AU775429B2 - A method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulins in therapy and in vivo diagnosis - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: 0.00 .0:0 .0 0 .000 *.oo 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): -1s06q 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 ivivo 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 inmmune system for therapeutic use. Immunoglobulin (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: neDespeci39688-dtvsonl.do inmunoglobulin irnmunothcrapy 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 Jiseased ceils (Slavin-Chiorini. et al..

Int. J. Cancer 97-103 i 1993)).

The constant region can oromote cell death through antibody dependent cell mediated cytotoxicity (A.DCC) or by complement dependent cyrotoxiciry (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)).

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 0 the L chain through hydrophobic interactions and generally a disulfide bond, depending on isorype. 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 functions.

*o* Today many anzribodics in clinical trials are directed against tumor associated antigens. Mosi rumor associated antigens are not turnor specific but are also oeneraily found on the cell surface of some normal, non-tumorigernic cells. The clinical use of some antibodies directed against tumor associated antigens are limited because of the tcxicity associated with their use. Therefore, there is a need for methods for inhibitine toxictv associated with inmuino!obulin use in the fieid of disease therapy therapy for rumors. kidney disease. and the ike)and 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 vive diagnosis, wherein the immunoglobulin recognizes both diseased and normal cells.

Our discovery involves generating imrmunoglobulin molecules or Ig fusion proteins having structurally altered constant regions which inhibit or reduce 1 immunoglobulin-induced toxicity.

The above discussion of background art is included to explain the context of the **invention. It is not to be taken as an admission or suggestion that any of the material *referred to was published, known or part of the common general knowledge in Australia at the priority date of any of the claims of this specification.

Throughout the description and claims of this specification the word "comprise" and variations of that word such as "comprises" and "comprising" are not intended to 25 exclude other additives, components, integers or steps.

St1 1ARY OF THE INVENTION Thne Freset in\'viion Droxides methods !Or iriJ-iti n immu-Ininoglobuilli-m7,duccd 1ox:citv by using knowTin imrunodobulin or I, sion, procein molecules which are strucurally altered in their constan re2ions so that the resulting structurally altered imnmunoiobulin or I [usion protein moiccules exhibit reduced or inhibited :oxicitv in compared to Lheir on2inal unmodifled counterarts.

Su-ucruiral alteration of the constant re21on ma';3, be e.ffected in a ni-mber of- %ays a-s long as it results in reCucinc or irnloitirn2 imimunoglobuiin-induceo tixc!1111 In one apct the present ivetion provides a method for ihibiting intmgobuini-ixuced toxicity resulting from immunoglobulin immunotherapy in a subject comprising 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 11 structurally altering multiple toxicity-associated domains in the CH2 domain so that immunoglobulin-induced toxicity is inhibited.

In accordance With tLhe practzice of onie embodiment of the invtntion. struc-tural alteration of the constant region, is effected bv deletion of the enrtire constant re2ion1.

In a:-other e-mbodimrent. only the CH:~ domain is delered. i another enibodi-Ment.

onl%- that por-tioni of the C'14 domain that binds the Fc receptor is deieted. IP vet another emnbodiment. orilv thai portion of The ClH-, domain that binds zhe complement comconent Clcl is deleted. Alze-nativtoiv. in another embodimrent. muitirole deietions :in discrete Fc receptor and connpieme rnt component binding domnains are effected.

A~emt~e:,str'uccLLrai 2!iterat;;in is ettectec or mu- mutaticon-s in !ne *c--domainl Such as am :o .*nzeru-,.ons, c ruoiszir-utions. Themuato or ~mutaz:ons mus reutn~tICItn rrango;:-nuc2oxcr.Byw: examole. the am *io acids in- muliile toxicitw. z:soc'-ar-ed docmains mtecoi-szanre2i~n can be altered So as to render the constant Tregton unabie to m-eci-ate a D-CC resconse or activate complement ther-eby iruotttng IMunI-2obuiin. !nouced toxic rv resulting from imrntinotheraov. Alternatively. multinie amino ac:c S m a singie toxtc associatedi domai*n in the constant reo a eatrc 00- Further, altemnativeiv. strucairai aittration can be effected by isorvpe switcatung resulting in an altered iraunoglobulin molecule that either does not induce- toxicirv or induces some limited toxicirv but does not cause a harmful effect. For example.

isorvocL~ switching can result In the constant req!on be~ng 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 of cBR96-2.

Figure 2 is a schematic diagram of a plasmid designated pTWD-cJVK.L1 including the chimeric (c)BR96-light chain (SEQ ID NO. 11).

Figure 3 is a schematic diagram of a plasmid designated pD16hJl.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 pDl7-J-dCH2.I-I, 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 pD17-cJ.Hl. 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' Figure 8 is a line graph showing the results of an ELISA assay of(l) BR96-A-Dox 30 to Le (closed diamond), chiBR96 to Le y (closed square), cBR96-A to Le y (96:0003 RJA)(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, domain point mutations.

Figure 1 1 is a schematic diagram showing the construction of the pNg1/14 vector.

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

Figure 13 is a schematic diagram showing the construction of pDI7-hJr.l4dCH2.H 1.

Figures 14A-J are the nucleic acid sequence of pDl7-cJ-dCH-2.HI, the plasmid shown in Figure S. chimeric BR96 having the CH, deletion.

Figure 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 diaeram of a lasmid desi2nated pD I 7-hG lb.

Figures 18 A-F are the nucleic acid sequence of pDl7-himi4.H1.

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

nfl..: Figure 20 is a line graph showing complement dependent ctIotoxicir:. In :he legend. the closed square is hBR96-l; closed diamond is hBR96-2B: closed crcLe is hBR96-2C; closed triangle is hBR96-2D; open square is hBR96-21H- open circle is hBR96-2A and open triangle is 2B8, anti-Pseudonomas aeruginosa flagella type b mAb negative control.

II

Figure 21 is a line graph showing antibody dependent cell-mediated cytotoxitv. 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 triangli 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 heavw chain region showing the hinge CH, and CH 3 domains as boxed regions. Site-specific mutations to be introduced into

CH

2 positions L1, 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 1-2) are simultaneously; introduced by inresngte number of recombining PCP, produces as is shown in the four-way re-combination ofe RsA2, AIB2. BIRa w, ith vector.

Figure 25 is a sgei showving Eco-47-li1 restriction enidonuclease anal1ysis of DN.-s prepared from colonies generated by multiple PCR frapgment RPCR. Lane M: lkb ladder DNA marker (GIBCOiBR-L Life Science Technology). Lanes 1-12: Twve~ve randomly selected colonies resulting 1from quadruple homologous recombiniation events were used to prepare plasmid and digested wvith E:co47-1II. Clones 1. 6 1 0 and 9 contain the fully assembled 1 .4 krb insert.

Figure 26 provides the amino acid sequence for hLBR96-2 heavy-chain varible re~ion and the human IgG1 constant region.

Figuz re 2priesthe amino acid seau,cce or hSR96-2-A heavy-%-chain varIble reaion)r and the huma,'- 12G: constant reegion -vnich the C-.2 region has bCeen deleted.

Fig-urez 23 provide-,s the amino acid sequence for c"1i BR96 heavy'-ai-n van~abie re2ion and the human lri conszant re:-c ,w.ithout ZheCH dman DETAILED DESCRIPTION OF THET IN-VEN-TON a.....DEFINITIONS *As used herein the term 'inh-ibiting immunoglobulin-induced toxicity" me-ars to reduce or allev-iate symnptoms geeal;associated wvith toxicirv causec imamunoqlobulin or [sg fusion protein therapy, toxicity, mediated by elTtctor .:functions of the Flc receptor. For exam, pie. BR96 anti'body recognizes and binds ,0 BR96 antiaen wh-ich 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 monoclonai 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 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 CH2 domain nay 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 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 IgG 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-dehydrotestosterone.

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 monoclonai 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.

As used herein, "treating" means to provide tumor regression so that the tumor is 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 carriers 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 and 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 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 descnption 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'. In another embodiment, the immunoglobulin recognizes and binds Lex.

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.: HB 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 CH3 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 inununoglobulin 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 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'. 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, 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, 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.

0000 oooo 20 Techniques for conjugating therapeutic agents to immunoglobulins are well knovn (see, Arnon et al.. "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclona! Antibodies And Cancer Therapy, Reisfeld et al.

pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstr6m et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Robinson et al. pp. 623-53 25 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In 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 131I; 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 drug 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 al., "Anti-Tumor Effects Of Antibody-alkaline Phosphatase", Proc. Natl. Acad. Sci. USA, 85:4842-46 (1988); "Enhancement of the in vitro and in viv Antitumor Activities of Phosphorylated Mitomycin 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 c 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 protarine sulfate.

20 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 carrer 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 The present invention provides structurally altered BR96 or BR96 Ig ifsion 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 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 mutated to alanine Tao et al., "Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation" J. Exp.

Med. 178:661-667 (1993); Y. Xu et al., "Residue at position 331 in the IgG1 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 lysine residue 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 therapeutis or diagnostic agent, will determine the different forms of structurally altered BR96 which is made.

Several options exists for antibody expression. Immunoexpression libraries can be combined with transfectoma technology, the genes for the Fab molecules derived from the immunoglobulin gene expression library can be connected to the S"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: glutamine 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 alarine and valine Methionine which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine and arginine (R) 25 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, 25 GCC, GCA, or GCG.

In hBR96-2G, serine at positions 318, 320, 322 is encoded by UCU, UCC, UCA, 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

Immunoconjugates (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" Immunol. Rev., 65:5- 27 (1982)), and morholinodoxorubicin (Mueller et al., "Antibody Conjugates With Morpholinodcxorubicin and Acid-Cleavabie Linkers", Bioconjugate Chem., 1:325- 330 (1990)).

BR96 has been conjugated to doxorubicin and has been shown to be effective in 25 therapy of certain cancers or carcinomas (Trail, Willner, Lasch, S.J., Henderson, Casazza, Firestone, Hellstrom, 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.

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

Alkylating agents include mechlorethamine, thiotepa chlorambucil, melphalan, car-mustine (BSNU) and tomustine (CCNU), cvclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C. and cis-dichlorodiamine platinum (II) (DDP) cisplatin.

Anthracyciines include daunonibicin (formerly daunomycin) and doxorubicin (also referred to herein as adriamycin). Additional examples include mitozantrone and bisantrene.

Antibiotics include dactinomycin (formerly actinomycin), bleomycin. mithramyvcin, 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 of mechlorethamine 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 CH2 domain with the mutations followed by homologous recombination of the 20 mutated CH 2 into the vector containing the desired irmmunoglobulin, 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 IgGl 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 (10x); 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')2 Fragment Goat A.nti Human IgG Fc Fragment specific (Jackson Imununo Research), Goat Anti Human Kappa-UNLB (Southern Biotechnoiogy Associates), Le-HSA (Alberta Research Council).

Methods: Dilute primary antibody or antigen to 1.0 g/m! in 0.05M Carb/Bicarb 20 buffer. Add 100pl 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°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°C. Wash plates 3x with saline/Tween.

For conjugation add 100 pl/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 pl/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 H 2

SO

4 100 .l/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 2 deleted BR96 molecules, the hinge, CH 2 and CH 3 domains were removed from chimeric BR96 and humanized BR9696-2 IgGI 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 IgGI (pNyl.14) molecule lacking the C-I 2 domain. Two oligonucleotides (Sense 49mer, Antisense 50mer) homologous to the sequences of S 20 IgG1 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 IgGI molecules were verified by restriction mapping and sequencing.

A sewing PCR strategy was used for the construction of CH 2 deleted human igG1 (pNy l.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 IgGI constant region vector (pNy The PCR fragment extends from the 5' end of the Hind-III site (in bold) through the Cel-II, Sal-I, Dra- III, Kpn-I, 6 bp nucleotide spacer and Mro-I sites (in bo!d) 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 IgG1 constant region vector (pNy1.7). A PCR fragment (about 150 bp) with Sal-I, Dra-III, Kpn-1, 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 CH- partial PCR fragments were combined in a PCR without any primer. The reaction was run through two full cycles of denaruration 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 po!ymerase extends the DNA with primer A and primer D, yielding a full- length (660 bp) PCR fragment. The newly extended PCR 20 fragment is arranged from the 5' end to the 3' end in the foilowing order: Hind-III CHI Cel-II Sal-! Dra-III Kpn-I 6 bp spacer Mro-I CHI partial Xba-1.

The combined PCR fragment, with the CHI and partial CH 3 domains, was then 25 cloned by a blunt end ligation into a Sma-I site on a pEMBL18 vector and the sequence was confirmed by dideoxy sequencing (Sanger et al. (1977) PNAS (USA) 74:5463-5466).

To transfer the CHI and partial CH 3 into a mammalian expression vector, both the pEMBL18 and pN1y.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 CHI 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-llI cloning site at each end. These sites also exist between the CH 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 18 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-IIl linearized sites on the vector. The new construct, now carrying the CHI, hinge and CH 3 domains, was designated pN 1.11.

To make the final CH 2 deleted human igG1 construct, both the pNyi.11 construct 20 and pNl 1.11 vector were digested with BamH1 and HindIII. A fragment containing the CHI, hinge and CH 3 domains was cloned into the linearized pNy1l.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-b and CH 3 domains was identified on the constant region sequence ofBR96 IgGI i 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 IgG1 molecule. The hinge, CH 2 and CH 3 domains (!.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 pDI7-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 CH3 were amplified from a CH 2 deleted L6 IgG (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 pNy 1.14 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 IgGI vector was digested with Eco47-III and the hinge, CH 2 and CH3 domains were removed. The linearized pDI7-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 CH2 domain of BR96 IgG1 molecules, and is designated pD17- BR96-dCH2 (Figure 13).

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

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

Three dogs received 400 mg/m 2 of cBR96-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 dimerization 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 I 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 CH2 deleted cBR96 was delivered to the GI tract in these dogs.

Table 5. Localization of cBR96 to GI tissue.

Group Animal Specific mean Localization

C

C C #271 155 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/mn), 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 CH2 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 Le

Y

expressing dogs. The study used chimeric versus constant region mutant of cBR96- 2.

SCBR96-2 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 O 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 CH2 domain would result in immunoglobuiininduced toxicity inhibition.

Toxicology studv of hBR96-2B 20 The toxicology study of hBR96-2B in high Lewis Y expressor dogs showed that a dose of 400 m:/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

*S

The polymerase chain reaction (PCR) is a widely used and versatile technique for the amplification and subsequent modification ofimmunoglobulin genes. The rapidity 30 and accuracy with which antibody genes can be modified in vitro has produced an assortment 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 anti-HLA-DR is necessary for Clq, FcyRI and FcyRIII binding. Immunology. 86:319-324; Duncan, 20 A.R. and G. Winter. 1988. The binding site for Clq on IgG. Nature 332:738-740; Tao, R.I.F. Smith and S.L. Morrison. 1993. Structural features of human S •0 immunoglobulin G that determine isotype-specific differences in complement Sactivation. 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 L 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; LI; and L2; L1, L2 and L3; etc.).

S 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.,

II

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 CH 2 domain.

20 Humanized BR96 variable region hcavy and light chain genes, previously cloned and co-expressed as an assembled active Fab fragment in an M 13 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 S 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 CH, 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 antisense 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 i* 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

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72 0 C for 1 min/kb, followed by a final extension at 72°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.

To evaluate the expression of Lef -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 S 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 advantage of this approach is the ability to simultaneously introduce multiple distaily-located mutations with PCR products synthesized by a single round of PCR.

Recoibinant 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 S 25 complexly mutated protein domains as well as broadening the number and location of desired mutations.

Analysis of transformants generated by multiple-fragment RPCR.

Mutant IgGs PCR HR' events Colonies Cloning Constructed Fragments in Analyzed Efficiencyb reaction 2 2 triple 24 2 3 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 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 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. He!lstrom. 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 (H 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 nrimers 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-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 u! 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 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°C for 45 sec, extension at 72 0 C for 1 min/kb, and a final extension at 72 0 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 37° 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 CH2 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. PD17hG Ib 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 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

II

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.Hl. The pD17-hJml4.H 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 supernarants by protein A chromatography.

The oligonucleotide primers homoiogous 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

II

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 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 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 IgGI constant region. Mutations in the constant region are marked.

SEQUENCE LISTING GENEIRAL INFORMATION APP'-ICANT: Yelzon, Dale E.

Rosok, Mae Joanne TITLE OF THE !NVENTION: A METHOD FOR INHIBITING !%21UNOGLOBULIN-INDUCED TOXICITY RESULTING 'FROM THE USE OF I'AM~UNOGLOBULINS IN THERAPY AND IN VIVO DIAGNOSIS (iii) NUMBER OF SEQUENCES: 27 (iv) CORRESCNDENCE ADDRESS: ADDRESSEE: Merchant, Gould, Smith, Edell, Welter Schmidt STREET: 11150 Santa Monica Boulevard, Suite 400 CITY: Los Angeles STATE: CA CCUNTRY: USA 90025 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SC7-WARE: FascSEQ for W4indows Version CURRENT APPLICATION DATA: APP-LICATION NUMBER: PCT US97/13562 FILING DATE: 01-AUG-1997

CLASSIFI-CATION:

(v RO APPLICATION DATA: APPLICATION NUMBER: 60/023,033 FILING DATE: 02-AUG-1996 ~v~)ATTORNEY/AGENT INFORMATION: kA; NA4E Canadv, Karen S.

REZ:ISTPATION NUMBER: 39,927 t REE-.FNC-/!CXET NUMBER: 30436.43WCUl 7_LZ.CCXYMUN!CAT:CN INFORMATION: TELEPHO:NE: 310-445-1140 TELEF7AX: 310-445-9031

TELEX:

INFORIMATION FOR SEQ ID NO-.l: SEQUENCE C?_ARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid C) STRAIIDEDNESS: single (2)TOPOLOGY: linear (ii) MOLECULE TYPE: cONA SEQUENCE DESCRIPTION: SEQ ID NO:l: 3 :TGGCACCGA AGCTTTCTGG GOAGGOCAG GCCTGA 3 INFORM-ATION FO0R SEQ ID NO:2: SEQUENCE CHA-RACTERISTICS LENGTH: 57 base pairs TYPE: nuclei4c acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cCNA (xi) SEQUENCE OESCRIPT -CN: SE-Q I-D NO:2: TCCGGACATG TTGGTACCCA CGTGGTGGTC GACGCTGAGC CTGGCTTCGA GCAc.ACA INFORMATION TOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ IDC NO:3: GTCGACCACC ACGTGGGTAC CAACATGTCC GGAGCCACAT GGACAGAGGC CGC-CT INFORKATION FOR SEQ ID NO:4: ()SEQUENCE CHARACTERITSTICS: LE7NGTH: 30 base =airs TYPE: nucleic acid S71R.31CEDNESS: sincle TOPOLOGY: linear )iiJ) MOLECULE TYPE: crDNA SEQUE-NCE DESCRIPTION: SEQ ID NO:4: CTGTTCTG TTCATCTCCT, CTCTAGATGG 7 7CRYAT ION FOR SEQ 1D NO: :)SEQUENCE CHAMR.AC-ER:-ST:-CS: LEN=T-: 36 b-ase pairs (C3 S:PA:4DEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cCNA (xi) SEQUENCE DESCRITION: SEQ ID ACCATGGTCG ACCTCAGACC TGCCAAGAGC CATATC INFOCRYATION FOR SEQ 1D NO: 6: i) SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic ac, STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CATGGTCACG TSGTGTS3TCC C-GGATGCAG GCTAC7CTAG -2 INFORIMATION £CR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs -YPE: nucleic acid STRAtiDECNESS: s:ngle TOPOLOGY: IL-iear OLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTICN: SEQ ID NO:7: CAGGGAGGGA GGGTGTCTSC TGGAGCCAG GCTCAGCGCT GACCTCAGA !NFO.ATION FOR SEQ ID NO:3: SEQUENCE CHPRCTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STPRA-NCEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGAAAGAACC ATC7ACAGTCT CGCAGGGGCC CAGGGCAGCG CTGGTGCTT INFORMATION FOR SEQ -D NO:9: SEQUENCE CHAkcRCTERIST:-CS: EGH 6691 base pairs TYP-'E: nucleic acid STR.ANDEDNESS: single TOPOLOGY:. linear: 'ii NLE7CULE TYPE: rN lxi) SEQ UENCE DESCRIPTION: SEQ ID NO:9: A7-C=TG G-ACTC7C 7TCCCOV'GC TTGTG7-:TG

AGAG

=7CTCGCGA -rGTACD-GGCC AGTAATCAAT TACGGGT-A T-ACGGTAAA TGGCCG TG3ACGTATGT =CCATAGTA ATTTACGGTA AAkCTGCCCAC CTATTGACGT CAATGACGGT GGGACTTTCC TACTTGGCAG GGTTTTGGCA G-tCATCAT TOCACCCCAT TGACGTCAAT A-.7GTCGTAA CAACTCCGCC T=.!-ATAAG CAGA G=T T-AATACGAC TCACTAT7AGG AGGO'TCGAG TOTIGATA C '!'CCATGGAG TTGTGGTTAA 60 G-1GAAGT GAA T'TGGTGGAG T=TCTGTGT AA.CCTCTGGA CTCCAGAGAA GAGGCTGGAG ATrCCAGACAC TGTA;AAGGGT ACCTGCAAAT GAGCCG-C-rG 7GGACGACGG GGCCT'GGTTTT rCTAGCACCAA SGGC2CATCG

AGGTC-ACCTG

.AG-AC.ATC-T

GAGGTCGCTG

AATT GCAT GA

AGATATACGC

TO AGTTCATA

GGCTGACCGC

ACGCCAATAG

TTGGCAGTAC

AAATGGCCCG

TACATCTACG

GGGCGTGGAT

GGGAGTTTGT

CCATTGACGC

TGGCTAACTA

GAGACCCAAG

ACCGGTCAAT

GCTTGGTCC'T

TCTGGGGGAG

TTCACTTTCA

TGGGTCGCAT

CGATTCACCA

AAGTCTGAGG

GCTTACTGGG

GTCTTCCCCC

AGGC-GC-CG

7TTGAGATGG

CCGA=G

AGTAGTGCGC

AGAATCTGCT

GTTGACATTG

GCCCATATAT

CCAACGACCC

GGACTTTCCA

ATCAAGTGTA

CCTGGCATTA

TATTAGT CAT

AGCGGTTTGA

TTTGGCACCA

.AAATGGGCGG

GAGAACC TAC

CTTGGTACCA

CGATTGGAAT

TCCTTGT OCT

GCTTAGTGCA

GTGACTATTA

ACATTAGTCA

TCTCCAGAGA

ACACAGCCAT

GCCAAGGGAC

TGGCACC CT C ACTACTT CCC

GCTTCGAATA

AGTTTGGCGC

OGOATAG-TA

GAGCPAATT

TAGGGTTAGG

ATTATTGACT

GGAGTTCCGC

CCGCCCATTG

TTGACGTCAA

TCATATGCCA

TGCCCAGTAC

CGCTATTACC

CTCACGGGGA

AAATCAACGG

TAGGCGTGTA

TGCTTCTGG

ATTTAAATTG

TCTTGCGGCC

TGTTTTMAAA

GCCTGGAGGG

CATGTATTGG

AGGTGGTGAT

CAATGCCAAG

GTATTACTGT

TCTGGTCACG

CTCCAAGAGC

CGIAACC G GCCAGAG7AA

CGATOO!'CCG

AGCCAGTAT 0 TAAGCTnACAA

CGTT-TGCOC

AGTTATTAA T

GTTACATAAC

ACGTCAATAA

TGGGTGGACT

AGTACGCCCC

ATGACCTTAT

ATGGTGATGC

TTTCCAAGTC

GACTTTCCAA

CGGTGGGAGG

CTTATCGlAA

ATATCTCCTT

GCTTGCTAGC

GGTGTCCAGT

TCCCTGAAAG

GTTCGCCAGA

ATAACCGACT

AACACCCTGT

GCAAGAGGCC

GTCTCTGTAG

ACCTCTGGGG

ACGGTG7CG 240 '00 360 480 540 600 660 '720 730 340 ?6.3 1200 1260 1320 1380 144 0 560 -320

S..

S.

5@

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

AGCAGCACC-

CA.GCCCCTGC

CATGCCCACT

CTACCCCCAC

AACCGACTCC

CACACACTCA

CACCACACAC

CCCAGACCAG

CCCCACGCGG

TCAGACAAAC

GGATCACACA

CAGGACGGAT

CCCGTCCTT

GAAATTCGCATr 45 GACAGCAA.GG

ATGGCTTCTG

AC-CGGCGCAT

AGCGCCCTAG

CCTCTCAAAA

CTAACTCCGC

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATTGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

60 GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGT

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

CGCCCTGACC

CCTCAGCAGC

CGTGAATCAC

GGGAGGGAGG

ATGCAGCCCC

TGCCCGCCCC

GGCACAGGCT

GACCTGCCA.A

CAAACTCTCC

ATCTTCTCTC

GTAAGCCAGC

GCATCCAGGG

CCTGAACTCC

ATGATCTCCC

GAGGTCAAGT

CGGGAGGAGC

GACTGGCTGA

ATCGAGAAAA

CAGAGGCCGG

TACAGGGCAG

CAAGAACCAG

GGAGTGGGAG

CTCCGACGGC

GGGGAkCGTC

GAGCCTCTCC

CTCTCGCGGT

AGCATGGAAA

CCACGGGTCA

GTCCCCACAC

AGGGGCTGCC

GCCCTGG;GCT

CTCTGTAGGA

CGGGGGCATG

GGCACTAACC

C-GGGACATGC

GCCCAGAC-C

ACACGTGCAC

AGCAAGGTCC

CACCTCAAGG

CCAGCCCTCC

CCACGTCACG

Cm'GCr-TCGAC

COTTGACCCT

CGCATTGCT

GGGAC-GATTG

AGGCGGAAAG

T.AAGCGCGGC

CGCCCGCTCC

AAGGGAAAAA

CCATCCCGCC

TTTTTATTTA

GAGGCTTTTT

CGCGCCAAAC

TCATGGTTCG

ACGGAGACCT

CAACCTCTTC

CCATTCCTGA

TCAAAGAACC

TTATTGAACA

CTGTTTACCA

TGCAGGAATT

TCCCAGAATA

TTGAAGTCTA

TAAAGCTATG

GGAACCTTAC

TAAGGTAAAT

TGTATTTTAG

AGCGGCGTGC

GTGGTCACCG

AAGCCCAGCA

GTGTCTGCTG

AGTCCAGGGC

ACTCATGCTC

AGGTGCCCCT

GAGCCATATC

ACTCCCTCAG

TGCAGAGCCC

CCAGGCCTCG

ACAGGCCCCA

TGGGGGGACC

GGACCCCTGA

TCAACTGGTA

AGTACAACAG

ATGGCALAGGA

CCATCTCCAA

CTCGGCCCAC

CCCCGAGAAC

GTCAGCCTGA

AGCAATGGGC

TCCTTCTTCC

TTCTCATGC'.

CTGTCTCCGG

CGCACGAGGA

T.AAAGCACCC

GGCCGAGTCT

TC-GCCCAGGC

CTCGGCAGGG

GGGCCACGGG

GACTGTCCTG

CC~TGTCCAT

CCTGGCTGCC

ACTCTCGGGC

GTTCAACAAA

GCCTCACACA

TCGC-ACACGT

CC-CACGAGCC

TCTCACAAGG

T CCCTGGCCC

TGTGCCTTCT

GGAAGGTGCC

GAGTAGGTGT

GGAAGACA-AT

AACCAGCTGG

GGGTGTGGTG

TTTCGCTTTC

AAGCATGCAT

CCTAACTCCG

TGCAGAGGCC

TGGAGGCCTA

TTGACGGCAA

ACCATTGAAC

ACCCTGGCCT

AGTGGAAGGT

GAAGAATCGA

ACCACGAGC-A

ACCGGAATTG

GGAAGCCATG

TGAAAGTGAC

CCCAGGCGTC

CGAGAAGAAA

CATTTTTATA

TTCTGTGGTG

ATXAAATTTT

ATTCCAACCT

ACACCTTCCC

TGCCCTCCAG

ACACCAAGGT

GAAGCCAGGC

AGCAAGGCAG

AGGGAGAGGG

AACCCAGGCC

CGGGAGGACC

CTCGGACACC

AAATCTTGTG

CCCTCCAGCT

GCCGGGTGCT

GTCAGTCTTC

GGTCACATGC

CGTGGACGGC

CACGTACCGT

GTACAAGTGC

AGCCAAAGGT

CCTCTGCCCT

CACAGGTGTA

CCTGCCTGGT

AGCCGGAGAA

TCTACAGCAA

CCGTGATGCA

GTAAATGAGT

TGCTTGGCAC

AGCGCTGCCC

GAGGCCTGAG

TGTGCAGGTG

TGGGGGATTT

AAGCCCTAGG

TTCTGTGAGC

-TCGCGTAGGG

CTGCCCAGCC

CCTGTC-GAGG

CCCCGCACTG

CGC-AGCCTCA

GAACACTCCT

TCTCGGCAGC

GTGCCCC7IGC

TGC-CACTT

AGTTGCCACC

ACTCCCACTG

CATTCTATTC

AGCAGGCATG

GGCTCTAGGG

GTTACGCGCA

TTCCCTTCCT

CTCAATTAGT

CCCAGTTCCG

GAGGCCGCCT

GGCTTTTGCA

TCCTAGCGTG

TGCATCGTCG

CCGCTCAGGA

AAACAGAATC

CCTTTAAAGG

GCTCATTTTC

GCAAGTAAAG

AATCAACCAG

ACGTTTTTCC

CTCTCTGAGG

GACTAACAGG

AGACCATGGG

TGACATAATT

TAAGTGTATA

ATGGAACTGA

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

TGC-GCCCCTG

TGGCATGAGG

TGCCTGGGCC

GCCAGCGTGG

AGCC-CTGGG

GCCCCTGTCC

ACAGGCCCTC

TCGCACCCGC

GACTGGTGCA

AGGTTGGCCG

CCCGGGCGAA

CGGACACAGG

TTCTCCACAT

AGCCGCCACA

fCCAGTGCCG

CATCTGT.TGT

TCCTTTCCTA

TGGGGGGTGG

CTGGGGATGC

GGTATCCCCA

GCGTGACCGC

-ITCTCGCCAC

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

CGAGACTC.TG

GAGGCAGAGC

CCCTAGGGTG

CCCTCCCTCCI

GACAGACACA

TCCCGACCTC

CCTCACCCAT

ATGGGGACAC

GATGCCCACA

GCCACACGGC

CTGCACAGCA

CCCCCACGAG

GCTGACCTGC

CACAkCACAGG

CCCTTCCCTG

TTGCCCCTCC

ATAAAATGAG

GGTGGGGCAG

C-GTGGGCTCT

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 00*0*0 *too.* S S S @5

ATGCCTTTAA

CTACTGCTGA

AGGACTTTCC

TTGCTTGCTT

TGGAAAAATA

TTTTTCTTAC

GTACCTTTAG

TGACTAGAGA

CTCCCACACC

TTTATTGCAG

GCATTTTTTT

GTCTGGATCG

CCCAACTTGT

ACAAATAAAG

TCTTATCATG

CTGTTTCCTG

ATAAAGTGTA

TCACTGCCCG

CGCGCGGGGA

CTGCGCTCGG

TTATCCACAG

GCCAGGAACC

GAGCATCACA

TACCAGGCGT

ACCGGATACC

TGTAGGTATC

CCCGTTCAGC

AGACACGACT

GTAGGCGGTG

GTATTTGGTA

TGATCCGGCA

ACGCGCAGAA

CAGTGGAACG

ACCTAGATCC

ACTTGGTCTG

TTTCGTTCAT

TTACCATCTG

TTATCAGCAA

TCCGCCTCCA

40 AATAGTTTGC

GGTATGGCTT

TTGTGCAAAA

GCAGTGTTAT

GTAAGATGCT

45 CGGCGACCGA

ACTTTAAAAG

CCGCTGTTGA

TTTACTTTCA

GGAATAAGGG

50 AGCATTTATC

AAACAAATAG

TGAGGAAAAC

CTCTCAACAT

TTCAGAATTG

TGCTATTTAC

TTCTGTAACC

TCCACACAGG

CTTTTTAATT

TCATAATCAG

TCCCCCTGA\

CTTATAATGG

CACTGCATTC

GCTGGATGAT

TTATTGCAGC

CATTTTTTTC

TCTGTATACC

TGTGAAATTG

AAGCCTGGGG

CTTTCCAGTC

GAGGCGGTTT

TCGTTCGGCT

AATCAGGGGA

GTAAAAAGGC

AAAATCGACG

TTCCCCCTGG

TGTCCGCCTT

TCAGTTCGGT

CCGACCGCTG

TATCGCCACT

CTACAGAGTT

TCTGCGCTCT

AACAAACCAC

AAAAAGGATC

AAAACTCACG

TTTTAAATTA

ACAGTTCA

CCATAGTTGC

GCCCCAGTGC

TAAACCAGCC

TCCAGTCTAT

GCAACGTTGT

CATTCAGCTC

AAGCGGTTAG

CACTCATGGT

TTTCTGTGAC

C-TTGCTCTTG

TGCTCATCAT

GATCCAGTTC

CCAGCGTTTC

CGACACGGAA

AGGGTTATTG

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

TCAAGAAGAT

TTA.AGGGATT

AAAATGAAGT

ATGCTTAATC

CTGACTCCCC

TGCAATGATA

AG2CCGGAAGG

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

AGGTGGCGrA

GTGCGCTCTC

GGAAGCGTGG

CGCTCCAAGC

GGTAACTATC

ACTGGTAACA

TGGCCTAACT

GTTACCTTCG

GGTGGTTTTT

CCTTTC-ATCT

TTGGTCATGA

TTTAAATCAA

AGTGAGGCAC

GTCGTGTAGA

C-CGCGAGACC

GCCGAC-CGCA

CGGGAAGCTA

ACAGGCATCG

CGATCAAGGC

CCTCCGATCG

CTGCATAA-T

TCAACCA AGT

ATACGGGATA

TCTTCGGGGC

?LCTCGTGCAC

AAAACAGGAA

CTCATACTCT

GGATACATAT

CGA.AAAGTGC

GCCATCTAGT

GAGAAAGGTA

TGTGTTTAGT

ACTGCTATAC

TTATAATCAT

TAACTATGCT

ATATTTGATG

TTTTACTTGC

CAATTGTTGT

TCACAAATTT

TCATCAATGT

TGCTGGAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTAATG

CTTCCTCGCT

ACTCAAAGGC

GAGCAAAAGG

ATAGGCTCCG

ACCCGACAGG

CTGTTCCGAC

CGCTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAAA.AAZGAGT

TTGTTTGCAA

TTTCTACGGG

GATTATCAAA

TCTA-AAGTAT

CTATCTCAGC

TAACTACGAT

CACGCTCACC

GAAGTGGTCC

GAGTAAGTAG

TGGTGTCACG

GAGTTACATG

TTGTCAGAAG

CTCTTACTGT

CATTCTGAGA

ATACCGCGCC

GAAAACTCTC

CCAACTGATC

GGCAAAATGC

TCCTTTTTCA

TTGAATGTAT

CACCTGACGT

GATGATGAGG

GAAGACCCCA

AATAGAACTC

AAGAAAATTA

AACATACTGT

CAAAAATTGT

TATAGTGCCT

TTTAAAA-AAC

TGTTAACTTG

CACAAATAAA

ATCTTATCAT

CTTCGCCCAC

CACAAATTTC

CATCAATGTA

ATGGTCATAG

AGCCGGAAGC

TGCGTTGCGC

AATCGGCCAA

CACTGACTCG

GGTAATACGG

CCAGCAAAAG

CCCCCCTGAC

ACTATAAAGA

CCTGCCGCTT

AT GCTCAkCGC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGAGTAA

GATCTGTCTA

ACGGGAGGGC

GGCTCCAGAT

TGCAACTTTA

TTCGCCA-TT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTA-G

ACATAGCAGA

AAGGATCTTA

TTCAGCATCT

CGCAAAAAAG

ATATTATTGA

TTAGAAA-AAT

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 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8691

C.

6 0 6006 0 0 0S6@ 06~~S SS Ce 0O 0 0 0@ OS S

C

mm..

S

*6 Ce 30

C

0 60 6 0 0O S em S0 INFORMATION FOR SEQ ID SEQUENCE CHAR~ACTERISTICS: LENGTH: 8321 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GACGGATCGG GAGATCTGCT AGGTGACCTG AGGCGCGCCG GCTTCGAATA GCCAGAGTAA CCTTTTTTTT TAATTTTATT TTATTTTATT TTTGAGATGG AGTTTGGCGC CGATCTCCCG ATCCCCTATG GTCGACTCTC AGTACAATCT GCTCTGATGC CGCATAC-TTA AGCCAGTATC

TGCTCCCTGC

CAAGGCALAGG

TGCTTCGCGA

AGTAATCAAT

T'rACGGTAAA

TGACGTATGT

ATTTACGGTA

CTATTGACGT

GGGACTTTCC

GGTTTTGGCA

TCCACCCCAT

AATGTCGTAA

TCTATATAAG

TTAATACGAC

AGGTCTCGAG

CACCATGGAG

GTGALAGTGAA

TCTCCTGTGT

CTCCAGAGAA

ATCCAGACAC

ACCTGCAAAT

TGGACGACGG

CTAGCACCAA

GCACAGCGGC

GGAACTCAGG

GACTCTACTC

ACATCTGCAA

GGCCAGCACA

CATCCCGGCT

CGGAGGCCTC

GCTCTGGGCA

GCTGGGCTCA

CCCCAAAGGC

GTAACTCCCA

CCGTGCCCAG

AGAGTAGCCT

CAGAGGCCGG

TACAGGGCAG

CAAGAACCAG

GGAGTGGGAG

CTCC-GACGGC

GGGGAACGTC

GAGccTrcTcc

CTCTCGCGGT

45 AGCATGGAAA

CCACGGGTCA

GTCCCCACAC

AGGGGCTGCC

GCCCTGGGCT

50 CTCTGTAGGA

CGGGGGCATG

GGCACTAACC

GGGGACATGC

GCCCAGACCC

55 ACACGTGCAC

AGCAAGGTCC

CACCTCAAGG

CCAGCCCTCC

CCACGTCACG

CAGCCTCGAC

CCTTGACCCT

CGCATTGTCT

GGGAGGATTG

AGGCGGAAAG

TAAGCGCGGC

CGCCCGCTCC

AAGGGAAAAA

TTGTGTGTTG

CTTGACCGAC

TGTACGGGCC

TACGGGGTC.A

TGGCCCGCCr

TCCCATAGTA

AACTGCCCAC

CAATGACGGT

TACTTGGCAG

GTACATCAAT

TGACGTCAAT

CAACTCCGCC

CAGAGCTCTC

TCACTATAGG

TCTCTAGATA

TTGTGGTTAA

TCTGGTGGAG

AACCTCTGGA

GAGGCTGGAG

TGTAAAGGGT

GAGCCGTCTG

GGCCTGGTTT

GGGCCCATCG

CCTGGGCTGC

CGCCCTGACC

CCTCAGCAGC

CGTGA.ATCAC

GGGAGGGAGG

ATGCAGCCCC

TGCCCGCCCC

GGCACAC-GCT

GACCTGCCA-A

CAAACTCTCC

ATCTTCTCTC

GTAAGCCAGC

GCATCCAGGG

CTCGGCCCAC

CCCCGAC-AAC

GTCAGCCTGA

AGCAATGGGC

TCCTTCTTCC

TTCTCATGCT

CTGTCTCCCG

CGCACGAGGA

TAAAGCACCC

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

C-AGGCCTCG

ACCACCACG

CCTCTGCCCTi

CACAGGTGTA

CCTGCCTGGT

AGCCGGAGAA

TCTACAGCAA

CCGTGATGCA

GTAAATGAGT

TGCTTGGCAC

AG CGCTGCC C

GAGGCCTGAG

TGTGCAGGTG

TGGGGGATTT

AAGCCCTAGG

TTCTGTGAGC

GTGCGTAGGG

CTGCCCAGCC

CCTGTGGAGG

CCCCGCACTG

CGGAGCCTCA

GAACACTCCT

TCTCGGCAGC

GTGCCCCTGC

TGGCCCACTT

AGTTGCCAGC

ACTCCCACTG

CATTCTATTC

AGCAGGCATG

GGCTCTAGGG

GTTACGCGCA

TTCCCTTCCT

CTCAATTAGT

AGTAGTGCGC

AGAATCTGCT

GTTGACATTG

GCCCATATAT

CCAACGACCC

GGACTTTCCA

ATCALAGTGTA

CCTGGCATTA

TATTAGTCAT

AGCGGTTTGA

TTTGGCACCA

AAATGGGCGG

GAGAACCCAC

CTTGGTACCA

CGATTGGAAT

TCCTTGTCCT

GCTTAGTGCA

GTGACTATTA

ACATTAGTCA

TCTCCAGAGA

ACACAGCCAT

GCCAAGGGAC

TGGCACCCTC

ACTACTTCCC

ACACCTTCCC

TGCCCTCCAG

ACACCAAGGT

GAAGCCAC-GC

AGCAAGGCAG

AGGGAGAGGG

AACCCAGGCC

CGGGAGGACC

CTCGGACACC

AAATCTTGTG

CCCTCCAGCT

TC-GGTACCAA

GAGAGTGACC

CACCCTGCCC

CA-AAC-GCTTC

CAACTACAAG

GCTCACCGG

TGAGGCTCTG

GCGACGGCCG

GTACCCCCTG

TGGGCCCCTG

TGGCATGAGG

TGCCTGGGCC

GCCAGCGTGG

AGCCCCTGGG

GCCCCTGTCC

ACAGGCCCTC

TCGCACCCGC

GACTGGTGCA

AGGTTGGCCG

CCCGGGCGAA

CGGACACAGG

TTCTCCACAT

AGCCGCCACA

CCCAGTGCCG

CATCTGTTGT

TCCTTTCCTA

TGGGGGGTGG

CT GGGGATG C

GGTATCCCCA

GCGTGACCGC

TTCTCGCCAC

CAGCAACCAT

51

GAGCAAAATT

TAGGGTTAGG

ATTATTrGACT

GGAGTTCCGC

CCGCCCATTG

TTGACGTCAA

TCATATGCCA

TGCCCAGTAC

CGCTATTACC

CTCACGGGGA

AAATCAACGG

TAGGCGTGTA

TGCTTACTGG

ATTTAAATTG

TCTTGCGGCC

TGTTTTAAAA

GCCTGGAGGG

CATGTATTGG

AGGTGGTGAT

CAATGCCAAG

GTATTACTGT

TCTGGTCACG

CTCCAAGAGC

CGAACCGGTG

GGCTGTCCTA

CAGCTTGGGC

GGACAAGAAA

TCAGCGCTCC

GCCCCGTCTG

TCTTCTGGCT

CTGCACACAA

CTGCCCCTGA

TTCTCTCCTC

ACAAPAACTCA

-AAGGCGGGA

CATGTCCGGA

GCTGTACCAA

CCATCCCGGG

TATCCCAGCG

ACCACGCCTC

GACAJAGAGCA

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

GTTGGTGAGA

TGCCTG%'AC-G

CCTCTTCACC

TTTTCCCCAG

AGGGGCAGGT

CCTAAGCCCA

CCAGATTCCA

CACATGCCCA

CAGGTGCC-T

GCCACATGGA

CCTC-T,CCC

ATGAGCTGAC

ACATCGCCGT

CCGTGCTLGGA

GGTGGCAGCA

ACACGCAGAA

GCTCCCCGGG

CCGGGCGCCC

ATGGTTCTTT

GGGTCCCACT

GGGCTCAGCC

AGCAGCACCT

CAGCCCCTGC

CATGCCCACT

CTACCCCCAC

AACCGACTCC

CACACACTCA

CACCACACAC

CCCAGACCAG

CCCCACGCGG

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 1690 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

CTCTCAACAT

TTCAGAATTG

TGCTATTTAC

TTCTGTAACC

TCCACACAGG

CTTTTTAATT

TCATAATCAG

TCCCCCTGAA

CTTATAATGG

CACTGCATTC

GCTGGATGAT

TTATTGCAGC

CA-ITTTTTTC

TCTGTATACC

TGTGAAATTG

AAGCCTGGGG

CTTTCCAGTC

GAGGCGCGTTT

TCGTTCGGCT

AATCAGGGGA

GTAAAAAGGC

AAAATCGACG

TTCCCCCTGG

TGTCCGCCrlT

TCAGTTC'GT

CCGACCGCTG

TATCGCCACT

CTACAGAGTT

TCTGCGCTCT

AACAAACCAC

50 AAAAAGGATC

AAAACTCACG

TTTTAAATTA

ACAGTTACCA

CCATAGTTGC

55 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

TTATA.ATGGT

ACTGCATTCT

GTCGACCTCT

TTATCCGCTC

TGCCTAATGA

-GGAAACCTG3

GCGTATTGGG

GCGGCGAGCG

TAACGCAGGA

CGCGTTGCTG

CTCAAGTCAG

AAGCTCCCTC

TCTCCCTTCG

GTAGGTCGTT

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

GCAAGTA.AAG

AATCAACCAG

ACGTTTTTCC

CTCTCTGAGG

GACTAACAGG

AGACCATGGG

TGACATAATT

TAAGTGTATA

ATGGAACTGA

CAGAAGAAAT

CAAAAAAGAA

TGAGTCATGC

AAAAAGCTGC

GGCATAACAG

CTGCTATTAA

TTAATAAGGA

TTTGTAGAGG

AAAATGAATG

AGCAATAGCA

TTGTCCAAAC

GGGGATCTCA

TACAAATAAA

AGTTGTGGTT

AGCTAGAGCT

ACAATTCCAC

GTGAGCTAAC

TCGTGCCAGC

CGCTCTTCCG

GTATCAGCTC

AJAGAACATGT

GCGTTTTTCC

AGGTGGCGAA

GTGC-GCTCTC

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

CCCATTCTCC

CGGCCTCTGA

AA.AAGCTTGG

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

TCATCA-ATGT

TGCTGGAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTAAG

CTTCCTCGCT

ACT CAAAC-GC

GAGCAAAAGG

ATAGGCTCCG

ACCCGACAGG

CTGTTCCGAC

CGOTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAAAAAGAGT

TTGTTTGCAA

TTTCTACGGG

GATTATCAAA

TCTAAAGTAT

CTATCTCAGC

TAACTACGAT

CACGCTCACC

GAAGTGGTCC

GAGTAAGTAG

TGGTGTCACG

GAGTTACATG

TTGTCAGAAG

CTCTTACTGT

CATTCTGAGA

ATACCGCGCC

GAAAACTCTC

CCAACTGATC

CC CAAAATG C

TCCTTTTTCA

GCCCCATGGC

GCTATTCCAG

ACAGCTCACG

GGATTTTATC

AAATATGGGG

GTACTTCCAA

GGGTAGGAAA

TATAGTTCTC

TTTGGATGAT

TTGGATAGTC

ACTCTTTGTG

TTTGGGGAAA

AAAAGGCATC

CA.AGTTCTCT

GCTTTAGATC

CCTACAGAGA

CTACTGATTC

CAGTGGTGGA

GATGATGAGG

GAAGACCCCA

AATAGAACTC

AAGAAAATTA

AACATACTGT

CAAAAATTGT

TATAGTGCCT

TTTAAAAAAC

TGTTAACTTG

CACAAATAAA

ATCTTATCAT

CTTCGCCCAC

CACAAATTTC

CATCAATGTA

ATGGTCATAG

AGCCGC-AAGC

TGCGTTGCGC

AATCGGCCZA

CACTGACTCG

GGTAATACGG

CCAGCAAAAG

CCCCCCTGAC

ACTATAAAGA

CCTGCCGCTT

ATGCTCACC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGAGTAA

GATCTGTCTA

ACGGGAGGC

GGCTCCAGAT

TGCAACTTTA

TTCGCCAGTT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTATG

ACATAGGAGA

AAGGATCTTA

TTCAGCATCT

CGCAAAAAAG

ATATTATTGA

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATTGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGT

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

ATGCCTTTAA

CTACTGCTGA

AGGACTTTCC

TTGCTTGCTT

TGGAAAAATA

TTTTTCTTAC

GTACCTTTAG

TGACTAGAGA

CTCCCACACC

TTTATTGCAG

GCATTTTTT1

GTCTGGATCG

CCCAACTTGT

ACAAATAA-AG

TCTTATCATG

CTGTTTCCTG

ATAAAGTGTA

TCACTGCCCG

CGCGCGGGGA

CTGCGCTCGG

TTATCCACAG

GCCAGGAACC

GAGCATCACA

TACCAGGCGT

ACCGGATACC

TGTAGGTATC

CCCGTTCAGC

AGACAGGACT

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

C

AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAkATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT C INFORM.ATION FOR SEQ ID NO:ll: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 8897 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1l: 8280 8321

GGTACCAATT

TGTTGGTGCT

CAGTCTCCCT

TCATTGTACA

CTCCACAGCT

GCGGCAGTGG

TGGGAGTTTA

AGTTGGAAAT

AAACTCTGAG

AGGTCAGAAA

AGAACTTTAT

TACGCTTCTT

CCTAACATGC

CATCCTGTTT

CATCTGATGA

ATCCCAGAGA

AGGAGAGTGT

CGCTGAGCAA

GCCTGAGCT-

CCCACCTGCT

CCACAGGGGA

CCTCCTTGGC

CACCTGTGGT

CA.ATTTCTCT

AATCATCCTT,

ACAAGCCTTC

TCCCCTCCTC

TCCTTTGA-T

TATAAAGAGA

45 ATAAACAAAC

ATGCCTTATT

GAGTACTTTC

AAATGTTGCA

ACTTCTAGAT

50 CTTTATTTAC

TTAAACTGTG

TATACCTACT

AAAAGATATG

TAGAAATTTG

55 ACAAGAAGGG

ATGATCTGTG

TCTTATACCC

GTAAGTGGGG

TGAGTCTGCC

CATCTGTGCC

CTTCAGCAAG

AACACTCACA

ATGGGGCACT

ACCTCTCTCT

CAAATGACTG

TGGGGAAGGA

GAATGTTGAT

TAAATTGATA

GATGTTCTGG

GCCTGTCAGT

TAATAATGGC

CCTGATCTAC

ATCAGGGACA

TTACTGCTTT

AAAACGTAAG

GGGGTCGGAT

AGCATGCAAA

TA.AGGAAT.;G

GGTCTCCTTG

CCTTATCCGC

GCTTCTTTCC

CCAGTTCAAA

GGCCAAAGTA

CACAGAGCAG

AGCAGACTAC

GCCCGTCACA

CCTCAGTTCC

CCTACCCCTA

TTTAATTATG

TTCTOTCTTT

TATAAGGGAC

C3Z;TTCTAT-T

TGTCCTCACA

AGCAAGCCCT

CAATTCCCTG

ATCATTCATT

AATAGGGAAA

TACATTTTTA

CACAACCTAA

AAGGTTCTAT

GACTGAGTGT

AAAAGCCAAA

GTATGTTTAT

CACACAGATG

TTCTGTATGT

GATGGAAATT

GCTTCTGGGG

CACTGTTCTG

AGTTAATAGA

GCCTGGGATC

TTCCAGGGCT

CTGTTTGGCT

GGGACAGAGG

TGTTTGGGA.A

CTGGCCCTGC

GCCTACACTC

ACAATCCCTT

CAGTCATGGA

GAGTATCAAA

TCTCCTTAGG

ATTCCTGCTT

CTTGGAGATC

AACACCTATT

AAAGTTTCCA

GATTTCACAC

CAAGGTTCAC

TCTCGAGTCT

GACGTGGCCA

GCCCTCAGAA

GGGGAAGCTA

CTATAATTAT

AAACAACACA

TCAGGAACTG

TC-TGGAACTG

CAGTGGAAGG

GAGAC-CAAGG

GAGAAACACA

AAGAGCTTCA

AGCCTGACCC

TTGCGGTCCT

CTAATGTTGG

CCTCATTTAA

TAAAT1ATGTA

TACCCTATCA

GTCCCCTGGG

CATAGTCCTT

AGAJATCAACC

GCAACATC-AT

TGTTTAAGTT

AACAGGTACT

TTTAATCCAC

AAAGCTGAGA

CCCCACCCAC

AATTGGAAAT

ACATTAGAAT

AATCTCATAA

TTTCATCCAT

ACTCTTAGCT

TCTTGGTAAT

TATACACATT

TAGAAGAGGA

AAATAGCTAC

CAAGGTGCTC

AGCTAGGAGC

ACAGAATTAA

GGGGGAAGGG

CCCTCTCAGC

TGAAGGGGTT

TGTCCTGCTT

GAAACTACAT

TCTTTCAAAC

TCTCGAGCAC

CCAGCAGTGA

AAGCGTCCAT

TAGAATGGTA

ACCGATTTTC

TCAAGATCAG

ATGTTCCATT

CTAGATAACC

TTCTTTGCCT

TGGCTGCAAA

GGAAGAAACT

CTGGGATAAG

CCCAAGGGCA

TGGCTGCACC

CCTCTGTTGT

TGGATAACGC

ACAGCACCTA

AAGTCTACGC

ACAGGGGAGA

CCTCCCATCC

CCAGCTC.ATC

AGGAGAATGA

TAATTATTAT

GTCATCCTA-A

TCCTCTGCAA

CCATGGTAGG

TTTA.AGGGTG

AAAGCAAAZTT

ATAAAATAAC

CATCATGGTA

GAGGGACTCC

ACTATACTGT

GACAAATATA

CAAAAAACTA

AGCCCGATTG

ACCCAATGAG

AAATAATGTT

ATA.AAGTTCA

GGGGGTGGGC

GTTCTGTTCC

ATGCTTCAAA

ATAAGTAATA

CTGCCTAATC

AACAAAACAA

ACACATACAT

CCTTGCCCAG

CACATGTAAA

TACTCATCCA

CAGGAGTAAC

TGTTTTTCTT

AAGGAAGCAC

TTTGGAGGTT

53

CATGAAGTTG

TGTTTTGATG

CTCTTGCAGA

CCTGCAGAAA

TGGGGTCCCA

CAGAGTGGAG

CACGTTCGGC

GGTCAATCGA

AAAGCATTGA

GAGCTCCAAC

CAAAACATCA

CATGCTGTTT

GAACTTTGTT

ATCTGTCTTC

GTGCCTGCTG

CCTCCAATCG

CAGCCTCAGC

CTGCGAAGTC

GTGTTAGAGG

TTTGGCCTCT

TTTCACCTCA

ATAAATAAAG

CTGTTGTTTT

GGCACGTAAC

CACAGTCCTC

AGAGACTTGC

ACAGGTCfTA

TTTCJAAAAGA

AACACAATAA

CTTAGACTTA

TGTCTGCCAA

GAGATTAAAA

TTCTATAACT

TGCAAGAATG

TCCAAC-ZATA

GAGAATTAAC

ACATAAGAGA

AAACCAGGTA

GAGTTAGTGC

TCGTGTGGGG

ATAACTTCAC

GGTCAAGACC

CTGCCCWCTT

CAGGCCTGCT.

AGAAATTAAA

ACACTGGAAA

TGAGGACTCT

TCCAACACAC

TAACACAGCA

TCCAGTCAGT

CTTGCC-CTTC

TGAGTAGGGG

CCTGTTAGGC

ACCCAAATTC

TCTAGTCAGA

CCAGGCCAGT

GACAGGTTCA

GCTGAGGATC

TCGGGGACAA

TTGGAATTCT

GTTTACTGCA

AAAACAATTT

AGATTTTAAA

TCTGTCTGTC

ACTTAAACAC

ATCTTCCCGC

AATAACTTCT

GGTAACTCCC

AGCACCCTGA

ACCCATCAGG

GAGAAGTGCC

GACCCTTTTT

CCCCCCTCCT

TGA.ATCTTTG

ACCAACTACT

CATTTATAAA

CC-TCAAACCC

TTC-CTTGTTT

CAGTCATATA

AG.AALACCTGC

AAGCAATTAA

ATGGAATGTC

GGGCCGTATT

ACATTCATTA

CAGCAA.TCCC

TTCAAAGCAG

GAATGAGTTA

AAGCTACAAC

AACTCAATGC

AAAATAAAGT

CTGGC-AGAA.G

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

ATCCALACCGC

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

T.ATTTTAGAT

AGGAAAACCT

CTCAACATTC

CAGAATTGCT

CTATTTACAC

CTGTAACCTT

CACACAGGCA

TTTTALATTTG

ATAATCAGCC

CCCCTGAACC

TATAATGGTT

CTGCATTCTA

TGGATGATCC

ATTGCAGCTT

TTTTTTTCAC

TGTATACCGT

TGAAATTGTT

GCCTGGGGTG

TTCCAGTCGG

GGCGGTTTGC

GTTCGGCTGC

50 TCAGGGGATA

AAAAAGGCCG

ALATCGACGCT

CCCCCTGGAA

TCCGCCTTTC

55 AGTTCGGTGT

GACCGCTGCG

TCGCCACTGG

ACAGAGTTCT

TGCGCTCTGC

CAAACCACCG

AAAGGATCTC

AACTCACGTT

TTAAATTAA.A

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

G-TTTTGCTCA

TACTCCTCC-A

AAGTTTTTTG

CACAAAGGAA

TATAAGTAGG

TAGA-TCTCT

TAAAGGGGTT

ATACCACATT

TGAAACATAA

ACAAAT.AAAG

GTTGTGGTTT

TCCAGCGCGG

ATA.ATGGTTA

TGCATTCTAG

CGACCTCTAG

ATCCGCTCAC

CCTAATGA-T

GAA:ACCTGTC

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

PLAAGCTGCAC

CATAACAGTT

GCTATTAATA

AATAAGGAAT

TGTAGA-BGGTT

AATGAATGCA

CAATAGCATC

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

GCAACCATAG

CATTCTCCGC

GCCTCTGAGC

AAGCTTGGAC

GGCTGGTAGG

GTGTCCCAAA

GAGTTCAAGT

GTGATTATGG

AGAATTAATA

GCCAAAAGTT

GACATGGTTT

CACCTTAGAC

GAAATTGATT

CAGGAGGAAA

GATGCTTTCA

TTTTGCTGGC

ACAAACTACC

GTGTTAAACT

AATGGGAGCA

CATCTAGTGA

GAAAGGTAGA

TGTTTAGTAA

TGCTATACAA

ATAATCATAA

ACTATGCTCA

ATTTGATGTA

TTACTTGCTT

ATTGTTGTTG

ACAAATTTCA

ATCAATGTAT

CTGGAGTTC-T

AATAGCATCA

TCC.AAACTCA

GCGTAATCAT

AACATACGAG

ACATTAATTIG

CATTAATGAA

TCCTCGCTCA

TCAAAGGCGG

GCAAAAGGCC

AGGCTCCGCC

CCGACAGGAC

GTTCCGACCC

CTTTCTCAA-A

GGCTGTGTGC

CTTGAGTCCA

ATTAGCAGAG

GGCTACACTA

AAAAGAGTTG

GTTTGCALAGC

TCTACGGGGT

TTATCAAAAA

TAAAGTATAT

ATCTCAGCGA

ACTAGGATAC

CGCTCACCGG

AGTGGTCCTG

TGGGGGCCAA

TTGCTAGCTT

ATGCTAGAGC

GCCCCTCCCC

AAAATGAGGA

TGGGGCAGGA

TGGGCTCTAT

CGCCCTGTAG

CACTTGCCAG

TCGCCGGGC

TCCCGCCCCT

CCCATGGCTG

TATTCCAGAA

AGCTCAGGGC

ATTTTATCCC

ATATGGGGAT

ACTTCCAAAG

GTAGGAAAAC

TAGTTCTCAG

TGGATGATGC

GGATAGTCGG

TCTTTGTGAC

TGGGGAAATA

AAGGCATCA.A

AGTTCTCTGC

TTTAGATCTC

TACAGAGATT

ACTGATTCTA

GTGGTGGAAT

TGATGAGGCT

AGACCCCAAG

TAGA-ACTCTT

GAAAATTATG

CATACTGTTT

AAATTGTGT

TAG'TGCCTTG

TAAXa.AACCT

TTAACTTGTT

CAAATA?-AGC

CTTATCATGT

TCGCCCACCC

CAAA;VTTTCAC

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

ATTGTTTGTC,

GCCTTTAATG

ACTGCTGACT

GACTTTCCTT

GCTTGCTTTG

GALAAAAZTATT

TTTCTTACTC

ACCTTTAGCT

ACTAGAGATC

CCCACACCTC

TATTGCAGCT

AT7TTTTTTCA

CTGC-ATCGGC

CAACTTG.-TT

AAPATAAAGCA

TTATCATGTC

GTTTCCTGTG

AAAGTGTAPA

ACTGCCCGCT

CGCGGGGAGA

GCGCTCGGTC

ATCCACAGAA

CAGGAACCGT

GCATCACAAA

CCAGGCGTTT

CGGATACCTG

TAGGTATCTC

CGTTCAGCCC

ACACGACTTA

AGGCGGTGCT

ATTTGGTATC

ATCCGGCAA-A

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 708 0 7140

CAGTCTATTA

AACGTTGTTG

TTCAGCTCCG

GCGGTTAGCT

CTCATGGTTA

TCTGTGACTG

TGCTCTTGCC

CTCATCATTG

TCCAGTTCGA

AGCGTTTCTG

ACACGGAAAT

GGTTATTGTC

GTTCCGCGCA

GCCCGGGTGA

TATTTTATTT

TCTCAGTACA

GTTGGAGGTC

CGACA.ATTGC

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

A.ACCAAGTCA

ACGGGATAAT

TTCGGGGCGA

TCGTGCACCC

AACAGGAAGG

CATACTCTTC

ATACATATTT

AAAAGTGCCA

GCCGGCTTCG

ATGGAGTTTG

ATGCCGCATA

GCGCGAGCAA

TGCTTAGGGT

ATTGATTATT

ATATGGAGTT

ACCCCCGCCC

TCCATTGACG

TGTATCATAT

ATTATGCCCA

TCATCGCTAT

TTGACTCACG

ACCAAAATCA

GCGGTAGGCG

GTAAGTAGTT

GTGTCACGCT

GTTACATGAT

GTCAGAAGTA

CTTACTGTCA

TTCTGAGAAT

ACCGCGCCAC

AAACTCTCAA

AACTGATCTT

CAAAATGCCG

CTTTTTCAAT

GAATGTATTT

CCTGACGTCG

AATAGCCAGA

GCGCCGATCT

GTTALAGCCAG

AATTTAAGCT

TAGGCGTTTT

GACTAGTTAT

CCGCGTTACA

ATTGACGTCA

TCAATGGGTG

GCCAAGTACG

GTACATGACC

TACCATGGTG

GGGATTTCCA

ACGGGACTTT

TGTACGGTGG

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

TTCCTACT1TG

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 CCACTGCTTA CTGGCTTATC INFORM4ATION FOR SEQ ID NO:12: SEQUENCE CHLARACTERISTICS: LENGTH: 8321 base pairs TYPE: nucleic acid STRAkNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: S. 55 S. S

GGTACCAATT

TTGGAATTCT

TGTCCTTGTT

AGTGCAGCCT

CTATTACATG

TAGTCAAGAT

CAGAGACAAT

AGCCGTGTAT

AGGGACTCTG

ACCCTCCTCC

CTTCCCCGAA

55 CTTCCCGGCT

CTCCAGCAGC

CAAGGTGGAC

CCAGGCTCAG

AGGCAGGCCC

AGAGGGTCTT

CAGGCCCTGC

AGGACCCTGC

GACACCTTCT

CTTGTGACAA

65 CCAGCTCAAG

TACCAACATG

GTGACCGCTG

TA-AATTG AT-1A

TGCGGCCGCT

TTAAAAGGTG

GGAGGGTCCC

TATTGGGTTC

GGTGATATAA

GCAAAGAACA

TACTGTGCAA

GTCACGGTCT

AAGAGCACCT

CCGGTGACGG

GTCCTACAGT

TTGGGCACCC

AAGAAAGTTG

CGCTCCTGCC

CGTCTGCCTC

CTGGCTTTTT

ACACAAAGGG

CCCTGACCTA

CTCCTCCCAG

AACTCACACA

GCGGGACAGG

TCCGGAGCCA

TACCAACCTC

TCTCCTTAGG

TC-CTAGCCAC

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

TGGTCTTCCTl

GGGGAGGCTT

CGTTCAGTGA

TCTCATACAT

TCACCATCTC

ACGAGGAC.AC

ACTGGGGCCA

TCCCCCTGGC

TCAAGGACTA

GCGTGCACAC

TCACCGTGCC

CCAGCAACAC

CTGCTGGAAG

CAGGGCAGCA

ATGCTCAGGG

GCCCCTAACC

CATATCCGGG

CCTCAGCTCG

GAGCCCAAAT

GCCTCGCCCT

ACCACGTGGG

TGCCCTGAGA

GGTGTACACC

1.20 180 240 300 360 420 480 540 600 660 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 CTGCCCCCAT CCCGGGATGA GGCTTCTATC CCAGCGACAT TACAAGACCA CGCCTCCCGT ACCGTGGACA AGAGCAGGTG GCTCTGCACA ACCACTACAC CGGCCGGCAA GCCCCCGCTC CCCCTGTACA TACTTCCCGG CCCCTGCGAG ACTGTGATGG ATGAGGGAGG CAGAGCGGGT TGGGCCCCCT AGGGTGGGGC GCGTGGCCCT CCCTCCAGCA CCTGGGGACA GACACACAGC CTGTCCTCCC GACCTCCATG GCCCTCCCTC ACCCATCTAC ACCCGCATGG GGACACAACC GGTGCAGATG CCCACACACA TGGCCGGCCA CACGGCCACC GGCGAAC'rGC ACAGCACCCA CACAGGCCCC CACGAGCCCC CCACATGCTG ACCTGCTCAG GCCACACACA CACAGGGGAT GTGCCGCCCT TCCCTGCAGG TGTTGTTTGC CCCTCCCCCG TTCCTAATAA AATGAGGAAA GGGTGGGGTG GGGCAGGACA GGATGCGGTG GGCTCTATGG TCCCCACGCG CCCTGTACCG GACCGCTACA CTTGCCAGCG CGCCACGTTC GCCGGGCCTC AACCATAGTC CCGCCCCTAA TTCTCCGCCC CATGGCTGAC CTCTGAGCTA TTCCAGAAGT GCTTGGACAG CTCAGGGCTG CTGGTAGGAT TTTATCCCCG GTCCCAAAAT A:TGGGGATTG GTTCAA.GTAC TTCCAAAGAA GATTA;'TGGGT AGGAAAACCT AATTAATATA GTTCTCAGTA CAAAAGTTTG C-ATGATGCCT CATC-GTTTGG ATAGTCGGALG CCTTAC-ACTC TTTGTGACA-A G A TT TG GGC-AAkATATA GGAGGA-AAAAs GGCATCAAGT TGCTrTTCAAG TTCTCTGCTC TTGC7GGCTT TAGATCTCTT AAACTACCTA CAGAGATTTA GTTAAACTAC TGATTCTAAT TGGGAGCAGT GGTGGAATGC TCTAGTGATG ATGAGGCTAC 50 AAGGTAGAAG ACCCCAAGGA TTTAGTAATA GAACTCTTGC CTATACAAGA AAATTATGGA AATCATAACA TACTGTTTTT TATGCTCAAA AATTGTGTAC 55 TTGATGTATA GTGCCTTGAC ACTTGCTTTA AAAAACCTCC TGTTGTTGTT AACTTGTTTA AAATTTCACA AATAAAGCAT CAATGTATCT =ACATCTCT GGAGTTCTTC GC CCCCA TAGCATCACA AATTTCACAA CAAACTCATC AATGTATCTT GTAATCATGG TCATAGCTGT CATACGAGCC GGAAGCATAA 65 ATTAATTGCG TTGCGCTCAC TTAATGAATC GGCCAACGCG CTCGCTCACT 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

GCGCATTAPAG

CCCTAGCGCC

TCAAAAAAGG

CTCCGCCCAT

TALATTTTTTT

AGTGAGGAGG

CGATTTCGCG

CTGCCAT CAT

GCAAGA-ACGG

TGACCACAAC

GGTTCTCCAT

GAGAACTCA-A

TAAGACTTAT

GCAGTTCTGT

GGATCATGCA

AACTTCTCCC

ATAA-GTTTGA

CCCTCCTAAA

TGTGA.AGGA

AAGCTCTAAG

TGTTTGTGTA

CTTTAATGAG

TGCTGACTCT

CTTTCCTTCA

TTGCTTTGCT

AAAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

GGATCGGCTG

ACTTGTTTAT

ATAAAGCATT

ATCATGTCTG

TTCCTGTGTG

AGTGTAAAGC

TGCCCGCTTT

CGGGGAGAGG

GCTCGGTCGT

AACCAGGTCA

TGGGAGAGCA

GACGGCTCCT

AACGTCTTCT

CTCTCCCTGT

CGCGGTCGCA

TGGAAATAAA

GGGTCAGGCC

CCACACTGGC

GCTGCCCTCG

TGGGCTGGGC

GTAGGAGACT

GGCATGCCTA

CTAACCCCTG

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

AATCAGCCAT

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

AACAAAC CCC

CACACACGGA

ACACGTGAAC

CGAGCCTCTC

ACAAGGGTGC

TGGCCCTGGC

CCTTCTAGTT

GGTGCCACTC

AGGTGTCATT

GACAATAGCA

AGCTGGGGCT

GTGGTGGTTA

GCTTTCTTCC

ATGCATCTCA

ACTCCGCCCA

GAGGCCGAGG

GGCCTAGGCT

CGGCAATCCT

TTGAACTGCA

TC-GCCTCCGC

GAAGGTAAAC

AATCGACCTT

CGAGGAGCTC

GAATTGGCAA

GCCATGAATC

AGTGACACGT

GGCGTCCTCT

AAGAAAGACT

TTTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGCA

GAGTGTCTGC

AAGGGGTTAA

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAGCGCGGGG

AATGGTTACA

CATTCTAGTT

AC CTCTAGCT

CCGCTCACAA

TAATGAGTGA

AACCTGTCGT

ATTGGGCGCT

CGAGCGGTAT

CCTGGTCAA-A

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

CCGCCTCGGC

TTTGCAAA-ZA.

AGCGTGAAGG

TCGTCGCCGT

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

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 49S0 5040 5100 5160 5220 5280 5340 5400 5460 a a

AAAGGCGGTA

AAAAGGCCAG

GCTCCGCCCC

GACAGGACTA

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

TCTCCCC-ATC

CAGTATCTGC

GCTACAACAA

TTTGCGCT.GC

TATTAATAGT

ACATAACTTA

TCA-ATAATGP.

GTGGACTATT

ACG-CCCCCTA

ACCTTATGGG

GTGATGCGGT

CCA.AGTCTCC

TTTCCAAAAT

TGGGAGGTCT

ATCGAAATTA

(A

(B

(C

(D

(ii) (x i)

GACGGATCGG

AGTAACCTTT

TCCCGATCCC

GTATCTGCTC

TACAACAAGG

TGCGCTGCTT

ATACGGTTAT

CAAAAGGCCA

CCTGACGAGC

TAAAGATACC

CCGCTTACCG

TCACGCTGTA

GAACCCCCCG

CCGGTAAGAC

AGGTATGTAG

AGGACAGTAT

AGCTCTTGAT

CAGATTACGC

GACGCTCAGT

ATCTTCACCT

GAGTAAACTT

TGTCTATTTC

GAGGGCTTAC

CCAGATTTAT

ACTTTATCCG

CCAGTTAATA

TCGTTTGGTA

CCCATGTTGT

TTGGCCGCAG

CCATCCGTAA

TGTATGCGGC

AGCAGAACTT

ATCTTACCGC

GCATCTTTTA

AAAAAGGGAA

TATTGAAGCA

AAAATAAAC

GGATCGGGAG

TTTTTTTTAA

CCCTATC-GTC

TCCCTGCTTG

GGC.AAGGCTT

TTCGCGATGT

AATACAA'TAC

CGGTAAATGG

CGTATGTTCC

TACGGTAAAC

TT-ACC-TCA

ACTTTC'CTAI:

TTTGGCAGTA

ACCCCATTGA

GTf-GTAACAA

ATATAAGCAG

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

AAATAGGGGT

ATCTGCTAGG

TTTTATTTTA

GACTCTCAGT

TGTGTTGGAG

GACCGACAAT

ACGGGCCAGA

GGGGTCATTA

CCCGCCTGGC

CA.TAGTAADCG

TGCCOACTTG

TGACGGTA-AA

TTGGCAGTAC

CATCAATGGG

CGTCAATGGG

CTCCGCCCCA

AGCTCTCTGG

CTATAGGGAG

AGGGGATAAC

AAAGGCCGCG

TCGACGCTCA

CCCTGGAAGC

CGCCTTTCTC

TTCGGTGTAG

CCGCTGCGCC

GCCACTGGCA

AGAGTTCTTG

CGCTCTGCTG

AACCACCGCT

AGGATCTCAA

CTCACGTTAA

A.AATTAAAAA

TTACCAATGC

AGTTGCCTGA

CAGTGCTGCA

CCAGCCAGCC

GTCTATTAAT

CGTTGTTGCC

CAGCTCCGGT

GGTTAGCTCC

CATGGTTATG

TGTGACTGGT

CTCTTGCCCG

CATCATTGGA

CAGTTCGATG

CGTTTCTGGG

ACGGA.AATGT

TTATTGTCTC

TCCGCGCACA

TGACCTGAGG

TTTTATTTTT

ACA.ATCTGCT

GTCGCTGAGT

TGCATGAAGA

TATACGCGTT

GTTCATAGCC

TGACCGCCCA

CrAATAGGGA

GCAGTACATC

TGC-CCCGCCT

ATCTACGTA',

CGTGGATAGC

AGTTTrGTTTT

TTGACGCAAA

CTAACTAGAG

ACCCAAGCTT

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

TTTCCCCGAA

CGCGCCGGCT

GAGATGGAGT

C'rGATGCCGC

AGTGCGCGAG

Al CTGCCTTAG

GACATTGATT

CATATATGGA

ACGACCCCCG

CTTTC'CATTG

AAGTGTATCA

GGCATTATGC

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

GTGCACCCAAb

CAGGAAGGCA

TACTCTTCCT

ACATATTTGA

AAGTGCCACC

TCGA.ATAGCC

TTGGCGCCGA

ATAGTTAAGC

CAAA-ATTTAA

GGTTAGGCGT

ATTGACTAGT

GTTCCGCGTT

CCCATTGACG

ACGTCAATGG

TATGCCAAGT

CCAGTACATG

TATTACCATG

ACGGGGATTT

TCAACGGGAC

GCG'TGTACGG

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 2) INFORMATION FOR SEQ ID NO:13: SEQUENCE CH-ARACTERISTICS: LENGTH: 8897 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO:13:

GAGATCTGCT

TTTTTTAATT

CTATGGTCGA

CCTGCTTGTG

CAAGGCTTGA

CGCGATGTAC

AGCCCGGGTG

TTATTTTATT

CTCTCAGTAC

TGTTGGAGGT

CCGACAATTG

GGGCCAGATA

ACCTGAGGCG

TTATTTTTGA

AATCTGCTCT

CGCTGAGTAG

CATGAAGAAT

TACGCGTTGA

CGCCGGCTTC

GATGGAGTTT

GATGCCGCAT

TGCGCGAGCA

CTGCTTAGGG

CATTGATTAT

GAATAGCCAG

GGCGCCGATC

AGTTAAGCCA

AAATTTAAGC

TTAGGCGTTT

TGACTAGTTA

TTAATAGTAA

ATAACTTACG

AATAATGACG

GGACTATTTA

GCCCCCTATT

CTTATGGGAC

GATGCGGTTT

AAGTCTCCAC

TCCAAAATGT

GGAGGTCTAT

CGAAATTAAT

TCCTTAGGTC

TCCTGCTTCC

TGGACAACCT

CACCTATCTG

AGTTTCCAAC

TTTCACACTC

GGGTTCACAT

TCGAGTCTCT

CGTGGCCATT

CCTCAGAATG

GGAAGCTAGG

ATAATTATCT

ACAACACACC

AGGAACTGTG

TGGAACTGCC

GTGGAAGGTG

GAGCAAGGAC

GAAACACAAA

GAGCTTCAPAC

CCTC-ACCCCC

GCGGTCCTCC

AATGTTGGAG

TCATTTAATA

AATATGTAGT

CCCTATCATC

CCCCTGGGCC

TAGTCCTTTT

?.ATCAACCAA

AACATGATAT

TTITAAGTTCA

CAGGTACTGA

TA.ATCCACAC

AGCTGAGAGA

CCACCCACCA

TTGGAAATAG

ATTAGAATAC

TCTCATAAAA

TCATCCATAT

TCTTAGCTGG

TTGGTAATGT

TACACATTAT

GAAGAGGAAT

ATAGCTACCT

AGGTGCTCAA

CTAGGAGCAC

AGAATTAACC

GGGAAGGGCA

CTCTCAGCTA

AAGGGGTTCA

TCCTGCTTTG

AACTACATAA

TTTCAAACTT

AACTTGCTCA

65 ATGAATTCTT

TTCTATAGTG

GCCAGCCATC

TCAATTACGG

GTAAATGGCC

TATGTTCCCA

CGGTAAACTG

GACGTCAATG

TTTCCTACTT

TGGCAGTACA

CCCATTGACG

CGTAACAACT

ATAAGCAGAG

ACGACTCACT

TCGAGCACCA

AGCAGTGATG

GCGTCCATCT

GAATGGTACC

CGATTTTCTG

AAGATCAGCA

GTTCCATTCA

AGATAACCGG

CTTTGCCTAA

GCTGCAAAGA

AAGAAACTCA

GGGATAAGCA

CAAGGGCAGA

GCTGCACCAT

TCTGTTGTGT

GATAACC-CCC

AGCACCTACA

GTCTACGCCT

AGGGG~AGAGT

TCCCATCCTT

AGCTCATC-7

GAGAATGAAT

ATTATTATCT

CATCCTAAGG

CTCTGCAAGA

ATGC-TAGGAG

TAAGGGTGAC

AGCAAAT--T

AAAATAACAA

TCATGGTACT

GGGACTCCTG

TATACTGTGA

CAAATATATT

ZJAAACTATG

CCCGATTGTC

CCAATGAGGA

ATAATGTTAC

AAAGTTCAAA

GGGTGGGCGA

TCTGTTCCTC

GCTTCAAAAT

AAGTAATAGG

GCCTAATCCT

CAAAACAACA

ACATACATAG

TTGCCCAGAC

CATGTAAATG

CTCATCCATC

GGAGTAACTA

TTTTTCTTTC

GGAAGCACCT

TGGAGGTTTG

CTCATCCCTG

GCGGCCGCTT

TCACCTAAAT

TGTTGTTTGC

GGTCATTAGT

CGCCTGGCTG

TAGTAACGCC

CCCACTTGGC

ACGGTAAATG

GGCAGTACAT

TCAATGGGCG

TCAATGGGAG

CCGCCCCATT

CTCTCTGGCT

ATAGGGAGAC

TGAAGTTGCC

TTGTCATGAC

CTTGCAGATC

AGCAGAGACC

GGGTCCCAGA

GAGTGGAGGC

CGTTCGGCCA

TCAATCGATT

AGCATTGAGT

GCTCCAACAA

AAACATCAAG

TGCTGTTTTC

ACTT'rGTTAC

CTGTCTTCAT

GCCTGCTGAA

TCCAATCGGG

GCCTCAGCAG

GCGAAGTCAC

GTTAGAGGGA

TGGCCTCTGA

TCACCTCACC

AAATAAAGTG

GTTGTTTTAC

CACGTAACCA

CAGTCCTCCC

AGACTTGCTT

AGGTCTTACA

TCAAAAGAAG

CACAATAAAA

TAGACTTAAT

TCTGCCAAGG

CATTAAAAAC

CTATAACTCA

CAAGAATGTT

CAACAATAGA

GAATTAACAA

ATAAGAGAAA

ACCAGGTAAA

GTTAGTGCCT

GTGTGGGGTT

AACTTCACAT

TCAAGACCAA

GCCC'RCTTGA

GGCCTGCTAT

AAATTAAATG

ACTGGAAACC

AGGACTCTTC

CAACACACCT

ACACAGCATC

CAGTCAGTAC

TGCCCTTCTG

AGTAGGGGTG

GGGGCCAAAT

GCTAGCTTCA

GCTAGAGCTC

CCCTCCCCCG

TCATAGCCCA

ACCGCCCAAC

AATAGGGACT

AGTACATCAA

GCCCGCCTGG

CTACGTATTA

TGGATAGCCG

TTTGTTTTGG

GACGCAAATG

AACTAGAGAA

CCAAGCTTGG

TGTTAGGCTG

CCAAACCCCA

TAGTCAGATC

AGGGCAGTCT

CAGGTTCAGC

TGAGGATGTG

AGGGACAAAG

GGAATTCTAA

TTACTGCAAG

AACAATTTAG

ATTTTAAATA

TGTCTGTCCC

TTAAACACCA

CTTCCCGCCA

TAACTTCTAT

TAACTCCCAG

CACCCTGACG

C-ATCAGGGC

GAAGTGCCCC

CCCTTTTTCC

CCCCTCCTCC

AAkTCTTTGCA

CAACTACTCA

TTTATAAAPA

TCAAACCCAC

CCTTGTTTTC

GTCATATIATC

AAACCTGCTA

GCAATTAAAT

GGAATGTCAT

GCCGTATGA

ATTCATTAAA

GCAATCCCAC

CAAAGCAGCT

ATGAGTTATT

GCTACAACTA

CTCAATGCAA

AATAAAGTTA

GGGAGAAC-AC

GTGCAGTTAT

AAAGAACATC

CGCAGCTGGT

GCCCTGAATG

TTTCCTG(CA

AAACAGACCT

CATGTATGAA

CTCATTCTAT

TTCTAAGTAC

CCTTCCCTCA

TGGGAAAGTG

CCTCTTGAGA

AGACTCAGTA

TGAACAATCA

CGTGTTGGAT

GCTGATCAGC

TGCCTTCCTT

TATATGGAGT

GACCCCCGCC

TTCCATTGAC

GTGTATCATA

CATTATGCCC

GTCATCGCTA

TTTGACTCAC

CACCAAAATC

GGCGGTAGGC

CCCACTGCTT

TACCAATTTA

TTGGTGCTGA

CTGTCCAGTC

ATTGTACATA

CCACGGC'rCC

GGCAGTGGAG

GGAGTTTACT

TTGGAAATCA

ACTCTGAGGG

GTCAGAAAAG

AACTTTATTA

CGCTTCTTGG

TAACATGCCC

TCCTGTTTGC

TCTGATGAGC

OCCAGAGAGG

GAGAGTGTCA

CTGAC-CAAAG

CTGAGCTCGC

CACCTGCTCC

ACAGGGGACC

TCCTTGGCTT

CCTGTGGTTT

ATTTCTCTTA

TCATCCTTCA

AAGCCTTCTG

CCCTCCTCAG

CTTTGATTCA

TAAAGAGAAT

AAACAAACAA

GCCTTATTTA

GTACTTTCCA

ATGTTGCAA.A

TTCTAGATGA

TTATTTACAA

AAACTGTGGT

TACCTACTCA

AAGATATGTT

GAAATTTGGA

AAGAAGGGGC

GATCTGTGCA

TTATACCCAG

AAGTGGGGGC

AGTCTGCCTT

TCTGTGCCCT

TCAGCA.AGGG

CACTCACATG

GGGGCACTCT

CTCTCTCTGC

AATGACTGAC

GGGAAGGACA

ATGTTGATGA

ATGTCCCTTC

AAGGCAGGCA

CCAACCGCGG

CTCGACTGTG

GACCCTGGAA

TCCGCGTTAC

CATTGACGTC

GTCAATGGGT

TGCCAAGTAC

AGTACATGAC

TTACCATGGT

GGGGATTTCC

ALACGGGACTT

GTGTACGGTG

ACTGGCTTAT

AATTGATATC

TGTTCTGGAT

CTGTCACGCT

ATAATGGCAA

TGATCTACAA

CTGGGACAGA

ACTGCTTCCA

AACGTAAGTC

GGTCGGATGA

CATGCAAAGC

AGGAATAGGG

TCTCCTTGCT

TTATCCGCAA

TTCTT'TCCTC

AGTTGAAATC

CCAAAGTACA

CAGAGCAGGA

CAGACTACGZL

CCGTCACAAA

TCAGTTCCAG

TACCCCTATT

TAATTATGCT

CTCTCTTTCC

TAAGGGACTA

TTCTATTTTA

TCC-TCACArT

CAAGCCCTCA

ATTCCCTGAG

CATTCATTGC

TAGGGAAATG

CATTTTTAAA

CAACCTAATT

GGTTCTATA.A

CTGAGTGTCC

AAGCCAAAAA

ATGTTTATAC

CACAGATGAA

CTGTATGTTT

TGGAAATTAC

TTCTGGGGTC

CTGTTCTGTA

TTAATAGATA

CTGGGATCAA

CCAGGGCTCA

GTTTGGCTAG

GACAGAGGAC

TTTGGGAAGG

GGCCCTGC-C

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 294C 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 I. 4 4 4 4 CCACTGTCCT TTCCTAATAA CTATTCTGGG GGGTGGGGTG GGCATGCTGG GGATGCGGTG CTAGGGGGTA TCCCCACGCG CGCGCAGCGT GACCGCTACA CTTCCTTTCT CGCCACGTTC ATTAGTCAGC AACCATAGTC GTTCCGCCCA TTCTCCGCCC CCGCCTCGGC CTCTGAGCTA TTTGCAAAA GCTTGGACAG AGCGTGAAGG CTGGTAGGAT TCGTCGCCGT

GTCCCAAAAT

TCAGGAACGA

GTTCAAGTAC

AGAATCTGGT GATTATGGGT TAAAGGACAG AATTAATATA ATTTTCTTGC CAAAAGTTTG GTAAAGTAGA CATGGTTTGG AACCAGGCCA CCTTAGACTC TTTTCCCAGA AATTGATTTG CTGAGGTCCA GGAGGAAAAA AACAGGAAGA TGCTTTCAAG CATGGGACTT TTGCTGGCTT ATAATTGGAC AAACTACCTA TGTATAATGT GTTAAACTAC AACTGATGAA TGGGAGCAG1 AGAAATGCC-A TCTAGTGATG AAAGAAGAGA AAGGTAGAAG TCATGCTGTG TTTAGTAATA AGCTGCACTG CTATACAA.GA TAACAGTTAT AATCATAACA TATTAATAAC TATGCTCAAA TAAGGAATAT TTGATC-TATA TAGAGGTTTT ACTTGCTTTA TGAATGCAAT TGTTGTTGTT ATAGCATCAC AAATTTCACA CCAA.ACTCAT CAATGTATCT ATCT".CATGCT GGAGTTCTTC AATAAAC-CPAA TAGCATCACA C-TGGTTTGTC CAAACTCATC AGAGCTTC-GC GTAATCA'rGG TTCCACACAA CATACGAGCC GCTAACTCAC ATTAATTGCG GCCAGCTGCA TTAATGAATC cTrTCCGCTTC CTCGCTCACT CAGCTCACTC kAAGGCGGTA ACATGT'GAGC' AAAAGGCCAG TTTTCCATAG GCTCCGCCCC GGCGAAACCC GACAGGACTA GCTCTCCTGT TCCGACCCTG 50 GCGTGGCGCT TTCTCAATGC CCAAGCTGGG CTGTGTGCAC ACTATCGTCT TGAGTCCAAC GTAACAGGAT TAGCAGAGCG CTAACTACGG CTACACTAGA 55 CTT:GAAA AAGAGTTGGT GTTTTTTTGT TTGCAAGCAG TGATCTTTTC TACGGGGTCT TCATGAGATT ATCAAAAAGG AATCAATCTA AAGTATATAT AGGCACCTAT CTCAGCGATC TGTAGATAAC TACGATACGG GAGACCCACG CTCACCGGCT AGCGCAGAAG TGGTCCTGCA AAGCTAGAGT AAGTAGTTCG 65 GCATCGTGGT GTCACGCTCG CAAGGCGAGT TACATGATCC CGATCGTTGT CAGAAGTAAG

AATGAGGAA

GGGCAGGACA

GGCTCTATGG

CCCTGTAGCG

CTTGCCAGCG

GCCGGGCCTC

CCGCCCCTAA

CATGGCTGAC

TTCCAGAAGT

CTCAGGGCTG

TTTATCCCCG

ATGGGGATTG

TTCCAAAGAA

AGGAAAACCT

GTTCTCAGTA

GATGATGCCT

ATAGTCGGAG

TTTGTGACAA

GGGAAATATA

GGCATCAAGT

TTCTCTGCTC

TAGATCTCTT

CAGAGATTTA

TGATTCTAAT

GGTGGAATGC

ATGAGGCTAC

ACCCCAAGGA

.GAZACTCTTGC

AAATTATC-GA

TACTGTTTTT

AATTGTGTAC

GT GCCTTGAC

AAAAACCTCC

.AACTTGTTTA

AATAAAGCAT

TATCATGTCT

GCCCACCCCA

AATTT CACAA

AATGTATCTT

TCATAGCTGT

GGAAGCATAA

TTGCGCTCAC

GGCCAACGCG

GACT'-GCTGC

ATACGGTTAT

CAAAAGGCCA

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

TGC-TGACTCT

CTTTCCTTCA

TTGCTTTGCT

AAAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

GGATCGGCTG

ACTTGTTTAT

ATAJAAGCATT

ATCA.TCTCTG

TTCCTGTGTG

AGTGTA-BAGC

TGCCC:C:CTTT

CGC-GGAGAGG

GCTCGGTCGT

CCACAGAATC

GGAACCGTAA

ATCACAAAAA

AGGCGTTTCC

GATACCTGTC

GGTATCTCAG

TTCAGCCCGA

ACGACTTATC

GCGGTGCTAC

TTGGTATCTG

CCGGCAAACA

GCAGAAAAAA

GGAACGAAAA

AGATCCTTTT

GGTCTGACAG

GTTCATCCAT

CATCTGGCCC

CAGCAATAAA

CCTCCATCCA

GTTTGCGCAA

TGGCTTCATT

GCAAAAAAGC

TGTTATCACT

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

AATCAGCCAT

CCTGAACCTG

TA-ATGGTTAC

GCATTCTAGT

GATGATCCTC

TGCAGCTTAT

TTTTTCACTG

TATACCGTCG

AAATTGTTAT

CTGGGGTGCC

CCAGTC-GGGA

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

GGCGT-CTCT

AAGAAAACT

TTTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCC-AA

GTTTTTTGAG

CAAAGGAAA.A

TAAGTAGGCA

GAGTGTCTGC

AAGGC-GTT'A

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAGCGCCGGG

AATGGTTACA

CATTCTAGTT

ACCTCTAGCT

=CCTCACAA4

TAATGAGTGA

AACCTGTCGT

ATT-GcCGCr

CGAGCGGTAT

GCAGGAAAc3A

TTGCTGGCGT

AGTCAGAGGT

TCCCTCGTGO

CCTTCGG-A.A

GTCGTTCGCT

TTATCCGGTA

GCAGCCACTG

AAGTGGTGGC

AAGCCAGTTA

GGTAGCGGTG

GAAGATCCTT

GGGATTTTGG

TGAAGTTTTA

TTAATCAGT'

CTCCCCGTC3

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 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 1620 7680 ;740 7800 7860 '7920 7980 8040 8100 8160 8220 8280 8340 8400 9 9 9 9* 9 *.*999 9

U.

ATAATTCTC'r TACTGTCATG CCATCCGTA GATGCTTTTC TGTGACTGGT

GAGTACTCAA

CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG

GCGTCAATAC

GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCr CATCATTGGA

AAACGTTCTT

CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG

TAACCCACTC

GTGCACCCAA CTGATCrTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG

TGAGCAAAA

CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT

TGAATACTCA

TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC

ATGAGCGGAT

ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA

TTTCCCCGA

AAGTGCCACC

TGACGTC

INFORMIATION 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:l4: GAAGAGGAAG ACTGACGGTG CCCCCGCGAG TTCAGGTGCT

GAGG

INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 44 base pairs TYPE: nucleic acid STRAkNEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID CCTCAGCACC TGACTCGCG GGGGCACCGT CAGTCTTCCT

CTTC

INFORMATION FOR SEQ ID NO:16: SEQUENCE CHAkRACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGGGAGGGC TTTGTTGGAG ACCGAGCACG AGTACGACTT GCCATTCAGC

C

INFORKATION FOR SEQ ID NO 55 SEQUENCE

CHARACTERISTICS:

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: 8460 8520 8580 8640 8700 8760 8820 8880 8897 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: 91 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GATGGTTTTC TCGATGGCGG CTGGGAGGGC TTTGTTGGAG ACCGAGCACG AGTACGACTT GCCATTCAGC CAGTCCTGGT G 81 S. 2) INFORMATION FOR SEQ ID NO:21: 45 SEQUENCE CHARACTERISTICS: LENGTH: 81 base oairs TYPE: nucleic acid STRANDEDNESS: single S(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: o""o 55 CACCAGGACT GGCTGAATGG CAAGTCGTAC TCGTGCTCGG TCTCCAACAA AGCCCTCCCA GCCGCCATCG AGAAAACCAT C 81 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 8690 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S (ii) MOLECULE TYPE: cDNA *i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GGTACCAATT

TTGGAATTCT

TGTCCTTGTT

AGTGCAGCCT

CTATTACATG

TAGTCAAGAT

CAGAGACAAT

AGCCGTGTAT

AGGGACTCTG

ACCCTCCTCC

CTTCCCCGAA

CTTCCCGGCT

CTCCAGCAGC

CAAGGTGGAC

CCAGGCTCAG

AGGCAGGCCC

AGAGGGTCTT

CAGGCCCTGC

AGGACCCTGC

GACACCTTCT

CTTGTGACAA

CCAGCTCAAG

GGTGCTGACA

GTCTTCCTCT

ACATGCGTGG

GACGGCGTGG

TACCGTGTGG

AAGTGCAAGG

AAAGGTGGGA

TGCCCTGAGA

GGTGTACACC

CCTGGTCAAA

GGAGAACAAC

CAGCAAGCTC

C-ATGCATGAG

ATGAGTGCGA

TGGCACGTAC

CTGCCCTGGG

CCTGAGTGGC

CAGGTGTGCC

GGATTTGCCA

CCTAGGAC-CC

GTGAGCGCCC

GTAGGGACAG

CCAGCCTCGC

TGGAGGGACT

GCACTGAGGT

50 GCCTCACCCG

ACTCCTCGGA

GGCAGC7TCT

CCCTGCAGCC

CCACTTCCCA

55 GCCAGCCATC

CCACTGTCCT

CTATTCTGGG

GGCATGCTGG

CTAGGGGGTA

CGCGCAGCGT

CTTCCTTTCT

ATTAGTCAGC

GTTCCGCCCA

CCGCCTCGGC

65 TTTGCAAAAA

AGCGTGAAGG

TCGTCGCCGT

TAAATTGATA

TGCGGCCGCT

TTAAAAGGTG

GGAGGGTCCC

TATTGGGTTC

GGTGATATAA

GCAAAGAACA

TACTGTGCAA

GTCACGGTCT

AAGAGCACCT

CCGGTGACGG

GTCCTACAGT

TTGGGCACCC

AAGAAAGTTG

CGCTCCTGCC

CGTCTGCCTC

CTGGCTTTTT

ACACAAAGGG

CCCTGACCTA

CTCCTCCCAG

AACTCACACA

GCGGGACAGG

CGTCCACCTC

TCCCCCCAAA

TGGTGGACGT

AGGTGCATAA

TCAGCGTCCT

TCTCCAACAA

CCCGTGGGGT

GTGACCGCTG

CTGCCCCCAT

GGCTTCTATC

TACAAGACCA

ACCGTGGACA

GC-TCTGCACA

CGGCCGGCAA

CCCCTlGTACA

CCCCTGCGAC-

ATGAGGGAGG

TGGGCCCCCT

GCGTGOGCCCT

CCTGGGGACA

CTGTCCTCCC

GCCCTCCCTC

ACCCGCATGG

GGTGCAGATG

TGGCCGGCCA

GGCGAACTGC

CACAGGCCCC

CCACATGCTG

GCCACACACA

GTGCCGCCCT

TGTTGTTTGC

TTCCTAATAA

GGGTGGGGTG

GGATGCGGTG

TCCCCACGCG

GACCGCTACA

CGCCACGTTC

AACCATAGTC

TTCTCCGCCC

CTCTGAGCTA

GCTTGGACAG

CTGGTAGGAT

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

CCAGCGACAT

CGCCTCCCGT

AGAGCA(GG

ACCACTACAC

C-CCCCCGCTC

TACTTCCCGG

A'CTGTGATGG

CAGAGCGGGT

AGGGTGGGGC

CC CT CCAGCA

GACACACAGC

GACCTCCATG

ACCCATCTAC

GGACACAACC

CC-CACACACA

CACGGCCACC

ACAGCACCCA

CACGAGCCCC

ACCTGCTCAG

CACAGGGGAT

TCCCTGCAGG

CCCTCCCCCG

AATGAGGAAA

GGGCAGGACA

GGCTCTATGG

CCCTGTAGCG

CTTGCCAGCG

GCCGGGCCTC

CCGCCCCTAA

CATGGCTGAC

TTCCAGAAGT

CTCAGGGCTG

TTTATCCCCG

ATGGGGATTG

TCTCGAGTCT

CATGGAGTTG

AGTGCAACTG

CTGTGCTGCA

AGGCAAGGGN

AGACTCCGTA

GCAAATGAAC

GGACGGGGCC

CACCAAGGGC

AGCGGCCCTG

CTCAGGCGCC

CTACTCCCTC

CTGCAACGTG

AGCACAGGGA

CCGGCTATGC

GGCCTCTGCC

TGGGCAGGCA

GGCTCAGACC

AAAGGCCAAA

CTCCCAATCT

GCCCAGGTAA

TAGCCTGCAT

TCAGCACC",G

ACCCTCATGA

GACCCTGAGG

AAGCCGCGGG

CACCAGGACT

GCCCCCATCG

CATGGACAGA

TC-TCCCTACA

GCTGACCAAG

CGCCGTGGAG

GCTLGGACT.CC

GCAGCAGGGG

GCAC-AAGAGC

CCCGGGCTCT

GCGCCCAGCA

TTCTTTCCAC

CCCACTGTCC

TCAC-CCAGGG

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

TGGTTAAGCT

GTGGAGTCTG

TCTGGATTCC

CTGGAGTGGG

AAGGGTCGAT

AGCCTGAGGG

TGGTTTGCTT

CCAT CGGT CT

GGCTGCCTGG

CTGACCAGCG

AGCAGCGTGG

AATCACAAGC

GGGAGGCTGT

AGCCCCAGTC

CGCCCCACTC

CAGGCTAGGT

TGCCAAGAGC

CTCTCCACTC

TCTCTCTGCA

GCCAGCCCAG

CCAGGGACAG

AACTCCTGGG

TCTCCCGGAC

TCAAGTTCAA

AGGAGCAGTA

GGCTGAATGG

AGAAAACCAT

GGCCGGCTCG

GGGCAGCCCC

AACCAGGTCA

TGGGAGAGCA

GACGGCTCCT

AACGTCTTCT

CTCTCCCTGT

CGCGGTCGCA

TGGAAATA-AA

GGGTCAGGCC

CCACACTGGC

GCTGCCCTCG

TGC-GCTGGGC

GTAGGAGACT

GGCATGCCTA

CTAACCCCTG

ACATGCACTC

AGACCCGTTC

GTGCACGCCT

AGGTCCTCGC

TCAAGGCCCA

CCCTCCTCTC

GTCACGTCCC

CTCGACTGTG

GACCCTGGAA

TTGTCTGAGT

GGATTGGGAA

GGAAAGAACC

CGCGGCGGGT

CGCTCCTTTC

GAAAAAAAGC

CCCGCCCCTA

TATTTATGCA

CTTTTTTGGA

CCAAACTTGA

GGTTCGACCA

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

C.AACAGCACG-

CAAGGAGTAC

CTCCAAAGCC

GCCCACCCTC

GAGAACCACA

GCCTGACCTG

ATGGGCAGCC

TCTTCCTCTA

CATGCTCCGT

CTCCGGG-.A

CGAGGATGCT

GCACCCAGCG

GAGTCTGG

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

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 1960 1920 1980 2040 2100 2160 2220 2290 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

TA.AAGGACAG

ATTTTCTTGC

GTAAAGTAGA

AACCAG4GCCA

TTTTCCCAGA

CTGAGGTCCA

AACAGGAAGA

CATGGGACTT

ATAATTGGAC

TGTATAATGT

AACTGATGAA

AGAAATGCCA

AAAGAAGAGA

TCATGCTGTG

AGCTGCACTG

TAACAGTTAT

TATTAATAAC

TAAGGAATAT

TAGAGGTTTT

TGAATGCAAT

ATAGCATCAC

CCAAACTCAT

ATCTCATGCT

AATAAAGCA

GTGGTTTGTC

AGAGCTTGGC

TTCCACACAA

GCTAACTCAC

GCCAGCTGCA

CTTCCGCTTC

C.AGCT CACTC

ACATGTGAGC

TTTTCCATAG

GGCGAAACCC

GCTCTCCTGT

GCGTGGCGCT

CCAAGCTGC-G

ACTATCGTCT

GTAACAGGAT

CTAANCTACGG

CCTTCGGAAA

GTTTTTTTGT

TGCATCT-TTTC

TCATGAGATT

AATCAATCTA

AGGCACCTAT

TGTAGATAAC

50 GAGACCCACG

AGCGCAGAAG

AAGCTAGAGT

GCATCGTGGT

CAAGGCGAGT

55 CGATCGTTGT

ATAATTCTCT

CCAAGTCATT

GGGATAATAC

CGGGGCGAAA

GTGCACCCAA

CAGGAAGGCA

TACTCTTCCT

ACATATTTGA

AAGTGCCACC

65 TCGAATAGCC

TTGGCGCCGA

ATAGTTAAGC

GTTCAAGTAC

GATTATGGGT

AATTAATATA

CAAAAGTTTG

CATGGTTTGG

CCTTAGACTC

AATTGATTTG

GGAGGAAAAA

TGCTTTCA6AG

TTGCTGGCTT

AAACTACCTA

GTTAAACTAC

TGGGAGCAGT

TCTAGTGATG

AAGGTAGAAG

TTTAGTAATA

CTATACAAGA

AATCATAACA

TATGCTCAAA

TTGATGTATA

ACTTGCTTTA

TGTTGTTGTT

AAATTTCACA

CAATGTATCT

GGAGTTCTTC

TAGCATCACA

CAAACTCATC

GTAATCATGG

CATACGAGCC

ATTAATTGCG

TTAATGAATC

CTCGCTCACT

AAAr2GC-G'rA

AAAAGGCCAG

GCTLCCGCCCC

GACAGGACTA

TCCGACCCTG

TTCTCAATGC

CTGTGTGCAC

TGAGTCCAAC

T.AGCAGAGCG

CTACACTAGA

ANAGAGTTGGT

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

AAAAACC'rCC

AACTTGTTTA

AATAAAGCAT

TATCATGTCT

GCCCACCCCA

AATTTCACAA

AATGTATCTT

TCATAGCTGT

GGAAGCATAA

T'rGCGCTCAC

GGCCAACG:CG

GACTCGCTGC

ATACGGTTAT

CAAAAGGCCA

CCTGACGAGC

TAAAGATACC

CCGCTTACCG

TCACGCTGTA

GAACCCOCCG

CCGGTPAAGAC

tAGGTATGTAG

AGGACAGTAT

AGCTCTTGA-,

CAGATTACGC

GACGCTCAGT

AT-TTCACCT

GAGTAAACTT,

TGTCTATTTC

GAGGGCTTAC

CCAGATTTAT

ACTTTATCCG

CCAGTTAATA

TCGTTTGGTA

CCCATC.TTGT

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

TGCTGACTC'r

CTTTCCTTCA

TTGCTTTGCT

A.AAATATTCT

TCTTACTCCA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCAC1'

GGATCGGCTG

ACTTGTTTAT

ATAAAGCATT

ATCATGTCrG

TTCCTGTGTG

AGTGTAAAGC

TGCCCGCTTT

CGGGGAGAGG

GCTCGGTCGT

CCACAGAATC

CGGAACCGTAA

ATCACAA-kAA

AGGCGTTTCC

GATACCTGTC

GGTATCTCAG

TTCAC-CCCGA

ACGACTTATC

GCGGTGCTAC

TTGGTATCTG

CCGGCAAACA

GCAGAAAAAA

GGAACGAAAA

AGATCCTTTT

GGTC-TGACAG

GTTCATCCAT

CATCTGGCCC

CAGCAATAAA

CCTC-ATCCA

GTTTGCGCAA

TGGCTTCATT

GC.PAAAAGC

TGTTATCACT

GATGCTTTTC

GACCGAGTTG

TAAAAGTGCT

TGTTGAGATC

CTTTCACCAG

TAAGGGCGAC

TTTATCAGGG

AAATAGGGGT

ATCTGCTAGG

TTTTATTTTA

GACTCTCAGT

TGTGTTGGAG

CTCTTCAGTG

TCCTGAGAAG

AGAACCACCA

TGAACAACCG

TTACCAGGAA

GGAATTTGAA

AGAATACCCA

AGTCTACGAG

GCTATGCATT

CCTTACTTCT

GTAAATATAA

TTTTAGATTC

GAAAACCTGT

CAACATTCTA

GAATTGCTAA

ATTTACACCA

GTAACCTTTA

CACAGGCATA

TTAATTTGTA

AATCAGCCAT

CCTGAACCTG

TAATGGTTAC

GCATTCmAGT

GATGATCCTC

TGCAGCTTAT

TTTTTCACTG

TATACCGTCG

AAATTGTTAT

CTGGGGTGCC

CCAGTCGGGA

CGGTTTGCGT

TCGGCTGCGG

AGGGGATAAC

AAAGGCCGCG

TCGACGCTCA

CCCTGGAADGC

CGCCTTTCTC

TTCGGTGTAG

CCGCTGCGCC

GCCACTGGCA

AGAGTTCTTG

CGCTCTGCTG

AACCACCGCT

AGGATCTCkPL

CTCACGTTA

AAATTAAAAA

TTACCAATGC

AGTTGCCTGA

CAGTGCTC-CA

CCAGCCAGCC

GTCTATTAAT

CGTTGTTGCC

CAGCTCCGGT

GGTTAGCTCC

CATGGTTATG

TGTGACTGGT

CTCTTGCCCG

CATCATTGGA

CAGTTCC.ATG

CGTTTCTGGG

ACGGAAATGT

TTATTGTCTC

TCCGCGCACA

TGACCTGAGG

TTTTATTTTT

ACAATCTGCT

GTCGCTGAGT

GAAGGTAAAC

AATCGACCTT

CGAGGAGCTC

GAATTGGCAA

GCCATGAATC

AGTGACACGT

GGCGTCCTCT

AAGAAAGACT

TTTATAAGAC

GTGGTGTGAC

AATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGCA

GAGTGTCTGC

AAGGGGTTAA

ACCACATTTG

AAACATAAAA

AAATAAAGCA

TGTGGTTTGT

CAGCGCC-GGG

AATGGTTACA

CATTCTAGTT

ACCTCTAGCT

CCGCTCACAAL

TAATGAGTGA

AACCTGTCGT

ATTGGGCGCT

CGAGCGGTAT

GCAGGAAAGA

TTGCTGGCGT

AGTCAGAGGT

TCCCTCGTGC

CCTTCGGGAA

GTCGTTCGCT

TTA-CCGGTA

GCAGCCACTG

AAGTGGTGGC

AAGCCAGTTA.

GGTAGCGGTG

C-AAGATCCTT

GGGATTTGG

TGAAGTTTTA

TTAATCAGTG

CTCCCCGTCG

ATGATACCGC

GGAAGC-GCCG

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 S.1.

7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920

S*

CAAAA

GGTTAG

ATTGAC

GTTCCG

CCCATT

ACGTC

TATGCC

CCAGTA

TATTAC

ACGGGG

TCAACG

GCGTGT

TTACTG

'TTAA

;GCGT

,TAGT

;CGTT

GACG

LATGG

:AAGT

.CATG

: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

TGACCGCCCA

CCAATAGGGA

GCAGTACATC

TGGCCCGCCT

ATCTACGTAT

CGTGGATAGC

AGTTTGTTTT

TTGACGCAAA

CTAACTAGAG

ACCCAAGCTT

ATCTGCTTAG

GACATTGATT

CATATATGGA

ACGACCCCCG

CTTTCCATTG

AAGTGTATCA

GGCATTATGC

TAGTCATCGC

GGTTTGACTC

GGCACCAAA

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: a a. a.

a a a a a GGTACCAATT TAAATTGATA TTGGAATTCT TGCGGCCGCT TCCTCCAAGA GCACCTCTGG CCCGAACCGG TGACGGTGTC CCGGCTGTCC TACAGTCCTC AGCAGCTTGG GCACCCAGAC GTGGACAAGA AAGTTGGTGA GCTCAC-CGCT CCTGCCTGGA AGGCCCCGTC TGCCTCTTCA GGTCTTCTGG CTTTTTCCCC CCCTGCACAC AAAGGGGCAG CCCTGCC-CCT GACCTAAGCC CCTTCTCTCC TCCCAGATTC TGACAAAACT CACACATGCC CTCAAGGCGG GACAGGTGCC CTGACACGTC CACCTCCATC TCCTCTTCCC CCCAAAACCC GCGTGGTGGT GGACGTGAGC GCGTGGAGGT GCATAATGCC GTGTGGTCAG CGTCCTCACC GCAAGGTCTC CAACAAAGCC GTGGGACCCG TGGGGTGCGA CTGAGAGTGA CCGCTGTACC 50 TACACCCTGC CCCCATCCCG GTCAAAGGCT TCTATCCCAG AACAACTACA AGACCACGCC AAGCTCACCG TGGACAAGAG CATGAGGCTC TGCACAACCA 55 GTGCGACGGC CGGCAAGCCC ACGTACCCCC TGTACATACT CCTGGGCCCC TGCGAGACTG AGTGGCATGA GGGAGGCAGA TGTGCCTGGG CCCCCTAGGG TTGCCAGCGT GGCCCTCCCT GGAGCCCCTG GGGACAGACA GCGCCCCTGT CCTCCCGACC CTATGGCTTC TGAGGCGGAA GTAGCGGCGC ATTAAGCGCG 65 CCAGCGCCCT AGCGCCCGCT GCTTTCCCCG TCAAGCTCTA GGCACCTCGA CCCCAAAAAA

TCTCCTTAGG

TGCTAGCACC

GGGCACAGCG

GTGGAACTCA

AGGACTCTAC

CTACATCTGC

GAGGCCAGCA

CGCATCCCGG

CCCGGAGGCC

AGGCTCTGGG

GTGCTGGGCT

CACCCCAAAG

CAGTAACTCC

CACCGTGCCC

CTAGAGTAGC

TCTTCOTCAG

AAGGACACCC

CACGAAGACC

AAGACAAAGC

GTCCTGCACC

CTCCCAGCCC

GGGCCACATG

AACCTC1'GTC

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

-^'GACCTGCC

GCCAAACTCT

CAATCTTCTC

AGGTAAGCCA

CTGCATCCAG

CACCTGAAC T

TCATGATCTC

crGAGGTCAA

CGCGGGAGGA

AGGACTGGCT

CCATCGAGAA

GACAGAGGCC

CCTACAGGGC

ACCAAGAACC

GTGGAGTGGG

GACTCCGACG

CAGGGGAACG

AAGAGCCTCT

GGCTCTCGCG

CCAGCATGGA

TTCCACGGGT

CTGTCCCCAC

CCAGGGGCTG

CTGCCCTGCGG

GCCTCTGTAG

CTCGGGGGCA

GGGGCTCTAG

TGGTTACGCG

TCTTCCCTTC

TCCCTTTAGG

GTGATGGTTC

CTAGATAACC

CGGTCTTCCC

GCCTGGTCAA

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

GGTCAATCGA

CCTGGCACCC-

GGACTACTTC

GCACACCTTC

CGTGCCC TCC

CAACACCAAG

TGGAAGCCAG

GCAGCAAzGGC

TCAGGGAGAG

CTAACCCAGG

TCCGGGAGGA

AGCTCGGACA

CCAAATCTTG

CC-CCCTCCAG

CAGCCGGGTG

CCGTCAGTCT

GAGGTCACAT

TACGTGGACG

AGCACGTACC

GAGTACAAGT

AAAGCCAA-AG

ACCCTCTGCC

ACCACAGGTG

GACCTGCCTG

GCAGCCGGAG

CCTCTACAGC

CTCCGTGATG

GGGTAAATGA

GATGCTTGGC

CCAGCGCTGC

CTGAGGCCTG

GCTGTGCAGG

GGTGGGGGAT

GGAAGCCCTA

TGTTCTGTGA

GCGGTGGGCT

CACGCGCCCT

GCTACACTTG

ACGTTCGCCG

AGTGCTTTAC

CCATCGCCCT

120 190 240 300 360 420 480 540 600 660 720 780 84U 900 960 102C 1080 1140 1200 1.260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 i980 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

APAGCC:TGGG

CTTTCCAGTC

23AGC-CGGTTT TcGTrCGGCT

PATCAGGGGA

45 GTAAAAAGGC

AAAATCGACG

TTCCCCCTGG

TGTCCGCCTT

TCAGTTCGGT

CCGACCGCTG

TATCGCCACT

CTACAGAGTT

TCTGCGCTCT

AACAAACCAC

AAAAAGGATC

AAAACTCACG

TTTTAAATTA

ACAGTTACCA

CCATAGTTGC

60 GCCCCAGTGC

TAAACCAGCC

TCCAGTCTAT

GCAACGTTGT

CATTCAGCTC

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

CC'TCCAGCGC

TTATAATGGT

ACTGCATTCT

GTCGACCTCT

TTATCCGCTC

TGCCTAATGA

GGGAAACCTG

GCGTATTGGG

GCGGCGAGCG

TAACGCAGGA

CGCGTTGCTG

CTCAAGTCAG

AAGCTCCCTC

TCTCCCTTCG

GTAGGTCGTT

CGCCTTATCC

GGCAGCAGCC

CTTGAAGTGG

GCTGAAGCCA

CGCTGGTAGC

TCAAGAAGAT

TTAAGGGATT

AAAATGALAGT

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

CAGA.AGAAAT

CAAAAAAGAA

TGAGTCATGC

AAAAAGCTGC

GGCATAACAG

CTGCTATTAA

TTAATAAGGA

TTTGTAGAGG

AAAATGAATG

AGCAATAGCA

TTGTCCAAAC

GGGGATCTCA

TACAAATA-A-

AGTTGTGGTT

AGCTAGAGCT

ACAATTCCAC

GTGAGCTAAC

TCGTGCCAGC

CGCTCTTCCG

GTATCAC-CTC

AAGAACATGT

GCGTTTTTCC

AGGTGGCGA-A

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

GAGAAAG4GTA

TGTGTTTAGT

ACTGCTATAC

TTATAATCAT

TAACTATGCT

ATATTTGATG

TTTTACTTGC

CAATITGTTGT

TCACAAATTT

TCATCAATGT

TGCTC-GAGTT

GCAATAGCAT

TGTCCAAACT

TGGCGTAATC

ACAACATACG

TCACATTAAT

TGCATTAATG

CTTCCTCGCT

ACTCAJAAGGC

GAGCAAAAGG

ATAGGCTCCG

ACCCGACAGG

CTGTTCCGAC

CGCTTTCTCA

TGGGCTGTGT

GTCTTGAGTC

GGATTAGCAG

ACGGCTACAC

GAAAAAGAGT

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

TTTAAAJAAAC

TGTTAACTTG

CACAAATAAA

ATCTTATCAT

CTTCGCCCAC

CAPCAAAT TTC

CATCAATGTA

ATC-GTCATAG

AGCCGGAAGC

TGCGTTGCGC

AATCGGCCAA

CACTGACTCG

GGTA-ATACGG

CCAGCAA.kAG

CCCCCCTGAC

ACTATAAAGA

CCTGCCGCTT

ATGCTCACGC

GCACGAACCC

CAACCCGGTA

AGCGAGGTAT

TAGAAGGACA

TGGTAGCTCT

GCAGCAGATT

GTCTGACGCT

AAGGATCTTC

ATATGAGTAA

GATCTGTCTA

ACGGGAGGGC

GGCTCCAGAT

TGCAACTTTA

TTCGCCAGTT

CTCGTCGTTT

ATCCCCCATG

TAAGTTGGCC

CATGCCATCC

ATAGTGTATG

GGACTCTTGT

TA.AGGGATTT

AACGCGAATT

CAGGCAGGCA

CTAACTCCGC

TGACTAATTT

AAGTAGTGAG

GCTGCGATTT

CCCGCTGCCA

ATGGCAAGA

AGAATGACCA

ACCTGGTTCT

AGTAGAGAAC

GCCTTAAGAC

GGAGGCAGTT

ACAAGGATCA

TATAAACTTC

AAGTATAAGr

GCTCCCCTCC

TCTTTGTGAA

TTTAAAGCTC

TAATTGTTTG

ATGCC7LTTAA

CTACTGCTGA

AGGACTTTCC

TTGC1-TTGCTT

TGGAAAAATA

TTTTTCTTAQ,

GTACCTTTAG

TGACTAGAGA

CTCCCACACC

TTTATTGCAG

GCATTTTTTT

GTCTGGATCG

CCCAACTTGT

ACAAATAAAG

TC-lrATCATG

CTGTTTCCTG

ATAG-AGT'A

TCA.CTGCCCG

CGCGCGGGGA

CTGCGCTCGG

TTATCCACAG

GCCAGGAACC

GAGCATCACA

TACCAGGCGT

ACCGGATACC

TGTAGGTATC

CCCGTTCAGC

AGACACGACT

GTAGGCGGTG

GTATTTGGTA

TGATCCGGCA

ACGCGCAGAA

CAGTGGAACG

ACCTAGATCC

ACTTGGTCTG

TTTCGTTCAT

TTACCATCTG

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 0740 4800 4860 4920 4980 5040 52 00 5160 5220 5280 5340 5400 5460 5520 5580 5640 C5700or 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 see.

0

GTTGCTCTTG

TGCTCATCAT

GATCCAGTTC

CCAGCGTTTC

CGACACGGAA

AGGGTTATTG

GGGTTCCGCG

TAGGTGACCT

TTTATTTTAT

CAGTACAATC

GGAGGTCGCT

CAATTGCATG

CAGATATACG

ATTAGTTCAT

TGGCTGACCG

AACGCCAATA

CTTGGCAGTA

TAAATGGCCC

GTACATCTAC

TGGGCGTGGA

TGGGAGTTTG

CCCATTGACG

CTGGCTAACT

GGAGACCCAA

S

(A)

(B)

(C)

(D)

(xi)

CCCGGCGTCA

TGGAAAACGT

GATGTAACCC

TGGGTGAGCA

ATGTTGAATA

TCTCATGAGC

CACATTTCCC

GAGGCGCGC

TTTTGAGATG

TGCTCTGATG

GAGTAGTGCG

AAGAATCTGC

CGTTGACATT

AGCCCATATA

CCCAACGACC

GGGACTTTCC

CATCAAGTGT

GCCTGGCATT

GTATTAGTCA

TAGCGGTTTG

TTTTGGCACC

CAAATGGGCG

AGAGAACCCA

GCTT

ATACGGGATA

TCTTCGG4GGC

ACTCGTGCAC

AAAACAGGAA

CTCATACTCT

GGATACATAT

CGAAAAGTGC

GGCTTCGAAT

GAGTTTGGCG

CCGCATAGTT

CGAGCAAAAT

TTAGGGTTAG

GATTATTGAC

TGGAGTTCCG

CCCGCCCATT

ATTGACGTCA

ATCATATGCC

ATGCCCAGTA

TCGCTATTAC

ACTCACGGGG

AAAATCAACG

GTAGGCGTGT

CTGCTTACTG

ATACCGCGCC

GAAAACTCTC

CCAACTGATC

GGCAAAATGC

TCCTTTTTCA

TTGAATGTAT

CACCTGACCT

AGCCAGAGTA

CCGATCTCCC

AAGCCAGTAT

TTAAGCTACA

GCGTTTTGCG

TAGTTATTAA

CGTTACATAA

GACGTCAATA

ATGGGTGGAC

AAGTACGCCC

CATGACCTTA

CATGGTGATG

ATTTCCAAGT

GGACTTTCCA

ACGGTGGGAG

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

GGAGATCTGC

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 71.40 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7874 INFORMATION FOR SEQ ID NO:24: ;EQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRA24DEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO:24: Val Gin Leu Val Glu Ser Gly Gly Cys Ala Ala Ser Leu Val GIn Phe Pro Phe Ser Leu Arg Tyr Met Tyr Ser Tyr Ile 50 Lys Gly Arg 65 Leu Gln Met Leu Ser Pro Gly Gly is Ser Asp Tyr Glu Trp Val Val Arg Gin Gly Lys Gly Leu 0O 4@ S 0@*O 0@O*

S

5555 SeeS SS @5 5 S 0 0*O@

S.

5* 0

S

0 Sc..

*OOS@@

S

55505.

0 @5 S S 0 *0 0 Oe Ser Gin Asp Gly 55 Phe Thr Ile Ser 70 Asn Ser Leu Arg ile Thr Arg Asp Asn Asp Glu Asp Asp Tyr Ala Lys 75 Thr Ala Ala Tyr Ala Asp Ser Val Asn Ser Leu Tvr 85 90 Asp Gly Ala Trp Phe Val Tyr Trp Gly 110 Tyr Cys Gin Gly Ala Arg Gly Leu 50 100 Ala Thr Leu Val Thr Val Ser Ser 115 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 15 10 Ser Phe Gly Leu Tyr Lys Pro Lys Val1 145 Tyr Glu His Lys Gin.

225 Leu Pro As n Leu Val 305 Gin Thr Pro ValI Ser Ile ValI Ala Pro 130 ValI Val Gin Gin Ala 2130 Pro Thr Ser Tyr Tyr 290 Phe Lys Gly Pro Thr ValI As n Pro 100 Giu Asp Asp Gly Asn 180 Trp Pro Giu As n Ile 260 Thr L-vs Cys Leu Giy Vali Phe ValI ValI 85 Lys Leu Thr ValI Val1 165 Ser Leu Ala Pro Gin 245 Al a Th r Leu Ser Se r 325 Leu T rp Leu Se r Pro Lys 105 Pro Ser Asp As n ValI 185 Giu Lys Thr Thr Glu 265 Leu Lys Gi y Gly Cy Asn Se Gin Se Ser Se Ser As Thr Hi Ser va Arg Th Pro Gi Ala Ly 170 Val Se Tyr Ly Thr Ii Leu Pr 23 Cys Le 250 Ser As Asp Se Ser Ar Ala Le 31 Lys 330 0O: 26: INFORMATION FOR SEQ ID 50 Ala 1 Ser Phe Gly Leu 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 Gly Thr Ala Ala Leu Gly 20 Pro Glu Pro Val Thr Val Ser Trp Asn Val His Thr Phe Pro Ala Val Leu Gin s0 55 Ser Ser Val Val Thr Val 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 Asp Phe 145 Glu Phe Gly Tyr Giu 1 Ser Tyr Ala Lys Leu Ala Thr Pro 45 Gly 145 As n Gin Se r Ser Th r 225 Tyr Leu Trp Val1 Asp 305 Gly Gin Pro Arg Giu Pro Gin Val Tyr Thr Le 115 120 Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cy 130 135 14 Tyr Pro Ser Asp Ile Ala Val Giu Trp Glu Se 150 155 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu As 165 170 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Se 180 185 Asn Val Phe Ser Cys Ser Val Met His Glu Al 195 200 Thr Gin Lys Ser Leu Ser Leu Ser Pro Giy Ly 210 215 22 INFORMATION FOR SEQ ID NO:27: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 339 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Val Asn Leu Giu Ser Giy Gly Cys Val Thr Ser Ara Gin Thr Pro 40 Giy Gly Asp Ile Ile Ser Arg Asp 7 0 Leu Lys Ser Giu Asp Gly Ala Trp 105 Ser Val Ala Ser 120 Ser Lys Ser Thr 135 Asp Tyr Phe Pro 150 Thr Ser Giy Vai Tyr Ser Leu Ser 185 Gin Thr Tyr Ile 200 Asp Lys Lys Val 215 Pro Cys Pro Gly 230 Ser Arg Asp Giu Lys Gly Phe Tyr 265 Gin Pro Giu Asn 280 Gly Ser Phe Phe 295 Gin Gin Gly Asn Gly Giy Glu Thr Asn Asp Phe Thr Ser Glu His 170 Se r Cys Giu Gin Leu 250 Pro Asn Leu Val1 Gin Phe Leu Pro Asn Met T rp Pro 125 Th r Thr Pro Th r As n 205 Se r Giu As n Ile Thr 285 Lys Cys Gly Tyr ValI Val1 Tyr Cys Gly Phe Le u T rp 160 Leu Ser Pro Lys Val1 240 Se r Glu Pro ValI Met 320 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 325 330 335 Pro Gly Lys 69

Claims (51)

1. A method for inhibiting immunoglobulin-induced toxicity resulting from immunoglobulin immunotherapy in a subject comprising 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 CH2 domain so that immunoglobulin-induced toxicity is inhibited.

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 20 protein to the subject, the Ig fusion protein having multiple structurally altered toxicity-associated domains in the CH2 domain, and the Ig fusion protein being derived from an IgGI immunoglobulin molecule. 25 4. The method of claim 1 or claim 2 wherein the method prevents immunoglobulin-induced toxicity resulting fiom immunotherapy for a disease, and the immunoglobulin molecule 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. The method of claim 3 '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.

6. The method of any one of claims 1, 2 or 4, wherein the immunoglobulin molecule is IgG.

7. The method of any one of claims 1, 2 or 4, wherein the immunoglobulin molecule is IgM.

8. The method of any one of claims 1, 2 or 4, wherein the immunoglublin molecule is IgA.

9. The method of claim 2, wherein the S* immunoglobulin molecule recognizes and binds to the antigen Le y The method of claim 2, wherein the immunoglobulin molecule recognizes and binds to the antigen Le.

11. The method of claim 2 wherein the immunoglobulin molecule administered is an antibody modified from a monoclonal antibody 30 having the amino acid sequence o of the antibody BR96, produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC.

12. The method ofclaim 2, wherein the immunoglobulin molecule 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.

13. The method of claim 1 or claim 4, wherein the immunoglobulin recognizes and binds to the antigen Le y

14. The method of claim 1 or claim 4, wherein the immunoglobulin recognizes and binds to the antigen Le".

15. The method of claim 1 or claim 4, 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.

16. The method of claim 1 or claim 4, 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. 25 17. The method of claim 3 or claim 5, wherein the Ig fusion protein recognizes and binds to the antigen Le y

18. The method of claim 3 or claim 5, wherein the Ig fusion protein recognizes and binds to the antigen Lex.

19. The method of claim 3 or claim 5, wherein the Ig fusion protein has an amino acid sequence altered from the amino acid sequence of a monoclonal 72 ooo o antibody BR96 produced by the hybridoma having the identifying characteristics of HB 10036 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. The method of claim 3 or claim 5, wherein the Ig fusion protein has an 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.

21. 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.

22. A pharmaceutical composition comprising a pharmaceutically 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 CH 2 domain altered to inhibit immunoglobulin- induced toxicity resulting from administration of the structurally altered fusion protein. *73 *oooo

23. A method of blocking or inhibiting the growth of carcinomas in vivo comprising .administering to a subject a pharmaceutically effective amount of the composition of claim 21 or claim 22.

24. The composition of claim 21, 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.

25. The composition of claim 22, 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.

26. The method of any one of claims 2, 4 or 6 to 16, wherein the immunoglobulin molecule is conjugated to a cytotoxic agent.

27. The method of claim 1, wherein the immunoglobulin is conjugated to a cytotoxic agent.

28. The method of any one of claims 3, 5 or 17 to 22, wherein the IG fusion protein is conjugated to a cytotoxic agent.

29. The method of any one of claims 26 to 28, wherein 25 the cytotoxic agent is selected from the group consisting of alkylating agents, anthracyclines, antibiotics, and anti-mitotic agents. A method for killing cancer cells, the cancer cells being characterized as a group of cells having a tumor associated antigen on their cell 30 surface, which method comprises of administering to a subject harboring the cancer an amount of the composition of claim 21 or claim 22 joined to a cytotoxic agent in an amount sufficient to kill cancer cells under conditions that permit the molecule so joined to bind the tumour associated antigen on the cell surface in order to kill the cells.

31. A pharmaceutical composition comprising a pharmaceutically effective amount of an antibody having an amino acid sequence structurally altered from an antibody designated BR96 antibody produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC, the structurally altered antibody having a CH2 domain altered to inhibit immunoglobulin-induced toxicity, and an acceptable carrier.

32. A method for killing cells associated with a proliferative type disease, the disease being characterized by cells having an antigen capable of being specifically bound by monoclonal antibody BR96 produced by the 'ybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC on the cell surface, which comprises administering to a subject harboring such cells an effective amount of a composition of claim 31 joined to doxorubicin such that the composition of claim 31 'joined to doxorubicin binds the antigen on the cell surface and kills the cells.

33. A method of inhibiting toxicity induced by a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC resulting from 1: immunoglobulin immunotherapy in a subject comprising administering a modified BR96 molecule to the subject, the modification comprising the deletion or 25 substitution of at least one amino acid residue in the toxicity associated domain localized to amino acids 310-331 and the deletion or substitution of at least one •amino acid residue in the toxicity associated domain localized to amino acids 231- 238 so that complement and Fc receptor mediated toxicity is inhibited. Si' 30 34. A method for preventing toxicity induced by a monoclonal antibody designated BR96 as produced by the hybridoma having the identifying O*O O characteristics of HB10036 as deposited with the ATCC resulting from immunotherapy for cancer in a subject comprising: mutating the BR96 polypeptide by the deletion or substitution of at least one amino acid residue in the toxicity associated domain localized to amino acids 310-331 and the deletion or substitution of at least one amino acid residue in the toxicity associated domain localized to amino acids 231-238 so the complement and Fc receptor mediated immunoglobulin-induced toxicity is inhibited in the mutated BR96 polypeptide; and administering the mutated BR96 polypeptide of step to the subject under conditions that the peptide recognized the binds cancer associated Le y antigens, thereby alleviating symptoms associated with the cancer, the mutation of the toxicity associated domains thereby preventing BR96 toxicity in the subject. A chimeric antibody whose amino acid sequence is modified from the amino acid sequence of the chimeric antibody ChiBR96 produced by the hybridoma having identifying characteristics of HB10460 as deposited with the ATCC, the modified chimeric antibody having the CH1 and CH 3 domains but not the CH 2 domain, the antibody being designated CbR96-A.

36. The chimeric antibody of claim 35 which is expressed by the plasmid having the sequence shown in SEQ ID

37. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of the monoclonal antibody designated BR96 and produced by the hybridoma having the identifying characteristics of HB10036 as deposited with the ATCC, wherein the constant region has been structurally altered so that Cu 1 and the CH 3 domains are present but the CH 2 domain is not, the anti body designated hBR96- S**2A.

38. The monoclonal antibody of claim 37 which is expressed by the plasmid having the sequence shown in SEQ ID NO:12.

39. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC, the antibody having a structurally altered constant region wherein leucine at amino acid position 235 is mutated to alanine and glycine in amino acid position 237 is mutated to alanine, the antibody designated hBR96-2B. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC, the antibody having a structurally altered constant region wherein glutamic acid at amino acid position 318 is mutated to serine; lysine at amino acid position 320 is mutated to serine; and lysine at amino acid position 322 is mutated to serine, the antibody designated hBR96-2C.

41. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of the monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC, the antibody having a structurally altered constant region wherein proline at amino acid position 331 is mutated to alanine, the antibody designated hBR96-2D.

42. A monoclonal antibody whose amino acid sequence is S: modified from the amino acid sequence of the monoclonal antibody designated 25 BR96 produced by the hybridoma having the identifying characteristics of HB10036 i as deposited with the ATCC, the antibody having a structurally altered constant region wherein leucine at amino acid position 235 is mutated to alanine; glycine at amino acid position 237 is mutated to alanine; glutamic acid at amino acid position 318 is mutated to serine, lysine at amino acid position 320 is mutated to serine, and lysine at amino acid position 322 is mutated to serine, the antibody being designated hBR96-2E. 77 II 4J. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of the monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of IHB 10036 as deposited with the ATCC, the antibody having a structurally altered constant region wherein leucine at amino acid position 235 is mutated to alanine; glycine at amino acid position 237 is mutated to alanine; and proline at amino acid position 331 is mutated to alanine, the antibody being designated hBR96-2F.

44. A monoclonal antibody whose amino acid sequence is modified from the amino acid sequence of a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC having a structurally altered constant region wherein glutamic acid at amino acid position 318 is mutated to serine; lysine at amino acid position 320 is mutated to serine; lysine at amino acid position 322 is mutated to serine; and proline at amino acid position 331 is mutated to alanine, the antibody designated hBR96-2G. A monoclonal antibody whose amino acid sequence is modified from a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC, having a structurally altered constant region wherein leucine at amino acid position 235 is mutated to alanine; glycine at amino acid position 237 is mutated to alanine; o ~glutamic acid at amino acid position 318 is mutated to serine; lysine at amino acid S: position 320 is mutated to serine; lysine at amino acid position 322 is mutated to 25 serine; and proline at amino acid position 331 is mutated to alanine, the antibody being designated hBR§6-2H.

46. A nucleic acid molecule encoding the antibody of claim claim 37, or any of claims 39 to

47. The nucleic acid molecule of claim 46 wherein the nucleic S acid molecule is a cDNA. 78

48. A plasmid which comprises a nucleic acid molecule of claims 46 or 47.

49. A host sector system comprising the plasmid of claim 48 in a suitable host cell. A method for producing a protein comprising allowing the host cell of the host vector system of claim 49 to replicate under conditions that the protein encoded by the plasmid of claim 48 is expressed so as to produce the protein in the host and then recovering the protein so produced.

51. Use of an immunoglobulin molecule as defined in any one of claims 1, 2, 4, 8 to 16, 26 or 27 in the manufacture of a pharmaceutical composition for inhibiting immunoglobulin-induced toxicity resulting from immunoglobulin immunotherapy.

52. Use of an Ig fusion protein as defined in any one of claims 3, 5, 7 to 20 or 28 in the manufacture of a pharmaceutical 20 composition for inhibiting immunoglobulin-induced toxicity resulting from immunotherapy.

53. Use of a structurally altered immunoglobulin as defined in claim 21 in the manufacture of a pharmaceutical composition for blocking or inhibiting the growth of carcinomas in vivo.

54. Use of a structurally altered Ig fusion protein as defined in claim 22 in the manufacture of a pharmaceutical composition for blocking or inhibiting the growth of carcinomas in vivo.

55. Use of a structurally altered immunoglobulin or a structurally altered Ig fusion protein as defined in claims 21 or 22 respectively in the manufacture of a pharmaceutical composition for killing cancer cells having a tumor associated antigen on their cell surface, Y:\Vilet-Grac o DeeteN57417.doc the pharmaceutical composition being joined to a cytotoxic agent in an amount sufficient to kill cancer cells under conditions that permit the molecule so joined to bind the tumor associated antigen on the cell surface in order to kill the cells.

56. Use of an antibody as defined in claim 31 in the manufacture of a pharmaceutical composition for killing cells associated with a proliferative type disease characterised by cells having an antigen capable of being specifically bound by monoclonal antibody BR96 produced by the hybridoma having the identifying characteristics of HB10036 as deposited with the ATCC on the cell surface, the pharmaceutical composition being joined to doxorubicin such that the composition binds the antigen on the cell surface and kills the cells.

57. Use of a modified BR96 molecule as defined in claim 33 in the manufacture of a pharmaceutical composition for inhibiting toxicity induced by a monoclonal antibody designated BR96 produced by the hybridoma having the identifying characteristics of HB 10036 as de posited with the ATCC resulting from immunoglobulin immunotherapy.

58. Use of a mutated B96 polypeptide resulting from step of claim 34 in the manufacture of a pharmaceutical composition for preventing toxicity induced by a monoclonal antibody designated BR96 as produced by the hybridoma having the identifying characteristics of HB 10036 as deposited with the ATCC resulting from immunotherapy for cancer. o

59. An immunoglobulin molecule substantially as °hereinbefore described with reference to the examples. ce De*et 577 Y:\Violt-GracaNo Delete657417.doc A plasmid substantially as hereinbefore described as illustrated in the figures.

61. A method according to claim 1 or claim 3 substantially as hereinbefore described.

62. A pharmaceutical composition according to any one of claims 21, 22 or 32 substantially as hereinbefore described.

63. A method for inhibiting toxicity according to claim 33 or claim 34 substantially as hereinbefore described.

64. A chimeric antibody according to claim substantially as hereinbefore described. A monoclonal antibody according to any one of claims 37 to 45 substantially as hereinbefore described. DATED: 17 March, 2004 PHILLIPS ORMONDE FITZPATRICK Attorneys for: Bristol-Myers Squibb Company and Mae Joanne Rosok oo* *oooo* YWVolet-GraceWo Delete657417.oc