Intracellular domain of the her-2/neu protein for preventionor treatment of malignancies

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Description

INTRACELLULAR DOMAIN OF THE HER-2/NEU PROTEIN FOR PREVENTION OR TREATMENT OF MALIGNANCIES

Technical Field

The present invention is generally directed toward polypeptides, and nucleic acid molecules encoding such polypeptides, for eliciting or enhancing an immune response to HER-2/neu protein, including for use in the treatment of malignancies in which the HER-2/neu oncogene is associated.

Background of the Invention

Despite enormous investments of financial and human resources, cancer remains one of the major causes of death. For example, cancer is the leading cause of death in women between the ages of 35 and 74. Breast cancer is the most common malignancy in women and the incidence for developing breast cancer is on the rise. One in nine women will be diagnosed with the disease. Standard approaches to cure breast cancer have centered around a combination of surgery, radiation and chemotherapy. These approaches have resulted in some dramatic successes in certain malignancies. However, these approaches have not been successful for all malignancies and breast cancer is most often incurable when attempting to treat beyond a certain stage. Alternative approaches to prevention and therapy are necessary.

A common characteristic of malignancies is uncontrolled cell growth. Cancer cells appear to have undergone a process of transformation from the normal phenotype to a malignant phenotype capable of autonomous

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PCT/US96/01689 growth. Amplification and overexpression of somatic cell genes is considered to be a common primary event that results in the transformation of normal cells to malignant cells

The malignant phenotypic characteristics encoded by the to the oncogenic progeny of

Ongoing genes are passed on during cell division the transformed cells.

research involving oncogenes has identified at least forty oncogenes operative in malignant cells and different location certain regard, protein degree responsible for, or associated with,

Oncogenes have groups based of their gene by the oncogene)

Oncogenes are on the products believed aspects of normal cellular the HER-2/neu oncogene is a putative function or to be essential for physiology.

In this member of the tyrosine kinase family of oncogenes and of homology with the epidermal receptor .

growth induce result shares growth a high factor

HER-2/neu presumably plays a and/or differentiation.

role in cell

HER-2/neu appears to malignancies through quantitative mechanisms that from increased or deregulated expression of essentially normal gene product.

HER-2/neu (pl85)

HER-2/neu oncogene.

The the HER-2/neu protein is cancers including prostate cancer.

transformation. It an is the protein product of.

HER-2/neu gene is amplified overexpressed in a breast, ovarian, colon,

HER-2/neu is related to the and variety lung of and malignant is found in 50%-60% of ductal in carcinoma and 20%-40% of all breast cancers, as well situ as a substantial fraction of adenocarcinomas arising in the ovaries _r prostate, colon and lung.

HER-2/neu is intimately associated not only with the malignant phenotype_r but also with the aggressiveness of the

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PCT/US96/01689 malignancy, being found in one-fourth of all invasive breast cancers.

HER-2/neu overexpression is correlated with a poor prognosis in both breast and ovarian cancer.

HER-2/neu is a transmembrane protein with a relative molecular mass acids (aa) in of 185 kd that is approximately 1255 amino length.

It has an extracellular binding domain (ECD) of approximately 645 epidermal growth factor hydrophobic transmembrane carboxyterminal

580 aa with 80%

Due cytoplasmic homology to aa, with 40% homology to receptor (EGFR), a highly anchor domain (TMD), and a domain (CD) of approximately

EGFR.

to the difficulties in the current approaches to therapy of cancers in which the HER-2/neu oncogene improved invention is associated, there is a need in the art for compounds and compositions.

fulfills this need, related advantages .

Summary of the Invention

Briefly stated, the polypeptides, nucleic acid

The present and further provides other present invention provides molecules (directing the expression of (directing the such polypeptides) and viral vectors expression for the immunization, or for immunization, of a malignancy in

A polypeptide invention may of such polypeptides) for use the manufacture of a warm-blooded animal which the HER-2/neu oncogene is or be pharmaceutically medicament against a associated nucleic acid molecule according to this present in acceptable polypeptide, nucleic acid a composition that includes carrier or diluent.

Such molecule, viral vector pharmaceutical composition may be used for immunization or on a one-time basis (e.g., when a or on a periodic basis (e.g., for an individual with an

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A medicament for immunization may be useful in the treatment of an existing tumor or to prevent tumor occurrence or reoccurrence.

In one embodiment, the present invention provides a polypeptide encoded by a DNA sequence selected from: (a) nucleotides 2026 through 3765 of SEQ ID NO:1; and (b) DNA sequences that hybridize to a nucleotide sequence complementary to nucleotides 2026 through 3765 of SEQ ID NO:1 under moderately stringent conditions, wherein the DNA sequence encodes a polypeptide that produces an immune response to HER-2/neu protein. In a preferred embodiment, a polypeptide has the amino acid sequence of SEQ ID NO:2 from lysine, amino acid 676, through valine, amino acid 1255, or a variant thereof that produces at least an provided invention equivalent immune that comprises a in combination response . polypeptide with

A composition is of the present pharmaceutically acceptable carrier or diluent.

In another embodiment, composition of the present invention immunization of a warm-blooded animal polypeptide or provided for the against is a malignancy in which the HER-2/neu oncogene is associated.

In another embodiment, such a polypeptide or composition is used for the manufacture of a medicament for immunization of a warm-blooded animal against a malignancy in which the HER-2/netz oncogene is associated.

In another embodiment, a nucleic acid molecule directing the expression of a polypeptide according to the present invention is provided for immunization by transfecting the cells of a warm-blooded animal with the nucleic acid molecule. In another embodiment, such a nucleic acid molecule is used for the manufacture of a medicament for immunization of a warm-blooded animal

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PCT/ÜS96/01689 against a malignancy in which the HER-2/neu oncogene is associated.

In another embodiment, a viral vector directing the expression of a polypeptide according to the present invention is provided for immunization by infecting the cells of a warm-blooded animal with the vector. In another embodiment, such a viral vector is used for the manufacture of a medicament for immunization of a warm blooded animal against a malignancy in which the HER-2/neu oncogene is associated.

These and other aspects of the present will become evident upon reference to the invention following detailed description and attached drawings.

Brief Description of the Drawings

Figure 1 shows the results of the priming of naive T lymphocytes to HER-2/neu polypeptide by dendritic cells .

Bone marrow-derived DC were generated with GM-CSF and IL6 from

CD34 + stem cells.

DC pulsed with HER-2/neu polypeptide induced protein-specific proliferation of autologous

CD4+/CD45RA+ T lymphocytes after 7 days of culturing T cells with

DC.

Bone marrow-derived CD34 + stem cells cultured for one week in serum-free medium containing GM-CSF and

IL-6 were used as APC.

APC were plated into 96-well round-bottomed plates

Corning, NY, USA) at various concentrations and incubated for 16-18 hours with

20-25 gg/ml of recombinant

HER-2/neu polypeptide .

CD4 +

T lymphocytes were isolated from autologous peripheral blood mononuclear, cells by positive selection using immunoaffinity columns (CellPro, Inc.,

Bothell, WA, USA). Antigen-pulsed APC were irradiated (10 Gy), and CD44- T lymphocytes were added at 10⁵ per well.

Proliferative response of T cells was measured by the uptake of (³H) thymidine (^Ci/well) added on day 7 for 16CA 02216601 1997-09-25

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PCT/US96/01689 hours.

Proliferation assays were performed in serumand cytokine-free medium in well replicates.

The symbols represent:

DC

HER-2/neu polypeptide +

CD4+/CD45RA+ T cells;

DC

CD4+/CD45RA+ T cells; and

DC + HER-2/reu polypeptide.

Figure 2 shows the response of CD4+ cells to

HER-2/neu polypeptide. Using the priming assay described for for

The

Figure 1, responses symbols

CD4+ T cells from normal donors to recombinant human HER-2/neu represent :

SC+CD4;

were tested polypeptide .

and

SC+CD4+HER-2/neu polypeptide .

SC is stem cells .

Figure 3 shows that rats immunized with rat

HER-2/neu polypeptide develop rat neu specific antibodies .

Rats were immunized with recombinant rat HER-2/neu polypeptide 25 ug in MPL or vaccel adj uvant.

Three immunizations were given, each 20 days apart. Twenty days after the final immunization rats were assessed for antibody responses to rat neu. Animals immunized with rat

HER-2/neu polypeptide and the vaccel adjuvant showed high titer rat neu specific responses.

The control was an animal immunized with human HER-2/neu protein).

100 ug develop shown).

In separate experiments, rats immunized and 300 ug of purified ’whole rat neu did detectable neu specific antibodies

Data represents the mean and standard of 3 animals. The symbols represent:

rat with not (data not deviation

HER-2/neu polypeptide/MPL;

rat HER-2/neu polypeptide/vaccel ;

MPL alone; —O— vaccel alone; and---O---control.

MPL

Neu¹¹ and vaccel are adjuvants (Ribi, Bozeman, MT, USA) .

is HER-2/neu

Figure 4 protein . shows that breast cancer patients have preexistent immunity to

HER-2/neu polypeptide.

Patient

PBMC were evaluated by tritiated thymidine incorporation

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PCT/US96/01689 in 24 well replicates. Responsive wells are scored as greater than the mean and 3 standard deviations (372 cpm) of the control wells. This HER-2/neu positive-stage II breast cancer patient has a significant response to recombinant human HER-2/neu polypeptide. The symbols p represent peptides for HER-2/neu protein, tt represents tetanus toxoid, and hHNP represents recombinant human HER-2/neu polypeptide.

Detailed Description of the Invention

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.

HER-2/neu polypeptide - as used herein, refers to a portion of the HER-2/neu protein also known as of SEQ pl85 or

ID NO:2 valine, synthetically functionally or more amino c-erbB2) having the from lysine, amino amino acid 1255; and may be produced, genetically equivalent variant thereof, e.g., acids are replaced by other amino acid(s) amino acid sequence acid

676, through derived, naturally engineered, or where one or non-amino acid(s) which do not substantially affect elicitation or enhancement of an immune response to HER2/neu protein (e.g., variant stimulates a response by helper T cells or cytotoxic T cells).

Proliferation of T cells - as used herein, includes the multiplication of T cells as well as the stimulation of T cells leading to multiplication, i.e., the initiation of events leading to mitosis and mitosis itself. . Methods for detecting proliferation of T cells are discussed below.

As noted above, the present invention is directed toward compounds and compositions to elicit or enhance immunity to the protein product expressed by the

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HER-2/neu oncogene, including for malignancies in a warmblooded animal wherein an amplified HER-2/neu gene is associated with the malignancies. Association of an amplified HER-2/neu gene with a malignancy does not require that the protein expression product of the gene be present on the tumor. For example, overexpression of the protein expression product may be involved with initiation of a tumor, but the protein expression may subsequently be lost. A use of the present invention is to elicit or enhance an effective autochthonous immune response to convert a HER-2/neu positive tumor to HER-2/neu negative.

More specifically, the disclosure of the present invention, in one aspect, shows that a polypeptide based on a particular portion protein expression product (HER-2/neu polypeptide) of the of the HÊR-2/neu gene can be recognized by thymus-dependent lymphocytes T cells) and, therefore, the autochthonous (hereinafter immune T cell response can be utilized prophylactically or to treat malignancies in which such a protein is or has been overexpressed.

The disclosure of the present invention also .shows, in directing the alone or in a another aspect, that nucleic acid expression of such a peptide may be viral vector for immunization.

molecules used

In general, considered to function

CD4 + T cell populations as helpers/inducers through are the release of lymphokines when stimulated by a specific antigen; however, a subset of

CD4 ⁺ cells can act as cytotoxic T lymphocytes (CTL). are considered to function by

Similarly, CD8⁺

T ceils targets; however, under a variety of circumstances they can secrete lymphokines to provide helper or DTH function. Despite the potential of overlapping function, the phenotypic CD4 and CD8 markers are linked to the recognition of peptides bound to class II or class I MHC

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PCT/US96/01689 antigens. The recognition of antigen in the context of class II or class I MHO mandates that CD4 ⁺ and CD8 ⁺ T cells respond to different antigens or the same antigen presented under different circumstances. The binding of immunogenic peptides to class II MHC antigens most commonly occurs for antigens ingested by antigen presenting cells. Therefore, CD4⁴· T cells generally recognize antigens that have been external to the tumor cells. By contrast, under normal circumstances, binding of peptides to class I MHC occurs only for proteins present in the itself, proteins cytosol and synthesized by the target external environment are excluded.

in the

An exception to this is the binding of exogenous peptides with a precise class I binding motif which are present outside the cell in high concentration. Thus, CD4 ⁺ and

CD8⁺ T cells have broadly different functions and tend to recognize different antigens as a reflection of where the antigens normally reside.

As disclosed within the present invention, a polypeptide portion of the protein product expressed by the HER-2/neu oncogene is recognized by T cells.

Circulating HER-2/neu polypeptide is degraded to peptide fragments. Peptide fragments from the polypeptide bind to major histocompatibility complex (MHC) antigens. By display of a peptide bound to MHC antigen on the cell surface and recognition by host T cells of the combination of peptide plus self MHC antigen, HER-2/neu polypeptide (including that expressed on a malignant ceil) will be immunogenic to T cells. The exquisite specificity of the T cell receptor enables individual T cells to discriminate between peptides which differ by a single amino acid residue .

During the immune response to a peptide fragment from the polypeptide, T cells expressing a T cell receptor

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PCT/US96/01689 with high affinity binding of the peptide-MHC complex will bind to the peptide-MHC complex and thereby become activated and induced to proliferate.

In the first encounter with a peptide, small numbers of immune T cells will secrete lymphokines, proliferate and differentiate into effector and memory T cells. The primary immune response will occur in vivo but has been difficult to detect in vitro. Subsequent encounter with the same antigen by the memory T cell will lead to a faster and more intense immune response. The secondary response will occur either in vivo or in vitro. The in vitro response is easily gauged by measuring the degree of proliferation, the degree of cytokine production, or the generation of cytolytic activity of the T cell population re-exposed in the antigen. Substantial proliferation of the T cell population in response to a particular antigen is considered to be indicative of 'prior exposure or priming to the antigen.

The compounds of this invention generally comprise HER-2/neu polypeptides or DNA molecules that direct the expression of such peptides, wherein the DNA molecules may be present in a viral vector. As noted above, the polypeptides of the present invention include variants of the polypeptide of SEQ ID NO : 2 from amino acid 676 through amino acid 1255, that retain the ability to stimulate an immune response. Such variants include various structural forms of the native polypeptide. Due to the presence of ionizable amino and carboxyl groups, for example, a HER-2/neu polypeptide may be in the form of an acidic or basic salt, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.

Variants within the scope of this invention also include polypeptides in which the primary amino acid

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PCT/US96/01689 structure native HER-2/neu polypeptide is modified by forming covalent or aggregative conjugates with other peptides or polypeptides, or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-terminus.

The present invention also includes HER-2/neu polypeptides with or without glycosylation. Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. For instance, expression of DNA encoding polypeptides in bacteria such as E. coli typically provides non-glycosylated molecules. Nglycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-Ai-Z, where Ai is any amino acid except Pro, and Z is Ser or Thr. Variants of HER-2/neu polypeptides having inactivated Nglycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide . synthesis and ligation or . site-specific mutagenesis techniques, and are within the scope of this invention. Alternatively, N-linked glycosylation sites can be added to a HER-2/neu polypeptide.

The polypeptides of this invention also include variants of the SEQ ID NO:2 polypeptide (i.e., variants of a polypeptide having the amino acid sequence of SEQ ID NO: 2 from amino acid 67 6 through amino acid 1255) that have an amino acid sequence different from this sequence because of one or more deletions, insertions, substitutions or other modifications. In one embodiment, such variants are substantially homologous to the native HER-2/neu polypeptide and retain the ability to stimulate

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PCT/US96/01689 an immune response. Substantial homology, as used herein, refers to amino acid sequences that may be encoded by DNA sequences that are capable of hybridizing under moderately stringent conditions to a nucleotide sequence complimentary to a naturally occurring DNA sequence encoding the specified polypeptide portion of SEQ ID NO:2 herein (i.e., nucleotides 2026 through 3765 of SEQ ID NO:1). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC (containing 0.1% SDS) . Such hybridizing DNA sequences are also within the scope of this invention. The effect of any such modifications on the ability of a HER-2/neu polypeptide to produce an immune response may be readily determined (e.g., by analyzing the ability of the mutated HER-2/neu polypeptide to induce a T cell response using, for example, the methods described herein).

in a variety of variants within example,

Generally, amino acid substitutions may be made ways to provide other embodiments of the present invention.

acid substitutions may

i.e., a substitute amino acid amino conservatively; amino acid that has skilled in the art of secondary polypeptide the following changes :

structure (1) (2) (4) cys, lys,

First, for be made replaces an similar properties, such that peptide chemistry would expect and hydropathic nature of to be substantially unchanged.

groups of ala, pro, one the the

In general, amino acids represent conservative ser, arg, tyr, his ;

thr ;

and gly, glu, asp, gin, asn, (3) val, ile, leu, met, ser, thr;

ala, phe;

An example of of a non-conservative change is to replace an amino acid one group with an amino acid from another group.

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Another way to make amino acid substitutions to produce variants of the present invention is to identify and replace amino acids in T cell motifs with potential to bind to class II MHC molecules (for CD4 + T cell response) or class I MHC molecules (for CD8+ T cell response) . Peptide segments (of a HER-2/neu polypeptide) with a motif with theoretical potential to bind to class II MHC molecules may be identified by computer analysis. For example, a protein sequence analysis package, T Sites, that incorporates several computer algorithms designed to distinguish potential sites for T cell recognition can be used (Feller and de la Cruz, Nature 345:720-721, 1991) .

Two searching algorithms are used: (1) the AMPHI algorithm described by Margalit (Feller and de la Cruz, Nature 349:720-721, 1991; Margalit et al·., J. Immunol,

233:2213-2229, 1987) identifies epitope motifs according to alpha-helical periodicity and amphipathicity; (2) the

Rothbard and Taylor algorithm identifies epitope motifs according to charge and polarity pattern (Rothbard and Taylor, EMBO 7:93-100, 1988). Segments with both motifs are most appropriate for binding to class II MHC molecules. CD8⁺ T cells recognize peptide bound to class I MHC molecules. Falk et al. have determined that peptides binding to particular . MHC molecules share discernible sequence motifs (Falk et al., Nature 351:290296, 1991). A peptide motif for binding in the groove of HLA-A2.1 has been defined by Edman degradation of peptides stripped from

HLA-A2.1 molecules of a cultured cell line (Table 2, from

Falk et al., supra). The method identified the typical or average

HLA-A2.1 binding peptide as being 9 amino acids in length with dominant anchor residues occurring at positions (L) and (V) .

Commonly occurring strong binding residues have been identified at positions (M), 4 (E,K), (V) ,

The

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PCT/US96/01689 identified motif represents the average of many binding peptides .

The HLA-A2.1 Restricted Motif

<td></td><td> Amino Acid Position 123456789</td><td> Point Assignment</td>

<td> Dominant Binding Anchor Residue</td><td> L V</td><td> + 3</td>

<td> Strong Binding Residue</td><td> M E V K K</td><td> 4-2</td>

<td> Weak Binding Residue</td><td> I AGIIAEL L Y P K L Y S F F D Y T H K P T N Μ M G Y S V H</td><td> + 1</td>

The derived peptide motif as currently defined is not particularly stringent. Some HLA-A2.1 binding peptides do not contain both dominant anchor residues and the amino acids flanking the dominant anchor residues play ma j or roles in allowing or disallowing binding.

Not every peptide with the current described binding motif will bind,. and some peptides without the motif will bind.

However, the current motif is valid enough to identification of some peptides capable of binding.

allow

Of note, the current HLA-A2.1 motif places 6 amino acids between the dominant anchor amino acids at residues 2 and 9 .

Following identification of peptide motifs within a HER-2/neu polypeptide, amino acid substitutions may be made conservatively or non-conservatively. The latter- type of substitutions are intended to produce an improved polypeptide that is more potent and/or more

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PCT/US96/01689 broadly cross-reactive (MHC polymorphism). An example of a more potent polypeptide is one that binds with higher affinity to the same MHC molecule as natural polypeptide, without affecting recognition by T cells specific for natural polypeptide. An example of a polypeptide with broader cross-reactivity is one that induces more broadly cross-reactive immune responses (i.e., binds to a greater range of MHC molecules) than natural polypeptide.

Similarly, one or more amino acids residing between peptide motifs and having a spacer function (e.g., do not may be substituted conservatively or non-conservatively.

It will be evident to those of ordinary skill in the art that polypeptides containing one or more amino acid substitutions may be tested for beneficial or adverse immunological interactions by a variety of assays, including those described herein for the ability to stimulate

T cell recognition.

Variants within the scope of this invention may also, or alternatively, contain other modifications, including the deletion or addition of amino acids, that have minimal influence on the desired immunological properties of the polypeptide.

It will be appreciated by those of ordinary skill in the art that truncated forms or non-native extended forms of a HER-2/neu polypeptide may be used, provided the desired immunological properties are at least roughly equivalent to that of full length, native

HER-2/neu polypeptide .

Cysteine residues may be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present.

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obtained

A HER-2/neu polypeptide using a genomic or cDNA may generally be clone encoding the protein.

HER-2/neu

A genomic sequence that encodes full length is shown in SEQ ID NO:1, and the deduced amino acid sequence is presented in SEQ ID NO:2. Such clones may be isolated. by screening an appropriate expression library for clones that express HER-2/neu protein. The library preparation and screen may generally be performed using methods known to those of ordinary skill in the art, such as methods described in Sambrook et al., Molecular

Cloning: A

Laboratory

Manual,

Cold

Spring Harbor

Laboratories,

Cold Spring

Harbor,

N.Y.

1989*

Briefly, bacteriophage expression library may be plated and transferred to filters. The filters may then be incubated with a detection reagent.

In the context of this invention, a detection reagent is any compound capable of binding to HER-2/neu protein, which may then be detected by any of a variety of means known to those of ordinary skill in the art. Typical detection reagents contain a binding agent, such as Protein A,

Protein G,

IgG or a lectin, coupled to a reporter group.

Preferred reporter groups include enzymes, substrates, cofactors , inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. More preferably, the reporter group is horseradish peroxidase, which may be detected by incubation with a substrate such as tetramethylbenzidine or 2,2’-azino-di-3-ethylbenzthiazoline sulfonic acid. Plaques containing genomic or cDNA sequences that express HER-2/neu protein are isolated and purified by techniques known to those of ordinary skill in the art. Appropriate methods may be found, for example, in Sambrook et al., Molecular Cloning: A

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Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.

Variants of . the polypeptide that retain the ability to stimulate an immune response may generally be identified by modifying the sequence in one or more of the aspects described above and . assaying the resulting polypeptide for the ability to stimulate an immune response, e.g., a T cell response. For example, such assays may generally be performed by contacting T cells with the modified polypeptide and assaying the response. Naturally occurring variants of the polypeptide may also be isolated by, for example, screening an appropriate cDNA or genomic library with a DNA sequence encoding the polypeptide or a variant thereof.

The above-described sequence modifications may be introduced using standard recombinant techniques or by automated synthesis of the modified polypeptide. For example, mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analogue having the desired amino acid insertion, substitution, or deletion .

Alternatively, oligonucleotide-directed sitespecific mutagenesis procedures can be employed to provide a gene in which particular codons are altered according to

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January

<img file="CA2216601C_D0005.tif" />

Principles and Methods, Plenum Press, 1981; and U.S.

Patent Nos. 4,518,584 and 4,737,462.

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Mutations in nucleotide sequences constructed for expression of such HER-2/neu polypeptides must, of course, preserve the reading frame of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the mRNA.

Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined.

For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed HER-2/neu polypeptide mutants screened for the desired activity.

Not all mutations in a nucleotide sequence which encodes a HER-2/neu polypeptide will be expressed in the final product. For example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see,

e.g._z European Patent Application 75,444A), or to provide codons that are more readily translated by the selected host, such as the well-known E. coll preference codons for

E. coll expression.

The polypeptides of the present invention, both naturally occurring and modified, are preferably produced by recombinant DNA methods. Such methods include inserting a DNA sequence encoding a HER-2/neu polypeptide into a recombinant expression vector and expressing the DNA sequence in a recombinant microbial, mammalian or insect cell expression system under conditions promoting expression. DNA sequences encoding the polypeptides provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being inserted in a recombinant

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PCT/US96/01689 expression vector and expressed in a recombinant transcriptional unit

Recombinant expression vectors contain a DMA sequence encoding a HER-2/neu polypeptide operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes.

Such regulatory elements include transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.

An origin facilitate recognition of transformants may additionally be incorporated.

DNA regions are operably linked when they are functionally related to each other.

a signal peptide (secretory leader)

For example, DNA for is operably linked to

DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, in reading frame. DNA sequences encoding HER-2/neu polypeptides which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA.

Expression vectors for bacterial use may comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017)- Such commercial vectors

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PCT7US96/01689 include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA) . These pBR322 backbone sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose

1978; and promoter system (Chang et al·., Nature 275:615,

Goeddel et al

Nature 281:544,

1979), the tryptophan Acids Res.

(trp) promoter

6:4057, 1980;

system (Goeddel et and European

Patent al., Nucl.

the tac promoter (Maniatis,

Application

Molecular Cloning :

Laboratory, p.412,

A Laboratory Manual, Cold Spring Harbor

1982). A particularly useful bacterial expression system employs the phage λ P_L promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American. Type Culture Collection which incorporate derivatives of the λ P_L promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3phosphoglycerate kinase (Hitzeman et al., C. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 27:4900, 1978), such as enolase, glyceraldehyde3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,

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PCT/US96/01689 triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., European Patent Application 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli. (Amp^r gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and cx-factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258z2674, 1982) and Beier et al. (Nature 300:7 24, 1982) . The yeast cc-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed (see, e.g., Kurjan et al., Coll 30:933, 1982; and Bitter et al., Proc. Natl. Acad.

Sci. USA 37:5330, 1984) . The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from polyoma, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from

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PCT/US96/01689 the Hind III site toward the Bgl II site located in the viral origin of replication is included. Further, viral genomic promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen. Exemplary vectors can be constructed as disclosed by Okayama and Berg, Mol. Cell. Biol. 3:280, 1983.

A useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by

Cosman et al. (Mol. Immunol. 23:935, 1986).

A preferred eukaryotic vector for expression of LbeIF4A protein DNA is pDC406 (McMahan et al.,

EMBO J. 10:2821,

1991) , and includes regulatory sequences derived from

SV40, human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV) . Other preferred vectors include pDC409 and pDC410, which are derived from pDC406. pDC410 was derived from pDC406 by substituting the EBV origin of replication with sequences encoding the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl II restriction site outside of the multiple cloning site has been deleted, making the Bgl II site within the multiple cloning site unique .

A useful cell line that allows for episomal replication of expression vectors, such as pDC406 and pDC409, which contain the EBV origin of replication, is CV-l/EBNA (ATCC CRL 10478). The CV-L/EBNA cell line was derived by transfection of the CV-1 cell line with a gene encoding Epstein-Barr virus nuclear . antigen-I (EBNA-1) and constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter.

Transformed host cells are cells which have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and which

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PCT/ÜS96/01689 contain sequences encoding a HER-2/neu polypeptide of the present invention. Transformed host cells may express the desired HER-2/neu polypeptide, but host cells transformed for purposes of cloning or amplifying HER-2/neu DNA do not need to express the HER-2/neu polypeptide. Expressed polypeptides will preferably be secreted into the culture supernatant, depending on the DNA selected, but may also be deposited in the cell membrane.

Suitable host cells for recombinant eukaryotic proteins include prokaryotes, cells under the control expression of yeast or higher of appropriate include gram negative example E. coli or promoters .

positive organisms, for

Higher eukaryotic cells include established cell insect or mammalian origin as described below.

Prokaryotes

E.

or gram

Bacilli .

lines of

Cell-free translation systems could also be employed to produce HER-2/neu polypeptides using RNAs derived from DNA constructs. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, by Pouwels et al·., Cloning Vectors: A Laboratory Manual_r Elsevier, New York, 1985.

Prokaryotic expression hosts may be used for expression of HER-2/neu polypeptides that do not require extensive proteolytic and disulfide processing. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and· an origin of replication recognized by the host to ensure amplification within the host. Suitable prokaryotic hosts for transformation include E. coll, Bacillus subtilis_rSalmonella typhimurium, and various species within the

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PCT/ÜS96/01689 genera Pseudomonas, Streptomyces, and Staphylococcus_falthough other hosts may also be employed.

Recombinant HER-2/neu polypeptides may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisise. Yeast of other genera, such as Plchla or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2μ yeast autonomously replicating sequence (ARS), encoding the polyadenylation plasmid or a promoter, an

DNA

HER-2/neu polypeptide , sequences for and transcription termination and selection gene.

origin of replication

Preferably, yeast vectors will and selectable marker transformation of both ampicillin resistance cerevisiae trpl gene, for a mutant strain of in tryptophan, and a expressed structural lesion in effective growth in known to technique include an permitting yeast and gene which yeast

E.

coll _r of . E.

provides lacking coll and the S.

the selection ability marker to grow highly promoter derived to induce -transcription sequence downstream. The presence of the trpl yeast gene from a of a the yeast host cell genome then provides environment for detecting transformation the absence of tryptophan.

Suitable yeast those of skill described by Hind

USA 75:1929,

1978), an by transformation in the art.

et al. {Proc.

protocols are

An

Natl.

involves selecting exemplary

Acad,

Scl.

for

Trp⁺ transformants in a selective medium consisting of base, 0.5% casamino acids,

0.67% yeast nitrogen mg/ml adenine and 2 0 mg/ml uracil.

2% glucose, 10

Host strains promoter may be vectors comprising for expression in a rich transformed by the ADH2 grown yeast with medium consisting of 1%

1% glucose supplemented extract, 2% peptone, and mg/ml adenine and 80 mg/ml uracil. Derepression

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PCT/US96/01689 of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4°C prior to further purification.

Various mammalian or insect (e.g., Spodoptera or Trichoplusia) cell culture systems can also be employed to express recombinant polypeptide. Baculovirus systems for production of heterologous polypeptides in insect cells are reviewed, for example, by Luckow and Summers, Bio/Technology 6:47, 1988. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other including, for example, cell lines capable of expressing an appropriate vector CV-l/EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO),

COS, NS-1,

HeLa and BHK cell lines.

comprise nontranscribed replication, a

Mammalian expression elements such as an vectors may of suitable the gene to be nontranscribed origin promoter and enhancer linked ' or 3 ' flanking to sequences, such polyadenylation transcriptional expressed, and other sequences, and 5’ or

3’ nontranslated binding sites, a as necessary ribosome site, splice donor and acceptor sites, and termination sequences .

Purified HER-2/neu polypeptides may be prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supernatants from systems which secrete recombinant polypeptide into culture media may be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate may be applied to a suitable purification matrix. For example, a suitable affinity matrix may comprise a counter structure protein * trademark

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PCT/US96/01689 (i.e., a protein to which a HER-2/neu polypeptide binds in a specific interaction based on antibody molecule bound to Alternatively, an for . example, a diethylaminoethyl acrylamide, agarose, dextran, cellulose structure) or a suitable anion matrix (DEAE) exchange resin or . substrate groups .

The lectin or support. employed, having pendant matrices can be can be or other types commonly employed in protein purification, a cation exchange step can be employed.

exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl· groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying a HER-2/neu.

Affinity chromatography is a preferred method of purifying HER-2/neu polypeptides. For example, monoclonal antibodies against the HER-2/neu polypeptide may also be useful in affinity chromatography purification, by utilizing methods that are well-known in the art. Finally, one or .more reverse-phase high performance liquid chromatography (RP-HPLC) , steps employing hydrophobic RP-HPLC media (e.g._z silica gel having pendant methyl or other aliphatic groups) may be employed to further purify a HER-2/neu polypeptide composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant polypeptide.

Recombinant HER-2/neu polypeptide produced in bacterial culture is preferably isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) may be employed for final purification steps. Microbial cells employed in expression of recombinant LbeIF4A protein can be disrupted by any

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PCT/ÜS96/01689 convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents .

Fermentation of yeast which express

HER-2/neu polypeptide as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:111, 1984). This reference describes two sequential, reverse-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column.

Preparations of HER-2/neu polypeptides synthesized in recombinant culture may contain nonHER-2/neu cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the HER-2/neu polypeptide from the culture. These components ordinarily will be of yeast, prokaryotic or non-human eukaryotic origin. Such preparations are typically free of other proteins which may be normally associated with the HER-2/neu protein as it is found in nature in its species of origin.

Automated synthesis provides an alternate method for preparing polypeptides of this invention. For example, any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See

Merrifield, J. Am. Chem. Soc. &lt;95:214 9-214 6, 1963.)

Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. of Foster City, CA, and may generally be operated according to the manufacturer's instructions.

Within one aspect of the present invention, use of_a HER-2/neu polypeptide (or a DNA molecule that directs

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PCT/TJS96/01689 the expression of such a peptide) to generate an immune response to the HER-2/neu protein (including that expressed on a malignancy in which a HER-2/neu oncogene is associated) may be detected. Representative examples of such malignancies include breast, ovarian, colon, lung and prostate cancers. An immune response to the HER-2/neu protein, once generated by a HER-2/neu polypeptide, can be long-lived and can be detected long after immunization, regardless of whether the protein is present or absent in the body at the time of testing. An immune response to the HER-2/neu protein generated by reaction to a HER-2/neu polypeptide can be detected by examining for the presence or absence, or enhancement, of specific activation of CD4 ⁺or CD8⁺ T cells. More specifically, T cells isolated. from an immunized individual by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes) are incubated with HER-2/neu

<td> protein .</td><td> For example, T cells may be incubated in vitro</td>

<td> for 2-9</td><td> days (typically 4 days) at 37°C with HER-2/neu</td>

<td> protein</td><td> (typically, 5 pg/ml of whole protein or graded</td>

numbers of cells synthesizing HER-2/neu protein). It may be desirable to incubate another aliquot of a T cell sample in the absence of HER-2/neu protein to serve as a control.

of CD4⁺ or CD8 ⁺ T cells may ways. Methods for detecting include detecting the production of cytokines generation of cytolytic cytotoxic T cells specific CD4⁺ T cells, a preferred T cell activation is the of T cells . For CD8 ⁺detecting specific T cell

Specific activation be detected in a variety of specific T cell activation proliferation of T cells, the (e.g., lymphokines), or the activity (i.e., generation of for HER-2/neu protein). For method for detecting specific detection of the proliferation T cells, a preferred method for

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PCT7US96/01689 activation is the detection of the generation of cytolytic activity.

Detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis. T cells which have been stimulated to proliferate exhibit an increased rate of DNA synthesis. A typical way to measure the rate of DNA synthesis is, for example, by pulse-labeling cultures of

T cells with tritiated thymidine, a nucleoside precursor which is incorporated into newly synthesized DNA.

The amount of tritiated thymidine incorporated can be determined using a liquid scintillation spectrophotometer.

Other ways to detect T cell proliferation include measuring increases in interleukin-2 (IL-2) production, Ca²⁺ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferon-gamma) can be measured or the relative number of T cells that can respond to intact p!8 5^HER^2/neu protein may be quantified.

By use or expression of a HER-2/neu polypeptide, T cells which recognize the HER-2/neu protein can be proliferated in vivo. For example, a medicament for immunization with a HER-2/neu peptide (i.e., as a vaccine) can induce continued expansion in the number of cells necessary for therapeutic attack against a tumor in which the HER-2/neu oncogene is associated.

Typically, about

0.01 μg/kg to about

100 mg/kg body weight will be administered by the intradermal, subcutaneous or intravenous route. A preferred dosage is about 1 μ9^5 -to about 1 mg/kg, with about 5 μg/kg to about 200 μg/kg particularly preferred. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the

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PCT/ÜS96/01689 patient. It may be desirable to administer the HER-2/neu polypeptide repetitively. It will be evident ' to those skilled in this art that more than one HER-2/neu polypeptide may be administered, either simultaneously or sequentially. Preferred peptides for use in a medicament for immunization are those that include the amino acid sequence of SEQ ID NO: 2 beginning at about the lysine residue at amino acid position 676 and extending to about the valine residue at amino acid position 1255. It will be appreciated by those in the art that the present invention contemplates the use of an intact HER-2/neu polypeptide as well as division of such a polypeptide into a plurality of peptides. Neither intact pl85^HER“^2/neuprotein nor a peptide having the amino acid seguence of its entire extracellular domain (i.e., a peptide having an amino acid sequence of SEQ ID NO: 2 from amino acid position 1 up to amino acid position 650, plus or minus’ about one to five positions, and with or without the first 21 amino acid positions) are used alone for immunization.

A HER-2/neu polypeptide (or nucleic acid) is preferably formulated for use in the above methods as a pharmaceutical composition (e.g., vaccine). Pharmaceutical compositions generally comprise one or more polypeptides in combination with a pharmaceutically acceptable carrier, excipient or diluent. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. The use of a HER-2/neu polypeptide in conjunction with chemotherapeutic agents is also contemplated.

In addition to the HER-2/neu polypeptide (which functions as an antigen) , it may be desirable to include other components in the vaccine, such as a vehicle for antigen delivery and immunostimulatory substances designed to enhance the protein's immunogenicity. Examples of

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PCT/US96/01689 vehicles for antigen delivery include aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oilin-water emulsions, biodegradable microcapsules, and

5.

liposomes .

isoglutamine

IL-12, evident / neu

Examples of immunostimulatory substances include

N—acetylmuramyl-L-alanine-D(MDP), lipopoly-saccharides (LPS), glucan,

GM-CSF, gamma interferon and to those of polypeptide synthetically or

While ordinary skill be any in

IL-15.

It will be ordinary skill'in this art that a HERfor a vaccine may be prepared naturally suitable derived.

the art pharmaceutical compositions carrier will vary depending and whether a sustained of carrier known to those of on may this the be employed in the invention, the type of mode of administration release is desired.

For parenteral administration, such as subcutaneous injection, the fat, the carrier preferably comprises water, saline, alcohol, a a wax or a buffer above lactose, talcum,

For .oral administration, any of carriers or a solid carrier, such as mannitol, starch, magnesium stearate, sodium saccharine, cellulose, glucose, sucrose, and magnesium carbonate, may be (e.g.> polylactic carriers invention .

disclosed,

5,075,109.

for the for within the the surface employed .

Biodegradable .microspheres may also be employed as pharmaceutical compositions of

Suitable biodegradable microspheres example, in U.S.

Patent Nos. 4,897,268

HER-2/neu polypeptide biodegradable microsphere of the microsphere.

preferred embodiment, a polypeptide sequence of

SEQ ID NO : 2 from amino may or

For thi s are and be encapsulated associated example, having the amino acid 676 through with in a acid amino acid 1255 is encapsulated within a biodegradable microsphere .

In this regard, it is preferable that the microsphere be larger than approximately 25 microns .

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PCT/ÜS96/01689 may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 proteins, amino acids, carbohydrates including glucose, chelating agents such as EDTA, stabilizers and excipients.

sucrose or glutathione

Neutral buffered dextrins , and other saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.

Preferably, product is formulated as a lyophilizate using appropriate excipient solutions

As an diluents .

alternative to the presentation of

HER-2/neu polypeptides, the subject invention includes compositions capable of delivering nucleic acid molecules encoding a HER-2/neu polypeptide.

Such compositions include recombinant viral vectors (e.g., retroviruses (see

WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO

6·. 616627, 1988;

Li et al. ,

Hum. Gene Ther. 4:403-409,

1993;

Vincent et al., Nat. Genet. 5:130-134, 1993; and Rolls et al., Proc.

Natl.

Acad. Scl.

virus (see

U.S.

Patent No.

4,769,330; U.S. Patent No.

5,017,487;

and WO naked DNA (see WO 90/11092), nucleic acid molecule complexed to a polycationic molecule (see WO

93/03709), and nucleic acid associated with liposomes (see Wang et al., Proc. Natl.

Acad. Scl.

USA

34:7851,

1987).

In certain embodiments, the DNA may be linked to killed or inactivated adenovirus (see Curiel et al., Hum. Gene Ther.

3:147-154,

1992; Cotton et al., Proc.

Natl. Acad. Scl. USA 33:6094,

1992).

Other suitable compositions include DNA-ligand ( see Wu et al. ,

J. Biol.

Chem,

64:1698 5-16 987, 1989) and lipid-DNA combinations

Feigner et al., Proc. Natl. Acad. Scl.

USA 34:74137417,

1989)

In addition, the efficiency of naked DNA

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PCT/ÜS96/01689 uptake into cells may be increased by coating the DNA onto biodegradable beads.

In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from' an animal, modified, and placed into the same or another animal. It will be evident that one can utilize any of the compositions noted above for introduction of HER-2/neu nucleic acid molecules into tissue cells in an ex vivo context. Protocols for viral, physical and chemical methods of uptake are well known in the art.

Accordingly, the present invention is useful for enhancing or eliciting, in a patient or cell culture, a cellular immune response (e.g., the generation of antigenspecific cytolytic T cells) . As used herein, the term patient refers to any warm-blooded animal, preferably a human. A patient may be afflicted with cancer, such as breast cancer, or may be normal (i.e./ free of detectable disease and infection). A cell culture is any preparation of T cells or isolated component cells (including, but not limited to, macrophages, monocytes, B cells and dendritic cells). Such cells may be isolated by any of a variety of techniques well known to those of ordinary skill in the art (such as Ficoll-hypaque density centrifugation).

The cells may (but need not) have been isolated from a patient afflicted with a HER-2/neu associated malignancy, and may be reintroduced into a patient after treatment.

The present invention also discloses that HER2/neu polypeptide, in addition to being immunogenic to

T cells, appears to stimulate

B-cells to produce antibodies capable of recognizing

HER-2/neu polypeptide .

Antibodies (i.e., which exhibit a binding affinity of specific about 10⁷ liters/mole or better) for HER-2/neu protein may be found in a variety of body fluids including

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PCT/US96/01689 sera and ascites. Briefly, a body fluid sample is isolated from a warm-blooded animal, such as a human, for whom it is desired to determine whether antibodies specific for HER-2/neu polypeptide are present. The body fluid is incubated with HER-2/neu polypeptide under conditions and for a time sufficient to permit immunocomplexes to form between the polypeptide and antibodies specific for the protein. For example, a body fluid and HER-2/neu polypeptide may be incubated at 4°C for 24-48 hours. Following the incubation, the reaction mixture is tested for the presence of immunocomplexes.

Detection of one or more immunocomplexes formed between

HER-2/neu polypeptide and antibodies specific for HER2/neu polypeptide may be accomplished by a variety of known enzyme techniques, such as radioimmunoassays (RIA) and linked immunosorbent assays (ELISA).

Suitable immunoassays include the double monoclonal antibody sandwich immunoassay technique of

David et al.

(U.S

Patent ; monoclonalpolyclonal antibody sandwich assays (Wide et al., in

Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and

Ξ. Livingstone, Edinburgh, 1970); the western blot method of Gordon et al. (U.S. Patent 4,452,901);

immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem. 255:4980-4983, 1980); enzyme-linked immunosorbent assays as described by, for example, Raines and Ross (J. Biol. Chem. 257:5154-5160, 1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); and neutralization of activity [Bowen-Pope et al., Proc. Natl. Acad. Sci. USA 91:2396-2400 (1984)].

In addition to the immunoassays described above, a number of other immunoassays are available, including those described in

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PCT7US96/01689 ³⁵

U.S. Patent Nos.: 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876,

For detection purposes, HER-2/neu polypeptide (antigen) may either be labeled or unlabeled. When unlabeled, the antigen finds 'yse in agglutination assays. In addition, unlabeled antigen can be used in combination with labeled- molecules’ that are reactive with immunocomplexes, or in combination with labeled antibodies (second antibodies) that are reactive with the antibody directed against HER-2/neu polypeptide, such as antibodies specific for immunoglobulin. Alternatively, the antigen can be directly labeled. Where it is labeled, the reporter group can include radioisotopes, fluorophores, enzymes, iuminescers, or dye particles. These and other labels are well known in the art and are described, for example, in the following U.S. patents: 3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402 .

Typically in an ELISA assay, antigen is adsorbed to the surface of a microtiter well. Residual proteinbinding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent). The well is then incubated with a sample suspected of containing specific antibody. The sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1%“5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. After incubating for a sufficient length of time to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with an anti-species specific immunoglobulin antibody labeled with a reporter

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PCT/ÜS96/01689 group. The reporter group can be chosen from a variety of enzymes, including horseradish peroxidase, betagalactosidase, alkaline phosphatase, and glucose oxidase. Sufficient time is allowed for specific binding to occur, then the well is again washed to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally.

In one preferred embodiment of this aspect of the present invention, a reporter group is bound to HER2/neu protein. The step of detecting immunocomplexes involves removing substantially any unbound HER-2/neu protein and then detecting the presence or absence of the reporter group.

In another preferred embodiment, a reporter group is bound to a second antibody capable of binding to the antibodies specific for HER-2/neu protein. The step of detecting immunocomplexes involves (a) removing substantially any unbound antibody, (b) adding the second antibody, (c) removing substantially any unbound second antibody and then (d) detecting the presence or absence of the reporter group. Where the antibody specific for HER2/neu protein is derived from a human, the second antibody is an anti-human

In a antibody .

third preferred embodiment reporter group is bound to the immunocomplexes.

(a) adding the molecule.

for detecting molecule to a

The immunocomplexes, capable of binding detecting involves substantially any unbound molecule, and the presence or absence of the reporter of a molecule capable of binding to the then step of removing (c) detecting group. An example immunocomplexes is protein A.

It will be evident to one skilled in the art that variety of methods for detecting the

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WO 96/30514 PCT/US96/01689 immunocomplexes may be employed within the present invention. Reporter groups suitable for use in any of the methods include radioisotopes, fluorophores, enzymes, luminescers/ and dye particles.

In a related aspect of the present invention, detection of immunocomplexes formed between HER-2/neu polypeptide and antibodies in body fluid which are specific for HER-2/neu polypeptide may be used to monitor the effectiveness of cancer therapy, which involves a 10 HER-2/neu polypeptide, for a malignancy in which the HER2/neu oncogene is associated. Samples of body fluid taken from an individual prior to and subsequent to initiation of therapy may be analyzed for the immunocomplexes by the methodologies described above. Briefly, the number of 15 immunocomplexes detected in both samples are compared. A substantial change in the number of immunocomplexes in the second sample (post-therapy initiation) relative to the first sample (pre-therapy) reflects successful therapy.

The following examples are “ offered by way of 20 illustration and not by way of limitation.

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PCT/ÜS96/01689

EXAMPLES

EXAMPLE 1

Expression and Purification of Recombinant Human HER-2/neu Polypeptide

The human HER-2/neu polypeptide was recovered by the PCR method (e.g., U.S.

Patent Nos. 4,683, 195;

4,683,202; 4,800,159) from a plasmid prepared according to

Di Fiore et al. (King et al.,

Science 229:974-976,

1985;

Di Fiore et al. ,

Science

237:178-182, using oligonucleotide primers that additionally introduced a

BssHII restriction site and an enterokinase protease site on the 5’ end and an EcoRI site for the

5’-end was

5’-TCTGGCGCGCTGGATGACGATGACAAGAAACGACGGCAGCAGAAGATC-3’ (SEQ ID NO:3) while the primer for the 3’-end was

5’-TGAATTCTCGAGTCATTACACTGGCACGTCCAGACCCAG-3¹ (SEQ

ID

NO: 4) . The resulting 1.8 kb PCR fragment was subcloned into the T-vector from Novagen (Madison, W1, USA) and the sequence of selected clones was determined on the ABI 373 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA) using overlapping sequencing primers. PCR fragments with sequence that corresponded to the published DNA sequence for the human HER-2/neu cDNA (SEQ ID NO:1; Coussens et al., Science 230:1132, 1985; Yamamoto et al.. Nature 319:230, 1986)were then connected in the correct reading frame via the BssHII site to a modified E. coli thioredoxin reductase. A 6Xhistidine affinity tag employed in Ni-NTA affinity purification of the expressed fusion protein was incorporated into the thioredoxin reductase fusion partner. This cDNA for the trxA-human HER-2/neu polypeptide fusion protein was subcloned into a modified pET expression vector for expression in E. coli.

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While thioredoxin reductase has been reported to stabilize and solubilize other heterologous proteins expressed in E.

coll, it did not appear to offer any significant advantage for human

HER-2/neu polypeptide expression in E. coll.

While a significant proportion of the trxA-HER-2/neu polypeptide fusion protein was soluble, a majority was expressed in inclusion bodies. The fusion protein was also subjected to degradation during expression in E. coll. The presence of the thioredoxin reductase fusion partner may, however, stabilize the protein during purification. The availability of monoclonal antibodies to thioredoxin reductase provides a convenient marker to follow during purification.

For purification of the human HER-2/neu polypeptide with the thioredoxin reductase fusion partner containing the 6XHis affinity tag, the E. coll pellet was resuspended with protease inhibitors and lysozyme and sonicated. The inclusion bodies were isolated by centrifugation, and are washed 3X with deoxycholate, the last wash being overnight to remove LPS. The washed inclusion bodies are solubilized in GuHCl for Ni purification. The Ni column was eluted with Imidazole in urea and dialyzed against 10 mM Tris pH8. The recovery of HER-2/neu polypeptide using this protocol was from 80%-95% pure full length protein with the main contaminant being degraded protein. From 500 ml of fermentation, 20 mg were recovered. It was >98% HER-2/neu polypeptide. The techniques used herein are well known to those in the art and have been described, for example, in J. Sambrook et al.. Molecular. Cloning: Ά Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, Cold Spring

Harbor, New York, USA.

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EXAMPLE 2

Dendritic Cells Can Prime Human HER-2/neîv Polypeptide

A. Generation of DC Cultures From Bone Marrow

DC cultures were generated from CD34 + hematopoietic progenitor cells (HPC). CD34+ cells were purified from bone marrow of normal donors using the cell separation system Ceprate LC Kit* (CellPro, Bothell, WA, USA) . Purity of recovered CD34+ cells was determined by flow cytometric analysis to be 80% to 95%. CD34+ cells were cultured in serum-free medium (X-VIVO 10, Biowhittaker, Inc., Walkersville, MD, USA) supplemented with L-glutamine (584 μς/1), penicillin (10 IU/ml) , streptomycin (100 μg/ml), 100 ng/ml human rGM-CSF and 50 ng/ml human rIL-6 (Immunex, Seattle, WA, USA). After 0 to 17 days of culture time, cells were harvested and used for phenotyping and T cell stimulation assays. GM-CSF alone and in combination with IL-4 or TNFa have been described to induce the in vitro growth of DC. ⁴ In experiments using

KLH and OVA as antigens to prime naive T cells, GM-CSF plus IL-6 consistently gave a comparable total stimulation, but with a lower background and thus a higher stimulation index as compared to GM-CSF plus IL-4 or TNFa.

B. T Cell Priming Assay

Bone marrow derived CD34+ HPC cultured in serumfree medium containing GM-CSF and IL-6 were used as APC after a culture period of 0-17 days. Priming ability of DC was determined by culturing them with autologous, naive T lymphocytes in the presence or absence of the protein antigen recombinant human HER-2/neu polypeptide (hHNP) (10 μς/ιηΐ) . CD4+ T lymphocytes were isolated from peripheral * trademark

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PCT/US96/01689 blood mononuclear cells by positive selection using immunoaffinity columns (CellPro, Inc., Bothell, WA, USA) . CD4 + CD45RA+ (naive) T lymphocytes were selected from CD4 + T lymphocytes using an anti-CD45RA mAb directly conjugated to FITC (Immunotech, Westbrook, ME, USA) by flow cytometric sorting. The CD4 + CD45RA+ T cells obtained were 99% pure. DC cultures were plated into 96-well round-bottomed plates (Corning, Corning, NY, USA) at various concentrations and incubated for 16-18 hours with hHNP 10 pg/ml final concentration. Antigen-pulsed DC were

<td> irradiated (10</td><td> Gy) ,</td><td> and</td><td> autologous</td><td> CD44- CD45RA+</td><td> T</td>

<td> lymphocytes were</td><td> added</td><td> (5</td><td> X 10⁴/well).</td><td colspan="2"> Proliferative</td>

<td> response of T</td><td> cells</td><td> was</td><td> measured by</td><td> the uptake</td><td> of</td>

(³H)thymidine (1 pCi/well) added on day 6 for 16-18 hours. Proliferation assays were performed in serum-free and cytokine-free medium. The results are shown in Figure 1. Figure 2 shows the results of testing CD4 + T cells, from a normal donor, for responses to hH’NP. Similar data was obtained . with T cells from nine out of ten normal individuals .

EXAMPLE 3

Assay for Detecting Low Frequency Lymphocyte Precursors

Three assays can be used for the detection of CD4⁺ responses: a standard proliferation assay, a screening method for low frequency events, and a limiting dilution assay (LDA). Conventional proliferative assays are capable of readily detecting primed responses. The proliferative response stimulation index provides a rough correlation with precursor frequency of antigen-reactive T cells. Any specific proliferative response detected from PEL is considered to be a primed response.

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To provide a more quantitative interpretation of

CD4⁺ T cell responses, the assay system developed for detecting low lymphocyte precursor frequency responses (described

This assay is simple and costeffective .

In circumstances in which more precision is needed, the precursor frequency is validated by limiting dilution assays (Bishop and Orosz,

Transplantation 47z671677, 1989).

Responses greater than detected in normal individuals are defined as a primed response and imply existent immunity. Low responses, detectable . only by

LDA conditions are considered to be unprimed responses.

An absent response by LDA or a response lower than that defined by the normal population analysis is considered to be tolerance/anergy.

In general, primed CD4⁺ T cell responses can be detected in conventional proliferative assays, whereas unprimed responses are not detectable in the same assays .

Detection of small numbers of unprimed T cells is limited by confounding background thymidine uptake including the autologous mixed lymphocyte response (AMLR) to self MHC antigen plus responses to’ processed self serum proteins and exogenously added serum proteins.

To elicit and detect unprimed

T cells, an assay system for low frequency responses based on

Poisson sampling statistics was used

Pinnacles,

Chiron

This type of analysis applies specifically to low frequency events in that, if the precursor frequency is less than the number of cells in one replicate culture, many replicates are required to detect a statistically significant number of positives.

Theoretically, the analysis will correct for autologous responses by setting up a known positive control (such as

PHA or tetanus toxoid) and known negative control (no

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PCT/US96/01689 antigen) and evaluating all data points from lowest to highest irrespective of the experimental group to which they belong. A cutoff value is calculated based on the equation cutoff = M + (F + SD) , where M = arithmetic mean, F = 3.29, a factor from tables of standardized normal distribution chosen so not more than 0.1% of the true negatives of a normally distributed background will be above the cutoff, and SD = standard deviation. In this screening assay, wells above the cutoff are considered true positives that potentially contain a lymphocyte that is specifically proliferating to the antigen of interest. Although estimations of lymphocyte precursor frequency is possible using this method, precise determination requires formal LDA analysis.

EXAMPLE 4

HER-2/ΝΕσ Polypeptide Based Vaccine Elicits

Immunity to HER-2/weu Protein

A. Animals

Rats used in this study were Fischer strain 344 (CDF (F-344)/CrlBR) (Charles River Laboratories, Portage MT). Animals were maintained at the University of

Washington Animal facilities under specific pathogen free conditions and routinely used for experimental studies between 3 and 4 months of age.

B. Immunization

Fischer rats were immunized with recombinant rat HER-2/neu polypeptide (rHNP) in a variety of adjuvants (MPL, Vaccel; Ribi, Bozeman, MT, USA) . Animals received 50 μg of rHNP mixed with adjuvant subcutaneously. Twenty days later the animals were boosted with a second

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PCT/US96/01689 immunization of 50 μς of rHNP administered in the same fashion. Twenty days after the booster immunization animals were tested for the presence of antibodies directed against rat HER-2/neu protein (neu).

<td> C. Cell</td><td> Lines</td><td></td><td></td><td></td><td></td>

<td></td><td> Two cell lines</td><td> were used as</td><td> a source</td><td> of</td><td> neu</td>

<td> proteins .</td><td> SKBR3, a human</td><td> breast cancer</td><td> cell line</td><td> that</td><td> is</td>

a marked overexpressor of HER-2/neu (American Type Culture Collection, Rockville, MD) , was maintained in culture in 10% fetal bovine serum (FBS) (Gemini Bioproducts_r Inc., Calabasas, CA) and RPMI . DHFR-G8, an NIH/3T3 cell line cotransfected with cneu-p and pSV2-DHFR (American Type Culture Collection, Rockville, MD) , was used as a source of non-transforming rat neu protein (Bernards et al., Proc. Natl. Acad. Sci. USA 54:6854-6858, 1987). This cell line was maintained in 10% FBS and Dulbecco's modified Eagle's medium with 4.5g/L glucose. DHFR-G8 cells were passaged through the same medium supplemented with 0.3 μΜ methotrexate at every third passage to maintain the neu transfectant.

D. Preparation of Cell Lysates

Lysates of both SKBR3 and DHFR-G8 were prepared and used as a source of neu protein. Briefly, a lysis buffer consisting of tris base, sodium chloride and

Triton-X* (1%) pH 7.5 was prepared.

Protease inhibitors were added; aprotinin (lpg/ml), benzamidine (lmM) and PMSF ( lmM) . 1 ml of the lysis buffer was used to suspend 10⁷cells. The cells were vortexed for 15 seconds every 10 minutes for an hour until disrupted. All procedures were performed on ice in a 4 °C cold room.

After disruption the cells were microfuged at 4°C for 20 minutes. Supernatant was removed from cell debris and stored in small aliquots * trademark

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PCT/ÜS96/01689 at -70°C until used. Presence of human and rat neu in the lysates was documented by Western blot analysis.

E. ELISA for Rat neu Antibody Responses well Immulon 4* plates (Baxter SP, Redmond, WA: Dynatech Laboratories) were incubated overnight at 4°C with a rat neu specific monoclonal antibody (Oncogene Science), 7.16.4, at a concentration of 10 μς/ml diluted in carbonate buffer (equimolar concentrations of Na₂CO₃ and NaHCO3 pH 9.6). After incubation, all wells were blocked with PBS-1% BSA (Sigma Chemical, St. Louis, MO, USA), 100 μΐ/well for 3 hours at room temperature. The plate was washed with PBS-0.5% Tween and lysates of DHFRG8, a murine cell line transfected with rat neu ONA (American Type Culture Collection, Rockville, MD, USA); a source of rat neu protein, were added to alternating rows. The plate was incubated overnight at 4°C. The plate was then washed with PBS-0.5% Tween*and experimental sera was added at the following dilutions: 1:25 to 1:200. The sera was diluted in PBS-1% BSA-1% FBS-25 μς/ιηΐ mouse IgG-0.01% NaN₃ and then serially into PBS-1% BSA. 50 μΐ of diluted sera was added/well and incubated 1 hour at room temperature. Each experimental sera was added to a well with rat neu and a well without rat neu. Sheep anti-rat Ig F(ab')₂ horseradish peroxidase (HRP) was added to the wells at a 1:5000 dilution in PBS-1% BSA and incubated for 45 minutes at room temperature (Amersham Co., Arlington Heights, IL, USA). Following the final wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, MD) developing reagent was added. Color reaction was read at an optical density of 450 nm. The OD of each serum dilution was calculated as the OD of the rat neu coated wells minus the OD of the PBS-1% BSA coated wells. Sera from animals immunized with * trademark

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PCT/ÜS96/01689 the adjuvants alone and an animal immunized with hHNP (foreign protein) were also evaluated in a similar manner. The results are shown in Figure 3.

F. T Cell Proliferation Assays

For analysis of HER-2/neu polypeptide specific responses: Fresh spleen or lymph node cells are harvested by mechanical disruption and passage through wire mesh and washed. 2 x 10⁵ spleen cells/well and 1 x 10⁵ lymph node cells/well are plated into 96-well round bottom microtiter plates (Corning, Corning, NY) with 6 replicates per experimental group. The media consists of ΞΗΑΑ 120 (Biofluids) with L-glutamine, penicillin/streptomycin, 2mercaptoethanol, and 5% FBS. Cells are incubated with polypeptides. After 4 days, wells are pulsed with 1 pCi of [³H] thymidine for 6-8 hours and counted. Data is expressed as a stimulation index (SI) which is defined as the mean of the experimental wells divided by the mean of the control wells (no antigen). For analysis of HER-2/neu protein specific responses: Spleen or lymph node cells are cultured for 3 in vitro stimulations. At the time of analysis 1 x 10⁵ cultured spleen or lymph node T cells are plated into 96 well microtiter plates as described above. Cells are incubated with lpg/ml immunoaffinity column purified rat neu (from DHFR-G8 cells as the source of rat neu) . After 4 days, wells were pulsed with 1 pCi of [³HJthymidine for 6-8 hours and counted. Data is expressed as a stimulation index which is defined as the mean of the experimental wells divided by the mean of the control wells (no antigen).

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EXAMPLE 5

Primed Responses to Human HER-2/neu Polypeptide

Can Be Detected in patients With Breast Cancer

Heparinized blood was obtained from a patient with stage

II HER-2/neu overexpressing breast cancer.

Peripheral blood mononuclear cells (PBMC) were separated by Ficoll plated at

Hypaque density centrifugation.

x 10⁵/well into 96-well a concentration of 2 round-bottomed plates (Corning,

PBMC were

Corning, NY, USA) .

well replicates were performed for each experimental group .

Antigens consisting of HER-2/neu derived peptides ' (15-20 amino acids in length with number of first amino acid in sequence listed) pg/ml, human HER-2/neu polypeptide (hHNP) pg/ml.

tetanus toxoid 1 pg/ml, and p30 a peptide derived from tetanus 25 pg/ml were added to each 24 well replicate. The assay was performed in media containing 10% human sera. Proliferative response of T cells was measured by the uptake of (³H) thymidine (1 pCi/well) added on day 4 for 10 hours. Positive wells, antigen reactive wells, were scored as positive if the cpm was greater than the mean and 3 standard deviations of the no antigen wells. The results are shown in Figure 4. This stage II breast cancer patient has a significant response to recombinant hHNP.

From the foregoing, it will be evident that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

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Sequence Listing (1) GENERAL INFORMATION:

(1) APPLICANT: University of Washington (if) TITLE OF INVENTION: COMPOUNDS FOR ELICITING OR ENHANCING IMMUNE REACTIVITY TO HER-2/neu PROTEIN FOR PREVENTION

OR TREATMENT OF MALIGNANCIES IN WHICH THE HER-2/neu ONCOGENE IS ASSOCIATED (ill) NUMBER OF SEQUENCES: 4 (Iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SEED and BERRY LLP (B) STREET: 6300 Columbia Centen, 701 Fifth Avenue (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: USA (F) ZIP: 98104-7092 (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: 28-MAR-1996 (C) CLASSIFICATION:

tviil) ATTORNEY/AGENT INFORMATION:

(A) NAME: Sharkey, Richard G.

(B) REGISTRATION NUMBER: 32,629 (C) REFERENCE/DOCKET NUMBER: 920010.448PC (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 622-4900 (B) TELEFAX: (206) 682-6031 (2) INFORMATION FOR SEQ ID NO:1:

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<td> 49</td><td></td>

<td> (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3768 base pairs (B) TYPE: nucleic acid</td><td></td>

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

(A) NAME/KEY: CDS (B) LOCATION: 1..3765 (xl) SEQUENCE DESCRIPTION: SEQ ID N0:l:

<td rowspan="2"> ATG Met 1</td><td colspan="3"> GAG CTG GCG</td><td colspan="4"> GCC TTG TGC CGC</td><td colspan="6"> TGG GGG CTC CTC CTC GCC</td><td rowspan="2"> CTC Leu 15</td><td rowspan="2"> TTG Leu</td><td rowspan="2"> 48</td>

<td> Glu</td><td colspan="2"> Leu Ala</td><td> Ala 5</td><td> Leu</td><td colspan="2"> Cys Arg</td><td> Trp</td><td> Gly 10</td><td> Leu</td><td> Leu</td><td> Leu</td><td> Ala</td>

<td> ccc</td><td> CCC</td><td> GGA</td><td> GCC</td><td> GCG</td><td> AGC</td><td> ACC</td><td> CAA</td><td> GTG</td><td> TGC</td><td> ACC</td><td> GGC</td><td> ACA</td><td> GAC</td><td> ATG</td><td> AAG</td><td> 96</td>

<td> Pro</td><td> Pro</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Ser</td><td> Thr</td><td> Gin</td><td> Vai</td><td> Cys</td><td> Thr</td><td> Gly</td><td> Thr</td><td> Asp</td><td> Met</td><td> Lys</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td>

<td> CTG</td><td> CGG</td><td> CTC</td><td> CCT</td><td> GCC</td><td> AGT</td><td> CCC</td><td> GAG</td><td> ACC</td><td> CAC</td><td> CTG</td><td> GAC</td><td> ATG</td><td> CTC</td><td> CGC</td><td> CAC</td><td> 144</td>

<td> Leu</td><td> Arg</td><td> Leu</td><td> Pro</td><td> Ala</td><td> Ser</td><td> Pro</td><td> Glu</td><td> Thr</td><td> His</td><td> Leu</td><td> Asp</td><td> Met</td><td> Leu</td><td> Arg</td><td> His</td><td></td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td> CTC</td><td> TAC</td><td> CAG</td><td> GGC</td><td> TGC</td><td> CAG</td><td> GTG</td><td> GTG</td><td> CAG</td><td> GGA</td><td> AAC</td><td> CTG</td><td> GAA</td><td> CTC</td><td> ACC</td><td> TAC</td><td> 192</td>

<td> Leu</td><td> Tyr</td><td> Gin</td><td> Gly</td><td> Cys</td><td> Gin</td><td> Vai</td><td> Vai</td><td> Gin</td><td> Gly</td><td> Asn</td><td> Leu</td><td> Glu</td><td> Leu</td><td> Thr</td><td> Tyr</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td> CTG</td><td> CCC</td><td> ACC</td><td> AAT</td><td> GCC</td><td> AGC</td><td> CTG</td><td> TCC</td><td> TTC</td><td> CTG</td><td> CAG</td><td> GAT</td><td> ATC</td><td> CAG</td><td> GAG</td><td> GTG</td><td> 240</td>

<td> Leu</td><td> Pro</td><td> Thr</td><td> Asn</td><td> Ala</td><td> Ser</td><td> Leu</td><td> Ser</td><td> Phe</td><td> Leu</td><td> Gin</td><td> Asp</td><td> lie</td><td> Gin</td><td> Glu</td><td> Vai</td><td></td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td>

<td> CAG</td><td> GGC</td><td> TAC</td><td> GTG</td><td> CTC</td><td> ATC</td><td> GCT</td><td> CAC</td><td> AAC</td><td> CAA</td><td> GTG</td><td> AGG</td><td> CAG</td><td> GTC</td><td> CCA</td><td> CTG</td><td> 288</td>

<td> Gin</td><td> Gly</td><td> Tyr</td><td> Vai</td><td> Leu</td><td> lie</td><td> Ala</td><td> His</td><td> Asn</td><td> Gin</td><td> Vai</td><td> Arg</td><td> Gin</td><td> Vai</td><td> Pro</td><td> Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td>

<td> CAG</td><td> AGG</td><td> CTG</td><td> CGG</td><td> ATT</td><td> GTG</td><td> CGA</td><td> GGC</td><td> ACC</td><td> CAG</td><td> CTC</td><td> TTT</td><td> GAG</td><td> GAC</td><td> AAC</td><td> TAT</td><td> 336</td>

<td> Gin</td><td> Arg</td><td> Leu</td><td> Arg</td><td> He</td><td> Vai</td><td> Arg</td><td> Gly</td><td> Thr</td><td> Gin</td><td> Leu</td><td> Phe</td><td> Glu</td><td> Asp</td><td> Asn</td><td> Tyr</td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

<td> GCC</td><td> CTG</td><td> GCC</td><td> GTG</td><td> CTA</td><td> GAC</td><td> AAT</td><td> GGA</td><td> GAC</td><td> CCG</td><td> CTG</td><td> AAC</td><td> AAT</td><td> ACC</td><td> ACC</td><td> CCT</td><td> 384</td>

<td> Ala</td><td> Leu</td><td> Ala</td><td> Vai</td><td> Leu</td><td> Asp</td><td> Asn</td><td colspan="2"> Gly Asp</td><td> Pro</td><td> Leu</td><td> Asn</td><td> Asn</td><td> Thr</td><td> Thr</td><td> Pro</td><td></td>

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115 120 125

<td rowspan="2"> GTC Vai</td><td rowspan="2"> ACA Thr 130</td><td colspan="11"> GGG GCC TCC CCA GGA GGC CTG CGG GAG CTG CAG</td><td colspan="2"> CTT CGA</td><td rowspan="2"> AGC Ser</td><td rowspan="2"> 432</td>

<td> Gly</td><td colspan="4"> Ala Ser Pro Gly 135</td><td> Gly</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Leu’ 140</td><td> Gin</td><td> Leu</td><td> Arg</td>

<td> CTC</td><td> ACA</td><td> GAG</td><td> ATC</td><td> TTG</td><td> AAA</td><td> GGA</td><td> GGG</td><td> GTC</td><td> TTG</td><td> ATC</td><td> CAG</td><td> CGG</td><td> AAC</td><td> CCC</td><td> CAG</td><td> 480</td>

<td> Leu</td><td> Thr</td><td> Glu</td><td> He</td><td> Leu</td><td> Lys</td><td> Gly</td><td> Gly</td><td> Vai</td><td> Leu</td><td> lie</td><td> Gin</td><td> Arg</td><td> Asn</td><td> Pro</td><td> Gin</td><td></td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td>

<td> CTC</td><td> TGC</td><td> TAC</td><td> CAG</td><td> GAC</td><td> ACG</td><td> ATT</td><td> TTG</td><td> TGG</td><td> AAG</td><td> GAC</td><td> ATC</td><td> TTC</td><td> CAC</td><td> AAG</td><td> AAC</td><td> 528</td>

<td> Leu</td><td> Cys</td><td> Tyr</td><td> Gin</td><td> Asp</td><td> Thr</td><td> He</td><td> Leu</td><td> Trp</td><td> Lys</td><td> Asp</td><td> lie</td><td> Phe</td><td> His</td><td> Lys</td><td> Asn</td><td></td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td>

<td> AAC</td><td> CAG</td><td> CTG</td><td> GCT</td><td> CTC</td><td> ACA</td><td> CTG</td><td> ATA</td><td> GAC</td><td> ACC</td><td> AAC</td><td> CGC</td><td> TCT</td><td> CGG</td><td> GCC</td><td> TGC</td><td> 576</td>

<td> Asn</td><td> Gin</td><td> Leu</td><td> Ala</td><td> Leu</td><td> Thr</td><td> Leu</td><td> lie</td><td> Asp</td><td> Thr</td><td> Asn</td><td> Arg</td><td> Ser</td><td> Arg</td><td> Ala</td><td> Cys</td><td></td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td>

<td> CAC</td><td> ccc</td><td> TGT</td><td> TCT</td><td> CCG</td><td> ATG</td><td> TGT</td><td> AAG</td><td> GGC</td><td> TCC</td><td> CGC</td><td> TGC</td><td> TGG</td><td> GGA</td><td> GAG</td><td> AGT</td><td> 624</td>

<td> His</td><td> Pro</td><td> Cys</td><td> Ser</td><td> Pro</td><td> Met</td><td> Cys</td><td> Lys</td><td> Gly</td><td> Ser</td><td> Arg</td><td> Cys</td><td> Trp</td><td> Gly</td><td> Glu</td><td> Ser</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td>

<td> TCT</td><td> GAG</td><td> GAT</td><td> TGT</td><td> CAG</td><td> AGC</td><td> CTG</td><td> ACG</td><td> CGC</td><td> ACT</td><td> GTC</td><td> TGT</td><td> GCC</td><td> GGT</td><td> GGC</td><td> TGT</td><td> 672</td>

<td> Ser</td><td> Glu</td><td> Asp</td><td> Cys</td><td> Gin</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Arg</td><td> Thr</td><td> Vai</td><td> Cys</td><td> Ala</td><td> Gly</td><td> Gly</td><td> Cys</td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td></td>

<td> GCC</td><td> CGC</td><td> TGC</td><td> AAG</td><td> GGG</td><td> CCA</td><td> CTG</td><td> CCC</td><td> ACT</td><td> GAC</td><td> TGC</td><td> TGC</td><td> CAT</td><td> GAG</td><td> CAG</td><td> TGT</td><td> 720</td>

<td> Ala</td><td> Arg</td><td> Cys</td><td> Lys</td><td> Gly</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Thr</td><td> Asp</td><td> Cys</td><td> Cys</td><td> His</td><td> Glu</td><td> Gin</td><td> Cys</td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td>

<td> GCT</td><td> GCC</td><td> GGC</td><td> TGC</td><td> ACG</td><td> GGC</td><td> CCC</td><td> AAG</td><td> CAC</td><td> TCT</td><td> GAC</td><td> TGC</td><td> CTG</td><td> GCC</td><td> TGC</td><td> CTC</td><td> 768</td>

<td> Ala</td><td> Ala</td><td> Gly</td><td> Cys</td><td> Thr</td><td> Gly</td><td> Pro</td><td> Lys</td><td> His</td><td> Ser</td><td> Asp</td><td> Cys</td><td> Leu</td><td> Ala</td><td> Cys</td><td> Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td>

<td> CAC</td><td> TTC</td><td> AAC</td><td> CAC</td><td> AGT</td><td> GGC</td><td> ATC</td><td> TGT</td><td> GAG</td><td> CTG</td><td> CAC</td><td> TGC</td><td> CCA</td><td> GCC</td><td> CTG</td><td> GTC</td><td> 816</td>

<td> HIS</td><td> Phe</td><td> Asn</td><td> His</td><td> Ser</td><td> Gly</td><td> lie</td><td> Cys</td><td> Glu</td><td> Leu</td><td> His</td><td> Cys</td><td> Pro</td><td> Ala</td><td> Leu</td><td> Vai</td><td></td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td><td></td>

<td> ACC</td><td> TAC</td><td> AAC</td><td> ACA</td><td> GAC</td><td> ACG</td><td> TTT</td><td> GAG</td><td> TCC</td><td> ATG</td><td> CCC</td><td> AAT</td><td> CCC</td><td> GAG</td><td> GGC</td><td> CGG</td><td> 864</td>

<td> Thr</td><td> Tyr</td><td> Asn</td><td> Thr</td><td> Asp</td><td> Thr</td><td> Phe</td><td> Glu</td><td> Ser</td><td> Met</td><td> Pro</td><td> Asn</td><td> Pro</td><td> Glu</td><td colspan="2"> Gly Arg</td><td></td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td><td></td>

<td> TAT</td><td> ACA</td><td> TTC</td><td> GGC</td><td> GCC</td><td> AGC</td><td> TGT</td><td> GTG</td><td> ACT</td><td> GCC</td><td> TGT</td><td> CCC</td><td> TAC</td><td> AAC</td><td> TAC</td><td> CTT</td><td> 912</td>

<td> Tyr</td><td> Thr</td><td> Phe</td><td> Gly</td><td> Ala</td><td> Ser</td><td> Cys</td><td> Vai</td><td> Thr</td><td> Ala</td><td> Cys</td><td> Pro</td><td> Tyr</td><td> Asn</td><td> Tyr</td><td> Leu</td><td></td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td></td>

CA 02216601 1997-09-25

WO 96/30514 PCT/ÜS96/01689

<td colspan="3"> TCT ACG GAC</td><td colspan="3" rowspan="2"> GTG GGA TCC Vai Gly Ser 310</td><td rowspan="2"> TGC Cys</td><td colspan="3"> ACC CTC GTC</td><td colspan="4"> TGC CCC CTG CAC</td><td rowspan="2"> AAC Asn</td><td rowspan="2"> CAA Gin 320</td><td rowspan="2"> 960</td>

<td> Ser 305</td><td> Thr</td><td> Asp</td><td> Thr</td><td colspan="2"> Leu Vai</td><td> Cys 315</td><td> Pro</td><td> Leu</td><td> His</td>

<td> GAG</td><td> GTG</td><td> ACA</td><td> GCA</td><td> GAG</td><td> GAT</td><td> GGA</td><td> ACA</td><td> CAG</td><td> CGG</td><td> TGT</td><td> GAG</td><td> AAG</td><td> TGC</td><td> AGC</td><td> AAG</td><td> 1008</td>

<td> Glu</td><td> Vai</td><td> Thr</td><td> Ala</td><td> Glu</td><td> Asp</td><td> Gly</td><td> Thr</td><td> Gin</td><td> Arg</td><td> Cys</td><td> Glu</td><td> Lys</td><td> Cys</td><td> Ser</td><td> Lys</td><td></td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td>

<td> CCC</td><td> TGT</td><td> GCC</td><td> CGA</td><td> GTG</td><td> TGC</td><td> TAT</td><td> GGT</td><td> CTG</td><td> GGC</td><td> ATG</td><td> GAG</td><td> CAC</td><td> TTG</td><td> CGA</td><td> GAG</td><td> 1056</td>

<td> Pro</td><td> Cys</td><td> Ala</td><td> Arg</td><td> Vai</td><td> Cys</td><td> Tyr</td><td> Gly</td><td> Leu</td><td> Gly</td><td> Met</td><td> Glu</td><td> His</td><td> Leu</td><td> Arg</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td>

<td> GTG</td><td> AGG</td><td> GCA</td><td> GTT</td><td> ACC</td><td> AGT</td><td> GCC</td><td> AAT</td><td> ATC</td><td> CAG</td><td> GAG</td><td> TTT</td><td> GCT</td><td> GGC</td><td> TGC</td><td> AAG</td><td> 1104</td>

<td> Vai</td><td> Arg</td><td> Ala</td><td> Vai</td><td> Thr</td><td> Ser</td><td> Ala</td><td> Asn</td><td> lie</td><td> Gin</td><td> Glu</td><td> Phe</td><td> Ala</td><td> Gly</td><td> Cys</td><td> Lys</td><td></td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td>

<td> AAG</td><td> ATC</td><td> TTT</td><td> GGG</td><td> AGC</td><td> CTG</td><td> GCA</td><td> TTT</td><td> CTG</td><td> CCG</td><td> GAG</td><td> AGC</td><td> TTT</td><td> GAT</td><td> GGG</td><td> GAC</td><td> 1152</td>

<td> Lys</td><td> He</td><td> Phe</td><td> Gly</td><td> Ser</td><td> Leu</td><td> Ala</td><td> Phe</td><td> Leu</td><td> Pro</td><td> Glu</td><td> Ser</td><td> Phe</td><td> Asp</td><td colspan="2"> Gly Asp</td><td></td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td></td>

<td> CCA</td><td> GCC</td><td> TCC</td><td> AAC</td><td> ACT</td><td> GCC</td><td> CCG</td><td> CTC</td><td> CAG</td><td> CCA</td><td> GAG</td><td> CAG</td><td> CTC</td><td> CAA</td><td> GTG</td><td> TTT</td><td> 1200</td>

<td> Pro</td><td> Ala</td><td> Ser</td><td> Asn</td><td> Thr</td><td> Ala</td><td> Pro</td><td> Leu</td><td> Gin</td><td> Pro</td><td> Glu</td><td> Gin</td><td> Leu</td><td> Gin</td><td> Vai</td><td> Phe</td><td></td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td>

<td> GAG</td><td> ACT</td><td> CTG</td><td> GAA</td><td> GAG</td><td> ATC</td><td> ACA</td><td> GGT</td><td> TAC</td><td> CTA</td><td> TAC</td><td> ATC</td><td> TCA</td><td> GCA</td><td> TGG</td><td> CCG</td><td> 1248</td>

<td> Glu</td><td> Thr</td><td> Leu</td><td> Glu</td><td> Glu</td><td> lie</td><td> Thr</td><td> Gly</td><td> Tyr</td><td> Leu</td><td> Tyr</td><td> lie</td><td> Ser</td><td> Ala</td><td> Trp</td><td> Pro</td><td></td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td>

<td> GAC</td><td> AGC</td><td> CTG</td><td> CCT</td><td> GAC</td><td> CTC</td><td> AGC</td><td> GTC</td><td> TTC</td><td> CAG</td><td> AAC</td><td> CTG</td><td> CAA</td><td> GTA</td><td> ATC</td><td> CGG</td><td> 1296</td>

<td> Asp</td><td> Ser</td><td> Leu</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Ser</td><td> Vai</td><td> Phe</td><td> Gin</td><td> Asn</td><td> Leu</td><td> Gin</td><td> Vai</td><td> lie</td><td> Arg</td><td></td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td><td></td>

<td> GGA</td><td> CGA</td><td> ATT</td><td> CTG</td><td> CAC</td><td> AAT</td><td> GGC</td><td> GCC</td><td> TAC</td><td> TCG</td><td> CTG</td><td> ACC</td><td> CTG</td><td> CAA</td><td> GGG</td><td> CTG</td><td> 1344</td>

<td> Gly</td><td> Arg</td><td> lie</td><td> Leu</td><td> His</td><td> Asn</td><td> Gly</td><td> Ala</td><td> Tyr</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Gin</td><td> Gly</td><td> Leu</td><td></td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td>

<td> GGC</td><td> ATC</td><td> AGC</td><td> TGG</td><td> CTG</td><td> GGG</td><td> CTG</td><td> CGC</td><td> TCA</td><td> CTG</td><td> AGG</td><td> GAA</td><td> CTG</td><td> GGC</td><td> AGT</td><td> GGA</td><td> 1392</td>

<td> Gly</td><td> He</td><td> Ser</td><td> Trp</td><td> Leu</td><td> Gly</td><td> Leu</td><td> Arg</td><td> Ser</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Leu</td><td> Gly</td><td> Ser</td><td> Gly</td><td></td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td></td>

<td> CTG</td><td> GCC</td><td> CTC</td><td> ATC</td><td> CAC</td><td> CAT</td><td> AAC</td><td> ACC</td><td> CAC</td><td> CTC</td><td> TGC</td><td> TTC</td><td> GTG</td><td> CAC</td><td> ACG</td><td> GTG</td><td> 1440</td>

<td> Leu</td><td> Ala</td><td> Leu</td><td> lie</td><td> His</td><td> His</td><td> Asn</td><td> Thr</td><td> His</td><td> Leu</td><td> Cys</td><td> Phe</td><td> Vai</td><td> His</td><td> Thr</td><td> Vai</td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td><td></td>

CA 02216601 1997-09-25

WO 96/30514 PCT/US96/01689

<td colspan="14"> CCC TGG GAC CAG CTC TTT CGG AAC CCG CAC CAA GCT CTG CTC</td><td rowspan="2"> CAC His 495</td><td rowspan="2"> ACT Thr</td><td rowspan="2"> 1488</td>

<td> Pro</td><td colspan="2"> Trp Asp</td><td> Gin</td><td colspan="3"> Leu Phe Arg 485</td><td colspan="2"> Asn Pro</td><td> His 490</td><td> Gin</td><td> Ala</td><td> Leu</td><td> Leu</td>

<td> GCC</td><td> AAC</td><td> CGG</td><td> CCA</td><td> GAG</td><td> GAC</td><td> GAG</td><td> TGT</td><td> GTG</td><td> GGC</td><td> GAG</td><td> GGC</td><td> CTG</td><td> GCC</td><td> TGC</td><td> CAC</td><td> 1536</td>

<td> Ala</td><td> Asn</td><td> Arg</td><td> Pro</td><td> Glu</td><td> Asp</td><td> Glu</td><td> Cys</td><td> Vai</td><td> Gly</td><td> Glu</td><td> Gly</td><td> Leu</td><td> Ala</td><td> Cys</td><td> His</td><td></td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td><td></td>

<td> CAG</td><td> CTG</td><td> TGC</td><td> GCC</td><td> CGA</td><td> GGG</td><td> CAC</td><td> TGC</td><td> TGG</td><td> GGT</td><td> CCA</td><td> GGG</td><td> CCC</td><td> ACC</td><td> CAG</td><td> TGT</td><td> 1584</td>

<td> Gin</td><td> Leu</td><td> Cys</td><td> Ala</td><td> Arg</td><td> Gly</td><td> His</td><td> Cys</td><td> Trp</td><td> Gly</td><td> Pro</td><td> Gly</td><td> Pro</td><td> Thr</td><td> Gin</td><td> Cys</td><td></td>

<td></td><td></td><td> 515</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td><td></td>

<td> GTC</td><td> AAC</td><td> TGC</td><td> AGC</td><td> CAG</td><td> TTC</td><td> CTT</td><td> CGG</td><td> GGC</td><td> CAG</td><td> GAG</td><td> TGC</td><td> GTG</td><td> GAG</td><td> GAA</td><td> TGC</td><td> 1632</td>

<td> Vai</td><td> Asn</td><td> Cys</td><td> Ser</td><td> Gin</td><td> Phe</td><td> Leu</td><td> Arg</td><td> Gly</td><td> Gin</td><td> Glu</td><td> Cys</td><td> Vai</td><td> Glu</td><td> Glu</td><td> Cys</td><td></td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td><td></td>

<td> CGA</td><td> GTA</td><td> CTG</td><td> CAG</td><td> GGG</td><td> CTC</td><td> CCC</td><td> AGG</td><td> GAG</td><td> TAT</td><td> GTG</td><td> AAT</td><td> GCC</td><td> AGG</td><td> CAC</td><td> TGT</td><td> 1680</td>

<td> Arg</td><td> Vai</td><td> Leu</td><td> Gin</td><td> Gly</td><td> Leu</td><td> Pro</td><td> Arg</td><td> Glu</td><td> Tyr</td><td> Vai</td><td> Asn</td><td> Ala</td><td> Arg</td><td> His</td><td> Cys</td><td></td>

<td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td><td></td>

<td> TTG</td><td> CCG</td><td> TGC</td><td> CAC</td><td> CCT</td><td> GAG</td><td> TGT</td><td> CAG</td><td> CCC</td><td> CAG</td><td> AAT</td><td> GGC</td><td> TCA</td><td> GTG</td><td> ACC</td><td> TGT</td><td> 1728</td>

<td> Leu</td><td> Pro</td><td> Cys</td><td> His</td><td> Pro</td><td> Glu</td><td> Cys</td><td> Gin</td><td> Pro</td><td> Gin</td><td> Asn</td><td> Gly</td><td> Ser</td><td> Vai</td><td> Thr</td><td> Cys</td><td></td>

<td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td><td></td>

<td> TTT</td><td> G6A</td><td> CCG</td><td> GAG</td><td> GCT</td><td> GAC</td><td> CAG</td><td> TGT</td><td> GTG</td><td> GCC</td><td> TGT</td><td> GCC</td><td> CAC</td><td> TAT</td><td> AAG</td><td> GAC</td><td> 1776</td>

<td> Phe</td><td> Gly</td><td> Pro</td><td> Glu</td><td> Ala</td><td> Asp</td><td> Gin</td><td> Cys</td><td> Vai</td><td> Ala</td><td> Cys</td><td> Ala</td><td> His</td><td> Tyr</td><td> Lys</td><td> Asp</td><td></td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td><td></td>

<td> CCT</td><td> CCC</td><td> TTC</td><td> TGC</td><td> GTG</td><td> GCC</td><td> CGC</td><td> TGC</td><td> CCC</td><td> AGC</td><td> GGT</td><td> GTG</td><td> AAA</td><td> CCT</td><td> GAC</td><td> CTC</td><td> 1824</td>

<td> Pro</td><td> Pro</td><td> Phe</td><td> Cys</td><td> Vai</td><td> Ala</td><td> Arg</td><td> Cys</td><td> Pro</td><td> Ser</td><td> Gly</td><td> Vai</td><td> Lys</td><td> Pro</td><td> Asp</td><td> Leu</td><td></td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td><td></td>

<td> TCC</td><td> TAC</td><td> ATG</td><td> CCC</td><td> ATC</td><td> TGG</td><td> AAG</td><td> TTT</td><td> CCA</td><td> GAT</td><td> GAG</td><td> GAG</td><td> GGC</td><td> GCA</td><td> TGC</td><td> CAG</td><td> 1872</td>

<td> Ser</td><td> Tyr</td><td> Met</td><td> Pro</td><td> lie</td><td> Trp</td><td> Lys</td><td> Phe</td><td> Pro</td><td> Asp</td><td> Glu</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Cys</td><td> Gin</td><td></td>

<td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td><td></td>

<td> CCT</td><td> TGC</td><td> CCC</td><td> ATC</td><td> AAC</td><td> TGC</td><td> ACC</td><td> CAC</td><td> TCC</td><td> TGT</td><td> GTG</td><td> GAC</td><td> CTG</td><td> GAT</td><td> GAC</td><td> AAG</td><td> 1920</td>

<td> Pro</td><td> Cys</td><td> Pro</td><td> Ils</td><td> Asn</td><td> Cys</td><td> Thr</td><td> His</td><td> Ser</td><td> Cys</td><td> Vai</td><td> Asp</td><td> Leu</td><td> Asp</td><td> Asp</td><td> Lys</td><td></td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td><td></td>

<td> GGC</td><td> TGC</td><td> CCC</td><td> GCC</td><td> GAG</td><td> CAG</td><td> AGA</td><td> GCC</td><td> AGC</td><td> CCT</td><td> CTG</td><td> ACG</td><td> TCC</td><td> ATC</td><td> ATC</td><td> TCT</td><td> 1968</td>

<td> Gly</td><td> Cys</td><td> Pro</td><td> Ala</td><td> Glu</td><td> Gin</td><td> Arg</td><td> Ala</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Ser</td><td> lie</td><td> lie</td><td> Ser</td><td></td>

645 650 655

GCG GTG GTT GGC ATT CTG CTG GTC GTG GTC TTG GGG GTG GTC

TTT GGG 2016

CA 02216601 1997-09-25

WO 96/30514 PCT/US96/01689

<td> Ala</td><td> Vai</td><td colspan="2"> Veil Gly 660</td><td colspan="2"> lie Leu</td><td colspan="2"> Leu Vai</td><td> Vai 665</td><td> Vai</td><td> Leu</td><td> Gly</td><td> Vai</td><td> Vai 670</td><td> Phe</td><td colspan="2"> Gly</td>

<td> ATC</td><td> CTC</td><td> ATC</td><td> AAG</td><td> CGA</td><td> CGG</td><td> CAG</td><td> CAG</td><td> AAG</td><td> ATC</td><td> CGG</td><td> AAG</td><td> TAC</td><td> ACG</td><td> ATG</td><td> CGG</td><td> 2064</td>

<td> Ile</td><td> Leu</td><td> lie</td><td> Lys</td><td> Arg</td><td> Arg</td><td> Gin</td><td> Gin</td><td> Lys</td><td> lie</td><td> Arg</td><td> Lys</td><td> Tyr</td><td> Thr</td><td> Met</td><td> Arg</td><td></td>

<td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td><td></td>

<td> AGA</td><td> CTG</td><td> CTG</td><td> CAG</td><td> GAA</td><td> ACG</td><td> GAG</td><td> CTG</td><td> GTG</td><td> GAG</td><td> CCG</td><td> CTG</td><td> ACA</td><td> CCT</td><td> AGC</td><td> GGA</td><td> 2112</td>

<td> Arg</td><td> Leu</td><td> Leu</td><td> Gin</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Leu</td><td> Vai</td><td> Glu</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Ser</td><td> Gly</td><td></td>

<td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td><td></td>

<td> GCG</td><td> ATG</td><td> CCC</td><td> AAC</td><td> CAG</td><td> GCG</td><td> CAG</td><td> ATG</td><td> CGG</td><td> ATC</td><td> CTG</td><td> AAA</td><td> GAG</td><td> ACG</td><td> GAG</td><td> CTG</td><td> 2160</td>

<td> Ala</td><td> Met</td><td> Pro</td><td> Asn</td><td> Gin</td><td> Ala</td><td> Gin</td><td> Met</td><td> Arg</td><td> He</td><td> Leu</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Leu</td><td></td>

<td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td><td></td>

<td> AGG</td><td> AAG</td><td> GTG</td><td> AAG</td><td> GTG</td><td> CTT</td><td> GGA</td><td> TCT</td><td> GGC</td><td> GCT</td><td> TTT</td><td> GGC</td><td> ACA</td><td> GTC</td><td> TAC</td><td> AAG</td><td> 2208</td>

<td> Arg</td><td> Lys</td><td> Vai</td><td> Lys</td><td> Vai</td><td> Leu</td><td> Gly</td><td> Ser</td><td> Gly</td><td> Ala</td><td> Phe</td><td> Gly</td><td> Thr</td><td> Vai</td><td> Tyr</td><td> Lys</td><td></td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td><td></td>

<td> GGC</td><td> ATC</td><td> TGG</td><td> ATC</td><td> CCT</td><td> GAT</td><td> GGG</td><td> GAG</td><td> AAT</td><td> GTG</td><td> AAA</td><td> ATT</td><td> CCA</td><td> GTG</td><td> GCC</td><td> ATC</td><td> 2256</td>

<td> Gly</td><td> lie</td><td> Trp</td><td> lie</td><td> Pro</td><td> Asp</td><td> Gly</td><td> Glu</td><td> Asn</td><td> Vai</td><td> Lys</td><td> lie</td><td> Pro</td><td> Vai</td><td> Al a</td><td> lie</td><td></td>

<td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td><td></td><td></td><td></td>

<td> AAA</td><td> GTG</td><td> TTG</td><td> AGG</td><td> GAA</td><td> AAC</td><td> ACA</td><td> TCC</td><td> CCC</td><td> AAA</td><td> GCC</td><td> AAC</td><td> AAA</td><td> GAA</td><td> ATC</td><td> TTA</td><td> 2304</td>

<td> Lys</td><td> Vai</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Asn</td><td> Thr</td><td> Ser</td><td> Pro</td><td> Lys</td><td> Ala</td><td> Asn</td><td> Lys</td><td> Glu</td><td> He</td><td> Leu</td><td></td>

<td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td><td></td><td></td><td></td><td></td>

<td> GAC</td><td> GAA</td><td> GCA</td><td> TAC</td><td> GTG</td><td> ATG</td><td> GCT</td><td> GGT</td><td> GTG</td><td> GGC</td><td> TCC</td><td> CCA</td><td> TAT</td><td> GTC</td><td> TCC</td><td> CGC</td><td> 2352</td>

<td> Asp</td><td> Glu</td><td> Ala</td><td> Tyr</td><td> Vai</td><td> Met</td><td> Ala</td><td> Gly</td><td> Vai</td><td> Gly</td><td> Ser</td><td> Pro</td><td> Tyr</td><td> Vai</td><td> Ser</td><td> Arg</td><td></td>

<td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td><td></td><td></td>

<td> CTT</td><td> CTG</td><td> GGC</td><td> ATC</td><td> TGC</td><td> CTG</td><td> ACA</td><td> TCC</td><td> ACG</td><td> GTG</td><td> CAG</td><td> CTG</td><td> GTG</td><td> ACA</td><td> CAG</td><td> CTT</td><td> 2400</td>

<td> Leu</td><td> Leu</td><td> Gly</td><td> lie</td><td> Cys</td><td> Leu</td><td> Thr</td><td> Ser</td><td> Thr</td><td> Vai</td><td> Gin</td><td> Leu</td><td> Vai</td><td> Thr</td><td> Gin</td><td> Leu</td><td></td>

<td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td><td></td><td></td><td></td><td> 800</td><td></td>

<td> ATG</td><td> CCC</td><td> TAT</td><td> GGC</td><td> TGC</td><td> CTC</td><td> TTA</td><td> GAC</td><td> CAT</td><td> GTC</td><td> CGG</td><td> GAA</td><td> AAC</td><td> CGC</td><td> GGA</td><td> CGC</td><td> 2448</td>

<td> Met</td><td> Pro</td><td> Tyr</td><td> Gly</td><td> Cys</td><td> Leu</td><td> Leu</td><td> Asp</td><td> His</td><td> Vai</td><td> Arg</td><td> Glu</td><td> Asn</td><td> Arg</td><td> Gly</td><td> Arg</td><td></td>

<td></td><td></td><td></td><td></td><td> 805</td><td></td><td></td><td></td><td></td><td> 810</td><td></td><td></td><td></td><td></td><td> 815</td><td></td><td></td>

<td> CTG</td><td> GGC</td><td> TCC</td><td> CAG</td><td> GAC</td><td> CTG</td><td> CTG</td><td> AAC</td><td> TGG</td><td> TGT</td><td> ATG</td><td> CAG</td><td> ATT</td><td> GCC</td><td> AAG</td><td> GGG</td><td> 2496</td>

<td> Leu</td><td> Gly</td><td> Ser</td><td> Gin</td><td> Asp</td><td> Leu</td><td> Leu</td><td> Asn</td><td> Trp</td><td> Cys</td><td> Met</td><td> Gin</td><td> lie</td><td> Ala</td><td> Lys</td><td> Gly</td><td></td>

<td></td><td></td><td></td><td> 820</td><td></td><td></td><td></td><td></td><td> 825</td><td></td><td></td><td></td><td></td><td> 830</td><td></td><td></td><td></td>

<td> ATG</td><td> AGC</td><td> TAC</td><td> CTG</td><td> GAG</td><td> GAT</td><td> GTG</td><td> CGG</td><td> CTC</td><td> GTA</td><td> CAC</td><td> AGG</td><td> GAC</td><td> TTG</td><td> GCC</td><td> GCT</td><td> 2544</td>

<td> Met</td><td> Ser</td><td> Tyr</td><td> Leu</td><td> Glu</td><td> Asp</td><td> Vai</td><td> Arg</td><td> Leu</td><td> Vai</td><td> His</td><td> Arg</td><td> Asp</td><td> Leu</td><td> Ala</td><td> Ala</td><td></td>

CA 02216601 1997-09-25

WO 96/30514 PCT/ÜS96/01689

<td></td><td></td><td> 835</td><td></td><td></td><td></td><td></td><td> 840</td><td></td><td></td><td></td><td></td><td> 845</td><td></td><td></td><td></td><td></td>

<td> CGG</td><td> AAC</td><td> GTG</td><td> CTG</td><td> GTC</td><td> AAG</td><td> AGT</td><td> CCC</td><td> AAC</td><td> CAT</td><td> GTC</td><td> AAA</td><td> ATT</td><td> ACA</td><td> GAC</td><td> TTC</td><td> 2592</td>

<td> Arg</td><td> Asn</td><td> Val</td><td> Leu</td><td> Val</td><td> Lys</td><td> Ser</td><td> Pro</td><td> Asn</td><td> His</td><td> Val</td><td> Lys</td><td> Ile</td><td> Thr</td><td rowspan="2"> Asp</td><td> Phe</td><td></td>

<td></td><td> 850</td><td></td><td></td><td></td><td></td><td> 855</td><td></td><td></td><td></td><td></td><td> 860</td><td></td><td></td><td></td><td></td>

<td> GGG</td><td> CTG</td><td> GCT</td><td> CGG</td><td> CTG</td><td> CTG</td><td> GAC</td><td> ATT</td><td> GAC</td><td> GAG</td><td> ACA</td><td> GAG</td><td> TAC</td><td> CAT</td><td> GCA</td><td> GAT</td><td> 2640</td>

<td> Gly</td><td> Leu</td><td> Al a</td><td> Arg</td><td> Leu</td><td> Leu</td><td> Asp</td><td> Ile</td><td> Asp</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Tyr</td><td> His</td><td> Al a</td><td> Asp</td><td></td>

<td> 865</td><td></td><td></td><td></td><td></td><td> 870</td><td></td><td></td><td></td><td></td><td> 875</td><td></td><td></td><td></td><td></td><td> 880</td><td></td>

<td> GGG</td><td> GGC</td><td> AAG</td><td> GTG</td><td> CCC</td><td> ATC</td><td> AAG</td><td> TGG</td><td> ATG</td><td> GCG</td><td> CTG</td><td> GAG</td><td> TCC</td><td> ATT</td><td> CTC</td><td> CGC</td><td> 2688</td>

<td> Gly</td><td> Gly</td><td> Lys</td><td> Val</td><td> Pro</td><td> Ile</td><td> Lys</td><td> Trp</td><td> Met</td><td> Al a</td><td> Leu</td><td> Glu</td><td> Ser</td><td> Ile</td><td> Leu</td><td> Arg</td><td></td>

<td></td><td></td><td></td><td></td><td> 885</td><td></td><td></td><td></td><td></td><td> 890</td><td></td><td></td><td></td><td></td><td> 895</td><td></td><td></td>

<td> CGG</td><td> CGG</td><td> TTC</td><td> ACC</td><td> CAC</td><td> CAG</td><td> AGT</td><td> GAT</td><td> GTG</td><td> TGG</td><td> AGT</td><td> TAT</td><td> GGT</td><td> GTG</td><td> ACT</td><td> GTG</td><td> 2736</td>

<td> Arg</td><td> Arg</td><td> Phe</td><td> Thr</td><td> His</td><td> Gin</td><td> Ser</td><td> Asp</td><td> Val</td><td> Trp</td><td> Ser</td><td> Tyr</td><td> Gly</td><td> Val</td><td> Thr</td><td> Val</td><td></td>

<td></td><td></td><td></td><td> 900</td><td></td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td><td></td><td> 910</td><td></td><td></td><td></td>

<td> TGG</td><td> GAG</td><td> CTG</td><td> ATG</td><td> ACT</td><td> TTT</td><td> GGG</td><td> GCC</td><td> AAA</td><td> CCT</td><td> TAC</td><td> GAT</td><td> GGG</td><td> ATC</td><td> CCA</td><td> GCC</td><td> 2784</td>

<td> Trp</td><td> Glu</td><td> Leu</td><td> Met</td><td> Thr</td><td> Phe</td><td> Gly</td><td> Al a</td><td> Lys</td><td> Pro</td><td> Tyr</td><td> Asp</td><td> Gly</td><td> Ile</td><td> Pro</td><td> Al a</td><td></td>

<td></td><td></td><td> 915</td><td></td><td></td><td></td><td></td><td> 920</td><td></td><td></td><td></td><td></td><td> 925</td><td></td><td></td><td></td><td></td>

<td> CGG</td><td> GAG</td><td> ATC</td><td> CCT</td><td> GAC</td><td> CTG</td><td> CTG</td><td> GAA</td><td> AAG</td><td> GGG</td><td> GAG</td><td> CGG</td><td> CTG</td><td> CCC</td><td> CAG</td><td> CCC</td><td> 2832</td>

<td> Arg</td><td> Glu</td><td> Ile</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Leu</td><td> Glu</td><td> Lys</td><td> Gly</td><td> Glu</td><td> Arg</td><td> Leu</td><td> Pro</td><td> Gin</td><td> Pro</td><td></td>

<td></td><td> 930</td><td></td><td></td><td></td><td></td><td> 935</td><td></td><td></td><td></td><td></td><td> 940</td><td></td><td></td><td></td><td></td><td></td>

<td> CCC</td><td> ATC</td><td> TGC</td><td> ACC</td><td> ATT</td><td> GAT</td><td> GTC</td><td> TAC</td><td> ATG</td><td> ATC</td><td> ATG</td><td> GTC</td><td> AAA</td><td> TGT</td><td> TGG</td><td> ATG</td><td> 2880</td>

<td> Pro</td><td> Ile</td><td> Cys</td><td> Thr</td><td> Ile</td><td> Asp</td><td> Val</td><td> Tyr</td><td> Met</td><td> Ile</td><td> Met</td><td> Val</td><td> Lys</td><td> Cys</td><td> Trp</td><td> Met</td><td></td>

<td> 945</td><td></td><td></td><td></td><td></td><td> 950</td><td></td><td></td><td></td><td></td><td> 955</td><td></td><td></td><td></td><td></td><td> 960</td><td></td>

<td> ATT</td><td> GAC</td><td> TCT</td><td> GAA</td><td> TGT</td><td> CGG</td><td> CCA</td><td> AGA</td><td> TTC</td><td> CGG</td><td> GAG</td><td> TTG</td><td> GTG</td><td> TCT</td><td> GAA</td><td> TTC</td><td> 2928</td>

<td> Ile</td><td> Asp</td><td> Ser</td><td> Glu</td><td> Cys</td><td> Arg</td><td> Pro</td><td> Arg</td><td> Phe</td><td> Arg</td><td> Glu</td><td> Leu</td><td> Val</td><td> Ser</td><td> Glu</td><td> Phe</td><td></td>

<td></td><td></td><td></td><td></td><td> 965</td><td></td><td></td><td></td><td></td><td> 970</td><td></td><td></td><td></td><td></td><td> 975</td><td></td><td></td>

<td> TCC</td><td> CGC</td><td> ATG</td><td> GCC</td><td> AGG</td><td> GAC</td><td> CCC</td><td> CAG</td><td> CGC</td><td> TTT</td><td> GTG</td><td> GTC</td><td> ATC</td><td> CAG</td><td> AAT</td><td> GAG</td><td> 2976</td>

<td> Ser</td><td> Arg</td><td> Met</td><td> Al a</td><td> Arg</td><td> Asp</td><td> Pro</td><td> Gin</td><td> Arg</td><td> Phe</td><td> Val</td><td> Val</td><td> Ile</td><td> Gin</td><td> Asn</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td> 980</td><td></td><td></td><td></td><td></td><td> 985</td><td></td><td></td><td></td><td></td><td> 990</td><td></td><td></td><td></td>

<td> GAC</td><td> TTG</td><td> GGC</td><td> CCA</td><td> GCC</td><td> AGT</td><td> CCC</td><td> TTG</td><td> GAC</td><td> AGC</td><td> ACC</td><td> TTC</td><td> TAC</td><td> CGC</td><td> TCA</td><td> CTG</td><td> 3024</td>

<td> Asp</td><td> Leu</td><td> Gly</td><td> Pro</td><td> Al a</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Asp</td><td> Ser</td><td> Thr</td><td> Phe</td><td> Tyr</td><td> Arg</td><td> Ser</td><td> Leu</td><td></td>

<td></td><td></td><td> 995</td><td></td><td></td><td></td><td></td><td> 1001</td><td> 3</td><td></td><td></td><td></td><td> 100!</td><td> 5</td><td></td><td></td><td></td>

<td> CTG</td><td> GAG</td><td> GAC</td><td> GAT</td><td> GAC</td><td> ATG</td><td> GGG</td><td> GAC</td><td> CTG</td><td> GTG</td><td> GAT</td><td> GCT</td><td> GAG</td><td> GAG</td><td> TAT</td><td> CTG</td><td> 3072</td>

<td> Leu</td><td> Glu</td><td> Asp</td><td> Asp</td><td> Asp</td><td> Met</td><td> Gly</td><td> Asp</td><td> Leu</td><td> Val</td><td> Asp</td><td> Al a</td><td> Glu</td><td> Glu</td><td> Tyr</td><td> Leu</td><td></td>

1010 1015 1020

CA 02216601 1997-09-25

WO 96/30514 PCT/US96/01689

<td> GTA</td><td> CCC</td><td> CAG</td><td> CAG</td><td> GGC</td><td> TTC</td><td> TTC</td><td> TGT</td><td> CCA</td><td> GAC</td><td colspan="2"> CCT GCC</td><td> CCG</td><td> GGC</td><td> GCT</td><td> GGG</td><td> 3120</td>

<td> Vai</td><td> Pro</td><td> Gin</td><td> Gin</td><td> Gly</td><td> Phe</td><td> Phe</td><td> Cys</td><td> Pro</td><td> Asp</td><td> Pro</td><td> Ala</td><td> Pro</td><td> Gly</td><td> Ala</td><td> Gly</td><td></td>

<td colspan="2"> 1025</td><td></td><td></td><td></td><td colspan="2"> 1030</td><td></td><td></td><td></td><td colspan="2"> 1035</td><td></td><td></td><td></td><td> 1040</td><td></td>

<td> GGC</td><td> ATG</td><td> GTC</td><td> CAC</td><td> CAC</td><td> AGG</td><td> CAC</td><td> CGC</td><td> AGC</td><td> TCA</td><td> TCT</td><td> ACC</td><td> AGG</td><td> AGT</td><td> GGC</td><td> GGT</td><td> 3168</td>

<td> Gly</td><td> Met</td><td> Vai</td><td> His</td><td> His</td><td> Arg</td><td> His</td><td> Arg</td><td> Ser</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Arg</td><td> Ser</td><td> Gly</td><td> Gly</td><td></td>

<td></td><td></td><td></td><td></td><td colspan="2"> 1045</td><td></td><td></td><td></td><td colspan="2"> 1050</td><td></td><td></td><td></td><td colspan="2"> 1055</td><td></td>

<td> GGG</td><td> GAC</td><td> CTG</td><td> ACA</td><td> CTA</td><td> GGG</td><td> CTG</td><td> GAG</td><td> CCC</td><td> TCT</td><td> GAA</td><td> GAG</td><td> GAG</td><td> GCC</td><td> CCC</td><td> AGG</td><td> 3216</td>

<td> Gly</td><td> Asp</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Gly</td><td> Leu</td><td> Glu</td><td> Pro</td><td> Ser</td><td> Glu</td><td> Glu</td><td> Glu</td><td> Ala</td><td> Pro</td><td> Arg</td><td></td>

<td></td><td></td><td></td><td colspan="2"> 1060</td><td></td><td></td><td></td><td colspan="2"> 1065</td><td></td><td></td><td></td><td colspan="2"> 1070</td><td></td><td></td>

<td> TCT</td><td> CCA</td><td> CTG</td><td> GCA</td><td> CCC</td><td> TCC</td><td> GAA</td><td> GGG</td><td> GCT</td><td> GGC</td><td> TCC</td><td> GAT</td><td> GTA</td><td> TTT</td><td> GAT</td><td> GGT</td><td> 3264</td>

<td> Ser</td><td> Pro</td><td> Leu</td><td> Ala</td><td> Pro</td><td> Ser</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Vai</td><td> Phe</td><td> Asp</td><td> Gly</td><td></td>

<td></td><td></td><td colspan="2"> 1075</td><td></td><td></td><td></td><td colspan="2"> 1080</td><td></td><td></td><td></td><td colspan="2"> 1085</td><td></td><td></td><td></td>

<td> GAC</td><td> CTG</td><td> GGA</td><td> ATG</td><td> GGG</td><td> GCA</td><td> GCC</td><td> AAG</td><td> GGG</td><td> CTG</td><td> CAA</td><td> AGC</td><td> CTC</td><td> CCC</td><td> ACA</td><td> CAT</td><td> 3312</td>

<td> Asp</td><td> Leu</td><td> Gly</td><td> Met</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Lys</td><td> Gly</td><td> Leu</td><td> Gin</td><td> Ser</td><td> Leu</td><td> Pro</td><td> Thr</td><td> Hi s</td><td></td>

<td></td><td colspan="2"> 1090</td><td></td><td></td><td></td><td colspan="2"> 1095</td><td></td><td></td><td></td><td colspan="2"> 1100</td><td></td><td></td><td></td><td></td>

<td> GAC</td><td> CCC</td><td> AGC</td><td> CCT</td><td> CTA</td><td> CAG</td><td> CGG</td><td> TAC</td><td> AGT</td><td> GAG</td><td> GAC</td><td> CCC</td><td> ACA</td><td> GTA</td><td> CCC</td><td> CTG</td><td> 3360</td>

<td> Asp</td><td> Pro</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Gin</td><td> Arg</td><td> Tyr</td><td> Ser</td><td> Glu</td><td> Asp</td><td> Pro</td><td> Thr</td><td> Vai</td><td> Pro</td><td> Leu</td><td></td>

<td colspan="2"> 1105</td><td></td><td></td><td></td><td colspan="2"> 1110</td><td></td><td></td><td></td><td colspan="2"> 1115</td><td></td><td></td><td></td><td> 1120</td><td></td>

<td> ccc</td><td> TCT</td><td> GAG</td><td> ACT</td><td> GAT</td><td> GGC</td><td> TAC</td><td> GTT</td><td> GCC</td><td> CCC</td><td> CTG</td><td> ACC</td><td> TGC</td><td> AGC</td><td> CCC</td><td> CAG</td><td> 3408</td>

<td> Pro</td><td> Ser</td><td> Glu</td><td> Thr</td><td> Asp</td><td colspan="2"> Gly Tyr</td><td> Vai</td><td> Ala</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Cys</td><td> Ser</td><td> Pro</td><td> Gin</td><td></td>

<td></td><td></td><td></td><td></td><td colspan="2"> 1125</td><td></td><td></td><td></td><td colspan="2"> 1130</td><td></td><td></td><td></td><td colspan="2"> 1135</td><td></td>

<td> CCT</td><td> GAA</td><td> TAT</td><td> GTG</td><td> AAC</td><td> CAG</td><td> CCA</td><td> GAT</td><td> GTT</td><td> CGG</td><td> CCC</td><td> CAG</td><td> CCC</td><td> CCT</td><td> TCG</td><td> CCC</td><td> 3456</td>

<td> Pro</td><td> Glu</td><td> Tyr</td><td> Vai</td><td> Asn</td><td> Gin</td><td> Pro</td><td> Asp</td><td> Vai</td><td> Arg</td><td> Pro</td><td> Gin</td><td> Pro</td><td> Pro</td><td> Ser</td><td> Pro</td><td></td>

<td></td><td></td><td></td><td colspan="2"> 1140</td><td></td><td></td><td></td><td colspan="2"> 1145</td><td></td><td></td><td></td><td colspan="2"> 1150</td><td></td><td></td>

<td> CGA</td><td> GAG</td><td> GGC</td><td> CCT</td><td> CTG</td><td> CCT</td><td> GCT</td><td> GCC</td><td> CGA</td><td> CCT</td><td> GCT</td><td> GGT</td><td> GCC</td><td> ACT</td><td> CTG</td><td> GAA</td><td> 3504</td>

<td> Arg</td><td> Glu</td><td> Gly</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Ala</td><td> Ala</td><td> Arg</td><td> Pro</td><td> Ala</td><td> Gly</td><td> Ala</td><td> Thr</td><td> Leu</td><td> Glu</td><td></td>

<td></td><td></td><td colspan="2"> 1155</td><td></td><td></td><td></td><td colspan="2"> 1160</td><td></td><td></td><td></td><td colspan="2"> 1165</td><td></td><td></td><td></td>

<td> AGG</td><td> CCC</td><td> AAG</td><td> ACT</td><td> CTC</td><td> TCC</td><td> CCA</td><td> GGG</td><td> AAG</td><td> AAT</td><td> GGG</td><td> GTC</td><td> GTC</td><td> AAA</td><td> GAC</td><td> GTT</td><td> 3552</td>

<td> Arg</td><td> Pro</td><td> Lys</td><td> Thr</td><td> Leu</td><td> Ser</td><td> Pro</td><td> Gly</td><td> Lys</td><td> Asn</td><td> Gly</td><td> Vai</td><td> Vai</td><td> Lys</td><td> Asp</td><td> Vai</td><td></td>

<td></td><td colspan="2"> 1170</td><td></td><td></td><td></td><td colspan="2"> 1175</td><td></td><td></td><td></td><td colspan="2"> 1180</td><td></td><td></td><td></td><td></td>

<td> TTT</td><td> GCC</td><td> TTT</td><td> GGG</td><td> GGT</td><td> GCC</td><td> GTG</td><td> GAG</td><td> AAC</td><td> CCC</td><td> GAG</td><td colspan="2"> TAC TTG</td><td> ACA</td><td> CCC</td><td> CAG</td><td> 3600</td>

<td> Phe</td><td> Ala</td><td> Phe</td><td colspan="2"> Gly Gly</td><td> Ala</td><td> Vai</td><td> Glu</td><td> Asn</td><td> Pro</td><td> Glu</td><td colspan="2"> Tyr Leu</td><td> Thr</td><td> Pro</td><td> Gin</td><td></td>

1185 1190 1195 1200

CA 02216601 1997-09-25

WO 96/30514 PCT/ÜS96/01689

<td> GGA</td><td> GGA</td><td> GCT</td><td> GCC</td><td> CCT</td><td> CAG</td><td> CCC</td><td> CAC</td><td> CCT</td><td> CCT</td><td> CCT</td><td> GCC</td><td> TTC</td><td> AGC</td><td> CCA</td><td> GCC</td><td> 3648</td>

<td> Gly</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Pro</td><td> Gin</td><td> Pro</td><td> Hi s</td><td> Pro</td><td> Pro</td><td> Pro</td><td> Ala</td><td> Phe</td><td> Ser</td><td> Pro</td><td> Ala</td><td></td>

<td></td><td></td><td></td><td></td><td colspan="2"> 1205</td><td></td><td></td><td></td><td colspan="2"> 1210</td><td></td><td></td><td></td><td colspan="2"> 1215</td><td></td>

<td> TTC</td><td> GAC</td><td> AAC</td><td> CTC</td><td> TAT</td><td> TAC</td><td> TGG</td><td> GAC</td><td> CAG</td><td> GAC</td><td> CCA</td><td> CCA</td><td> GAG</td><td> CGG</td><td> GGG</td><td> GCT</td><td> 3696</td>

<td> Phe</td><td> Asp</td><td> Asn</td><td> Leu</td><td> Tyr</td><td> Tyr</td><td> Trp</td><td> Asp</td><td> Gin</td><td> Asp</td><td> Pro</td><td> Pro</td><td> Glu</td><td> Arg</td><td> Gly</td><td> Ala</td><td></td>

<td></td><td></td><td></td><td colspan="2"> 1220</td><td></td><td></td><td></td><td colspan="2"> 1225</td><td></td><td></td><td></td><td colspan="2"> 1230</td><td></td><td></td>

<td> CCA</td><td> CCC</td><td> AGC</td><td> ACC</td><td> TTC</td><td> AAA</td><td> GGG</td><td> ACA</td><td> CCT</td><td> ACG</td><td> GCA</td><td> GAG</td><td> AAC</td><td> CCA</td><td> GAG</td><td> TAC</td><td> 3744</td>

<td> Pro</td><td> Pro</td><td> Ser</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Gly</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Ala</td><td> Glu</td><td> Asn</td><td> Pro</td><td> Glu</td><td> Tyr</td><td></td>

<td></td><td></td><td colspan="2"> 1235</td><td></td><td></td><td></td><td colspan="2"> 1240</td><td></td><td></td><td></td><td colspan="2"> 1245</td><td></td><td></td><td></td>

<td> CTG</td><td> GGT</td><td> CTG</td><td> GAC</td><td> GTG</td><td> CCA</td><td> GTG</td><td> TGA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 3768</td>

<td> Leu</td><td> Gly</td><td> Leu</td><td> Asp</td><td> Vai</td><td> Pro</td><td> Vai</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

1250 1255 (2) INFORMATION FOR SEQ ID N0:2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1255 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (11) MOLECULE TYPE: protein (xl) SEQUENCE DESCRIPTION: SEQ ID NO:2:

<td> Met</td><td> Glu</td><td> Leu</td><td> Ala</td><td> Ala</td><td> Leu</td><td> Cys</td><td> Arg</td><td> Trp</td><td> Gly</td><td> Leu</td><td> Leu</td><td> Leu</td><td> Ala</td><td> Leu</td><td> Leu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td> Pro</td><td> Pro</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Ser</td><td> Thr</td><td> Gin</td><td> Vai</td><td> Cys</td><td> Thr</td><td> Gly</td><td> Thr</td><td> Asp</td><td> Met</td><td> Lys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td> Leu</td><td> Arg</td><td> Leu</td><td> Pro</td><td> Ala</td><td> Ser</td><td> Pro</td><td> Glu</td><td> Thr</td><td> His</td><td> Leu</td><td> Asp</td><td> Met</td><td> Leu</td><td> Arg</td><td> His</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td> Leu</td><td> Tyr</td><td> Gin</td><td> Gly</td><td> Cys</td><td> Gin</td><td> Vai</td><td> Vai</td><td> Gin</td><td> Gly</td><td> Asn</td><td> Leu</td><td> Glu</td><td> Leu</td><td> Thr</td><td> Tyr</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td> Leu</td><td> Pro</td><td> Thr</td><td> Asn</td><td> Ala</td><td> Ser</td><td> Leu</td><td> Ser</td><td> Phe</td><td> Leu</td><td> Gin</td><td> Asp</td><td> lie</td><td> Gin</td><td> Glu</td><td> Vai</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td> Gin</td><td> Gly</td><td> Tyn</td><td> Vai</td><td> Leu</td><td> lie</td><td> Ala</td><td> His</td><td> Asn</td><td> Gin</td><td> Vai</td><td> Arg</td><td> Gin</td><td> Vai</td><td> Pro</td><td> Leu</td>

90 95

CA 02216601 1997-09-25

WO 96/30514

PCT7US96/01689

<td colspan="5"> Gin Arg Leu Arg Ile</td><td colspan="3" rowspan="2"> Val Arg Gly</td><td colspan="2" rowspan="2"> Thr Gin 105</td><td colspan="2" rowspan="2"> Leu Phe</td><td colspan="2" rowspan="2"> Glu Asp 110</td><td rowspan="2"> Asn</td><td rowspan="2"> Tyr</td>

<td></td><td colspan="4"> 100</td>

<td> Al a</td><td> Leu</td><td> Al a</td><td> Val</td><td> Leu</td><td colspan="4"> Asp Asn Gly Asp</td><td> Pro</td><td> Leu</td><td> Asn</td><td> Asn</td><td> Thr</td><td> Thr</td><td> Pro</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td> Val</td><td> Thr</td><td> Gly</td><td> Al a</td><td> Ser</td><td> Pro</td><td colspan="2"> Gly Gly</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Leu</td><td> Gin</td><td> Leu</td><td> Arg</td><td> Ser</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td> Leu</td><td> Thr</td><td> Glu</td><td> Ile</td><td> Leu</td><td> Lys</td><td colspan="2"> Gly Gly</td><td> Val</td><td> Leu</td><td> Ile</td><td> Gin</td><td> Arg</td><td> Asn</td><td> Pro</td><td> Gin</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td> Leu</td><td> Cys</td><td> Tyr</td><td> Gin</td><td> Asp</td><td> Thr</td><td> Ile</td><td> Leu</td><td> Trp</td><td> Lys</td><td> Asp</td><td> Ile</td><td> Phe</td><td> His</td><td> Lys</td><td> Asn</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td> Asn</td><td> Gin</td><td> Leu</td><td> Al a</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Ile</td><td> Asp</td><td> Thr</td><td> Asn</td><td> Arg</td><td> Ser</td><td> Arg</td><td> Al a</td><td> Cys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td> Hi s</td><td> Pro</td><td> Cys</td><td> Ser</td><td> Pro</td><td> Met</td><td> Cys</td><td> Lys</td><td> Gly</td><td> Ser</td><td> Arg</td><td> Cys</td><td colspan="2"> Trp Gly</td><td> Glu</td><td> Ser</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td> Ser</td><td> Glu</td><td> Asp</td><td> Cys</td><td> Gin</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Arg</td><td> Thr</td><td> Val</td><td> Cys</td><td> Al a</td><td> Gly</td><td> Gly</td><td> Cys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td> Al a</td><td> Arg</td><td> Cys</td><td> Lys</td><td> Gly</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Thr</td><td> Asp</td><td> Cys</td><td> Cys</td><td> His</td><td> Glu</td><td> Gin</td><td> Cys</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td> Al a</td><td> Al a</td><td> Gly</td><td> Cys</td><td> Thr</td><td> Gly</td><td> Pro</td><td> Lys</td><td> His</td><td> Ser</td><td> Asp</td><td> Cys</td><td> Leu</td><td> Al a</td><td> Cys</td><td> Leu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td> His</td><td> Phe</td><td> Asn</td><td> His</td><td> Ser</td><td> Gly</td><td> Ile</td><td> Cys</td><td> Glu</td><td> Leu</td><td> His</td><td> Cys</td><td> Pro</td><td> Al a</td><td> Leu</td><td> Val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td> Thr</td><td> Tyr</td><td> Asn</td><td> Thr</td><td> Asp</td><td> Thr</td><td> Phe</td><td> Glu</td><td> Ser</td><td> Met</td><td> Pro</td><td> Asn</td><td> Pro</td><td> Glu</td><td colspan="2"> Gly Arg</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td> Tyr</td><td> Thr</td><td> Phe</td><td> Gly</td><td> Al a</td><td> Ser</td><td> Cys</td><td> Val</td><td> Thr</td><td> Al a</td><td> Cys</td><td> Pro</td><td> Tyr</td><td> Asn</td><td> Tyr</td><td> Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td> Ser</td><td> Thr</td><td> Asp</td><td> Val</td><td> Gly</td><td> Ser</td><td> Cys</td><td> Thr</td><td> Leu</td><td> Val</td><td> Cys</td><td> Pro</td><td> Leu</td><td> His</td><td> Asn</td><td> Gin</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

Glu Val Thr Al a Glu Asp Gly Thr Gin Arg Cys Glu Lys Cys Ser Lys

325 330 335

CA 02216601 1997-09-25

WO 96/30514

PCT/ÜS96/01689

<td colspan="4"> Pro Cys Ala Arg</td><td colspan="3" rowspan="2"> Val Cys Tyr</td><td rowspan="2"> Gly</td><td colspan="2" rowspan="2"> Leu Gly 345</td><td colspan="2" rowspan="2"> Met Glu</td><td rowspan="2"> His</td><td rowspan="2"> Leu 350</td><td rowspan="2"> Arg</td><td rowspan="2"> Glu</td>

<td colspan="2"></td><td colspan="2"> 340</td>

<td> Val</td><td> Arg</td><td> Ala</td><td> Val</td><td> Thr</td><td> Ser</td><td> Ala</td><td> Asn</td><td> He</td><td> Gin</td><td> Glu</td><td> Phe</td><td> Ala</td><td> Gly</td><td> Cys</td><td> Lys</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td> Lys</td><td> lie</td><td> Phe</td><td> Gly</td><td> Ser</td><td> Leu</td><td> Ala</td><td> Phe</td><td> Leu</td><td> Pro</td><td> Glu</td><td> Ser</td><td> Phe</td><td> Asp</td><td> Gly</td><td> Asp</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td> Pro</td><td> Ala</td><td> Ser</td><td> Asn</td><td> Thr</td><td> Ala</td><td> Pro</td><td> Leu</td><td> Gin</td><td> Pro</td><td> Glu</td><td> Gin</td><td> Leu</td><td> Gin</td><td> Val</td><td> Phe</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td> Glu</td><td> Thr</td><td> Leu</td><td> Glu</td><td> Glu</td><td> lie</td><td> Thr</td><td colspan="2"> Gly Tyr</td><td> Leu</td><td> Tyr</td><td> lie</td><td> Ser</td><td> Ala</td><td> Trp</td><td> Pro</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td> Asp</td><td> Ser</td><td> Leu</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Ser</td><td> Val</td><td> Phe</td><td> Gin</td><td> Asn</td><td> Leu</td><td> Gin</td><td> Val</td><td> lie</td><td> Arg</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td colspan="2"> Gly Arg</td><td> He</td><td> Leu</td><td> His</td><td> Asn</td><td> Gly</td><td> Ala</td><td> Tyr</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Gin</td><td> Gly</td><td> Leu</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td> Gly</td><td> lie</td><td> Ser</td><td> Trp</td><td> Leu</td><td> Gly</td><td> Leu</td><td> Arg</td><td> Ser</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Leu</td><td> Gly</td><td> Ser</td><td> Gly</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td> Leu</td><td> Ala</td><td> Leu</td><td> lie</td><td> His</td><td> Hi s</td><td> Asn</td><td> Thr</td><td> His</td><td> Leu</td><td> Cys</td><td> Phe</td><td> Val</td><td> His</td><td> Thr</td><td> Val</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td> Pro</td><td colspan="2"> Trp Asp</td><td> Gin</td><td> Leu</td><td> Phe</td><td> Arg</td><td> Asn</td><td> Pro</td><td> His</td><td> Gin</td><td> Ala</td><td> Leu</td><td> Leu</td><td> His</td><td> Thr</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td> Ala</td><td> Asn</td><td> Arg</td><td> Pro</td><td> Glu</td><td> Asp</td><td> Glu</td><td> Cys</td><td> Val</td><td> Gly</td><td> Glu</td><td> Gly</td><td> Leu</td><td> Ala</td><td> Cys</td><td> His</td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td>

<td> Gin</td><td> Leu</td><td> Cys</td><td> Ala</td><td> Arg</td><td> Gly</td><td> His</td><td> Cys</td><td> Trp</td><td> Gly</td><td> Pro</td><td> Gly</td><td> Pro</td><td> Thr</td><td> Gin</td><td> Cys</td>

<td></td><td></td><td> 515</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td>

<td> Val</td><td> Asn</td><td> Cys</td><td> Ser</td><td> Gin</td><td> Phe</td><td> Leu</td><td> Arg</td><td> Gly</td><td> Gin</td><td> Glu</td><td> Cys</td><td> Val</td><td> Glu</td><td> Glu</td><td> Cys</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td> Arg</td><td> Val</td><td> Leu</td><td> Gin</td><td> Gly</td><td> Leu</td><td> Pro</td><td> Arg</td><td> Glu</td><td> Tyr</td><td> Val</td><td> Asn</td><td> Ala</td><td> Arg</td><td> His</td><td> Cys</td>

545 550 555 550

Leu Pro Cys His Pro Glu Cys Gin Pro Gin Asn Gly Ser Vai Thr Cys

565 570 575

CA 02216601 1997-09-25

WO 96/30514

PCT/US96/01689

<td colspan="5"> Phe Gly Pro Glu Ala</td><td rowspan="2"> Asp</td><td rowspan="2"> Gin</td><td rowspan="2"> Cys</td><td rowspan="2"> Vai 585</td><td colspan="3" rowspan="2"> Ala Cys Ala</td><td rowspan="2"> His</td><td rowspan="2"> Tyr 590</td><td rowspan="2"> Lys</td><td rowspan="2"> Asp</td>

<td colspan="3"></td><td colspan="2"> 580</td>

<td> Pro</td><td> Pro</td><td> Phe</td><td> Cys</td><td> Vai</td><td> Ala</td><td> Arg</td><td> Cys</td><td> Pro</td><td> Ser</td><td> Gly</td><td> Vai</td><td> Lys</td><td> Pro</td><td> Asp</td><td> Leu</td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td>

<td> Ser</td><td> Tyr</td><td> Met</td><td> Pro</td><td> lie</td><td> Trp</td><td> Lys</td><td> Phe</td><td> Pro</td><td> Asp</td><td> Glu</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Cys</td><td> Gin</td>

<td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td>

<td> Pro</td><td> Cys</td><td> Pro</td><td> He</td><td> Asn</td><td> Cys</td><td> Thr</td><td> His</td><td> Ser</td><td> Cys</td><td> Vai</td><td> Asp</td><td> Leu</td><td colspan="2"> Asp Asp</td><td> Lys</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td>

<td> Gly</td><td> Cys</td><td> Pro</td><td> Ala</td><td> Glu</td><td> Gin</td><td> Arg</td><td> Ala</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Ser</td><td> He</td><td> He</td><td> Ser</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td> Ala</td><td> Vai</td><td> Vai</td><td> Gly</td><td> lie</td><td> Leu</td><td> Leu</td><td> Vai</td><td> Vai</td><td> Vai</td><td> Leu</td><td> Gly</td><td> Vai</td><td> Vai</td><td> Phe</td><td> Gly</td>

<td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td> lie</td><td> Leu</td><td> lie</td><td> Lys</td><td> Arg</td><td> Arg</td><td> Gin</td><td> Gin</td><td> Lys</td><td> lie</td><td> Arg</td><td> Lys</td><td> Tyr</td><td> Thr</td><td> Met</td><td> Arg</td>

<td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td> Arg</td><td> Leu</td><td> Leu</td><td> Gin</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Leu</td><td> Vai</td><td> Glu</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Ser</td><td> Gly</td>

<td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td>

<td> Ala</td><td> Met</td><td> Pro</td><td> Asn</td><td> Gin</td><td> Ala</td><td> Gin</td><td> Met</td><td> Arg</td><td> He</td><td> Leu</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Leu</td>

<td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td> Arg</td><td> Lys</td><td> Vai</td><td> Lys</td><td> Vai</td><td> Leu</td><td> Gly</td><td> Ser</td><td> Gly</td><td> Ala</td><td> Phe</td><td> Gly</td><td> Thr</td><td> Vai</td><td> Tyr</td><td> Lys</td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td>

<td> Gly</td><td> He</td><td> Trp</td><td> lie</td><td> Pro</td><td> Asp</td><td> Gly</td><td> Glu</td><td> Asn</td><td> Vai</td><td> Lys</td><td> He</td><td> Pro</td><td> Vai</td><td> Ala</td><td> lie</td>

<td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td><td></td><td></td>

<td> Lys</td><td> Vai</td><td> Leu</td><td> Arg</td><td> Glu</td><td> Asn</td><td> Thr</td><td> Ser</td><td> Pro</td><td> Lys</td><td> Ala</td><td> Asn</td><td> Lys</td><td> Glu</td><td> lie</td><td> Leu</td>

<td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td><td></td><td></td><td></td>

<td> Asp</td><td> Glu</td><td> Ala</td><td> Tyr</td><td> Vai</td><td> Met</td><td> Ala</td><td> Gly</td><td> Vai</td><td> Gly</td><td> Ser</td><td> Pro</td><td> Tyr</td><td> Vai</td><td> Ser</td><td> Arg</td>

<td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td><td></td>

<td> Leu</td><td> Leu</td><td> Gly</td><td> lie</td><td> Cys</td><td> Leu</td><td> Thr</td><td> Ser</td><td> Thr</td><td> Vai</td><td> Gin</td><td> Leu</td><td> Vai</td><td> Thr</td><td> Gin</td><td> Leu</td>

<td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td><td></td><td></td><td></td><td> 800</td>

<td> Met</td><td> Pro</td><td> Tyr</td><td> Gly</td><td> Cys</td><td> Leu</td><td> Leu</td><td> Asp</td><td> His</td><td> Vai</td><td> Arg</td><td> Glu</td><td> Asn</td><td> Arg</td><td> Gly</td><td> Arg</td>

810

805

815

CA 02216601 1997-09-25

WO 96/30514

PCT/US96/01689

<td colspan="10"> Leu Gly Ser Gin Asp Leu Leu Asn Trp Cys</td><td colspan="2" rowspan="2"> Met Gin</td><td rowspan="2"> Ile</td><td rowspan="2"> Ala 830</td><td rowspan="2"> Lys</td><td rowspan="2"> Gly</td>

<td colspan="2"></td><td colspan="3"> 820</td><td colspan="5"> 825</td>

<td> Met</td><td> Ser</td><td> Tyr</td><td> Leu</td><td> Glu</td><td> Asp</td><td> Val</td><td> Arg</td><td> Leu</td><td> Val</td><td> His</td><td> Arg</td><td> Asp</td><td> Leu</td><td> Ala</td><td> Ala</td>

<td></td><td></td><td> 835</td><td></td><td></td><td></td><td></td><td> 840</td><td></td><td></td><td></td><td></td><td> 845</td><td></td><td></td><td></td>

<td> Arg</td><td> Asn</td><td> Val</td><td> Leu</td><td> Val</td><td> Lys</td><td> Ser</td><td> Pro</td><td> Asn</td><td> His</td><td> Val</td><td> Lys</td><td> Ile</td><td> Thr</td><td> Asp</td><td> Phe</td>

<td></td><td> 850</td><td></td><td></td><td></td><td></td><td> 855</td><td></td><td></td><td></td><td></td><td> 860</td><td></td><td></td><td></td><td></td>

<td> Gly</td><td> Leu</td><td> Al a</td><td> Arg</td><td> Leu</td><td> Leu</td><td> Asp</td><td> Ile</td><td> Asp</td><td> Glu</td><td> Thr</td><td> Glu</td><td> Tyr</td><td> His</td><td> Ala</td><td> Asp</td>

<td> 865</td><td></td><td></td><td></td><td></td><td> 870</td><td></td><td></td><td></td><td></td><td> 875</td><td></td><td></td><td></td><td></td><td> 880</td>

<td> Gly</td><td> Gly</td><td> Lys</td><td> Val</td><td> Pro</td><td> Ile</td><td> Lys</td><td> Trp</td><td> Met</td><td> Ala</td><td> Leu</td><td> Glu</td><td> Ser</td><td> Ile</td><td> Leu</td><td> Arg</td>

<td></td><td></td><td></td><td></td><td> 885</td><td></td><td></td><td></td><td></td><td> 890</td><td></td><td></td><td></td><td></td><td> 895</td><td></td>

<td> Arg</td><td> Arg</td><td> Phe</td><td> Thr</td><td> Hi s</td><td> Gin</td><td> Ser</td><td> Asp</td><td> Val</td><td> Trp</td><td> Ser</td><td> Tyr</td><td> Gly</td><td> Val</td><td> Thr</td><td> Val</td>

<td></td><td></td><td></td><td> 900</td><td></td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td><td></td><td> 910</td><td></td><td></td>

<td> Trp</td><td> Glu</td><td> Leu</td><td> Met</td><td> Thr</td><td> Phe</td><td> Gly</td><td> Al a</td><td> Lys</td><td> Pro</td><td> Tyr</td><td> Asp</td><td> Gly</td><td> Ile</td><td> Pro</td><td> Ala</td>

<td></td><td></td><td> 915</td><td></td><td></td><td></td><td></td><td> 920</td><td></td><td></td><td></td><td></td><td> 925</td><td></td><td></td><td></td>

<td> Arg</td><td> Glu</td><td> Ile</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Leu</td><td> Glu</td><td> Lys</td><td> Gly</td><td> Glu</td><td> Arg</td><td> Leu</td><td> Pro</td><td> Gin</td><td> Pro</td>

<td></td><td> 930</td><td></td><td></td><td></td><td></td><td> 935</td><td></td><td></td><td></td><td></td><td> 940</td><td></td><td></td><td></td><td></td>

<td> Pro</td><td> Ile</td><td> Cys</td><td> Thr</td><td> Ile</td><td> Asp</td><td> Val</td><td> Tyr</td><td> Met</td><td> Ile</td><td> Met</td><td> Val</td><td> Lys</td><td> Cys</td><td> Trp</td><td> Met</td>

<td> 945</td><td></td><td></td><td></td><td></td><td> 950</td><td></td><td></td><td></td><td></td><td> 955</td><td></td><td></td><td></td><td></td><td> 960</td>

<td> Ile</td><td> Asp</td><td> Ser</td><td> Glu</td><td> Cys</td><td> Arg</td><td> Pro</td><td> Arg</td><td> Phe</td><td> Arg</td><td> Glu</td><td> Leu</td><td> Val</td><td> Ser</td><td> Glu</td><td> Phe</td>

<td></td><td></td><td></td><td></td><td> 965</td><td></td><td></td><td></td><td></td><td> 970</td><td></td><td></td><td></td><td></td><td> 975</td><td></td>

<td> Ser</td><td> Arg</td><td> Met</td><td> Al a</td><td> Arg</td><td> Asp</td><td> Pro</td><td> Gin</td><td> Arg</td><td> Phe</td><td> Val</td><td> Val</td><td> Ile</td><td> Gin</td><td> Asn</td><td> Glu</td>

<td></td><td></td><td></td><td> 980</td><td></td><td></td><td></td><td></td><td> 985</td><td></td><td></td><td></td><td></td><td> 990</td><td></td><td></td>

<td> Asp</td><td> Leu</td><td> Gly</td><td> Pro</td><td> Al a</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Asp</td><td> Ser</td><td> Thr</td><td> Phe</td><td colspan="2"> Tyr Arg</td><td> Ser</td><td> Leu</td>

<td></td><td></td><td> 995</td><td></td><td></td><td></td><td></td><td colspan="2"> 1000</td><td></td><td></td><td></td><td colspan="2"> 1005</td><td></td><td></td>

<td> Leu</td><td> Glu</td><td colspan="3"> Asp Asp Asp</td><td> Met</td><td> Gly</td><td> Asp</td><td> Leu</td><td> Val</td><td> Asp</td><td> Ala</td><td> Glu</td><td> Glu</td><td> Tyr</td><td> Leu</td>

<td></td><td colspan="2"> 1010</td><td></td><td></td><td></td><td colspan="2"> 1015</td><td></td><td></td><td></td><td colspan="2"> 1020</td><td></td><td></td><td></td>

<td> Val</td><td> Pro</td><td> Gin</td><td colspan="2"> Gin Gly</td><td> Phe</td><td colspan="2"> Phe Cys</td><td> Pro</td><td> Asp</td><td> Pro</td><td> Ala</td><td> Pro</td><td> Gly</td><td> Ala</td><td> Gly</td>

1025 1030 1035 1040

Gly Met Val Hi s Hi s Arg Hi s Arg Ser Ser Ser Thr Arg Ser Gly Gly

1045 1050 1055

CA 02216601 1997-09-25

WO 96/30514

PCT/US96/01689

<td> Gly</td><td> Asp</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Gly</td><td> Leu</td><td> Glu</td><td> Pro</td><td> Ser</td><td> Glu</td><td> Glu</td><td> Glu</td><td> Ala</td><td> Pro</td><td> Arg</td>

<td></td><td></td><td></td><td colspan="2"> 1060</td><td></td><td></td><td></td><td colspan="2"> 1065</td><td></td><td></td><td></td><td colspan="2"> 1070</td><td></td>

<td> Ser</td><td> Pro</td><td> Leu</td><td> Ala</td><td> Pro</td><td> Ser</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Vai</td><td> Phe</td><td> Asp</td><td> Gly</td>

<td></td><td></td><td colspan="2"> 1075</td><td></td><td></td><td></td><td colspan="2"> 1080</td><td></td><td></td><td></td><td colspan="2"> 1085</td><td></td><td></td>

<td> Asp</td><td> Leu</td><td> Gly</td><td> Met</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Lys</td><td> Gly</td><td> Leu</td><td> Gin</td><td> Ser</td><td> Leu</td><td> Pro</td><td> Thr</td><td> Hi s</td>

<td></td><td colspan="2"> 1090</td><td></td><td></td><td></td><td colspan="2"> 1095</td><td></td><td></td><td></td><td colspan="2"> 1100</td><td></td><td></td><td></td>

<td> Asp</td><td> Pro</td><td> Ser</td><td> Pro</td><td> Leu</td><td> Gin</td><td> Arg</td><td> Tyr</td><td> Ser</td><td> Glu</td><td> Asp</td><td> Pro</td><td> Thr</td><td> Val</td><td> Pro</td><td> Leu</td>

<td colspan="2"> 1105</td><td></td><td></td><td></td><td colspan="2"> 1110</td><td></td><td></td><td></td><td colspan="2"> 1115</td><td></td><td></td><td></td><td> 1120</td>

<td> Pro</td><td> Ser</td><td> Glu</td><td> Thr</td><td> Asp</td><td> Gly</td><td> Tyr</td><td> Vai</td><td> Ala</td><td> Pro</td><td> Leu</td><td> Thr</td><td> Cys</td><td> Ser</td><td> Pro</td><td> Gin</td>

<td></td><td></td><td></td><td></td><td colspan="2"> 1125</td><td></td><td></td><td></td><td colspan="2"> 1130</td><td></td><td></td><td></td><td colspan="2"> 1135</td>

<td> Pro</td><td> Glu</td><td> Tyr</td><td> Vai</td><td> Asn</td><td> Gin</td><td> Pro</td><td> Asp</td><td> Vai</td><td> Arg</td><td> Pro</td><td> Gin</td><td> Pro</td><td> Pro</td><td> Ser</td><td> Pro</td>

<td></td><td></td><td></td><td colspan="2"> 1140</td><td></td><td></td><td></td><td colspan="2"> 1145</td><td></td><td></td><td></td><td colspan="2"> 1150</td><td></td>

<td> Arg</td><td> Glu</td><td> Gly</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Ala</td><td> Ala</td><td> Arg</td><td> Pro</td><td> Ala</td><td> Gly</td><td> Ala</td><td> Thr</td><td> Leu</td><td> Glu</td>

<td></td><td></td><td colspan="2"> 1155</td><td></td><td></td><td></td><td colspan="2"> 1160</td><td></td><td></td><td></td><td colspan="2"> 1165</td><td></td><td></td>

<td> Arg</td><td> Pro</td><td> Lys</td><td> Thr</td><td> Leu</td><td> Ser</td><td> Pro</td><td> Gly</td><td> Lys</td><td> Asn</td><td> Gly</td><td> Vai</td><td> Val</td><td> Lys</td><td> Asp</td><td> Val</td>

<td></td><td colspan="2"> 1170</td><td></td><td></td><td></td><td colspan="2"> 1175</td><td></td><td></td><td></td><td colspan="2"> 1180</td><td></td><td></td><td></td>

<td> Phe</td><td> Ala</td><td> Phe</td><td> Gly</td><td> Gly</td><td> Ala</td><td> Vai</td><td> Glu</td><td> Asn</td><td> Pro</td><td> Glu</td><td> Tyr</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Gin</td>

<td colspan="2"> 1185</td><td></td><td></td><td></td><td colspan="2"> 1190</td><td></td><td></td><td></td><td colspan="2"> 1195</td><td></td><td></td><td></td><td> 1200</td>

<td> Gly</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Pro</td><td> Gin</td><td> Pro</td><td> Hi s</td><td> Pro</td><td> Pro</td><td> Pro</td><td> Al a</td><td> Phe</td><td> Ser</td><td> Pro</td><td> Ala</td>

<td></td><td></td><td></td><td></td><td colspan="2"> 1205</td><td></td><td></td><td></td><td colspan="2"> 1210</td><td></td><td></td><td></td><td colspan="2"> 1215</td>

<td> Phe</td><td> Asp</td><td> Asn</td><td> Leu</td><td> Tyr</td><td> Tyr</td><td> Trp</td><td> Asp</td><td> Gin</td><td> Asp</td><td> Pro</td><td> Pro</td><td> Glu</td><td> Arg</td><td> Gly</td><td> Ala</td>

<td></td><td></td><td></td><td colspan="2"> 1220</td><td></td><td></td><td></td><td colspan="2"> 1225</td><td></td><td></td><td></td><td colspan="2"> 1230</td><td></td>

<td> Pro</td><td> Pro</td><td> Ser</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Gly</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Ala</td><td> Glu</td><td> Asn</td><td> Pro</td><td> Glu</td><td> Tyr</td>

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid

CA 02216601 1997-09-25

WO 96/30514

PCT/US96/01689 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

TCTGGCGCGC TGGATGACGA TGACAAGAAA CGACGGCAGC AGAAGATC (2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

TGAATTCTCG AGTCATTACA CTGGCACGTC CAGACCCAG