant). All mutants were purified from CHO culture supernatants by protein A chromatography.

The oligonucleotide primers homologous to the vector and used to introduce the constant regions mutations were as follows: 20 Oligonucleotides homologous to vector sequences: Sens(sense)CH2 E47-3-5: CAG GGA GGG AGG GTG TCT GCT GGA AGC CAG GCT CAG CGC TGA CCT CAGA D CH2 E47-3 A (antisense): GGA AAG AAC CAT CAC AGT CTC GCA GGG GCC CAG GGC AGC GCT GGG TGC TT Oligonucleotides to mutate Leu235 to Ala and Gly237 to Ala (underlined sequences show sites of mutation): Antisense CH2 L235-G237/aa: GAA GAG GAA GAC TGA CGG TGC CCC CGC GAG TTC AGG TGC TGA GG 30 SensCH2 L235-G237/AA: CCT CAG CAC CTG AAC TCG CGG GGG CAC CGT CAG TCT TCC TCT TC Oligonucleotides to mutate Glu318, Lys320, Lys322 to Ser Antis(antisense)CH2 EKK/SSS-2: CTG GGA GGG CTT TGT TGG AGA CCG AGC ACG AGT ACG ACT TGC CAT TCA GCC Oligonucleotides to mutate Pro331 to Ala: Antis CH2 P331/A/3: GAT GGT TTT CTC GAT GGC GGC TGG GAG GGC Sense CH2 P33/A: GCC CTC CCA GCC GCC ATC GAG AAA ACC ATC Alternative antisense oligo to introduce Ala at 331 by site-directed mutation: CH2P331A: GAT GGT TTT CTC GAT AGC GGC TGG GAG GGC TTT G Oligonucleotides to mutate Glu318 to Ser, Lys320 to Ser, Lys322 to Ser, and Pro331 to Ala: Antis CH2 EKKP/SSA-6: GAT GGT TTT CTC GAT GGC GGC TGG GAG GGC TTT GTT GGA GAC CGA GCA CGA GTA CGA CTT GCC ATT CAG CCA GTC CTG GTG Sense CH2 EKKP/SSA-6: CAC CAG GAC TGG CTG AAT GGC AAG TCG TAC TCG TGC TCG GTC TCC AAC AAA GCC CTC CCA GCC GCC ATC GAG AAA ACC ATC 20 In vitro Assays of the Mutants Results of the CDC demonstrate that mutant hBR96-2B has approximately 10 fold less activity than the control hBR96-1 (two affinity mutations, one in H2 and one in H3, refer to previous patent (Figure The mutants that have the least ability to kill cells in the presence of complement is hBR96-2C with the triple mutations at positions 318, 320, and 322 and the hBR96-2H mutant (least cytotoxic antibodies in the panel) which contains all six mutations at the three different locations. ADCC activity was most affected by the CH2 deleted hBR96-2 molecule (Figure 21).

hBR96-2B and -2H lost between 100 and 1000 fold activity to kill in the presence 30 of effector cells. In the ADCC assay the hBR96-2B molecule also lost approximately 10 fold activity (Figure 21).

Figures 26-28 provide the amino acid sequences for the heavy chain variable region for both chimeric and humanized BR96 having the HI, H2, and H3 mutations. The amino acid sequence for the light chain variable region is known and methods for generating it are found in PCT Application No. 95/305444. Additionally provided is the amino acid sequence for the IgG1 constant region. Mutations in the constant region are marked.

*4 *o*o Page(s)'IC' are claims pages they appear after the sequence listing SEQUENCE LISTING GEN4ERAL INFORM.ATION )iJ) APP7LI-CANT: Yelzon, Dale E.

Rosok, Mae Joanne (ii) TITLE OF THE INVENTION: A METHOD FOR INHIBITING I>MMUNOGLOBUL IN- INDUCED TOXICITY RESULTING 'FROM THE USE OF I-MMUNOGLOBULINS IN THERAPY AND IN VIVO DIAGNOSIS (iii) NUMBER OF SEQUENCES: 27 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Merchant, Gould, Smith, Edell, Welter Schmidt STREET: 11150 Santa Monica Boulevard, Suite 400 CITY: Los Angeles STATE: CA COUNTRY: USA ZIP: 90025 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastrSEQ for Windows Version (vi CUR-ENT APPLICATION

DATA:

APPLICATION NUMBER: PCT US97/13562 FIL7ING DATE: 0l1-AUG-1997 CLASSIFI7CATICN: (v44) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 60/023,033 FI7LING DATE: 02-AUG-1996 tzii ATTORNEY/AGENT

INFORMATION:

CA, NAM.E: Canady, Karen S.

RE3I1S7RATION NUMBER: 39,927 REFERENCE/DOCCKET NUMB ER: 30436.43WoUl T L iz =2CMUN !CAT: ON INFORMATION: TELE7PuONE: 310-445-1140 TELEFAX: 310-445-9031

TELE-X:

INFORMATION FOR SEQ ID NO:1: 0*.0 SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs ()TYPE: nucleic aci STRANDEDNESS: single ID) TOPOLOGY: linear 9 900 Mi)SOLECUJLE TYPE: cDNA :00. 60 SEQUENCE DESCRI7PTION: SEQ ID NO:l1: *.TGGCACCGAA AGCTTTCTGG GGCAGGCCAG GCCTGA 36 65(2) INFORY-ATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CCNA (Xi) SEQUENCE DESCRIPT-CN: SEQ TZ) NO:2: TCCGGACATG TTGGTACCCA CGTGGTGGTC GACGCTGAGC CTGGCTTCGA GCAGACA 57 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ TO NO:3: GTCGACCACC ACGTGGGTAC CAACATGTCC GGAGCCACAT GGACAGAGGC COOCT INFORM-ATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TY?E: nucleic acid STRP14CEDNESS: single TOPOLOGY: linear ,ii) MOLECULE TYPE: cDnNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTGGTTCTTG_ TTCATCTCCT CTCTAGATGG 7NFORMATO FOR SEQ 1D NO S i)SEQUENCE CHA;RAC-ER:STICS: LENGTH: 36 base pairs 7Y7E: nucleic acia S-RNDEDNESS: singl.e TC=?O''GY linear M~OLECULE TYPE: CCNA (xi) SEQUENCE DESCRITION: SEQ ID ACCATGGTCG ACCTCAGACC TGCCAAGAGC CATATC 36 INFOMAIO FR SEQ ID NO: 6: iSEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TY?7: nucleic acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CATGGTCACG TGGTGTS7CC CTGGATGCAG GCTACTCTAG !NFORMATION FCR SEQ I D NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs nucleic acid STRANDECNESS: single TOPOLCGY: linear (ii) MOLECJLE TYPE: cDNA (Xi) SEQUENCE DESCRIPTICN: SEQ 1D 140:7: CAGGGAGGGA GGGTGTCTGC TGGAAGCCAG GCTCAGCGCT GACCTCAGA :NFORAMATION FOR SEQ 1D NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nuclei;c acid STRANDEDNESS: single TOPOLCGY: linear (11i) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: GGAAAGAACC ATCACAGTCT Cc-CAGGGGCC CAGGGCAGCG CTGGGTGCTT (2,1 INFCRMAT7ION OR SEQ 7D NO:9: SEQUENCE CHARACTERTST:S: -ENGT 8691 base pai4rs YPE:nucleic acid STRANLDEDNESS: single TOPOLOGY: linear ii)' X-GLECULE TYPE: cDNA (xi) SZEQUZNCE DESCRIPTION: SEQ ID NO:9: GAC-?C- C 45 CAAnooC;A'-1.

TGC:TCGCOA

AGTAIATCA.AT

T-ACGGTAAA

TGACGTATGT

50 ATTTACGGTA

CTATTGACGT

GGGACTTTCC

GGTTT-TGGCA

TCOACCCCAT

55 AATGTCGT.AA T zrA:ATAAG

TTAATACGAC

AC OT GAG 60 GTGAAGTGAA

TCTCCTGTGT

CTCCAGAGAA

ATCCAC-ACAC

ACCTGCAAAT

7GGACGACGG

CTAGCACCAA

GC:.AGCSGC

G=3CTCTC TAC GGTOA

-CCA:'AGTA

AACTGCCCAC

CAATGACGGT

TACTTGGCAG

=TCATCAAT

TGACGTCAAT

CAACTCCGCC

TOAC7ATACGG 7CTCGA-A

TTG-TGGTTAA

T'r TGGTGAG

GAGGCTGGAG

TGTAAAGGGT

CAGCCGTCTG

GGCCTC-'T

OGCCAT CO

AG-GTGACCTG

AG-ACAATCT

GAGGTCGCTG

AATTGCATG.:

AGATATACGC

TT-AGTTCATAk

GGCTGACCGC

ACGCCATAG

TTGGCAGTAC

AA.ATGGCCCG

TACATCTACG

GCGTGGAT

GGGAGTTTGT

CCATTGACGC

TGGCTAACTA

GAGACCCAAG

ACCGGTCAAT

GCTTGGTCCT

TCTGGGGGAG

TTCACTTTCA

TGGGTCGCAT

CGATTCACCA

AAGTCTGAOG

GCTTACTGGG

GTCTTCCCCC

CTGGTCAAGG

AGCCGC C-C

TTTGACOATG

GCT CTCATGC

AGTAGT-GOC

AGAATCTGCT

GTTGACA-TTG

GCCCATATAT

CCAACGACCC

GGACTTTCCA

ATCAAGTGTA

CCTGGCATTA

TATTAGTCAT

AGCGGTTTGA

TTTGGCACCA

AZAATGGGCGG

GAGAACCO-AC

CTT GGTACCA

CGATTGGAAT

TCCTTGTCCT

GCTTAGTGCA

GTGACTATTA

ACATTAGTCA

TCTCCAGAGA

ACACAGCCAT

GCCAAGGGAC

TGGCACCCTC

ACT ACTTCCC

GCTTCGAATA

AGITTTGGCGC

0CCATAG'TA

GAGCAAAATT

TAGGGTTAGG

ATTATT GACT

C-GAGTTCCGC

CCGCCCATTG

TTGACGTCAA

TCATATGCCA

TGCCCAGTAC

CGCTATTACC

CTCACGGGGA

AAATCAACGG

TAGGCGTGTA

TGCTTACTGG

ATTTAAATTG

TCTTGCGGCC

TGTTTTAA.AA

GCCTGGAGGG

CATGTATTGG

AGGTGGTGAT

CAATGCCAAG

GTATTACTGT

TCTGGTCACG

CTCCAAGAOC

C::XACCGGTG

GCCAGAGT A.A

CGATCTCC

AGCCAGTATC

TAAGCTrACAA

CGTTT-TGCGO

AGTT.MTTAA'T

GTTACATAAC

ACGTCAATAA

TGGOTGGACT

AGTACGCCCC

ATGACCTTAT

ATGGTOATGC

TTTCCAAGTC

GACTTTCCAA

CGGTGGGAGG

C T T AT CGAAA

ATATCTCCTT

GCTTGCTAGC

OGTGTCCAGT

TCCCTGAAAG

GTTCGCCAGA

ATAACCGACT

AACACCCTGT

GCAAGA-GCC

GTCTCTGTAG

ACCTCTOGO

ACGGTGTCGT

2410 320 360 420 480 540 600 660 720 940 9 00 960 :32C 138 0 200 1260 "320 2.380 1440 15 00 156 0 :620

GGAACTCAGG

GACTCTACTC

ACATCTGCAA

GGCCAGCACA

CATCCCGGCT

CGGAGGCCTC

GCTCTGGGCA

GCTGGGCTCA

CCCCAAAGGC

GTAACTCCCA

CCGTGCCCAG

AGAGTAGCCT

TTCCTCAGCA

GGACACCCTC

CGAAGACCCT

GACAAAGCCG

CCTGCACCAG

CCCAGCCCCC

GCCACATGGA

CCTCTGTCCC

ATGAGCTGAC

ACATCGCCGT

CCGTGCTGGA

GGTGGCAGCA

ACACGCAGAA

GCTCCCCGGG

CCGGGCGCCC

ATGGTTCTTT

GGGTCCCACT

GGGCTCAGCC

AGCAGCACCT

CAGCCCCTGC

CATGCCCACT

CTACCCCCAC

AACCGACTCC

CACACACTCA

CACCACACAC

CCCAGACCAIG

CCCCACGCGG

TCAGACAAAC

GGATCACACA

CAGGACGGAT

CCCGTGCCTT

GAAATTGCATr

GACAGCAAGG

ATGGCTTCTG

AGCGGCGCAT

AGCGCCCTAG

CCTCTCAAAA

CTAACTCCGC

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATTGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGT

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

CGCCCTGACC AGCGGCGTGC CCTCAGCAGC GTGGTCACCG CGTGAATCAC AAGCCCAGCA GGGAGGGAGG GTGTCTGCTG ATGCAGCCCC AGTCCAGGGC TGCCCGCCCC

ACTCATGCTC

GGCACAGGCT

AGGTGCCCCT

GACCTGCCAA GAGCCATATC CAAACTCTCC ACTCCCTCAG ATCTTCTCTC

TGCAGAGCCC

GTAAGCCAGC CCAGGCCTCG GCATCCAGGG ACAGGCCCCA CCTGAACTCC TGGGGGGACC ATGATCTCCC

GGACCCCTGA

GAGGTCAAGT TCAACTGGTA CGGGAGGAGC AGTACAACAG GACTGGCTGA ATGGCAAGGA ATCGAGAAAA CCATCTCCAA CAGAGGCCGG CTCGGCCCAC TACAGGGCAG CCCCGAGAAC CAAGAACCAG GTCAGCCTGA GGAGTGGGAG AGCAATGGGC CTCCGACGGC TCCTTCTTCC GGGGAACGTC TTCTCATGC*.

GAGCCTCTCC

CTGTCTCCGG

CTCTCGCGGT

CGCACGAGGA

AGCATGGA.AA T.AAAGCACCC CCACGGGTCA GGCCGAGTCT 6 0*@ :.,so

GTCCCCACAC

AGGGGCTGCC

GCCCTGGGCT

CTCTGTAGGA

CGGC-GGCATG

GGCACTAACC

GGC-GACATGC

GCCCAGACCC

ACACGTGCAC

AGCAAGGTCC

CACCTCAAGG

CC-AGCCCTCC

C-ACGTCACG

CAGCfCTCGAC

CCTTGACCCT

CGCATTGTCT

GGGAGGATTG

AGGCGGAAAG

TAkAGCGCGGC

CGCCCGCTCC

AAGGGAAAAA

CCATCCCGCC

TTTTTATTTA

GAGGCTTTTT

CGCGCCAAAC

TCATGGTTCG

ACGGAGACCT

CAACCTCTTC

CCATTCCTGA

TCAAAGAACC

TTATTGAACA

CTGTTTACCA

TGCAGGAATT

TCCCAGAATA

TTGAAGTCTA

TAAAGCTATG

GGAACCTTAC

TAAGGTAAAT

TGTATTTTAG

TGGCCCAGGC

CTCGGCAGGG

GGGCCACGGG

GACTGTCCTG

CCTAGTCCAT

CCTGGCTGCC

ACTCTCGGGC

GTTCAACAAA

GCCTCACACA

TCGCAC.NCG-T

CCCACGAGCC

TCTCACAAGG

T'CCCTGGCCC

TGTGCCTTCT

GGAAGGTGCC

GAGTAGGTGT

GGAAGACA.AT

AACCAGCTGG

GGGTGTGGTG

TTTCGCTTTC

AAGCATGCAT

CCTAACTCCG

TGCAGAGGCC

TGGAGGCCTA

TTGACGGCAA

ACCATTGAAC

ACCCTGGCCT

AGTGGAAGGT

GAAGAATCGA

ACCACGAGGA

ACCGGAATTG

GGAAGCCATG

TGAAAGTGAC

CCCAGGCGTC

CGAGAAGAAA

CATTTTTATA

TTCTGTGGTG

ATAAAATTTT

ATTCCAACCT

ACACCTTCCC

TGCCCTCCAG

ACACCAAGGT

GAAGCCAGGC

AGCAAGGCAG

AGGGAGAGGG

AACCCAGGCC

CGGGAGGACC

CTCGGACACC

AAATCTTGTG

CCCTCCAGCT

GCCGGGTGCT

GTCAGTCTTC

GGTCACATGC

CGTGGACGGC

CACGTACCGT

GTACAAGTGC

AGCCAAAGGT

CCTCTGCCCT

CACAGGTGTA

CCTGCCTGGT

AGCCGGAGAA

TCTACAGCAA

CCC-TGATGCA

GTAAATGAGT

TGCTTGGCAC:

AGCGCTGCCC

GAGGCCTGAG

TGTGCAGGTG

TGGGGGATTTr

AAGCCCTAGG

TTCTGTGAGC

GTGCGTAGGG

CTGCCCAGCC

CCTGTGGAGG

CCCCGCACTG

CGGAGCCTCA

GAACACTCCT

TCTCGGCAGC

GTGCCCCTGQC

TGC-CC-CACTT

AGTTGCCAGC

ACTCCCACTG

CATTCTATTC

AGCAGGCATG

GGCTCTAGGG

GTTACGCGCA

TTCCCTTCCT

CTCAATTAGT

CCCAGTTCCG

GAGGCCGCCT

GGCTTTTGCA

TCCTAGCGTG

TGCATCGTCG

CCGCTCAGGA

AAACAGAATC

CCTTTAAAGG

GCT CATTTTC

GCAAGTAAAG

AATCAACCAG

ACGTTTTTCC

CTCTCTGAGG

GACTAACAGG

AGACCATGGG

TGACATAATT

TAAGTGTATA

ATGGAACTGA

49

GGCTGTCCTA

CAGCTTGGGC

GGACAAGAAA

TCAGCGCTCC

GCCCCGTCTG

TCTTCTGGCT

CTGCACACAA

CTGCCCCTGA

TTCTCTCCTC

ACAAAACTCA

CAAGGCGGGA

GACACGTCCA

CTCTTCCCCC

GTGGTGGTGG

GTGGAGGTGC

GTGGTCAGCG

AAGGTCTCCA

GGGACCCGTG

GAGAGTGACC

CACCCTGCCC

CAAAGGCTTC

CAACTACAAG

GCTCACCGTG

TGAGGCTCTG

GCGACGGCCG

GTACCCCCTG

TGGGCCCCTG

TGGCATGAGG

TGCCTGGGCC

GCCAGCGTGG

AGCCCCTGGG

GCCCCTGTC-

ACAGGCCCTC

TCGCACCCGC

GACTGGTGCA

AGGTTGGCCG

CCCGGGCGAA

CGGACACAGG

TTCT-CACAT

AGCCGCCACA

!ZC-CAGTGCCG

CAT CTGTrTGT

TCCTTTCCTA

TGGGGGGTGG

CTIGGGGATGC

GGTATCCCCA

GCGTGACCGC

T TCTCGCCAC

CAGCAACCAT

CCCATTCTCC

CGGCCTCTGA

AAAAGCTTGG

AAGGCTGGTA

CCGTGTCCCA

ACGAGTTCAA

TGGTGATTAT

ACAGAATTAA

TTGCCAAAAG

TAGACATGGT

GCCACCTTAG

CAGAAATTGA

TCCAGGAGGA

AAGATGCTTT

ACTTTTGCTG

GGACAAACTA

ATGTGTTAAA

TGAATGGGAG

CAGTCCTCAG

ACCCAGACCT

GTTGGTGAGA

TGCCTGGACG

CCTCTTCACC

TTTTCCCCAG

AGGGGCAGGT

CCTAAGCCCA

CCAGATTCCA

CACATGCCCA

CAGGTGCCCT

CCTCCATCTC

CAAAACCCAA

ACGTGAGCCA

ATAATGCCAA

TCCTCACCGT

ACAAAGCCCT

GGGTGCGAGG

GCTGTACCAA

CCATCCCGGG

TATCCCAGCG

ACCACGCCTC

GACAAGAGCA

CACAACCACT

GCAAGCCCCC

TACATACTTC

CGAGACTGTG

GAGGCAGAGC

CCCTAGGGTG

CCCTCCCTCC

GACAGACACA

TCCCGACCTC

CCTCACCCAT

ATGGGGACAC

GATGCCCACA

GCCACACGGC

CTGCACAGCA

CCCCCACGAG

GCTGACCTGC

CACJACACAGG

CCCTTCCCTG

TTGCCCCTCC

ATAAAATGAG

GGTGGGGCAG

GGTGGGCTCT

CGCGCCCTGT

TACACTTGCC

GTTCGCCGGG

AGTCCCGCCC

GCCCCATGGC

GCTATTCCAG

ACAGCTCAGG

GGATTTTATC

AAATATGGGG

GTACTTCCAA

GGGTAGGAAA

TATAGTTCTC

TTTGGATGAT

TTGGATAGTC

ACTCTTTGTG

TTTGGGGAAA

AAAAGGCATC

CAAGTTCTCT

GCTTTAGATC

CCTACAGAGA

CTACTGATTC

CAGTGGTGGA

1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 ATGCCTTT.

TGAGGAAAAC

CTACTGCTGA

CTCTCAACAT

AGGACTTTCC

TTCAGAATTG

TTGCTTGCTT

TGCTATTTAC

TGGAAAAATA

TTCTGTAACC

TTTTTCTTAC

TCCACACAGG

GTACCTTTAG

CTTTTTAATT

TGACTAGAGA

TCATAATCAG

CTCCCACACC

TCCCCCTGA

TTTATTGCAG

CTTATAATGG

GCATTTTTTT

CACTGCATTC

GTCTGGATCG

GCTGGATGAT

CCCAACTTGT

TTATTGCAGC

ACAAATAAAG

CATTTTTTTC

TCTTATCATG

TCTGTATACC

CTGTTTCCTG

TGTGAAATTG

ATAAAGTGTA

AAGCCTGGGG

TCACTGCCCG

CTTTCCAGTC

CGCGCGGGGA

GAGGCGGTTT

CTGCGCTCGG

TCGTTCGGCT

TTATCCACAG

AATCAGGGGA

GCCAGGAACC

GTAAAAAGGC

GAGCATCACA

AAAATCGACG

TACCAGGCGT

TTCCCCCTGG

ACCGGATACC

TGTCCGCCTT

TGTAGGTATC

TCAGTTCGGT

CCCGTTCAGc

CCGACCGCTG

AGACAGGACT

TATCGCCACT

GTAGGCGGTG

CTACAGAGTT

GTATTTGGTA

TCTGCGCTCT

TGATCCGGCA

AACAAACCAC

ACGCGCAGAA JAAAAAGGATC CAGTGGAACG

AAAACTCACG

ACCTAGATCC

TTTTAAATTA

ACTTGGTCTG

ACAGTTACCA

TTTCGTTCAT

CCATAGTTGC

TTACCATCTG

GCCCCAGTGC

TTATCAGCAA

TAAACCAGCC

TCCGCCTCCA.

TCCAGTCTAT

40 AATAGTTTGC

GCAACGTTGT

GGTATGGCTT

CATTCAGCTC

TTGTGCAAAA

AAGCGGTTAG

GCAGTGTTAT CACTCATG3GT GTAAGATGCT

TTTCTGTGAC

45 CGGCGACCGA

GTTGCTCTTG

ACTTTAAAAG

TGCTCATCAT

CCGCTGTTGA

GATCCAGTTC

TTTACTTTCA

CCAGCGTTTC

GGAATAAGGG CGACACGGAA AGCATTTATC

AGGGTTATTG

AAACAAATAG GGGTTCCGCG

CTGTTTTGCT

TCTACTCCTC

CTAAGTTTTT

ACCACAAAGG

TTTATAAGTA

CATAGAGTGT

TGTAAAGGGG

CCATACCACA

CCTGAAACAT

TTACAAATAA

TAGTTGTGGT

CCTCCAGCGC

TTATAATGGT

ACTGCATTCT

GTCGACCTCT

TTATCCGCTC

TGCCTAATGA

GGGAAACCTG

GCGTATTGGG

GCGGCGAGCG

TAACGCAGGA

CGCGTTGCTG

CTCAAGTCAG

AAGCTCCCTC

TCTCCCTTCG

GTAGGTCGTT

CGCCTTATCC

GGCAGCAGCC

CTTGAAGTGG

GCTGAAGCCA

CGCTGGTAGC

TCAAGA.AGAT

TTAAGGGATT

AAAATGAAGT

ATGCTTAATC

CTGACTCCCC

TGCAATGATA

AGCCGGA.AGG

TAATTGTTGC

TGCCATTGCT

CGGTTCCCAA

CTCCTTCG-T

TATGGCAGCA

TGGTGAGTAC

CCCGGCGTCA

TGGAAAACGT

GATGTAACCC

TGGGTGAGCA

ATGTTGAATA

TCTCATGAGC

CACATTTCCC

CAGAAGAAAT

CAAAAAAGAA

TGAGTCATGC

AAAAAGCTGC

GGCATAACAG

CTGCTATTAA

TTAATAACGA

TTTGTAGAGG

AAAATGAATG

AGCAATAGCA

TTGTCCAAAC

GGGGATCTCA

TACAAATAAA

AGTTGTGGTT

AGCTAGAGCT

ACAATTCCAC

GTGAGCTAAC

TCGTGCCAGC

CGCTCTTCCG

GTATCAGCTC

AAGAACATGT

GCGTTTTTCC

AGGTGGCGA

GTGCGCTCTC

GGAAGCGTGG

CGCTCCAAGC

GGTAACTATC

ACTGGTAACA

TGGCCTAACT

GTTACCTTCG

GGTGGTTTTT

CCTTTGATCT

TTGGTCATGA

TTTAAATCAA

AGTGAGGCAC

GTCGTGTAGA

CCGCGAGACC

GCCGAGCGCA

CGGGAAGCTA

ACAGGCATCG

CGATCAAGGC

CCTCCGATCG

CTGCATAATT

TCAACCAAGT

ATACGGGATA

TCTTCGGGGC

ACTCGTGCAC

AAAACAGGAA

CTCATACTCT

GGATACATAT

CGAA.AAGTGC

GCCATCTAGT

GAGAAAGGTA

TGTGTTTAGT

ACTGCTATAC

TTATAATCAT

TAACTATGCT

ATATTTGATG

TTTTACTTGC

CAATTGTTGT

TCACAAATTT

TCATCAATGT

TGCI'GGAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTAATG

CTTCCTCGCT

ACTCAAAGGC

GAGCAAAAGG

ATAGGCTCCG

ACCCGACAGG

CTGTTCCGAC

CGCTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAAAAAzGADGT

TTGTTTGCAA

TTTCTACGGG

GATTATCAAA

TCTA-AAGTAT

CTATCTCAGC

TA-ACTACGAT

CACGCTCACC

GAAGTGGTCC

GAGTAAGTAG

TGGTGTCACG

GAGTTACATG

TTGTCAGAAZG

CTCTTACTGT

CATTCTGAGA

ATACCGCGCC

GAAAACTCTC

CCAACTGATC

GGC:AAA-ATGC

TCCTTTTTCA

TTGAATGTAT

CACCTGACGT

GATGATGAGG

GAAGACCCCA~

AATAGAACTC

AAGAAAATTA

AACATACTGT

CAAAAATTGT

TATAGTGCCT

TTTAAAAAAC

TGTTAACTTG

CACAAATWA

ATCTTATCAT

CTTCGCCCAC

CACAAATTTC

CATCAATGTA

ATGGTCATAG

AGCCGGAAGC

TGCGTTGCGC

AATCGGCCAA

CACTGACTCG

GGTA.ATACGG

CCAGCAAAAG

CCCCCCTGAC

ACTATAAA-A

CCTGCCGCTT

ATGCTCACGC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGACTAA

GATCTGTCTA

ACGGGAGGGC

GGCTCCAGAT

TGCAACTTTA

TTCGCCAGTT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTATG

ACATAGCAGA

AAGGATCTTA

TTCAGCATCT

CGCAAAAAAG

ATATTATTGA

TTAGAAAAAT

C

5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 '744 0 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8691 6O S. t 000S 6O*w es..

0 *See 0S OS 0 0 0* S. 0

S

0@055@

S

S

0 S. eS

S

0 0O S *5 0O INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 8321 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GACGGATCGG GAGATCTGCT AGGTGACCTG AGGCGCGCCG GCTTCGAATA GCCAGAGTAA CCTTTTTTTT TAATTTTATT TTATTTTATT TTTGAGATGG AGTTTGGCGC CGATCTCCCG ATCCCCTATG GTCGACTCTC AGTACAATCT GCTCTGATGC CGCATAC-TTA AGCCAGTATC

TGCTCCCTGC

CAAGGCAAGG

TGCTTCGCGA

AGTAATCAAT

TTACGGTAAA

TGACGTATGT

ATTTACGGTA

CTATTGACGT

GGGACTTTCC

GGTTTTGGCA

TCCACCCCAT

AATGTCGTAA

TCTATATAAG

TTAATACGAC

AGGTCTCGAG

CACCATGGAG

GTGAAGTGAA

TCTCCTGTGT

CTCCAGAGAA

ATCCAGACAC

ACCTGCAAAT

TGGACGACGG

CTAGCACCAA

GCACAGCGGC

GGALACTCAGG

GACTCTACTC

ACATCTGCAA

GGCCAGCACA

CATCCCGGCT

CGGAGGCCTC

GCTCTGGGCA

GCTGGGCTCA

CCCCAAAGGC

GTAACTCCCA

CCGTGCCCAG

AGAGTAGCCTr

CAGAGGCCGG

TACAGGGCAG

CAAGAACCAG

GGAGTGGGAG

CTCCGACGGC

GGGGAACGTC

CAGcc~rTcc

CTCTCGCGGT

45 AGCATGGAAA

CCACGGGTCA

GTCCCCACAC

AGGGGCTGCC

GCCCTGGGCT

50 CTCTGTAGGA

CGGGGGCATG

GGCACTAACC

GGGGACATGC

GCCCAGACCC

55 ACACGTGCAC

AGCAAGGTCC

CACCTCAAGG

CC-AGCCCTCC

CCACGTCACG

60 CAGCCTCGAC

CCTTGACCCT

CGCATTGTCT

GGGAGGATTG

AGGCGGAAAG

65 TAAGCGCGGC

CGCCCGCTCC

AAGGGAAAAA

TTGTGTGTTG

CTTGACCGAC

TGTACGGGCC

TACGGGGTCA

TGGCCCGCCT

TCCCATAGTA

AACTGCCCAC

CAATGACGGT

TACTTGGCAG

GTACATCAAT

TGACGTCAjAT

CAACTCCGCC

CAGAGCTCTC

TCACTATAGG

TCTCTAGATA

TTGTGGTTAA

TCTGGTGGAG

AACCTCTGGA

GAGGCTGGAG

TGTAAAGGGT

GAGCCGTCTG

GGCCTGGTTT

GGGCCCATCG

CCTGGGCTGC

CGCCCTGACC

CCTCAGCAGC

CGTGAATCAC

GGGAGGGAGG

ATGCAGCCCC

TGCCCGCCCC

GGCACACGCT

GACCTGCCAA

CAAACTCTCC

ATCTTCTCTC

GTAAGCCAGC

GCATCCAGGG

CTCGGCCCAC

CCCCGAGAAC

-TCAGCCT-A

AGCAATGGGC

TCCTTCTTCC

T-ICTCATGCT

CTGTCTCCGG

CGCACGAGGA

TAA.AGCACCC

GGCCGAGTCT

TGGCCCAGGC

CTCGGCAGGG

GGGCCACGGG

GACTGTCCTG

CCTAGTCCAT

CCTGGCTGCC

ACTCTCGGGC

GTTCAACAAA

GCCTCACACA

TCGCACACGT

CCCACGAGCC

TCTCACAAGG

TCCCTGGCCC

TGTGCCTTCT

GGAAGGTGCC

GAGTAGGTGT

GGAAGACAAT

AACCAGCTGG

GGGTGTGGTG

TTTCGCTTTC

AAGCATGCAT

GAGGTCGCTG

AATTGCATGA

AGATATACGC

TTAGTTCATA

GGCTGACCGC

ACGCCAATAG

TTGGCAGTAC

AAATGGCCCG

TACATCTACG

GGGCGTGGAT

GGGAGTTTGT

CCATTGACGC

TGGCTAACTA

GAGACCCAAG

ACCGGTCAAT

GCTTGGTCCT

TCTGGGGGAG

TTCACTTTCA

TGGGTCGCAT

CGATTCACCA

AAGTCTGAGG

GCTTACTGGG

GTCTTCCCCC

CTGGTCAAGG

AGCGGCGTGC

GTGGTCACCG

AAGCCCAGCA

GTGTCTGCTG

AGTCCAGGGC

ACTCATGCTC

AGGTGCCCCT

GAGCCATATC

ACTCCCTCAG

TGCAGAGCCC

CCAGGCCTCG

ACACACCACG

CCTCTGCCCT

CAC-AGGTGTA

CCTGCCTGGT

AGCCGGAGAA

TCTACAGCAA

CCGTGATGCA

GTAAATGAGT

TGCTTGGCAC

AG CGCTGCC C

GAGGCCTGAG

TGTGCAGGTG

TGGGGGATTT

AAG-CCTAGG

TTCTGTGAGC

GTGCGTAGGG

CTGCCCAGCC

CCTGTGGAGG

CCCCGCACTG

CGGAGCCTCA

GAACACTCCT

TCTCGGCAGC

GTGCCCCTGC

TGGCCCACTT

AGTTGCCAGC

ACTCCCACTG

CATTCTATTC

AGCAGGCATG

GGCTCTAGGG

GTTACGCGCA

TTCCCTTCCT

CTCAATTAGT

AGTAGTGCGC

AGAATCTGCT

GTTGACATTG

GCCCATATAT

CCAACGACCC

GGACTTTCCA

ATCAAGTGTA

CCTGGCAT-TA

TATTAGTCAT

AGCGGTTTGA

TTTGGCACCA

AAATGGGCGG

GAGA.ACCCAC

CTTGGTACCA

CGATTGGAAT

TCCTTGTCCT

GCTTAGTGCA

GTGACTATTA

ACATTAGTCA

TCTCCAGAGA

ACACAGCCAT

GCCAAGGGAC

TGGCACCCTC

ACTACTTCCC

ACACCTTCCC

TGCCCTCCAG

ACACCAAGGT

GAAGCCAGGC

AGCAAGGCAG

AGGGAGAGGG

AACCCAGGCC

CGGGAGGACC

CTCGGACACC

AAATCTTGTG

CCCT;CCAGCT

TGGGTACCAA

GAGAGTGACC

CACCCTGCCC

CAAAGGCTTC

CAACTACAAG

GCTCACCGTG

TGAGGCTCTG

GCGACGGCCG

GTACCCCCTG

TGGGCCCCTG

TGGCATGAGG

TGCCTGGGCC

GCCAGCGTGG

AGCCCCTGGG

GCCCCTGTCC

ACAGGCCCTC

TCGCACCCGC

GACTGGTGCA

AGGTTGGCCG

CCCGGGCGAA

CGGACACAGG

TTCTCCACAT

AGCCGCCACA

CCCAGTGCCG

CATCTGTTGT

TCCTTTCCTA

TGGGGGGTGG

CTGGGGATGC

GGTATCCCCA

GCGTGACCGC

TTCTCGCCAC

CAGCAACCAT

GAGCAAAATT

TAGGGTTAGG

ATTATTGACT

GGAGTTCCGC

CCGCCCATTG

TTGACGTCAA

TCATATGCCA

TGCCCAGTAC

CGCTATTACC

CTCACGGGGA

AAATCAACGG

TAGGCGTGTA

TGCTTACTGG

ATTTAAATTG

TCTTGCGGCC

TGTTTTAAAA

GCCTGGAGGG

CATGTATTGG

AGGTGGTGAT

CAATGCCAAG

GTATTACTGT

TCTGGTCACG

CTCCAAGAGC

CGAACCGGTG

GGCTGTCCTA

CAGCTTGGGC

GGACAAGAAA

TCAGCGCTC

GCCCCGTCTG

TCTTCTGGCT

CTGC.:CA2CAA

CTGCCCCTGA

TTCTCTCCTC

ACAP.AACTCA

C-AAGGCGGGA

CATGTCCGGA

GCTGTACCAA

CCATCCCGGG

TATCCCAGCG

ACCACGCCTC

GACAAGAGCA

CACAACCACT

GCAAGCCCCC

TACATACTTC

CGAGACTGTG

GAGGCAGAGC

CCCTAGGGTG

CCCTCCCTCC

GACAGACACA

TCCCGACCTC

CCTCACCCAT

ATGGGGACAC

GATGCCCACA

GCCACACGGC

CTGCACAGCA

CCCCCACGAG

GCTGACCTGC

CACACACAGG

CCCTTCCCTG

TTGCCCCTCC

ATAAAATGAG

GGTGGGGCAG

GGTGGGCTCT

CGCGCCCTGT

TACACTTGCC

GTTCGCCGGG

AGTCCCGCCC

TAAGCTACAA

CGTTTTGCGC

AGTTATTAAT

GTTACATAAC

ACGTCAATAA

TGGGTGGACT

AGTACGCCCC

ATGACCTTAT

ATGGTGATGC

TTTCCAAGTC

GACTTTCCAA

CGGTGGGAGG

CTTATCGAAA

ATATCTCCTT

GCTTGCTAGC

GGTGTCCAGT

TCCCTGAAAG

GTTCGCCAGA

ATAACCGACT

AACACCCTGT

GCAAGAGGCC

GTCTCTGTAG

ACCTCTGGGG

ACGGTGTCGT

CAGTCCTCAG

ACCCAGACCT

GTTGGTGAG-A

TGCCTGGACG

CCTCTTCACC

TTTTCCCCAG

AGGGGCAGGT

CCTAAGCCCA

CCAGATTCCA

CACATGCCCA

CAGGTGCCCT

GCCA.CATGGA

CCTCTGTCCC

ATGAGCTGAC

ACATCGCCGT

CCGTGCTGGA

GGTGGCAGCA

ACACGCAGAA

GCTCCCCGGG

CCGGGCGCCC

ATGGTTCT'T

GGGTCCCACT

GGGCTCAGCC

AGCAGCACCT

CAGCCCCTGC

CATGCCCACT

CTACCCCCAC

AACCGACTCC

CACACACTCA

CACCACACAC

CCCAGACCAG

CC-CCACGCGG

TCAGACAAAC

GGATCACACA

CAGGACGGAT

CCCGTGCCTT

GAAATTGCAT

GACAGCAAGG

ATGGCTTCTG

AGCGGCGCAT

AGCGCCCTAG

CCTCTCAAAA

CTAACTCCGC

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200

CCATCCCGCC

TTTTTATTTA

GAGGCTTTTT

CGCGCCAAAC

TCATGGTTCG

ACGGAGACCT

CAACCTCTTC

CCATTCCTGA

TCAAAGAACC

TTATTGAACA

CTGTTTACCA

TGCAGGAATT

TCCCAGAATA

TTGAAGTCTA

TAAAGCTATG

GGAACCTTAC

TAAGGTAAAT

TGTATTTTAG

TGAGGAAAAC

cCTTCAACAT

TTCAGAATTG

TGCTATTTAC

TTCTGTAACC

TCCACACAGG

CTTTTTAATT

TCATAATCAG

TCCCCCTGAA

CTTATAATGG

CACTGCATTC

GCTGGATGAT

TTATTGCAGC

CATTTTTTTC

TCTGTA:TACC

TGTGAAATTG

AAGCCTGGGG

CTTTCCAGTC

GAC-GCGGTTT

TCGTTCGGCT

AATCAGGGGA

GTAAAAAGGC

AAAATCGACG

TTCCCCCTGG

TGTCCGCCTT

TCAGTTCGGT

CCGACCGCTG

TATCGCCACT

CTACAGAGTT

TCTGCGCTCT

AACAAACCAC

50 AAAAAGGATC

AAAACTCACG

TTTTAAATTA

ACAGTTACCA

CCATAGTTGC

GCCCCAGTGC

TAAACCAGCC

TCCAGTCTAT

GCAACGTTGT

CATTCAGCTC

AAGCGGTTAG

CACTCATGGT

TTTCTGTGAC

GTTGCTCTTG

TGCTCATCAT

65 GATCCAGTTC

CCAGCGTTTC

CGACACGGAA

CCTAACTCCG

TGCAGAGGCC

TGGAGGCCTA

TTGACGGCAA

ACCATTGAAC

ACCCTGGCCT

AGTGGAAGGT

GAAGAATCGA

ACCACGAGGA

ACCGGAATTG

GGAAGCCATG

TGAAAGTGAC

CCCAGGCGTC

CGAGAAGAAA

CATTTTTATA

TTCTGTGGTG

ATAAAATTTT

ATTCCAACCT

CTGTTTTGCT

TCTACTCCTC

CTAAGTTTTT

ACCACAAAGG

TTTATAAGTA

CATAGAGTGT

TGTAAAGGGG

CCATACCACA

CCTGAAACAT

TTACAAATAA

TAGTTGTGGT

CCTCCAGCGC

TTATAATGGT

ACTGCATTCT

GTCGACCTCT

TTATCCGCTC

TGCCTAATGA

GGGAhAACCT-G

GCGTATTGGG

GCGGCGAGCG

TAACGCAGGA

CGCGTTGCTG

C-.CAAr.TCAG AAG CTC CCT C

TCTCCCTTCG

GTAkGGTCGTT

CGCCTTATCC

GGCAGCAGCC

CTTGAAGTGG

GCTGAAGCCA

CGCTGGTAGC

TCAAGAAGAT

TTAAGGGATT

AAAATGAAGT

ATGCTTAATC

CTGACTCCCC

TGCAATGATA

AGCCGGAAGG

TAATTGTTGC

TGCCATTGCT

CGGTTCCCAA

CTCCTTCGGT

TATGGCAGCA

TGGTGAGTAC

CCCGGCGTCA

TGGAAAACGT

GATGTAACCC

TGGGTGAGCA

ATGTTGAATA

CCCAGTTCCG

GAGGCCGCCT

GGCTTTTGCA

TCCTAGCGTG

TGCATCGTCG

CCGCTCAGGA

AAACAGAATC

CCTTTAAAGG

GCTCATTTTC

GCAAGTAAAG

AATCAACCAG

ACGTTTTTCC

CTCTCTGAGG

GACTAACAGG

AGACCATGGG

TGACATAATT

TAAGTGTATA

ATGGAACTGA

CAGAAGAAAT

CAAAAAAGAA

TGAGTCATGC

AAAAAGCTGC

GGCATAACAG

CTGCTATTAA

TTAATAAGGA

TTTGTAGAGG

AAAATGAATG

AGCAATAGCA

TTGTCCAAAC

GGGGATCTCA

TACAAATAAA

AGTTGTGGTT

AGCTAGAGCT

ACAATTCCAC

GTGAGCTAPC

TCGTGCCAGC

CGCTCTTCCG

GTATCAGCTC

A-AGAACATGT

GCGTTTTTCC

AGGTGGCGAA

GTGCGCTCTC

GGAAGCGTGG

CGCTCCAAGC

GGTAACTATC

ACTGGTAACA

TGGCCTAACT

GTTACCTTCG

GGTGGTTTTT

CCTTTGATCT

TTGGTCATGA

TTTAAATCAA

AGTGAGGCAC

GTCGTGTAGA

CCGCGAGACC

GCCGAGCGCA

CGGGAAGCTA

ACAGGCATCG

CGATCAAGGC

CCTCCGATCG

CTGCATAATT

TCAACCAAGT

ATACGGGATA

TCTTCGGGGC

ACTICGTGCAC

AAAACAGGAA

CTCATACTCT

CCCATTCTCC

CGGCCTCTGA

AAAAGCrroo

AAGGCTGGTA

CCGTGTcCCCA

ACGAGT"ICA

TGGTGATTAT

ACAGAATvL

TTGCCAAAAG

TAGACATGGT

GCCACCTTAG

CAGAAATTGA

TCCAGGAGGA

AAGATGCTTT

ACTTTTGCTG

GGACAA-ACTA

ATGTGTTAAA

TGAATGGGAG

GCCATCTAGT

GAGAAAGGTA

TGTGTTTAGT

ACTGCTATAC

TTATAATCAT

TAACTATGCT

ATATTTGATG

TTTTACTTGC

CAATTGTTGT

TCACAAATTT

TCATCAAkTGT

TGCTGGAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTALATG

CTTCCTCGCT

ACTCAAAGGC

GAGCAAAAGG

ATAGGC'rCCG

ACCCGACAGG

CTGTTCCGAC

CGCTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAAAAAGAGT

TTGTTTGCAA

TTTCTACGGG

GATTATCAAA

TCTAAAGTAT

CTATCTCAGC

TAACTACGAT

CACGCTCACC

GAAGTGGTCC

GAGTAAGTAG

TGGTGTCACG

GAGTTACATG

TTGTCAGAAG

CTCTTACTGT

CATTCTGAGA

ATACCGCGCC

GAAAACTCTC

CCAACTGATC

GGCAAAATGC

TCCTTTTTCA

GCCCCATGGC

GCTATTCCAG

ACAGCTCAGG

GGATTTTATC

AAATATGGGG

GTACTTCCAA

GGGTAGGAAA

TATAGTTCTC

TTTGGATGAT

TTGGATAGTC

ACTCTTTGTG

TTTGGGGAAA

AAAAGGCATC

CAAGTTCTCT

GCTTTAGATC

CCTACAGAGA

CTACTGATTC

CAGTGGTGGA

GATGATGAGG

GAAGACCCCA

AATAGAACTC

AAGAAAATTA

AACATACTGT

CAAAAATTGT

TATAGTGCCT

TTTAAAAAAC

TGTTAACTTG

CACAAATAAA

ATCTTATCAT

CTTCGCCCAC

CACA-AATTTC

CATCAATGTA

ATGGTCATAG

AGCCGC-AAGC

TGCGTTGCGC

AATCGGCCAA

CACTGACTCG

GGTAATACGG

CCAGCAAAAG

CCCCCCTGAC

ACTATAAAGA

CCTGCCGCTT

ATGCTCACGC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGAGTAA

GATCTGTCTA

ACGGGAGGGC

GGCTCCAGAT

TGCAACTTTA

TTCGCCAGTT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTATG

ACATAGCAGA

AAGGATCTTA

TTCAGCATCT

CGCAAAAAAG

ATATTATTGA

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATTGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGT

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

ATGCCTTTAA

CTACTGCTGA

AGGACTTTCC

TTGCTTGCrT

TGGAAAAATA

TTTTTCTTAC

GTACCTTTAG

TGACTAGAGA

CTCCCACACC

TTTATTGCAG

GCATTTTTT1

GTCTGGATCG

CCCAACTTGT

ACAAATAAJAG

TCTTATCATG

CTGTTTCCTG

ATAAAGTGTA

TCACTGCCCG

CGCGCGGGGA

CTGCGCTCGG

TTATCCACAG

GCCAGGAACC

GAGCATCACA

TACCAGGCGT

ACCGGATACC

TGTAGGTATC

CCCGTTCA.GC

AGACACGACT

GTAGGCGGTG

GTATTTGGTA

TGATCCGGCA

ACGCGCAGAA

CAGTGGAACG

ACCTAGATCC

ACTTGGTCTG

TTTCGTTCAT

TTACCATCTG

TTATCAGCAA

TCCGCCTCCA

AATAGTTTGC

GGTATGGCTT

TTGTGCAAAA

GCAGTGTTAT

GTAAGATGCT

CGGCGACCGA

ACTTTAAAAG

CCGCTGTTGA

TTTACTTTCA

GGAATAAGGG

AGCATTTATC

4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220

S

~4 AGGGTTATTG TCTCATGAGC GGATACATAT TTGAALTGTAT TTAGAAAAAT

AAACAAATAG

GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT

C

INFORMATION FOR SEQ ID NO:11: SEQUENCE

CHAR.ACTERISTICS:

LENGTH: 8897 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID No:l1: 8280 8321

GGTACCAATT

TGTTGGTGCT

CAGTCTCCCT

TCATTGTACA

CTCCACAGCT

GCGGCAGTGG

TGGGAGTTTA

AGTTGGAAAT

AAACTCTGAG

AGGTCAGAAA

AGAACTTTAT

TACGCTTCTT

CCTAACATGC

CATCCTGTTT

CATCTGATGA

ATCCCAGAGA

AGGAGA.GTGT

CGCTGAGCAA.

GCCTGAGCTC-

CCCACCTGCT

CCACAGGGGA

CCTCCTTGGC

CACCTGTGGT

CAATTTCTCT

kATCATCCTT

ACAAGCCTTC

TCCCCTCCTC

TCCTTTGATT

TATAAAGAGA

45 ATAAZACAAAC

ATGCCTTATT

GAGTACTTTC

AAATGTTGCA

ACTTCTAGAT

50 CTTTATTTAC

TTAA.ACTGTG

TATACCTACT

AAAAGATATG

TAGAAATTTG

55 ACAAGAAGGG

ATGATCTGTG

TCTTATACCC

GTAAGTGGGG

TGAGTCTGCC

60 CATCTGTGCC

CTTCAGCAAG

AACACTCACA

ATGGGGCACT

ACCTCTCTCT

65 CAAATGACTG

TGGGGAAGGA

GAATGTTGAT

TAAATTGATA

GATGTTCTGG

GCCTGTCAGT

TAATAATGGC

CCTGATCTAC

ATCAGGGACA

TTACTGCTTT

AAAACGTAAG

GGGGTCGGAT

AGCATGCAAA

TAJAGGAATAG

GGTCTCCTTG

CCTTATCCGC

GCTTCTrTTCC

GCAGTTGAAA

GGCCAAAGTA

CACAGAGCAG

AGCAGACTAC

GCCCGTCACA

CCTCAGTTCC

CCTACCCCTA

TTTAATTATG

TTCTCTCTTT

TATAAGGGAC

CATTCTATTT

TGTCCTCACA

AGCAAGCCCT

CAATTCCCTG

ATCATTCATT

ATAGGGGAAA

TACATTTTTA

CACAACCTA

AAGGTTCTAT

GACTGAGTGT

AAAAGCCAAA

GTATGTTTAT

CACACAGATG

TTCTGTATGT

GATGGAAATT

GCTTCTGGGG

CACTGTTCTG

AGTTAATAGA

GCCTGGGATC

TTCCAGGGCT

CTGTTTGGCT

GGGACAGAGG

TGTTTGGGAA

CTGGCCCTGC

GCCTACACTC

ACAATCCCTT

CAGTCATGGA

GAGTATCAAA

TCTCCTTAGG

ATTCCTGCTT

CTTGGAGATC

AACACCTATT

AAAGTTTCCA

GATTTCACAC

CAAGGTTCAC

TCTCGAGTCT

GACGTGGCCA

GCCCTCAGAA

GGGGAAGCTA

CTATA.ATTAT

AAACAACACA

TCAGGAACTG

TCTGGAACTG

CAGTGGAAGG

GAGAGCAA.GG

GAGA.AACACA

AAGAGCTTCA

AGCCTGACCC

TTGCGGTCCT

CTAATGTTGG

CCTCATTTAA.

i AA.ATATGTA

TACCCTATCA

GTCCCCTGGG

CATAGTCCTT

AGAATCAACC

GCAACATGAT

TGTTTAAGTT

AACAGGTACT

TTTAATCCAC

AAAGCTGAGA

CCCCACCCAC

AATTGGAAAT

ACATTAGAAT

AATCTCATAA

TTTCATCCAT

ACTCTTAGCT

TCTTGGTAAT

TATACACATT

TAGAAGAGGA

AAATAGCTAC

CAAGGTGCTC

AGCTAGGAGC

ACAGAATTAA

GGGGGAAGGG

CCCTCTCAGC

TGAAGGGGTT

TGTCCTGCTT

GAAACTACAT

TCTTTCAA.AC

TCTCGAGCAC

CCAGCAGTGAL

AAGCGTCCAT

TAGAATGGTA

ACCGATTTTC

TCAAGATCAG

ATGTTCCATT

CTAGATAACC

TTCTTTGCCT

TGGCTGCAAA

GGA.AGAAACT

CTGGGAZTAAG

CCCAAGGGCA

TGGCTGCACC

CCTCTGTTGT

TGGATAACGC

ACAGCACCTA

AAGTCTACGC

ACAGGizGAGA

CCTCCCATCC

CCAGCTCATC

AGGAGAATGA

TAATTATTAT

GTCATCCTA.A

TCCTCTGCAA

CCATGGTAGG

TTTA.A-GGGTG

AAAGCAAAzTT

ATAAAATAAC

CATCATGGTA

GAGGGACTCC

ACTATACTGT

GACAAA-TATA

CAAAAAACTA

AGCCCGATTG

ACCCAATGAG

AAATA.ATGTT

ATAAAGTTCA

GGGGGTGGGC

GTTCTI:TTCC

ATGCTTCAAA

ATAAGTAATA

CTGCCTAATC

AACAAAACAA

ACACATACAT

CCTTGCCCAG

CACATGTAAA

TACTCATCCA

CAGGAGTAAC

TGTTTTTCTT

AAGGAAGCAC

TTTGGAGGTT

CATGAAGTTG

TGTTTTGATG

CTCTTGCAGA

CCTGCAGAA.

TGGGGTCCCA

CAGAGTGGAG

CACGTTCGGC

GGTCAATCGA

AP.AGCATTGA

GAGCTCCAAC

CAAAACATCA.

CATGCTGTTT

GAACTTTGTT

ATCTGTCTTC

GTGCCTGCTG

CCTCCAATCG

CAGCCTCAGC

CTGCGAAGTC

GTGTTAGAGG

TTTGGCCTCT

TT.TCACCTCA

ATA-AATAAAG

CTGTTGTTTT

GGCACGTAAC

GACAGTCCTC

AGAGACTTCC

ACAGGTCTTA

TTTCAAkAAGA

AJACACAATA-A

CTTAGACTTA

TGTCTGCCAA

GAGATTAAAA

TTCTATAACT

TGCAAGAATG

TCCAACAATA

GAGAATTAAC

ACATAAGAGA

AAACCAGGTA

GAGTTAGTGC

TCGTGTGGGG

ATAACTTCAC

GGTCAAGACC

CTGCCCWCTT

CAGGCCTGCT

AGAAATTAAA

ACACTGGAAA

TGAGGACTCT

TCCAACACAC

TAACACAGCA

TCCAGTCAGT

CTTGCCCTTC

TGAGTAGGGG

CCTGTTAGGC

ACCCAAATTC

TCTAGTCAGA

CCAGGCCAGT

GACAGGTTCA

GCTGAGGATC

TCGGGGACAA

TTGGAATTCT

GTTTACTGCA

AAAACAATTT

AGATTTTAAA

TCTGTCTGTC

ACTTAAACAC

ATCTTCCCGC

AATAACTTCT

GGTAACTCCC

AGCACCCTGA

ACCCATCAGG

GAGAAGTGCC

GACCCTTTTT

CCCCCCTCCT

TGAATCTTTG

ACCAACTACT

CATTTATAAA

CCTCAAACCC

TTCC-TTGTTT

CAGTCATATA

AGAAACCTGC

AAGCAATTAA

ATGGAATGTC

GGGCCGTATT

ACATTCATTA

CAGCAATCCC

TTCAAAGCAG

GAATGAGTTA

AAGCTACAAC

AACTCAATGC

AAAATAAkAGT

CTGGGAGAAG

TTGTGCAGTT

ATAAAGAACA

AACGCAGCTG

GAGCCCTGAA

ATTTTCCTGG

TGAAACAGAC

CCCATGTATG

TCCTCATTCT

CTTTCTAAGT

TCCCTTCCCT

ACTGGGAAAG

TGCCTCTTGA

TGAGACTCAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1 620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120

TAATGTCCCT

CAAAGGCAGG

ATCCAACCGC

GCCTCGACTG

TTGACCCTGG

CATTGTCTGA

GAGGATTGGG

GCGGAAAGAA

AGCGCGGCGG

CCCGCTCCTT

GGGAAAAAAA

ATCCCGCCCC

TTTATTTATG

GGCTTTTTTG

CGCCAAACTT

ATGGTTCGAC

GGAGACCTAC

ACCTCTTCAG

ATTCCTGAGA

AAAGAACCAC

ATTGAACAAC

GTTTACCAGG

CAGGAATTTG

CCAGAATACC

GAAGTCTACG

AAGCTATGCA

AACCTTACTT

AGGTAAATAT

TATTTTAGAT

AGGAAAACCT

CTCAACATTC

CAGAATTGCT

CTATTTACAC

CTGTAACCTT

CACACAGGCA

TTTTAATTTG

ATAATCAGCC

CCCCTGAACC

TATAATGGTT

CTGCATTCTA

TGGATGATCC

ATTGCAGCTT

TTTTTTTCAC

TGTATACCGT

45 TGAAATTGTT

GCCTGGGGTG

TTCCAGTCGG

GGCGGTTTGC

GTTCGGCTGC

50 TCAGGGGATA

AAAAAGGCCG

AATCGACGCT

CCCCCTGGAA

TCCGCCTTTC

55 AGTTCGGTGT

GACCGCTGCG

TCGCCACTGG

ACAGAGTTCT

TGCGCTCTGC

60 CAAACCACCG

AAAGGATCTC

AACTCACGTT

TTAAATTAAA

AGTTACCAAT

65 ATAGTTGCCT

CCCAGTGCTG

AACCAGCCAG

TCCAATGACA

CATAATCCAG

GGAAGGGCCC

TGCCTTCTAG

AAGGTGCCAC

GTAGGTGTCA

AAGACAATAG

CCAGCTGGGG

GTGTGGTGGT

TCGCTTTCTT

GCATGCATCT

TAACTCCGCC

CAGAGGCCGA

GAGGCCTAGG

GACGGCAATC

CATTGAACTG

CCTGGCCTCC

TGGAAGGTAA

AGAATCGACC

CACGAGGAGC

CGGAATTGGC

AAGCCATGAA

AAAGTGACAC

CAGGCGTCCT

AGAAGAAAGA

TTTTTATAAG

CTGTGGTGTG

AAAATTTTTA

TCCAACCTAT

GTTTTGCTCA

TACTCCTCC-A

AAGTTTTTTG

CACAAAGGAA

TATAAGTA.GG

TAGAGTGTCT

TIAAAGGGGTT

ATACCACATT

TGAAACATAA

ACAAATAAAG

GTTGTGGTTT

TCCAGCGCGG

ATA-ATGGTTA

TGCATTCTAG

CGACCTCTAG

ATCCGCTCAC

CCTLATGAGT

G-AAACCTGTC

GTATTGGGCG

GGCGAGCGGT

ACGCAGGAAA

CGTTGCTGGC

CAAGTCAGAG

GCTCCCTCGT

TCCCTTCGGG

AGGTCGTTCG

CCTTATCCGG

CAGCAGCCAC

TGAAGTGGTG

TGAAGCCAGT

CTGGTAGCGG

AAGAAGATCC

AAGGGATTTT

AATGAAGTTT

GCTTAATCAG

GACTCCCCGT

CAATGATACC

CCGGAAGGGC

TGAACTTGCT

TTATGAATTC

TATTCTATAG

TTGCCAGCCA

TCCCACTGTC

TTCTATTCTG

CAGGCATGCT

CTCTAGGGGG

TACGCGCAGC

CCCTTCCTTT

CAATTAGTCA

CAGTTCCGCC

GGCCGCCTCG

CTTTTGCAAA

CTAGCGTGAA

CATCGTCGCC

GCTCAGGAAC

ACAGAATCTG

TTTAAAGGAC

TCATTTTCTT

AAGTAAAGTA

TCAACCAGGC

GTTTTTCCCA

CTCTGAGGTC

CTAACAGGAA

ACCATGGGAC

ACATAATTGG

AGTGTATAAT

GGAACTGATG

GAAGAAATGC

AAAAAGAAGA

AGTCATGCTG

AAAGCTGCAC

CATAACAGTT

GCTATTAATA

AATAAGGAAT

TGT-ACAGGTT

AATGAATGCA

CAAT.AGCATC

GTCCAAACTC

GGATCTCATG

CAAATAAAGC

TTGTGGTTTG

CTAGAGCTTG

AATTCCACAC

GAGCTAACTC

GTGCCAGCTG

CTCTTCCGCT

ATCAGCTCAC

GAACATGTGA

GTTTTTCCAT

GTGGCGAAAC

GCGCTCTCCT

AAGCGTGGCG

CTCCAAGCTG

TAACTATCGT

TGGTAACAGG

GCCTAACTAC

TACCTTCGGA

TGGTTTTTTT

TTTGATCTTT

GGTCATGAGA

TAAATCAATC

TGAGGCACCT

CGTGTAGATA

GCGAGACCCA

CGAGCGCAGA

CACTCATCCC

TTGCGGCCGC

TGTCACCTAA

TCTGTTGTTT

CTTTCCTAAT

GGGGGTGGGG

GGGGATGCrG

TATCCCCACG

GTGACCGCTA

CTCGCCACGT

GCALACCATAG

CATTCTCCGC

GCCTCTGAGC

AAGCTTGGAC

GGCTGGTAGG

GTGTCCCAAA

GAGTTCAAGT

GTGATTATGG

AGAATTAATA

GCCAAAAGTT

GACATGGTTT

CACCTTAGAC

GAAATTGATT

CJAGGAGGAAA

GATGCTTTCA

TTTTGCTGGC

ACAAACTACC

GTGTTAAACT

AATGGGAGCA

CATCTAGTGA

GAAAGGTAGA

TGTTTAGTAA

TGCTATACAA

ATAATCATAA

ACTATGCTCA

ATTTGATGTA

TTACTTGCTT

ATTGTTGTTG

ACAAA-TTTCA

ATCAATGTAT

CTGGAGTTCT

AATACICATCA

TCCAAACTCA

GCGTAATCAT

AACATACGAC-

ACATTAATTG

CATTAATGAA

TCCTCGCTCA

TCAAAGGCGG

GCAAAAGGCC

AGGCTCCGCC

CCGACAGGAC

GTTCCGACCC

CTTTCTCAAT

GGCTG7rGTGC

CTTGAGTCCA

ATTAGCAGAG

GGCTACACTA

AAAAGAGTTG

GTTTGCAAGC

TCTACGGGGT

TTATCAAAAA

TAAAGTATAT

ATCTCAGCGA

ACTACGATAC

CGCTCACCGG

AGTGGTCCTG

TGGGGGCCAA

TTGCTAGCTT

ATGCTAGAGC

GCCCCTCCCC

AAAkATGAGGA

TGGGGCAGGA

TGGGCTCTAT

CGCCCTGTAG

CACTTGCCAG

TCGCCGGGCC

TCCCGCCCCT

CCCATGGCTG

TATTCCAGAA

AGCTCAGGGC

ATTTTATCCC

ATATGGGGAT

ACTTCCAAAG

GTAGGAAAAC

TAGTTCTCAG

TGGATGATGC

GGATAGTCGG

TCTTTGTGAC

TGGGGAAATA

AAGGCATCA.A

AGTTCTCTGC

TTTAGATCTC

TACAGAGATT

ACTGATTCTA

GTGGTGGAAT

TGATGAGGCT

AGACCCCAAG

TAGAJACTCTT

GAAAATTATG

CATACTGTTT

AP.AATTGTGT

TAGTGCCTTG

TAAAAAACCT

TTAACTTGTT

rC.A.ATAP.GC

CTTA-TCATGT

TCG-CCCACCC

C-AAATTTCAC

TCAATGTATC

GGTCATAGCT

CCGGAAGCAT

CGTTGCGCTC

TCGGCCAACG

CTGACTCGCT

TAATACGGTT

AGCAAAAGGC

CCCCTGACGA

TATAAAGATA

TGCCGCTTAC

GCTCACGCTG

ACGAACCCCC

ACCCGGTAAG

CGAGGTATGT

GAAGGACAGT

GTAGCTCTTG

AGCAGATTAC

CTGACGCTCA

GGATCTTCAC

ATGAGTAAAC

TCTGTCTATT

GGGAGGGCTT

CTCCAGATTT

CAACTTTATC

ATTGAACAAT

CACGTGTTGG

TCGCTGATCA

CGTGCCTTCC

AATTGCATCG

CAGCAAGGGG

GGCTTCTGAG

CGGCGCATTA

CGCCCTAGCG

TCTCAAAAAA

AACTCCGCCC

ACTAATTTTT

GTAGTGAGGA

TGCGATTTCG

CGCTGCCATC

TGGCAAGAAC

AATGACCACA

CTGGTTCTCC

TAGAGAACTC

CTTAAGACTT

AGGCAGTTCT

AAGGATCATG

TAAACTTCTC

GTATAAGTTT

TCCCCTCCTA

TTTGTGAAGG

TAAAGCTCTA

ATTGTTTGTG

GCCTTTAATG

ACTGCTGACT

GACTTTCCTT

GCTTGCTTTG

GAAAAATATT

TTTCTTACTC

ACCTTTAGCT

ACTAGAGATC

CCCACACCTC

TATTGCAGCT

ATTTTTTTCA

CTGGATCGGC

CA.ACT?-GLTT

AAATAAAGCA

TTATCATGTC

GTTTCCTGTG

AAAGTG-iAAA

ACTGCCCGCT

CGCGGGGAGA

GCGCTCGGTC

ATCCACAGAA

CAGGAACCGT

GCATCACAAA

CCAGGCGTTT

CGGATACCTG

TAGGTATCTC

CGTTCAGCCC

ACACGACTTA

AGGCGGTGCT

ATTTGGTATC

ATCCGGCAAA

GCGCAGAAAA

GTGGAACGAA

CTAGATCCTT

TTGGTCTGAC

TCGTTCATCC

ACCATCTGGC

ATCAGCAATA

CGCCTCCATC

3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140

CAGTCTATTA

AACGTTGTTG

TTCAGCTCCG

GCGGTTAGCT

CTCATGGTTA

TCTGTGACTG

TGCTCTTGCC

CTCATCATTG

TCCAGTTCGA

AGCGTTTCTG

ACACGGAAAT

GGTTATTGTC

GTTCCGCGCA

GCCCGGGTGA

TATTTTATTT

TCTCAGTACA

GTTGGAGGTC

CGACAATTGC

GGCCAGATAT

GTCATTAGTT

GCCTGGCTGA

AGTAACGCCA

CCACTTGGCA

CGGTAAATGG

GCAGTACATC

CAATGGGCGT

CAATGGGAGT

CGCCCCATTG

TCTCTGGCTA

TAGGGAGACC

ATTGTTGCCG

CCATTGCTAC

GTTCCCAACG

CCTTCGGTCC

TGGCAGCACT

GTGAGTACTC

CGGCGTCAAT

GAAAACGTTC

TGTAACCCAC

GGTGAGCAAA

GTTGAATACT

TCATGAGCGG

CATTTCCCCG

CCTGAGGCGC

TATTTTTGAG

ATCTGCTCTG

GCTGAGTAGT

ATGAAGAATC

ACGCGTTGAC

CATAGCCCAT

CCGCCCAACG

ATAGGGACTT

GTACATCAAG

CCCGCCTGGC

TACGTATTAG

GGATAGCGGT

TTGTTTTGGC

ACGCAAATGG

ACTAGAGAAC

CAAGCTT

GGAAGCTAGA

AGGCATCGTG

ATCAAGGCGA

TCCGATCGTT

GCATAATTCT

AACCAAGTCA

ACGGGATAAT

TTCGGGGCGA

TCGTGCACCC

AACAGGAAGG

CATACTCTTC

ATACATATTT

AAAAGTGCCA

GCCGGCTTCG

ATGGAGTTTG

ATGCCGCATA

GCGCGAGCAA

TGCTTAGGGT

ATTGATTATT

ATATGGAGTT

ACCCCCGCCC

TCCATTGACG

TGTATCATAT

ATTATGCCCA

TCATCGCTAT

TTGACTCACG

ACCAAAATCA

GCGGTAGGCG

CCACTGCTTA

GTAAGTAGTT

GTGTCACGCT

GTTACATGAT

GTCAGAAGTA

CTTACTGTCA

TTCTGAGAAT

ACCGCGCCAC

AAACTCTCAA

AACTGATCTT

CAAAATGCCG

CTTTTrCAAT

GAATGTATTT

CCTGACGTCG

AATAGCCAGA

GCGCCGATCT

GTTAAGCCAG

AATTTAAGCT

TAGGCGTTTT

GACTAGTTAT

CCGCGTTACA

ATTGACGTCA

TCAATGGGTG

GCCAAGTACG

GTACATGACC

TACCATGGTG

GGGATTTCCA

ACGGGACTTT

TGTACGGTGG

CTGGCTTATC

CGCCAGTTAA

CGTCGTTTGG

CCCCCATGTT

AGTTGGCCGC

TGCCATCCGT

AGTGTATGCG

ATAGCAGAAC

GGATCTTACC

CAGCATCTTT

CAAAAAAGGG

ATTATTGAAG

AGAAAAATAA

ACGGATCGGG

GTAACCTTTT

CCCGATCCCC

TATCTGCTCC

ACAACAAGGC

GCGCTGCTTC

TAATAGTAAT

TAACTTACGG

ATAATGACGT

GACTATTTAC

CCCCCTATTG

TTATGGGACT

ATGCGGTTTT

AGTCTCCACC

CCAAAATGTC

GAGGTCTATA

GAAATTAATA

TAGTTTGCGC

TATGGCTTCA

GTGCAAAAAA

AGTGTTATCA

AAGATGCTTT

GCGACCGAGT

TTTAAAAGTG

GCTGTTGAGA

TACTTTCACC

AATAAGGGCG

CATTTATCAG

ACAAATAGGG

AGATCTGCTA

TTTTTAATTT

TATGGTCGAC

CTGCTTGTGT

AAGGCTTGAC

GCGATGTACG

CAATTACGGG

TAAATGGCCC

ATGTTCCCAT

GGTAAACTGC

ACGTCAATGA

TTCCTACTTG

GGCAGTACAT

CCATTGACGT

GTAACAACTC

TAAGCAGAGC

CGACTCACTA

7200 7260 7320 7380 7440 7500 7560 '7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 9280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8897 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 832'1 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GGTACCAATT

45 TTGGAATTCT

TGTCCTTGTT

AGTGCAGCCT

CTATTACATG

TAGTCAAGAT

50 CAGAGACAAT

AGCCGTGTAT

AGGGACTCTG

ACCCTCCTCC

CTTCCCCGAA

55 CTTCCCGGCT

CTCCAGCAGC

CAAGGTGGAC

CCAGGCTCAG

AGGCAGGCCC

60 AGAGGGTCTT

CAGGCCCTGC

AGGACCCTGC

GACACCTTCT

CTTGTGACAA

65 CCAGCTCAAG

TACCAACATG

GTGACCGCTG

TA-A.TTGATA

TGCGGCCGCT

TTAAAAGGTG

GGAGGGTCCC

TATTGGGTTC

GGTGATATAA

GCAAAGAACA

TACTGTGCAA

GTCACGGTCT

AAGAGCACCT

CCGGTGACGG

GTCCTACAGT

TTGGGCACCC

AAGAAAGTTG

CGCTCCTGCC

CGTCTGCCTC

CTGGCTTTTT

ACACAAAGGG

CCCTGACCTA

CTCCTCCCAG

AACTCACACA

GCGGGACAGG

TCCGGAGCCA

TACCAACCTC

TCTCCTTAGG

TGCTAGCCAC

TCCAGTGTGA

TGCGACTTTC

GCCAGGCTCC

CCGACTATGC

GCCTGTACCT

GAGGCCTGGC

CTTCCGCTAG

CTGGGGGCAC

TGTCGTGGAA

CCTCAGGACT

AGACCTACAT

GTGAGAGGCC

TGGACGCATC

TTCACCCGGA

CCCCAGGCTC

GCAGGTGCTG

AGCCCACCCC

ATTCCAGTAA

TGCCCACCGT

TGCCCTAGAG

CATGGACAGA

TGTCCCTACA

TCTCGAGTCT

CATGGAGTTG

AGTGCAACTG

CTGTGCTGCA

AGGCAAGGGA

AGACTCCGTA

GCAAATGAAC

GGACGGGGCC

CACCAAGGGC

AGCGGCCCTG

CTCAGGCGCC

CTACTCCCTC

CTGCAACGTG

AGCACAGGGA

CCGGCTATGC

GGCCTCTGCC

TGGGCAGGCA

GGCTCAGACC

AAAGGCCAAA

CTCCCAATCT

GCCCAGGTAA

TAGCCTGCAT

GGCCGGCTCG

GGGCAGCCCC

CTAGATAACC

TGGTTAAGCT

GTGGAGTCTG

TCTGGATTCC

CTGGAGTGGG

AAGGGTCGAT

AGCCTGAGGG

TGGTTTGCTT

CCATCGGTCT

GGCTGCCTGG

CTGACCAGCG

AGCAGCGTGG

AATCACAAGC

GGGAGGGTGT

AGCCCCAGTC

CGCCCCACTC

CAGGCTAGGT

TGCCAAGAGC

CTCTCCACTC

TCTCTCTGCA

GCCAGCCCAG

CCAGGGACAC

GCCCACCCTC

GAGAACCACA

C-GTCAATCGA

TGGTCTTCCT

GGGGAGGCTT

CGTTCAGTGA

TCTCATACAT

TCACCATCTC

ACGAGGACAC

ACTGGGGCCA

TCCCCCTGGC

TCAAGGACTA

GCGTGCACAC

TCACCGTGCC

CCAGCAACAC

CTGCTGGAAG

CAGGGCAGCA

ATGCTCAGGG

GCCCCTAACC

CATATCCGGG

CCTCAGCTCG

GAGCCCAAAT

GCCTCGCCCT

ACCACGTGGG

TGCCCTGAGA

GGTGTACACC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440

CTGCCCCCAT

GGCTTCTATC

TACAAGACCA

ACCGTGGACA

GCTCTGCACA

CGGCCGGCAA

CCCCTGTACA

CCCCTGCGAG

ATGAGGGAGG

TGGGCCCCCT

GCGTGGCCCT

CCTGGGGACA

CTGTCCTCCC

GCCCTCCCTC

ACCCGCATGG

GGTGCAGATG

TGGCCGGCCA

GGCGAACTGC

CACAGGCCCC

CCACATGCTG

GCCACACACA

GTGCCGCCCT

TGTTGTTTGC

TTCCTAATAA

GGGTGGGGTG

GGATGCGGTG

TCCCCACGCG

GACCGCTACA

CGCCACGTTC

AACCATAGTC

TTCTCCGCCC

CTCTGAGCTA

GCTTGGACAG

CTGGTAGGAT

GTC:CCAAAAT

GTTCA-AGTAC

GATTA.TGGGT

AATTIAAATA

CAAAAGTTTG

CATC-GTTTGG

CCTTAC-ACTC

AATTGATTTG

GGAGGA-AA.AA

TGCTTTCAAG

TTGCTGGCTT

AAACTACCTA

GTTAAACTAC

TGGGAGCAGT

TCTAGTGATG

50 AAGGTAGAAG

TTTAGTAATA

CTATACAAGA

AATCATAACA

TATGCTCAAA

55 TTGATGTATA

ACTTGCTTTA

TGTTGTTGTT

AAATTTCACA

CAATGTATCT

60 GGAGTTCTTC

TAGCATCACA

CAAACTCATC

GTAATCATGG

CATACGAGCC

65 ATTAATTGCG

TTAATGAATC

CTCGCTCACT

CCCGGGATGA

CCAGCGACAT

CGCCTCCCGT

AGAGCAGGTG

ACCACTACAC

GCCCCCGCTC

TACTTCCCGG

ACTGTGATGG

CAGAGCGGGT

AGGGTGGGGC

CCCTCCAGCA

GACACACAGC

GACCTCCATG

ACCCATCTAC

GGACACAAC

CCCACACACA

CACGGCCACC

ACAGCACCCA

CACGAGCCCC

ACCTGCTCAG

CACAGGGGAT

TCCCTGCAGG

CCCTCCCCCG

AATGAGGAAA

GGGCAGGACA

GGCTCTATGG

CCCTGTAGCG

CTTGCCAGCG

GCCGGGCCTC

CCGCCCCTAA

C.ATGGCTGAC

TTCCAGAAGT

CTCAGGGCTG

TTTATCCCCG

ATGGGGATTG

TTCC:AAAGAA

AGGAAAACCT

GTTCTCAGTA

GATGATGCCT

ATAGTCGGAG

TTTGTGACAA

GGC-AAATATA

GGCATCAAGT

TTCTCTGCTC

TAGATCTCTT

CAGAGATTTA

TGATTCTAAT

GGTGGAATGC

ATGAGGCTAC

ACCCCAAGGA

GAACTCTTGC

AAATTATGGA

TACTGTTTTT

AATTGTGTAC

GTGCCTTGAC

AAAAACCTCC

AACTTGTTTA

AATAAAGCAT

TATCATGTCT

GCCCACCCCA

AATTTCACAA

AATGTATCTT

TCATAGCTGT

GGAAGCATAA

TTGCGCTCAC

GGCCAACGCG

GACTCGCTGC

GCTGACCAAG

CGCCGTGGAG

GCTGGACTCC

GCAGCAGGGG

GCAGAAGAGC

CCCGGGCTCT

GCGCCCAGCA

TTCTTTCCAC

CCCACTGTCC

TCAGCCAGGG

GCACCTGCCC

CCCTGCCTCT

CCCACTCGGG

CCCCACGGCA

GACTCCGGGG

CACTCAGCCC

ACACACACAC

GACCAGAGCA

ACGCGGCACC

ACAAACCCAG

CACACACCAC

ACGGATCAGC

TGCCTTCCTT

TTGCATCGCA

GCAAGGGGGA

CTTCTGAGGC

GCGCATTAAG

CCCTAGCGCC

TCAAAAAAGG

CTCCGCCCAT

TAATTTTTTT

AGTGAGGAGG

CGATTTCGCG

CTGCCATCAT

GCAAGAACGG

TGACCACAAC

GGTTCTCCAT

GAGAACTCAA

TAAGACTTAT

GCAGTTCTGT

GGATCATGCA

AACTTCTCCC

ATAAGTTTGA

CCCTCCTAAA

TGTGAAGGAA

AAGCTCTAAG

TGTTTGTGTA

CTTTAATGAG

TGCTGACTCT

CTTTCCTTCA

TTGCTTTGCT

AAAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

GGATCGGCTG

ACTTGTTTAT

ATAAAGCATT

ATCATGTCTG

TTCCTGTGTG

AGTGTAAAGC

TGCCCGCTTT

CGGGGAGAGG

GCTCGGTCGT

AACCAG4GTCA

TGGGAGAGCA

GACGGCTCCT

AACGTCCT

CTCTCCCTGT

CGCGGTCGCA

TGGAAATAAA

GGGTCAGGCC

CCACACTGGC

GCTGCCCTCG

TGGGCTGGGC

GTAGGAGACT

GGCATGCCTA

CTAACC:CCTG

ACATGCACTC

AGACCCGTTC

GTGCACGCCT

AGGTCCTCGC

TCAAGGCCCA

CCCTCCTCTC

GTCACGTCCC

CTCGACTGTG

GACCCTGGAA

TTGTCTGAGT

GGATTGGGAA

GGAAAGAACC

CGCGGCGGGT

CGCTCCTTTC

GAAAAAAAGC

CCCGCCCCTA

TATTTATGCA

CTTTTTTGGA

CCAAACTTGA

GGTTCGACCA

AGACCTACCC

CTCTTCAGTG

TCCTGAGAAG

AGAACCACCA

TGAACAACCG

TTACCAGGAA

GGAATTTGAA

AGAATACCCA

AGTCTACGAG

GCTATGCATT

CCTTACTTCT

GTAAATATAA

TTTTAGATTC

GAAAACCTGT

CAACATTCTA

GAATTGCTAA

ATTTACACCA

GTAACCTTTA

CACAGGCATA

TTAATTTGTA

ALATCAGCCAT

CCTGAACCTG

TAATGGTTAC

GCATTCTAGT

GATGATCCTC

TGCAGCTTAT

TTTTTCACTG

TATACCGTCG

AAATTGTTAT

CTGGGGTGCC

CCAGTCGGGA

CGGTTTGCGT

TCGGCTGCGG

GCCTGACCTG

ATGGGCAGCC

TCTTCCTCTA

CATGCTCCGT

CTCCGGGTAA

CGAGGATGCT

GCACCCAGCG

GAGTCTGAGG

CCAGGCTGTG

GCAGGGTGGG

CACGGGAAGC

GTCCTGTTCT

GTCCATGTGC

GCTGCCCTGC

TCGGGCCCTG

AACAAACCCC

CACACACGGA

ACACGTGAAC

CGAGCCTCTC

ACAAGGGTGC

TGGCCCTGGC

CCTTCTAGTT

GGTGCCACTC

AGGTGTCATT

GACAATAGCA

AGCTGGGGCT

GTGGTGGTTA

GCTTTCTTCC

ATGCATCTCA

ACTCCGCCCA

GAGGCCGAGG

GGCCTAGGCT

CGGCAATCCT

TTGAACTGCA

TGGCCTCCGC

GAAGGTAAAC

AATCGACCTT

CGAGGAGCTC

GAATTGGCAA

GCCATGAATC

AGTGACACGT

C-GCGTCCTCT

AAGAAAGACT

TTTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGCA

GAGTGTCTGC

AAGGGGTTAA

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAGCGCGGGG

AATGGTTACA

CATTCTAGTT

ACCTCTAGCT

CCGCTCACAA

TAATGAGTGA

AACCTGTCGT

ATTGGGCGCT

CGAGCGGTAT

CCTGGTCAAA

GGAGAACAAC

CAGCAAGCTC

GATGCATGAG

ATGAGTGCGA

TGGCACGTAC

CTGCCCTGGG

CCTGAGTGGC

CAGGTGTGCC

GGATTTGCCA

CCTAGGAGCC

GTGAGCGCCC

GTAGGGACAG

CCAGCCTCGC

TGGAGGGACT

GCACTGAGGT

GCCTCACCCG

ACTCCTCGGA

GGCAGCTTCT

CCCTGCAGCC

CCACTTCCCA

GCCAGCCATC

CCACTGTCCT

CTATTCTGGG

GGCATGCTGG

CTAGGGGGTA

CGCGCAGCGT

CTTCCTTTCT

ATTAGTCAGC

GTTCCGCCCA

CCGCCTCGGO

TTTGCAAAA

AGCGTGAAGG

TCGTCGCCGT

TCAGGAACGA

AGAATLCTGGT

TAAAGGACAG

ATTTTCTTGC

GTAAAGTAGA

AACCAGGCCA

TTTTCCCAGA

CTGAGGTCCA

AACAGGAAGA

CAT GGGACTT

ATAATTGGAC

TGTATAATGT

AACTGATGAA

AGAAATGCCA

AAAGAAGAGA

TCATGCTGTG

AGCTGCACTG

TAACAGTTAT

TATTAATAAC

TAAGGAATAT

TAGAGGTTTT

TGAATGCAAT

ATAGCATCAC

CCAAACTCAT

ATCTCATGCT

AATAAAGCAA

GTGGTTTGTC

AGAGCTTGGC

TTCCACACAA

GCTAACTCAC

GCCAGCTGCA

CTTCCGCTTC

CAGCTCACTC

1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 a a a a. a.

a a a. a a.

a a.

AALAGGCGGTA

AAAAGGCCAG

GCTCCGCCCC

GACAGGATA

TCCGACCCTG

TTCTCAATGC

CTGTGTGCAC

TGAGTCCAAC

TAGCAGAGCG

CTACACTAGA

AAGAGTTGGT

TTGCAAGCAG

TACGGGGTCT

ATCAAAAAGG

AAGTATATAT

CTCAGCGATC

TACGATACGG

CTCACCGGCT

TGGTCCTGCA

AAGTAGTTCG

GTCACGCTCG

TACATGATCC

CAGAAGTAAG

TACTGTCATG

CTGAGAATAG

CGCGCCACAT

ACTCTCAAGG

CTGATCTTCA

AAATGCCGCA

TTTTCAATAT

ATGTATTTAG

TGACGTCGAC

AGAGTAACCT

TCTCCCGATC

CAGTATCTGC

GCTACA.ACAA

TTTGCGCTGC

TATTAATAGT

ACATAACTTA

TCA,.AATGA.

GTGGACTATT

ACGCCCCCTA

ACCTTATGGG

GTGATGCGGT

45 CCAAGTCTCC

TTTCCAAA.AT

TGGGAGGTCT

ATCGAAATTA

ATACGGTTAT

CAAAAGGCCA

CCTGACGAGC

TAAAGATACC

CCGCTTACCG

TCACGCTGTA

GAACCCCCCc

CCGGTAAGAC

AGGTATGTAG

AGGACAGTAT

AGCTCTTGAT

CAGATTACGC

GACGCTCAGT

ATCTTCACCT

GAGTAAACTT

TGTCTATTTC

GAGGGCTTAC

CCAGATTTAT

ACTTTATCCG

CCAGTTAATA

TCGTTTGGTA

CCCATGTTGT

TTGGCCGCAG

CCATCCGTAA

TGTATGCGGC

AGCAGAACTT

ATCTTACCGC

GCATCTTTTA

AAAAAGGGA.A

TATTGAAGCA

AAAAATAAAC

GGATCGGGAG

TTTTTTTTAA.

CCCTATGGTC

TCCCTGCTTG

GGCAAGGCTT

TTCGCGATGT

A!ATCAATk1TAC

CGGTAAATGG

CGTATGTTCC

TACGGTAAAC

TTGACC.TCAA

ACTTTCCTAC

TTTGGCAGTA

ACCCCATTGA

GTCGTAACAA

ATATA.AGCAG

ATACGACTCA

CCACAGAATC

GGAACCGTAA

ATCACAAAAA

AGGCGTTTCC

GATACCTGTC

GGTATCTCAG

TTCAGCCCGA

ACGACTTATC

GCGGTGCTAC

TTGGTATCTG

CCGGCAAACA

GCAGAAAAAA

GGAACGAAAA

AGATCCTTTT

GGTCTGACAG

GTTCATCCAT

CATCTGGCCC

CAGCAATAAA

CCTCCATCCA

GTTTGCGCAA

TGGCTTCATT

GCAAAAAAGC

TGTTATCACT

GATGCTTTTC

GACCGAGTTG

TAAAAGTGCT

TGTTGAGATC

CTTTCACCAG

TAAGGGCGAC

TTTATCAGGG

AAATAGGG-T

ATCTGCTAGG

TTTTATTTTA

GACTCTCAC-T

TGTGTTGGAG

GACCGACAAT

ACGGGCCAGA

GGGGTCATTA

CCCGCCTGGC

CATAGTAAMCG

TGCCOACTTG

TGACGGrA-AA

TTGGCAGTAC

CATCAATGGG

CGTCA.ATGGG

CTCCGCCCCA

AGCTCTCTGG

CTATAGGGAG

AGGGGATAAC

AAAGGCCGCG

TCGACGCTCA

CCCTGGAAGC

CGCCTTTCTC

TTCGGTGTAG

CCGCTGCGCC

GCCACTGGCA

AGAGTTCTTG

CGCTCTGCTG

AACCACCGCT

AGGATCTCAA

CTCACGTTAA

AAATTAAAAA

TTACCAATGC

AGTTGCCTGA

CAGTGCTGCA

CCAGCCAGCC

GTCTATTAAT

CGTTGTTGCC

CAGCTCCGGT

GGTTAGCTCC

CATGGTTATG

TGTGACTGGT

CTCTTGCCCG

CATCATTGGA

CAGTTCGATG

CGTTTCTGGG

ACGGAAATGT

TTATTGTCTC

TCCGCGCACA

TGACCTGAGG

TTTTATTTTT

ACAATCTGCT

GTCGCTGAGT

TGCATGAAGA

TATACGCGT-1

GTTCATAGCC

TGACCGCCCA

CCAATAGGGA

GCAGTACATC

TGGCCCGCCT

ATCTACGTAT

CGTGGATAGC

AGTTT-GTTTT

TTGACGCAAA

CTAACTAGAG

ACCCPLAGCTT

GCAGGAAAGA

TTGCTGGCGT

AGTCAGAGGT

TCCCTCGTGC

CCTTCGGGAA

GTCGTTCGCT

TTATCCGGTA

GCAGCCACTG

AAGTGGTGGC

AAGCCAGTTA

GGTAGCGGTG

GAAGATCCTT

GGGATTTTGG

TGAAGTTTTA

TTAATCAGTG

CTCCCCGTCG

ATGATACCGC

GGAAGGGCCG

TGTTGCCGGG

ATTGCTACAG

TCCCAACGAT

TTCGGTCCTC

GCAGCACTGC

GAGTACTCAA

GCGTCAATAC

AAACGTTCTT

TAACCCACTC

TGAGCAAAAA

TGAATACTCA

ATGAGCGGAT

TTTCCCCGA.A

CGCGCCGGCT

GAGATGGAGT

CTGATGCCGC

AGTGCGCGAG

ATCTAGCTTAG

GACATTGATT

CATATATGGA

ACGACCCCCG

CTTTC^-ATTG

AAGTGTATCA

GjGCAT-TATGC

TAGTCATCGC

GGTTTGACTC

GGCACCAAAA

TGGGCGGTAG

AACCCACTGC

G

ACATGTGAGC

TTTTCCATAG

GGCGAAACCC

GCTCTCCTGT

GCGTGGCGCT

CCAAGCTGGG

ACTATCGTCT

GTAACAGGAT

CTAACTACGG

CCTTCGGAA.A

GTTTTTTTGT

TGATCTTTTC

TCATGAGATT

AATCAATCTA

AGGCACCTAT

TGTAGATAAC

GAGACCCACG

AGCGCAGAAG

AAGCTAGAGT

GCATCGTGGT

CAAGGCGAGT

CGATCGTTGT

ATAATTCTCT

CCAAGTCATT

GGGATAATAC

CGGGGCGAAA

GTGCACCCA.A

CAGGAAGGCA

TACTCTTCCT

ACATATTTGA

AAGTGCCACC

TCGA.ATAGCC

TTGGCGCCGA

ATAGTTAAGC

CAAA.ATTTAA

GGTTAGGCGT

ATTGACTAGT

GTTCCGCGTT

CCCATTGACG

ACGTCAATGG

TATGCCAAGT

CCAGTACATG

TATTACCATG

ACGGGGATTT

TCAACGGGAC

GCGTGTACGG

TTACTGGCTT

5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8321 9 999.

*99* .9 *9 9 INFORMATION FOR SEQ ID NO:l13: SEQUENCE CHARACTERISTICS: LENGTH: 8897 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 9 9999 9 9. *9 9 9 9 .9 9 99

GACGGATCGG

AGTAACCTTT

TCCCGATCCC

65 GTATCTGCTC

TACAACAAGG

TGCGCTGCTT

GAGATCTGCT

TTTTTTAATT

CTATGGTCGA

CCTGCTTGTG

CAAGGCTTGA

CGCGATGTAC

AGCCCGGGTG

TTATTTTATT

CTCTCAGTAC

TGTTGGAGGT

CCGACAATTG

GGGCCAGATA

ACCTGAGGCG

TTATTTTTGA

AATCTGCTCT

CGCTGAGTAG

CATGAAGA.AT

TACGCGTTGA

57

CGCCGGCTTC

GATGGAGTTT

GATGCCGCAT

TGCGCGAGCA

CTGCTTAGGG

CATTGATTAT

GAATAGCCAG

GGCGCCGATC

AGTTAAGCCA

AAATTTAAGC

TTAGGCGTTT

TGACTAGTTA

TTAATAGTAA TCAATTACGG ATAACTTACG GTAAATGGCC AATAATGACG TATGTTCCCA GGACTATTTA CGGTAAACTG GCCCCCTATT GACGTCAATG CTTATGGGAC TTTCCTACTT GATGCGGTTT TGGCAGTACA AAGTCTCCAC CCCATTGACG TCCAAAATGT CGTAACAACT GGAGGTCTAT ATAAGCAGAG CGAAATTAAT ACGACTCACT TCCTTAGGTC TCGAGCACCA TCCTGCTTCC AGCAGTGATG TGGACAACCT GCGTCCATCT CACCTATCTG GAATGGTACC AGTTTCCAAC CGATTTTCTG TTTCACACTC AAGATCAGCA GGGTTCACAT GTTCCATTCA TCGAGTCTCT AGATAACCGG CGTGGCCATT CTTTGCCTlAA CCTCAGAATG GCTGCAAAGA GGAAGCTAGG AAGAAACTCA ATAATTATCT GGGATAAGCA ACAACACACC CAAGGGCAGA AGGAACTGTG GCTGCACCAT TGGAACTGCC TCTGTTGTGT GTGGAAGGTG GATAACGCCC GAGCAAGGAC AGCACCTACA GAAACACAAA GTCACGCCT %GAGCTTCAAC AC-GGGAGAGT CCTC-ACCCCC TCCCATCCTT GCGGTCCTCC AGCTCATCTT AATGTTGGAG GAGAATGAkAT TCATTTA.ATA ATTATTATCT PLATATGTAGT CATCCTAAGG CCCTATCATC CTCTGCAAGA CCCCTGGGCC ATGGTAGGAG TAGTCCTTTT TAAGGGTGAC AATCALACCAA AGCAAATTTT AACATGATAT AAAATAACAA TTTAAGTTCA TCATGGTACT CAGGTACTGA GGGACTCCTG TAATCCACAC TATACTGTGA AGCTGAGAGA CAAATATATT 45 CCACCCACCA AV.AAACTATG TTGGAAATAG CCCGATTGTC ATTAGAATAC CCAATGAGGA TCTCATAAAA ATAATGTTAC TCATCCATAT AAAGTTCAAA 50 TCTTAGCTGG GGGTGGGCGA TTGGTAATGT TCTGTTCCTC TACACATTAT GCTTCAAAAT GAAGAGGAAT AAGTAATAGG ATAGCTACCT GCCTAATCCT 55 AGGTGCTCAA CAAAACAACA CTAGGAGCAC ACATACATAG AGAATTAACC TTGCCCAGAC GGGAAGGGCA CATGTAAATG CTCTCAGCTA CTCATCCATC 60 AAGGGGTTCA GGAGTAACTA TCCTGCTTTG TTTTTCTTTC AACTACATAA GGAAGCACCT TTTCAAACTT TGGAGGTTTG ALACTTGCTCA CTCATCCCTG 65 ATGAATTCTT GCGGCCGCTT TTCTATAGTG TCACCTAAAT GCCAGCCATC TGTTGTTTGC

GGTCATTAGT

CGCCTGGCTG

TAGTAACGCC

CCCACTTGGC

ACGGTAAATG

GGCAGTACAT

TCAATGGGCG

TCAATGGGAG

CCGCCCCATT

CTCTCTGGCT

ATAGGGAGAC

TGAAGTTGCC

TTGTCATGAC

CTTGCAGATC

AGCAGAGACC

GGGTCCCAGA

GAGTGGAGGC

CGTTCGGCCA

TCAATCGATT

AGCATTGAGT

GCTCCAACAA

AAACATCAAG

TGCTGTTTTC

ACTTTGTTAC

CTGTCTTCAT

GCCTGCTGAA

TCCAATCGGG

GCCTCAGCAG

GCGAAGTCAC

GTTAGAGGGA

TGGCCTCTGA

TCACCTCACC

AAkATAAAGTG

GTTGTTTTAC

CACGTAACCA

CAGTCCTCCC

AGACTTGCTT

AGGTCTTACA

TCAAAAGAAG

CACAATAAAA

TAGAC-TTAAT

TCTGCCAAGG

C-ATTAAAAAC

CTATAACTCA

CAAGAATGTT

CAACAATAGA

GAATTAACAA

ATAAGAGAAA

ACCAGGTAAA

GTTAGTGCCT

GTGTGGGGTT

AACTTCACAT

TCAAGACCAA

GCCCWRCTTGA

GGCCTGCTAT

AAATTAAATG

ACTGGAAACC

AGGACTCTTC

CAACACACCT

ACACAGCATC

CAGTCAGTAC

TGCCCTTCTG

AGTAGGGGTG

GGGGCCAAAT

GCTAGCTTCA

GCTAGAGCTC

CCCTCCCCCG

TCATAGCCCA

ACCGCCCAAC

AATAGGGACT

AGTACATCAA

GCCCGCCTGG

CTACGTATTA

TGGATAGCCG

TTTGTTTTGG

GACGCAAATG

AACTAGAGPA

CCAAGCTTGG

TGTTAGGCTG

CCAAACCCCA

TAGTCAGATC

AGGGCAGTCT

CAGGTTCAGC

TGAGGATGTG

AGGGACAAAG

GGAATTCTAA

TTACTGCAAG

AACAATTTAG

ATTTTAAATA

TGTCTGTCCC

TTAAACACCA

CTTCCCGCCA

TAACTTCTAT

TAACTCCCAG

CACCCTGACG

C-CATCAGGGC

GAAGTGCCCC

CCCTTTTTCC

CCCCTCCTCC

AATCTTTGCA

CAACTACTCA

TTTATAAA.AA

TCAAACCCAC

CCTTGTTTTC

GTCATATATC

AAACCTGCTA

GCAATTAAMT

GGAATGTCAT

GCCGTATTGA

ATTCATTAA

GCAATCCCAC

CAAAGCAGCT

ATGAGTTATT

GCTACAACTA

CTCAATGCAA

AATAAAGTTA

GGGAGAAGAC

GTGCAGTTAT

AAAGAACATC

CGCAGCTGGT

GCCCTGAATG

TTTCCTGGCA

AAACAGACCT

CATGTATGAA

CTCATTCTAT

TTCTAAGTAC

CCTTC:CCTCA

TGGGAAAGTG

CCTCTTGAGA

AGACTCAGTA

TGAACAATCA

CGTGTTGGAT

GCTGATCAGC:

TGCCTTCCTT

58

TATATGGAGT

GACCCCCGCC

TTCCATTGAC

GTGTATCATA

CATTATGCCC

GTCATCGCTA

TTTGACTCAC

CACCAAAATC

GGCGGTAGGC

CCCACTGCTT

TACCAATTTA

TTGGTGCTGA

CTGTCCAGTC

ATTGTACATA

CCACGGCTCC

GGCAGTGGAG

GGAGTTTACT

TTGGAAATCA

ACTCTGAGGG

GTCAGAAAAG

AACTTTATTA

CGCTTCTTGG

TAACATGCCC

TCCTGTTTGC

TCTGATGAGC

CCCAGAGAGG

GAGAGTGTCA

CTGAGCAAAG

CTGAGCTCGC

CACCTGCTCC

ACAC-GGGACC

TCCTTGGCTT

CCTGTGGTTT

ATTTCTCTTA

TCATC'CTTCA

AAGCCTTCTG

CCCTCCTCAG

CTTTGATTCA

TAAAGAGAAT

AAACAAACAA

GCCTTATTTA

GTACTTTCCA

ATGTTGCAA-A

TTCTAGATGA

TTATTTACA

AAACTGTGGT

TACCTACTCA

AAGATATGTT

GAA.ATTTGGA

AAGAAGGGGC

GATCTGTGCA

TTATACCCAG

AAGTGGGGGC

AGTCTGCCTT

TCTGTGCCCT

TCAGCAAGGG

CACTCACATG

GGGGCACTCT

CTCTCTCTGC

AATGACTGAC

GGGAAGGACA

ATGTTGATGA

ATGTCCCTTC

AAGGCAGGCA

CCAACCGCGG

CTCGACTGTG

GACCCTGGAA

TCCGCGTTAC

CATTGACGTC

GTCAATGGGT

TGCCAAGTAC

AGTACATGAC

TTACCATGGT

GGGGATTTCC

AACGGGACTT

GTGTACGGTG

ACTGGCTTAT

AATTGATATC

TGTTCTGGAT

CTGTCACGCT

ATAATGGCAA

TGATCTACAA

CTGGGACAGA

ACTGCTTCCA

AACGTAAGTC

GGTCGGATGA

CATGCAAAGC

AGGAATAGGG

TCTCCTTGCT

TTATCCGCAA

TTCTTTCCTC

AGTTGAAATC

CCAAAGTACA

CAGAGCAGGA

CAGACTAGGA

CCGTCACAAA

TCAGTTCCAG

TACCCCTATT

TAATTATGCT

CTCTCTTTCC

TAAGGGACTA

TTCTATTTTA

TCCTCACAGT

CAAGCCCTCA

ATTCCCTGAG

CATTCATTGC

TAGGGAAATG

CATTTTTAAA

CAACCTAA-T

GGTTCTATAA

CTGAGTGTCC

AAGCCAAAAA

ATGTTTATAC

CACAGATGAA

CTGTATGTTT

TGGAAATTAC

TTCTGGGGTC

CTGTTCTGTA

TTAATAGATA

CTGGGATCAA

CCAGGGCTCA

GTTTGGCTAG

GACAGAGGAC

TTTGGGAAGG

GGCCCTGCCC

CTACACTCTG

AATCCCTTTG

GTCATGGAGA

GTATCAAATC

CAATGACATG

TAATCCAGTT

AAGGGCCCTA

CCTTCTAGTT

GGTGCCACTC

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380

C

a a a. a.

a a a a.

CCACTGTCCT

TTCCTAATAA

CTATTCTGGG

GGGTGGGGTG

GGCATGCTGG

GGATGCGGTG

CTAGGGGGTA

TCCCCACGCG

CGCGCAGCGT

GACCGCTACA

CTTCCTTTCT

CGCCACGTTC

ATTAGTCAGC

AACCATAGTC

GTTCCGCCCA

TTCTCCGCCC

CCGCCTCGGC

CTCTGAGCTA

TTTGCAAAAA

GCTTGGACAG

AGCGTGAAGG

CTGGTAGGAT

TCGTCGCCGT

GTCCCAAAAT

TCAGGAACGA

GTTCAAGTAC

AGAATCTGGT

GATTATGGGT

TAAAGGACAG

AATTAATATA

ATTTTCTTGC

CAAAAGTTTG

GTAAAGTAGA

CATGGTTTGG

AACCAGGCCA

CCTTAGACTC

TTTTCCCAGA

AATTGATTTG

CTGAGGTCCA

GGAGGAAA

AACAGGAAGA

TGCTTTCAAG

CATGGGACTT

TTGCTGGCTT

ATAATTGGAC

AAACTACCTA

TGTATAATGT

GTTAAACTAC

AACTGATGAA

TGGGAGCAGT

AGAAATGCC-A

TCTAGTGATG

AAAGAAGAGA

AAGGTAGAAG

TCATGCTGTG

TTTAGTAATA

AGCTGCACTG

CTATACA.GA

TAACAGTTAT AATCATlAACA TATTAATAAC

TATGCTCAA

TAAGGAATAT

TTGATGTATA

TAGAGGTTTT

ACTTGCTTTA

TGAATGCAAT

TGTTGTTGTT

ATAGCATCAC

AAATTTCACA

CCAAACTCAT

CAATGTATCT

ATCTCATGCT

GGAGTTCTTC

AATAAACAA-Z

TAGCATCACA

GTGGTTTGTC

CAAACTCATC

AGAGCTTGGC GTAiATCATGG TTCCACACAA

CATACGAGCC

GCTAACTCAC

ATTAATTGCG

GCCAGCTGCA

TTAATGAATC

CTTCCGCTTC

CTCGCTCACT

45 CAGCTCACTC

AAAGGCGGTA

ACATGTGAGC-

AAAAGGCCAG

TTTTCCATAG

GCTCCGCCCC

GGCGAACCC

GACAGGACTA

GCTCTCCTGT

TCCGACCCTG

50 GCGTGGCGCT

TTCTCAATGC

CCAAGCTGGG

CTGTGTGCAC

ACTATCGTCT

TGAGTCCAAC

GTAACAGGAT

TAGCAGAGCG

CTAACTACGG

CTACACTAGA

CCTTCGGAAA

AAGAGTTGGT

GTTTTTTTGT

TTGCAAGCAG

TGATCTTTTC

TACGGGGTCT

TCATGAGATT

ATCAAAAAGG

AATCAATCTA

AAGTATATAT

60 AGGCACCTAT

CTCAGCGATC

TGTAGATAAC

TACGATACGG

GAGACCCACG

CTCACCGGCT

AGCGCAGAAG

TGGTCCTGCA

AAGCTAGAGT

AAGTAGTTCG

65 GCATCGTGGT

GTCACGCTCG

CAAGGCGAGT

TACATGATCC

CGATCGTTGT

CAGAAGTAAG

*AATGAGGWAA

GGGCAGGACA

GGCTCTATGG

CCCTGTAGCG

CTTGCCAGCG

GCCGGGCCTC

CCGCCCCTA

CATGGCTGAC

TTCCAGAAGT

CTCAGGGCTG

TTTATCCCCG

ATGGGGATTG

TTCCAAAGAA

AGGAAAACCT

GTTCTCAGTA

GATGATGCCT

ATAGTCGGAG

TTTGTGACAA

GGGAAATATA

GGCATCAAGT

TTCTCTGCTC

TAGATCTCTT

CAGAGATTTA

TGATTCTAAT

GGTGGAATGC

ATGAGGCTAC

ACCCCAAGGA

GAkACTCTTGC

AAATTATGGA

TACTGTTTTT

AATTGTGTAC

GTGCCTTGAC

AAAAACCTCC

AACTTGTTTA

AATAAAGCAT

TATCATGTCT

GCCCACCCCA

AATTTCACAA

AATGTATCTT

GCTAGCTGT

-GGAGCATMA

TTGCGCTCAC

GGCCAACGCG

GACTGCTGC

ATACGGTTAT

AAAAGGCCA

CCTGACGAGC

TAAAGATACC

CCGCTTACCG

TCACGCTGTA

GAACCCCCCG

CCGGTAAGAC

AGGTATGTAG

AGGACAGTAT

AGCTCTTGAT

CAGATTACGC

GACGCTCAGT

ATCTTCACCT

GAGTAAACTT

TGTCTATTTC

GAGGGCTTAC

CCAGATTTAT

ACTTTATCCG

CCAGTTAATA

TCGTTTGGTA

CCCATGTTGT

TTGGCCGCAG

TTGCATCGCA

GCAAGGGGGA

CTTCTGAGGC

GCGCATTAAG

CCCTAGCGCC

TCAAAAAAGG

CTCCGCCCAT

TAATTTTTTT

AGTGAGGAGG

CGATTTCGCG

CTGCCATCAT

GCAAGAACGG

TGACCACAAC

GGTTCTCCAT

GAGAACTCAA

TAAGACTTAT

GCAGTTCTGT

GGATCATGCA

AACTTCTCCC

ATAAGTTTGA

CCCTCCTAAA

TGTGAAGGAA

AAGCTCTAAG

TGTTTGTGTA

CTTTAATGAG

TGCTGACTCT

CTTTCCTTCA

TTGCTTTGCT

-AAAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

GGATCGGCTG

ACTTGTTTAT

ATAAAGCATT

ATCATC-TCTG

TTCCTGTGTG

AGTGT-AAIAGC

TGC'CC3CTTT

CGGGGAGAGG

GCTCGGTCGT

CCACAGAATC

GGAACCGTAA

ATCACAAAAA

AGGCGTTTCC

GATACCTGTC

C-GTATCTCAG

TTCAGCCCGA

ACGACTTATC

GCGGTGCTAC

TTGGTATCTG

CCGGCAAACA

GCAGAAAAAA

GGAACGAAAA

AGATCCTTTT

GGTCTGACAG

GTTCATCCATr

CATCTGGCCC

CAGCAATAAA

CCTCCATCCA

GTTTGCGCAA

TGGCTTCATT

GCAAAAAAGC

TGTTATCACT

*TTGTCTGAGT

GGATTGGGAA

GGAAAGAACC

CGCGGCGGGT

*CGCTCCTTTC

GAAAAAAAGC

CCCGCCCCTA

TATTTATGCA

CTTTTTTGGA

CCAAACTTGA

GGTTCGACCA

AGACCTAcCCC

CTCTTCAGTG

TCCTGAGAAG

AGAACCACCA

TGAA.CAACCG

TTACCAGGA

GGAATTTGA

AGAATACCCA

AGTCTACGAG

GCTATGCATT

CCTTACTTCT

GTAAATATAA

TTTTAGATT-

GAAAACCTGT

CAACATTCTA

GAATTGCTAA

ATTTACACCA

GTAACCTTTA

CACAGGCATA

TTAATTTGTA

AATCAGCCAT

CCTGAACCTG

TAATGGTTAC

GCATTCTAGT

GATG-ATCCTC

TGCAGCrTTAT

TTTTTC-ACTG

TATACCGTCG

AAATTGTTAT

CTGGGGTGCC

CCAGTCGGGA

CGGTTTGCGT

TCGGCTGCGG

AGGGGATAAC

AAA-GGCCGCG

TCGACGCTCA

CCCTGGAAGC

CGCCTTTCTC

TTCGGTGTAG

CCGCTGCGCC

GCCACTGGCA

AGAGTTCTTG

CGCTCTGCTG

A-ACCACCGCT

AGGATCTCAA

CTCACGTTAA

AAATTAAAAA

TTACCAATGC

AGTTGCCTGA

CAGTGCTGCA

CCAGCCAGCC

GTCTATTAAT

CGTTGTTGCC

CAGCTCCGGT

GGTTAGCTCC

CATGGTTATG

AGGTGTCATT

GACAATAGCA

AGCTGGGGCT

GTGGTGGTTA

GCTTTCTTCC

ATGCATCTCA

ACTCCGCCCA

GAGGCCGAGG

GGCCTAGGCT

CGGCAATCCT

TTGAACTGCA

TGGCCTCCGC

GAAGGTAAAC

AATCGACCTT

CGAGGAGCTC

GAATTGGCAA

GCCATGAATC

AGTGACACGT

GGCGTCCTCT

AAGAAAGACT

ITTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGC&A

GAGTGTCTGC

AAGGGGTTA-A

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAC-CGCC-GGG

AATGGTTACA

CATTCTAGTT

ACCTCTAGCT

CCGCT.CAC.A

TA-ATC-AGTGA

AACCTGTCGT

ATTCGGCc:r

CGAGCGGTAT

GCAGGAAAGA

TTGCTGGCGT

AGTCAGAGGT

TCCCTCGTGC

CCTTCGGGPA

GTCGTTCGCT

TTATCCGGTA

GCAGCCACTG

AAGTGGTGGC

AAGCCAGTTA

GGTAGCGGTG

GAAGATCCTT

GGGATTTTGG

TGAAGTTTTA

TTAATCAGT'

CTCCCCGTCG

ATGATACCGC

GGAAGGGCCG

TGTTGCCGGG

ATTGCTACAG

TCCCAACGAT

TTCGGTCCTC

GCAGCACTGC

4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6-380 6840 6900 6960 7020 708 0 7140 7200 7260 7320 7380 7440 7500 7560 7620 7,680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 ATAATTCTCT TACTGTCATG CCATCCGTA GATGCTTTTC TGTGACTGGT GAGTACTCAA 8460 CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG GCGTCAATAC 8520 GGGATAATAC CGCGCCACAT AGCAGACTT TAAAAGTGCr CATCATTGGA AAACGTTCTT 8580 CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC C-AGTTCGATG TAACCCACTC 8640 GTGCACCCA CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA 8700 CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA 8760 TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT 8820 ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA 8880 AAGTGCCACC TGACGTC 8897 INFORMATION FOR SEQ ID NO:14: SEQUENCE

CHARACTERISTICS:

LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAAGAGGAAG ACTGACGGTG CCCCCGCGAG TTCAGGTGCT GAGG 44 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCTCAGCACC TGA"CCCG GGGGCACCGT CAGTCTTCCT CTTC 44 INFORM-ATION FOR SEQ ID NO: 16: SEQUENCE

CH.ARACTERISTICS:

LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single 45 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGGGAGGGC TTTGTTGGAG ACCGAGCACG AGTACGACTT GCCATTCAGC C 51 INFORMATION FOR SEQ ID NO:17: 555 SEQUENCE CHARACTERISTICS: S LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: .65 GATGGTTTTC TCGATGGCGG CTGGGAGGGC INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCCCTCCCAG CCGCCATCGA GAAAACCATC INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GATGGTTTTC TCGATAGCGG CTGGGAGGGC TTTG 34 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MCLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GATGGTTTTC TCGATGGCGG CTGGGAGGGC TTTGTTGGAG ACCGAGCACG AGTACGACTT GCCATTCAGC CAGTCCTGGT G 81 INFORMATION FOR SEQ ID NO:21: 45 SEQUENCE CHARACTERISTICS: e* LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CACCAGGACT GGCTGAATGG CAAGTCGTAC TCGTGCTCGG TCTCCAACAA AGCCCTCCCA GCCGCCATCG AGAAAACCAT C 81 INFORMATION FOR SEQ ID NO:22: 60 SEQUENCE CHARACTERISTICS: LENGTH: 8690 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GGTACCAATT TAAATTGATA TTGGAATTCT TGCGGCCGCT TGTCCTTGTT TTAAAAGGTG AGTGCAGCCT GGAGGGTCCC CTATTACATG TATTGGGTTC TAGTCAAGAT GGTGATATAA CAGAGACAAT GCAAAGAACA AGCCGTGTAT TACTGTGCAA AGGGACTCTG GTCACGGTCT ACCCTCCTCC AAGAGCACCT CTTCCCCGAA CCGGTGACGG CTTCCCGGCT GTCCTACAGT CTCCAGCAGC TTGGGCACCC CAAGGTGGAC AAGAAAGTTG CCAGGCTCAG CGCTCCTGCC AGGCAGGCCC CGTCTGCCTC AGAGGGTCTT CTGGCTTTTT CAGGCCCTGC ACACAAAGGG AGGACCCTGC CCCTGACCTA GACACCTTCT CTCCTCCCAG CTTGTGACAA AACTCACACA CCAGCTCAAG GCGGGACAGG GGTGCTGACA CGTCCACCTC GTCTTCCTCT TCCCCCCAAA ACATGCGTGG TGGTGGACGT GACGGCGTGG AGGTGCATAA TACCGTGTGG TCAGCGTCCT AAGTGCAAGG TCTCCAACAA AAAGGTGGGA CCCGTGGGGT TGCCCTGAGA GTGACCGCTG GGTGTACACC CTGCCCCCAT CCTGGTCAAA GGCTTCTATC GGAGAACAAC TACAAGACCA CAGCAAGCTC ACCGTGGACA G-ATGCATG-AG GCTCTGCACA ATGAGTGCGA CGGCCGGCAA TGGC.ACGTAC CCCCTGTACA CTGCCCTGGG CCCCTGCGAC- CCTGAGTGGC ATGAGGGAGG CAGGTGTGCC TGGGCCCCCT GGATTTGCCA GCGTGGCCCT CCTAGGAC-CC CCTGGGGACA 45 GTGAGCGCCC CTGTCCTCCC GTAGGGACAG GCCCTCCCTC CCAGCCTCGC ACCCGCATGG TGGAGGGACT GGTGCAGATG GCACTGAGGT TGGCCGGCCA 50 GCCTCACCCG GGCGAACTGC ACTCCTCGGA CACAGGCCCC GGCAGCTTCT CCACATGCTG CCCTGCAGCC GCCACACACA CCACTTCCCA GTGCCGCCCT GCCAGCCATC TGTTGTTTGC CCACTGTCCT TTCCTAATAA CTATTCTGGG GGGTGGGGTG GGCATGCTGG GGATGCGGTG CTAGGGGGTA TCCCCACGCG 60 CGCGCAGCGT GACCGCTACA CTTCCTTTCT CGCCACGTTC ATTAGTCAGC AACCATAGTC GTTCCGCCCA TTCTCCGCCC CCGCCTCGGC CTCTGAGCTA 65 TTTGCAAAAA GCTTGGACAG AGCGTGAAGG CTGGTAGGAT TCGTCGCCGT GTCCCAAAAT

TCTCCTTAGG

TGCTAGCCAC

TCCAGTGTGA

TGCGACTTTC

GCCAGGCTCC

CCGACTATGC

GCCTGTACCT

GAGGCCTGGC

CTTCCGCTAG

CTGGGGGCAC

TGTCGTGGAA

CCTCAGGACT

AGACCTACAT

GTGAGAGGCC

TGGACGCATC

TTCACCCGGA

CCCCAGGCTC

GCAGGTGCTG

AGCCCACCCC

ATTCCAGTAA

TGCCCACCGT

TGCCCTAGAG

CATCTCTTCC

ACCCAAGGAC

GAGCCACGAA

TGCCAAGACA

CACCGTCCTG

AGCCCTCCCA

GCGAGGGCCA

TACCAACCTC

CCCGGGATGA

CC.NGCGACAT

CGCCTCCCGT

AGAGCAGGTG

ACCACTACAC

GCCCCCGCTC

TACTTCCCGG

ACTGTGATGG

CAGAGCGGGT

AGGGTGGGGC

CCCTCCAGCA

GACACACAGC

GACCTCCATG

ACCCATCTAC

GGACACAACC

CCCACACACA

CACGGCCACC

ACAGCACCCA

CACGAGCCCC

ACCTGCTCAG

CACAGGGGAT

TCCCTGCAGG

CCCTCCCCCG

AATGAGGAAA

GGGCAGGACA

GGCTCTATGG

CCCTGTAGCG

CTTGCCAGCG

GCCGGGCCTC

CCGCCCCTAA

CATGGCTGAC

TTCCAGAAGT

CTCAGGGCTG

TTTATCCCCG

ATGGGGATTG

TCTCGAGTCT

CATGGAGTTG

AGTGCAACTG

CTGTGCTGCA

AGGCAAGGC-M

AGACTCCGTA

GCAAATGAAC

GGACGGGGC

CACCAAGGGC

AGCGGCCCTG

CTCAGGCGCC

CTACTCCCTC

CTGCAACGTG

AGCACAGGGA

CCGGCTATGC

GGCCTCTGCC

TGGGCAGGCA

GGCTCAGACC

AAAGGCCAAA

CTCCCAATCT

GCCCAGGTA-A

TAGCCTGCAT

TCAGCACCTG

ACCCTCATGA

GAC-CCTGAGG

AAGCCGCGGG

CACCAGGACT

GCCCCCATCG

CATGGACAGA

TGTCCCTACA

GCTGACCAAG

CGCCGTGGAG

GCTGGACTCC

GCAGCAGGGG

GCAGAAGAGC-

CCCGGGCTCT

GCGCCCAZGCA

TTCTTTCCAC

CCCACTGTC-C

TCAGCCAGGG

GCACCTGCCC

CCCTGCCTCT

CCCACTCGGG

CCCCACGGCA

GACTCCGGGG

CACTCAGCCC

ACACACACAC

GACCAGAGCA

ACGCGGCACC

ACAAACCCAG

CACACACCAC

ACGGATCAGC

TGCCTTCCTT

TTGCATCGCA

GCAAGGGGGA

CTTCTGAGGC

GCGCATTAAG

CCCTAGCGCC

TCAAAAAAGG

CTCCGCCCAT

TAATTTTTTT

AGTGAGGAGG

CGATTTCGCG

CTGCCATCAT

GCAAGAACGG

CTAGATAACC

1'GGTTAAGCT

GTGGAGTCTG

TCTGGATTCC

CTGGAGTGGG

AAGGGTCGAT

AGCCTGAGGG

TGGTTTGCTT

CCATCGGTCT

GGCTGCCTGG

CTGACCAGCG

AGCAGCGTGG

AATCACAAGC

GGGAGGGTGT

AGCCCCAGTC

CGCCCCACTC

CAGGCTAGGT

TGCCAAGAGC

CTCTCCACTC

TCTCTCTGCA

GCCAGCCCAG

CCAGGGACAG

AACTCCTGGG

TCTCCCGGAC

TCAAGTTCAA

AGGAGCAGTA

GGCTGAATGG

AGAAAACCAT

GGCCGGCTCG

GGGCAGCCCC

AACCAGGTCA

TGGGAGAGCA

GACGGCTCCT

AACGTCTTCT

CTC7CCCTGT

CGCGGTCGCA

TGGAAATAAA

GGGTCAGGCC

CCACACTGGC

GCTGCCCTCG

TGC-GCTGGGC

GTAGGAGACT

GGCATGCCTA

CTAACCCCTG

ACATGCACTC

AGACCCGTTC

GTGCACGCCT

AGGTCCTCGC

TCAAGGCCCA

CCCTCCTCTC

GTCACGTCCC

CTCGACTGTG

GACCCTGGAA

TTGTCTGAG-T

GGATTGGGAA

GGAAAGAACC

CGCGGCGGGT

CGCTCCTTTC

GAAAAAAAGC

CCCGCCCCTA

TATTTATGCA

CTTTTTTGGA

CCAAACTTGA

GG-TTCGACCA

AGACCTACCC

GGTCAATCGA

TGGTCTTCCT

GGGGAGGCTT

CGTTCAGTGA

TCTCATACAT

TCACCATCTC

ACGAGGACAC

ACTGGGGCCA

TCCCCCTGGC

TCAAGGACTA

GCGTGCACAC

TCACCGTGCC

CCAGCAACAC

CTGCTGGAAG

CAGGGCAGCA

ATGCTCAGGG

GCCCCTAACC

CATATCCGGG

CCTCAGCTCG

GAGCCCAAAT

GCCTCGCCCT

GCCCCAGCCG

GGGACCGTCA

CCCTGAGGTC

CTGGTACGTG

CAACAGCACG-

CAAGGAGTAC

CTCCAAAGCC

GCCCACCCTC

GAGAACCACA

GCCTGACCTG

ATGGGCAGCC

TCTTCCTCTA

CATGCTCCGT

CTCCGGGTA-A

CGAGGATGCT

GCACCCAGCG

GAGTCTG-AGG

CCAGGCTGTG

GCAGGGT'GG

CACGGGAAGC

GTCCTGTTCT

GTCCATGTGC

GCTGCCCTGC

TCGGGCCCTG

AACAAACCCC

CACACACGGA

ACACGTGAAC

CGAGCCTCTC

ACAAGGGTGC

TGGCCCTGGC

CCTTCTAGTT

GGTGCCACTC

AGGTGTCATT

GACAATAGCA

AGCTGGGGCT

GTGGTGGTTA

GCTTTCTTCC

ATGCATCTCA

ACTCCGCCCA

GAGGCCGAGG

GGCCTAGGCT

,CGGCAATCCT

TTGAACTGCA

TGGCCTCCGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900

TCAGGAACGA

AGAATCTGGT

TAAAGGACAG

ATTTTCTTGC

GTAAAGTAGA

AACCAGGCCA

TTTTCCCAGA

CTGAGGTCCA

AACAGGAAGA

CATGGGACTT

ATAATTGGAC

TGTATAATGT

AACTGATGAA

AGAAATGCCA

AAAGAAGAGA

TCATGCTGTG

AGCTGCACTG

TAACAGTTAT

TATTAATAAC

TAAGGAATAT

TAGAGGTTTT

TGAATGCAAT

ATAGCATCAC

CCAAACTCAT

ATCTCATGCT

AATAAAGCAA

GTGGTTTGTC

AGAGCTTGGC

TTCCACACAA

GCTAACTCAC

GCCAGCTGCA

CTTCCGCTTC

CAGCTCACTC

ACATGTGAGC

TTTTCCATAG

GGCGAAACCO

GCTC-TCCTGT

GCGTGGCGCT1

CCAAGCTGG

ACTATCGTCT

GTAACAGGAT

CTAACTACGG

CCTTCGGAAA

GTTTTTTTGT

T-GATCTTTTC

TCATGAGATT

AATCAATCTA

AGGCACCTAT

TGTAGATAAC

GAGACCCACG

AGCGCAGAAG

AAGCTAGAGT

GCATCGTGGT

CAAGGCGAGT

CGATCGTTGT

ATAATTCTCT

CCAAGTCATT

GGGATAATAC

CGGGGCGAAA

60 GTGCACCCAA

CAGGAAGGCA

TACTCTTCCT

ACATATTTGA

AAGTGCCACC

TCGAATAGCC

TTGGCGCCGA

ATAGTTALAGC

GTTCAAGTAC

GATTATGGGT

AATTAATATA

CAAAAGTTTG

CATGGTTTGG

CCTTAGACTC

AATTGATTTG

GGAGGAAAAA

TGCTTTCAG

TTGCTGGCTT

AAACTACCTA

GTTAAACTAC

TGGGAGCAGT

TCTAGTGATG

AAGGTAGAAG

TTTAGTAATA

CTATACAAGA

AATCATAACA

TATGCTCAAA

TTGATGTATA

ACTTGCTTTA

TGTTGTTGTT

AAATTTCACA

CAATGTATCT

GGAGTTCTTC

TAGCATCACA

CAAACTCATC

GTAATCATGG

CATACGAGCC

ATTAATTGCG

TTAATGAATC

CTCGCTCACT

AAAG.GCGGTrA

AAAAGGCCAG

GCTCCGCCCC

GACAGGACTA

TCCGACCCTG

TTCTCAATGC

CTGTGTGCAC

TGAGTCCAAC

TAC-CAG.AGCG

CTACACTAGA

AAGAGTTGGT

TTGCAAGCAG

TACGGGGTCT

ATCAAAAAGG

AAGTATATAT

CTCAGCGATC

TACGATACGG

CTCACCGGCT

TGGTCCTGCA

AAGTAGTTCG

GTCACGCTCG

TACATGATCC

CAGAAGTAAG

TACTGTCATG

CTGAGAATAG

CGCGCCACAT

ACTCTCAAGG

CTGATCTTCA

AAATGCCGCA

TTTTCAATAT

ATGTATTTAG

TGACGTCGAC

AGAGTAACCT

TCTCCCGATC

CAGTATCTGC

TTCCAAAGAA

AGGAAAACCT

GTTCTCAGTA

GATGATGCCT

ATAGTCGGAG

TTTGTGACAA

GGGAAATATA

GGCATCAAGT

TTCTCTGCTC

TAGATCTCTT

CAGAGATTTA

TGATTCTAAT

GGTGGAATGC

ATGAGGCTAC

ACCCCAAGGA

GAACTCTTGC

AAATTATGGA

TACTGTTTTT

AATTGTGTAC

GTGCCTTGAC

AAAAACCTCC

AACTTGTTTA

AATAAAGCAT

TATCATGTCT

GCCCACCCCA

AATTTCACAA

AATGTATCTT

TCATAGCTGT

GGAAGCATAA

TTGCGCTCAC

GGCCAACGC-G

GACTCGCTGC

ATACGGTTAT

CAAAAGGCCA

CCTGACGAGC

TJAAAGATACC

CCGCTTACCG

TCACGCTIGTA

GAACCCCCCG

CCGGTA.AGAC

A~GATGTAG

AGGACAGTAT

AGCTCTTGAT

CAGATTACGC

GACGCTCAGT

ATCTTCACCT

GAGTAAACTT

TGTCTATTTC

GAGGGCTTAC

CCAGATTTAT

ACTTTATCCG

CCAGTTAATA

TCGTTTGGTA

CCCATGTTGT

TTGGCCGCAG

CCATCCGTAA

TGTATGCGGC

AGCAGAACTT

ATCTTACCGC

GCATCTTTTA

AAAAAGGGAA

TATTGAAGCA

AAAAATAAAC

GGATCGGGAG

TTTTTTTTAA

CCCTATGGTC

TCCCTGCTTG

TGACCACAAC

GGTTCTCCAT

GAGAACTCAA

TAAGACTTAT

GCAGTTCTGT

GGATCATGCA

AACTTCTCCC

ATAAGTTTGA

CCCTCCTAAA

TGTGAAGGAA

AAGCTCTAAG

TGTTTGTGTA

CTTTAATGAG

TGCTGACTCT~

CTTTCCTTCA

TTGCTTTGCT

AAAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

GGATCGGCTG

ACTTGTTTAT

ATAAAGCATT

ATCATGTCTG

TTCCTGTGTG

AGTGTAAAGC

TGCCCGCTTT

CGGGGAGAGG

GCTCGGTCGT

CCACAGAATC

C-GAACCGTAA

ATCACAAAAA

AGGCGTTTCC

GATACCTGTC

GGTATCTCAG

TTCAG-CCCGA

ACGACTTATC

GCGGTIGCTAC

TTGGTATCTG

CCGGCAAACA

GCAGAAAAAA

GGAACC-AAAA

AGATCCTTTT

GGTC-TGACAG

GTTCATCCAT

CATCTGGCCC

CAGCAATAAA

CCTCCATCCA

GTTTGCGCAA

TGGCTTCATT

GCAAAAAAGC

TGTTATCACT

GATGCTTTTC

GACCGAGTTG

TAAAAGTGCT

TGTTGAGATC

CTTTCACCAG

TAAGGGCGAC

TTTATCAGGG

AAATAGGGGT

ATCTGCTAGG

TTTTATTTTA

GACTCTCAGT

TGTGTTGGAG

CTCTTCAGTG GAAGGTAAAC TCCTGAGAAG AATCGACCTT AGAACCACCA CGAGGAGCTC TGAACAACCG GAATTGGCAA TTACCAGGAA GCCATGAATC

GGAATTTGAA

AGAATACCCA

AGTCTACGAG

GCTATGCATT

CCTTACTTCT

GTAAATATAA

TTTTAGATTC

GAAAACCTGT

CAACATTCTA

GAATTGCTAA

ATTTACACCA

GTAACCTTTA

CACAGGCATA

TTAATTTGTA

AATCAGCCAT

CCTGAACCTG

TAATGGTTAC

GCATTC7AGT

GATGATCCTC

TGCAGCTTAT

TTTTTCACTG

TATACCGTCG

AAATTGTTAT

CTGGGGTGCC

CCAGTCGGGA

CGGTTTGCGT

TCGGCTGCGG

AGGGGATAAC

AAAGGCCGCG

TCGACGCTCA

CCCTGGAADGC

CGCCTLTTCTC

TTCGGTGTAG

CCGCTGCGCC

GCCACTGGCA

AGAGTTCTTG

CGCTCTGCTG

AACCACCGCT

AGGATCTCAA

CTCACGTTAA

AAATTAAAAA

TTACCAATGC

AGTTGCCTGA

CAGTGCTGCA

CCAGCCAGCC

GTCTATTAAT

CGTTGTTGCC

CAGCTCCGGT

GGTTAGCTCC

CATGGTTATG

TGTGACTGGT

CTCTTGCCCG

CATCATTGGA

CAGTTCGATG

CGTTTCTGGG

ACGGAAATGT

TTATTGTCTC

TCCGCGCACA

TGACCTGAGG

TTTTATTTTT

ACALATCTGCT

GTCGCTGAGT

AGTGACACGT

GGCGTCCTCT

AAGAAAGACT

TTTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGCA

GAGTGTCTGC

AAGGGGTTAA

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAGCGCGGGG

AATGGT'TACA

CATTCTAGTT

ACCTCTAGCT

CCGCTCACAA

TAATGAGTGA

AACCTGTCGT

ATTGGGCGCT

CGAGCGGTAT

GCAGGAAAGA

TTGCTGGCGT

AGTCAGAGGGT

TCCCTCGTGC

CCTTCGGGAA

GTCGTTCGCT

TTATCGGTA

GCAGCCACTG

AAGTGGTGGC

AAGCCAGTTA.

GGTAGCGGTG

C-AAGATCCTT

GGGATTTrGG

TGAAGTTT;TA

TTAATCAGTG

CTCCCCGTCG

ATGATACCGC

GGALAGCGCCG

TGTTGCCGGG

ATTGCTACAG

TCCCAACGAT

TTCGGTCCTC

GCAGCACTGC,

GAGTACTCAA

GCGTCAATAC

AAACGTTCTT

TAACCCACTC

TGAGCAAAAA

TGAATACTCA

ATGAGCGGAT

TTTCCCCGAA

CGCGCCGGCT

GAGATGGAGT

CTGATGCCGC

AGTGCGCGAG

3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 618C 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 be.* 0..0 a. CAAAA1

GGTTAC

ATTGAC

GTTCCG

CCCATT

ACGTC

TATGCC

CCAGTA

TATTAC

ACGGGG

TCAACG

GCGTGI

TTACTG

TTAA

~GCGT

'TAGT

ICGTT

GACG

.ATGG

:AAGT

LCATG

.CATG

~ATTT

GGAC

ACGG

;GCTT

GCTACAACAA

TTTGCGCTGC

TATTAATAGT

ACATAACTTA

TCAATAATGA

GTGGACTATT

ACGCCCCCTA

ACCTTATGGG

GTGATGCGGT

CCAAGTCTCC

TTTCCAAAAT

TGGGAGGTCT

ATCGAAATTA

GGCAAGGCTT

TTCGCGATGT

AATCAATTAC

CGGTAAATGG

CGTATGTTCC

TACGGTAAAC

TTGACGTCAA

ACTTTCCTAC

TTTGGCAGTA

ACCCCATTGA

GTCGTAACAA

ATATAAGCAG

ATACGACTCA

GACCGACAAT

ACGGGCCAGA

GGGGTCATTA

CCCGCCTGGC

CATAGTAACG

TGCCCACTTG

TGACGGTAAA

TTGGCAGTAC

CATCAATGGG

CGTCAATGGG

CTCCGCCCCA

AGCTCTCTGG

CTATAGGGAG

TGCATGAAGA

TATACGCGTT

GTTCATAGCC

TGACCGCCA

CCAATAGGGA

GCAGTACATC

TGGCCCGCCT

ATCTACGTAT

CGTGGATAGC

AGTTTGTTTT

TTGACGCAA

CTAACTAGAG

ACCCAAGCTT

ATCTGCTTAG

GACATTGATT

CATATATGGA

ACGACCCCCG

CTTTCCATTG

AAGTGTATCA

GGCATTATGC

TAGTCATCGC

GGTTTGACTC

GGCACCAAAA

TGGGCGGTAG

AACCCACTGC

7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8690 INFORMATION FOR SEQ ID NO:23: SEQUENCE

CHARACTERISTICS:

LENGTH: 7874 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: me:* to 0 0*

GGTACCAATT

TTGGAATTCT

-o TCCTCCAAGA

CCCGAACCGG

CCGGCTGTCC

AGCAGCTTGG

GTGGACAAGA

35 GCTCAGCGCT

AGGCCCCGTC

GGTCTTCTGG

CCCTGCACAC

CCCTGCCCCT

CCTTCTCTCC

TGACAAAACT

CTCAAGGCGG

CTGACACGTC

TCCTCTTCCC

GCGTGGTGGT

45 GCGTGGAGGT

GTGTGGTCAG

GCAAGGTCTC

GTGGGACCCG

CTGAGAGTGA

50 TACACCCTGC

GTCAAAGGCT

AACAACTACA

AAGCTCACCG

CATGAGGCTC

GTGCGACGGC

ACGTACCCCC

CCTGGGCCCC

AGTGGCATGA

TGTGCCTGGG

TTGCCAGCGT

GGAGCCCCTG

GCGCCCCTGT

CTATGGCTTC

GTAGCGGCGC

CCAGCGCCCT

GCTTTCCCCG

GGCACCTCGA

TAAATTGATA

TGCGGCCGCT

GCACCTCTGG

TGACGGTGTC

TACAGTCCTC

GCACCCAGAC

AAGTTGGTGA

CCTGCCTGGA

TGCCTCTTCA

CTTTTTCCCC

AAAGGGGCAG

GACCTAAGCC

TCCCAGATTC

CACACATGCC

GACAGGTGCC

CJACCTCCATC

CCCAAAACCC

GGACGTGAGC

GCATAATGCC

CGTCCTCACC

CAACAAAGCC

TGGGGTGCGA

CCGCTGTACC

CCCCATCCCG

TCTATCCCAG

AGACCACGCC

TGGACAAGAG

TGCACAACCA

CGGCAAGCCC

TGTACATACT

TGCGAGACTG

GGGAGGCAGA

CCCCCTAGGG

GGCCCTCCCT

GGGACAGACA

CCTCCCGACC

TGAGGCGGAA

ATTAAGCGCG

AGCGCCCGCT

TCAAGCTCTA

CCCCAAAAAA

TCTCCTTAGG

TGCTAGCACC

GGGCACAGCG

GTGGAACTCA

AGGACTCTAC

CTACATCTGC

GAGGCCAGCA

CGCATCCCGG

CCCGGAGGCC

AGGCTCTGGG

GTGCTGGGCT

CACCCCAAAG

CAGTAACTCC

CACCGTGCCC

CTAGAGTAGC

TCTTCCTCAG

AAGGACACCC

CACGAAGACC

AAGACAAAGC

GTCCTGCACC

CTCCCAGCCC

GGGCCACATG

AACCTCTGTC

GGATGAGCTG

CGACATCGCC

TCCCGTGCTG

CAGGTGGCAG

CTACACGCAG

CCGCTCCCCG

TCCCGGGCGC

TGATGGTTCT

GCGGGTCCCA

TGGGGCTCAG

CCAGCAGCAC

CACAGCCCCT

TCCATGCCCA

AGAACCAGCT

GCGGGTGTGG

CCTTTCGCTT

AATCGGGGCA

CTTGATTAGG

TCTCGAGTCT

AAGGGCCCAT

GCCCTGGGCT

GGCGCCCTGA

TCCCTCAGCA

AACGTGAATC

CAGGGAGGGA

CTATGCAGCC

TCTGCCCGCC

CAGGCACAGG

-AGACCTGCC

GCCAAACTCT

CAATCTTCTC

AGGTAAGCCA

CTGCATCCAG,

CACCTGAACT

TCATGATCTC

CrGAGGTCAA

CGCGGGAGGA

AGGACTGGCT

CCATCGAGAA

GACAGAGGCC

CCTACAGGGC

ACCAAGAACC

GTGGAGTGGG

GACTCCGACG

CAGGGGAACG

AAGAGCCTCT

GGCTCTCGCG

CCAGCATGGA

TTCCACGGGT

CTGTCCCCAC

CCAGGGGCTG

CTGCCCTGGG

GCCTCTGTAG

CTCGGGGGCA

GGGGCTCTAG

TGGTTACGCG

TCTTCCCTTC

TCCCTTTAGG

GTGATGGTTC

CTAGATAACC

CC-GTCTTCCC

GCCTGGTCA

CCAGCGGCGT

GCGTGGTCAC

ACAAGCCCAG

GGGTGTCTGC

CCAGTCCAGG

CCACTCATGC

CTAGGTGCCC

AAGAGCCATA

CCACTCCCTC

TCTGCAGAGC

GCCCAGGCCT

GGACAGGCCC

CCTGGGGGGA

CCGGACCCCT

GTTCAACTGG

GCAGTACAAC

GAATGGCAAG

AACCATCTCC

GGCTCGGCCC

AGCCCCGAGA

AGGTCAGCCT

AGAGCAATGG

GCTCCTTCTT

TCTTCTCATG

CCCTGTCTCC

GTCGCACGAG

AATAAAGCAC

CAGGCCGAGT

ACTGGCCCAG

CCCTCGGCAG

CTGGGCCACG

GAGACTGTCC

TGCTGGGGAT

GGGGTATCCC

CAGCGTGACC

CTTTCTCGCC

GTTCCGATTT

ACGTAGTGGG

GGTCAAkTCGA

CCTGGCACCO

GGACTACTTC

GCACACCTTC

CGTGCCCTCC

CAACACCAAG

TGGAAGCCAG

GCAGCAAGGC

TCAGGGAGAG

CTAACCCAGG

TCCC-GGAGGA

AGCTCGGACA

CCAAATCTTG

CGCCCTCCAG

CAGCCGGG-G

CCGTCAGTCT

GAGGTCACAT

TACGTGGACG

AGCACGTACC

GAGTACAAGT

AAAGCCAA-AG

ACCCTCTGCC

ACCACAGGTG

GACCTGCCTG

GCAGCCGGAG

CCTCTACAGC

CTCCGTGATG

GGGTAALATGA

GATGCTTGGC

CCAGCGCTGC

CTGAGGCCTG

GCTGTGCAGG

GGTGGGGGAT

GGAAGCCCTA

TGTTCTGTGA

GCGGTGGGCT

CACGCGCCCT

GCTACACTTG

ACGTTCGCCG

AGTGCTTTAC

CCATCGCCCT

120 180 240 300 360 420 480 540 600 660 720 780 84U 900 960 102 0 1080 1140 1200 ,260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460

GATAGACGGT

TCCAAACTGG

TGGGGATTTC

AATTCTGTGG

GAAGTATGCA

CCATCCCGCC

TTTTTATTTA

GAGGCTTTTT

CGCGCCAAAC

TCATGGTTCG

ACGGAGACCT

CAACCTCTTC

CCATTCCTGA

TCAAAGAACC

TTATTGAACA

CTGTTTACCA

TGCAGGAATT

TCCCAGAATA

TTGAAGTCTA

TAAAGCTATG

GGAACCTTAC

TAAGGTAAAT

TGTATTTTAG

TGAGGAAAAC

CTCTCAACAT

TTCAGAATTG

TGCTATTTAC

TTCTGTAACC

TCCACACAGG

CTTTTTAATT

TCATAATCAG

TCCCCCTGAA

CTTATAATGG

CACTGCATTC

GCTGGATGAT

TTATTGCAGC

CATTTTTTTC

-TCTGTATACC

TGTGAAATTG

APAGCCTGCGGG

CTTTCCAGTC

GAGC-CGGTTT

TCGTrCGGCT

P-ATCAGGGGA

45 GTAAAAAGGC AkAAATCGACG

TTCCCCCTGG

TGTCCGCCTT

TCAGTTCGGT

50 CCGACCGCTG

TATCGCCACT

CTACAGAGTT

TCTGCGCTCT

AACAAACCAC

55 AAAAAGGATC

AAAACTCACG

TTTTAAATTA

ACAGTTACCA

CCATAGTTGC

60 GCCCCAGTGC

TAAACCAGCC

TCCAGTCTAT

GCAACGTTGT

CATTCAGCTC

65 AAGCGGTTAG

CACTCATGGT

TTTCTGTGAC

TTTTCGCCCT

AACAACACTC

GGCCTATTGG

AATGTGTGTC

AAGCATGCAT

CCTAACTCCG

TGCAGAGGCC

TGGAGGCCTA

TTGACGGCAA

ACCATTGAAC

ACCCTGGCCT

AGTGGAAGGT

GAAGAATCGA

ACCACGAGGA

ACCGGAATTG

GGAAGCCATG

TGAAAGTGAC

CCCAGGCGTC

CGAGAAGAAA

CATTTTTATA

TTCTGTGGTG

ATAAAATTTT

ATTCCAACCT

CTGTTTTGCT

TCTACTCCTC

CTAAGTTTTT

ACCACAAAGG

TTTATAAGTA

CATAGAGTGT

TGTAAAGGGG

CCATACCACA

CCTGAAACAT

TTACAAATAA

TAG7TTGTGGT

CCTCCAGCGC

TTATAATGGT

ACTGCATTCT

GTCGACCTCT

TTATCCGCTC

TGCCTAATGA

GGGAAACCTG

GCGTATTGGG

GCGGCG.AGCG

TAACGCAGGA

CGCGTTGCTG

CTCAAGTCAG

AAGCTCCCTC

TICTCCCTTCG

GTAGGTCGTT

CGCCTTATCC

GGCAGCAGCC

CTTGAAGTGG

GCTGAAGCCA

CGCTGGTAGC

TCAAGAAGAT

TTAAGGGATT

AAAATGAAGT

ATGCTTAATC

CTGACTCCCC

TGCAATGATA

AGCCGGAAGG

TAATTGTTGC

TGCCATTGCT

CGGTTCCCAA

CTCCTTCGGT

TATGGCAGCA

TGGTGAGTAC

TTGACGTTGG

AACCCTATCT

TTAAAAAATG

AGTTAGGGTG

CTCAATTAGT

CCCAGTTCCG

GAGGCCGCCT

GGCTTTTGCA

TCCTAGCGTG

TGCATCGTCG

CCGCTCAGGA

AAACAGAATC

CCTTTAAAGG

GCTCATTTTC

GCAAGTAAAG

AATCAACCAG

ACGTTTTTCC

CTCTCTGAGG

GACTAACAGG

AGACCATGGG

TGACATAATT

TAAGTGTATA

ATGGAACTGA

CAGAAGAAAT

CAAAAAAGAA

TGAGTCATGC

AAAAAGCTGC

GGCATAACAG

CTGCTATTAA

TTAATAAGGA

TTTGTAGAGG

AAAATGAATG

AGCAATAGCA

TTGTCCAAAC

GGGGATCTCA

TACAA.ATAAA

AGTTGTGGTT

AGCTAGAGCT

ACAATTCCAC

GTGAGCTAAC

TCGTGCCAGC

CGCTCTTCCG

GTATCAGCTC

AAGAACATGT

GCGTTTTTCC

AGGTGGCGAA

GTGCGCTCTC

GGAAGCGTGG

CGCTCCAAGC

GGTAACTATC

ACTGGTAACA

TGGCCTAACT

GTTACCTTCG

GGTGGTTTTT

CCTTTGATCT

TTGGTCATGA

TTTAAATCAA

AGTGAGGCAC

GTCGTGTAGA

CCGCGAGACC

GCCGAGCGCA

CGGGAAGCTA

ACAGGCATCG

CGATCAAGGC

CCTCCGATCG

CTGCATAATT

TCAACCAAGT

AGTCCACGTT

CGGTCTATTC

AGCTGATTTA

TGGAAAGTCC

CAGCAACCAT

CCCATTCTCC

CGGCCTCTGA

AAAAGCTTGG

AAGGCTGGTA

CCGTGTCCCA

ACGAGTTCAA

TGGTGATTAT

ACAGAATTAA

TTGCCAAAAG

TAGACATGGT

GCCACCTTAG

CAGAAATTGA

TCCAGGAGGA

AAGATGCTTT

ACTTTTGCTG

GGACAAACTA

ATGTGTTAAA

TGAATGGGAG

GCCATCTAGT

GAGAAAGGTA

TGTGTTTAGT

ACTGCTATAC

TTATAATCAT

TAACTATGCT

ATATTTGATG

TTTTACTTGC

CAATTGTTGT

TCACAAATTT

TCATCAATGT

TGCTGGAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTAATG

CTTCCTCGCT

ACTCAAAGGC

GAGCAAAAGG

ATAGGCTCCG

ACCCGACAGG

VI'GTTCCGAC

CGCTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAP.AAAGAGT

TTGTTTGCAA

TTTCTACGGG

GATTATCAAA

TCTAAAGTAT

CTATCTCAGC

TAACTACGAT

CACGCTCACC

GAAGTGGTCC

GAGTAAGTAG

TGGTGTCACG

GAGTTACATG

TTGTCAGAAG

CTCTTACTGT

CATTCTGAGA

CTTTAATAGT

TTTTGATTTA

ACAAAAATTT

CCAGGCTCCC

AGTCCCGCCC

GCCCCATGGC

GCTATTCCAG

ACAGCTCAGG

GGATTTTATC

AAATATGGGG

GTACTTCCAA

GGGTAGGAAA

TATAGTTCTC

TTTGGATGAT

TTGGATAGTC

ACTCTTTGTG

TTTGGGGAAA

AAAAGGCATC

CAAGTTCTCT

GCTTTAGATC

CCTACAGAGA

CTACTGATTC

CAGTGGTGGA

GATGATGAGG

GAAGACCCCA

AATAGAACTC

AAGAAA-ATTA

AACATACTGT

CAAAAATTGT

TATAGTGCCT

TTTAAAAAAC

TGTTAAkCTTG

CACAAATAAA

ATCTTATCAT

CTTCGCCCAC

CACAAATTTC

CATCAATGTA

ATGGTCATAG

AGCCGGikAGC

TGCGTTGCGC

AATCGGCCAA

CACTGACTCG

GGTAATACGG

CCAGCAAAPAG

CCCCCCTGAC

ACTATAAAGA

CCTGCCGCTT

ATGCTCACGC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGAGTAA

GATCTGTCTA

ACGGGAGGGC

GGCTCCA.GAT

TGCAACTTTA

TTCGCCAGTT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTATG

GGACTCTTGT

TAAGGGATTT

AACGCGAATT

CAGGCAGGCA

CTAACTCCGC

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATTGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGT

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

ATGCCTITTAA

CTACTGCTGA

AGGACTTTCC

TTGCTTCTT

TGGAAAAATA

TTTTTCTTAQ-

GTACCTTTAG

TGACTAG;ZA

CTCCCACACC

TTTATTGCAG

GCATTTTTTT

GTCTGGATCG

CCCAACTT-T

ACAAATA,4AG

TCTATCATG

CTGTTTCCTG

ATAAAC-TGTA

TCACTGCCCG

CGCGCGGGGA

CTGCGCTCG

TTATCCACAG

GCCAGGAACC

GAGCATCACA

TACCAGGCGT

ACCGGATACC

TGTAGGTATC

CCCGTTCAGC

AGACACGACT

GTAGGCGGTG

GTATTTGGTA

TGATCCGGCA

ACGCGCAGAA

CAGTGGAACG

ACCTAGATCC

ACTTGGTCTG

TTTCGTTCAT

TTACCATCT-

TTATCAGCAA

TCCGCCTCCA

AATAGTTTGC

GGTATGGCTT

TTGTGCAAAA

GCAGTGTTAT

GTAAGATGCT

CGGCGACCGA

2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 0O

S

S

S. St S S

S

*5SS

S*

S. S 55*555

S

t*5*

S

.55.

SSSSS

S

555555

S

*5 @5 5 5 S S 55 5 S S

S.

GTTGCTCTTG

TGCTCATCAT

GATCCAGTTC

CCAGCGTTTC

CGACACGGAA

AGGGTTATTG

GGGTTCCGCG

TAGGTGACCT

TTTATTTTAT

CAGTACAATC

GGAGGTCGCT

CAATTGCATG

CAGATATACG

ATTAGTTCAT

TGGCTGACCG

AACGCCAATA

CTTGGCAGTA

TAAATGGCCC

GTACATCTAC

TGGGCGTGGA

TGGGAGTTTG

CCCATTGACG

CTGGCTACT

GGAGACCCAA

CCCGGCGTCA

TGGAAAACGT

GATGTAACCC

TGGGTGAGCA

ATGTTGAATA

TCTCATGAGC

CACATTTCC

GAGGCGCGCC

TTTTGAGATG

TGCTCTGATG

GAGTAGTGCG

AAGAATCTGC

CGTTGACATT

AGCCCATATA

CCCAACGACC

GGGACTTTCC

CATCAAGTGT

GCCTGGCATT

GTATTAGTCA

TAGCGGTTTG

TTTTGGCACC

CAAATGGGCG

AGAGAACCCA

GCTT

ATACGGGATA ATACCGCGCC TCTTCGGGGC GAAAACTCTC ACTCGTGCAC CCAACTGATC AAAACAGGAA GGCAAAATGC CTCATACTCT TCCTTTTTCA GGATACATAT TTGAATGTAT CGAAAAGTGC CACCTGAcCCT GGCTTCGAAT AGCCAGAGTA GAGTTTGGCG CCGATCTCCC CCGCATAGTT AAGCCAGTAT CGAGCAAAAT TTAAGCTACA TTAGGGTTAG GCGTTTTGCG GATTATTGAC TAGTTATTAA TGGAGTTCCG CGTTACATA CCCGCCCATT GACGTCAATA ATTGACGTCA ATGGGTGGAC ATCATATGCC AAGTACGCCC ATGCCCAGTA CATGACCTTA TCGCTATTAC CATGGTGATG ACTCACGGGG ATTTCCAAGT AAAATCAACG GGACTTTCCA GTAGGCGTGT ACGGTGGGAG CTGCTTACTG GCTTATCGAA

ACATAGCAGA

AAGGATCTTA

TTCAGCATCT

CGCAAAAAAG

ATATTATTGA

TTAGAAAAAT

CGACGGATCG

ACCTTTTTTT

GATCCCCTAT

CTGCTCCCTG

ACAAGGCAAG

CTGCTTCGCG

TAGTAATCAA

CTTACGGTAA

ATGACGTATG

TATTTACGGT

CCTATTGACG

TGGGACTTTC

CGGTTTTGGC

CTCCACCCCA

AAATGTCGTA

GTCTATATAA

ATTAATACGA

ACTTTAAAAG

CCGCTGTTGA

TTTACTTTCA

GGAATAAGGG

AGCATTTATC

AAACAAATAG

GGAGATCrGC

TTAATTTTAT

GGTCGACTCT

CTTGTGTGTT

GCTTGACCGA

ATGTACGGGC

TTACGGGGTC

ATGGCCCGCC

TTCCCATAGT

AAACTGCCCA

TCAATGACGG

CTACTTGGCA

AGTACATCAA

TTGACGTCAA

ACAACTCCGC

GCAGAGCTCT

CTCACTATAG

6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7874 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Val Gln Letu Val Glu Ser Gly Leu Arg Leu Ser Cys Ala Ala Gly Gly 10 Ser Gly 25 Pro Gly ile Thr Leu Val GIn Pro Phe Pro Phe Ser Lys Gly Leu Giu Asp Tyr Ala Asp Gly Asp Trp Tyr Met Tyr Ser Tvr Ile Trp Val Arg Gin Ala 40 Ser Gin Asp Gly Asp Ser Val 0@ a. a 0@O@ 0 S. 00 S 0 ~0 a. 0 45 Lys 65 Leu Arg Phe Thr Ile Ser Arg Asp Asn Ala 70 75 Leu Arg Asp Glu Asp Thr Lys Asn Ser Leu Ala Val Tyr Tyr Gin Met Asn Ty r Cys Gly Ala Arg Gly Leu 50 100 Thr Leu Val Thr 115 Asp Gly Ala Ala Tyr Trp Gin Val Ser Ser INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 330 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 0O a@ 0 S 6 S. S

OS

a.

Se r Phe Gly Leu Tyr Lys Pro Lys Val1 145 Tyr Giu His Lys Gin.

225 Leu Pro As n Leu Val1 305 Gin Thr Ser Pro Glu Val His Ser Ser Ile Cys Val Giu Ala Pro 115 Pro Lys 130 Val Val Val Asp Gin Tyr Gin Asp 195 Ala Leu 210 Pro Arg Thr Lys Ser Asp Tyr Lys 275 Tyr Ser 290 Phe Ser Lys Ser Gly Gly Pro Val Thr Phe Val Val Asn Val 85 Pro Lys 100 Glu Leu Asp Thr Asp Vai Gly Val 165 Asn Ser 180 Trp Leu Pro Ala Giu Pro Asn G 1.n 245 Ile Ala 260 Thr Thr Lys Leu Cys Ser Leu Ser 325 Ala Se r 40 Val1 Pro Lys Asp Gly 120 Ile Giu His Arg Lys 200 Giu Tyr Leu T rp Val1 280 Asp His Pro Leu Gly 25 Trp Asn Leu Gin Ser Ser Pro Ser 90 Lys Thr 105 Pro Ser Ser Arg Asp Pro Asn Aia 170 Vai Val 185 Glu Tyr Lys Thr Thr Leu Thr Cys 250 Giu Ser 265 Leu Asp Lys Ser GIu Ala Gly Lys 330 Cys Ser Ser Ser 75 Asn His Val Thr Giu 155 Lys Ser Lys Ile Pro 235 Leu Asn Ser Arg Leu 315 Leu Gly Ser 60 Leu Thr Thr Phe Pro 140 Val Thr Val1 Asp Ser 220 Pro Val Gly Asp T rp 300 His Ty r Ser Se r Thr Lys Cys Pro Cys T rp 160 Giu Leu Asn Gly Giu 240 Tyr As n ?he Asn Thr 320 INFORMATION FOR SEQ ID \0:26: 000.

0000* Ala 1 Ser ?he Gly Leu 65 Tyr Lys SEQUENCE CHARACTERISTICS: LENGTH: 220 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Thr Lys Gly Pro Ser Val Phe Pro 5 Thr Ser Gly Giy Thr Aia Ala Leu Gly 25 Pro Giu Pro Val Thr Val Ser Trp Asn 40 Val His Thr Phe Pro Ala Val Leu Gin 50 55 Ser Ser Val Val Thr Vai Pro Ser Ser 70 Ile Cys Asn Val Asn His Lys Pro Ser 85 Val Giu Pro Lys Ser Cys Asp Lys Thr 100 105 NO: 2 6: Leu Ala Cys Leu Ser Gly Ser Ser Ser Leu Asn Thr His Thr Pro Gly Gin Pro Arg Giu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125 Asp Giu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 339 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Giu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 Ser Leu Lys Val Ser Cys Val Thr Ser Gly Phe Thr Phe Ser Asp Tyr 25 Tyr Met Tyr Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Trp Val 40 Ala Tyr Ile Ser Gin Gly Gly Asp Ile Thr Asp Tyr Pro Asp Thr Val 50 55 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 7 0 75 Leu Gin Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 90 Ala Arg Gly Leu Asp Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gin Gly 100 105 1.10 Thr Leu Val Thr Val Ser Val Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Giu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Val Thr Val Pro Ser *180 185 190 *Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Gly Gin Pro Arg Glu Pro Gin Val *225 230 235 240 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 245 250 255 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Vai Glu *260 265 270 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 275 280 285 *Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 290 295 300 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met *305 310 315 320 :68 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 325 330 335 Pro Gly Lys 69

Claims (13)

1. 21-- 0aiim Ifinin JTi Irrti~c Are As RO1kQ, 1. A method of inhibiting immunoglobulin-induced toxicity resulting from immunoglobulin immunotherapy in a subject comprising a9 administering an immunoglobulin molecule to the subject, the immunoglobulin molecule having a variable region and a constant region, the immunoglobulin molecule being modified prior to administration by structurally altering multiple toxicity-associated domains in the constant regions of the immunoglobulin so that immunoglobulin-induced toxicity is inhibited, the modified immunoglobulin molecule including at least a portion of the Fc region that is lacking in F(ab') 2 or Fab fragments. 2. The method of claim 1 wherein the multiple toxicity associated domains in the constant region are modified so as to render the constant region unable to mediate an ADCC response or activate complement. 3. A method for inhibiting immunoglobulin-induced toxicity resulting from immunotherapy in a subject comprising administering an Ig fusion protein to the subject, the Ig fusion protein having multiple structurally altered Stoxicity-associated domains in the constant region, .the Ig fusion protein being derived from an IgGI immunoglobulin molecule and including at least a portion of the Fc region that is lacking in F(ab') 2 or Fab fragments. 25 4. The method of claim 3 wherein the multiple structurally altered toxicity associated domains are located within the CH 2 domain. The method of claim 1 wherein the method prevents immunoglobulin-induced toxicity resulting from immunotherapy for a disease, and 30 the antibody is administered to the subject under conditions such that the structurally altered immunoglobulin recognizes and binds a target, thereby alleviating symptoms associated with the disease. 6. The method of claim 4 wherein immunoglobulin-induced toxicity resulting from immunotherapy for a disease is prevented, the Ig fusion protein administered is selected to recognize and bind the target, the target being associated with a disease, and the structurally altered Ig fusion protein is administered to the subject under conditions that the structurally altered Ig fusion protein recognizes and binds the target, thereby alleviating symptoms associated with the disease. 7. The method of claim 1, 2, 3, or 5, wherein the portion of the constant region that is structurally altered is the CH 2 domain. 8. The method of claim 1 or 5, wherein the immunoglobulin molecule is IgG. 9. The method of claim i or 5, wherein the immunoglobulin molecule is IgM. The method of claim 1 or 5, wherein the immunoglobulin molecule is IgA. 11. The method of claim 2, wherein the antibody recognizes and 25 binds to the antigen Le y 12. The method of claim 2, wherein the antibody recognizes and binds to the antigen Lex. 13. The method of claim 2 wherein the antibody administered is an antibody modified from a monoclonal antibody having the amino acid sequence 71 of the antibody BR96, produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC. 14. The method of claim 2, wherein the antibody administered is an antibody modified from an antibody having the amino acid sequence of chimeric antibody ChiBR96 produced by the hybridoma having the identifying characteristics of HB 10460 as deposited with the ATCC. The method of claim 1 or 5, wherein the immunoglobulin recognizes and binds to the antigen Le y 16. The method of claim 1 or 5, wherein the immunoglobulin recognizes and binds to the antigen Lex. 17. The method of claim 1 or 5, wherein the immunoglobulin is an antibody modified from a monoclonal antibody having the amino acid sequence of monoclonal antibody BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC. 18. The method of claim 1 or 5, wherein the immunoglobulin is an antibody modified from a chimeric antibody having the amino acid sequence of ChiBR96 produced by the hybridoma having the identifying characteristics of HB10460 as deposited with the ATCC. 19. The method of claim 3, 4, or 6, wherein the Ig fusion protein recognizes and binds to the antigen Le y 20. The method of claim 3, 4, or 6, wherein the Ig fusion protein recognizes and binds to the antigen Lex o* 21. The method of claims3, 4, or 6, wherein the Ig fusion protein has an amino acid sequence altered from the amino acid sequence of a monoclonal 72 antibody BR96 produced by the hybridoma having the identifying characteristics of HB10036 as deposited with the ATCC by one or more conservative amino acid substitutions, the conservative amino acid substitutions being selected from the group consisting of: any of isoleucine, valine, and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa; (3) glutamine for asparagine and vice versa; and serine for threonine and vice versa.
2. 22. The method of claim 3, 4, or 6, wherein the Ig fusion protein has ah amino acid sequence altered from the amino acid sequence of chimeric antibody ChiBR96 produced by the hybridoma having the identifying characteristics of HB 10460 as deposited with the ATCC by one or more conservative amino acid substitutions, the conservative amino acid substitutions being selected from the group consisting of: any of isoleucine, valine, and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa; (3) glutamine for asparagine and vice versa; and serine for threonine and vice versa.
3. 23. A pharmaceutical composition comprising a pharmaceutically effective amount of a structurally altered immunoglobulin and an acceptable carrier, wherein the structurally altered immunoglobulin: recognizes and binds a target associated with cancer and has a CH2 domain altered to inhibit immunoglobulin- induced toxicity resulting from administration of the immunoglobulin in the pharmaceutical composition.
4. 24. A pharmaceutical composition comprising a pharmaceutically 25 effective amount of a structurally altered Ig fusion protein and an acceptable carrier, wherein the structurally altered Ig fusion protein: recognizes and binds a target associated with cancer and has a CH2 domain altered to inhibit immunoglobulin- induced toxicity resulting from administration of the structurally altered fusion protein. 73 A method of blocking or inhibiting the growth of carcinomas in vivo comprisingapd'administering to a subject a pharmaceutically effective amount of the composition of claim 23 or 24.
5. 26. The composition of claim 23, wherein the immunoglobulin in the composition is labeled so as to directly or indirectly produce a detectable signal with a compound selected from the group consisting of a radiolabel, an enzyme, a chromophore, a chemiluminescer, and a fluorescer.
6. 27. The composition of claim 24, wherein the Ig fusion protein in the composition is labeled so as to directly or indirectly produce a detectable signal with a compound selected from the group consisting of a radiolabel, an enzyme, a chromophore, a chemiluminescer, and a fluorescer.
7. 28. The method of claim 2 or 5, wherein the antibody is conjugated to a cytotoxic agent.
8. 29. The method of claim 1, wherein the immunoglobulin is conjugated to a cytotoxic agent. The method of claim 3, 4, or 6, wherein the Ig fusion protein is conjugated to a cytotoxic agent.
9. 31. The method of claim 28, 29, or 30, wherein the cytotoxic 25 agent is selected from the group consisting of alkylating agents, anthracyclines, antibiotics, and anti-mitotic agents.
10. 32. A method for killing cancer cells, the cancer cells being characterized as a group of cells having a tumor associated antigen on their cell surface, which method comprises of administering to a subject harboring the cancer an amount of the composition of claim 23 or 24 joined to a Eytotoxic agent in an S* amount sufficient to kill cancer cells under conditions that permit the molecule so 74 L_ A plasmid which comprises a nucleic acid molecule of claim s
11. 48.
12. 51. A host vector system comprising the plasmid of claim 50 in a suitable host cell.
13. 52. A method for producing a protein comprising allowing the host cell of the host vector system of claim 50 to replicate under conditions that the protein encoded by the plasmid of claim 50 is expressed so as to produce the protein in the host and then recovering the protein so produced. DATED: 23 November 2001 PHILLIPS CRMONDE FITZPATRICK Attorneys for: BRISTOL-MYERS SQUIBB COMPANY MAE JOANNE RSOK a .o C...0 oC... o4 a. 4e 'eeg C•