IMPROVED METHOD FOR TREATMENT AND DIAGNOSIS OF IL-5

MEDIATED DISORDERS

Cross Reference to Related Applications This application is a continuation-in-part of PCT/US95/17082 filed December

22, 1995, which is a continuation-in-part of U S Serial Nos 08/470,1 10 and 08/467,420, both filed June 6, 1995, which are continuation-in-parts of U S Serial No 08/363.131 filed December 23, 1994

Field of the Invention

The present invention relates generally to treatment and diagnosis of conditions mediated by IL-5 and excess eosinophil production, and more specifically to mAbs and other altered antibodies such as Fabs, chimeric, human and humanized antibodies

Background of the Invention

Eosinophils have been implicated in the pathogenesis of a wide variety of inflammatory disease states including allergic disorders associated with hypersensitivity reactions in the lung tissue (Butterfield et al , In Immunopharmacology of Eosinophils. H Smith and R Cook. Eds., p.151-192, Academic Press, London (1993)) A notable example is asthma, a disease characterized by reversible obstruction of the airways leading to non-specific bronchial hyperresponsiveness This in turn is dependent upon the generation of a chronic inflammatory reaction at the level of the bronchial mucosa and a characteristic infiltration by macrophages, lymphocytes and eosinophils The eosinophil appears to play a central roie in initiating the mucosal damage typical of the disease (Comgan et al . Immunol Today. 13 501-507 (1992)) Increased numbers of activated eosinophils have been reported in the circulation, bronchial secretions and lung parenchyma of patients with chronic asthma, and the seventy of the disease, as measured by a variety of lung function tests, correlates with blood eosinophil numbers (Gπffen et al , J_ Aller Clm Immunol . 67.548-557 (1991)). Increased numbers of eosinophils, often in the process of degranulation, have also been recovered in bronchoalveolar lavage (BAL) fluids of patients undergoing late asthmatic reactions, and reducing eosinophil numbers, usually as a consequence of steroid therapy, is associated with improvements in clinical symptoms (Bousquet et al., N Eng J Med . 323 1033-1039 ( 1990)) Interleukin 5 (IL-5) is a homodimeπc glycoprotem produced predominantly by activated CD4-I- T lymphocytes. In man, LL-5 is largely responsible for controlling the growth and differentiation of eosinophils Elevated levels of DL-5 are detected in the bronchoalveolar lavage washings of asthmatics (Motojima et al , Allergy. 48 98

( 1993)). Mice which are transgenic for IL-5 show a marked eosinophilia in peripheral blood and tissues in the absence of antigenic stimulation (Dent et al., J. Exp. Med.. 172: 1425 ( 1990)) and anti-murine IL-5 monoclonal antibodies have been shown to have an effect in reducing eosinophilia in the blood and tissues of mice (Hitoshi et al., Int. Immunol.. 3: 135 (1991)) as well as the eosinophilia associated with parasite infection and allergen challenge in experimental animals (Coffman et al.. Science. 245:308-310 (1989), Sher et al.. Proc. Natl. Acad. Sci.. 83:61-65 (1990). Chand et aL. Eur. J. Pharmacol.. 21 1 : 121-123 ( 1992)).

Although corticosteroids are extremely effective in suppressing eosinophil numbers and other inflammatory components of asthma, there are concerns about their side effects in both severe asthmatics and more recently in mild to moderate asthmatics. The only other major anti-inflammatory drug therapies - cromoglycates (cromolyn sodium and nedocromil) - are considerably less effective than corticosteroids and their precise mechanism of action remains unknown. More recent developments have focused on new inhaled steroids, longer acting bronchodilators and agents acting on novel biochemical or pharmacological targets (e.g., potassium channel activators, leukotriene antagonists, 5-lipoxygenase (5-LO) inhibitors etc.). An ideal drug would be one that combines the efficacy of steroids with the safety associated with cromolyn sodium, yet has increased selectivity and more rapid onset of action. Neutralizing IL-5 antibodies may potentially be useful in relieving eosinophila-related symptoms in man.

Hence there is a need in the art for a high affinity IL-5 antagonist, such as a neutralizing monoclonal antibody to human interleukin 5, which would reduce eosinophil differentiation and proliferation (i.e., accumulation of eosinophils) and thus eosinophil inflammation.

Summary of the Invention

In a first aspect, the present invention provides an improved method for treating conditions associated with excess eosinophil production wherein the improvement comprises the step of administering a neutralizing monoclonal antibody for human LL- 5, which does not block binding of human LL-5 to the human IL-5 receptor a-chain. Exemplary of such monoclonal antibodies is rat monoclonal antibody 4A6.

In yet another aspect of the invention is a method to assess the presence or absence of a human IL-5 soluble receptor α-chain/human IL-5 complex in a human which comprises obtaining a sample of biological fluid from a patient and allowing a monoclonal antibody for human BL-5 which does not block binding of human LL-5 to the α-chain of the human LL-5 receptor to come in contact with such sample under

conditions such that a human IL-5 soluble receptor α-cham/human IL-5/monoclonal antibody complex can form and detecting the presence or absence of said human IL-5 soluble receptor α-chain/human LL-5/ monoclonal antibody complex This method can be used to diagnose conditions associated with excess eosinophil production in a human and also to track progress and treatment of such disorders.

In a further aspect, the present invention provides a method of screening compounds to identify those compounds which antagonize binding of LL-5, LL-5/LL-5 receptor α-chain complex, or LL-5/IL-5 receptor α-chain/mAb complex to a human IL-5 receptor β-chain which comprises contacting the human LL-5 receptor β-cham with a plurality of candidate compounds under conditions to permit binding to the LL-5 receptor β-chain and identifying those candidate compounds that antagonize binding of said LL-5, LL-5/IL-5 receptor α-chain complex, or LL-5/IL-5 receptor α-cham/mAb complex to the LL-5 receptor β-chain

Other aspects and advantages of the present invention are described further in the detailed description and the preferred embodiments thereof

Brief Description of the Drawings

FIG 1 [SEQ ID NOs 1 and 15] illustrates the heavy chain variable region for the muπne antibody 2B6, and the muπne/human 2B6 chimeric antibody The boxed areas indicate the CDRs

FIG. 2 [SEQ ID NOS 2 and 16] illustrates the light chain variable region for the muπne antibody 2B6, and the muπne/human 2B6 chimeric antibody. The boxed areas indicate the CDRs

FIG. 3 [SEQ ID NO 3] illustrates the heavy chain variable region for the muπne antibody 2F2. The boxed areas indicate the CDRs

FlG 4 [SEQ ID NO 4] illustrates the light chain variable region for the muπne antibody 2F2 The boxed areas indicate the CDRs.

FlG 5 [SEQ ID NO 5] illustrates the heavy chain variable region for the muπne antibody 2E3. The boxed areas indicate the CDRs. FlG 6 [SEQ ID NO 6] illustrates the light chain variable region for the muπne antibody 2E3 The boxed areas indicate the CDRs.

FlG 7 [SEQ ID NOS:7-14] illustrates the heavy and light chain CDRs from muπne antibodies 2B6, 2F2 and 2E3.

FlG 8 [SEQ ID NOs: 18, 19] illustrates the heavy chain variable region for the humanized antibody 2B6. The boxed areas indicate the CDRs.

FlG 9 [SEQ ID NOs: 20, 21] illustrates the light chain variable region for the humanized antibody 2B6 The boxed areas indicate the CDRs

FIG. 10 IS a schematic drawing of plasmid pCDIL5HZHCl 0 employed to express a humanized heavy chain gene in mammalian cells The plasmid contains a beta lactamase gene (BETA LAC), an SV-40 origin of replication (SV40), a cytomegalovirus promoter sequence (CMV), a signal sequence, the humanized heavy chain, a poly A signal from bovine growth hormone (BGH), a betagiobm promoter (beta glopro), a dihydrofolate reductase gene (DHFR), and another BGH sequence poly A signal in a pUC 19 background

FlG 1 1 IS a schematic drawing of plasmid pCNIL5HZLCl 0 employed to express a humanized light chain gene in mammalian cells FlG. 12 [SEQ ID NOs 61, 62] illustrates the NewM heavy chain variable region for the humanized antibody 2B6 The boxed areas indicate the CDRs

FlG 13 [SEQ ID NOs 69, 70] illustrates the REI light chain variable region for the humanized antibody 2B6 The boxed areas indicate the CDRs

Detailed Description of the Invention

The present invention provides a variety of antibodies, altered antibodies and fragments thereof, which are characterized by human LL-5 binding specificity, neutralizing activity, and high affinity for human LL-5 as exemplified in muπne monoclonal antibody 2B6 or rat antibody 4A6 The antibodies of the present invention were prepared by conventional hybridoma techniques, phage display combinatorial libraries, immunoglobulin chain shuffling, and humanization techniques to generate novel neutralizing antibodies These products are useful in therapeutic and pharmaceutical compositions for treating LL-5-medιated disorders, e g , asthma These products are also useful in the diagnosis of IL-5-medιated conditions by measurement (e g , enzyme linked immunosorbent assay (ELISA)) of endogenous LL-5 levels in humans or IL-5 released ex vivo from activated cells.

Preferably the antibodies of the invention bind to human LL-5, but do not block the interaction between human LL-5 and LL-5 receptor α-chain That is, the preferred antibodies are non-competitive with the LL-5 receptor α-cham for human LL-5 The preferred antibodies of the invention also bind to an LL-5/LL-5 receptor α-chain complex A naturally-occurring soluble form of the LL-5 receptor α-chain has been observed in vitro (see, e.g , Tavernier et al , Cell. 66- 1 175-1184 ( 1991)) but it was not known, prior to the this invention, whether a soluble form of the LL-5 receptor α-chain was produced in vivo Applicants have identified a soluble form of the LL-5 receptor α-chain in vivo Thus, an antibody that binds to complexed IL-5/IL-5 receptor α-chain would be a more effective therapeutic for it would bind "free" or uncomplexed LL-5 as well as complexed IL-5 In addition, the soluble form of the LL-5 receptor α-chain

may be a natural antagonist of human IL-5 Hence, an antibody that does not compete with the soluble receptor is a more desirable and effective antagonist of LL-5, and thus it is an improved therapeutic (relative to mAbs that do compete with the IL-5 receptor α-chain for binding to LL-5) for treating IL-5 mediated conditions such as excess eosinophil production

/ Definitions.

"Altered antibody" refers to a protein encoded by an altered immunoglobulin coding region, which may be obtained by expression in a selected host cell Such altered antibodies are engineered antibodies (e.g , chimeric or humanized antibodies) or antibody fragments lacking all or part of an immunoglobulin constant region, e.g., Fv, Fab, or F(ab) ₂ and the like.

"Altered immunoglobulin coding region" refers to a nucleic acid sequence encoding altered antibody of the invention When the altered antibody is a CDR- grafted or humanized antibody, the sequences that encode the complementarity determining regions (CDRs) from a non-human immunoglobulin are inserted into a first immunoglobulin partner comprising human variable framework sequences Optionally, the first immunoglobulin partner is operatively linked to a second immunoglobulin partner.

"First immunoglobulin partner" refers to a nucleic acid sequence encoding a human framework or human immunoglobulin variable region in which the native (or naturally-occurring) CDR-encoding regions are replaced by the CDR-encodmg regions of a donor antibody The human variable region can be an immunoglobulin heavy chain, a light chain (or both chains), an analog or functional fragments thereof. Such CDR regions, located withm the variable region of antibodies (lmmunoglobuhns) can be determined by known methods in the art. For example Kabat et al (Sequences of Proteins of Immunological Interest. 4th Ed., U.S Department of Health and Human Services, National Institutes of Health (1987)) disclose rules for locating CDRs. In addition, computer programs are known which are useful for identifying CDR regions/structures "Neutralizing' refers to an antibody that inhibits IL-5 activity by preventing the binding of human LL-5 to its specific receptor or by inhibiting the signaling of LL-5 through its receptor, should binding occur. A mAb is neutralizing if it is 90% effective, preferably 95% effective and most preferably 100% effective in inhibiting LL-5 activity as measured in the B 13 cell bioassay (LL-5 Neutralization assay, see Example 2C)

The term "high affinity" refers to an antibody having a binding affinity characteπzed by a K _d equal to or less than 3.5 x 10 " M for human IL-5 as determined by optical biosensor anaylsis (see Example 2D).

By "binding specificity for human LL-5" is meant a high affinity for human, not muπne, LL-5.

"Second immunoglobulin partner" refers to another nucleotide sequence encoding a protein or peptide to which the first immunoglobulin partner is fused in frame or by means of an optional conventional linker sequence (i.e., operatively linked) Preferably it is an immunoglobulin gene. The second immunoglobulin partner may include a nucleic acid sequence encoding the entire constant region for the same (1 e., homologous - the first and second altered antibodies are derived from the same source) or an additional (i.e., heterologous) antibody of interest It may be an immunoglobulin heavy chain or light chain (or both chains as part of a single polypeptide). The second immunoglobulin partner is not limited to a particular immunoglobulin class or isotype. In addition, the second immunoglobulin partner may comprise part of an immunoglobulin constant region, such as found in a Fab, or F(ab) ₂ (i.e., a discrete part of an appropriate human constant region or framework region) Such second immunoglobulin partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, e.g., as part of a phage display library, or a sequence encoding a protein for analytical or diagnostic detection, e.g., horseradish peroxidase, β-galactosidase, etc.

The terms Fv, Fc, Fd, Fab, or F(ab) ₂ are used with their standard meanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual. Cold Spπng Harbor Laboratory, (1988)). As used herein, an "engineered antibody" describes a type of altered antibody, i.e , a full-length synthetic antibody (e.g., a chimeric or humanized antibody as opposed to an antibody fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope. For example, such molecules may include antibodies characterized by a humanized heavy chain associated with an unmodified light chain (or chimeric light chain), or vice versa Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity. These antibodies can comprise replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein.

A "chimeric antibody refers to a type of engineered antibody which contains naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody A "humanized antibody" refers to a type of engineered antibody having its

CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-deπved parts of the molecule being derived from one (or more) human ιmmunoglobulm(s) In addition, framework support residues may be altered to preserve binding affinity (see, e g , Queen et al , Proc Natl Acad Sci USA. 86 10029- 10032 ( 19891. Hodgson et al . Biotechnology. 9 421 (1991 ))

The term "donor antibody" refers to an antibody (monoclonal, or recombinant) which contributes the nucleic acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibodv with the antigenic specificity and neutralizing activity characteristic of the donor antibody One donor antibody suitable for use in this invention is a non-human neutralizing monoclonal antibody (1 e , muπne) designated as 2B6 The antibody 2B6 is defined as a high affinity, human-IL-5 specific (I e , does not recognize muπne LL- 5), neutralizing antibody of isotype IgG, having the variable light chain DNA and amino acid sequences of SEQ ID NOs 2 and 16, respectively, and the variable heavy chain DNA and amino acid sequences of SEQ ID NOs 1 and 15, respectively, on a suitable muπne IgG constant region

The term "acceptor antibody" refers to an antibody (monoclonal, or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but preferably all) of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner Preferably a human antibody is the acceptor antibody

"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervaπable regions of immunoglobulin heavy and light chains See, e g , Kabat et al , Sequences of Proteins of Immunological Interest.4th Ed , U S Department of Health and Human Services, National Institutes of Health (1987) There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin Thus, "CDRs" as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropπate)

CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived

By 'sharing the antigen binding specificity or neutralizing ability' is meant, for example, that although mAb 2B6 may be characterized by a certain level of antigen affinity, a CDR encoded by a nucleic acid sequence of 2B6 m an appropriate structural environment may have a lower, or higher affinity It is expected that CDRs of 2B6 in such environments will nevertheless recognize the same epιtope(s) as 2B6 Exemplary heavy chain CDRs of 2B6 include SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and exemplary light chain CDRs of 2B6 include SEQ ID NO 10, SEQ ID NO 11 , and SEQ ID NO 12 A "functional fragment' is a partial heavy or light chain variable sequence

(e g , minor deletions at the amino or carboxy terminus of the immunoglobulin variable region) which retains the same antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived

An "analog" is an amino acid sequence modified by at least one amino acid, wherein said modification can be chemical or a substitution or a rearrangement of a few amino acids (i e , no more than 10), which modification permits the amino acid sequence to retain the biological characteristics, e g , antigen specificity and high affinity, of the unmodified sequence For example, (silent) mutations can be constructed, via substitutions, when certain endonuclease restriction sites are created within or surrounding CDR-encoding regions

Analogs may also arise as allelic variations An "allelic variation or modification" is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention Such vaπations or modifications may be due to degeneracy in the genetic code or may be deliberately engineered to provide desired characteristics These vaπations or modifications may or may not result in alterations in any encoded amino acid sequence.

The term "effector agents" refers to non-protem carrier molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments of the donor antibody may be associated by conventional means Such non-protein carriers can include conventional earners used in the diagnostic field, e.g , polystyrene or other plastic beads, polysaccharides, e.g., as used in the BIAcore [Pharmacia] system, or other non-protem substances useful in the

medical field and safe for administration to humans and animals. Other effector agents may include a macrocycle, for chelating a heavy metal atom, or radioisotopes. Such effector agents may also be useful to increase the half-life of the altered antibodies, e.g., polyethylene glycol.. "Conditions associated with excess eosinophil production" refer to allergic and/or atopic responses, or to responses associated with eosinophilia, such as but not limited to, allergic rhinitis and asthma.

//. High Affinity IL-5 Monoclonal Antibodies

For use in constructing the antibodies, altered antibodies and fragments of this invention, a non-human species (for example, bovine, ovine, monkey, chicken, rodent (e.g., murine and rat), etc.) may be employed to generate a desirable immunoglobulin upon presentment with native human LL-5 or a peptide epitope therefrom. Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human mAb to LL-5. Such hybridomas are then screened for binding using IL-5 coated to 96-well plates, as described in the Examples section, or alternatively with biotinylated LL-5 bound to a streptavidin coated plate.

One exemplary, high affinity, neutralizing mAb of this instant invention is mAb 2B6, a murine antibody which can be used for the development of a chimeric or humanized antibody, described in more detail in Example 1 below. The 2B6 mAb is characterized by an antigen binding specificity for human LL-5, with a K, of less than 3.5 x 10 " M (about 2.2 x 10 " M) for IL-5. The K, for LL-5 of a Fab fragment from 2B6 (see, Example 3H) is estimated to be about 9 x 10 " M as determined by optical biosensor. MAb 2B6 appears to block the binding interaction between human LL-5 and the α-chain of the human LL-5 receptor. Another desirable donor antibody is the murine mAb, 2E3. This mAb is characterized by being isotype IgG _2a , and having a dissociation constant for hLL-5 of less than 3.5 x 10 ^" " M (about 2.0 x 10 " M).

Yet, another desirable donor antibody is the rat mAb, 4A6. This mAb is characterized by having a dissociation constant for hLL-5 of less than 3.5 x 10 " M (about 1.8 x 10 " M). In addition, mAb 4A6 appears to block the binding interaction between human LL-5 and the β-chain of the IL-5 receptor. MAb 4A6 does not block binding of human LL-5 to the α-chain of the LL-5 receptor. Thus, mAb 4A6 binds human LL-5 and an LL-5/LL-5 receptor α-chain complex.

This invention is not limited to the use of the 2B6 mAb, the 2E3 mAb, or its hypervariable (i.e., CDR) sequences. Any other appropriate high affinity LL-5 antibodies characterized by a dissociation constant equal or less than 3.5 x 10 " M for human LL-5 and corresponding anti-LL-5 CDRs may be substituted therefor. Wherever

in the following description the donor antibody is identified as 2B6 or 2E3, this designation is made for illustration and simplicity of description only. ///. Antibody Fragments

The present invention also includes the use of Fab fragments or F(ab') ₂ fragments derived from mAbs directed against human LL-5. These fragments are useful as agents protective in vivo against IL-5 and eosinophil-mediated conditions or in vitro as part of an LL-5 diagnostic. A Fab fragment contains the entire light chain and amino terminal portion of the heavy chain; and an F(ab') ₂ fragment is the fragment formed by two Fab fragments bound by disulfide bonds. MAbs 2B6, 2E3, and other similar high affinity, LL-5 binding antibodies, provide sources of Fab fragments and F(ab') ₂ fragments which can be obtained by conventional means, e.g., cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin, or by recombinant methods. These Fab and F(ab') ₂ fragments are useful themselves as therapeutic, prophylactic or diagnostic agents, and as donors of sequences including the variable regions and CDR sequences useful in the formation of recombinant or humanized antibodies as described herein.

The Fab and F(ab') ₂ fragments can be constructed via a combinatorial phage library (see, e.g.. Winter et al., Ann. Rev. Immunol.. 12:433-455 ( 1994)) or via immunoglobulin chain shuffling (see, e.g., Marks et al., Bio/Technology. 10:779-783 (1992), which are both hereby incoφorated by reference in their entirety) wherein the Fd or V _H immunoglobulin from a selected antibody (e.g., 2B6) is allowed to associate with a repertoire of light chain immunoglobulins, V _L (or v _κ ), to form novel Fabs. Conversely, the light chain immunoglobulin from a selected antibody may be allowed to associate with a repertoire of heavy chain immunoglobulins, v _H (or Fd), to form novel Fabs. Neutralizing IL-5 Fabs were obtained when the Fd of mAb 2B6 was allowed to associate with a repertoire of light chain immunoglobulins, as described in more detail in the Examples section. Hence, one is able to recover neutralizing Fabs with unique sequences (nucleotide and amino acid) from the chain shuffling technique. IV. Anti-IL-5 Amino Acid and Nucleotide Sequences of Interest The mAb 2B6 or other antibodies described above may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various altered antibodies which are characterized by the antigen binding specificity of the donor antibody. As one example, the present invention thus provides variable light chain and variable heavy chain sequences from the LL-5 murine antibody 2B6 and sequences derived therefrom. The heavy chain variable region of 2B6 is illustrated by FlG. 1.

„„,,„„„ PCT/US97/10769 O 97/48418

The CDR-encoding regions are indicated by the boxed areas and are provided in SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9. The light chain clone variable region of 2B6 is illustrated by FlG. 2. The CDR-encoding regions are provided in SEQ ID NO: 10; SEQ ID NO: 1 1; and SEQ ID NO: 12. A humanized heavy chain variable region is illustrated in Fig. 8 [SEQ ID NOs:

18 and 19]. The signal sequence is also provided in SEQ ID NO: 17. Other suitable signal sequences, known to those of skill in the art, may be substituted for the signal sequences exemplified herein. The CDR amino acid sequences of this construct are identical to the native murine and chimeric heavy chain CDRs and are provided by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. An exemplary (synthetic) humanized light chain variable sequence is illustrated in Fig. 9 [SEQ ID NOs: 20 and 21],

The nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incoφoration of the resulting modified or fusion nucleic acid sequence into a plasmid for expression. For example, silent substitutions in the nucleotide sequence of the framework and CDR-encoding regions were used to create restriction enzyme sites which facilitated insertion of mutagenized CDR (and/or framework) regions. These CDR-encoding regions were used in the construction of a humanized antibody of this invention.

Taking into account the degeneracy of the genetic code, various coding sequences may be constructed which encode the variable heavy and light chain amino acid sequences, and CDR sequences of the invention as well as functional fragments and analogs thereof which share the antigen specificity of the donor antibody. The isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs can be used to produce altered antibodies, e.g., chimeric or humanized antibodies, or other engineered antibodies of this invention when operatively combined with a second immunoglobulin partner. It should be noted that in addition to isolated nucleic acid sequences encoding portions of the altered antibody and antibodies described herein, other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions. Useful DNA sequences include those sequences which hybridize under stringent hybridization conditions [see, T. Maniatis et al, Molecular Cloning (A Laboratory Manuall. Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences. An n

example of one such stringent hybridization condition is hybridization at 4XSSC at 65° C, followed by a washing in 0 1XSSC at 65°C for an hour Alternatively an exemplary stringent hybridization condition is m 50% formamide, 4XSSC at 42°C Preferably, these hybridizing DNA sequences are at least about 18 nucleotides in length, i.e., about the size of a CDR

V. Altered immunoglobulin molecules and Altered antibodies

Altered immunoglobulin molecules can encode altered antibodies which include engineered antibodies such as chimeric antibodies and humanized antibodies. A desired altered immunoglobulin coding region contains CDR-encoding regions that encode peptides having the antigen specificity of an LL-5 antibody, preferably a high affinity antibody such as provided by the present invention, inserted into a first immunoglobulin partner (a human framework or human immunoglobulin variable region).

Preferably, the first immunoglobulin partner is operatively linked to a second immunoglobulin partner The second immunoglobulin partner is defined above, and may include a sequence encoding a second antibody region of interest, for example an Fc region Second immunoglobulin partners may also include sequences encoding another immunoglobulin to which the light or heavy chain constant region is fused in frame or by means of a linker sequence Engineered antibodies directed against functional fragments or analogs of IL-5 may be designed to elicit enhanced binding with the same antibody.

The second immunoglobulin partner may also be associated with effector agents as defined above, including non-protein carrier molecules, to which the second immunoglobulin partner may be operatively linked by conventional means Fusion or linkage between the second immunoglobulin partners, e.g., antibody sequences, and the effector agent may be by any suitable means, e.g , by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctionai cross-linkers, e.g , carbodiimide, glutaraldehyde, and the like Such techniques are known in the art and readily described in conventional chemistry and biochemistry texts. Additionally, conventional linker sequences which simply provide for a desired amount of space between the second immunoglobulin partner and the effector agent may also be constructed into the altered immunoglobulin coding region The design of such linkers is well known to those of skill m the art.

In addition, signal sequences for the molecules of the invention may be modified to enhance expression As one example the 2B6 humanized antibody having the signal sequence and CDRs derived from the murine heavy chain sequence, had the original signal peptide replaced with another signal sequence [SEQ ID NO 17].

An exemplary altered antibody contains a variable heavy and/or light chain peptide or protein sequence having the antigen specificity of mAb 2B6, e.g., the V _H and V _L chains. Still another desirable altered antibody of this invention is characterized by the amino acid sequence containing at least one, and preferably all of the CDRs of the variable region of the heavy and/or light chains of the murine antibody molecule 2B6 with the remaining sequences being derived from a human source, or a functional fragment or analog thereof. See, e.g., the humanized V _H and V _L regions (Figs. 8 and 9).

In still a further embodiment, the engineered antibody of the invention may have attached to it an additional agent. For example, the procedure of recombinant DNA technology may be used to produce an engineered antibody of the invention in which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule (i.e., a polypeptide effector or reporter molecule). The second immunoglobulin partner may also be operatively linked to a non- immunoglobulin peptide, protein or fragment thereof heterologous to the CDR- containing sequence having the antigen specificity of murine 2B6. The resulting protein may exhibit both anti-LL-5 antigen specificity and characteristics of the non- immunoglobulin upon expression. That fusion partner characteristic may be, e.g., a functional characteristic such as another binding or receptor domain, or a therapeutic characteristic if the fusion partner is itself a therapeutic protein, or additional antigenic characteristics.

Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains, or any discrete fragment thereof, such as the Fab or F(ab'), fragments, a heavy chain dimer, or any minimal recombinant fragments thereof such as an F _v or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor mAb, e.g., mAb 2B6 or 2E3. Such protein may be used in the form of an altered antibody, or may be used in its unfused form. Whenever the second immunoglobulin partner is derived from an antibody different from the donor antibody, e.g., any isotype or class of immunoglobulin framework or constant regions, an engineered antibody results. Engineered antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from one source, e.g., the acceptor antibody, and one or more (preferably all) CDRs from the donor antibody, e.g., the anti-LL-5 antibody described herein. In addition, alterations, e.g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region at the nucleic acid or amino acid levels, or

the donor CDR regions may be made in order to retain donor antibody antigen binding specificity

Such engineered antibodies are designed to employ one (or both) of the variable heavy and/or light chains of the LL-5 mAb (optionally modified as described) or one or more of the below-identified heavy or light chain CDRs (see also FlG 7) The engineered antibodies of the invention are neutralizing, i.e , they desirably block binding to the receptor of the IL-5 protein and they also block or prevent proliferation of LL-5 dependent ceils

Such engineered antibodies may include a humanized antibody containing the framework regions of a selected human immunoglobulin or subtype, or a chimeric antibody containing the human heavy and light chain constant regions fused to the LL-5 antibody functional fragments A suitable human (or other animal) acceptor antibody may be one selected from a conventional database, e g , the KABAT® database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected m a similar manner It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody

Desirably the heterologous framework and constant regions are selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA, and IgE However, the acceptor antibody need not comprise only human immunoglobulin protein sequences For instance a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding a non-immunoglobuhn amino acid sequence such as a polypeptide effector or reporter molecule

One example of a particularly desirable humanized antibody contains CDRs of 2B6 inserted onto the framework regions of a selected human antibody sequence. For neutralizing humanized antibodies, one, two or preferably three CDRs from the LL-5 antibody heavy chain and/or light chain vaπable regions are inserted into the framework regions of the selected human antibody sequence, replacing the native CDRs of the latter antibody Preferably, in a humanized antibody, the variable domains in both human heavy and light chains have been engineered by one or more CDR replacements It is possible to use all six CDRs, or various combinations of less than the six CDRs.

Preferably all six CDRs are replaced It is possible to replace the CDRs only in the human heavy chain, using as light chain the unmodified light chain from the human acceptor antibody Still alternatively, a compatible light chain may be selected from another human antibody by recourse to the conventional antibody databases The remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin

The engineered humanized antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e g , treatment of LL-5 mediated inflammatory diseases in man, or for diagnostic uses

As another example, an engineered antibody may contain three CDRs of the variable light chain region of 2E3 [SEQ ID NO 10, 1 1 and 13] and three CDRs of the variable heavy chain region of 2B6 [SEQ ID NO 7, 8 and 9] The resulting humanized antibody should be characterized by the same antigen binding specificity and high affinity of mAb 2B6

It will be understood by those skilled in the art that an engineered antibody may be further modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (i e , an analog) It is anticipated that heavy and light chain ammo acids may be substituted by other amino acids either in the vaπable domain frameworks or CDRs or both

In addition, the constant region may be altered to enhance or decrease selective properties of the molecules of the instant invention For example, dimeπzation, binding to Fc receptors, or the ability to bind and activate complement (see, e g , Angal et al , Mol Immunol. 30 105- 108 (1993), Xu et al , J Biol Chem. 269 3469-3474 (1994), Winter et al , EP 307,434-B)

An aitered antibody which is a chimeric antibody differs from the humanized antibodies described above by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions, in association with human immunoglobulin constant regions for both chains It is anticipated that chimeric antibodies which retain additional non-human sequence relative to humanized antibodies of this invention may elicit a significant immune response in humans

Such antibodies are useful in the prevention and treatment of LL-5 mediated disorders, as discussed below

VI Production of Altered antibodies and Engineered Antibodies Preferably, the vaπable light and/or heavy chain sequences and the CDRs of rnAb 2B6 or other suitable donor mAbs (e g., 2E3, 2F2, 4A6, etc ), and their encoding nucleic acid sequences, are utilized in the construction of altered antibodies, preferably

humanized antibodies, of this invention, by the following process. The same or similar techniques may also be employed to generate other embodiments of this invention.

A hybridoma producing a selected donor mAb, e.g., the murine antibody 2B6, is conventionally cloned, and the DNA of its heavy and light chain variable regions obtained by techniques known to one of skill in the art, e.g., the techniques described in Sambrook et al., CMolecular Cloning (A Laboratory Manual). 2nd edition, Cold Spring Harbor Laboratory (1989)). The variable heavy and light regions of 2B6 containing at least the CDR-encoding regions and those portions of the acceptor mAb light and/or heavy variable domain framework regions required in order to retain donor mAb binding specificity, as well as the remaining immunoglobulin-deπved parts of the antibody chain derived from a human immunoglobulin are obtained using polynucleotide primers and reverse transcriptase. The CDR-encoding regions are identified using a known database and by comparison to other antibodies

A mouse/human chimeric antibody may then be prepared and assayed for binding ability Such a chimeric antibody contains the entire non-human donor antibody V _H and V _L regions, in association with human Ig constant regions for both chains.

Homologous framework regions of a heavy chain variable region from a human antibody were identified using computerized databases, e.g., KABAT®, and a human antibody having homology to 2B6 was selected as the acceptor antibody. The sequences of synthetic heavy chain vaπable regions containing the 2B6 CDR-encoding regions within the human antibody frameworks were designed with optional nucleotide replacements in the framework regions to incoφorate restriction sites. This designed sequence was then synthesized using long synthetic oligomers. Alternatively, the designed sequence can be synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (PCR), and corrected for errors

A suitable light chain vaπable framework region was designed in a similar manner.

A humanized antibody may be deπved from the chimeric antibody, or preferably, made synthetically by inserting the donor mAb CDR-encoding regions from the heavy and light chains appropriately within the selected heavy and light chain framework. Alternatively, a humanized antibody of the invention made be prepared using standard mutagenesis techniques. Thus, the resulting humanized antibody contains human framework regions and donor mAb CDR-encoding regions. There may be subsequent manipulation of framework residues. The resulting humanized antibody can be expressed in recombinant host cells, e.g., COS, CHO or myeloma

cells. Other humanized antibodies may be prepared using this technique on other suitable IL-5-specific, neutralizing, high affinity, non-human antibodies.

A conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the altered antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences, which can be derived from other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain. Preferably this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the altered antibody may reside on a single vector. A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention. The humanized antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other altered antibodies and molecules of this invention.

Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the conventional pUC series of cloning vectors, may be used. One vector used is pUC 19, which is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden). Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.

Similarly, the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vector. The vectors also contain selected regulatory sequences (such as CMV promoters) which direct the replication and expression of heterologous DNA sequences in selected host cells. These vectors contain the above described DNA

sequences which code for the engineered antibody or altered immunoglobulin coding region. In addition, the vectors may incoφorate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betagiobm promoter sequence (betaglopro) The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art

The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this puφose

The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobulin molecules thereof. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. colt are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention.

Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably mammalian cells such as CHO, COS, a fibrobiast cell (e.g., 3T3), and myeloid cells, and more preferably a CHO or a myeloid cell. Human cells may be used, thus enabling the molecule to be modified with human glycosyiation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and puπfication are known in the art. See, e.g., Sambrook et al., cited above.

Bacteπal cells may prove useful as host cells suitable for the expression of the recombinant Fabs of the present invention (see, e.g., Plϋckthun, A., Immunol. Rev.. 130: 151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacteπal cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacteπal cell was produced in a properly folded form, that bacterial cell would be a desirable host. For

example, various strains of E coli used for expression are well-known as host cells in the field of biotechnology Various strains of B subtihs, Streptomyces, other bacilli and the like may also be employed in this method

Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e g Drosophila and Lepidoptera and viral expression systems See, e g Miller et al , Genetic Engineering. 8 277-298, Plenum Press (1986) and references cited therein

The general methods by which the vectors of the invention may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the altered antibody of the invention from such host cell are all conventional techniques Likewise, once produced, the altered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like Such techniques are withm the skill of the art and do not limit this invention

Yet another method of expression of the humanized antibodies may utilize expression in a transgenic animal, such as described in U S Patent No 4,873,316 This relates to an expression system using the animal's casein promoter which when transgenically incoφorated into a mammal permits the female to produce the desired recombinant protein in its milk

Once expressed by the desired method, the engineered antibody is then examined for in vitro activity by use of an appropriate assay Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the engineered antibody to LL-5 Additionally, other in vitro assays may also be used to \ eπfy neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the engineered antibody in the body despite the usual clearance mechanisms

Following the procedures described for humanized antibodies prepared from 2B6, one of skill in the art may also construct humanized antibodies from other donor LL-5 antibodies, variable region sequences and CDR peptides descπbed herein

Engineered antibodies can be produced with variable region frameworks potentially recognized as "self by recipients of the engineered antibody Minor modifications to the variable region frameworks can be implemented to effect large increases in antigen binding without appreciable increased immunogenicity for the recipient Such engineered antibodies may effectively treat a human for LL-5 mediated conditions Such antibodies may also be useful in the diagnosis of such conditions VII Therapeutic/Prophylactic/Diagnostic Uses

This invention also relates to a method of treating humans experiencing eosinophilia-related symptoms, i.e., conditions associated with excess eosinophil production, such as asthma, which comprises administering an effective dose of antibodies including one or more of the engineered antibodies or altered antibodies described herein, or fragments thereof.

The therapeutic response induced by the use of the molecules of this invention is produced by binding to human LL-5 and thus subsequently blocking eosinophil stimulation. Preferably, the molecules of this invention are non-competitive with the LL-5 receptor alpha-chain for binding human LL-5. That is, the preferred molecules of this invention do not block the binding of human IL-5 to the α-chain of the human IL- 5 receptor. Thus, the molecules of the present invention, when in preparations and formulations appropriate for therapeutic use, are highly desirable for those persons ^• experiencing an allergic and/or atopic response, or a response associated with eosinophilia, such as but not limited to, allergic rhinitis, asthma, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, coeliac disease, eosinophilic gastroenteritis, Churg-Strauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, oedematous reactions including episodic angiodema, helminth infections, where eosinophils may have a protective role, onchocercal dermatitis and atopic dermatitis. The altered antibodies, antibodies and fragments thereof of this invention may also be used in conjunction with other antibodies, particularly human mAbs reactive with other markers (epitopes) responsible for the condition against which the engineered antibody of the invention is directed.

The therapeutic agents of this invention are believed to be desirable for treatment of allergic conditions from about 2 days to about 3 weeks, or as needed. For example, longer treatments may be desirable when treating seasonal rhinitis or the like. This represents a considerable advance over the currently used infusion protocol with prior art treatments of LL-5 mediated disorders. The dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient.

The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host. The altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, or intranasally.

Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the engineered (e.g., humanized) antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier. In the prophylactic agent of the invention, an aqueous suspension or solution containing the engineered antibody, preferably buffered at physiological pH, in a form ready for injection is preferred. The compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an pharmaceutically acceptable carrier, preferably an aqueous carrier A variety of aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter These solutions may be sterilized by conventional, well known sterilization techniques (e g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an engineered antibody of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 and preferably 5 mg to about 25 mg of an engineered antibody of the invention. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail m, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. It is preferred that the therapeutic agent of the invention, when in a pharmaceutical preparation, be present in unit dose forms. The appropriate therapeutically effective dose can be determined readily by those of skill in the art. To effectively treat an inflammatory disorder in a human or other animal, one dose of approximately 0.1 mg to approximately 20 mg per 70 kg body weight of a protein or an antibody of this invention should be administered parenterally, preferably i.v. or i.m. (intramuscularly). Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician during the inflammatory response.

The antibodies, altered antibodies and engineered antibodies of this invention may also be. used in diagnostic regimens, such as for the determination of LL-5 mediated disorders or tracking progress of treatment of such disorders As diagnostic reagents, these antibodies may be conventionally labeled for use in ELISA's and other conventional assay formats for the measurement of LL-5, and/or LL-5/LL-5 receptor α- chain complex levels in serum, plasma or other appropriate tissue, or the release by human cells in culture The nature of the assay in which the altered antibodies are used are conventional and do not limit this disclosure

Thus, one embodiment of the present invention relates to a method for aiding the diagnosis of allergies and other conditions associated with excess eosinophil production in a patient which comprises the steps of determining the amount of human LL-5 and/or LL-5/IL-5 receptor α-chain complex in sample (plasma or tissue) obtained from said patient and comparing said determined amount to the mean amount of human LL-5 in the normal population whereby the presence of a significantly elevated amount of LL-5 and/or IL-5/TL-5 receptor α-chain complex in the patient s sample is an indication of allergies and other conditions associated with excess eosinophil production

In the compound screening embodiment of this invention, the human LL-5 receptor β-chain is isolated in a membrane fraction, or in cell bound form, and is contacted with a plurality of candidate molecules from which candidates are selected which bind to and interact with the receptor The candidate compounds can be subjected to a competition screening assays, in which a known ligand, i e, human LL-5 or LL-5/IL-5 receptor α-chain complex, preferably labeled with an analytically detectable reagent, most preferably radioactivity, is introduced with the drug to be tested and the compound's capacity to inhibit or enhance the binding of the labeled ligand is measured Alternatively, the binding or interaction can be measured directly by using radioactively labeled candidate compounds of interest or by the second messenger effect resulting from the interaction or binding of the candidate compounds Compounds are screened for their increased affinity and selecϋvity to the receptor interest Molecules that bind gratuitously, I e , without inducing effects on the human IL-5 receptor β-chain, are most likely to be good antagonists Potential antagonists include small organic molecules, peptides, polypeptides and antibodies specific for the LL-5 receptor β-chain and thereby inhibit or exUnguish its activity

The antibodies, altered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier pπor to use This technique has been shown to be effective with conventional immunoglobulins and art- known lyophihzation and reconstitution techniques can be employed

The following examples illustrate various aspects of this invention including the construction of exemplary engineered antibodies and expression thereof in suitable vectors and host cells, and are not to be construed as limiting the scope of this invention All amino acids are identified by conventional three letter or single letter codes All necessary restriction enzymes, plasmids, and other reagents and materials were obtained from commercial sources unless otherwise indicated All general cloning hgation and other recombinant DNA methodology were as performed in T Maniatis et al , cited above, or the second edition thereof (1989), eds Sambrook et al , by the same publisher ("Sambrook et al ")

Example 1 - Production of MAbs to hIL-5

Human LL-5 was expressed in Drosophila Schneider 2 (S2) cells and purified to homogeneity. Murine IL-5 was expressed in Baculovirus using Spodoptera frugiperda 21 (Sf21) cells and purified to homogeneity Monoclonal antibody TRFK-5 (a neutralizing rat anti-mouse LL-5 antibody) was obtained from Genzyme Coφ. (Cambridge, MA).

A. Immunization Procedure

Recombinant human LL-5 (LL-5) was used as the immunogen for a panel of seven CAFl female mice (Charles River, Wilmington, MA). The animals received three subcutaneous injections of IL-5 in phosphate buffered saline (PBS) emulsified with a one to one ratio of TiterMAX™ (CytoRx Coφ., Norcross, GA) over a period of four months. The priming antigen dose was 50 μg (micrograms) and boosts were 25 and 10 μg (micrograms). After the boosts, serum samples were collected and assayed both for binding to IL-5 and for neutralization activity via the receptor binding inhibition assay and B 13 proliferation assay (or LL-5 neutralization assay (Example 2C)). All of the mice produced serum samples that bound to IL-5 Animals selected as spleen donors were boosted intravenously with 10 μg (micrograms) of recombinant human LL-5 three days prior to euthanasia

B . Hybridoma Development: The fusion procedure, first reported by Kohler et al., (Nature, 256:495 (1975)), was used with modifications to perform the technique using a cell monolayer (Kennet et al., Eds., "Hybridomas: A new dimension in biological analysis", pp. 368-377, Plenum Press, New York) Spleen cells from two donor mice were pooled and fusions performed using a ratio of 50 million spleen cells to ten million SP2/0/Agl4 myeloma cells. Supematants from fusion-positive welis were assayed for binding to LL-5 by ELISA. Wells containing cells producing antibody to LL-5 were expanded and supematants screened in an LL-5 receptor binding inhibition assay, and a B 13 (neutralization) proliferation assay (described below).

Sixteen hybridomas were isolated which secreted mAbs reactive with LL-5. The hybridoma supematants were mixed with iodinated IL-5, added to a membrane extract prepared from Drosophila cells expressing the α-chain of the LL-5 receptor (IL- 5R), and assayed for inhibition of receptor binding. Eleven of the hybridoma supematants inhibited by greater than 60% the binding of iodinated LL-5 to the LL-5 receptor α-chain. Three of the mAbs, 2B6, 2E3 and 2F2, also inhibited by greater than 70% the proliferation of murine B 13 cells in response to human but not murine LL-5. Five of the hybridomas, four of which blocked binding and/or proliferation ( 1 C6, 2B6, 2E3 and 2F2) and 1 of which was non-neutralizing (24G9), were repeatedly subcioned

in soft agar to generate stable clonal cell lines Supematants from the cloned lines were screened for cross-reactivity by ELISA and did not bind to human IL-lα, IL-l β,

IL-4, LL-8, M-CSF or TGFα The mAbs were purified and binding affinities were estimated from optical biosensor (BIAcore) analysis to range from 10 to 100 pM Supematants from the lines were isotyped using murine isotyping reagents

(PharMingen, San Diego, CA) A summary of the affinities and IC50 for neutralizing activities of the mAbs is presented in Table I (Example 2)

By similar methods, rat hybridomas were derived from immunized rats, using a comparable immunization protocol and rat myelomas for the fusion as described for the mouse Two rat hybridomas, 4A6 and 5D3, were identified that produced mAbs which bound to LL-5 Like mAbs 2B6, 2E3 and 2F2, mAbs 4A6 and 5D3 were found to be neutralizing in the B 13 assay described below

C Hybridoma Deposit

The hybridoma cell line SKI 19-2B6 206 75(1 ) producing monoclonal antibody 2B6 was deposited with the American Type Culture Collection (ATCC), Rockville,

MD, USA, under accession number HB 1 1783, and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

The hybridoma cell line SKI 19-2E3.39 40 2 producing monoclonal antibody 2E3 was deposited with the Ameπcan Type Culture Collection (ATCC), Rockville,

MD, USA, under accession number HB 1 1782, and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

The hybridoma cell line SKI 19-2F2 37.80 12 producing monoclonal antibody 2F2 was deposited with the American Type Culture Collection (ATCC), Rockville,

MD, USA, under accession number HB 1 1781 , and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

The hybπdoma cell line SKI I9-24G9.8.20.5 producing monoclonal antibody 24G9 was deposited with the American Type Culture Collection (ATCC), Rockville,

MD, USA, under accession number HB 11780, and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

The hybridoma cell line 4A6(1)G1F7 producing monoclonal antibody 4A6 was deposited with the Ameπcan Type Culture Collection (ATCC), Rockville, MD, USA, under accession number HB 11943, and has been accepted as a patent deposit, in

accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

The hybridoma cell line 5D3(1)F5D6 producing monoclonal antibody 5D3 was deposited with the Ameπcan Type Culture Collection (ATCC), Rockville, MD, USA, under accession number HB 11942, and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the puφoses of patent procedure

Example 2 - Assays A ELISA

Individual wells of MaxiSorb™ immuno plates (Nunc, Naperville, IL) were coated with 0 2 ug IL-5 in 0 05M carbonate buffer pH 9 6 After incubating overnight at 4°C, the plates were rinsed with PBS containing 0 025% Tween® 20, and blocked with 1 % BSA in PBS with 0 025% Tween® 20 for two hours at room temperature Undiluted hybrid supematants were added to the LL-5 coated wells and incubated at room temperature for two hours After the plates were rinsed, peroxidase labeled goat anti-mouse IgG & IgM (Boehπnger Mannheim, Indianapolis, IN) was added at 1/7500 dilution in PBS containing 1% BSA and 0.025% Tween® 20 Two hours later the plates were washed and 0 2 ml of 0 1M citrate buffer pH 4 75 containing 0 1 % urea peroxide and 1 mg/ml orthophenylenediamine was added After 15 min the plates were read at 450nm on a VMax™ Microplate Reader (Molecular Devices, Menlo Park, CA) B Receptor Binding Inhibition Assay

Membrane extracts of Drosophila S2 cells expressing the α-chain of the human LL-5 Receptor (LL-5R) were used to measure the effect of antibody on LL-5 binding to receptor To prepare the membranes, 109 cells were pelleted at 1000 x g at 4°C for 10 min The cell pellet was frozen in a dry ice/ethanol bath for 15 min The pellet was thawed, resuspended in 10 ml PBS at 4°C and pelleted at 1000 x g for 10 min The cell pellet was washed 2X in PBS and resuspended in 13 5 ml Hypotomc buffer (10 mM Tns pH 7 5, 3 mM MgCl2, 1 mM dithiothreitol, ImM phenylmethylsulfonyl fluoride, 1 uM leupeptin, 1 uM pepstatin A) and incubated on ice for 5 min The cell suspension was homogenized in a 15 ml Dounce homogenizer and brought to a final concentration of 0 25 M sucrose with a solution of 2 5 M sucrose Cell debris was removed by a 15 min centrifugation at 1000 x g Cell membranes were pelleted at 100,000 x g at 4°C for 90 min and resuspended in 50 ml of 10 mM Tns pH 7 5, 3 mM MgCl2, 250 mM sucrose, and stored at -70°C

Assays with Drosophila membranes containing receptor were performed in MultiscreenGV™ plates (Milhpore Coφ., Bedford, MA) using Drosophila tissue

O 97/48418

culture medium M3 (Lindquist et al, Drosophila Inf. Serv.. 58. 163 (1982)) containing 25 mM HEPES buffer pH 7.2 and 0.1% BSA (Binding Buffer) Wells were pre- blocked with 0.1 ml binding buffer. 50 ul of the test sample, in triplicate, was added to wells followed by 25 ul iodinated ( ^{I 25} I) LL-5 After 20 minutes incubation at room temperature, 25 ul of the membrane extract of Drosophila S2 cells expressing the α- chain of the human IL5R was added to the wells. After 1 hour further incubation the membranes were collected by vacuum filtration and washed 3X with binding buffer. Filters were dried and counted. C. IL-5 Neutralization Assay. The murine IL-5/IL-3 dependent cell line LyH7.B13 (B 13) was obtained courtesy of R Palacios, Basel Institute of Immunology, Switzerland Cells were subcultured twice weekly in RPMI 1640 medium (GibcoBRL, Renfrewshire, UK), supplemented with L-Glutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin (all GibcoBRL), plus 2-mercaptoethanol (5 x 10 ^" ^ M, Sigma), 10% fetal bovine serum (Globepharm, Surrey, UK ) and 1- 10 units murine IL-5 For assays, cells were cultured for 48 hours in triplicate (5000 cells/well) in 96-well round bottom plates in the presence of appropriately diluted test samples and pulsed with 0.5 uCi ^H-thymιdιne (Amersham, Bucks, UK ) for the final 4 hours They were processed for scintillation counting in a 1205 Betaplate (LKB Wallac, Beds, UK). D. Optical Biosensor

Kinetic and equilibrium binding properties with immobilized hIL-5 and antibodies were measured using a BIAcore optical biosensor (Pharmacia Biosensor, Uppsala, Sweden) Kinetic data were evaluated using relationships described previously (Karlsson et al . J. Immunol. Meth , 145:229-240 (1991)) and which is incoφorated by reference in its entirety

Three of the neutralizing mAbs, namely 2B6, 2E3 and 2F2, had very similar potencies of inhibition of ^^I-LL-5 binding to membrane receptor and neutralization of B cell proliferation and also very similar affinities for LL-5 (see Table I). The nucleotide sequences of the VH and VL from these three mAbs, 2 IgGl and 1 IgG2a, respectively, were determined. The sequences obtained were very similar, differing only at a few residues

TABLE I Affinity and neutralizing activity of mAbs reactive with human IL-5

mAb Kd (pM) ^a Neutralization Binding ICsn(nM)b Proliferation ICsrf 100%Inhibition ^c

2B6 22 1 70 200

2E3 20 1 90 600

2F2 13 1 150 340

1C6 86 43 12,200 ND

24G9 ND >133 > 100,000 ND

4A6 18 >88 28 100

5D3 ND NT) 10Q 10,000 ^a Determined by optical biosensor (BIAcore) analysis (25°C) ^h Inhibition of ' ^I-IL-5 binding to IL-5R(α chain) from Drosophila membranes ^c Inhibition of proliferation (in pM) of B 13 cells in response to 8 pM human IL-5 ND = No data

Example 3-Isolation and Characterization of IL-5 Fabs from Combinatorial Library A. PCR and Combinatorial Library Construction:

RNA purified from the spleens of three mice was reverse transcribed with a cDNA kit (Boehringer Mannheim, Indianapolis, IN) using either the primer (dT) i5 supplied with the kit or the 3' Fd (IgGl, IgG2a & IgG3) and kappa light chain primers as described by Huse et al. (Science. 246: 1275 (1989)) and Kang, S.A. (Methods: Companion Methods Enzvmol., 2: 1 1 1 ( 1991 )) which are hereby incoφorated by reference in their entirety. Immunoglobulin cDNAs were amplified by PCR using the primers and the thermal cycling conditions described (Huse et al. supra). The Hot Start technique using AmpliWax™ PCR Gem 100 (Perkin Elmer Cetus, Norwalk, CT) beads and the manufacturer's protocol was used for all of the reactions. The PCR products were gel purified, digested, and ligated into the pMKFabGene3 vector (Ames et al., J. Immunol.. 152:4572 (1994)). The library titer following ligation with the Fd cDNAs was 5.1 X 10^ CFU and following ligation with the kappa cDNAs was 1.5 X 10 ⁶ CFU. XL 1 -Blue cells (Stratagene, La Jolla, CA) transformed with the phagemid library were infected with helper phage VCSM13 (Stratagene) and phage were prepared as described by Barbas and Lemer (Methods: Companion Methods Enzvmol.. 2: 1 19 ( 1991)).

B Biopanning

Four microtiter wells (Immulon II Removawell Strips, Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4°C with IL-5 (lug/well) in 0.1M bicarbonate, pH 8 6 The wells were washed with water and blocked with PBS containing 3% BSA at 37°C for 1 hour The blocking solution was removed, and the library was added to microtiter wells (50 ul/well) and incubated at 37°C for 2 hours. Wells were washed 10 times with TBS/Tween® (50mM Tris-HCl, pH 7 5, 150 mM NaCl, 0 5% Tween® 20) and once with H ₂ O prior to elution of the adherent phage with 0 1 M HCl, adjusted to pH 2 2 with glycine, containing 1 mg/ml BSA C Colony Lifts

Colony lifts from clones isolated from the third and fourth rounds of biopanning were processed as described (Barbas and Lerner, supra) Filters were incubated for 1 hour at room temperature with 0 5-1 0 uCi ^"^\-ΪL-5, which had been iodinated using Bolton-Hunter reagent (NEN, Billeπca, MA) following the manufacturers recommended procedure, in PBS containing 1 % BSA, washed with PBS 0 25% Tween, and exposed to Kodak XAR film Colonies expressing LL-5-reactιve Fabs were detected by autoradiography D Preparation of Soluble FABs

Phagemid DNAs were digested with Nhel and Spel to remove gene III and self- ligated XL 1 -Blue cells were transformed, and isolated clones were grown overnight at 37°C in 5.0 ml super broth (SB) medium (30 g tryptone, 20 g yeast extract, 10 g 3-[N- Mθφhohno]propanesulfonιc acid, MOPS with pH adjusted to 7) containing 1% glucose and 50 ug/ml carbenicillm Cells from 1 ml of this culture were pelleted at 3500 φm for 10 min in Beckman GS-6R centπfuge and used to inoculate 5 ml SB containing 50 ug/ml carbenicillm Cultures were shaken for 1 hour at 37°C, Isopopyl-b-D- thiogalactopyranoside (IPTG, 1 mM) was added and the cultures were transferred to 28°C overnight Soluble Fab was prepared from peπplasmic extracts by lysing the cell pellet for 20 min at 4°C in 20% sucrose suspended in 30 mM Tns pH 8 0, followed by centrifugation in a Microfuge for 10 mm Fab concentrations were estimated by westem blot by comparison to samples containing known amounts of murine Fab The different bacteπal peπplasmic extracts contained similar concentrations of Fab, ranging from 1 to 20 ug/ml, as estimated by westem blot analysis E Purification of FABs

A chelating peptide was engineered onto the carboxy-terminal end of the heavy chain to aid in protein purification Following removal of the M 13 genelll coding region, via digesϋon with Nhel and Spel, a pair of overlapping oligonucleotides [SEQ ID NO 43] S'-CTAGCCACCACCACCACCACCACTAA-S',

[SEQ ID NO: 44] 3'-GGTGGTGGTGGTGGTGGTGATTGATC-5' encoding six histidine residues were subcioned into the Fab expression vector. Induction of Fab expression was performed as described above. Following overnight induction at 28°C periplasmic lysate of the cell pellet was prepared by 30 min incubation at 4°C in 20% sucrose, 30 mM TRIS pH 8.0. Urea and Brij-35 detergent were added to the clarified supernatant to final concentrations of 2M and 1 % respectively. After stirring at room temperature for 1 hour, the treated and clarified supernatant was loaded at 0.5 ml/min directly onto a 5 ml Nickel-NTA metal chelating column ( 1.5 x 3 cm) equilibrated with buffer A ( 100 mM Na-Phosphate, 10 mM Tris, 0.3 M NaCl, 2 M urea, pH 8.0). After a 4 column volume (20 ml) wash bound materials were eluted with a 6 column volume (30 ml) reverse pH gradient from pH 8 to pH 4 in the same buffer as above. The purified Fabs eluted from the column in a shaφ symmetrical peak at pH 5.5. They were >90% pure and free of DNA.

F. FAB ELISA: Immulon II plates (Dynatech) were coated overnight at 4°C with protein suspended ( 1 mg/ml; 50 mi per well) in 0. 1 M bicarbonate buffer, pH 8.6. Dilutions and washes were performed in PBS containing 0.05% Tween™ 20. Plates were washed and blocked for 1 hour with PBS containing 1 % BSA at room temperature. Various dilutions of the bacterial supematants containing soluble Fabs, or purified Fabs, were added to the plates. Following a one hour incubation plates were washed and biotinylated goat anti-mouse kappa (Southern Biotechnology Associates, Inc., Birmingham, AL) was added (1 :2000 dilution; 50 ulΛvell) for 1 hour. The plates were washed and streptavidin labeled horseradish peroxidase was added (1:2000 dilution; 50 ul/well) for 1 hour. The plates were washed, ABTS peroxidase substrate was added ( 100 ul/well; Kirkegaard & Perry Laboratories, Gaithersburg, MD) and the optical density at 405 nm was read on a UVmax™ (Molecular Devices) microplate reader.

G. Isolation and Characterization of Fabs from a Combinatorial Library:

Phage bearing Fabs to LL-5 were selected from the library by multiple rounds of biopanning against microtiter wells coated with LL-5. After 4 rounds of selection LL-5 reactive Fabs were identified by a colony lift assay using ^^l-TL-5. Thirty four colonies from the third round and 4 colonies from the fourth round were identified which bound labeled LL-5. Binding to LL-5 was confirmed by direct binding ELISA using culture supematants expressing the Fab-geneHI fusion protein. DNA was isolated from these colonies and, after removing the coding region of Ml 3 gene III, soluble Fab expression was induced. Periplasmic fractions were prepared and assayed by ELISA for binding to LL-5. The Fabs bound specifically to LL-5 with no demonstrable binding to an another protein, rC5a.

The undiluted periplasmic extracts (containing 1 to 20 ug/ml Fab) were assayed in the IL-5R binding inhibition assay (Example 2) None of the Fabs inhibited binding of iodinated IL-5 to the IL-5Rα by more than 35% H Conversion of Neutralizing mAb to a FAB The Fd and K cDNAs of mAb (2B6) were isolated by PCR using the conditions described above The gel-puπfied fragments were subcioned into the pMKFabGene3 vector which had been modified to include the hexa-His sequence 3' of the gene in cDNA, resulting in the plasmid pMKFabGene3H A functional, LL-5 binding Fab clone containing the 2B6 heavy and light chains was identified by a colony lift assay Upon removal gene Ul via Nhe I/Spel I digestion and self-ligation the heavy chain was fused in frame to the hexa-His, allowing purification as described above In a dose dependent manner, this Fab inhibited receptor binding with an IC50 of approximately 7 5 ug/ml, similar to that of the parent mAb, murine 2B6

I Construction and Screening of Chain-Shuffled Ltbran. The cDNA encoding the Fd of the neutralizing mAb 2B6 was subcioned as an

Xhol/Spel fragment into pMKFabGene3H which contained a Sstl/Xbal fragment in lieu of a light chain cDNA This phagemid was digested with SstI and Xbal and ligated with the Sstl/Xbal digested light chain PCR product deπved from the LL-5 immunized mice (described above) The library titer following ligation was 4 X 10^ CFU Biopanning, and colony lift assay was performed as described above for the combinatorial library The library was constructed by pairing the cDNA encoding the Fd of the neutralizing mAb 2B6 with the same light chain repertoire, recovered from the IL-5 immunized mice, used to generate the combinatorial library This chain shuffled library was subjected to 4 rounds of biopanning vs immobilized LL-5 and the resultant colonies were assayed for LL-5 reactivity using the colony lift assay Positive colonies, which bound iodinated IL-5, were further assayed by ELISA and the LL-5Rα binding assay Two of the Fabs, 2 & 15, recovered from the chain shuffled library blocked binding of LL-5 to the IL-5Rα and inhibited LL-5 dependent proliferation in the B 13 assay The sequences of these 2 Vks were similar to the sequence of the 2B6 Vk, the original light chain partner for the 2B6 VH The light chain sequences for Fab 2 & 15 are SEQ ID NOs 45 and 46, respectively For Fab 2, CDRs 1-3 are SEQ ID NOs 10,

I I and 47, respectively. For Fab 15, CDRs 1-3 are SEQ ID NOs 10, 11 and 48, respectively

All antibody amino acid sequences listed below in Examples 4 and 5 use the

KABAT numbering system which allows variability in CDR and framework lengths That is, key amino acids are always assigned the same number regardless of the actual

number of amino acids preceding them. For example, the cysteine preceding CDRl of all light chains is always KABAT position 23 and the tryptophan residue following CDRl is always KABAT position 35 even though CDRl may contain up to 17 amino acids.

Example 4 - Humanized Antibody

One humanized antibody was designed to contain murine CDRs within a human antibody framework. This humanized version of the LL-5 specific mouse antibody 2B6, was prepared by performing the following manipulations. A. Gene Cloning: mRNA was isolated from each of the respective 2B6, 2F2 and 2E3 hybridoma cell lines (see Example 1) with a kit obtained from Boehringer Mannheim (Indianapolis, LN) and then reverse transcribed using the primer (dT) i5 supplied with a cDNA kit (Boehringer Mannheim) to make cDNA. PCR primers specific for mouse immunoglobulin were used to amplify DNA coding for domains extending from amino acid #9 (KABAT numbering system) of the heavy chain variable region to the hinge region and from amino acid #9 (KABAT numbering system) of the light chain variable region to the end of the constant region. Several clones of each antibody chain were obtained by independent PCR reactions. The mouse gamma 1 hinge region primer used is [SEQ ID NO: 22]: 5' GTACATATGCAAGGCTTACAACCACAATC 3'. The mouse gamma 2a hinge region primer used is [SEQ ID NO: 23]:

5' GGACAGGGCTTACTAGTGGGCCCTCTGGGCTC 3' The mouse heavy chain variable region primer used is [SEQ ID NO: 24]: 5' AGGT(C or G)(C or A)A(G or A)CT(G or T)TCTCGAGTC(T or A)GG

3'

The mouse kappa chain constant region primer used is [SEQ ID NO: 25]: 5' CTAACACTCATTCCTGTTGAAGCTCTTGACAATGGG 3' The mouse light chain variable region primer is [SEQ ID NO: 26]: 5" CCAGATGTGAGCTCGTGATGACCCAGACTCCA 3'

The PCR fragments were cloned into plasmids pGEM7f+ (Promega) that were then transformed into E. coli DH5a (Bethesda Research Labs). B. DNA Sequencing:

The heavy and light chain murine cDNA clones from Part A above were sequenced. The results of sequencing of the variable regions of these clones are shown in SEQ ID NOs: 1-6 (Fig. 1-6). Each clone contained amino acids known to

be conserved among mouse heavy chain variable regions or light chain variable regions The CDR ammo acid sequences are listed below

The CDR regions for the 2B6 heavy chain are SEQ ID NOs 7, 8 and 9 See Fig 7 These sequences are encoded by SEQ ID NO 1 The CDR regions for the light chain are SEQ ID NOs 10, 11 and 12. See Fig 7 These sequences are encoded by SEQ ID NO.2

The CDR regions for the 2F2 heavy chain are SEQ ID NOs 7, 8 and 9 See Fig 7 These sequences are encoded by SEQ ID NO 3 The CDR regions for the light chain are SEQ ID NOs 10, 11 and 13 See Fig 7 These sequences are encoded by SEQ ID NO 4

The CDR regions for the 2E3 heavy chain are SEQ ID NOs 7, 8 and 14 See Fig 7 These sequences are encoded by SEQ ID NO 5 The CDR regions for the light chain are SEQ ID NOs 10, 1 1 and 13 See Fig 7 These sequences are encoded by SEQ ID NO 6 C Selection of Human Frameworks

Following the cloning of 2B6, the amino acid sequences of the variable region heavy and light chains (Figs 1 and 2) (SEQ ID NOs 15 and 16, respectively) were compared with the known murine immunoglobulin sequences in the KABAT and SWISS-PROT (Nuc Acids Res . 20 2019-2022 (1992)) protein sequence databases in order to assign amino acids to the N-terminal residues The 2B6 heavy and light chain variable region deduced amino acid sequences were then compared with the human immunoglobulin protein sequence databases in order to identify a human framework for both the heavy and light chains which would most closely match the muπne sequence In addition, the heavy and light chains were evaluated with a positional database generated from structural models of the Fab domain to assess potential conflicts due to amino acids which might influence CDR presentation Conflicts were resolved during synthesis of the humanized variable region frameworks by substitution of the corresponding mouse amino acid at that location The heavy chain framework regions of an antibody obtained from a human myeloma immunoglobulin (COR) was used (E M Press and N M Hogg, Biochem J . 1 17 641 -660 ( 1970)) The human heavy chain framework amino acid sequence was found to be approximately 66% homologous to the 2B6 framework For a suitable light chain variable region framework, the light chain variable framework sequence of the Bence- Jones protein, (LEN) (Schneider et al , Hoppe-Seyler's Z Phvsiol Chem . 356-507-557 (1975)), was used The human

light chain framework regions were approximately 82% homologous to the murine 2B6 light chain framework regions, at the amino acid level.

The selected human frameworks were back translated to provide a DNA sequence. D. Construction of Humanized MAb Genes:

Given the 2B6 heavy chain CDRs [Fig. 7 and SEQ ID NOs: 1-2] and the framework sequences of the human antibodies, a synthetic heavy chain variable region was made [SEQ ID NO: 18]. This was made using four synthetic oligonucleotides [SEQ ID NOs:27 and 28] [ SEQ ID NOs: 29 and 30] which, when joined, coded for amino acids #21 -#106 (KABAT numeration). The oligonucleotides were then ligated into the Hpal-Kpnl restriction sites of a pUC18 based plasmid containing sequences derived from another humanized heavy chain based on the COR framework (supra). This plasmid provides a signal sequence [SEQ ID NO: 17] and the remaining variable region sequence. Any eπors in the mapped sequence were corrected by PCR with mutagenic primers or by the addition of synthetic linkers into existing restriction sites.

The signal sequence and humanized heavy chain variable region were excised from the pUC based plasmid as a EcoRI-Apal fragment and ligated into the expression vector pCD that contained an IgG _] human constant region. The synthetic heavy chain variable region nucleotide and amino acid sequences are provided in Fig. 8 [SEQ ID NOs: 18 and 19]. The human framework residues are amino acids 1-30, 36-49, 66-97 and 109-1 19 of SEQ ID NO: 19. The amino acid sequences of the CDRs are identical to the murine 2B6 CDRs. The resulting expression vector, pCDIL5HZHC1.0, is shown in Fig. 10. Given the 2B6 light chain CDRs [Fig. 7 and SEQ ID NOs: 10, 1 1 and 12] and the framework sequence of the human antibody, a synthetic light chain variable region was made [SEQ ID NO: 20]. Four synthetic oligonucleotides coding for amino acids #27-#58 (KABAT numeration) [SEQ ID NOs:31 and 32] and amino acids #80-#109 [SEQ ID NOs: 33 and 34] of the humanized V _L with Sacl-Kpnl and Pstl-Hindlll ends respectively, were inserted into a pUCIS based plasmid containing sequences derived from another human light chain framework (B 17) (Marsh et al. Nuc. Acids Res.. 13:6531-6544 (1985)) which shares a high degree of homology to the LEN framework. This plasmid provides the remaining variable region sequence. Any errors in the mapped sequence and the single amino acid difference between the LEN and B 17 frameworks were corrected by PCR with mutagenic primers or by the addition of synthetic linkers into existing restriction sites.

The humanized light chain variable region was isolated from the pUC plasmid as a EcoRV-Narl fragment and ligated into the expression vector pCN that contained a signal sequence [SEQ ID NO 17] along with a kappa human constant region The synthetic light chain variable region nucleotide and ammo acid sequences are provided in Fig 9 [SEQ ID NOs 20 and 21 ] The human framework residues are ammo acids 1-23, 41-55, 63-94 and 104-113 of SEQ ID NO 21 The amino acid sequences of the CDRs are identical to the murine 2B6 CDRs However, the coding sequences for these CDRs differ from the murine 2B6 coding sequences to allow creation of restriction enzyme sites One of the resulting expression vectors, pCNLL5HZLCl 0, is shown in Fig I I These synthetic variable light and/or heavy chain sequences are employed in the construction of a humanized antibody

E Expression of Humanized MAb

The humanized heavy chain, derived from an IgG _] isotype, utilizes a synthetic heavy chain variable region as provided in SEQ ID NO 19 This synthetic VH containing the 2B6 heavy chain CDRs was designed and synthesized as described above

The humanized light chain, a human kappa chain, utilizes a synthetic light chain variable region as provided in SEQ ID NO 21 This synthetic V _L containing the 2B6 light chain CDRs was designed and synthesized as descπbed above The DNA fragments coding for the humanized variable regions were inserted into pUC19-based mammalian cell expression plasmids that utilize a signal sequence and contain CMV promoters and the human heavy chain or human light chain constant regions of the chimera produced in Example 5 below, by conventional methods (Maniatis et al , cited above) to yield the plasmids pCDLL5HZHCl 0 (heavy chain) [SEQ ID NO 49, see also FlG 10] and pCNIL5HZLCl 0 (light chain) [SEQ ID NO 50, see also FIG 11 ] The plasmids were co-transfected into COS cells and supematants assayed after three and five days, respectively, by the ELISA descπbed in Example 5 for the presence of human antibody The above example describes the preparation of an exemplary engineered antibody Similar procedures may be followed for the development of other engineered antibodies, using other antι-LL-5 antibodies (e g , 2F2, 2E3, 4A6, 5D3, 24G9, etc ) developed by conventional means F Purification Purification of CHO expressed chimeric and humanized 2B6 can be achieved by conventional protein A (or G) affinity chromatography followed by ion exchange and molecular sieve chromatography Similar processes have been successfully

employed for the purification to >95% purity of other mAbs (e g , to respiratory syncytial virus, ιnterleukιn-4 and malaria circumsporozoite antigens) G Additional Humanized mAbs and Expression Plasmids

Given the plasmid pCDIL5HZHCl 0 [SEQ ID NO 49] the expression plasmid ρCDIL5HZHC 1 1 was made that substitutes an Asparagine for Threonine at framework position 73 This was done by ligatmg a synthetic linker with EcoRV and Xhol ends [SEQ ID NO 51 and SEQ ID NO 52] into identically digested pCDLL5HZHCl 0 Similarly, the expression plasmid pCDLL5HZHCl 2 substitutes an Isoleucine for Valine at framework position 37 This was accomplished by hgating a synthetic linker with Hpal and Xbal ends [SEQ ID NO 53 and SEQ ID NO 54] into identically digested pCDIL5HZHCl 0 The expression plasmid pCDIL5HZHCl 3 was also made by hgating a synthetic linker with Hpal and Xbal ends [SEQ ID NO 53 and SEQ ID NO 54] into identically digested pCDIL5HZHCl 1

Given the pUC18 based plasmid described previously which contains DNA sequences of four synthetic oligonucleotides [SEQ ID NOs 31 , 32, 33 and 34], a humanized light chain variable region was made where framework position #15 is changed from a Leucine to Alanine This plasmid was digested with Nhel and Sad restriction endonucleases and a synthetic linker [SEQ ID NOs 55 and 56] was inserted An EcoRV-Narl fragment was then isolated and ligated into the identically digested expression vector pCNLL5HZLC 1 0 to create pCNLL5HZLCl 1

A synthetic vaπable region was made using the heavy chain framework regions obtained from immunoglobulin (NEW) (Saul et al. J Biol Chem 253 585-597(1978)) and the 2B6 heavy chain CDRs [Fig 7 and SEQ ID NOs 1-2] Framework ammo acids which might influence CDR presentation were identified and substitutions made using methods described previously Four overlapping synthetic oligonucleotides were generated [SEQ ID NOs 57, 58, 59 and 60] which, when annealed and extended, code for amino acids representing a signal sequence [SEQ ID NO 17] and a heavy chain variable region This synthetic gene was then amplified using PCR primers [SEQ ID NOs 63 and 64] and ligated as a BstXI-HindLTI restriction fragment into a pUC18 based plasmid containing sequences derived from another humanized heavy chain based on the COR framework A phenylalanine to tyrosine framework substitution was made at ammo acid position 91 (Kabat numbering system) (equivalent to position 94 of Figure 12) by inserting a synthetic oligonucleotide linker [SEQ ID NOs 75 and 76] into SacII and Kpnl restπction sites The resulting heavy chain vaπable region [Fig 12 and SEQ ID NOs. 61 , 62] is referred to as the NEWM humanized heavy chain

_{Λ IM Λt o} PCT/US97/10769 O 97/48418

Any errors in the mapped sequence were corrected by PCR with mutagenic primers or by the addition of synthetic linkers into existing restriction sites The signal sequence and humanized heavy chain vaπable region were excised from the pUC based plasmid as a EcoRI-Apal fragment and ligated into the expression vector pCD that contained a human IgGj constant region to create the plasmid pCDLL5NEWM The amino acid sequences of the CDRs are identical to the murine 2B6 heavy chain CDRs

A synthetic variable region was made using the light chain framework regions obtained from immunoglobulin (REI) (Palm et al, Hoppe-Seyler's Z Physiol Chem 356 167-191(1975)) and the 2B6 light chain CDRs [Fig 7 and SEQ ID NOs 10, 11 and 12] Framework amino acids which might influence CDR presentation were identified and substitutions made using methods described previously Four overlapping synthetic oligonucleotides were generated [SEQ ID NOs 65, 66, 67 and 68] which, when annealed and extended, code for amino acids representing a light chain variable region [Fig 13 and SEQ ID NOs 69, 70] refeπed to as the REI humanized light chain This synthetic gene was then amplified using PCR primers [SEQ ID NOs 71 and 72] and ligated as an EcoRI-Hindlll restriction fragment into pGEM-7Zf(+) (Promega Coφoration, Madison, WI)

Any eπors in the mapped sequence were corrected by PCR with mutagenic primers or by the addition of synthetic linkers into existing restriction sites The humanized light chain variable region was excised from the pGEM-7Zf(+) based plasmid as an EcoRV-Narl fragment and ligated into the expression vector pCN that contained a signal sequence [SEQ ID NO 17] along with a human Kappa constant region to create the plasmid pCNIL5REI The amino acid sequences of the CDRs are identical to the murine 2B6 light chain CDRs However, the coding sequences for these CDRs differ from the murine 2B6 coding sequences to allow creation of restriction enzyme sites These synthetic vaπable light and/or heavy chain sequences are employed in the construction of a humanized antibody

Given the pGEM-7Zf(+) based plasmid described above, a humanized light chain vaπable region can be made where framework position #15 is changed from a Valine to Alanine This plasmid may be digested with Nhel and Sad restπction endonucleases and a synthetic linker [SEQ ID NOs 73 and 74] is inserted An EcoRV-Narl fragment may then be isolated and ligated into the identically digested expression vector pCNLL5HZREI to create the plasmid pCNLL5REIvi5A

Example 5 - Construction of a Chimeric Antibody

DNA coding for ammo acids #9-#104 (KABAT numeration) of the murine mAb 2B6 heavy chain variable region was isolated as a Avall-Styl restriction fragment from a pGEM7Zf+ based PCR clone of cDNA generated from the 2B6 hybridoma cell line (see Example 4). The flanking heavy chain variable region sequences and a signal sequence [SEQ ID NO: 17] were provided by combining this fragment along with four small synthetic oligomer linkers [SEQ ID NOs: 35 and 36] [SEQ ID NOs: 37 and 38] into a pUC18 based plasmid digested with BstX 1 -Hindlll. A consensus of N-terminal amino acids deduced from closely related murine heavy chains were assigned for the first eight Vpj residues and are coded within SEQ ID NOs: 35 and 36. The deduced amino acid sequence of the heavy chain was verified by the sequencing of the first 15 N-terminal amino acids of the 2B6 heavy chain. An EcoRI-Apal fragment containing sequence for signal and VH regions was isolated and ligated into plasmid pCD that already encodes the human IgGl constant region.

DNA coding for amino acids #12-#99 (KABAT nomenclature) of the murine mAb 2B6 light chain variable region was isolated as a Ddel-Aval restriction fragment from a pGEM7Zf+ based PCR clone of cDNA generated from the 2B6 hybridoma cell line (see Example 4). The flanking light chain variable region sequences were provided by combining this fragment along with four small synthetic oligomer linkers [SEQ ID NOs: 39 and 40] [SEQ ID NOs: 41 and 42] into a pUC18 based plasmid digested with EcoRV-Hindlll. A consensus of N- terminal amino acids deduced from closely related murine light chains were assigned for the first eight VL residues and are coded within SEQ ID NOs: 39 and 40. The deduced amino acid sequence of the light chain was verified by the sequencing of the first 15 N-terminal amino acids of the 2B6 light chain. This variable region was then isolated as a EcoRV-Narl fragment and ligated into the expression vector pCN that already contains the human kappa region and a signal sequence.

Expression of a chimeric antibody was accomplished by co-transfection of the pCD and pCN based plasmids into COS cells. Culture supematants were collected three and five days later and assayed for immunoglobulin expression by ELISA described as follows: Each step except for the last is followed by PBS washes. Microtiter plates were coated overnight with 100 ng/50 ul/well of a goat antibody specific for the Fc region of human antibodies. The culture supematants

were added and incubated for 1 hour Horseradish peroxidase conjugated goat anti-human IgG antibody was then added and allowed to incubate for 1 hour This was followed by addition of AJ3TS peroxidase substrate (Kirkegaard & Perry Laboratories Inc , Gaithersburg, MD) After 1 hour incubation, the absorbance at 405 nm was read on a microtiter plate reader (Molecular Devices Coφoration, Menlo Park, CA) Expression of the chimeric antibody was detected In a similar ELISA, the COS cell supematants, containing the chimeric antibody, bound specifically to microtiter wells coated with human LL-5 protein This result confirmed that genes coding for an antibody to LL-5 had been synthesized and expressed

The above example describes the preparation of an exemplary engineered antibody Similar procedures may be followed for the development of other engineered antibodies, using other antι-IL-5 donor antibodies (e g , 2F2, 2E3, 4A6, 5D3, 24G9, etc ) developed by conventional means

Example 6 - Human IL-5/hIL-5 Receptor α-chain/MAb Complex A ELISA

The following antibodies were evaluated 24G9 (non-neutralizing), 4A6 (neutralizing) and 2B6 (neutralizing), at concentrations between 2ng/mJ - 32ng/ml Flat bottomed ELISA plate wells (Wallac) were coated overnight with lOOul

Protein-A (5ug/ml) at 4° C Following aspiration, wells were blocked with 200ul blocking buffer containing 1% BSA and incubated for 60 min at 37° C Plates were washed 4x and lOOul soluble LL-5Ralpha-Fc (see Johnson et al , (1995) J Biol Chem. 270 9459-9471) (2.25ug/ml) added to each well and incubated at 37° C for 30 min Plates were washed lx and increasing concentrations of recombinant human LL-5 added to each well. Following a 30 min incubation at 37° C, wells were washed once and lOOul antibody (2ug/ml) added and incubated for 60 mm at 37° C. Plates were washed 4x and lOOul biotin-labelled goat anti-mouse Ig or goat anti-rat Ig (Sigma) added to each well. Plates were incubated for 60 mm at 37° C and washed 4x. Europium-labelled streptavidin (Wallac) was diluted to 1.1000 in europium buffer (Wallac) and lOOul volumes added to each well. Plates were incubated for 30 min at 37° C and washed 6x Enhancer solution (Wallac) was added to each well and plates read using a 1234 Delphia research fluorometer.

There was a dose-dependent increase in binding of mAbs 24G9 and 4A6 to the EL-5/LL-5 receptor complex. No such increase was seen using mAb 2B6 MAb 2B6 inhibits binding of LL-5 to the IL-5Ralpha chain. In contrast neither 24G9 or 4A6 inhibited binding of LL-5 to the IL-5Ralpha chain. See Table II.

TABLE II

Binding to hLL-5/IL-5 Receptor α-chain Complex (Counts)

B. Optical Biosensor

( 1 ) MAb 4A6 was immobilized onto a BIAcore chip (see Example 2D) hLL-5 (25ul) was passed onto the 4A6 surface at a flow rate of 5ul/mm. LL-5 receptor α- chain (@ 15, 30, 60, 120nM, in 25ul) was then injected The kinetics of IL-5 receptor α-chain (Ra) in binding to 4A6/LL-5 complex can be calculated as- Kon=6.3xl0 ⁵ (/M/s), Koff=2.3xlO" ³ {Is). The kinetics for IL-5-IL-5Pα interaction is. Kon=7.5xl0 ⁵ (/M7s), Koff=2 8xlO ^"3 (/s) Thus, mAb 4A6 has no significant effect on the interaction between LL-5 and LL-5 receptor α-chain.

(2) hLL-5 was immobilized on a BIAcore chip. MAb 4A6 (25ul, 32ug/ml) was injected onto the surface, followed by injection of LL-5 receptor α-chain (20ul, 90nM). As a control, the same amount of LL-5 receptor α-chain was injected directly onto the LL-5 surface Pre-bmding of mAb 4A6 to hLL5 did not block binding of IL-5 receptor α-chain to hIL-5.

(3) Protein A was immobilized on a BIAcore chip. IL-5Ra-Fc (30ul, 20ug/ml) was injected onto the protein A surface. LL-5 (25ul, 80nM) was then captured by IL-5Rα- Fc, followed by binding of mAb 24G9 (25ul, 32ug/ml) MAb 24G9 bound to the hlL- 5/IL-5Rα complex. (4) MAb 24G9 was immobilized on a BIAcore chip. LL-5 was captured to the 24G9 surface, followed by injections of different mAbs. Mabs 2B6, TRFK5 and CMX5-2 bind to the LL-5/24G9 complex, while binding of mAb 4A6 to LL-5 was blocked. This indicates that 4A6 and 24G9 share binding epitopes.

C. Sedimentation Velocity

A three component mixture of hLL-5, mAb 4A6, and a soluble IL-5 receptor α-chain was analyzed by sedimentation velocity after 14 hours (20°C) A

complex corresponding to the same size as a soluble IL-5 receptor α-chain/hlL- 5/mAb complex was observed.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(l) APPLICANT Cook, Richard M.

Appelbaum, Edward R

(ii) TITLE OF INVENTION Improved Method for Treatment and Diagnosis of IL-5 Mediated Disorders

(in) NUMBER OF SEQUENCES. 76

(iv) CORRESPONDENCE ADDRESS (A) ADDRESSEE SmithKlme Beecham Corp /Corporate

(B) STREET P O Box 1539-UW2220

(C) CITY King of Prussia

(D) STATE Pennsylvania

(E) COUNTRY USA (F) ZIP 19406-0939

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE- Floppy disk

(B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS

(D) SOFTWARE Patentin Release #1 0, Version #1 30

(vi) CURRENT APPLICATION DATA. (A) APPLICATION NUMBER. (B) FILING DATE

(C) CLASSIFICATION

(Vll) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/470110 (B) FILING DATE 06-JUN-1995

(Vll) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER- US 08/467420

(B) FILING DATE 06-JUN-1995

(vii) PRIOR APPLICATION DATA (A) APPLICATION NUMBER: US 08/363131

(B) FILING DATE. 23-DEC-1994

(vill) ATTORNEY/AGENT INFORMATION: (A) NAME: Sutton, Jeffrey A (B) REGISTRATION NUMBER. 34,028

(C) REFERENCE/DOCKET NUMBER P50282-2

(ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE- 610-270-5024 (B) TELEFAX 610-270-5090

(2) INFORMATION FOR SEQ ID NO 1

(1) SEQUENCE CHARACTERISTICS

(A) LENGTH: 334 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE- DNA (genomic)

(ix) FEATURE. (A) NAME/KEY: mιsc_feature

(B) LOCATION: 1..334

(D) OTHER INFORMATION: /note= "First base corresponds to Kabat position 24"

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ACCTGGCCTG GTGGCGCCCT CACAGAGCCT GTCCATCACT TGCACTGTCT CTGGGTTTTC 60

ATTAACCAGC TATAGTGTAC ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT 120

GGGAGTAATA TGGGCTAGTG GAGGCACAGA TTATAATTCG GCTCTCATGT CCAGACTGAG 180

CATCAGCAAA GACAACTCCA AGAGCCAAGT TTTCTTAAAA CTGAACAGTC TGCAAACTGA 240

TGACACAGCC ATGTACTACT GTGCCAGAGA TCCCCCTTCT TCCTTACTAC GGCTTGACTA 300

CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCA 334

(2) INFORMATION FOR SEQ ID NO:2 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 315 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(IX) FEATURE: (A) NAME/KEY: mιsc_feature

(B) LOCATION: 1..315

(D) OTHER INFORMATION: /note= "First base corresponds to Kabat position 25"

( Xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

TCCTCCCTGA GTGTGTCAGC AGGAGAGAAG GTCACTATGA GCTGCAAGTC CAGTCAGAGT 60

CTGTTAAACA GTGGAAATCA AAAGAACTAC TTGGCCTGGT ACCAGCAGAA ACCAGGGCAG 120

CCTCCTAAAC TTTTGATCTA CGGGGCATCC ACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180

ACAGGCAGTG GATCTGGAAC CGATTTCACT CTTTCCATCA GCAGTGTGCA GGCTGAAGAC 240

CTGGCAGTTT ATTACTGTCA GAATGTTCAT AGTTTTCCAT TCACGTTCGG CTCGGGGACA 300

GAGTTGGAAA TAAAA 315

(2) INFORMATION FOR SEQ ID NO:3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 334 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

in) MOLECULE TYPE- DNA (genomic)

(ix) FEATURE: (A) NAME/KEY: mιsc_feature

(B) LOCATION: 1..334

(D) OTHER INFORMATION: /note= "First base corresponds to Kabat position 24"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

ACCTGGCCTG GTGGCGCCCT CACAGAGCCT GTCCATCACT TGCACTGTCT CTGGGTTTTC 60

ATTAACCAGT TATAGTGTAC ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT 120

GGGAGTAATA TGGGCTAGTG GAGGCACAGA TTATAATTCG GCTCTCATGT CCAGACTGAG 180

CATCAGCAAA GACAACTCCA AGAGCCAAGT TTTCTTAAAA CTGAACAGTC TGCGAACTGA 240

TGACACAGCC ATGTACTACT GTGCCAGAGA TCCCCCTTCT TCCTTACTAC GGCTTGACTA 300

CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCA 334

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 315 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(IX) FEATURE:

(A) NAME/KEY: mιsc_teature

(B) LOCATION: 1..315

(D) OTHER INFORMATION: /note= 'First base corresponds to Kabat 25"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

TCCTCCCTGA GTGTGTCAGC AGGAGAGAAG GTCACTATGA GCTGCAAGTC CAGTCAGAGT 60

CTATTAAACA GTGGAAATCA AAAGAACTAC TTGGCCTGGT ACCAACAGAA ACCAGGGCAG 120

CCTCCTAAAC TTTTGATCTA CGGGGCATCC ACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180

ACAGGCAGTG GATCTGGAAC CGATTTCACT CTTACCATCA GCAGTGTGCA GGCTGAAGAC 240

CTGGCAGTTT ATTACTGTCA GAATGATCAT AGTTTTCCAT TCACGTTCGG CTCGGGGACA 300

GAGTTGGAAA TAAAA 315

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 334 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

iii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE: (A) NAME/KEY: misc_feature

(B) LOCATION: 1..334

(D) OTHER INFORMATION. /note= "First base corresponds to Kabat position 24"

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 5

ACCTGGCCTG GTGGCGCCCT CACAGAGCCT GTCCATCACT TGCACTGTCT CTGGGTTTTC 60

ATTAACCAGC TATAGTGTAC ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT 120

GGGAGTAATC TGGGCTAGTG GAGGCACAGA TTATAATTCG GCTCTCATGT CCAGACTGAG 180

CATCAGCAAA GACAACTCCA AGAGCCAAGT TTTCTTAAAA CTGAACAGTC TGCAAACTGA 240

TGACGCAGCC ATGTACTACT GTGCCAGAGA TCCCCCTTTT TCCTTACTAC GGCTTGACTT 300

CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCA 334

(2) INFORMATION FOR SEQ ID NO:6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 315 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE DNA (genomic)

(ix) FEATURE (A) NAME/KEY mιsc_feature

(B) LOCATION 1 .315

(D) OTHER INFORMATION /note= "First base corresponds to Kabat position 25"

(xi) SEQUENCE DESCRIPTION SEQ ID NO ^• 6

TCCTCTCTGA GTGTGTCAGC AGGAGAGAAG GTCACTATGA GCTGCAAGTC CAGTCAGAGT 60

CTGTTAAACA GTGGAAATCA AAAAAACTAC TTGGCCTGGT ACCAGCAGAA ACCAGGGCAG 120

CCTCCTAAAC TTTTGATCTA CGGGGCATCC ACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180

ACAGGCAGTG GATCTGGAAC CGATTTCACT CTTACCATCA GCAGTGTGCA GGCTGAAGAC 240

CTGGCAGTTT ATTACTGTCA GAATGATCAT AGTTTTCCAT TCACGTTCGG CTCGGGGACA 300

GAGTTGGAAA TAAAA 315

(2) INFORMATION FOR SEQ ID NO 7

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 5 ammo acids

(B) TYPE amino acid (C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7 -

Ser Tyr Ser Val Hi s

1 5

(2) INFORMATION FOR SEQ ID NO:8:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Val lie Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met Ser 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:9:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO.10

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 17 ammo acids

(C) STRANDEDNESS: Single (D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(Xl) SEQUENCE DESCRIPTION SEQ ID NO 10

Lys Ser Ser Gin Ser Leu Leu Asn Ser Gly Asn Gin Lys Asn Tyr Leu 1 5 10 15

Ala

(2) INFORMATION FOR SEQ ID NO: 11

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 7 ammo acids

(C) STRANDEDNESS: single (D) TOPOLOGY linear

(ii) MOLECULE TYPE, protein

(Xl) SEQUENCE DESCRIPTION. SEQ ID NO: 11:

Gly Ala Ser Thr Arg Glu Ser 1 5

( 2 ) INFORMATION FOR SEQ ID NO : 12 .

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH- 9 ammo acids

(C) STRANDEDNESS single (D) TOPOLOGY linear

(n) MOLECULE TYPE- protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO 12

Gin Asn Val His Ser Phe Pro Phe Thr 1 5

(2) INFORMATION FOR SEQ ID NO: 13.

(l) SEQUENCE CHARACTERISTICS- (A) LENGTH 9 ammo acids

(C) STRANDEDNESS single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE: protein

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 13

Gin Asn Asp His Ser Phe Pro Phe Thr 1 5

(2) INFORMATION FOR SEQ ID NO.14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH 11 amino acids (B) TYPE- amino acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(XI) SEQUENCE DESCRIPTION SEQ ID NO:14:

Asp Pro Pro Phe Ser Leu Leu Arg Leu Asp Phe 1 5 10

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO.15:

Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin 1 5 10 15

Ser Leu Ser lie Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30

Ser Val His Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45

Gly Val lie Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60

Ser Arg Leu Ser lie Ser Lys Asp Asn Ser Lys Ser Gin Val Phe Leu 65 70 75 80

Lys Leu Asn Ser Leu Gin Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Gin Gly 100 105 110

Thr Thr Leu Thr Val Ser Ser 115

(2) INFORMATION FOR SEQ ID NO: 16.

(l) SEQUENCE CHARACTERISTICS (A) LENGTH 113 amino acids

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE protein

(Xl ) SEQUENCE DESCRIPTION SEQ ID NO- 16

Asp lie Val Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30

Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu He Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80

He Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn

85 90 95

val His Ser Phe Pro Phe Thr Phe Gly Ser Gly Thr Glu Leu Glu He 100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO.17-

(l) SEQUENCE CHARACTERISTICS (A) LENGTH. 60 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 17

ATGGTGTTGC AGACCCAGGT CTTCATTTCT CTGTTGCTCT GGATCTCTGG TGCCTACGGG 60

(2) INFORMATION FOR SEQ ID NO: 18

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 357 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lΣ

CAGGTTACCC TGCGTGAATC CGGTCCGGCA CTAGTTAAAC CGACCCAGAC CCTGACGTTA 60

ACCTGCACCG TCTCCGGTTT CTCCCTGACG AGCTATAGTG TACACTGGGT CCGTCAGCCG 120

CCGGGTAAAG GTCTAGAATG GCTGGGTGTA ATATGGGCTA GTGGAGGCAC AGATTATAAT 180

TCGGCTCTCA TGTCCCGTCT GTCGATATCC AAAGACACCT CCCGTAACCA GGTTGTTCTG 240

ACCATGACTA ACATGGACCC GGTTGACACC GCTACCTACT ACTGCGCTCG AGATCCCCCT 300

TCTTCCTTAC TACGGCTTGA CTACTGGGGT CGTGGTACCC CAGTTACCGT GAGCTCA 357

(2) INFORMATION FOR SEQ ID NO: 19-

(ii SEQUENCE CHARACTERISTICS

(A) LENGTH 119 amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE- protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO 19

Gin Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gin

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Ser Val His Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val He Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60

Ser Arg Leu Ser He Ser Lys Asp Thr Ser Arg Asn Gin Val Val Leu 65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95

Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Arg Gly 100 105 110

Thr Pro Val Thr Val Ser Ser 115

(2) INFORMATION FOR SEQ ID NO:20:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GATATCGTGA TGACCCAGTC TCCAGACTCG CTAGCTGTGT CTCTGGGCGA GAGGGCCACC 60

ATCAACTGCA AGAGCTCTCA GAGTCTGTTA AACAGTGGAA ATCAAAAGAA CTACTTGGCC 120

TGGTATCAGC AGAAACCCGG GCAGCCTCCT AAGTTGCTCA TTTACGGGGC GTCGACTAGG 180

GAATCTGGGG TACCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC 240

ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTATACTACT GTCAGAATGT TCATAGTTTT 300

CCATTCACGT TCGGCGGAGG GACCAAGTTG GAGATCAAA 339

(2) INFORMATION FOR SEQ ID NO: 21.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE, protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO.21

Asp He Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15

Glu Arg Ala Thr He Asn Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu He Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

He Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95

Val His Ser Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu He

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO 22

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS. single (D) TOPOLOGY linear

(ii) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO.22.

GTACATATGC AAGGCTTACA ACCACAATC 29

(2) INFORMATION FOR SEQ ID NO 23

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 32 base pairs

(B) TYPE nucleic acid (C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE. DNA (genomic)

(Xl) SEQUENCE DESCRIPTION SEQ ID NO:23.

GGACAGGGCT TACTAGTGGG CCCTCTGGGC TC 32

(2) INFORMATION FOR SEQ ID NO 24-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

AGGTSMARCT KTCTCGAGTC WGG 23

(2) INFORMATION FOR SEQ ID NO:25:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 36 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

CTAACACTCA TTCCTGTTGA AGCTCTTGAC AATGGG 36

(2) INFORMATION FOR SEQ ID NO: 26:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

Iii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:26:

CCAGATGTGA GCTCGTGATG ACCCAGACTC CA 32

(2) INFORMATION FOR SEQ ID NO-27:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 140 base pairs

(B) TYPE, nucleic acid (C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27.

AACCTGCACC GTCTCCGGTT TCTCCCTGAC GAGCTATAGT GTACACTGGG TCCGTCAGCC 60

GCCGGGTAAA GGTCTAGAAT GGCTGGGTGT AATATGGGCT AGTGGAGGCA CAGATTATAA 120

TTCGGCTCTC ATGTCCCGTC 140

(2) INFORMATION FOR SEQ ID NO: 28:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 149 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE- DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

ATATCGACAG ACGGGACATG AGAGCCGAAT TATAATCTGT GCCTCCACTA GCCCATATTA 60

CACCCAGCCA TTCTAGACCT TTACCCGGCG GCTGACGGAC CCAGTGTACA CTATAGCTCG 120

TCAGGGAGAA ACCGGAGACG GTGCAGGTT 149

(2) INFORMATION FOR SEQ ID NO:29:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 139 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY, linear

(il) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.29:

TGTCGATATC CAAAGACACC TCCCGTAACC AGGTTGTTCT GACCATGACT AACATGGACC 60

CGGTTGACAC CGCTACCTAC TACTGCGCTC GAGATCCCCC TTCTTCCTTA CTACGGCTTG 120

ACTACTGGGG TCGTGGTAC 139

(2) INFORMATION FOR SEQ ID NO:30:

(ι) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 126 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

CACGACCCCA GTAGTCAAGC CGTAGTAAGG AAGAAGGGGG ATCTCGAGCG CAGTAGTAGG 60

TAGCGGTGTC AACCGGGTCC ATGTTAGTCA TGGTCAGAAC AACCTGGTTA CGGGAGGTGT 120

CTTTGG 126

(2) INFORMATION FOR SEQ ID NO 31.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 117 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION. SEQ ID NO: 31:

CTCAGAGTCT GTTAAACAGT GGAAATCAAA AGAACTACTT GGCCTGGTAT CAGCAGAAAC 60

CCGGGCAGCC TCCTAAGTTG CTCATTTACG GGGCGTCGAC TAGGGAATCT GGGGTAC 117

(2) INFORMATION FOR SEQ ID NO:32:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 117 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.32:

CCCAGATTCC CTAGTCGACG CCCCGTAAAT GAGCAACTTA GGAGGCTGCC CGGGTTTCTG 60

CTGATACCAG GCCAAGTAGT TCTTTTGATT TCCACTGTTT AACAGACTCT GAGAGCT 117

(2) INFORMATION FOR SEQ ID NO.33

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 102 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GCTGAAGATG TGGCAGTATA CTACTGTCAG AATGTTCATA GTTTTCCATT CACGTTCGGC 60

GGAGGGACCA AGTTGGAGAT CAAACGTACT GTGGCGGCGC CA 102

(2) INFORMATION FOR SEQ ID NO:34:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 111 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:34:

AGCTTGGCGC CGCCACAGTA CGTTTGATCT CCAACTTGGT CCCTCCGCCG AACGTGAATG 60

GAAAACTATG AACATTCTGA CAGTAGTATA CTGCCACATC TTCAGCCTGC A 111

(2) INFORMATION FOR SEQ ID NO: 35

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 82 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 35

ATGGTGTTGC AGACCCAGGT CTTCATTTCT CTGTTGCTCT GGATCTCTGG TGCCTACGGG 60

CAGGTTCAAC TGAAAGAGTC AG 82

(2) INFORMATION FOR SEQ ID NO: 36 :

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

GTCCTGACTC TTTCAGTTGA ACCTGCCCGT AGGCACCAGA GATCCAGAGC AACAGAGAAA 60

TGAAGACCTG GGTCTGCAAC ACCATGTTG 89

(2) INFORMATION FOR SEQ ID NO:37:

'ι) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:37:

CAAGGCACCA CTCTCACAGT CTCCTCAGCT AGTACGAAGG GCCCA 45

(2) INFORMATION FOR SEQ ID NO:38:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 38

AGCTTGGGCC CTTCGTACTA GCTGAGGAGA CTGTGAGTGG TGC 43

(2) INFORMATION FOR SEQ ID NO 39

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 28 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 39

ATCGTGATGA CCCAGTCTCC ATCCTCCC 28

(2) INFORMATION FOR SEQ ID NO 40

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH 31 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS single (D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 40

TCAGGGAGGA TGGAGACTGG GTCATCACGA T 31

(2) INFORMATION FOR SEQ ID NO: 41:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE- DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO : 4 i

TCGGGGGACA GAGTTGGAAA TAAAACGTAC TGTGGCGGCG CCA 43

(2) INFORMATION FOR SEQ ID NO: 42.

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH- 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single (D) TOPOLOGY linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

AGCTTGGCGC CGCCACAGTA CGTTTTATTT CCAACTCTGT CC 42

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:43:

CTAGCCACCA CCACCACCAC CACTAA 26

(2) INFORMATION FOR SEQ ID NO:44:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

CTAGTTAGTG GTGGTGGTGG TGGTGG 26

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : protein

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 45 :

Glu Leu Val Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin

35 40 45

Pro Pro Lys Leu Leu He Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

He Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn 85 90 95

Asp His Ser Tyr Pro Phe Thr Pne Gly Ser Gly Thr Lys Leu Glu He

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:46.

Glu Leu Val Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30

Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu He Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

He Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn 85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu He 100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO.47.

(l) SEQUENCE CHARACTERISTICS. (A) LENGTH: 9 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY linear

(11) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:47.

Gin Asn Asp His Ser Tyr Pro Pne Thr 1 5

(2) INFORMATION FOR SEQ ID NO:48-

(l) SEQUENCE CHARACTERISTICS (A) LENGTH 9 ammo acids

(B) TYPE, amino acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE- protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO.4?

Gin Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5

(2) INFORMATION FOR SEQ ID NO:49

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6285 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: circular

(ll) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG AATAGCTCAG 60

AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA TGCATGGGGC 120

GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGG GCGGAGTTAG GGGCGGGACT 180

ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 240

GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT TCTGCCTGCT 300

GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACAT TCCACAGAAT TAATTCCCGG 360

GGATCGATCC GTCGACGTAC GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT 420

CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 480

CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 540

ATAGGGACTT TCCATTGACG TCAATGGGTG GACTATTTAC GGTAAACTGC CCACTTGGCA 600

GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 660

CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 720

TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 780

GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 840

TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 900

ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TGGGTACGTG 960

AACCGTCAGA TCGCCTGGAG ACGCCATCGA ATTCGAGGAC GCCAGCAACA TGGTGTTGCA 1020

GACCCAGGTC TTCATTTCTC TGTTGCTCTG GATCTCTGGT GCCTACGGGC AGGTTACCCT 1080

GCGTGAATCC GGTCCGGCAC TAGTTAAACC GACCCAGACC CTGACGTTAA CCTGCACCGT 1140

CTCCGGTTTC TCCCTGACGA GCTATAGTGT ACACTGGGTC CGTCAGCCGC CGGGTAAAGG 12D0

TCTAGAATGG CTGGGTGTAA TATGGGCTAG TGGAGGCACA GATTATAATT CGGCTCTCAT 1260

GTCCCGTCTG TCGATATCCA AAGACACCTC CCGTAACCAG GTTGTTCTGA CCATGACTAA 1320

CATGGACCCG GTTGACACCG CTACCTACTA CTGCGCTCGA GATCCCCCTT CTTCCTTACT 1380

ACGGCTTGAC TACTGGGGTC GTGGTACCCC AGTTACCGTG AGCTCAGCTA GTACCAAGGG 1440

CCCATCGGTC TTCCCCCTGG CACCCTCCTC CAAGAGCACC TCTGGGGGCA CAGCGGCCCT 1500

GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA ACTCAGGCGC 1560

CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG TCCTCAGGAC TCTACTCCCT 1620

CAGCAGCGTG GTGACCGTGC CCTCCAGCAG CTTGGGCACC CAGACCTACA TCTGCAACGT 1680

GAATCACAAG CCCAGCAACA CCAAGGTGGA CAAGAGAGTT GAGCCCAAAT CTTGTGACAA 1740

AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT 1800

CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT 1860

GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT 1920

GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT 1980

GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA 2040

GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA 2100

GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA 2160

GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA 2220

GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG 2280

CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT 2340

CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC 2400

CCTGTCTCCG GGTAAGTGAG TGTAGTCTAG ATCTACGTAT GATCAGCCTC GACTGTGCCT 2460

TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT 2520

GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG 2580

TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 2640

AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGAAC CAGCTGGGGC TCGACAGCGC 2700

TGGATCTCCC GATCCCCAGC TTTGCTTCTC AATTTCTTAT TTGCATAATG AGAAAAAAAG 2760

GAAAATTAAT TTTAACACCA ATTCAGTAGT TGATTGAGCA AATGCGTTGC CAAAAAGGAT 2820

GCTTTAGAGA CAGTGTTCTC TGCACAGATA AGGACAAACA TTATTCAGAG GGAGTACCCA 2880

GAGCTGAGAC TCCTAAGCCA GTGAGTGGCA CAGCATTCTA GGGAGAAATA TGCTTGTCAT 2940

CACCGAAGCC TGATTCCGTA GAGCCACACC TTGGTAAGGG CCAATCTGCT CACACAGGAT 3000

AGAGAGGGCA GGAGCCAGGG CAGAGCATAT AAGGTGAGGT AGGATCAGTT GCTCCTCACA 3060

TTTGCTTCTG ACATAGTTGT GTTGGGAGCT TGGATAGCTT GGACAGCTCA GGGCTGCGAT 3120

TTCGCGCCAA ACTTGACGGC AATCCTAGCG TGAAGGCTGG TAGGATTTTA TCCCCGCTGC 3180

CATCATGGTT CGACCATTGA ACTGCATCGT CGCCGTGTCC CAAAATATGG GGATTGGCAA 3240

GAACGGAGAC CTACCCTGGC CTCCGCTCAG GAACGAGTTC AAGTACTTCC AAAGAATGAC 3300

CACAACCTCT TCAGTGGAAG GTAAACAGAA TCTGGTGATT ATGGGTAGGA AAACCTGGTT 3360

CTCCATTCCT GAGAAGAATC GACCTTTAAA GGACAGAATT AATATAGTTC TCAGTAGAGA 3420

ACTCAAAGAA CCACCACGAG GAGCTCATTT TCTTGCCAAA AGTTTGGATG ATGCCTTAAG 3480

ACTTATTGAA CAACCGGAAT TGGCAAGTAA AGTAGACATG GTTTGGATAG TCGGAGGCAG 3540

TTCTGTTTAC CAGGAAGCCA TGAATCAACC AGGCCACCTT AGACTCTTTG TGACAAGGAT 3600

CATGCAGGAA TTTGAAAGTG ACACGTTTTT CCCAGAAATT GATTTGGGGA AATATAAACT 3660

TCTCCCAGAA TACCCAGGCG TCCTCTCTGA GGTCCAGGAG GAAAAAGGCA TCAAGTATAA 3720

GTTTGAAGTC TACGAGAAGA AAGACTAACA GGAAGATGCT TTCAAGTTCT CTGCTCCCCT 3780

CCTAAAGCTA TGCATTTTTA TAAGACCATG GGACTTTTGC TGGCTTTAGA TCAGCCTCGA 3840

CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC 3900

TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC 3960

TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT 4020

GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGGAACCA GCTGGGGCTC 4080

GATCGAGTGT ATGACTGCGG CCGCGATCCC GTCGAGAGCT TGGCGTAATC ATGGTCATAG 4140

CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC 4200

ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC 4260

TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA 4320

CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG 4380

CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG 4440

TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG 4500

GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC 4560

GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA 4620

TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT 4680

ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA ATGCTCACGC 4740

TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC 4800

CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA 4860

AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT 4920

GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA 4980

GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT 5040

TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT 5100

ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT 5160

CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC 5220

ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA 5280

ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA 5340

TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC 5400

TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT 5460

TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA 5520

TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT 5580

AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT 5640

GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG 5700

TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC 5760

GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC 5820

GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG 5880

CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA 5940

ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA 6000

CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT 6060

TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG 6120

GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA 6180

AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT 6240

AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCT 6285

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5703 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(11) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION. SEQ ID NO: 50:

GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG AATAGCTCAG 60

AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA TGCATGGGGC 120

GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGG GCGGAGTTAG GGGCGGGACT 180

ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 240

GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT TCTGCCTGCT 300

GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACAT TCCACAGAAT TAATTCCCGG 360

GGATCGATCC GTCGACGTAC GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT 420

CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 480

CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 540

ATAGGGACTT TCCATTGACG TCAATGGGTG GACTATTTAC GGTAAACTGC CCACTTGGCA 600

GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 660

CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 720

TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 780

GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 840

TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 900

ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TGGGTACGTG 960

AACCGTCAGA TCGCCTGGAG ACGCCATCGA ATTCATTGAT AGGATCCAGC AAGATGGTGT 1020

TGCAGACCCA GGTCTTCATT TCTCTGTTGC TCTGGATCTC TGGTGCCTAC GGGGATATCG 1080

TGATGACCCA GTCTCCAGAC TCGCTAGCTG TGTCTCTGGG CGAGAGGGCC ACCATCAACT 1140

GCAAGAGCTC TCAGAGTCTG TTAAACAGTG GAAATCAAAA GAACTACTTG GCCTGGTATC 1200

AGCAGAAACC CGGGCAGCCT CCTAAGTTGC TCATTTACGG GGCGTCGACT AGGGAATCTG 1260

GGGTACCTGA CCGATTCAGT GGCAGCGGGT CTGGGACAGA TTTCACTCTC ACCATCAGCA 1320

GCCTGCAGGC TGAAGATGTG GCAGTATACT ACTGTCAGAA TGTTCATAGT TTTCCATTCA 1380

CGTTCGGCGG AGGGACCAAG TTGGAGATCA AACGTACTGT GGCGGCGCCA TCTGTCTTCA 1440

TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA 1500

ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG 1560

GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC AGCCTCAGCA 1620

GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA 1680

CCCATCAGGG CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAATTCT 1740

AGATCCGTTA TCTACGTATG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT 1800

GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 1860

TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 1920

GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 1980

GCGGTGGGCT CTATGGAACC AGCTGGGGCT CGACAGCTCG AGCTAGCTTT GCTTCTCAAT 2040

TTCTTATTTG CATAATGAGA AAAAAAGGAA AATTAATTTT AACACCAATT CAGTAGTTGA 2100

TTGAGCAAAT GCGTTGCCAA AAAGGATGCT TTAGAGACAG TGTTCTCTGC ACAGATAAGG 2160

ACAAACATTA TTCAGAGGGA GTACCCAGAG CTGAGACTCC TAAGCCAGTG AGTGGCACAG 2220

CATTCTAGGG AGAAATATGC TTGTCATCAC CGAAGCCTGA TTCCGTAGAG CCACACCTTG 2280

GTAAGGGCCA ATCTGCTCAC ACAGGATAGA GAGGGCAGGA GCCAGGGCAG AGCATATAAG 2340

GTGAGGTAGG ATCAGTTGCT CCTCACATTT GCTTCTGACA TAGTTGTGTT GGGAGCTTGG 2400

ATCGATCCAC CATGGTTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG 2460

AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT 2520

TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC 2580

TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT 2640

GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG 2700

TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG 2760

CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG 2820

CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG 2880

ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC 2940

GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA 3000

TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC 3060

GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG 3120

CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT 3180

ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG TTCGAAATGA CCGACCAAGC 3240

GACGCCCAAC CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG 3300

CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT 3360

GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA 3420

TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC 3480

CAAACTCATC AATGTATCTT ATCATGTCTG GATCGCGGCC GCGATCCCGT CGAGAGCTTG 3540

GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC 3600

AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC 3660

ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG 3720

CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT 3780

TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 3840

TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA 3900

GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT 3960

AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC 4020

CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 4080

GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 4140

CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 4200

GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 4260

CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 4320

ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 4380

GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 4440

AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 4500

GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 4560

TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 4620

TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 4680

TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 4740

ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 4800

ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 4860

CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 4920

AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 4980

GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG 5040

GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 5100

GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 5160

GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 5220

CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 5280

TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT 5340

ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 5400

AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 5460

AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 5520

CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 5580

CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 5640

GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 5700

CCT 5703

(?) INFORMATION FOR SEQ ID NO 51

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 81 base pairs

(B) TYPE nucleic acid (C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO.51

ATCCAAAGAC AACTCCCGTA ACCAGGTTGT TCTGACCATG ACTAACATGG ACCCGGTTGA 60

CACCGCTACC TACTACTGCG C 81

(2) INFORMATION FOR SEQ ID NO:52.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH 85 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

TCGAGCGCAG TAGTAGGTAG CGGTGTCAAC CGGGTCCATG TTAGTCATGG TCAGAACAAC 60

CTGGTTACGG GAGTTGTCTT TGGAT

(2) INFORMATION FOR SEQ ID NO: 53

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single (D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

AACCTGCACC GTCTCCGGTT TCTCCCTGAC GAGCTATAGT GTACACTGGA TCCGTCAGCC 60

GCCGGGTAAA GGT 73

(2) INFORMATION FOR SEQ ID NO: 54:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

CTAGACCTTT ACCCGGCGGC TGACGGATCC AGTGTACACT ATAGCTCGTC AGGGAGAAAC 60

CGGAGACGGT GCAGGTT 77

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

CTAGCTGTGT CAGCTGGCGA GAGGGCCACC ATCAACTGCA AGAGCT 46

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56

CTTGCAGTTG ATGGTGGCCC TCTCGCCAGC TGACACAG 38

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 140 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

I n ) MOLECULE TYPE : DNA ( genomi c )

( XI ) SEQUENCE DESCRI PTION : SEQ ID NO : 57 :

TTCGAGGACG CCAGCAACAT GGTGTTGCAG ACCCAGGTCT TCATTTCTCT GTTGCTCTGG 60

ATCTCTGGTG CCTACGGGCA GGTCCAACTG CAGGAGAGCG GTCCAGGTCT TGTGAGACCT 120

AGCCAGACCC TGAGCCTGAC 140

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

GTGCCTCCAC TAGCCCATAT TACTCCAAGC CACTCTAGAC CTCGTCCAGG TGGCTGTCTC 60

ACCCAGTGTA CACTATAGCT GGTGAGGGAG AAGCCCGAGA CGGTGCAGGT CAGGCTCAGG 120

GTCTGGCTAG GTCTCACA 138

(2) INFORMATION FOR SEQ ID NO: 59.

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH- 143 base pairs (B) TYPE nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY- linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION. SEQ ID NO:59

GGCTTGGAGT AATATGGGCT AGTGGAGGCA CAGATTATAA TTCGGCTCTC ATGTCCAGAC 60

TGAGTATACT GAAAGACAAC AGCAAGAACC AGGTCAGCCT GAGACTCAGC AGCGTGACAG 120

CCGCCGACAC CGCGGTCTAT TTC 143

(2) INFORMATION FOR SEQ ID NO:60:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 136 base pairs (B) TYPE, nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

In) MOLECULE TYPE DNA (genomic)

(XI) SEQUENCE DESCRIPTION. SEQ ID NO: 60:

CCAGTGCCAA GCTTGGGCCC TTGGTGGAGG CGCTCGAGAC GGTGACCGTG GTACCTTGTC 60

CCCAGTAGTC AAGCCGTAGT AAGGAAGAAG GGGGATCTCG AGCACAGAAA TAGACCGCGG 120

TGTCGGCGGC TGTCAC 136

(2) INFORMATION FOR SEQ ID NO 61

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 357 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS double (D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(Xl) SEQUENCE DESCRIPTION SEQ ID NO 61

CAGGTCCAAC TGCAGGAGAG CGGTCCAGGT CTTGTGAGAC CTAGCCAGAC CCTGAGCCTG 60

ACCTGCACCG TCTCGGGCTT CTCCCTCACC AGCTATAGTG TACACTGGGT GAGACAGCCA 120

CCTGGACGAG GTCTAGAGTG GCTTGGAGTA ATATGGGCTA GTGGAGGCAC AGATTATAAT 180

TCGGCTCTCA TGTCCAGACT GAGTATACTG AAAGACAACA GCAAGAACCA GGTCAGCCTG 240

AGACTCAGCA GCGTGACAGC CGCCGACACC GCGGTCTATT ACTGTGCTCG GGATCCCCCT 300

TCTTCCTTAC TACGGCTTGA CTACTGGGGA CAAGGTACCA CGGTCACCGT CTCGAGC 357

(2) INFORMATION FOR SEQ ID NO: 62:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 119 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE- protein

(xi) SEQUENCE DESCRIPTION- SEQ ID NO-62.

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Ser Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp Leu 35 40 45

Gly Val He Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60

Ser Arg Leu Ser He Leu Lys Asp Asn Ser Lys Asn Gin Val Ser Leu 65 70 75 80

Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Gin Gly

100 105 110

Thr Thr Val Thr Val Ser Ser 115

(2) INFORMATION FOR SEQ ID NO:63

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH 28 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS. single (D) TOPOLOGY linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION. SEQ ID NO- 63:

AGGACGCCAG CAACATGGTG TTGCAGAC 28

(2) INFORMATION FOR SEQ ID NO.64

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 36 base pairs

(B) TYPE- nucleic acid (C) STRANDEDNESS. single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

TGCCAAGCTT GGGCCCTTGG TGGAGGCGCT CGAGAC 36

(2) INFORMATION FOR SEQ ID NO:65:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 121 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

In) MOLECULE TYPE: DNA (genomic)

(xi ) SEQUENCE DESCRIPTION SEQ ID NO: 65

GACCATGATT ACGAATTCGT AGTCGGATAT CGTGATGACC CAGAGCCCAA GCAGCCTGAG 60

CGCTAGCGTG GGTGACAGAG TGACCATCAC CTGTAAGAGC TCTCAGAGTC TGTTAAACAG 120

121

(2) INFORMATION FOR SEQ ID NO: 66

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 116 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. single (D) TOPOLOGY linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66":

AGATTCCCTA GTCGATGCCC CGTAGATCAG CAGCTTTGGA GCCTTACCGG GTTTCTGCTG 60

ATACCAGGCC AAGTAGTTCT TTTGATTTCC ACTGTTTAAC AGACTCTGAG AGCTCT 116

(2) INFORMATION FOR SEQ ID NO: 67:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH 116 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS- Single

(D) TOPOLOGY linear

(11) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 67

TCTACGGGGC ATCGACTAGG GAATCTGGGG TACCAGATAG ATTCAGCGGT AGCGGTAGCG 60

GAACCGACTT CACCTTCACC ATCAGCAGCC TGCAGCCAGA GGACATCGCC ACCTAC 116

(2) INFORMATION FOR SEQ ID NO 68

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 117 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION. SEQ ID NO 68

TCGATGCCAA GCTTGGCGCC GCCACAGTAC GTTTGATCTC CACCTTGGTC CCTTGTCCGA 60

ACGTGAATGG AAAACTATGA ACATTCTGGC AGTAGTAGGT GGCGATGTCC TCTGGCT 117

(2) INFORMATION FOR SEQ ID NO: 69

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

GATATCGTGA TGACCCAGAG CCCAAGCAGC CTGAGCGCTA GCGTGGGTGA CAGAGTGACC 60

ATCACCTGTA AGAGCTCTCA GAGTCTGTTA AACAGTGGAA ATCAAAAGAA CTACTTGGCC 120

TGGTATCAGC AGAAACCCGG TAAGGCTCCA AAGCTGCTGA TCTACGGGGC ATCGACTAGG 180

GAATCTGGGG TACCAGATAG ATTCAGCGGT AGCGGTAGCG GAACCGACTT CACCTTCACC 240

ATCAGCAGCC TGCAGCCAGA GGACATCGCC ACCTACTACT GCCAGAATGT TCATAGTTTT 300

CCATTCACGT TCGGACAAGG GACCAAGGTG GAGATCAAA 339

(2) INFORMATION FOR SEQ ID NO:70:

<l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 ammo acids (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( XI ) SEQUENCE DESCRI PTION : SEQ ID NO : 70 :

Asp He Val Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr He Thr Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys 35 40 45

Ala Pro Lys Leu Leu He Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 65 70 75 80

He Ser Ser Leu Gin Pro Glu Asp He Ala Thr Tyr Tyr Cys Gin Asn 85 90 95

Val His Ser Phe Pro Phe Thr Phe Gly Gin Gly Thr Lys Val Glu He

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 71

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 71:

GATTACGAAT TCGTAGTCGG ATAT 24

(2) INFORMATION FOR SEQ ID NO:72:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi ) SEQUENCE DESCRIPTION SEQ ID NO.72

TGCCAAGCTT GGCGCCGCCA CAGT 24

(2) INFORMATION FOR SEQ ID NO:73-

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE- nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

CTAGTGCGGG TGACCGAGTG ACCATCACCT GTAAGAGCT 39

(2) INFORMATION FOR SEQ ID NO:74:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 31 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 74

CTTACAGGTG ATGGTCACTC GGTCACCCGC A 31

(2) INFORMATION FOR SEQ ID NO.75

(l) SEQUENCE CHARACTERISTICS. (A) LENGTH 66 base pairs (B) TYPE, nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 75

GGTCTATTAC TGTGCTCGGG ATCCCCCTTC TTCCTTACTA CGGCTTGACT ACTGGGGACA 60

AGGTAC 66

(2) INFORMATION FOR SEQ ID NO:76:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 64 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-76-

CTTGTCCCCA GTAGTCAAGC CGTAGTAAGG AAGAAGGGGG ATCCCGAGCA CAGTAATAGA 60

CCGC 64