Method for treating gefitinib resistant cancer

ES 2 364 495 T3

DESCRIPTION

Method of treating cancer resistant to gefitinib

BACKGROUND

Epithelial cell cancers, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, spleen cancer, testicular cancer, thymus cancer, etc., are diseases characterized by accelerated, abnormal growth of epithelial cells. This accelerated growth initially causes a tumor to form. Finally, metastasis to different organ sites can also occur. Although advances have been made in the diagnosis and treatment of various cancers, these diseases still result in significant mortality.

Lung cancer remains the leading cause of cancer death in industrialized countries. Cancers that start in the lungs are divided into two main types, non-small cell lung cancer and small cell lung cancer, depending on how the cells appear under a microscope. Non-small cell lung cancer (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) generally spreads to other organs more slowly than small cell lung cancer does. About 75 percent of lung cancer cases are classified as non-small cell lung cancer (for example, adenocarcinomas), and the other 25 percent are small cell lung cancer. Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths in the United States, Japan, and Western Europe. For patients with advanced disease, chemotherapy provides a modest survival benefit, but at the cost of significant toxicity, underscoring the need for therapeutic agents that specifically target the critical genetic lesions that drive tumor growth (Schiller JH et al. al., N Engl J Med, 346: 92-98, 2002).

The epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) membrane-bound protein expressed on the surface of epithelial cells. EGFR is a member of the protein tyrosine kinase growth factor receptor family, a class of cell cycle regulatory molecules. (WJ Gullick et al., 1986, Cancer Res., 46: 285-292). EGFR is activated when its ligand (either EGF or TGF-α) binds to the extracellular domain, resulting in autophosphorylation of the receptor's intracellular tyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem. , 255: 4834-4842; AB Schreiber et al., 1983, J. Biol. Chem., 258: 846-853).

EGFR is the protein product of a growth promoting oncogene, erbB or ErbB1, which is only one member of one family, i.e. the ERBB family of proto-oncogenes, which are believed to play fundamental roles in the development and evolution of many cancers. humans. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. The ERBB family of oncogenes codes for four structurally related transmembrane receptors, namely EGFR, HER-2 / neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4). Clinically, ERBB oncogene amplification and / or receptor overexpression in tumors have been reported to correlate with disease recurrence and poor patient prognosis, as well as receptivity to therapy. (L. Harris et al., 1999, Int. J. Biol. Markers, 14: 815; and J. Mendelsohn and J. Baselga, 2000, Oncogene, 19: 6550-6565).

EGFR is composed of three main domains, namely, the extracellular domain (ECD), which is glycosylated and contains the ligand-binding pocket with two cysteine-rich regions; a short transmembrane domain and an intracellular domain that has intrinsic tyrosine kinase activity. The transmembrane region joins the ligand-binding domain with the intracellular domain. DNA and amino acid sequence analysis, as well as studies of non-glycosylated forms of EGFR, indicate that the protein backbone of EGFR has a mass of 132 kDa, with 1,186 amino acid residues (AL Ullrich et al., 1984, Nature, 307: 418-425; J. Downward et al., 1984, Nature, 307: 521-527; C. R: Carlin et al., 1986, Mol. Cell. Biol., 6: 257-264; and FLV Mayes and MD Waterfield, 1984, The EMBO J., 3: 531-537).

The binding of EGF or TGF-α to EGFR activates a signal transduction pathway and results in cell proliferation. Dimerization, conformational changes, and internalization of EGFR molecules function by transmitting intracellular signals that lead to the regulation of cell growth (G. Carpenter and S. Cohen, 1979, Ann. Rev. Biochem., 48: 193-216) . Genetic alterations that affect the regulation of growth factor receptor function, or that lead to receptor and / or ligand overexpression, result in cell proliferation. Furthermore, EGFR has been determined to play a role in cell differentiation, enhancement of cell mobility, protein secretion, neovascularization, invasion, metastasis, and resistance of cancer cells to chemotherapeutic agents and radiation. (M.-J. Oh et al., 2000, Clin. Cancer Res., 6: 4760-4763).

A variety of EGFR inhibitors have been identified, including several ongoing clinical trials for the treatment of various cancers. For a recent summary, see de Bono, JS and Rowinsky, EK (2002), "The ErbB Receptor Family: A Therapeutic Target For Cancer," Trends in Molecular Medicine, 8, S 19-26.

ES 2 364 495 T3

One set of promising targets for therapeutic intervention in cancer treatment includes elements of the HER-kinase axis. They are frequently upregulated in solid epithelial tumors of, for example, the prostate, lung and breast, and are also upregulated in glioblastoma tumors. The epidermal growth factor receptor (EGFR) is an element of the HER-kinase axis, and has been the target of choice for the development of several different cancer therapies. EGFR tyrosine kinase inhibitors (EGFR-TKI) are among these therapies; since reversible phosphorylation of tyrosine residues is required for activation of the EGFR pathway. In other words, EGFR-TKIs block a cell surface receptor responsible for triggering and / or maintaining the cell signaling pathway that induces tumor cell growth and division. Specifically, these inhibitors are believed to interfere with the EGFR kinase domain, designated HER-1. Among the most promising EGFR-TKIs are three series of compounds: quinazolines, pyridopyrimidines, and pyrrolopyrimidines.

Two of the most advanced compounds in clinical development include gefitinib (compound ZD1839 developed by AstraZeneca UK Ltd .; available under the trade name IRESSA; hereinafter "IRESSA") and erlotinib (compound OSI-774 developed by Genentech, Inc. and OSI Pharmaceuticals, Inc .; available under the trade name TARCEVA; hereinafter "TARCEVA"); both have generated encouraging clinical results. Conventional cancer treatment with both IRESSA and TARCEVA involves oral, daily administration of no more than 500 mg of the respective compounds. In May 2003, IRESSA became the first of these products to reach the United States market when it was approved for the treatment of patients with advanced non-small cell lung cancer.

IRESSA is an orally active quinazoline that works by directly inhibiting tyrosine kinase phosphorylation on the EGFR molecule. It competes for the adenosine triphosphate (ATP) binding site, leading to suppression of the HER-kinase axis. The exact mechanism of the IRESSA response is not fully understood, however studies suggest that the presence of EGFR is a necessary prerequisite for its action.

A significant limitation in the use of these compounds is that their receptors may develop resistance to their therapeutic effects upon initial response to therapy, or may not respond to EGFRTKI to any measurable degree at all. The response rate to EGFR-TKI varies among different ethnic groups. At the low end of EGFR-TKI-responsive patients, in some populations, only 10-15 percent of patients with advanced non-small cell lung cancer respond to EGFR kinase inhibitors. Therefore, a better understanding of the molecular mechanisms underlying sensitivity to IRESSA and TARCEVA would be extremely beneficial in targeting therapy to those individuals who are most likely to benefit from such therapy. WO 03/103676 describes a method of treating cancer in individuals who have developed a resistance to anticancer therapy with conventional tyrosine kinase inhibitors (TKIs) or who do not respond to them from the start. The method includes administering to patients an amount that overcomes the resistance of a TKI, which can be administered less frequently than conventional TKI treatments.

There is a significant need in the art for a successful treatment of cancer, and specifically of epithelial cell cancers such as cancers of the lung, ovaries, breast, brain, colon and prostate, that incorporates the benefits of TKI therapy and overcomes the lack response shown by patients. Such a treatment could have a drastic impact on the health of individuals, and especially older individuals, among whom cancer is especially common.

SUMMARY

The present invention is defined in and by the appended claims. The inventors of the present invention have surprisingly discovered that irreversible EGFR inhibitors are effective in treating cancer in subjects no longer responsive to gefitinib and / or erlotinib therapies. Thus, in one embodiment, the present invention enables a method for treating cancer resistant to gefitinib and / or erlotinib. In this embodiment, the progression of cancer in a subject is monitored at a time point after the subject has initiated treatment with gefitinib and / or erlotinib. Cancer progression is indicative of cancer that is resistant to gefitinib and / or erlotinib treatment and a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor is administered to the subject.

In preferred embodiments, the irreversible EGFR inhibitor is EKB-569, HKI-272, or HKI-357. Alternatively, the irreversible EGFR inhibitor can be any compound that binds EGFR cysteine ​​773.

The progression of the cancer can be monitored by methods well known to those of skill in the art. For example, progress can be monitored by visual inspection of the cancer, such as, by means of X-rays, computed tomography, or MRI. Alternatively, evolution can be monitored by detection of tumor biomarkers.

In one embodiment, the patient is monitored at various time points throughout the cancer treatment. For example, the evolution of a cancer can be monitored by analyzing the evolution of the cancer in a

ES 2 364 495 T3 second time point and comparing this analysis with an analysis at a first time point. The first time point may be before or after the initiation of gefitinib and / or erlotinib treatment and the second time point is after the first. Increased cancer growth indicates cancer progression.

In one embodiment, the progression of the cancer is monitored by analyzing the size of the cancer. In one embodiment, the size of the cancer is analyzed by visual inspection of the cancer by means of X-rays, computed tomography, or MRI. In one embodiment, the size of the cancer is monitored by detection of tumor biomarkers.

In one embodiment, the cancer is epithelial cell cancer. In one embodiment, the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, cancer. kidney, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genito-urinary cancer and bladder cancer.

In one embodiment, the size of the cancer is monitored at additional time points, and the additional time points are after the second time point.

In one embodiment, the last time point is at least 2 months after the previous time point. In one embodiment, the last time point is at least 6 months after the previous time point. In one embodiment, the last time point is at least 10 months after the previous time point. In one embodiment, the last time point is at least one year after the previous time point.

In another embodiment, the present invention enables a method of treating cancer, comprising administering to a subject having a mutation in EGFR, namely, a substitution of a threonine for a methionine at position 790 (T790M) of SEQ ID. No. I, a pharmaceutical composition comprising an irreversible EGFR inhibitor. The T790M mutation confers resistance to treatment with gefitinib and / or erlotinib.

BRIEF DESCRIPTION OF THE FIGURES

Figures LA-IB show EGFR sequence analysis in recurrent metastatic lesions of two NSCLC patients with acquired resistance to gefitinib. Figure IA shows the sequence analysis for case I. The EGFR T790M mutation is present in a recurrent liver lesion following the development of clinical resistance to gefitinib. (Left) The mutation was not detected in the primary lung lesion at the time of diagnosis. (Right) Both the primary lung tumor and the recurrent liver lesion harbor the gefitinib sensitization mutation L858R. Note that the L858R mutation is present in the expected reason for a heterozygous mutation in both primary and recurrent lesions, while T790M is detectable at low levels compared to the wild-type allele. A polymorphism (G / A) is shown on the same plot to demonstrate the equivalent representation of the two alleles in the uncloned PCR product. Figure 1B shows the sequence analysis for case 2. The T790M mutation is present within a small minority of gefitinib resistant cells. (Left) The T790M mutation was undetectable in either the primary lung tumor or eight recurrent liver lesions from this case by sequencing the uncloned PCR products. Heterozygosity at an adjacent polymorphism (G / A) confirms the amplification of both EGFR alleles from these samples. The heterozygous gefitinib priming mutation, L861Q, was detected in the expected ratio within the primary lung tumor as well as all eight recurrent liver lesions.

Figures 2A-2C show acquired resistance to gefitinib in bronchoalveolar cancer cell lines and persistent sensitivity to irreversible inhibitors of the ERBB family. Figure 2A shows the inhibition by tyrosine kinase inhibitors of the proliferation of bronchoalveolar cancer cell lines with wild-type EGFR (NCI-HI 666), the activating EGFR mutation of 1E746-A750 (NCIH1650), or two subclones resistant to gefitinib. Representatives of NCI-H1650 (G7 and C11). The effect of the reversible inhibitor gefitinib is compared with that of the irreversible inhibitor HKI-357. Comparable results were observed with the other irreversible inhibitors. Cell numbers were measured by crystal violet staining, after culture in 5% FCS, with EGFR 100 ng / ml, at 72 h after exposure to indicated drug concentrations. Each data point represents the mean of four samples. Figure 2B shows the chemical structure of gefitinib, a reversible EGFR inhibitor; EKB-569, an irreversible EGFR inhibitor; and HKI-272 and HKI-357, two irreversible dual inhibitors of EGFR and ERBB2. Figure 2C shows the generation of drug resistant NCI-H1650 cells after treatment with varying concentrations of gefitinib or the irreversible ERBB inhibitor EKB-569. Colonies were stained after 12 days of culture in the presence of inhibitors.

Figures 3A-3D show the persistent dependence of EGFR and ERBB2 signaling in gefitinib resistant cells, and altered receptor trafficking. Figure 3A shows cell viability after siRNA-mediated gene silencing of EGFR and ERBB2 in bronchoalveolar cell lines with wild-type EGFR (NCI-H1666), compared to cells with the activating EGFR mutation of E746-A750 (NCI-H1650). and two gefitinib resistant derivatives (G7 and C11). Viable cells were counted 72 h after double-stranded RNA treatment and are shown as a fraction relative to cells treated with non-specific siRNA, with standard deviations

ES 2 364 495 T3 based on triplicate samples. Figure 3B shows the inhibition of EGFR (Y1068) autophosphorylation and phosphorylation of downstream effectors AKT and MAPK (ERK) in cells treated with increasing concentrations of gefitinib or the irreversible inhibitor HKI-357, followed by a 2 h pulse. with EGF. The parent cell line NCI-H1650 is compared to a representative gefitinib resistant line, G7. Total AKT and MAPK are shown as controls; Tubulin is used as a loading control for total EGFR levels, which are at the lower limit of detection in these cells. Figure 3C shows altered EGFR internalization in NCI-H1650 (G7) cells resistant to gefitinib, compared to the original sensitive cell line NCI-H1650. Rhodamine-tagged EGF is used to label EGFR at 5 and 20 min after the addition of the ligand. Increased EGFR internalization in NCI-H1650 (G7) cells is more evident at 20 min. (Zeiss microscope, 63x magnification). Figure 3D shows the immunoblot of internalized EGFR from parent NCI-H1650 cells and the resistant derivative G7 after pulse labeling of cell surface proteins by biotinylation and searching for 20 min. The increase in intracellular EGFR in NCI-H1650 (G7) cells is compared with the internalization of the unchanged transferrin receptor (TR).

Figures 4A-4B show the efficacy of irreversible ERBB inhibitors in suppressing the EGFR mutant T790M. Figure 4A shows the comparison of gefitinib and two irreversible inhibitors, HKI-357 and HKI-272, in their ability to suppress autophosphorylation of EGFR (Y1068) and phosphorylation of downstream effectors AKT and MAPK (ERK) in the cell line. bronchoalveolar NCI-H1975, which harbors both a sensitizing mutation (L858R) and a resistance-associated mutation (T790M). Total EGFR, AKT, and MAPK are shown as loading controls. Figure 4B shows the suppression of proliferation in NCI-H1975 cells harboring the L858R and T790M mutations by three irreversible inhibitors of the ERBB family, compared to gefitinib.

Figure 5 shows the nucleotide sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 1) of EGFR.

Figure 6 shows that like gefitinib, HKI 357 and EKB 569 (labeled "Wyeth") demonstrated increased cell killing of NSCLC cells harboring an EGFR mutation, but unlike gefitinib, clones resistant to these drugs were not easily generated in vitro and retained their efficacy against gefitinib resistant clones.

DETAILED DESCRIPTION

Gefitinib and erlotinib resistant cancers

Gefitinib (compound ZD 1839 developed by AstraZeneca UK Ltd .; available under the trade name IRESSA) and erlotinib (compound OSI-774 developed by Genentech, Inc. and OSI Pharmaceuticals, Inc .; available under the trade name TARCEVA) induce drastic clinical responses in non-small cell lung cancers (NSCLC) harboring activating mutations in the EGF receptor (EGFR) (1-3), which are targeted by these competitive ATP-binding inhibitors (4, 5). The efficacy of these tyrosine kinase inhibitors may result both from alterations in the ATP cleavage associated with these mutations, which lead to potentiated inhibition of the mutant kinase by these drugs, and from the biological dependence of these cancer cells on increased signals. transduced by mutant receptors, a phenomenon described as "oncogenic addiction" (6, 7).

Although therapeutic responses to both gefitinib and erlotinib may persist for as long as 2-3 years, the mean duration of response in most NSCLC cases is only 6-8 months (8-10). The mechanisms underlying acquired drug resistance are not well understood. By analogy with imatinib (GLEEVEC), which inhibits BCR-ABL kinase implicated in chronic myeloid leukemias (CML), C-KIT kinase implicated in gastrointestinal stromal tumors (GIST) and FIP1L1-PDGFR-a kinase in idiopathic hypereosinophilic syndrome (SHE), secondary kinase domain mutations can possibly suppress drug binding (1116). However, recurrent NSCLC is not easily biopsied; therefore only limited clinical samples are available for analysis. Recently, a single secondary mutation, T790M, within the EGFR kinase domain has been reported in three of six cases with recurrent disease after gefitinib or erlotinib therapy (17, 18). Codon 315 of BCR-ABL, which is analogous to codon 790 of EGFR, is frequently mutated in imatinib-resistant CML (11,12), and the corresponding residue mutation in C-KIT (codon 670) and FIP1L1-PDGFR- a (codon 674) is associated with imatinib-resistant GIST and BES, respectively (15,16). Early in vitro modeling of resistance to EGFR inhibitors indicated that codon 790 mutation within the wild-type receptor would similarly suppress inhibition by an EGFR tyrosine kinase inhibitor (19). Recently, transfected EGFR proteins containing activating mutations along with the T790M substitution were shown to show reduced inhibition by gefitinib and erlotinib (17, 18). Although the T790M mutation appears to contribute to acquired resistance in some cases of NSCLC, the mechanisms underlying treatment failure in cases lacking secondary EGFR mutations remain unexplained.

Unlike the cytoplasmic BCR-ABL kinase, membrane-bound EGFR signaling involves a complex ligand-binding pathway, receptor homodimerization, and heterodimerization with ERBB2 and other family members, followed by internalization and recycling of the ligand-bound receptor. or receptor mediated degradation

ES 2 364 495 T3 for ubiquitin (20). Significant EGF-dependent signaling is believed to occur during the internalization process, which is also associated with dissociation of EGFR complexes at the low pH of intracellular vesicles. As such, multiple factors modulate the intensity and quality of the receptor-translated signal, and alterations in EGFR trafficking have been closely linked to the regulation of EGF-dependent cellular responses (20).

The present invention is based on the discovery that gefitinib resistant cancers may include those in which the EGFR T790M mutation is only present in a subset of resistant tumor cells and those in which the T790M mutation is not observed, but is observed increased EGF internalization. The invention is further based on the discovery that irreversible EGFR inhibitors, which covalently cross-link the receptor, are effective in inhibiting cancers with the T790M mutation and cancers with altered EGFR trafficking that can render such cancers resistant. to treatment with gefitinib and / or erlotinib. Accordingly, the present invention enables a method of treating cancers resistant to gefitinib and / or erlotinib comprising administering irreversible EGFR inhibitors.

Method of treating a patient

In one embodiment, the invention enables a method of treating gefitinib / erlotinib resistant cancer. The method comprises administering to a patient in need of such treatment an effective amount of certain irreversible EGFR inhibitors, including Eke-569 (4-anilinoquinoline-3-carbonitrile; Greenberger et al., 11^to NCIEORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, November 7-10, 2000, abstract 388; Wyeth), HKI-357 (a derivative of 4-anilinoquinoline-3-carbonitrile; Tsou et al. J. Med. Chem. 2005, 48: 11071131; Wyeth) and / or HKI-272 (a derivative of 4-anilinoquinoline- 3-carbonitrile; Rabindran et al., Cancer Res. 2004, 64, 3958-3965; Wyeth). In a preferred embodiment, the invention enables a method comprising administering to a patient in need of such treatment an effective amount of EKB-569. In a preferred embodiment, the invention enables a method comprising administering to a patient in need of such treatment an effective amount of HKI-357.

Treatment can also involve a combination of treatments, including, but not limited to, a tyrosine kinase inhibitor in combination with other tyrosine kinase inhibitors, chemotherapy, radiation, etc.

Cancers can be initially diagnosed as sensitive to gefitinib / erlotinib or predicted to be sensitive to gefitinib / erlotinib using the methods described in Lynch et al., 2004; 350: 2129-2139. Sensitivity to gefitinib / erlotinib can be predicted by the presence in the tumor of EGFR mutations including, for example, deletion of residues 747 (lysine) to 749 (glutamic acid) combined with a mutation at 750 (alanine), deletion of residues 747 (lysine) to 750 (alanine), substitution of leucine for arginine at residue 858, substitution of leucine for glutamine at residue 861.

Cancers can be diagnosed as resistant to gefitinib and / or erlotinib after starting treatment with gefitinib and / or erlotinib. Alternatively, cancers can be diagnosed as resistant to gefitinib and / or erlotinib prior to initiation of treatment with such compounds. Resistance to gefitinib and / or erlotinib in the tumor can occur after, for example, 6 months or more of treatment with gefitinib and / or erlotinib. Alternatively, tumor resistance to gefitinib and / or erlotinib can be diagnosed less than 6 months after starting treatment with gefitinib and / or erlotinib. Diagnosis of resistance to gefitinib and / or erlotinib can be achieved by monitoring tumor progression during treatment with gefitinib and / or erlotinib. Tumor evolution can be determined by comparing tumor status between time points after treatment has started or by comparing tumor status between a time point after treatment has started and a time point before initiation of treatment. treatment with gefitinib and / or erlotinib. The evolution of the tumor can be monitored during treatment with gefitinib and / or erlotinib visually, for example, by means of radiography, for example x-rays, computerized tomography, or other monitoring methods known to the skilled person, including palpitation of the cancer. or methods for monitoring tumor biomarker levels. Cancer progression during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. An increase in the level of tumor biomarkers indicates tumor evolution. Therefore, an increase in tumor biomarker levels during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. Detection of new tumors or detection of metastases indicates tumor evolution. The cessation of tumor contraction indicates evolution of the tumor. Cancer growth is indicated by, for example, tumor enlargement, metastasis or detection of new cancer and / or increased levels of tumor biomarkers.

The development of resistance to gefitinib and / or erlotinib can be monitored by testing for the presence of a mutation associated with resistance to gefitinib and / or erlotinib in circulating tumor cells obtained from the circulation, or other body fluid, of the subject. The presence of mutations associated with resistance to gefitinib and / or erlotinib in tumor cells of the subject is indicative of a tumor resistant to gefitinib and / or erlotinib.

In one embodiment, the subject's tumor harbors mutations indicative of sensitivity to gefitinib and / or erlotinib, is still resistant to treatment with gefitinib and / or erlotinib. In one embodiment, the subject's tumor harbors mutations

ES 2 364 495 T3 indicative of sensitivity to gefitinib and / or erlotinib and harbors mutations indicative of resistance to gefitinib and / or erlotinib, for example, the T790M mutation, that is, when the native threonine residue is replaced by a methionine residue , in EGFR, for example increased EGFR internalization. In one embodiment, the subject's tumor does not harbor mutations indicative of sensitivity to gefitinib and / or erlotinib and does not harbor mutations indicative of resistance to gefitinib and / or erlotinib, eg, the T790M mutation in EGFR, eg, increased internalization. by EGFR.

With respect to drug administration, an "effective amount" indicates an amount that results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement in symptoms, a cure, a reduction in the burden of disease, reduction of tumor mass or cell numbers, extension of life, improvement of quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of the particular type of disease or condition.

The effective dosage of the active ingredient employed may vary depending on the particular compound employed, the mode of administration, and the severity of the condition to be treated. The expert is aware of the effective dose for each patient, which can vary with the severity of the disease, individual genetic variation or metabolic rate. However, in general, satisfactory results are obtained when the compounds of the invention are administered in a daily dosage of from about 0.5 to about 1000 mg / kg of body weight, optionally administered in divided doses two to four times daily. , or in a sustained release form. The total daily dosage is predicted to be from about 1 to 1000 mg, preferably from about 2 to 500 mg. Pharmaceutical forms suitable for internal use comprise from about 0.5 to 1000 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The route of administration can be intravenous (iv), intramuscular (im), subcutaneous (sc), intradermal (id), intraperitoneal (ip), intrathecal (it), intrapleural, intrauterine, rectal, vaginal, topical, intratumoral and the like. The compounds of the invention can be administered parenterally by injection or by gradual infusion over time and can be delivered by peristaltic means.

Administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrating agents appropriate to the barrier to be permeated are used in the formulation. Such penetrating agents are generally known in the art, and include, for example, for transmucosal administration derivatives of fusidic acid and bile salts. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be by nasal sprays, for example, or using suppositories. For oral administration, the compounds of the invention are formulated in conventional oral administration forms such as capsules, tablets and tonics.

For topical administration, the pharmaceutical composition (kinase activity inhibitor) is formulated into ointments, balms, gels, or creams, as is generally known in the art.

The therapeutic compositions of this invention, for example irreversible EGFR inhibitors, are conventionally administered intravenously, as for example, by injection of a unit dose. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically distinct units suitable as a unit dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluents; ie carrier or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount to be administered and the schedule depend on the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amounts of the required active ingredient to be administered depend on the judgment of the physician and are peculiar to each individual.

Therapeutic compositions useful for practicing the methods of the present invention, eg, irreversible EGFR inhibitors, are described herein. Any formulation or drug delivery system containing the active ingredients may be used that is suitable for the intended use, as is generally known to those of skill in the art. Pharmaceutically acceptable carriers suitable for oral, rectal, topical, or parenteral (including inhalation, subcutaneous, intraperitoneal, intramuscular, and intravenous) administration are known to those skilled in the art. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other components of the formulation and not deleterious to the recipient thereof.

As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and

ES 2 364 495 T3 reagents are used interchangeably and represent that the materials can be administered to a mammal without producing undesired physiological effects.

Formulations suitable for parenteral administration conveniently include a sterile aqueous preparation of the active compound that is preferably isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, 5% dextrose in distilled water, or saline. Useful formulations also include concentrated or solid solutions containing the compound which upon dilution with an appropriate solvent provide a suitable solution for above parenteral administration.

For enteric administration, a compound may be incorporated into an inert carrier in discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, for example, a syrup, an elixir, an emulsion or a potion. Suitable carriers can be starches or sugars and include lubricants, flavors, binders, and other materials of the same nature.

A tablet can be prepared by compression or molding, optionally with one or more accessory components. Compressed tablets may be prepared by compression in a suitable machine of the active compound in a free-flowing form, for example a powder or granules, optionally mixed with accessory components, for example binders, lubricants, inert diluents, or surfactants or dispersing agents. Molded tablets can be prepared by molding in a suitable machine from a mixture of the powdered active compound with any suitable carrier.

A syrup or suspension can be prepared by adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which any accessory component can be added. Such accessory components may include flavoring, an agent that retards the crystallization of sugar, or an agent that increases the solubility of any other component, for example, as a polyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppository with a conventional carrier, for example cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations for oral administration may be presented with an enhancer. Orally acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoyl-carnitine, Laureth-9, phosphatidylcholine, cyclodextrin and derivatives thereof, bile salts such as sodium deoxycholate, sodium taurocholate, sodium glycolate, and sodium fusidate; chelating agents including EDTA, citric acid, and salicylates; fatty acids (eg oleic acid, lauric acid, acylcarnitines, mono and diglycerides). Other oral absorption enhancers include benzalkonium chloride, benzethonium chloride, CHAPS (3- (3-cholamidopropyl) -dimethylammonium-1-propanesulfonate), Big-CHAPS (N, N-bis (3-D-gluconamidepropyl) -cholamide), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. An especially preferred oral absorption enhancer for the present invention is sodium lauryl sulfate.

Alternatively, the compound can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. US Patent No. 4,789,734 describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids are added, along with surfactants if required, and the material is dialyzed or sonicated as necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed by polymers or proteins are well known to those skilled in the art, and can be adapted for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound can be incorporated and the microspheres, or the microsphere composite, implanted for slow release over a period of time ranging from days to months. See, for example, US Patent Nos.^you 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19: 155-157 (1998).

In one embodiment, the tyrosine kinase inhibitor of the present invention can be formulated in a liposome or microparticle that is suitably sized to deposit in capillary beds after intravenous administration. When the liposome or microparticle is deposited in the capillary beds surrounding the ischemic tissue, the agents can be delivered locally to the site where they can be most effective. Suitable liposomes for targeting ischemic tissue are generally less than about 200 nanometers and are also typically unilamellar vesicles, as disclosed, for example, in US Patent No. 5,593,688 issued to Baldeschweiler, entitled "Liposomal targeting of ischemic tissue ”.

ES 2 364 495 T3

Preferred microparticles are those prepared from biodegradable polymers, such as polyglycolide, polylactide, and copolymers thereof. Those skilled in the art can easily determine an appropriate carrier system depending on various factors, including the desired drug release rate and the desired dosage.

In one embodiment, the formulations are delivered via a catheter directly into blood vessels. Administration can occur, for example, through holes in the catheter. In embodiments where the active compounds have a relatively long half-life (on the order of 1 day to a week or more) the formulations can be embedded in biodegradable polymeric hydrogels, such as those disclosed in US Patent No. 5,410. 016 issued to Hubbell et al. These polymeric hydrogels can be delivered into the lumen of a tissue and active compounds released over time as the polymer degrades. If desired, the polymeric hydrogels can have microparticles or liposomes that include the active compound dispersed therein, providing another mechanism for controlled release of the active compounds.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of putting the active compound in association with a carrier that constitutes one or more accessory components. In general, the formulations are prepared by uniformly and intimately placing the active compound in association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into the desired unit dosage form.

The formulations may further include one or more optional accessory component (s) used in the art of pharmaceutical formulations, for example diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants), and the like.

The compounds of the present methods (i.e., irreversible EGFR inhibitors) can be presented for administration to the respiratory tract as a snuff or an aerosol or a solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with a inert carrier such as lactose. In such a case, the active compound particles suitably have diameters of less than 50 microns, preferably less than 10 microns, more preferably between 2 and 5 microns.

Generally for nasal administration a slightly acidic pH will be preferred. Preferably, the compositions of the invention have a pH of from about 3 to 5, more preferably from about 3.5 to about 3.9, and most preferably 3.7. Adjustment of the pH is accomplished by adding an appropriate acid, such as hydrochloric acid.

The preparation of a drug composition containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited on the basis of formulation. Typically such compositions are prepared as injectable preparations or as liquid solutions or suspensions, however solid forms suitable for dissolution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Furthermore, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like that enhance the efficacy of the active ingredient.

The irreversible kinase inhibitors of the present invention can include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic and the like. . Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials other than the active ingredients and water, or contain a buffer such as sodium phosphate at a physiological pH value, physiological saline or both, such as buffered saline with phosphate. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol, and other solutes.

Liquid compositions can also contain liquid phases in addition to and excluding water. Examples of

ES 2 364 495 T3 such additional liquid phases are glycerin, vegetable oils such as cottonseed oil and water-oil emulsions.

Definitions:

The terms "ErbB 1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein and refer to the native sequence of EGFR as disclosed, for example, in Carpenter et al. to the. Ann. Rev. Biochem. 56: 881-914 (1987), including variants thereof (eg an EGFR deletion mutant as in Humphrey et al. PNAS (USA) 87: 4207-4211 (1990)). erbB1 refers to the gene that codes for the EGFR protein product. As used herein, the EGFR protein is disclosed as GenBank Accession No. NP_005219 (SEQ ID NO: 1) which is encoded by the erbB1 gene, GenBank Accession No. NM_005228 ( SEQ ID NO: 2). The nucleotide and amino acid sequences of erbB1 / EGFR can be found in Figure 5.

The term "nucleic acid variance that increases kinase activity" as used herein refers to a variance (ie, mutation) in the nucleotide sequence of a gene that results in increased kinase activity. . Increased kinase activity is a direct result of variance in nucleic acid and is associated with the protein for which the gene encodes.

The term "drug" or "compound" as used herein refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a person to treat or prevent or control a disease or a disease. condition. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but can also be a larger compound, for example, an oligomer of nucleic acids, amino acids or carbohydrates including without limitation proteins, oligonucleotides, ribozymes , DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and modifications and combinations thereof.

As used herein, the terms "effective" and "efficacy" include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and / or organism level (often referred to as side effects) that result from the administration of the treatment. "Less effective" means that the treatment results in a lower therapeutically significant level of pharmacological efficacy and / or a therapeutically higher level of adverse physiological effects.

Nucleic acid molecules can be isolated from a particular biological sample using any of a number of procedures, which are well known in the art, with the particular isolation procedure chosen being appropriate for the particular biological sample. For example, alkaline lysis and freeze-thaw procedures may be useful to obtain nucleic acid molecules from solid materials; Alkaline and heat lysis procedures may be useful to obtain nucleic acid molecules from urine, and proteinase k extraction can be used to obtain nucleic acid from blood (Rolff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)) .

As used herein, a "cancer" in a subject or patient refers to the presence of cells that have typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, proliferation rate, and rapid growth, and certain characteristic morphological characteristics. In some circumstances, the cancer cells will be in the form of a tumor, or such cells may exist locally within an animal, or circulate in the bloodstream as independent cells.

EXAMPLES

Compounds. Compounds used herein, including EKB-569, HK1-357, and HK1-272 as described in US Patent No. 6,002,008; Greenberger et al., Proc. eleven^to NCI EORTC-AACR Symposium on New Drugs in Cancer Therapy, Clinical Cancer Res. Supplement vol. 6, November 2000, ISSN 1078-0432; in Rabindran et al., Cancer Res. 64: 3958-3965 (2004); Holbro and Hynes, Ann. Rev. Pharm. Tox. 44: 195-217 (2004); and Tejpar et al., J. Clin. Oncol. ASCO Annual Meeting Proc. vol. 22, No. 14S: 3579 (2004).

Analysis of recurrent NSCLC and generation of gefitinib resistant NCI-H1650 cells. Clinical samples of recurrent NSCLC were obtained at autopsy after appropriate consent. The entire EGFR kinase domain was sequenced after analysis of uncloned PCR products. Multiple clones from exon 20 were sequenced to examine codon 790. Mutational analysis of EGFR (exons 1-28), ERBB2 (exons 1-24), PTEN (exons 1-9), Kras (codons 12, 13 and 61) and p53 (exons 5-8) was performed in clones resistant to gefitinib as well as the original NCI-H1650 cell line by automatic sequencing of individual exons and flanking intron sequence (PCR conditions available on request) with bidirectional sequencing using dye terminator chemistry (BIGDYE version 1.1, Applied Biosystems). Sequencing reactions were run on a

ES 2 364 495 T3 ABI3100 sequencer (Applied Biosystems) and the electropherograms were analyzed using SEQUENCE NAVIGATOR and INVOICE software (Applied Biosystems).

To generate resistant subclones of NCI-H1650 cells, these were treated with ethyl methanesulfonate (EMS; 600 pg / ml), allowed to recover for 72 h and then seeded at a density of 6 x 10⁴ cells per 10 cm plate² in 20 pM gefitinib. The relative resistance of these cells to gefitinib, compared to irreversible inhibitors, was achieved by seeding 5 x 10⁴ cells in six-well plates in 5% FCS and 100 ng / ml EGF (Sigma), in the presence of varying drug concentrations, followed after 72 h by fixation of the cells with 4% formaldehyde, 0 crystal violet staining , 1% and cell mass quantification using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). For gene silencing experiments with small interfering RNA (siRNA), cells were transfected with double-stranded RNA oligonucleotides targeting EGFR, ERBB2 (both SMARTpool from Dharmacon, Lafayette, CO), or non-specific control (LRT1B), using X-treme GENE transfection reagent (Roche Applied Science). After 72 h, cells were stained with crystal violet and analyzed on the Odyssey infrared scanner.

Immunoblotting and signaling studies. Inhibition of EGFR signaling was determined by increasing concentrations of gefitinib or the irreversible inhibitors by seeding 9 x 10⁴ cells in 24-well plates, adding the drugs to medium containing 5% FCS for 15 min, followed by a 2 hr pulse with 100 ng / ml EGF, and collecting the lysates. Lysates were prepared in 2x gel loading buffer, sonicated, boiled, and then separated by 10% SDS / PAGE, followed by electroblotting to polyvinylidene fluoride (PVDF) membranes, and immunoblotting. The antibodies used were phosphoEGFR Y1068 and mitogen-activated phospho-protein kinase (MAPK) (Cell Signaling Technology, Beverly, MA), phosphoAKT (BioSource International, Camarillo, CA), and total EGFR, MAPK, AKT, and tubulin (Santa Cruz Biotechnology).

Analysis of EGFR internalization. To demonstrate internalization of EGFR by fluorescence microscopy, cells were grown on coverslips and incubated with 1 ng / ml recombinant human EGF (hr) (Molecular Probes, Eugene, OR) for various ranges before fixing in 4% paraformaldehyde. for 10 min. Coverslips were washed in PBS and mounted in ProLong Gold Fluorescence Mounting Reagent (Molecular Probes). To quantify EGFR internalization by cell surface biotinylation, cells were grown to confluence, pretreated with cyclohexamide, incubated on ice for 1 h with sulfosuccinimidyl-2- (biotinamido) ethyl-1,3-dithiopropionate 1.5 mg / ml (sulfo-NHS-SS-biotin; Pierce) and washed with blocking buffer (NH4Cl 50 nM / MgCl2 1 mM / CaCl2 0.1 mM in pBs) until the free sulfo-NHSSS-biotin was extinguished. , followed by several additional washes with PBS. Then, the cells were incubated in the culture medium at 37 ° C for various intervals to allow the internalization of the biotinylated molecules, they were washed twice for 20 min. in a glutathione solution (50 mM glutathione / 75 mM NaCl / 75 mM NaOH / 1% BSA) on ice to remove all biotinyl groups from the cell surface, and then scraped and lysed in radioimmunoprecipitation assay buffer ( RIPA) 500 pM (25 mM Tris-HCl, pH 7.4, with 150 mM NaCl / 0.1% SDS / 1% Triton X-100) supplemented with NaF, Na orthovanadate and protease inhibitors. The cell extracts were centrifuged and the supernatants were incubated with streptavidin beads (Sigma) to collect the biotinylated proteins, which were then analyzed by SDS / PAGE and immunoblotting with anti-EGFR antibody (SC-03, Santa Cruz Biotechnology) or antibody against the receptor. transferrin (Santa Cruz Biotechnology).

Results and Discussion

Analysis of recurrent lung cancers with acquired resistance to gefitinib. Recurrent gefitinib-resistant NSCLC developed in two patients whose tumors had harbored an activating EGFR kinase mutation at the time of diagnosis and who had shown a drastic initial clinical response to the drug (1). In both cases, progressive metastatic disease in the liver led to the death of the patients, 1-2 years after initiation of treatment. In case 1, analysis of the major liver metastasis obtained at autopsy indicated the persistence of the sensitizing EGFR mutation (L858R), as well as the presence of a newly acquired T790M mutation (Figure 1A). Interestingly, analysis of uncloned PCR products showed that the initial L858R mutation was present in abundance consistent with a heterozygous mutation that is present in all tumor cells, while the secondary T790M mutation was observed at approximately one fifth of the abundance of the corresponding wild-type allele. Thus, this resistance-associated mutation appears to be present in only a fraction of cells within the recurrent tumor.

Case 2 involved eight different recurrent liver metastases after failure of gefitinib therapy. In all of these independent lesions, the sensitizing EGFR L861Q mutation was present at the expected reason for a heterozygous mutation. No secondary EGFR mutation could be detected by analysis of uncloned PCR products from any of these metastases. However, after subcloning the PCR products, the T790M mutation was found to be present at a very low frequency in two of the four metastatic tumors analyzed (T790M, 2 of 50 sequenced clones from lesion 1 and 1 from 56 from lesion 1). lesion 2), but not from two other recurrent metastases (0 out of 55 clones from lesion 3 and 0 out of 59 from lesion 4), or the primary tumor (0 out of 75 clones) (Figure 1B and Table 1). Taken together, these results agree with

ES 2 364 495 T3 earlier reports that the T790M mutation is present in some, but not all, cases of acquired gefitinib resistance (three out of seven tumors; see references 17, 18 and 21). Furthermore, as previously indicated (18), even in some cases with this resistance-associated mutation, it appears to be present in only a small fraction of tumor cells within a recurrent lesion. These observations suggest that additional resistance mechanisms are involved in cases without a secondary EGFR mutation and that such mechanisms coexist with the T790M mutation in other cases.

Generation of gefitinib resistant cell lines with susceptibility to irreversible inhibitors. Given the excellent correlation between the clinical receptivity of EGFR mutant NSCLC and the enhanced gefitinib sensitivity of NSCLC cell lines with these mutations (2, 6, 22, 23), and the limited availability of clinical samples from patients with relapse, Gefitinib resistance was modeled in vitro. The bronchoalveolar cancer cell line NCI-H1650, which has an in-frame deletion of EGFR kinase (delE746-A750), was cultured in 20 pM gefitinib, either with or without prior exposure to ethyl methanesulfonate mutagen. This cell line shows a 100-fold increased sensitivity to gefitinib, compared to some NSCLC lines expressing wild-type EGFR (6). While the vast majority of these cells are efficiently killed by 20 pM gefitinib, drug resistant colonies were readily observed at a frequency of '10'.⁵regardless of mutagen treatment. Forty-nine independent drug resistant clones were isolated, showing an average 50-fold decrease in sensitivity to gefitinib (Figure 2A). All of these showed persistence of the sensitizing mutation without altered EGFR expression, and none had acquired a secondary EGFR mutation or new mutations in ERBB2, p53, Kras, or PTEN. Clones resistant to gefitinib demonstrated comparable resistance to related inhibitors of the anilinoquinazoline class. Notably, however, they exhibited persistent sensitivity to three inhibitors of the ERBB family (Figure 2A): HKI-272 (24) and HKI-357 (compound 7f in reference 25), which are dual inhibitors of EGFR and ERBB2 (values IC50 of 92 and 34 nM, respectively, for EGFR and 59 and 33 nM, respectively, for ERBB2), and EKB-569 (26), a selective EGFR inhibitor (IC values₅₀ of 39 nM for EGFR and 1.3 pM for ERBB2) (Wyeth) (Figure 2B). All three drugs are irreversible inhibitors, most likely through covalent bonding to the cys773 residue within the catalytic domain of EGFR or the cys805 of ERBB2. Like gefitinib, these compounds demonstrated increased killing of NSCLC cells harboring an EGFR mutation, compared to cells expressing the wild-type receptor (Figure 2A). However, unlike gefitinib, against which resistant clones are easily generated, even at high drug concentrations, cell clones that were resistant to irreversible inhibitors at concentrations above 10 pM could not be established, even after mutagenesis with Ethyl methanesulfonate (Figure 2C).

Dependence of gefitinib-resistant cells on EGFR and ERBB2 expression. To gain a good understanding of the mechanisms underlying the acquisition of resistance to gefitinib and persistent sensitivity to irreversible inhibitors, we first determined whether resistant cell lines remain dependent on EGFR for their viability. EGFR siRNA-mediated gene silencing has previously been shown to trigger apoptosis in cells harboring mutant EGFr, but not in those with wild-type alleles (6). Significantly, the parent NCI-H1650 cells as well as their gefitinib resistant derivatives showed a comparable reduction in cell viability after transfection with siRNA targeting EGFR (Figure 3A). Thus, the acquisition of resistance to gefitinib does not imply EGFR-independent activation of downstream effectors. Because HKI-272 and HKI-357 both target EGFr and ERBB2, suppression of this related receptor was also tested. ERBB2 gene silencing in NCIH1650 and its gefitinib-resistant derivatives also caused loss of viability (Figure 3A), suggesting a role for EGFR-ERBB2 heterodimers in the translation of essential survival signals in tumor cells harboring EGFR mutations. . Inhibition of EGFR alone by an irreversible inhibitor appears to be sufficient to induce apoptosis in gefitinib-resistant cells, as demonstrated by the efficacy of EKB-569, which primarily targets EGFR (26). However, given the possibly complementary effects of targeting both EGFR and ERBB2 using siRNA and the availability of irreversible inhibitors that target both members of this family, the potential benefit of dual inhibition warrants consideration.

The ability of gefitinib and irreversible inhibitors of the ERBB family to suppress signaling by EGFR downstream effectors that mediate their proliferative and survival pathways was compared. HKI-357 was 10 times more effective than gefitinib in suppressing EGFR autophosphorylation (measured at residue Y1068), and phosphorylation of AKT and MAPK in parent NCI-H1650 cells harboring the EGFR mutation of 1E746-A750 (Figure 3B). In a gefitinib resistant derivative, NCI-H 1650 (G7), gefitinib showed significantly reduced efficacy in suppressing phosphorylation of AKT, a key effector of EGFR signaling linked to receptivity to gefitinib (6), whereas HKI-357 demonstrated persistent activity (Figure 3B).

Altered EGFR internalization in gefitinib resistant clones. Given the absence of secondary EGFR mutations and the persistent susceptibility of gefitinib-resistant cells to siRNA-mediated suppression of EGFR, the mechanism underlying differential inhibition of EGFR signaling in gefitinib-resistant cells by inhibitors was tested for reversible and irreversible could be correlated with alterations in receptor traffic, a well-documented modulator of EGFR-dependent signaling (20). In fact, analysis of EGFR trafficking in resistant cells derived from NCI-H1650 demonstrated a steady increase in the

ES 2 364 495 T3 internalization of EGFR, compared to parent drug responsive cells, as measured by both internalization of fluorescein-labeled EGF (Figure 3C) and quantification of biotinylated cytoplasmic EGFR (Figure 3D). No such effect was seen with the transferrin receptor, suggesting that this did not result from a generalized alteration in all receptor processing. Although additional work is required to define the precise mechanism for this alteration in EGFR trafficking, a complex process involving numerous regulatory proteins, these results suggest that the ability of gefitinib to inhibit EGFR activation is compromised in these cells, while the action of irreversible inhibitors is not detectably affected.

Inhibition of EGFR T790M signaling and enhanced cell destruction by irreversible inhibitors. Enhanced suppression of EGFR signaling by irreversible ERBB inhibitors raised the possibility that these drugs may also show persistent activity in the context of cells harboring the T790M secondary mutation in EGFR. Therefore, the effect of these inhibitors was tested on the bronchoalveolar cancer cell line NCI-H1975, which harbors both the L858R and T790M mutations in EGFR (18). Significantly, this cell line was derived from a patient who had not been treated with an EGFR inhibitor, indicating that this mutation is not solely associated with acquired drug resistance. Both HKI-357 and HKI-272 were considerably more effective than gefitinib in suppressing ligand-induced autophosphorylation of EGFR and its downstream signaling, as determined by phosphorylation of AKT and MAPK (Figure 4A). Similarly, all three irreversible inhibitors suppressed proliferation in this cell line under conditions where it is resistant to gefitinib (Figure 4B). Thus, irreversible inhibitors of ERBB appear to be effective in cells harboring EGFR T790M as well as cells with altered wild-type receptor trafficking.

The results confirm the report of T790M mutations in EGFR as secondary mutations arising in previously sensitive NSCLC harboring an activating mutation, associated with the appearance of acquired resistance (17, 18). However, this mutation is present in only a subset of cases, and even tumors harboring the T790M mutation may contain only a small fraction of cells with this mutation. These observations imply that multiple resistance mechanisms may coexist in recent tumors following an initial response to gefitinib or similar reversible EGFR inhibitors. Furthermore, these findings suggest that the independent resistance mechanisms of T790M may be equally, if not more, effective than the T790M substitution itself in conferring drug resistance and may explain why recurrent tumors rarely show clonality to T790M (17, 18). In vitro mechanisms of acquired gefitinib resistance do not involve secondary EGFR mutations at a significant frequency, but instead correlate with altered receptor trafficking. However, it should be noted that EGFR trafficking was not examined in all resistant clones that were established in vitro, and it remains possible that additional mechanisms may contribute to gefitinib resistance in some of the clones. However, virtually all gefitinib resistant clones showed comparable sensitivity to irreversible ERBB inhibitors.

The results indicate striking differences between competitive EGFR inhibitors such as gefitinib, whose efficacy is limited by the rapid development of drug resistance in vitro; and irreversible inhibitors, for which acquired resistance appears to be rare (Figure 2C). It is speculated that increased internalization of ligand-bound EGFR in resistant cells may be linked to dissociation of the gefitinib-EGFR complex at low pH from intracellular vesicles. In contrast, the irreversible cross-linking of the receptor would not be affected by such alterations in receptor traffic. Acquired resistance to gefitinib is stable after cell passage for up to 20 generations in the absence of the drug, suggesting that genetic or epigenetic alterations in genes that modulate EGFR turnover may underlie this phenomenon. Because receptor trafficking cannot be easily studied using available clinical samples, identification of such genomic alterations may be required before clinical correlations are possible. However, such a mechanism may contribute to in vivo acquired resistance to gefitinib in patients with recurrent disease who do not have secondary EGFR mutations.

Irreversible inhibitors of ERBB also appear to be effective in overcoming resistance to gefitinib mediated by the T790M mutation, an effect that presumably results from preservation of inhibitor binding despite alteration of this critical residue. While this work was ongoing, another irreversible EGFR inhibitor [CL-387,785, Calbiochem (27)] was shown to inhibit the kinase activity of EGFR T790M mutants (17). The efficacy of CL-387,785 in the context of T790M was proposed to result from the absence of a chloride at position 3 of the aniline group, which is present in gefitinib and was postulated to sterically interfere with binding to mutant methionine in codon 790. However, EKB-569, HKI-272, and HKI-357 all have chloride residues at that position on the aniline ring, suggesting that their shared ability to bind irreversibly to EGFR is likely to explain their efficacy, rather than of the absence of a specific steric interaction with T790M (24-26). Thus, these irreversible inhibitors can be shown to be broadly effective in circumventing a variety of resistance mechanisms, in addition to the T790M mutation.

ES 2 364 495 T3

Table 1: Presence of the EGFR T790M mutation at a very low frequency in recurrent tumors of case 2 No. of clones

<td>Tumor</td><td>T790M mutant</td><td>Natural type</td>

<td>Primary</td><td> 0</td><td> 75</td>

<td>Recurring 1</td><td> 2</td><td> 48</td>

<td>Recurring 2</td><td> 1</td><td> 55</td>

<td>Recurring 3</td><td> 0</td><td> 55</td>

<td>Recurring 4</td><td> 0</td><td> 59</td>

Sequencing of large numbers of cloned PCR products revealed that a minority of alleles within two or four liver lesions contain the T790M mutation.

BIBLIOGRAPHY

1. Schiller JH, Harrington D, Belani CP, et al. Comparison of four chemotherapy regimens for advanced non-small cell lung cancer. N Engl J Med 2002; 346: 92-98.

2. Druker BJ, Talpaz M, Resta DJ et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in Chronic Myeloid Leukemia. N Engl J Med 2001; 344: 1031-1037.

3. Arteaga CL. ErbB-targeted therapeutic approaches in human cancer. Exp Cell Res. 2003; 284: 122-30.

Four. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res 2003; 284: 31-53

5. Luetteke NC, Phillips HK, Qui TH, Copeland NG, Earp HS, Jenkins NA, Lee DC. The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev 1994; 8: 399-413.

6. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. Eur J Cancer. 2001; 37: S9-15

7. Wong AJ, Ruppert JM, Bigner SH, et al. Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci. 1992; 89: 2965-2969.

8. Ciesielski MJ, Genstermaker RA. Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function Oncogene 2000; 19: 810-820.

9. Frederick L, Wang WY, Eley G, James CD. Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res 2000; 60:13 83-13 87.

10. Huang H-JS, Nagane M. Klingbeil CK, et al. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phophorylation and unattenuated signaling. J Biol Chem 1997; 272: 2927-2935.

eleven. Pegram MD, Konecny ​​G, Slamon DJ. The molecular and cellular biology of HER2 / neu gene amplification / overexpression and the clinical development of herceptin (trastuzumab) therapy for breast cancer. Cancer Treat Res 2000; 103: 57-75.

12. Ciardiello F, Tortora G. A novel approach in the treatment of cancer targeting the epidermal growth factor receptor. Clin Cancer Res. 2001; 7: 2958-2970

13. Wakeling AE, Guy SP, Woodburn JR et al. ZD1839 (Iressa): An orally active inhibitor of Epidermal Growth Factor signaling with potential for cancer therapy. Cancer Res 2002; 62: 5749-5754.

14. Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF, Arteaga CL. Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2 / neu (erbB2) -overexpressing breast cancer cells in vitro and in vivo. Cancer Res 2001; 61: 8887-8895.

fifteen. Moasser MM, Basso A, Averbuch SD, Rosen N. The tyrosine kinase inhibitor ZD1839 ("Iressa) inhibits HER2driven signaling and suppresses the growth of HER-2 overexpressing tumor cells. Cancer Res 2001; 61: 7184-7188.

16. Ranson M, Hammond LA, Ferry D, et al. ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J Clin Oncol. 2002; 20: 2240-2250.

17. Herbst RS, Maddox AM, Rothernberg ML, et al. Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well tolerated and has activity in non-small cell lung cancer and other solid tumors:

ES 2 364 495 T3 results of a phase I trial. J Clin Oncol. 2002; 20: 3815-3825.

18. Baselga J, Rischin JB, Ranson M, et al. Phase I safety, pharmacokinetic and pharmacodynamic trial of ZD1839, a selective oral Epidermal Growth Factor Receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J Clin Onc 2002; 20: 4292-4302.

19. Albanell J, Rojo F, Averbuch S, et al. Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: histopathologic and molecular consequences of receptor inhibition. J Clin Oncol. 2001; 20: 110-124.

twenty. Kris MG, Natale RB, Herbst RS, et al. Efficacy of Gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: A randomized trial. JAMA 2003; 290: 21492158.

twenty-one. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 2003; 21: 2237-2246.

22. Giaccone G, Herbst RS, Manegold C, et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: A phase III trial-INTACT 1. J Clin Oncol 2004; 22: 777-784.

2. 3. Herbst RS, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: A phase III trial - INTACT 2. J Clin Oncol 2004; 22: 785-794.

24. Rich JN, Reardon DA, Peery T, et al. Phase II Trial of Gefitinib in recurrent glioblastoma. J Clin Oncol 2004; 22: 133-142

25. Cohen MH, Williams GA, Sridhara R, et al. United States Food and Drug Administration Drug Approval Summary: Gefitinib (ZD1839; Iressa) Tablets. Clin Cancer Res. 2004; 10: 1212-1218.

26. Cappuzzo F, Gregorc V, Rossi E, et al. Gefitinib in pretreated non-small-cell lung cancer (NSCLC): Analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or Metastatic NSCLC. J Clin Oncol. 2003; 21: 2658-2663.

27. Fitch KR, McGowan KA, van Raamsdonk CD, et al. Genetics of Dark Skin in mice. Genes &amp; Dev 2003; 17: 214228.

28. Nielsen UB, Cardone MH, Sinskey AJ, MacBeath G, Sorger PK. Profiling receptor tyrosine kinase activation by using Ab microarrays. Proc Natl Acad Sci USA 2003; 100: 9330-5.

29. Burgess AW, Cho H, Eigenbrot C, et al. An open-and-shut case? Recent insights into the activation of EGF / ErbB receptors. Mol Cell 2003; 12: 541-552.

30. Stamos J, Sliwkowski MX, Eigenbrot C. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J Biol Chem. 2002; 277: 46265-46272.

31. Lorenzato A, Olivero M, Patrane S, et al. Novel somatic mutations of the MET oncogene in human carcinoma metastases activating cell motility and invasion. Cancer Res 2002; 62: 7025-30.

32. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002; 417: 906-7.

33. Bardelli A, Parsons DW, Silliman N, et al. Mutational analysis of the tyrosine kinome in colorectal cancers. Science 2003; 300: 949.

3. 4. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr / abl gene of the Philadelphia chromosome. Science 1990; 247: 824-30.

35. Heinrich, MC, Corless CL, Demetri GD, et al. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 2003; 21: 4342-4349.

36. Li B, Chang C, Yuan M, McKenna WG, Shu HG. Resistance to small molecule inhibitors of epidermal growth factor receptor in malignant gliomas. Cancer Res 2003; 63: 7443-7450.

SEQUENCE LIST &lt;110&gt; THE GENERAL HOSPITAL CORPORATION WYETH

ES 2 364 495 T3 &lt;120&gt; METHOD TO TREAT CANCER RESISTANT TO GEFITINIB &lt;130&gt; Document 030258-57642-PCT &lt;140&gt; Document PCT / US06 / 003717 &lt;141&gt; 02-02-2006 &lt;150&gt; Document 60 / 649.483 &lt;151 &gt; 03-02-2005 &lt;150&gt; Document 60 / 671.989 &lt;151&gt; 15-04-2005 &lt;160&gt; 5 &lt;170&gt; PatentIn Ver. 3.3 &lt;210&gt; 1 &lt;211&gt; 3878 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt; 220&gt;

&lt;221&gt; CDS &lt;222&gt; (246) .. (3875) &lt;400&gt; 1

ES 2364495 T3 cccggcgcag cgcggccgca gcagcctccg ccccccgcac ggtgtgagcg cccgacgcgg 60 ccgaggcggc cggagtcccg agctagcccc ggcggccgcc ccggacgaca gccgcccaga 120 ggccacctcg tcggcgtccg cccgagtccc cgcctcgccg ccaacgccac aaccaccgcg 180 cacggccccc tgactccgtc cagtattgat cgggagagcc ggagcgagct cttcggggag 240 cagcg atg cga ccc aka ggg acg gcc ggg gca gcg etc ctg gcg ctg ctg 290

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu 15 10 15

<td rowspan="2">gct Wing</td><td rowspan="2">gcg Wing</td><td rowspan="2">etc Leu</td><td rowspan="2">tgc Cys</td><td rowspan="2">ccg Pro twenty</td><td colspan="2">gcg agt</td><td rowspan="2">cgg Arg</td><td rowspan="2">gct Wing</td><td rowspan="2">ctg Leu 25</td><td rowspan="2">gag Glu</td><td rowspan="2">gaa Glu</td><td rowspan="2">aag Lys</td><td rowspan="2">aaa Lys</td><td rowspan="2">gtt Val 30</td><td rowspan="2">tgc Cys</td><td rowspan="2"> 338</td>

<td>To</td><td>To be</td>

<td>caa</td><td>ggc</td><td>acg</td><td>agt</td><td>aac</td><td>aag</td><td>etc</td><td>acg</td><td>cag</td><td>ttg</td><td>ggc</td><td>act</td><td>ttt</td><td>gaa</td><td>gat</td><td>cat</td><td> 386</td>

<td>Gln</td><td>Gly</td><td>Thr</td><td>To be</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Gln</td><td>Leu</td><td>Gly</td><td>Thr</td><td>Phe</td><td>Glu</td><td>Asp</td><td>His</td><td></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>ttt</td><td>etc</td><td>age</td><td>etc</td><td>cag</td><td>agg</td><td>atg</td><td>ttc</td><td>aat</td><td>aac</td><td>tgt</td><td>gag</td><td>gtg</td><td>gtc</td><td>ctt</td><td>ggg</td><td> 434</td>

<td>Phe</td><td>Leu</td><td>To be</td><td>Leu</td><td>Gln</td><td>Arg</td><td>Met</td><td>Phe</td><td>Asn</td><td>Asn</td><td>Cys</td><td>Glu</td><td>Val</td><td>Val</td><td>Leu</td><td>Gly</td><td></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>aat</td><td>ttg</td><td>gaa</td><td>att</td><td>acc</td><td>tat</td><td>gtg</td><td>cag</td><td>agg</td><td>aat</td><td>tat</td><td>gat</td><td>ctt</td><td>tcc</td><td>ttc</td><td>tta</td><td> 482</td>

<td>Asn</td><td>Leu</td><td>Glu</td><td>lie</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Gln</td><td>Arg</td><td>Asn</td><td>Tyr</td><td>Asp</td><td>Leu</td><td>To be</td><td>Phe</td><td>Leu</td><td></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></td>

<td>aag</td><td>acc</td><td>ate</td><td>cag</td><td>gag</td><td>gtg</td><td>gct</td><td>ggt</td><td>tat</td><td>gtc</td><td>etc</td><td>att</td><td>gcc</td><td>etc</td><td>aac</td><td>here</td><td> 530</td>

ES 2 364 495 T3

<td colspan="2">Lys Thr 80</td><td colspan="4">lie Gln Glu Val 85</td><td colspan="4">Gly Tyr Val Wing</td><td>Leu 90</td><td colspan="2">lie Wing</td><td>Leu</td><td>Asn</td><td colspan="2">Thr 95</td>

<td>gtg</td><td>gag</td><td>cga</td><td>att</td><td>cct</td><td>ttg</td><td>gaa</td><td>aac</td><td>ctg</td><td>cag</td><td>ate</td><td>ate</td><td>aga</td><td>gga</td><td>aat</td><td>atg</td><td> 578</td>

<td>Val</td><td>Glu</td><td>Arg</td><td>lie</td><td>Pro 100</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Gln 105</td><td>lie</td><td>lie</td><td>Arg</td><td>Gly</td><td>Asn 110</td><td>Met</td><td></td>

<td>tac</td><td>tac</td><td>gaa</td><td>aat</td><td>tcc</td><td>tat</td><td>gcc</td><td>tta</td><td>gca</td><td>gtc</td><td>tta</td><td>tet</td><td>aac</td><td>tat</td><td>gat</td><td>gca</td><td> 626</td>

<td>Tyr</td><td>Tyr</td><td>Glu</td><td>Asn 115</td><td>To be</td><td>Tyr</td><td>To</td><td>Leu</td><td>To 120</td><td>Val</td><td>Leu</td><td>To be</td><td>Asn</td><td>Tyr 125</td><td>Asp</td><td>To</td><td></td>

<td>aat</td><td>aaa</td><td>acc</td><td>gga</td><td>ctg</td><td>aag</td><td>gag</td><td>ctg</td><td>ccc</td><td>atg</td><td>aga</td><td>aat</td><td>tta</td><td>cag</td><td>gaa</td><td>ate</td><td> 674</td>

<td>Asn</td><td>Lys</td><td>Thr 130</td><td>Gly</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Leu 135</td><td>Pro</td><td>Met</td><td>Arg</td><td>Asn</td><td>Leu 140</td><td>Gln</td><td>Glu</td><td>lie</td><td></td>

<td>ctg</td><td>cat</td><td>ggc</td><td>gcc</td><td>gtg</td><td>cgg</td><td>ttc</td><td>age</td><td>aac</td><td>aac</td><td>cct</td><td>gcc</td><td>ctg</td><td>tgc</td><td>aac</td><td>gtg</td><td> 722</td>

<td>Leu</td><td>His 145</td><td>Gly</td><td>To</td><td>Val</td><td>Arg</td><td>Phe 150</td><td>To be</td><td>Asn</td><td>Asn</td><td>Pro</td><td>To 155</td><td>Leu</td><td>Cys</td><td>Asn</td><td>Val</td><td></td>

<td>gag</td><td>age</td><td>ate</td><td>cag</td><td>tgg</td><td>cgg</td><td>gac</td><td>tie</td><td>gtc</td><td>age</td><td>agt</td><td>gac</td><td>ttt</td><td>etc</td><td>age</td><td>aac</td><td> 770</td>

<td>Glu 160</td><td>To be</td><td>lie</td><td>Gln</td><td>Trp</td><td>Arg 165</td><td>Asp</td><td>lie</td><td>Val</td><td>To be</td><td>To be 170</td><td>Asp</td><td>Phe</td><td>Leu</td><td>To be</td><td>Asn 175</td><td></td>

<td>atg</td><td>teg</td><td>atg</td><td>gac</td><td>ttc</td><td>cag</td><td>aac</td><td>falls off</td><td>ctg</td><td>ggc</td><td>age</td><td>tgc</td><td>caa</td><td>aag</td><td>tgt</td><td>gat</td><td> 818</td>

<td>Met</td><td>To be</td><td>Met</td><td>Asp</td><td>Phe 180</td><td>Gln</td><td>Asn</td><td>His</td><td>Leu</td><td>Gly 185</td><td>To be</td><td>Cys</td><td>Gln</td><td>Lys</td><td>Cys 190</td><td>Asp</td><td></td>

<td>cea</td><td>age</td><td>tgt</td><td>ccc</td><td>aat</td><td>ggg</td><td>age</td><td>tgc</td><td>tgg</td><td>ggt</td><td>gca</td><td>gga</td><td>gag</td><td>gag</td><td>aac</td><td>tgc</td><td> 866</td>

<td>Pro</td><td>To be</td><td>Cys</td><td>Pro 195</td><td>Asn</td><td>Gly</td><td>To be</td><td>Cys</td><td>Trp 200</td><td>Gly</td><td>To</td><td>Gly</td><td>Glu</td><td>Glu 205</td><td>Asn</td><td>Cys</td><td></td>

<td>cag</td><td>aaa</td><td>ctg</td><td>acc</td><td>aaa</td><td>ate</td><td>ate</td><td>tgt</td><td>gcc</td><td>cag</td><td>cag</td><td>tgc</td><td>tcc</td><td>ggg</td><td>cgc</td><td>tgc</td><td> 914</td>

<td>Gln</td><td>Lys</td><td>Leu 210</td><td>Thr</td><td>Lys</td><td>lie</td><td>lie</td><td>Cys 215</td><td>To</td><td>Gln</td><td>Gln</td><td>Cys</td><td>To be 220</td><td>Gly</td><td>Arg</td><td>Cys</td><td></td>

<td>cgt</td><td>ggc</td><td>aag</td><td>tcc</td><td>ccc</td><td>agt</td><td>gac</td><td>tgc</td><td>tgc</td><td>falls off</td><td>aac</td><td>cag</td><td>tgt</td><td>gct</td><td>gca</td><td>ggc</td><td> 962</td>

<td>Arg</td><td>Gly 225</td><td>Lys</td><td>To be</td><td>Pro</td><td>To be</td><td>Asp 230</td><td>Cys</td><td>Cys</td><td>His</td><td>Asn</td><td>Gln 235</td><td>Cys</td><td>To</td><td>To</td><td>Gly</td><td></td>

<td>tgc</td><td>here</td><td>ggc</td><td>ccc</td><td>cgg</td><td>gag</td><td>age</td><td>gac</td><td>tgc</td><td>ctg</td><td>gtc</td><td>tgc</td><td>cgc</td><td>aaa</td><td>ttc</td><td>cga</td><td> 1010</td>

<td>Cys 240</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Arg</td><td>Glu 245</td><td>To be</td><td>Asp</td><td>Cys</td><td>Leu</td><td>Val 250</td><td>Cys</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Arg 255</td><td></td>

<td>gac</td><td>gaa</td><td>gcc</td><td>acg</td><td>tgc</td><td>aag</td><td>gac</td><td>acc</td><td>tgc</td><td>ccc</td><td>cea</td><td>etc</td><td>atg</td><td>etc</td><td>tac</td><td>aac</td><td> 1058</td>

<td>Asp</td><td>Glu</td><td>To</td><td>Thr</td><td>Cys 260</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Cys</td><td>Pro 265</td><td>Pro</td><td>Leu</td><td>Met</td><td>Leu</td><td>Tyr 270</td><td>Asn</td><td></td>

<td>ccc</td><td>acc</td><td>acg</td><td>tac</td><td>cag</td><td>atg</td><td>gat</td><td>gtg</td><td>aac</td><td>ccc</td><td>gag</td><td>ggc</td><td>aaa</td><td>tac</td><td>age</td><td>ttt</td><td> 1106</td>

<td>Pro</td><td>Thr</td><td>Thr</td><td>Tyr 275</td><td>Gln</td><td>Met</td><td>Asp</td><td>Val</td><td>Asn 280</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Lys</td><td>Tyr 285</td><td>To be</td><td>Phe</td><td></td>

<td>ggt</td><td>gcc</td><td>acc</td><td>tgc</td><td>gtg</td><td>aag</td><td>aag</td><td>tgt</td><td>ccc</td><td>cgt</td><td>aat</td><td>tat</td><td>gtg</td><td>gtg</td><td>here</td><td>gat</td><td> 1154</td>

<td>Gly</td><td>To</td><td>Thr 290</td><td>Cys</td><td>Val</td><td>Lys</td><td>Lys</td><td>Cys 295</td><td>Pro</td><td>Arg</td><td>Asn</td><td>Tyr</td><td>Val 300</td><td>Val</td><td>Thr</td><td>Asp</td><td></td>

<td>falls off</td><td>ggc</td><td>teg</td><td>tgc</td><td>gtc</td><td>cga</td><td>gcc</td><td>tgt</td><td>ggg</td><td>gcc</td><td>gac</td><td>age</td><td>tat</td><td>gag</td><td>atg</td><td>gag</td><td> 1202</td>

<td>His</td><td>Gly 305</td><td>To be</td><td>Cys</td><td>Val</td><td>Arg</td><td>To 310</td><td>Cys</td><td>Gly</td><td>To</td><td>Asp</td><td>To be 315</td><td>Tyr</td><td>Glu</td><td>Met</td><td>Glu</td><td></td>

<td>gaa</td><td>gac</td><td>ggc</td><td>gtc</td><td>cgc</td><td>aag</td><td>tgt</td><td>aag</td><td>aag</td><td>tgc</td><td>gaa</td><td>ggg</td><td>cct</td><td>tgc</td><td>cgc</td><td>aaa</td><td> 1250</td>

<td>Glu</td><td>Asp</td><td>Gly</td><td>Val</td><td>Arg</td><td>Lys</td><td>Cys</td><td>Lys</td><td>Lys</td><td>Cys</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Cys</td><td>Arg</td><td>Lys</td><td></td>

ES 2 364 495 T3

320

325

330

335

<td rowspan="2">gtg Val</td><td rowspan="2">tgt Cys</td><td rowspan="2">aac Asn</td><td rowspan="2">gga Gly</td><td colspan="2" rowspan="2">ata ggt lie Gly 340</td><td rowspan="2">att lie</td><td rowspan="2">ggt Gly</td><td rowspan="2">gaa Glu</td><td rowspan="2">ttt Phe 3. 4. 5</td><td colspan="2">aaa gac</td><td rowspan="2">torch To be</td><td rowspan="2">etc Leu</td><td rowspan="2">tcc Ser 350</td><td rowspan="2">ata lie</td><td rowspan="2"> 1298</td>

<td>Lys</td><td>Asp</td>

<td>aat</td><td>gct</td><td>acg</td><td>aat</td><td>att</td><td>aaa</td><td>falls off</td><td>ttc</td><td>aaa</td><td>aac</td><td>tgc</td><td>acc</td><td>tcc</td><td>ate</td><td>agt</td><td>ggc</td><td> 1346</td>

<td>Asn</td><td>To</td><td>Thr</td><td>Asn</td><td>lie</td><td>Lys</td><td>His</td><td>Phe</td><td>Lys</td><td>Asn</td><td>Cys</td><td>Thr</td><td>To be</td><td>lie</td><td>To be</td><td>Gly</td><td></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>gat</td><td>etc</td><td>falls off</td><td>ate</td><td>ctg</td><td>ccg</td><td>gtg</td><td>gca</td><td>ttt</td><td>agg</td><td>ggt</td><td>gac</td><td>tcc</td><td>ttc</td><td>here</td><td>cat</td><td> 1394</td>

<td>Asp</td><td>Leu</td><td>His</td><td>lie</td><td>Leu</td><td>Pro</td><td>Val</td><td>To</td><td>Phe</td><td>Arg</td><td>Gly</td><td>Asp</td><td>To be</td><td>Phe</td><td>Thr</td><td>His</td><td></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>act</td><td>cct</td><td>cct</td><td>ctg</td><td>gat</td><td>cea</td><td>cag</td><td>gaa</td><td>ctg</td><td>gat</td><td>att</td><td>ctg</td><td>aaa</td><td>acc</td><td>gta</td><td>aag</td><td> 1442</td>

<td>Thr</td><td>Pro</td><td>Pro</td><td>Leu</td><td>Asp</td><td>Pro</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Asp</td><td>lie</td><td>Leu</td><td>Lys</td><td>Thr</td><td>Val</td><td>Lys</td><td></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></td>

<td>gaa</td><td>ate</td><td>here</td><td>ggg</td><td>ttt</td><td>ttg</td><td>ctg</td><td>att</td><td>cag</td><td>gct</td><td>tgg</td><td>cct</td><td>gaa</td><td>aac</td><td>agg</td><td>acg</td><td> 1490</td>

<td>Glu</td><td>lie</td><td>Thr</td><td>Gly</td><td>Phe</td><td>Leu</td><td>Leu</td><td>lie</td><td>Gln</td><td>To</td><td>Trp</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Arg</td><td>Thr</td><td></td>

<td> 400</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>gac</td><td>etc</td><td>cat</td><td>gcc</td><td>ttt</td><td>gag</td><td>aac</td><td>cta</td><td>gaa</td><td>ate</td><td>tie</td><td>cgc</td><td>ggc</td><td>agg</td><td>acc</td><td>aag</td><td> 1538</td>

<td>Asp</td><td>Leu</td><td>His</td><td>To</td><td>Phe</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Glu</td><td>lie</td><td>lie</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Thr</td><td>Lys</td><td></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>caa</td><td>cat</td><td>ggt</td><td>cag</td><td>ttt</td><td>tet</td><td>ctt</td><td>gca</td><td>gtc</td><td>gtc</td><td>age</td><td>ctg</td><td>aac</td><td>tie</td><td>here</td><td>tcc</td><td> 1586</td>

<td>Gln</td><td>His</td><td>Gly</td><td>Gln</td><td>Phe</td><td>To be</td><td>Leu</td><td>To</td><td>Val</td><td>Val</td><td>To be</td><td>Leu</td><td>Asn</td><td>lie</td><td>Thr</td><td>To be</td><td></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>ttg</td><td>gga</td><td>tta</td><td>cgc</td><td>tcc</td><td>etc</td><td>aag</td><td>gag</td><td>tie</td><td>agt</td><td>gat</td><td>gga</td><td>gat</td><td>gtg</td><td>tie</td><td>att</td><td> 1634</td>

<td>Leu</td><td>Gly</td><td>Leu</td><td>Arg</td><td>To be</td><td>Leu</td><td>Lys</td><td>Glu</td><td>lie</td><td>To be</td><td>Asp</td><td>Gly</td><td>Asp</td><td>Val</td><td>lie</td><td>lie</td><td></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>torch</td><td>gga</td><td>aac</td><td>aaa</td><td>aat</td><td>ttg</td><td>tgc</td><td>tat</td><td>gca</td><td>aat</td><td>here</td><td>tie</td><td>aac</td><td>tgg</td><td>aaa</td><td>aaa</td><td> 1682</td>

<td>To be</td><td>Gly</td><td>Asn</td><td>Lys</td><td>Asn</td><td>Leu</td><td>Cys</td><td>Tyr</td><td>To</td><td>Asn</td><td>Thr</td><td>lie</td><td>Asn</td><td>Trp</td><td>Lys</td><td>Lys</td><td></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></td>

<td>ctg</td><td>ttt</td><td>ggg</td><td>acc</td><td>tcc</td><td>ggt</td><td>cag</td><td>aaa</td><td>acc</td><td>aaa</td><td>att</td><td>tie</td><td>age</td><td>aac</td><td>aga</td><td>ggt</td><td> 1730</td>

<td>Leu</td><td>Phe</td><td>Gly</td><td>Thr</td><td>To be</td><td>Gly</td><td>Gln</td><td>Lys</td><td>Thr</td><td>Lys</td><td>lie</td><td>lie</td><td>To be</td><td>Asn</td><td>Arg</td><td>Gly</td><td></td>

<td> 480</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>gaa</td><td>aac</td><td>age</td><td>tgc</td><td>aag</td><td>gcc</td><td>here</td><td>ggc</td><td>cag</td><td>gtc</td><td>tgc</td><td>cat</td><td>gcc</td><td>ttg</td><td>tgc</td><td>tcc</td><td> 1778</td>

<td>Glu</td><td>Asn</td><td>To be</td><td>Cys</td><td>Lys</td><td>To</td><td>Thr</td><td>Gly</td><td>Gln</td><td>Val</td><td>Cys</td><td>His</td><td>To</td><td>Leu</td><td>Cys</td><td>To be</td><td></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>ccc</td><td>gag</td><td>ggc</td><td>tgc</td><td>tgg</td><td>ggc</td><td>ccg</td><td>gag</td><td>ccc</td><td>agg</td><td>gac</td><td>tgc</td><td>gtc</td><td>tet</td><td>tgc</td><td>cgg</td><td> 1826</td>

<td>Pro</td><td>Glu</td><td>Gly</td><td>Cys</td><td>Trp</td><td>Gly</td><td>Pro</td><td>Glu</td><td>Pro</td><td>Arg</td><td>Asp</td><td>Cys</td><td>Val</td><td>To be</td><td>Cys</td><td>Arg</td><td></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>aat</td><td>gtc</td><td>age</td><td>cga</td><td>ggc</td><td>agg</td><td>gaa</td><td>tgc</td><td>gtg</td><td>gac</td><td>aag</td><td>tgc</td><td>aac</td><td>ctt</td><td>ctg</td><td>gag</td><td> 1874</td>

<td>Asn</td><td>Val</td><td>To be</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Glu</td><td>Cys</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cys</td><td>Asn</td><td>Leu</td><td>Leu</td><td>Glu</td><td></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>ggt</td><td>gag</td><td>cea</td><td>agg</td><td>gag</td><td>ttt</td><td>gtg</td><td>gag</td><td>aac</td><td>tet</td><td>gag</td><td>tgc</td><td>tie</td><td>cag</td><td>tgc</td><td>falls off</td><td> 1922</td>

<td>Gly</td><td>Glu</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Phe</td><td>Val</td><td>Glu</td><td>Asn</td><td>To be</td><td>Glu</td><td>Cys</td><td>lie</td><td>Gln</td><td>Cys</td><td>His</td><td></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></td>

<td>cea</td><td>gag</td><td>tgc</td><td>ctg</td><td>cct</td><td>cag</td><td>gcc</td><td>atg</td><td>aac</td><td>ate</td><td>acc</td><td>tgc</td><td>here</td><td>gga</td><td>cgg</td><td>gga</td><td> 1970</td>

<td>Pro</td><td>Glu</td><td>Cys</td><td>Leu</td><td>Pro</td><td>Gln</td><td>To</td><td>Met</td><td>Asn</td><td>lie</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Arg</td><td>Gly</td><td></td>

<td> 560</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>

ES 2 364 495 T3

<td>cea Pro</td><td>gac Asp</td><td>aac Asn</td><td>tgt Cys</td><td>ate lie 580</td><td>cag Gln</td><td>tgt Cys</td><td>gee wing</td><td>falls His</td><td>tac Tyr 585</td><td>att lie</td><td>gac Asp</td><td>ggc Gly</td><td>ccc Pro</td><td>falls His 590</td><td>tgc Cys</td><td> 2018</td>

<td>gtc</td><td>aag</td><td>acc</td><td>tgc</td><td>ccg</td><td>gca</td><td>gga</td><td>gtc</td><td>atg</td><td>gga</td><td>gaa</td><td>aac</td><td>aac</td><td>acc</td><td>ctg</td><td>gtc</td><td> 2066</td>

<td>Val</td><td>Lys</td><td>Thr</td><td>Cys</td><td>Pro</td><td>To</td><td>Gly</td><td>Val</td><td>Met</td><td>Gly</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Val</td><td></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>tgg</td><td>aag</td><td>tac</td><td>gca</td><td>gac</td><td>gee</td><td>ggc</td><td>cat</td><td>gtg</td><td>tgc</td><td>falls off</td><td>ctg</td><td>tgc</td><td>cat</td><td>cea</td><td>aac</td><td> 2114</td>

<td>Trp</td><td>Lys</td><td>Tyr</td><td>To</td><td>Asp</td><td>To</td><td>Gly</td><td>His</td><td>Val</td><td>Cys</td><td>His</td><td>Leu</td><td>Cys</td><td>His</td><td>Pro</td><td>Asn</td><td></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>tgc</td><td>acc</td><td>tac</td><td>gga</td><td>tgc</td><td>act</td><td>ggg</td><td>cea</td><td>ggt</td><td>ctt</td><td>gaa</td><td>ggc</td><td>tgt</td><td>cea</td><td>acg</td><td>aat</td><td> 2162</td>

<td>Cys</td><td>Thr</td><td>Tyr</td><td>Gly</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Cys</td><td>Pro</td><td>Thr</td><td>Asn</td><td></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></td>

<td>ggg</td><td>cct</td><td>aag</td><td>ate</td><td>ccg</td><td>tcc</td><td>ate</td><td>gee</td><td>act</td><td>ggg</td><td>atg</td><td>gtg</td><td>ggg</td><td>gee</td><td>etc</td><td>etc</td><td> 2210</td>

<td>Gly</td><td>Pro</td><td>Lys</td><td>lie</td><td>Pro</td><td>To be</td><td>lie</td><td>To</td><td>Thr</td><td>Gly</td><td>Met</td><td>Val</td><td>Gly</td><td>To</td><td>Leu</td><td>Leu</td><td></td>

<td> 640</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>ttg</td><td>ctg</td><td>ctg</td><td>gtg</td><td>gtg</td><td>gee</td><td>ctg</td><td>ggg</td><td>ate</td><td>ggc</td><td>etc</td><td>ttc</td><td>atg</td><td>cga</td><td>agg</td><td>cgc</td><td> 2258</td>

<td>Leu</td><td>Leu</td><td>Leu</td><td>Val</td><td>Val</td><td>To</td><td>Leu</td><td>Gly</td><td>lie</td><td>Gly</td><td>Leu</td><td>Phe</td><td>Met</td><td>Arg</td><td>Arg</td><td>Arg</td><td></td>

<td></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>falls off</td><td>ate</td><td>gtt</td><td>cgg</td><td>aag</td><td>cgc_t</td><td>acg</td><td>ctg</td><td>cgg</td><td>agg</td><td>ctg</td><td>ctg</td><td>cag</td><td>gag</td><td>agg</td><td>gag</td><td> 2306</td>

<td>His</td><td>lie</td><td>Val</td><td>Arg</td><td>Lys</td><td>Arg</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Arg</td><td>Glu</td><td></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>ctt</td><td>gtg</td><td>gag</td><td>cct</td><td>ctt</td><td>here</td><td>ccc</td><td>agt</td><td>gga</td><td>gaa</td><td>gct</td><td>ccc</td><td>aac</td><td>caa</td><td>gct</td><td>etc</td><td> 2354</td>

<td>Leu</td><td>Val</td><td>Glu</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Pro</td><td>To be</td><td>Gly</td><td>Glu</td><td>To</td><td>Pro</td><td>Asn</td><td>Gln</td><td>To</td><td>Leu</td><td></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>ttg</td><td>agg</td><td>ate</td><td>ttg</td><td>aag</td><td>gaa</td><td>act</td><td>gaa</td><td>ttc</td><td>aaa</td><td>aag</td><td>ate</td><td>aaa</td><td>gtg</td><td>ctg</td><td>ggc</td><td> 2402</td>

<td>Leu</td><td>Arg</td><td>lie</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Phe</td><td>Lys</td><td>Lys</td><td>lie</td><td>Lys</td><td>Val</td><td>Leu</td><td>Gly</td><td></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></td>

<td>tcc</td><td>ggt</td><td>gcg</td><td>ttc</td><td>ggc</td><td>acg</td><td>gtg</td><td>tat</td><td>aag</td><td>gga</td><td>etc</td><td>tgg</td><td>ate</td><td>cea</td><td>gaa</td><td>ggt</td><td> 2450</td>

<td>To be</td><td>Gly</td><td>To</td><td>Phe</td><td>Gly</td><td>Thr</td><td>Val</td><td>Tyr</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Trp</td><td>lie</td><td>Pro</td><td>Glu</td><td>Gly</td><td></td>

<td> 72 0</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>gag</td><td>aaa</td><td>gtt</td><td>aaa</td><td>att</td><td>ccc</td><td>gtc</td><td>gct</td><td>ate</td><td>aag</td><td>gaa</td><td>tta</td><td>aga</td><td>gaa</td><td>gca</td><td>here</td><td> 2498</td>

<td>Glu</td><td>Lys</td><td>Val</td><td>Lys</td><td>lie</td><td>Pro</td><td>Val</td><td>To</td><td>lie</td><td>Lys</td><td>Glu</td><td>Leu</td><td>Arg</td><td>Glu</td><td>To</td><td>Thr</td><td></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>tet</td><td>ccg</td><td>aaa</td><td>gee</td><td>aac</td><td>aag</td><td>gaa</td><td>ate</td><td>etc</td><td>gat</td><td>gaa</td><td>gee</td><td>tac</td><td>gtg</td><td>atg</td><td>gee</td><td> 2546</td>

<td>To be</td><td>Pro</td><td>Lys</td><td>To</td><td>Asn</td><td>Lys</td><td>Glu</td><td>lie</td><td>Leu</td><td>Asp</td><td>Glu</td><td>To</td><td>Tyr</td><td>Val</td><td>Met</td><td>To</td><td></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>age</td><td>gtg</td><td>gac</td><td>aac</td><td>ccc</td><td>falls off</td><td>gtg</td><td>tgc</td><td>cgc</td><td>ctg</td><td>ctg</td><td>ggc</td><td>ate</td><td>tgc</td><td>etc</td><td>acc</td><td> 2594</td>

<td>To be</td><td>Val</td><td>Asp</td><td>Asn</td><td>Pro</td><td>His</td><td>Val</td><td>Cys</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gly</td><td>lie</td><td>Cys</td><td>Leu</td><td>Thr</td><td></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>tcc</td><td>acc</td><td>gtg</td><td>cag</td><td>etc</td><td>ate</td><td>acg</td><td>cag</td><td>etc</td><td>atg</td><td>ccc</td><td>ttc</td><td>ggc</td><td>tgc</td><td>etc</td><td>ctg</td><td> 2642</td>

<td>To be</td><td>Thr</td><td>Val</td><td>Gln</td><td>Leu</td><td>lie</td><td>Thr</td><td>Gln</td><td>Leu</td><td>Met</td><td>Pro</td><td>Phe</td><td>Gly</td><td>Cys</td><td>Leu</td><td>Leu</td><td></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></td>

<td>gac</td><td>tat</td><td>gtc</td><td>cgg</td><td>gaa</td><td>falls off</td><td>aaa</td><td>gac</td><td>aat</td><td>att</td><td>ggc</td><td>tcc</td><td>cag</td><td>tac</td><td>ctg</td><td>etc</td><td> 2690</td>

<td>Asp</td><td>Tyr</td><td>Val</td><td>Arg</td><td>Glu</td><td>His</td><td>Lys</td><td>Asp</td><td>Asn</td><td>lie</td><td>Gly</td><td>To be</td><td>Gln</td><td>Tyr</td><td>Leu</td><td>Leu</td><td></td>

<td> 800</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>

ES 2 364 495 T3

<td rowspan="2">aac Asn</td><td rowspan="2">tgg Trp</td><td rowspan="2">tgt Cys</td><td rowspan="2">gtg Val</td><td rowspan="2">cag Gln 820</td><td rowspan="2">ate lie</td><td colspan="2">gca aag</td><td rowspan="2">ggc Gly</td><td colspan="2">atg aac</td><td rowspan="2">tac Tyr</td><td rowspan="2">ttg Leu</td><td rowspan="2">gag Glu</td><td rowspan="2">gac Asp 830</td><td rowspan="2">cgt Arg</td><td rowspan="2"> 2738</td>

<td>To</td><td>Lys</td><td>Met 825</td><td>Asn</td>

<td>cgc</td><td>ttg</td><td>gtg</td><td>falls off</td><td>cgc</td><td>gac</td><td>ctg</td><td>gca</td><td>gcc</td><td>agg</td><td>aac</td><td>gta</td><td>ctg</td><td>gtg</td><td>aaa</td><td>here</td><td> 2786</td>

<td>Arg</td><td>Leu</td><td>val</td><td>His 835</td><td>Arg</td><td>Asp</td><td>Leu</td><td>To</td><td>To 840</td><td>Arg</td><td>Asn</td><td>Val</td><td>Leu</td><td>val 845</td><td>Lys</td><td>Thr</td><td></td>

<td>ccg</td><td>cag</td><td>cat</td><td>gtc</td><td>aag</td><td>ate</td><td>here</td><td>gat</td><td>ttt</td><td>ggg</td><td>ctg</td><td>gcc</td><td>aaa</td><td>ctg</td><td>ctg</td><td>ggt</td><td> 2834</td>

<td>Pro</td><td>Gln</td><td>His 850</td><td>Val</td><td>Lys</td><td>lie</td><td>Thr</td><td>Asp 855</td><td>Phe</td><td>Gly</td><td>Leu</td><td>To</td><td>Lys 860</td><td>Leu</td><td>Leu</td><td>Gly</td><td></td>

<td>gcg</td><td>gaa</td><td>gag</td><td>aaa</td><td>gaa</td><td>tac</td><td>cat</td><td>gca</td><td>gaa</td><td>gga</td><td>ggc</td><td>aaa</td><td>gtg</td><td>cct</td><td>ate</td><td>aag</td><td> 2882</td>

<td>To</td><td>Glu 865</td><td>Glu</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>His 870</td><td>To</td><td>Glu</td><td>Gly</td><td>Gly</td><td>Lys 875</td><td>Val</td><td>Pro</td><td>lie</td><td>Lys</td><td></td>

<td>tgg</td><td>atg</td><td>gca</td><td>ttg</td><td>gaa</td><td>torch</td><td>att</td><td>tta</td><td>falls off</td><td>aga</td><td>ate</td><td>tat</td><td>acc</td><td>falls off</td><td>cag</td><td>agt</td><td> 2930</td>

<td>Trp 880</td><td>Met</td><td>To</td><td>Leu</td><td>Glu</td><td>To be 885</td><td>lie</td><td>Leu</td><td>His</td><td>Arg</td><td>lie 890</td><td>Tyr</td><td>Thr</td><td>His</td><td>Gln</td><td>To be 895</td><td></td>

<td>gat</td><td>gtc</td><td>tgg</td><td>age</td><td>tac</td><td>ggg</td><td>gtg</td><td>acc</td><td>gtt</td><td>tgg</td><td>gag</td><td>ttg</td><td>atg</td><td>acc</td><td>ttt</td><td>gga</td><td> 2978</td>

<td>Asp</td><td>Val</td><td>Trp</td><td>To be</td><td>Tyr 900</td><td>Gly</td><td>Val</td><td>Thr</td><td>Val</td><td>Trp 9.05</td><td>Glu</td><td>Leu</td><td>Met</td><td>Thr</td><td>Phe 910</td><td>Gly</td><td></td>

<td>tcc</td><td>aag</td><td>cea</td><td>tat</td><td>gac</td><td>gga</td><td>ate</td><td>cct</td><td>gcc</td><td>age</td><td>gag</td><td>ate</td><td>tcc</td><td>tcc</td><td>ate</td><td>ctg</td><td> 3026</td>

<td>To be</td><td>Lys</td><td>Pro</td><td>Tyr 915</td><td>Asp</td><td>Gly</td><td>lie</td><td>Pro</td><td>To 920</td><td>To be</td><td>Glu</td><td>lie</td><td>To be</td><td>To be 925</td><td>lie</td><td>Leu</td><td></td>

<td>gag</td><td>aaa</td><td>gga</td><td>gaa</td><td>cgc</td><td>etc</td><td>cct</td><td>cag</td><td>cea</td><td>ccc</td><td>tie</td><td>tgt</td><td>acc</td><td>ate</td><td>gat</td><td>gtc</td><td> 3074</td>

<td>Glu</td><td>Lys</td><td>Gly 930</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Gln 935</td><td>Pro</td><td>Pro</td><td>lie</td><td>Cys</td><td>Thr 940</td><td>lie</td><td>Asp</td><td>Val</td><td></td>

<td>tac</td><td>atg</td><td>ate</td><td>atg</td><td>gtc</td><td>aag</td><td>tgc</td><td>tgg</td><td>atg</td><td>tie</td><td>gac</td><td>gca</td><td>gat</td><td>agt</td><td>cgc</td><td>cea</td><td> 3122</td>

<td>Tyr</td><td>Met 945</td><td>lie</td><td>Met</td><td>Val</td><td>Lys</td><td>Cys 950</td><td>Trp</td><td>Met</td><td>lie</td><td>Asp</td><td>To 955</td><td>Asp</td><td>To be</td><td>Arg</td><td>Pro</td><td></td>

<td>aag</td><td>ttc</td><td>cgt</td><td>gag</td><td>ttg</td><td>ate</td><td>ate</td><td>gaa</td><td>ttc</td><td>tcc</td><td>aaa</td><td>atg</td><td>gcc</td><td>cga</td><td>gac</td><td>ccc</td><td> 3170</td>

<td>Lys 960</td><td>Phe</td><td>Arg</td><td>Glu</td><td>Leu</td><td>lie 965</td><td>lie</td><td>Glu</td><td>Phe</td><td>To be</td><td>Lys 970</td><td>Met</td><td>To</td><td>Arg</td><td>Asp</td><td>Pro 975</td><td></td>

<td>cag</td><td>cgc</td><td>tac</td><td>ctt</td><td>gtc</td><td>att</td><td>cag</td><td>ggg</td><td>gat</td><td>gaa</td><td>aga</td><td>atg</td><td>cat</td><td>ttg</td><td>cea</td><td>agt</td><td> 3218</td>

<td>Gln</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>Val 980</td><td>I have</td><td>Gln</td><td>Gly</td><td>Asp</td><td>Glu 985</td><td>Arg</td><td>Met</td><td>His</td><td>Leu</td><td>Pro 990</td><td>To be</td><td></td>

<td>cct</td><td>here</td><td>gac</td><td>tcc</td><td>aac</td><td>ttc</td><td>tac</td><td>cgt</td><td>gcc</td><td>ctg</td><td>atg</td><td>gat</td><td>gaa</td><td>gaa</td><td>gac</td><td>atg</td><td> 3266</td>

<td>Pro</td><td>Thr</td><td>Asp</td><td>To be 995</td><td>Asn</td><td>Phe</td><td>Tyr</td><td colspan="2">Arg Wing 1000</td><td>Leu</td><td>Met</td><td>Asp</td><td colspan="2">Glu Glu 1005</td><td>Asp</td><td>Met</td><td></td>

<td>gac</td><td>gac</td><td>gtg</td><td>gtg</td><td>gat</td><td>gcc</td><td>gac</td><td>gag</td><td>tac</td><td>etc</td><td>ate</td><td>cea</td><td>cag</td><td>cag</td><td>ggc</td><td>ttc</td><td> 3314</td>

<td>Asp</td><td colspan="2">Asp Val 1010</td><td>Val</td><td>Asp</td><td>To</td><td colspan="2">Asp Glu 1015</td><td>Tyr</td><td>Leu</td><td>lie</td><td colspan="2">Pro Gln 1020</td><td>Gln</td><td>Gly</td><td>Phe</td><td></td>

<td>ttc</td><td>age</td><td>age</td><td>ccc</td><td>tcc</td><td>acg</td><td>torch</td><td>cgg</td><td>act</td><td>ccc</td><td>etc</td><td>ctg</td><td>age</td><td>tet</td><td>ctg</td><td>agt</td><td> 3362</td>

<td colspan="2">Phe Ser 1025</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr</td><td>To be 1030</td><td>Arg</td><td>Thr</td><td>Pro</td><td colspan="2">Leu Leu 1035</td><td>To be</td><td>To be</td><td>Leu</td><td>To be</td><td></td>

<td>gca</td><td>acc</td><td>age</td><td>aac</td><td>aat</td><td>tcc</td><td>acc</td><td>gtg</td><td>gct</td><td>tgc</td><td>att</td><td>gat</td><td>aga</td><td>aat</td><td>ggg</td><td>ctg</td><td> 3410</td>

<td colspan="2">Wing Thr 1040</td><td>To be</td><td>Asn</td><td colspan="2">Asn Ser 1045</td><td>Thr</td><td>Val</td><td>To</td><td colspan="2">Cys lie 1050</td><td>Asp</td><td>Arg</td><td>Asn</td><td colspan="2">Gly Leu 1055</td><td></td>

<td>caa</td><td>age</td><td>tgt</td><td>ccc</td><td>ate</td><td>aag</td><td>gaa</td><td>gac</td><td>age</td><td>ttc</td><td>ttg</td><td>cag</td><td>cga</td><td>tac</td><td>age</td><td>torch</td><td> 3458</td>

ES 2 364 495 T3

<td>Gln</td><td>To be</td><td>Cys</td><td colspan="2">Pro lie 1060</td><td>Lys</td><td>Glu</td><td>Asp</td><td colspan="2">Be phe 1065</td><td>Leu</td><td>Gln</td><td>Arg</td><td colspan="2">Tyr Ser 1070</td><td colspan="2">To be</td>

<td>gac</td><td>ccc</td><td>here</td><td>ggc</td><td>gcc</td><td>ttg</td><td>act</td><td>gag</td><td>gac</td><td>age</td><td>tie</td><td>gac</td><td>gac</td><td>acc</td><td>ttc</td><td>etc</td><td> 3506</td>

<td>Asp</td><td>Pro</td><td>Thr</td><td>Gly</td><td>To</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Asp</td><td>To be</td><td>lie</td><td>Asp</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Leu</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>cea</td><td>gtg</td><td>cct</td><td>gaa</td><td>tac</td><td>tie</td><td>aac</td><td>cag</td><td>tcc</td><td>gtt</td><td>ccc</td><td>aaa</td><td>agg</td><td>ccc</td><td>gct</td><td>ggc</td><td> 3554</td>

<td>Pro</td><td>Val</td><td>Pro</td><td>Glu</td><td>Tyr</td><td>lie</td><td>Asn</td><td>Gln</td><td>To be</td><td>Val</td><td>Pro</td><td>Lys</td><td>Arg</td><td>Pro</td><td>To</td><td>Gly</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>tct</td><td>gtg</td><td>cag</td><td>aat</td><td>cct</td><td>gtc</td><td>tat</td><td>falls off</td><td>aat</td><td>cag</td><td>cct</td><td>ctg</td><td>aac</td><td>ccc</td><td>gcg</td><td>ccc</td><td> 3602</td>

<td>To be</td><td>Val</td><td>Gln</td><td>Asn</td><td>Pro</td><td>Val</td><td>Tyr</td><td>His</td><td>Asn</td><td>Gln</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Pro</td><td>To</td><td>Pro</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></td><td></td>

<td>age</td><td>aga</td><td>gac</td><td>cea</td><td>falls off</td><td>tac</td><td>cag</td><td>gac</td><td>ccc</td><td>falls off</td><td>age</td><td>act</td><td>gca</td><td>gtg</td><td>ggc</td><td>aac</td><td> 3650</td>

<td>To be</td><td>Arg</td><td>Asp</td><td>Pro</td><td>His</td><td>Tyr</td><td>Gln</td><td>Asp</td><td>Pro</td><td>His</td><td>To be</td><td>Thr</td><td>To</td><td>Val</td><td>Gly</td><td>Asn</td><td></td>

<td colspan="2"> 1120</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>CCC</td><td>gag</td><td>tat</td><td>etc</td><td>aac</td><td>act</td><td>gtc</td><td>cag</td><td>ccc</td><td>acc</td><td>tgt</td><td>gtc</td><td>aac</td><td>age</td><td>here</td><td>ttc</td><td> 3698</td>

<td>Pro</td><td>Glu</td><td>Tyr</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Val</td><td>Gln</td><td>Pro</td><td>Thr</td><td>Cys</td><td>Val</td><td>Asn</td><td>To be</td><td>Thr</td><td>Phe</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>gac</td><td>age</td><td>cct</td><td>gcc</td><td>falls off</td><td>tgg</td><td>gcc</td><td>cag</td><td>aaa</td><td>ggc</td><td>age</td><td>falls off</td><td>caa</td><td>att</td><td>age</td><td>ctg</td><td> 3746</td>

<td>Asp</td><td>To be</td><td>Pro</td><td>To</td><td>His</td><td>Trp</td><td>To</td><td>Gln</td><td>Lys</td><td>Gly</td><td>To be</td><td>His</td><td>Gln</td><td>lie</td><td>To be</td><td>Leu</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>gac</td><td>aac</td><td>cct</td><td>gac</td><td>tac</td><td>cag</td><td>cag</td><td>gac</td><td>ttc</td><td>ttt</td><td>ccc</td><td>aag</td><td>gaa</td><td>gcc</td><td>aag</td><td>cea</td><td> 3794</td>

<td>Asp</td><td>Asn</td><td>Pro</td><td>Asp</td><td>Tyr</td><td>Gln</td><td>Gln</td><td>Asp</td><td>Phe</td><td>Phe</td><td>Pro</td><td>Lys</td><td>Glu</td><td>To</td><td>Lys</td><td>Pro</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>aat</td><td>ggc</td><td>ate</td><td>ttt</td><td>aag</td><td>ggc</td><td>tcc</td><td>here</td><td>gct</td><td>gaa</td><td>aat</td><td>gca</td><td>gaa</td><td>tac</td><td>cta</td><td>agg</td><td> 3842</td>

<td>Asn</td><td>Gly</td><td>lie</td><td>Phe</td><td>Lys</td><td>Gly</td><td>To be</td><td>Thr</td><td>To</td><td>Glu</td><td>Asn</td><td>To</td><td>Glu</td><td>Tyr</td><td>Leu</td><td>Arg</td><td></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></td><td></td>

<td>gtc</td><td>gcg</td><td>cea</td><td>caa</td><td>age</td><td>agt</td><td>gaa</td><td>ttt</td><td>att</td><td>gga</td><td>gca</td><td>tga</td><td></td><td></td><td></td><td></td><td> 3878</td>

<td>Val</td><td>To</td><td>Pro</td><td>Gln</td><td>To be</td><td>To be</td><td>Glu</td><td>Phe</td><td>lie</td><td>Gly</td><td>To</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> 1200</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></td><td></td><td></td>

&lt;210&gt; 2 &lt;211&gt; 1210 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 2

ES 2 364 495 T3

<td>Met 1</td><td>Arg</td><td>Pro</td><td>To be</td><td>Gly 5</td><td>Thr</td><td>To</td><td>Gly</td><td>To</td><td>To 10</td><td>Leu</td><td>Leu</td><td>To</td><td>Leu</td><td>Leu fifteen</td><td>To</td>

<td>To</td><td>Leu</td><td>Cys</td><td>Pro twenty</td><td>To</td><td>To be</td><td>Arg</td><td>To</td><td>Leu 25</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Lys</td><td>Val 30</td><td>Cys</td><td>Gln</td>

<td>Gly</td><td>Thr</td><td>To be 35</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Gln 40</td><td>Leu</td><td>Gly</td><td>Thr</td><td>Phe</td><td>Glu Four. Five</td><td>Asp</td><td>His</td><td>Phe</td>

<td>Leu</td><td>To be fifty</td><td>Leu</td><td>Gln</td><td>Arg</td><td>Met</td><td>Phe 55</td><td>Asn</td><td>Asn</td><td>Cys</td><td>Glu</td><td>Val 60</td><td>Val</td><td>Leu</td><td>Gly</td><td>Asn</td>

<td>Leu 65</td><td>Glu</td><td>lie</td><td>Thr</td><td>Tyr</td><td>Val 70</td><td>Gln</td><td>Arg</td><td>Asn</td><td>Tyr</td><td>Asp 75</td><td>Leu</td><td>To be</td><td>Phe</td><td>Leu</td><td>Lys 80</td>

ES 2 364 495 T3

<td colspan="2">Thr lie</td><td>Gln</td><td colspan="2">Glu Val 85</td><td>To</td><td colspan="2">Gly Tyr</td><td>Val</td><td>Leu 90</td><td colspan="3">lie Ala Leu</td><td colspan="2">Asn Thr 95</td><td>Val</td>

<td>Glu</td><td>Arg</td><td>lie</td><td>Pro</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Gln</td><td>lie</td><td>lie</td><td>Arg</td><td>Gly</td><td>Asn</td><td>Met</td><td>Tyr</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>Tyr</td><td>Glu</td><td>Asn</td><td>To be</td><td>Tyr</td><td>To</td><td>Leu</td><td>To</td><td>Val</td><td>Leu</td><td>To be</td><td>Asn</td><td>Tyr</td><td>Asp</td><td>To</td><td>Asn</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>Lys</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Leu</td><td>Pro</td><td>Met</td><td>Arg</td><td>Asn</td><td>Leu</td><td>Gln</td><td>Glu</td><td>lie</td><td>Leu</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>His</td><td>Gly</td><td>To</td><td>Val</td><td>Arg</td><td>Phe</td><td>To be</td><td>Asn</td><td>Asn</td><td>Pro</td><td>To</td><td>Leu</td><td>Cys</td><td>Asn</td><td>Val</td><td>Glu</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>To be</td><td>lie</td><td>Gln</td><td>Trp</td><td>Arg</td><td>Asp</td><td>lie</td><td>Val</td><td>To be</td><td>To be</td><td>Asp</td><td>Phe</td><td>Leu</td><td>To be</td><td>Asn</td><td>Met</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>To be</td><td>Met</td><td>Asp</td><td>Phe</td><td>Gln</td><td>Asn</td><td>His</td><td>Leu</td><td>Gly</td><td>To be</td><td>Cys</td><td>Gln</td><td>Lys</td><td>Cys</td><td>Asp</td><td>Pro</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>To be</td><td>Cys</td><td>Pro</td><td>Asn</td><td>Gly</td><td>To be</td><td>Cys</td><td>Trp</td><td>Gly</td><td>To</td><td>Gly</td><td>Glu</td><td>Glu</td><td>Asn</td><td>Cys</td><td>Gln</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>Lys</td><td>Leu</td><td>Thr</td><td>Lys</td><td>lie</td><td>lie</td><td>Cys</td><td>To</td><td>Gln</td><td>Gln</td><td>Cys</td><td>To be</td><td>Gly</td><td>Arg</td><td>Cys</td><td>Arg</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>Gly</td><td>Lys</td><td>To be</td><td>Pro</td><td>To be</td><td>Asp</td><td>Cys</td><td>Cys</td><td>His</td><td>Asn</td><td>Gln</td><td>Cys</td><td>To</td><td>To</td><td>Gly</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>Thr</td><td>Gly</td><td>Pro</td><td>Arg</td><td>Glu</td><td>To be</td><td>Asp</td><td>Cys</td><td>Leu</td><td>val</td><td>Cys</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Arg</td><td>Asp</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>Glu</td><td>To</td><td>Thr</td><td>Cys</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Leu</td><td>Met</td><td>Leu</td><td>Tyr</td><td>Asn</td><td>Pro</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>Thr</td><td>Tyr</td><td>Gln</td><td>Met</td><td>Asp</td><td>Val</td><td>Asn</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Lys</td><td>Tyr</td><td>To be</td><td>Phe</td><td>Gly</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>To</td><td>Thr</td><td>Cys</td><td>Val</td><td>Lys</td><td>Lys</td><td>Cys</td><td>Pro</td><td>Arg</td><td>Asn</td><td>Tyr</td><td>Val</td><td>Val</td><td>Thr</td><td>Asp</td><td>His</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>Gly</td><td>To be</td><td>Cys</td><td>Val</td><td>Arg</td><td>To</td><td>Cys</td><td>Gly</td><td>To</td><td>Asp</td><td>To be</td><td>Tyr</td><td>Glu</td><td>Met</td><td>Glu</td><td>Glu</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>

<td>Asp</td><td>Gly</td><td>Val</td><td>Arg</td><td>Lys</td><td>Cys</td><td>Lys</td><td>Lys</td><td>Cys</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Cys</td><td>Arg</td><td>Lys</td><td>Val</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>Cys</td><td>Asn</td><td>Gly</td><td>lie</td><td>Gly</td><td>lie</td><td>Gly</td><td>Glu</td><td>Phe</td><td>Lys</td><td>Asp</td><td>To be</td><td>Leu</td><td>To be</td><td>lie</td><td>Asn</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>To</td><td>Thr</td><td>Asn</td><td>lie</td><td>Lys</td><td>His</td><td>Phe</td><td>Lys</td><td>Asn</td><td>Cys</td><td>Thr</td><td>To be</td><td>lie</td><td>To be</td><td>Gly</td><td>Asp</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>Leu</td><td>His</td><td>lie</td><td>Leu</td><td>Pro</td><td>Val</td><td>To</td><td>Phe</td><td>Arg</td><td>Gly</td><td>Asp</td><td>To be</td><td>Phe</td><td>Thr</td><td>His</td><td>Thr</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>Pro</td><td>Leu</td><td>Asp</td><td>Pro</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Asp</td><td>lie</td><td>Leu</td><td>Lys</td><td>Thr</td><td>Val</td><td>Lys</td><td>Glu</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>lie</td><td>Thr</td><td>Gly</td><td>Phe</td><td>Leu</td><td>Leu</td><td>lie</td><td>Gln</td><td>To</td><td>Trp</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Asp</td>

ES 2 364 495 T3

405 410 415

<td colspan="4">Leu His Ala Phe</td><td colspan="3" rowspan="2">Glu Asn Leu</td><td rowspan="2">Glu</td><td rowspan="2">lie 425</td><td colspan="2" rowspan="2">lie Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td colspan="2" rowspan="2">Thr Lys 430</td><td rowspan="2">Gln</td>

<td colspan="2"></td><td colspan="2"> 420</td>

<td>His</td><td>Gly</td><td>Gln</td><td>Phe</td><td>To be</td><td>Leu</td><td>To</td><td>Val</td><td>Val</td><td>To be</td><td>Leu</td><td>Asn</td><td>lie</td><td>Thr</td><td>To be</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>Leu</td><td>Arg</td><td>To be</td><td>Leu</td><td>Lys</td><td>Glu</td><td>lie</td><td>To be</td><td>Asp</td><td>Gly</td><td>Asp</td><td>Val</td><td>lie</td><td>lie</td><td>To be</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>Gly</td><td>Asn</td><td>Lys</td><td>Asn</td><td>Leu</td><td>Cys</td><td>Tyr</td><td>To</td><td>Asn</td><td>Thr</td><td>lie</td><td>Asn</td><td>Trp</td><td>Lys</td><td>Lys</td><td>Leu</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>Phe</td><td>Gly</td><td>Thr</td><td>To be</td><td>Gly</td><td>Gln</td><td>Lys</td><td>Thr</td><td>Lys</td><td>lie</td><td>lie</td><td>To be</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glu</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>Asn</td><td>To be</td><td>Cys</td><td>Lys</td><td>To</td><td>Thr</td><td>Gly</td><td>Gln</td><td>Val</td><td>Cys</td><td>His</td><td>To</td><td>Leu</td><td>Cys</td><td>To be</td><td>Pro</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>Glu</td><td>Gly</td><td>Cys</td><td>Trp</td><td>Gly</td><td>Pro</td><td>Glu</td><td>Pro</td><td>Arg</td><td>Asp</td><td>Cys</td><td>Val</td><td>To be</td><td>Cys</td><td>Arg</td><td>Asn</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>To be</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Glu</td><td>Cys</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cys</td><td>Asn</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Gly</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>Glu</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Phe</td><td>Val</td><td>Glu</td><td>Asn</td><td>To be</td><td>Glu</td><td>Cys</td><td>lie</td><td>Gln</td><td>Cys</td><td>His</td><td>Pro</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>Glu</td><td>Cys</td><td>Leu</td><td>Pro</td><td>Gln</td><td>To</td><td>Met</td><td>Asn</td><td>lie</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Arg</td><td>Gly</td><td>Pro</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>Asp</td><td>Asn</td><td>Cys</td><td>lie</td><td>Gln</td><td>Cys</td><td>To</td><td>His</td><td>Tyr</td><td>I have</td><td>Asp</td><td>Gly</td><td>Pro</td><td>His</td><td>Cys</td><td>Val</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>Lys</td><td>Thr</td><td>Cys</td><td>Pro</td><td>To</td><td>Gly</td><td>Val</td><td>Met</td><td>Gly</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Val</td><td>Trp</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>Lys</td><td>Tyr</td><td>To</td><td>Asp</td><td>To</td><td>Gly</td><td>His</td><td>Val</td><td>Cys</td><td>His</td><td>Leu</td><td>Cys</td><td>His</td><td>Pro</td><td>Asn</td><td>Cys</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>Thr</td><td>Tyr</td><td>Gly</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Cys</td><td>Pro</td><td>Thr</td><td>Asn</td><td>Gly</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>Pro</td><td>Lys</td><td>lie</td><td>Pro</td><td>To be</td><td>lie</td><td>To</td><td>Thr</td><td>Gly</td><td>Met</td><td>Val</td><td>Gly</td><td>To</td><td>Leu</td><td>Leu</td><td>Leu</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>Leu</td><td>Leu</td><td>Val</td><td>Val</td><td>To</td><td>Leu</td><td>Gly</td><td>lie</td><td>Gly</td><td>Leu</td><td>Phe</td><td>Met</td><td>Arg</td><td>Arg</td><td>Arg</td><td>His</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>Val</td><td>Arg</td><td>Lys</td><td>Arg</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Leu</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>Val</td><td>Glu</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Pro</td><td>To be</td><td>Gly</td><td>Glu</td><td>To</td><td>Pro</td><td>Asn</td><td>Gln</td><td>To</td><td>Leu</td><td>Leu</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>Arg</td><td>lie</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Phe</td><td>Lys</td><td>Lys</td><td>lie</td><td>Lys</td><td>Val</td><td>Leu</td><td>Gly</td><td>To be</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>Gly</td><td>To</td><td>Phe</td><td>Gly</td><td>Thr</td><td>Val</td><td>Tyr</td><td>Lys</td><td>Gly</td><td>Leu</td><td>Trp</td><td>lie</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Glu</td>

725 730 735

ES 2 364 495 T3

<td colspan="2">Lys Val</td><td>Lys</td><td>lie 740</td><td colspan="2">Pro Val</td><td>To</td><td colspan="2">lie Lys 745</td><td>Glu</td><td>Leu</td><td>Arg</td><td colspan="2">Glu Wing 750</td><td>Thr</td><td>To be</td>

<td>Pro</td><td>Lys</td><td>To 755</td><td>Asn</td><td>Lys</td><td>Glu</td><td>lie</td><td>Leu 760</td><td>Asp</td><td>Glu</td><td>To</td><td>Tyr</td><td>Val 765</td><td>Met</td><td>To</td><td>To be</td>

<td>Val</td><td>Asp 770</td><td>Asn</td><td>Pro</td><td>His</td><td>Val</td><td>Cys 775</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Gly</td><td>lie 780</td><td>Cys</td><td>Leu</td><td>Thr</td><td>To be</td>

<td>Thr 785</td><td>Val</td><td>Gln</td><td>Leu</td><td>lie</td><td>Thr 790</td><td>Gln</td><td>Leu</td><td>Met</td><td>Pro</td><td>Phe 795</td><td>Gly</td><td>Cys</td><td>Leu</td><td>Leu</td><td>Asp 800</td>

<td>Tyr</td><td>Val</td><td>Arg</td><td>Glu</td><td>His 805</td><td>Lys</td><td>Asp</td><td>Asn</td><td>lie</td><td>Gly 810</td><td>To be</td><td>Gln</td><td>Tyr</td><td>Leu</td><td>Leu 815</td><td>Asn</td>

<td>Trp</td><td>Cys</td><td>Val</td><td>Gln 820</td><td>lie</td><td>To</td><td>Lys</td><td>Gly</td><td>Met 825</td><td>Asn</td><td>Tyr</td><td>Leu</td><td>Glu</td><td>Asp 830</td><td>Arg</td><td>Arg</td>

<td>Leu</td><td>Val</td><td>His 835</td><td>Arg</td><td>Asp</td><td>Leu</td><td>To</td><td>To 840</td><td>Arg</td><td>Asn</td><td>Val</td><td>Leu</td><td>Val 845</td><td>Lys</td><td>Thr</td><td>Pro</td>

<td>Gln</td><td>His 850</td><td>Val</td><td>Lys</td><td>lie</td><td>Thr</td><td>Asp 855</td><td>Phe</td><td>Gly</td><td>Leu</td><td>To</td><td>Lys 860</td><td>Leu</td><td>Leu</td><td>Gly</td><td>To</td>

<td>Glu 865</td><td>Glu</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>His 870</td><td>To</td><td>Glu</td><td>Gly</td><td>Gly</td><td>Lys 875</td><td>Val</td><td>Pro</td><td>lie</td><td>Lys</td><td>Trp 880</td>

<td>Met</td><td>To</td><td>Leu</td><td>Glu</td><td>To be 885</td><td>lie</td><td>Leu</td><td>His</td><td>Arg</td><td>lie 890</td><td>Tyr</td><td>Thr</td><td>His</td><td>Gln</td><td>To be 895</td><td>Asp</td>

<td>Val</td><td>Trp</td><td>To be</td><td>Tyr 900</td><td>Gly</td><td>Val</td><td>Thr</td><td>Val</td><td>Trp 905</td><td>Glu</td><td>Leu</td><td>Met</td><td>Thr</td><td>Phe 910</td><td>Gly</td><td>To be</td>

<td>Lys</td><td>Pro</td><td>Tyr 915</td><td>Asp</td><td>Gly</td><td>lie</td><td>Pro</td><td>To 920</td><td>To be</td><td>Glu</td><td>lie</td><td>To be</td><td>To be 925</td><td>lie</td><td>Leu</td><td>Glu</td>

<td>Lys</td><td>Gly 930</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Gln 935</td><td>Pro</td><td>Pro</td><td>lie</td><td>Cys</td><td>Thr 940</td><td>lie</td><td>Asp</td><td>Val</td><td>Tyr</td>

<td>Met 945</td><td>lie</td><td>Met</td><td>Val</td><td>Lys</td><td>Cys 950</td><td>Trp</td><td>Met</td><td>lie</td><td>Asp</td><td>To 955</td><td>Asp</td><td>To be</td><td>Arg</td><td>Pro</td><td>Lys 960</td>

<td>Phe</td><td>Arg</td><td>Glu</td><td>Leu</td><td>lie 965</td><td>lie</td><td>Glu</td><td>Phe</td><td>To be</td><td>Lys 970</td><td>Met</td><td>To</td><td>Arg</td><td>Asp</td><td>Pro 975</td><td>Gln</td>

<td>Arg</td><td>Tyr</td><td>Leu</td><td>Val 980</td><td>lie</td><td>Gln</td><td>Gly</td><td>Asp</td><td>Glu 985</td><td>Arg</td><td>Met</td><td>His</td><td>Leu</td><td>Pro 990</td><td>To be</td><td>Pro</td>

<td>Thr</td><td>Asp</td><td>To be 995</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Arg</td><td>To 1000</td><td>Leu</td><td>Met</td><td>Asp</td><td>Glu</td><td>Glu 1005</td><td>Asp</td><td>Met</td><td>Asp</td>

<td>Asp</td><td>Val 1010</td><td>Val</td><td>Asp</td><td>To</td><td>Asp</td><td>Glu 1015</td><td>Tyr</td><td>Leu</td><td>lie</td><td>Pro</td><td>Gln 1020</td><td>Gln</td><td>Gly</td><td>Phe</td><td>Phe</td>

<td colspan="2">Be be 1025</td><td>Pro</td><td>To be</td><td>Thr</td><td>To be 1030</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Leu</td><td>Leu 1035</td><td>To be</td><td>To be</td><td>Leu</td><td>To be</td><td>To 1040</td>

<td>Thr</td><td>To be</td><td>Asn</td><td>Asn</td><td>To be 1045</td><td>Thr</td><td>Val</td><td>To</td><td>Cys</td><td>lie 1050</td><td colspan="2">Asp Arg</td><td>Asn</td><td>Gly</td><td>Leu 1055</td><td>Gln</td>

ES 2 364 495 T3

<td>To be</td><td>Cys</td><td colspan="2">Pro lie 1060</td><td>Lys</td><td colspan="2">Glu Asp</td><td>To be</td><td>Phe 1065</td><td>Leu</td><td>Gln</td><td>Arg</td><td colspan="2">Tyr Ser 1070</td><td>To be</td><td>Asp</td>

<td>Pro</td><td>Thr</td><td>Gly</td><td>To</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Asp</td><td>To be</td><td>lie</td><td>Asp</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Leu</td><td>Pro</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>Val</td><td>Pro</td><td>Glu</td><td>Tyr</td><td>lie</td><td>Asn</td><td>Gln</td><td>To be</td><td>Val</td><td>Pro</td><td>Lys</td><td>Arg</td><td>Pro</td><td>To</td><td>Gly</td><td>To be</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>Val</td><td>Gln</td><td>Asn</td><td>Pro</td><td>Val</td><td>Tyr</td><td>His</td><td>Asn</td><td>Gln</td><td>Pro</td><td>Leu</td><td>Asn</td><td>Pro</td><td>To</td><td>Pro</td><td>To be</td>

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

<td>Arg</td><td>Asp</td><td>Pro</td><td>His</td><td>Tyr</td><td>Gln</td><td>Asp</td><td>Pro</td><td>His</td><td>To be</td><td>Thr</td><td>To</td><td>Val</td><td>Gly</td><td>Asn</td><td>Pro</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>Glu</td><td>Tyr</td><td>Leu</td><td>Asn</td><td>Thr</td><td>Val</td><td>Gln</td><td>Pro</td><td>Thr</td><td>Cys</td><td>Val</td><td>Asn</td><td>To be</td><td>Thr</td><td>Phe</td><td>Asp</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>To be</td><td>Pro</td><td>To</td><td>His</td><td>Trp</td><td>To</td><td>Gln</td><td>Lys</td><td>Gly</td><td>To be</td><td>His</td><td>Gln</td><td>lie</td><td>To be</td><td>Leu</td><td>Asp</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>Asn</td><td>Pro</td><td>Asp</td><td>Tyr</td><td>Gln</td><td>Gln</td><td>Asp</td><td>Phe</td><td>Phe</td><td>Pro</td><td>Lys</td><td>Glu</td><td>To</td><td>Lys</td><td>Pro</td><td>Asn</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>Gly</td><td>lie</td><td>Phe</td><td>Lys</td><td>Gly</td><td>To be</td><td>Thr</td><td>To</td><td>Glu</td><td>Asn</td><td>To</td><td>Glu</td><td>Tyr</td><td>Leu</td><td>Arg</td><td>Val</td>

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

<td>To</td><td>Pro</td><td>Gln</td><td>To be</td><td>To be</td><td>Glu</td><td>Phe</td><td>lie</td><td>Gly</td><td>To</td><td></td><td></td><td></td><td></td><td></td><td></td>

1205 1210 &lt;210&gt; 3 &lt;211&gt; 16 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of artificial sequence: synthetic oligonueleotide &lt;400&gt; 3 tgcarctcatcacgca 16 &lt;210&gt; 4 &lt;211&gt; 16 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Description of artificial sequence: synthetic oligonueleotide &lt;400&gt; 4 tgcarctcat caygca 16

ES 2 364 495 T3 &lt;210&gt; 5 &lt;211&gt; 16 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Description of artificial sequence: synthetic oligonucleotide &lt;400&gt; 5 tgcaactcat catgca 16