MODULATORS OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) PATHWAY FOR USE IN THE TREATMENT OR PREVENTION OF SUBSTANCE

ABUSE

1. RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional No. 60/990,274, filed on November 26, 2007 and entitled "Novel Methods of Using Erlotinib," the entire contents of which are hereby incorporated by reference herein.

2. FIELD OF THE INVENTION

[0002] The present invention provides methods of using modulators of the epidermal growth factor receptor (EGFR) pathway for treatment or prevention of substance abuse such as alcohol abuse. The modulators include small molecule inhibitors such as Erlotinib (TARCEV A ^® , N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolineamine) or a pharmaceutically acceptable salt, hydrate or prodrug thereof and Gefitinib [IRESSA ^® , N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4- ylpropoxy)quinazolin-4-amine] or a pharmaceutically acceptable salt, hydrate or prodrug thereof and macromolecular inhibitors of the EGFR pathway such as siRNAs and antibodies that target genes or proteins in the EGFR/ERK pathway such as the EGFR/ErbB protein or a homolog thereof.

3. BACKGROUND OF THE INVENTION

[0003] According to the National Survey on Drug Use and Health (2004), an estimated 76 million people worldwide have alcohol addiction, including harmful use and dependence. In the US, the number of people with alcohol addiction is estimated at 10 million.

[0004] Many people .who would like to quit use of abused agents cannot because they are addicted to one or more dependence-inducing components (e.g., alcohol, nicotine, morphine, cocaine, amphetamine, caffeine, methamphetamine, etc.). Moreover, treatment for substance abuse often involves transfer of dependence to an alternative, but also dependence-inducing agent. Even successful treatment often involves significant and unpleasant withdrawal symptoms.

[0005] For example alcohol dependence constitutes one of the most serious public health problems worldwide. To date, three medications are available for the treatment of alcohol dependence: disulfiram, acamprosate and naltrexone. The opioid antagonist naltrexone has demonstrated the most consistent effect in reducing alcohol consumption in the context of behavioral therapy (Anton et al., Jama 2006, 295, 2003-17). Naltrexone has been shown to decrease ethanol consumption in numerous animal studies (Altshuler et al., Life Sci. 1980, 26, 679-88; Froehlich et al., Pharmacol. Biochem. Behav. 1990, 35, 385-90; Stromberg et al., Alcohol Clin. Exp. Res. 1998, 22, 2186-91; Stromberg et al., Alcohol 2001, 23, 109-16; Volpicelli et al., Life Sci. 1986, 38, 841-7) and clinical studies (Anton et al., J Clin. Psychopharmacol. 2001, 21, 72-7; O'Malley et al., Arch. Gen. Psychiatry 1992, 49, 881-7; Oslin et al., Am. J. Geriatr. Psychiatry 1997, 5, 324-32; Volpicelli et al., Arch. Gen. Psychiatry 1992, 49, 876-80) and has been shown to be more effective in heavy or excessive drinkers (Pettinati et al., J Clin. Psychopharmacol. 2006, 26, 610-25). However, not all patients respond to naltrexone, and this is partly explained by genetic variations in the mu opioid receptor gene (Oslin et al., Addict. Biol. 2006, 11, 397-403). Alcohol dependence is a complex disorder that will require the use of different therapeutic approaches to effectively treat the disease. [0006] Clearly, there remains a need for improved therapies for alcohol abuse and dependency as well as for substance-related disorders in general.

4. SUMMARY OF THE INVENTION

[0007] The present disclosure provides methods for the treatment or prevention of an alcohol-related disorder in a subject having the disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to treat or prevent the alcohol- related disorder.

[0008] In one aspect provided herein are methods of ameliorating or eliminating an effect of an alcohol-related disorder in a subject having the disorder, comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to ameliorate or eliminate the effect of the alcohol-related disorder.

[0009] In one aspect provided herein are methods for diminishing, inhibiting or eliminating an addiction-related behavior in a subject having an alcohol-related disorder, comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to diminish, inhibit or eliminate the addiction-related behavior.

[0010] In one aspect provided herein are methods for alleviating or eliminating withdrawal symptoms in a subject having an alcohol-related disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to alleviate or eliminate the withdrawal symptoms.

[0011] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is a macromolecular inhibitor.

[0012] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is an

RNAi agent selected from the group consisting of small interfering RNA (siRNA), small or short hairpin RNA (shRNA) and microRNA (miRNA).

[0013] In one aspect provided herein the RNAi targets a gene associated with the

EGFR/ERK pathway.

[0014] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is an antibody or an antibody fragment targeting a protein associated with the EGFR/ERK pathway.

[0015] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is a ribozyme or an antisense oligonucleotide targeting a gene associated with the

EGFR/ERK pathway.

[0016] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is a small molecule inhibitor.

[0017] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is erlotinib (TARCEV A ^® , N-(3 -ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolineamine) or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

[0018] In one aspect provided herein the inhibitor of the EGFR/ERK pathway is

Gefitinib [IRESSA ^® , N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4- ylpropoxy)quinazolin-4-amine] or a pharmaceutically acceptable salt, hydrate or prodrug thereof. '

5. BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 provides the chemical structure of erlotinib (TARCEV A ^® , N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolineamine). [0020] FIG. 2A provides a synthetic scheme for the synthesis of erlotinib and erlotinib hydrochloride.

[0021] FIG 2B provides a synthetic scheme for the synthesis of gefitinib (IRES S A ^® , N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-moφholin-4-ylpro poxy)quinazolin-4- amine).

[0022] FIG. 3 A and 3B provide a graphical representation of experimental results obtained with erlotinib on ethanol consumption and reward in rats using the continuous- access-two-bottle-choice drinking paradigm. Erlotinib (5, 20 and 40 mg/kg i.p.) was administered 30 minutes prior to the start of the drinking session. 5, 20 and 40 mg/kg significantly and dose dependently inhibited 10% ethanol consumption 30 minutes (A) and 16 hours (B) following the start of the drinking session. The values are expressed as mean ethanol consumed (g/kg) ± SEM (repeated measures ANOVA followed by Newman-Keuls post hoc test); **P<0.01, *** PO.001 compared to vehicle; n=10. [0023] FIG. 4 provides a graphical representation of experimental results obtained with erlotinib on ethanol-mediated behavior using an operant self-administration model of drinking and reward seeking in rats. Erlotinib (20, 40, and 80 mg/kg i.p.) was administered 2 hours prior to the start of the session and significantly inhibited active lever presses for 10 % ethanol at the highest dose, 80 mg/kg. The values are expressed as mean number of active lever presses ± SEM (repeated measures ANOVA followed by Newman-Keuls post hoc test); *P<0.05 compared to vehicle; n=12. [0024] FIGS. 5A-5C provide a graphical representation of the experimental results obtained with erlotinib on ethanol, sucrose and water consumption after 24h; here, rats were already consuming relatively high amounts of ethanol prior to testing effects of erlotinib.

(A-C) Erlotinib (5, 20 or 40 mg/kg i.p.) was administered to rats 30 minutes prior to the start of the drinking session using the continuous-access to 10% ethanol or water drinking paradigm. Repeated measures ANOVA followed by post-hoc Newman-Keuls analysis revealed that erlotinib significantly decreased (A) 10% ethanol consumption O<0.0009; F ₃ ,i i=7.0) but not (B) 5% sucrose consumption O>0.05; F ₃₎ i ₀ =2.0) 24 hours

after onset of drinking. (C) Erlotinib treatment had no overall effect on water consumption n=10.

[0025] FIGS. 6A-6D provide graphical representations of the experimental results obtained with erlotinib and gefitinib on ethanol sensitivity in flies. (A-D) Samples of 25 flies each were fed a sucrose/yeast mixture containing either erlotinib (0.8 mg/ml; A, B) or gefitinib (0.5 mg/ml; C, D) dissolved in vehicle, or vehicle alone, for 40 or 41 hr before they were assayed in the ethanol LOR assay (100/50 E/ A). (A, C) Sedation profiles and (B, D) ST50 values are shown. One-way ANOVA revealed a significant difference between erlotinib- and vehicle-fed flies (p=0.006; Fi _{1I 5} = 10.341 ; n=8) and between gefitinib- and vehicle-fed flies (p=0.022;

[0026] FIGS. 7A-7F depict additional measures of effects of erlotinib in flies and rats. (A-C) Samples of 25 flies each were treated identically to when they were assayed in the ethanol LOR assay, and were fed a sucrose/yeast mixture containing either erlotinib (0.8 mg/ml) dissolved in vehicle or vehicle alone for 40 or 41 hr. (A) Flies fed erlotinib did not show altered ethanol pharmacokinetics. Flies were exposed to ethanol vapor (100/50 E/ A) for 30 min in the tracking apparatus and ethanol levels were measured in extracts of erlotinib- and vehicle-fed flies. One-way ANOVA revealed no significant effect of erlotinib (p=0.11; Fi _j i ₅ =2.85; n=8). (B) Blue dye (erioglaucine, Sigma 861146, 0.2%) was included in food for both erlotinib- and vehicle-fed flies. Feeding behavior of flies was quantified at the time of assay by comparing total gut volumes in drug- and vehicle-fed flies, as assessed by dye uptake measured from fly extracts (absorbance units at 630 run, AU). No significant effect of erlotinib on feeding was detected by one way ANOVA (p=0.5; Fi n=0.43; n=6). (C) Effect of erlotinib on locomotor behavior of flies was quantified in the tracking apparatus. Average locomotor velocities of flies pre-fed either erlotinib or vehicle were measured in air alone and during early times of exposure to ethanol vapor/humidified air (100/50 E/A; gray bar), before flies had begun to sedate. No effect of erlotinib on fly velocity was seen in the absence of ethanol, while a modest increase in velocities of drug-fed flies was seen during ethanol exposure; while consistent (data not shown), this effect was not significant by two-way repeated measures ANOVA (p=0λ3; Fi _>7 =3.02; n=4). (D, E) The effect of erlotinib on the latency and duration of loss of righting reflex

(LORR) was measured in adult Long-Evans rats. Vehicle or erlotinib (40 mg/kg i.p) was administered 30 min prior to ethanol (3.6 g/kg i.p). Both the latency and the duration of the LORR were measured, and the data, analyzed by Student's unpaired Mest, revealed that erlotinib did not effect either (D) the latency to LORR 0=0.665), n=6 or (E) the duration of the LORR (p=0.295), n=6). (F) The effect of erlotinib (40mg/kg i.p) on blood ethanol clearance was measured in adult Long-Evans rats (see Methods). Blood samples were collected from the lateral tail vein at 30, 60, 120, 240, and 480 min following ethanol injection (3.6 g/kg i.p). One-way ANOVA revealed that erlotinib did not affect blood ethanol clearance at any time point (p>0.05), n=5. [0027] FIG. 8A-8B. Effects on ethanol sedation of genetic manipulations that reduce EGFR signaling in flies. (A) Flies expressing RNAi against EGFR, UAS- egfr™ ^A \ under the control of the pan-neuronal driver elav-GAL4 ^cl55 exhibit increased sensitivity to ethanol-induced sedation. One-way ANOVA of times at which 50% of flies are sedated (ST50 values) revealed a significant difference between genotypes (p<0.003). Post-hoc Newman-Keuls tests revealed a significant difference between elav-GAL4 ^cl55 ; UAS-egfr™ ^Al and elav-GAL4 ^cl55 (p<0.05) as well as between elav- GAL4 ^cXSS ; UAS-egfr ^RNAi and UAS-egfr ^mM (pO.Ol) (n=12). (B) Expression of an RNAi against the EGFR, UAS-egfr™^, using the pan-organismal driver Tub-GAL4 results in decreased egfr levels. RNA was isolated from whole flies and QPCR was performed using a primer/probe set for egfr expression analysis (Applied Biosystems - see Supplemental Methods). Relative egfr mRNA levels for each genotype are expressed relative to ribosomal protein L32 (Rpl32) expression. One-way ANOVA revealed a significant difference between genotypes (/Kθ.0001). Post-hoc Newman-Keuls tests revealed a significant difference between Tub-GAL4 + UAS-egfr ^mAl and Tub-GAL4 (p<0.00\) as well as between Tub-GAL4 + UAS-egfr ^RNAi and UAS-egfr™ ^Ai (p<0.001) (n=6). (C) GMR-GAL4 driven expression of UAS-egfr ^RNM in the fly eye results in a rough eye phenotype that is enhanced by concurrent expression of UAS-hppy ^RBl . Age- and sex-matched adult GMR-GAL4 flies, GMR-GAL4 + UAS-egfr ^mAi flies, and GMR- GAL4 + UAS-hppy ^RB + UAS-egfr ^mAi flies were anesthetized using CO ₂ and photographed together using a digital camera mounted on a dissecting microscope. [0028] FIG. 9. Activation of EGFR/ERK signaling in insulin producing cells (IPCs) and dopaminergic cells decreases ethanol sensitivity as measured in the loss-of-righting

(LOR) sedation assay. (A) Flies expressing a wild-type form of the EGFR, UAS-egfr^ ⁷ , in IPCs under the control of the dilp2-GAL4 driver are relatively resistant to ethanol- induced sedation. One-way ANOVA of ST50 values revealed a significant difference between genotypes (p=0.0016). Post-hoc Newman-Keuls tests revealed a significant difference between dilp2-GAL4;UAS-egfr ^wτ and dilp2-GAL4 (p<0.01) as well as between dilp2-GAL4;UAS-egfr ^NJ and UAS-egfr* ¹ (p<0.01) (n=8). (B, C) The projection pattern of brain IPCs appears unaffected by expression of a wild-type form of the EGFR, UAS-egfr™ ¹ : compare confocal microscope images of representative adult brains of dilp2-GAL4 + UAS-GFP flies (B) versus dilp2-GAL4 + UAS-GFP + UAS-egfr^ flies (C, D). Flies expressing a wild-type form of the EGFR, UAS-egfr ^wτ , under the control of the dopaminergic driver TH-GAL4 are resistant to ethanol-induced sedation. One-way ANOVA of ST50 values revealed a significant difference between genotypes (pO.OOOl). Post-hoc Newman-Keuls tests revealed a significant difference between TH-GAL4; UAS-egfr ^wτ and TH-GAL4 (p<0.00l) as well as between TH-GAL4;UAS- egfr* ¹¹ and UAS-egfr ^wτ (pO.OOl) (n=8). (E, F) The projection pattern of dopaminergic cells in the brain appears to be unaffected by expression of EGFR: compare the confocal microscope images of adult brains of TH-GAL4 + UAS-GFP flies (E) versus TH-GAL4 + UAS-GFP + UAS-egfr^ flies (F). (B ₅ C, E, F) Expression of GFP is green and expression of the general neuropil marker Nc82 is purple. [0029] FIG. 10 depicts results from genetic interaction studies (A-H). Genetic interactions in the fly eye. GMR-GAL4-dήven hppy-KB expression suppresses and enhances the rough eye phenotype caused by overexpression of EGFR and Yan, respectively; hppy overexpression does not affect the rough eye of flies expressing an activated rolled transgene. Scanning electron micrographs of adult eyes of the following genotypes: (A) GMR-GAL4, (B) GMR-GAL4;UAS-hppy ^RBl , (C) GMR-GAL4;UAS- egfr ^ψτ , arrow points to blister, (D) GMR-GAL4; UAS-egfr" ^τ \UAS-hppy™ ¹ , (E) GMR- GAL4;UAS-rl ^ACτ , (F) GMR-GAL4; UAS-rl ^ACr ; (G) GMR-G AL4\UAS-yan, (H) GMR-GAL4;UAS-yan; UAS-hppy ^KQX . Flies are heterozygous for all transgenes. Anterior is to the right, dorsal is up. (I) Genetic interactions with regard to viability. Expression of hppy-RB enhances the semi-lethality induced by GMR-GAL4-drwen expression of a dominant negative form of the EGFR, UAS-egfr ^DN , as well as expression of an activated form of the EGFR/ERK pathway inhibitor Yan, UAS-yan ^ACT . Student's

paired t-test assuming equal variance revealed a significant difference between GMR- GAL4;UAS-egfr ^m and GMR-GAL4; UAS-egfr ^m ; UAS-hppy ^Rm (p=0.0027) as well as between GMR-GAL4; UAS-yan ^ACτ and GMR-GAL4; UAS-yan ^ACτ ; UλS-hppy™ ¹ (p=0.0064) (n=3). (J) Genetic interactions in ethanol-induced sedation. Flies expressing RNAi against EGFR, UAS-egfr ^mAl , under the control of the pan-neuronal driver elav- GAL4 ^cl55 in the hppy ^KG5531 homozygous mutant background do not display increased sensitivity to ethanol-induced sedation. One-way ANOVA of ST50 values revealed a significant difference between genotypes (p<0.0001). Post-hoc Newman-Keuls tests revealed a significant difference between elav-GAL4 ^cl5S ; hppy ^KG5537 /hppy™ ⁵⁵³⁷ ; UAS- egfr ^KNAl and Control (p<0.001), but failed to reveal a significant difference between elav-GAL4 ^cl55 ; hppy ^KG5531 /hppy ^KG553η ; UAS-egfr ^mAi and elav-GAL4 ^cl55 ; hppy ^KG5531 /hppy ^KG5531 (p>0.05), hppy ^KG5531 /hppy ^KG5537 ; UAS-egfr ^mAi (p>0.05), or hppy ^KG553η /hppy ^KG5531 (p>0.05) (n=8).

[0030] FIGS. 1 IA-11C depict the results from experimental manipulations that alter EGFR/ERK signaling level in the pathway in flies: activation of EGFR/ERK signaling in neuronal tissues decreases ethanol sensitivity as measured in the ethanol LOR assay. (A) Flies expressing a secreted form of the EGFR ligand Spitz, UAS-spi ^SEC , under the control of the pan-organismal driver Tub-GAL4 display increased resistance to ethanol- induced sedation. One-way ANOVA of the ST50 values revealed a significant difference between genotypes (p<0.0001). Post-hoc Newman-Keuls analysis revealed a significant difference between Tub-GAL4; UAS-spi ^SEC and Tub-GAL4 (p<0.01) as well as between Tub-GAL4; UAS-spi ^SEC and UAS-spi ^SEC (pO.OOl). n=8. (B) Flies expressing a wild-type form of the EGFR, UAS-egfr* ^{11 '} , under the control of the pan- neuronal driver elav-GAL4 ^cl5S are resistant to ethanol-induced sedation. One-way ANOVA of the ST50 values revealed a significant difference between genotypes (p<0.0001). Post-hoc Newman-Keuls analysis revealed a significant difference between elav-GAL4 ^c]55 ; UAS-egfr" ¹ and elav-GAL4 ^cl55 (p<0.001) as well as between elav- GAL4 ^clS5 ; UAS-egfr* ¹ and UAS-egfr™ ¹ (p<0.00\). n=8. (C) Flies expressing a constitutively active form of the ERK rolled, UAS-rl ^ACT , under the control of the pan- neuronal driver elav-GAL4 ^ci55 display increased resistance to ethanol-induced sedation. One-way ANOVA of the ST50 values revealed a significant difference between genotypes (p=0.0002). Post-hoc Newman-Keuls analysis revealed a significant

difference between elav-GAL4 ^cX55 ; UAS-rl ^ACτ and elav-GAL4 ^cl55 (p<0.01) as well as between elav-GAL4 ^cXS5 ; UAS-rl ^ACυ and UAS-rl ^ACT (/XO.001). n=7-8.

6. DETAILED DESCRIPTION OF THE INVENTION 6.1 Definitions

[0031] As used herein, the following terms shall have the following meanings: [0032] The terms "treat," "treating" or "treatment," as used herein, refer to a method of alleviating or abrogating a disorder and/or its attendant symptoms. The terms "prevent," "preventing" or "prevention," in certain embodiments, refer to a method of barring a subject from acquiring a disorder and/or its attendant symptoms. In certain embodiments, the terms "prevent," "preventing," or "prevention," refer to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms. [0033] The term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In preferred embodiments, the subject is a human. [0034] The term "substance-related disorder" refers to a Substance Use Disorder known to practitioners of skill in the art such as Substance Dependence, Substance Craving and Substance Abuse; Substance-Induced Disorders such as Substance Intoxication, Substance Withdrawal, Substance-Induced Delirium, Substance-Induced Persisting Dementia, Substance-Induced Persisting Amnestic Disorder, Substance- Induced Psychotic Disorder, Substance-Induced Mood Disorder, Substance-Induced Anxiety Disorder, Substance-Induced Sexual Dysfunction, Substance- Induced Sleep Disorder and Hallucinogen Persisting Perception Disorder (Flashbacks); Alcohol- Related Disorders such as Alcohol Dependence (303.90), Alcohol Abuse (305.00), Alcohol Intoxication (303.00), Alcohol Withdrawal (291.81), Alcohol Intoxication Delirium, Alcohol Withdrawal Delirium, Alcohol-Induced Persisting Dementia, Alcohol-Induced Persisting Amnestic Disorder, Alcohol-Induced Psychotic Disorder, Alcohol-Induced Mood Disorder, Alcohol-Induced Anxiety Disorder, Alcohol-Induced Sexual Dysfunction, Alcohol- Induced Sleep Disorder and Alcohol-Related Disorder Not Otherwise Specified (291.9); Amphetamine (or Amphetamine-like)-Related Disorders such as Amphetamine Dependence (304.40), Amphetamine Abuse (305.70), Amphetamine Intoxication (292.89), Amphetamine Withdrawal (292.0), Amphetamine

Intoxication Delirium, Amphetamine Induced Psychotic Disorder, Amphetamine- Induced Mood Disorder, Amphetamine- Induced Anxiety Disorder, Amphetamine- Induced Sexual Dysfunction, Amphetamine- Induced Sleep Disorder and Amphetamine- Related Disorder Not Otherwise Specified (292.9); a Caffeine Related Disorder such as Caffeine Intoxication (305.90), Caffeine- Induced Anxiety Disorder, Caffeine-Induced Sleep Disorder and Caffeine-Related Disorder Not Otherwise Specified (292.9); a Cannabis-Related Disorder such as Cannabis Dependence (304.30), Cannabis Abuse (305.20), Cannabis Intoxication (292.89), Cannabis Intoxication Delirium, Cannabis- induced Psychotic Disorder, Cannabis-induced Anxiety Disorder and Cannabis-Related Disorder Not Otherwise Specified (292.9); a Cocaine-Related Disorder such as Cocaine Dependence (304.20), Cocaine Abuse (305.60), Cocaine Intoxication (292.89), Cocaine Withdrawal (292.0), Cocaine Intoxication Delirium, Cocaine-Induced Psychotic Disorder, Cocaine-Induced Mood Disorder, Cocaine-Induced Anxiety Disorder, Cocaine-Induced Sexual Dysfunction, Cocaine- Induced Sleep Disorder and Cocaine- Related Disorder Not Otherwise Specified (292.9); Hallucinogen-Related Disorders such as Hallucinogen Dependence (304.50), Hallucinogen Abuse (305.30), Hallucinogen Intoxication (292.89), Hallucinogen Persisting Perception Disorder (Flashbacks) (292.89), Hallucinogen Intoxication Delirium, Hallucinogen-Induced Psychotic Disorder, Hallucinogen-Induced Mood Disorder, Hallucinogen-Induced Anxiety Disorder and Hallucinogen-Related Disorder Not Otherwise Specified (292.9); an Inhalant-Related Disorders such as Inhalant Dependence (304.60), Inhalant Abuse (305.90), Inhalant Intoxication (292.89), Inhalant Intoxication Delirium, Inhalant- Induced Persisting Dementia, Inhalant-Induced Psychotic Disorder, Inhalant- Induced Mood Disorder, Inhalant-Induced Anxiety Disorder and Inhalant-Related Disorder Not Otherwise Specified (292.9); Nicotine-Related Disorders such as Nicotine Dependence (305.1), Nicotine Withdrawal (292.0) and Nicotine-Related Disorder Not Otherwise Specified (292.9); Opioid-Related Disorders such as Opioid Dependence (304.00), Opioid Abuse (305.50), Opioid Intoxication (292.89), Opioid Withdrawal (292.0), Opioid Intoxication Delirium, Opioid-induced Psychotic Disorder, Opioid-induced Mood Disorder, Opioid-induced Sexual Dysfunction, Opioid-induced Sleep Disorder and Opioid- Related Disorder Not Otherwise Specified (292.9); a Phencyclidine (or Phencyclidine-Like)- Related Disorder such as Phencyclidine Dependence (304.60),

Phencyclidine Abuse (305.90), Phencyclidine Intoxication (292.89), Phencyclidine Intoxication Delirium, Phencyclidine-induced Psychotic Disorder, Phencyclidine- induced Mood Disorder, Phencyclidine-induced Anxiety Disorder and Phencyclidine- Related Disorder Not Otherwise Specified (292.9); Sedative-, Hypnotic-, or Anxiolytic- Related Disorders such as Sedative, Hypnotic, or Anxiolytic Dependence (304.10), Sedative, Hypnotic, or Anxiolytic Abuse (305.40), Sedative, Hypnotic, or Anxiolytic Intoxication (292.89), Sedative, Hypnotic, or Anxiolytic Withdrawal (292.0), Sedative, Hypnotic, or Anxiolytic Intoxication Delirium, Sedative, Hypnotic, or Anxiolytic Withdrawal Delirium, Sedative-, Hypnotic-, or Anxiolytic-Persisting Dementia, Sedative-, Hypnotic-, or Anxiolytic- Persisting Amnestic Disorder, Sedative-, Hypnotic-, or Anxiolytic-induced Psychotic Disorder, Sedative-, Hypnotic-, or Anxiolytic-induced Mood Disorder, Sedative-, Hypnotic-, or Anxiolytic-induced Anxiety Disorder Sedative- , Hypnotic-, or Anxiolytic-induced Sexual Dysfunction, Sedative-, Hypnotic-, or Anxiolytic-induced Sleep Disorder and Sedative-, Hypnotic-, or Anxiolytic-Related Disorder Not Otherwise Specified (292.9); Polysubstance- Related Disorder such as Polysubstance Dependence (304.80); and another (or Unknown) Substance-Related Disorder induced by Anabolic Steroids, Nitrate Inhalants and Nitrous Oxide. The terms describing the indications used herein are classified in the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, published by the American Psychiatric Association (DSM-IV); the "Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR)", Washington, DCl American Psychiatric Association, 2000; and/or the International Classification of Diseases, 10th Edition (ICD-10). The contents of all are hereby incorporated by reference in their entireties. The various subtypes of the disorders mentioned herein are contemplated as part of the present invention. Numbers in brackets after the listed diseases above refer to the classification code in DSM-IV.

[0035] In certain embodiments, the substance causing a substance-related disorder in a subject includes, but is not limited to alcohol, amphetamine or similarly acting sympathomimetics, caffeine, cannabis, cocaine, hallucinogens, inhalants, nicotine, opioids, phencyclidine (PCP) or similarly acting arylcyclohexylamines, sedatives, hypnotics, medications such as anesthetics, analgesics, antiparkinsonian medications, gastrointestinal medications, other over-the-counter medications, and antidepressant

medications. In another embodiment the substance causing the substance-related disorder in a subject includes but is not limited to pesticides containing nicotine, or ethylene glycol (antifreeze). In yet another embodiment the substance causing the substance-related disorder includes, but is not limited to volatile substances or "inhalants", such as fuel or glue, if they are used for the purpose of becoming intoxicated.

[0036] The term "withdrawal" as used herein refers to the development of a substance-specific maladaptive behavioral change, with physiological and cognitive concomitants, that is due to the cessation of, reduction in, heavy and prolonged substance use. This substance-specific syndrome can cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. The symptoms are not due to a general medical condition and are not accounted for by any other mental disorder. Withdrawal is usually, but not always, associated with Substance Dependence. Most (perhaps all) individuals with Withdrawal have a craving to readminister the substance to reduce the symptoms. The diagnosis of Withdrawal is recognized, but not limited to the following groups of substances: alcohol; amphetamines and other related substances; cocaine; nicotine; opioids; and sedatives, hypnotics, and anxiolytics. The dose and duration of use and other factors such as the presence or absence of additional illnesses also affect withdrawal symptoms. [0037] The term "addiction-related behavior" as used herein refers to behavior resulting from compulsive substance use and is characterized by apparent substance dependency.

[0038] The term "substance dependency" or "substance dependence" as used herein refers to a condition of a subject displaying a maladaptive pattern of substance use, leading to clinically significant impairment or distress, as manifested by three (or more) of the following apparent to a practitioner of skill in the art, occurring any time in the same 12-month period:

(1) tolerance, as defined by either of the following:

(a) a need for markedly increased amounts of the substance to achieve intoxication or desired effect

(b) markedly diminished effect with continued use of the same amount of the substance

(2) withdrawal, as manifested by either of the following:

(a) the characteristic withdrawal syndrome for the substance (development of a substance-specific syndrome due to the cessation of (or reduction in) substance use that has been heavy and prolonged, wherein the substance-specific syndrome causes clinically significant distress or impairment in social, occupational, or other important areas of functioning)

(b) the same (or a closely related) substance is taken to relieve or avoid withdrawal symptoms

(3) the substance is often taken in larger amounts or over a longer period than was intended

(4) there is a persistent desire or unsuccessful efforts to cut down or control substance use

(5) a great deal of time is spent in activities necessary to obtain the substance (e.g. visiting multiple doctors or driving long distances), use the substance (e.g. chain smoking), or recover from its effects

(6) important social, occupational, or recreational activities are given up or reduced because of substance use

(7) the substance use is continued despite the knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the substance (e.g. current cocaine use despite recognition of cocaine induced depression, or continued drinking despite recognition that an ulcer was made worse by alcohol consumption)

[0039] The term "alcohol" and "ethanol" as used herein are interchangeable. [0040] The term "alcohol abuse" as used herein refers to a condition of a subject displaying a maladaptive pattern of alcohol use leading to clinically significant impairment or distress, as manifested by one (or more) of the following apparent to a practitioner of skill in the art occurring within a 12-month period: recurrent alcohol use resulting in a failure to fulfill major role obligations at work, school, or home (e.g., school and job performance may suffer either from the aftereffects of drinking or from

actual intoxication on the job or at school; child care or household responsibilities may be neglected; and alcohol-related absences may occur from job or school); recurrent alcohol use in situations in which it is physically hazardous (e.g., driving an automobile or operating machinery while intoxicated); recurrent alcohol-related legal problems (e.g., arrests for intoxicated behavior or for driving under the influence); continued alcohol use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of the substance (e.g., violent arguments with spouse while intoxicated, child abuse). Alcohol abuse requires fewer symptoms and, thus, may be less severe than dependence and is only diagnosed once the absence of dependence has been established.

[0041] The term "alcohol withdrawal" as used herein refers to a condition of a subject fulfilling the following diagnostic criteria as judged by a practitioner of skill in the art:

(1) Cessation of (or reduction in) alcohol use that has been heavy and prolonged.

(2) Two (or more) of the following, developing within several hours to a few days after Criterion (1):

(a) autonomic hyperactivity (e.g. , sweating or pulse rate greater than 100)

(b) increased hand tremor

(c) insomnia

(d) nausea or vomiting

(e) transient visual, tactile, or auditory hallucinations or illusions

(f) psychomotor agitation

(g) anxiety

(h) grand mal seizures

(3) The symptoms in Criterion (2) cause clinically significant distress or impairment in social, occupational, or other important areas of functioning.

(4) The symptoms are not due to a general medical condition and are not better accounted for by another mental disorder.

[0042] The term "solvate" as used herein, refers to a compound, agent, or small molecule of the present invention that is complexed to a solvent. Solvents that can form solvates with the compounds, agents, and small molecules of the present invention include common organic solvents such as alcohols (methanol, ethanol, etc.), ethers, acetone, ethyl acetate, halogenated solvents (methylene chloride, chloroform, etc.), hexane and pentane. Additional solvents include water. When water is the complexing solvent, the complex is termed a "hydrate."

[0043] As used herein and unless otherwise indicated, the term "prodrug" means an EGFR inhibitor derivative that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly an EGFR inhibitor. Examples of prodrugs include, but are not limited to, derivatives and metabolites of an EGFR inhibitor that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. In certain embodiments, prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by Burger 's Medicinal Chemistry and Drug Discovery 6 ^th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmbh).

[0044] As used herein, the term "small molecule inhibitor" or "small molecule EGFR inhibitor" comprises, but is not limited to erlotinib, gefitinib, canertinib, PD169540, PD158780, AG1478, PD153035, CGP-59326, PKI-166, and GW-572016. [0045] As used herein, "gene expression regulator" refers to any agent that is capable of regulating the expression level of a given gene. The regulation may occur at different levels: during the transcription stage (synthesis of mRNA) or during the translation stage, e.g., through mechanisms such as RNA interference (RNAi). [0046] As used herein, "RNA interference (RNAi)" is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. Agents capable of exhibiting the effects of RNAi include but are not limited to a

small interfering RNA (siRNA), a microRNA (miRNA), and a short hairpin RNA (shRNA).

[0047] As used herein an " siRNA " refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene, for example the alk gene or a homolog or iso-form thereof. The double stranded RNA siRNA can be formed by the complementary strands. In one embodiment, a siRNA refers to a nucleic acid that can form a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-30 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length). [0048] As used herein "shRNA" or "small hairpin RNA" (also called stem loop) is a type of siRNA. In one embodiment, these shRNAs comprise a short (e.g. about 19 to about 25 nucleotides), antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, followed in turn by the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.

[0049] The terms "microRNA" or " miRNA " used interchangeably herein are RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. Endogenous microRNA are small RNAs naturally present in the genome which are capable of modulating the productive utilization of mRNA. The term artificial microRNA includes any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the productive utilization of mRNA. MicroRNA sequences have been described in publications such as Lim et al. , Genes & Development, 17, 991-1008 (2003), Lim et al. Science 299, 1540 (2003), Lee et al, Science, 294, 862 (2001), Lau et al, Science 294, 858-861 (2001), Lagos- Quintana et al., Current Biology, 12, 735-739 (2002), Lagos-Quintana et al, Science 294, 853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003), each of which is incorporated by reference in its entirety. Multiple microRNAs can also be

incorporated into a precursor molecule. Furthermore, miRNA-like stem-loops can be expressed in cells as a vehicle to deliver artificial miRNAs and short interfering RNAs ( siRNAs ) for the purpose of modulating the expression of endogenous genes through the miRNA and or RNAi pathways.

[0050] As used herein, "monoclonal antibodies (mAb or moAb)" are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell.

[0051] The term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Ro we et ah, Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0052] The term "therapeutically effective amount" are meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The term "therapeutically effective amount" also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.

6.2 Methods of Use

[0053] In one embodiment provided herein are methods for the treatment or prevention of an alcohol-related disorder in a subject having the disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to treat or prevent the alcohol-related disorder.

[0054] In one embodiment provided herein are methods for the treatment or prevention of a substance-related disorder in a subject having the disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to treat or prevent the alcohol-related disorder.

[0055] In one embodiment herein, the subject is a human.

[0056] In certain embodiments, the substance causing a substance-related disorder in a subject is selected from alcohol, amphetamine or similarly acting sympathomimetics, caffeine, cannabis, cocaine, hallucinogens, inhalants, nicotine, opioids, phencyclidine (PCP) or similarly acting arylcyclohexylamines, sedatives, hypnotics, anesthetics, analgesics, antiparkinsonian medications, gastrointestinal medications, other over-the- counter medications, and antidepressant medications.

[0057] In one embodiment, the substance causing a substance-related disorder in a subject is alcohol, nicotine, morphine, cocaine, methamphetamine, caffeine, or amphetamine. In certain embodiments, the substance causing a substance-related disorder in a subject is alcohol.

[0058] In certain embodiments, the substance-related disorder is alcohol abuse or alcohol withdrawal.

[0059] In one embodiment provided herein are methods of ameliorating or eliminating an effect of an alcohol-related disorder in a subject having the disorder, comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to ameliorate or eliminate the effect of the alcohol-related disorder. [0060] In one embodiment provided herein are methods of ameliorating or eliminating an effect of a substance-related disorder in a subject having the disorder, comprising administering to the subject an amount of an inhibitor of the EGFR/ERK

pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to ameliorate or eliminate the effect of the alcohol-related disorder.

[0061] In certain embodiments, the effect of an alcohol-related disorder includes, but is not limited to significant impairment or distress caused by a maladaptive pattern of alcohol use.

[0062] In certain embodiments, the effect of a substance-related disorder includes, but is not limited to significant impairment or distress caused by a maladaptive pattern of substance use.

[0063] The significant impairment or distress is manifested including, but not limited to recurrent substance use resulting in a failure to fulfill major role obligations at work, school, or home (e.g. repeated absences or poor work performance related to substance use; substance-related absences, suspensions, or expulsions from school; neglect of children or household); recurrent substance use in situations in which it is physically hazardous (e.g. driving an automobile or operating a machine when impaired by substance use); recurrent substance-related legal problems (e.g., arrests for substance- related disorderly conduct); continued substance use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of the substance (e.g. arguments with spouse about consequences of intoxication, physical fights).

[0064] In an additional embodiment, the effects of a substance-related disorder include, but are not limited to those biochemical or behavioral changes that occur as a result of and within a reasonable time frame following the administration of the substance. Different effects can be expected depending on the substance and the dose administered thereof. For example, the effects of low doses of ethanol include locomotor activation whereas the effects of high doses of ethanol include symptoms of alcohol intoxication (for definition of alcohol intoxication, see American Psychiatric Association, Diagnostic Criteria for DSM-IV, Washington D.C, 2000, p. 214f). [0065] In one embodiment provided herein are methods for diminishing, inhibiting or eliminating an addiction-related behavior in a subject having an alcohol-related disorder, comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to diminish, inhibit or eliminate the addiction-related behavior.

[0066] In one embodiment provided herein are methods for diminishing, inhibiting or eliminating an addiction-related behavior in a subject having a substance-related disorder, comprising administering to the subject an amount of an inhibitor of the

EGFR/ERK pathway or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to diminish, inhibit or eliminate the addiction-related behavior.

[0067] In one embodiment provided herein are methods for alleviating or eliminating withdrawal symptoms in a subject having an alcohol-related disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to alleviate or eliminate the withdrawal symptoms.

[0068] In one embodiment provided herein are methods for alleviating or eliminating withdrawal symptoms in a subject having a substance-related disorder comprising administering to the subject an amount of an inhibitor of the EGFR/ERK pathway, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, effective to alleviate or eliminate the withdrawal symptoms.

[0069] In another embodiment, representative withdrawal symptoms include, but are not limited to autonomic hyperactivity (e.g. sweating or pulse rate greater than 100); increased hand tremor; insomnia or hypersomnia; nausea or vomiting; transient visual, tactile, or auditory hallucinations or illusions; psychomotor agitation or retardation; anxiety; grand mal seizures; fatigue; vivid, unpleasant dreams; increased appetite or weight gain; dysphoric or depressed mood; irritability, frustration or anger; difficulty concentrating; restlessness; decreased heart rate; sweating; or muscle pain.

[0070] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is a macromolecular inhibitor.

[0071] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is an RNAi agent selected from the group consisting of siRNA, shRNA and miRNA.

[0072] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is an antibody or an antibody fragment targeting a protein associated with the

EGFR/ERK pathway.

[0073] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is a ribozyme or an antisense oligonucleotide targeting a gene associated with the EGFR/ERK pathway.

[0074] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is a small molecule inhibitor.

[0075] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is erlotinib (T ARCEV A®, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolineamine) or a pharmaceutically acceptable salt, hydrate or prodrug thereof. [0076] In certain embodiments provided herein the inhibitor of the EGFR/ERK pathway is Gefitinib [IRESSA®, N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3- morpholin-4-ylpropoxy)quinazolin-4-amine] or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

[0077] Erlotinib, a pharmaceutically acceptable salt, hydrate or prodrug thereof, can be administered in any form deemed suitable by a practitioner of skill in the art and by any technique deemed suitable by the same. Exemplary forms and techniques for administration are provided herein.

[0078] In one embodiment, erlotinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 500 mg per day.

[0079] In one embodiment, erlotinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 300 mg per day.

[0080] In one embodiment, erlotinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 150 mg per day.

[0081] In one embodiment, erlotinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 50 mg per day.

[0082] In one embodiment, erlotinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 20 mg per day.

[0083] Gefitinib, a pharmaceutically acceptable salt, hydrate or prodrug thereof, can be administered in any form deemed suitable by a practitioner of skill in the art and by any technique deemed suitable by the same. Exemplary forms and techniques for administration are provided herein.

[0084] In one embodiment, gefitinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 500 mg per day.

[0085] In one embodiment gefitinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 300 mg per day.

[0086] In one embodiment gefitinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 150 mg per day.

[0087] In one embodiment gefitinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 50 mg per day.

[0088] In one embodiment gefitinib is administered to a subject having a substance- related disorder in a dosage range of 0.1 to 20 mg per day.

6.3 Erlotinib

[0089] Erlotinib (Fig. 1, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolineamine) is a small molecule designed to target the human epidermal growth factor receptor (EGFR/HER1) pathway, which is one of the factors critical to cell growth in non-small cell lung (NSCLC) and pancreatic cancers. It is believed that

Erlotinib inhibits the tyrosine kinase activity of the EGFR signaling pathway inside the cell. In November 2004, the U.S. Food and Drug Administration (FDA) approved

TARCEV A ^® (150 mg; OSI Pharmaceuticals, Genentech), erlotinib hydrochloride, for the treatment of patients with locally advanced or metastatic non-small cell lung cancer

(NSCLC).

[0090] The present invention provides methods of treating or preventing a substance-related disorder using Erlotinib (T ARCEV A ^® , N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)-4-quinazolineamine) or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

[0091] In certain embodiments erlotinib may be provided as a pharmaceutically acceptable salt deemed suitable by one of skill in the art. (See, Berge et al., J. Pharm.

ScL 1977, 66, 1-19; and "Handbook of Pharmaceutical Salts, Properties, and Use," Stahl and Wermuth, Ed.; Wiley- VCH and VHCA, Zurich, 2002).

[0092] In one embodiment, the acid to form a pharmaceutically acceptable salt of erlotinib is hydrochloric acid or methanesulfonic acid.

[0093] In certain embodiments, erlotinib is used in form of a polymorph. Two polymorphic forms of erlotinib hydrochloride, polymorph A and B, have been described and characterized in U.S. patent no. 6,900,221 incorporated herein by reference in its

entirety. In one embodiment the pharmaceutical composition comprises erlotinib hydrochloride as polymorph A. In one embodiment the pharmaceutical composition comprises erlotinib hydrochloride as polymorph B. In another embodiment the pharmaceutical composition comprises erlotinib hydrochloride, wherein erlotinib hydrochloride is a mixture of polymorphs comprising at least 70%; at least 80% at least 85%; at least 90%; at least 95%; at least 97%; at least 98%; or at least 99% by weight of polymorph B.

6.3.1 Methods of Preparation

[0094] Erlotinib or a pharmaceutically acceptable salt, hydrate or prodrug thereof may be prepared by any method known to those of skill in the art. In one embodiment, erlotinib may be synthesized according to U.S. patent no. 6,900,221 or U.S. patent no. 5,747,498 the contents of which are hereby incorporated by reference in their entireties or any other method known in the literature or via methods know to the person of ordinary skill in the art. For example, the compound of the invention may be synthesized as shown in Fig. 2A. In the following description, the numbers given in bold represent the corresponding chemical structures shown in Fig. 1 and Fig. 2 of the disclosure. The following examples are presented by way of illustration, not limitation. [0095] The synthesis of erlotinib (1, Fig. 1) may be accomplished starting from compound 2 (Fig. 2A), which may be prepared according to Chandregowda et al. (Heterocycles 2007, 7/, 39). Reduction of the nitro group in compound 2 using a suitable reducing agent such as sodium dithionite, in a suitable solvent, such as water, at elevated temperature, such as 50°C, gives aniline 3. Upon treating aniline 3 with N ₅ N- dimethylformamide-dimethylacetal (DMF-DMA) in a suitable solvent, such as toluene, at elevated temperature, such as 105°C, formamidine 4 is obtained. Compound 4 is reacted with 3-ethynylaniline (from Sigma- Aldrich , St. Louis, MO 63178, USA) and a suitable acid, such as acetic acid, at elevated temperature, such as 130°C. After applying a purification method to the crude product, such as one or more recrystallizations in suitable solvents, such as ethyl acetate/methanol, erlotinib (1) is obtained. The free base of erlotinib (1) was transformed in the hydrochloric salt 1*HC1 by passing hydrochloric acid through a solution or suspension of 1 in a suitable solvent, such as methanol, keeping the temperature in a suitable range, such as 15-20 ⁰ C. It will be understood, that

any other salt can be obtained using different acids in processes similar to the one described hereinabove or other processes known in the literature or to the person skilled in the art. An actual example of the described synthesis and a detailed analytical characterization of intermediates 3 and 4 and the final product erlotinib can be found in

Chandregowda et al., Org. Proc. Res. Dev. 2007, 11, 813-816.

[0096] In one embodiment, the process for producing polymorph B of erlotinib hydrochloride using a solvent mixture comprising alcohol and water is a process analogous to the one disclosed in U.S. patent no. 6,900,221 (col 3, 11. 12-28; col 28,

Example 28).

[0097] In the process, the recrystallization may comprise the steps of:

(a) heating to reflux alcohol, water and the hydrochloride salt of N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine so as to form a solution;

(b) cooling the solution to between about 65°C and 70°C;

(c) clarifying the solution; and

(d) precipitating polymorph B by further cooling the clarified solution. [0098] In certain embodiments, the pharmaceutical composition comprises a homogeneous crystalline polymorph of the hydrochloride salt of N-(3-ethynylphenyl)- 6,7-bis(2-methoxyethoxy)-4-Quinazolinamine designated the B polymorph that exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2- theta at approximately 6.26, 12.48, 13.39, 16.96, 20.20, 21.10, 22.98, 24.46, 25.14, and 26.91.

[0099] In certain embodiments, the pharmaceutical composition comprises a polymorph of the hydrochloride salt of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)- 4-quinazolinamine that exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately the values shown in Table 1 below.

TABLE 1

Polymorph B

Anode: Cu - Wavelength 1 : 1.54056 Wavelength 2: 1.54439 (ReI Intensity: 0.500) Range #1 - Coupled: 3.000 to 40.040 StepSize: 0.040 Step Time: 1.00

Smoothing Width: 0.300 Threshold: 1.0

2-Theta I(rel) 2-Theta I(rel) 2-Theta I(rel) 2-Theta I(rel) 2-Theta I(rel)

6.255 100.0 17.668 2.5 22.982 4.8 27.534 0.9 32.652 1.7

7.860 3.2 18.193 0.7 23.589 2.3 28.148 1.5 33.245 1.7

9.553 3.9 18.749 1.5 23.906 3.0 28.617 4.3 34.719 1.5

1 1.414 1.5 19.379 1.0 24.459 6.8 29.000 1.4 35.737 0.8

12.483 6.4 20.196 14.4 25.138 10.0 29.797 2.1 36.288 1.0

13.385 9.6 20.734 4.2 25.617 3.7 30.267 0.9 36.809 0.6

14.781 2.1 21.103 14.4 25.908 3.9 30.900 1.6 37.269 1.1

15.720 2.9 21.873 4.7 26.527 2.8 31.475 2.2 37.643 1.4

16.959 5.5 22.452 4.5 26.911 5.6 31.815 2.4 38.1 14 1.7

[00100] In certain embodiments, a crystalline polymorph of the hydrochloride salt of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinami ne designated the B polymorph that exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 6.26, 12.48, 13.39, 16.96, 20.20, 21.10, 22.98, 24.46, 25.14 and, 26.91, which is free of the A polymorph. [00101] It is to be understood that the X-ray powder diffraction pattern is only one of many ways to characterize the arrangement of atoms comprising the compound N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine hydrochloride, and that other methods well known in the art, e.g. single crystal X-ray diffraction, may be used to identify in a sample, composition or other preparation the presence of polymorph B of the hydrochloride salt of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine.

6.3.2 Pharmaceutical Compositions

[00102] In certain embodiments, the pharmaceutical composition comprising erlotinib or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, is provided as a solid dosage form for oral administration.

[00103] In certain embodiments, the pharmaceutical composition comprising erlotinib or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, is provided as a film- coated tablet.

[0100] In certain embodiments, the pharmaceutical composition comprises erlotinib hydrochloride as the active ingredient.

[0101] In an certain embodiments, the pharmaceutical composition comprises polymorph B of erlotinib hydrochloride.

[0102] In certain embodiments, the pharmaceutical composition discussed herein comprises the following inactive ingredients: lactose monohydrate, hypromellose, hydroxypropyl cellulose, magnesium stearate, microcrystalline cellulose, sodium starch glycolate, sodium lauryl sulfate and titanium dioxide and trace amounts of color additives, including FD&C Yellow #6.

6.4 IRESSA

[0103] In certain embodiments, the small molecule inhibitor is gefitinib (also known as 201839 or IRESSA ^® ; Astrazeneca) (Woodburn, Proc. Am. Assoc. Cancer Res. 1997, 38, 633). Iressa is an orally active inhibitor which blocks signal transduction pathways implicated in promoting cancer growth (WO2002/28409; WO2002/0020; WO2002/005791; WO2002/002534; WO2001/076586; each of which are incorporated herein by reference. Iressa reportedly has antiangiogenic activity, it has antitumor activity against such cancers as colon, breast, ovarian, gastric, non-small lung cancer, pancreatic prostate, and leukemia, it eliminates EGFR, HER2, and HER3 phosphorylation, it inhibits human breast xenograft growth and it has been used in patients (Ciardiello, Clin. Cancer Res. 2001, 7, 1459-1465; Moulder, Cancer Res. 2001, 61, 8887-8895; Barker, Bioorg. Med. Chem. Lett. 2001, //, 1911-1914; Moasser, Cancer Res. 2001, 61, 7184-7188; Chan, Caner Res. 2002, 62, 122-128; and Ranson, J. Clinc. Oncol. 2002, 20, 2240-2250).

[0104] Iressa is a quinazoline and has the chemical name N-(3-chloro-4- fluorophenyl)-7-methoxy-6-(3 -morpholin-4-ylpropoxy)quinazolin-4-amine, and the chemical formula C ₂₂ H ₂₄ CIFN ₄ O ₃ . The agent is disclosed in International Patent Application WO 96/33980 (Example 1) has the structure of the formula:

[0105] The present invention provides methods of treating or preventing a substance-related disorder using gefitinib or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

[0106] In certain embodiments gefitinib may be provided as a pharmaceutically acceptable salt deemed suitable by one of skill in the art. (See, Berge et al., J. Pharm. ScL 1977, 66, 1-19; and "Handbook of Pharmaceutical Salts, Properties, and Use," Stahl and Wermuth, Ed.; Wiley-VCH and VHCA, Zurich, 2002).

6.4.1 Methods of Preparation

[0107] Gefitinib or a pharmaceutically acceptable salt, hydrate or prodrug thereof may be prepared by any method known to those of skill in the art. In one embodiment, erlotinib may be synthesized according to International Patent Application WO 96/33980 the content of which is hereby incorporated by reference in its entirety or by any other method known in the literature or via methods know to the person of ordinary skill in the art. For example, gefitinib may be synthesized as shown in Fig. 2B. In the following description, the numbers given in bold represent the corresponding chemical structures shown in Fig. 2B of the disclosure. The following examples are presented by way of illustration, not limitation.

[0108] The synthesis of gefitinib (8, Fig. 2B) may be accomplished starting from compound 5, which may be prepared according to Chandregowda et al. {Heterocycles 2007, 71, 39) which is herewith incorporated by reference in its entirety. Reduction of the nitro group in compound 5 using a suitable reducing agent such as sodium dithionite, in a suitable solvent, such as water, at elevated temperature, such as 50 ⁰ C, gives aniline 6. Upon treating aniline 6 with N,N-dirnethylformarnide-dimethylacetal (DMF-DMA) in a suitable solvent, such as toluene, at elevated temperature, such as 105 ⁰ C, formamidine 7 is obtained. Compound 7 is reacted with 3-chloro-4-fluoroaniline (from Sigma- Aldrich , St. Louis, MO 63178, USA) and a suitable acid, such as acetic acid, at

elevated temperature, such as 130 ⁰ C. The reaction is then cooled, quenched with ice- water and adjusted to ~pH 9 with a suitable base, such as ammonia solution. A suitable solvent, such as ethyl acetate, is added and upon stirring and a solid precipitate corresponding to crude gefitinib is obtained via filtration. The crude material is suspended in a suitable solvent, such as methanol. A suitable acid, such as concentrated hydrochloric acid, is added with efficient stirring. A precipitate is formed, which is collected and washed with a suitable solvent, such as chilled methanol, to give a salt of gefitinib, such as the hydrochloric salt. The solid is suspended in a suitable solvent, such as water, cooled, filtered and washed with the suitable solvent to give the solid gefitinib salt. The solid is suspended in a suitable solvent, such as water, and adjusted to pH ~8 using a suitable base, such as ammonia solution, filtered and dried at suitable elevated temperature, such as 50°C, to give the final product 8. It will be understood, that any other salt can be obtained using different acids in processes similar to the one described hereinabove or other processes known in the literature or to the person skilled in the art. An actual example of the described synthesis and a detailed analytical characterization of intermediates 6 and 7 and the final product gefitinib can be found in Chandregowda et al., Org. Proc. Res. Dev. 2007, 11, 813-816 which herewith incorporated by reference in its entirety.

6.4.2 Pharmaceutical Compositions

[0109] Gefitinib, a pharmaceutically acceptable salt, hydrate or prodrug thereof, can be administered in any form deemed useful by the practitioners of skill in the art. In certain embodiments, gefitinib is administered in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients or diluents.

[0110] In certain embodiments, the pharmaceutical composition provided herein is provided as a solid dosage form for oral administration.

[0111] In certain embodiments, the pharmaceutical composition provided herein is provided as a film-coated tablet.

[0112] In certain embodiments, the pharmaceutical composition comprises the free base of gefitinib as the active ingredient.

[0113] In certain embodiments the pharmaceutical composition comprises the following inactive ingredients: Lactose monohydrate, microcrystalline cellulose,

croscarmellose sodium, povidone, sodium lauryl sulfate and magnesium stearate. Coating: hypromellose, polyethylene glycol 300, titanium dioxide, red ferric oxide and yellow ferric oxide.

6.5 Additional Small Molecule Inhibitors of the EGFR/ERK Pathway

[0114] In one aspect provided herein are small molecule inhibitors of the EGFR/ERK pathway for use in the methods presented in the instant disclosure. In one embodiment the small molecule inhibitor is a inhibitor of human endothelial growth factor receptor (EGFR/HER1). The following embodiments provide a non-limiting list of exemplary small molecule inhibitors of the EGFR/ERK pathway used in the methods provided herein.

[0115] In a further embodiment useful small molecule inhibitors of the EGFR/ERK pathway are described in Gibson (WO 1996/33980). In one embodiment the compound is 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(2-pyrrolidin-l-y lethoxy) quinazoline, 4- (3'-chloro-4'-fluoroanilino)-7-methoxy-6-(2-mopholinoethoxy) quinazoline, 4-(3'- chloro-4 '-fluoroanilino)-6-(3 -diethylaminopropoxy)-7-methoxy quinazoline, 4-(3 '- chloro-4'-fluoroanilino)-7-methoxy-6-(3-pyrrolidin-l-ylpropo xy) quinazoline, 4-(3'- chloro-4'-fluoroanilino)-6-(3-dimethylamιnopropoxy)-7-metho xy quinazoline, or 4- (3',4'-difluoroanilino)-7-methoxy-6-(3-mopholinopropoxy) quianzoline. [0116] In a further embodiment useful small molecule inhibitors of the EGFR/ERK pathway are described in Schnur et al. (WOl 996/30347). In one embodiment the inhibitor is (6,7-dimethoxyquinazolin-4-yl)-(3-ethynylphenyl)-amine, (6,7- dimethoxyquinazolin-4-yl)-[3-(3'-hydroxypropyn-l-yl) phenyl]-amine, (6,7- dimethoxyquinazolin-4-yl)-{(3-(2'-(aminomethyl)-ethynyl)phen yl]-amine, [(3- ethynylphenyl)-(6-nitroquinazolin-4-yl)-amine, or (6,7-dimethoxyquinazolin-4-yl)-(4- ethynylphenyl)-amine.

[0117] In a further embodiment the EGFR inhibitor is selected from a group consiting of compounds described in Barker et al. (WO/1997/30034). In one embodiment the compound is 6-(3-furyl)-4-[3-methyl-4-(2-pyridylmethoxy) anilino]quinazoline, 6-bromo-4-3-methyl-4-(2-pyridylmethoxy) anilino) quinazoline, 6- (2-benzimidazolylthiomethyl)-4-(3-methylanilino) quinazoline, 4-[3-methyl-4-(2-

pyridylmethoxy) anilino]-6-(2-thienyl) quinazoline, or 4-(3methylanilino)-6-( 1,2,3 - triazol-4-ylthiomethyl) quinazoline.

[0118] In a further embodiment the EGFR inhibitor is selected from compounds described in Barker et al. (WO 1997/30044). In one embodiment the compound is 4-(3- chloro-4-fluoroanilino)-7-(3-thienyl) quinazoline, 4-(3-chloro-4-fluoroanilino)-7-(3- furyl) quinazoline, 4-(3-chloro-4-fluoroanilino)-7-(2-furyl) quinazoline, 4-(3-chloro4- fluoroanilino)-7-(l-imidazolyl) quinazoline or 4-(3-chloro-4fluoroanilino)-7-(5- morpholinomethylthien-3 -yl) quinazoline.

[0119] In a further embodiment the EGFR inhibitor is selected from compounds described in Gibson (WO 1997/38994). In one embodiment the compound is 4-(3'- chloro-4'-fluoroanilino)-7-methoxy-6-tetrahydrofuran-2-ylmet hoxyquinazoline, 4-(3'- chloro-4'-fluoroanilino)-6-( I,3-dioxolan-2ylmethoxy)-7-methoxyquinazoline, 4-(3 '- chloro-4'-fluoroanilino)-6-[2-(l,3-dioxolan-2-yl)ethoxy]-7-m ethoxyquinazoline, 4-(3'- chloro-4'-fluoroanilino)-6-[3-(l ,3 -dioxolan-2-yl)propoxy]-7-methoxy quinazoline, or 4- (3'-chloro-4' -fluoroanilino)-6-(l,3-dioxolan-4-ylmethoxy)-7-methoxyquinaz oline. [0120] In a further embodiment the EGFR inhibitor is selected from compounds described in Schnur (WO 1997/49688). In one embodiment the compound is 4-(3- ethynylanilino)-imidazo [4,5-g]quinazoline, (3- ethynyl-phenyl)-(3-methyl-3H- imidazo[4,5-g]quinazolin-8-yl)-amine, (3-ethynyl-phenyl)-(l-methyl-lH-imidazo[4,5- g]quinazolin-8-yl)-amine, or benzo[g]quinazolin-4-yl-(3-ethynyl-phenyl)-amine. [0121] In a further embodiment the EGFR inhibitor is selected from compounds described in Cockerill et al. (WO 1998/002434). In one embodiment the compound is 1- (3-(4-(l -benzyl- lH-indazol-5-ylamino)-quinazolin-6-yl)-l,2,4-oxadiazol-5- ylmethyl)piperidin-4-one, l-(3-(4-(l-benzyl-l H-indazol-5-ylamino)-quinazolin-6-yl)- l,2,4-oxadiazol-5-ylmethyl)-pyrrolidin-2-one, (1 -benzyl- lH-indazol-5-yl)-(6-(5-((2- methanesulphonyl-ethylamino)-methyl)-furan-2yl)-quinazolin-4 -yl)-amine, (4- benzyloxy-phenyl)-(6-(5-(((2-methanesulphonyl-ethyl)-methyl- amino)-methyl)-furan-2- yl)-quinazolin-4-yl)-amine, or N-(2-((5 -(4-(4-benzyloxy-phenylamino)-quinazolin-6-yl)- furan-2-ylmethyl)-amino)ethyl)-methanesulphonamide.

[0122] In a further embodiment the EGFR inhibitor is selected from compounds described in Bridges et al. (WO1997/38983). In one embodiment the compound is N- [4- (3-Bromo-phenylamino)-pyrido[4,3-d]pyrimidin-7yl]-N-(3-morph olin-4-yl-propyl)-

acrylamide, N- [4-(3-Bromo-phenylamino)-pyrido [3 ,4-d]pyrimidin-6yl] -N-(3 -moφholin-

4-yl-propyl)-acrylamide, N-[4-(3-Bromo-phenylamino)-quinazolin-7-yl]acrylamide, N-

[4-[(3-Bromophenyllamino]quinazolin-7-yl]-N-[3-morpholino propyl]acrylamide, or 3-

[4-(3-Bromo-phenylamino)-quinazolin-7-ylcarbamoyl] acrylic acid

[0123] In a further embodiment the EGFR inhibitor is selected from compounds described in Bridges et al. (WOl 995/19774). In one embodiment the compound is 4-(3- bromoanilino)-6-(piperidin-l-yl)pyrido[3,4d]pyrimidine, 7-amino-4-(4- methoxyanilino)pyrido[4,3-d]pyrimidine, 7-amino-4-(3-ethylanilino)pyrido[4,3- djpyrimidine, 4-benzylaminopyrrolo [2, 3-d]pyrimidine, or 4-benzylaminothieno[3,2- d]pyrimidine.

[0124] In a further embodiment the EGFR inhibitor is selected from compounds described in Bridges et al. (WO1995/19970). In one embodiment the compound is 4-(3- bromoanilino)-benzo[g]quinazoline, 4-([R]-I -phenylethylamino)-benzo[g]quinazoline,

4-(3 -bromoanilino)-pyrrolo [3 ,2g]quinazoline, 4-(3 -bromoanilino)-thiazolo [5 ,4- g]quinazoline, or 4-(3-Bromoanilino)oxazolo[5,4-g]quinazoline.

[0125] In a further embodiment the EGFR inhibitor is selected from compounds described in Cockerill et al. (WO 1997/ 13771). In one embodiment the compound is 4-

(4-benzyloxvanilino)-6-chloropyrido[3,4-d]pyrimidine, 4-(4-benzyloxvanilino)-6-(N- methylimidazol-5-yl)pyrido[3,4-d]pyrimidine, 4-(4-benzyloxvanilinol-6-(N- methylimidazol-2-yl)pyrido[3,4-d]pyrimidine, 4-(4-benzyloxvanilinol-6-(N- methylpyrazol-2-yl)pyrido [3 ,4-d]pyrimidine, or 4-(4-benzyloxvanilinol-6-(furan-2- yl)pyrido [3 ,4-d]pyrimidine.

[0126] In a further embodiment the EGFR inhibitor is selected from compounds described in Cockerill et al. (WO1998/02437). In one embodiment the compound is (4-

Benzyloxy-phenyl)-(6-(5-(4-methyl-piperazin-l-ylmethyl)-f uran-2- yl)pyrido[3,4d]pyrimidin-4-yl)-amine, (4-benzyloxy-phenyl)-(6-(5-((2- methanesulphonyl-ethylamino)methyl)-furan-2-yl)pyrido[3,4d]- pyrimidin-4-yl)-amine,

((5-(4-(4-benzyloxy-phenylamino}-pyrido[3,4-d]pyrimidin-6 -yl)-furan-2-ylmethyl) amino)-acetic acid methyl ester, (4-benzyloxy-phenyl)-(6-(5-(pyridin-3-ylaminomethyl)- furan-2-yl)-pyrido [3 ,4d]pyrimidin-4-yl)-amine, or (4-benzyloxy-phenyl)-(6-(5-

(dimethylaminomethyl)-furan-2-yl)-pyrido[3,4-d]pyrimidin- 4-yl)-amine.

[0127] In a further embodiment the EGFR inhibitor is selected from compounds described in Cockerill et al. (WO1998/02438). In one embodiment the compound is (1- benzyl-1 H-indazol-5-yl)-(6-chloro-pyrido[3,4-d]pyrimidin-4-yl)-amine , N4-(l -benzyl- 1 H-indazol-5-yl)-N6,N6-dimethyl-pyrido[3,4-d]pyrimidine-4,6-d iamine, (1-benzyl-l H- indazol-5 -yl)-6-(N-(2-hydroxyethyl)-N-methylamino)-pyrido [3 ,4-d]pyrimidin-4-yl)- amine, (1-benzyl-l H-indazol-5-yl)-(pyrido[3,4-d]pyrimidin-4-yl)-amine, or (2 -benzyl- 1 H-benzimidazol-5-yl)-(6-chloro-pyrido[3,4-d]pyrimidin-4-yl)- amine. [0128] In a further embodiment the EGFR inhibitor is selected from compounds described in Dahmann et al. (CA 2243994). In one embodiment the compound is 4-[(3- chloro-4-fl uoro-phenyl) amino]-7-(4-amino- 1 -piperidinyl)-pyrido [4,3 -d]pyrimidine, 4- [(3 -chloro-4-fluoro-phenyl) amino] -7- [(l-methyl-4-piperidinyl) aminoj-pyrido [4,3 - d]pyrimidine, 4- [(3 -chloro-4-fluoro-phenyl) amino]-7-[4-(4-piperidinyl) piperidin-1-yl]- pyrido[4,3-d]pyimidine, 4- [(3 -chloro-4-fluoro-phenyl) amino]-7-[(l -tert- butyloxycarbonyl-4-piperidinyl) amino]pyrido[4,3-d]pyrimidine, or 4-[(3-chloro-4- fluoro-phenyl) amino]-7-[[trans-4(methoxycarbonyl)cyclohexyl)]-amino]pyrido [4,3- d]pyrimidine.

[0129] In a further embodiment the EGFR inhibitor is selected from compounds described in Boschelli et al. (WO 1998/33798). In one embodiment the compound is 2- cyclohexylamino-8-ethyl-8H-pyrido[2,3-d]pyrimidin-7-one, 2-(biphenyl-4-ylamino)-8- ethyl-8H-pyrido[2,3-d]pyrimidin-7-one, 8-ethyl-2-(pyridin-4-ylamino)-8H-pyrido[2,3- d]pyrimidin-7-one, 8-ethyl-2-(4-methoxyphenylamino)-8H-pyrido[2,3-d]pyrimidin-7 -one, or 2-[4-(2-diethylaminoethoxy)-phenylamino]-8-ethyl-8H-pyrido[2 ,3-d]pyrimidin-7-one. [0130] In a further embodiment the EGFR inhibitor is selected from compounds described in Dahmann et al. (CA02248720). In one embodiment the compound is 4-[(3- chloro-4-fluorophenyl) amino]-6-[4-(4-piperidinyl)l-piperidinyl]pyrimido[5,4- d]pyrimidine, 4- [(3-chloro-4-fluorophenyl) amino] -6- [ 1 -( 1 -piperidinyl)2- propylamino]pyrimido[5,4-d]pyrimidine, 4-[(3-chloro-4-fluorophenyl) amino]-6[l - (N,N-diethylamino)-2-propylamino]pyrimido[5/4-d]pyrimidine, or 4-[(3-chloro-4- fluorophenyl) amino]-6-[4-(aminomethyl)-cyclohexylmethylamino]-pyrimido[5, 4-d]- pyrimidine.

[0131] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (WO 1997/02266). In one embodiment the compound is (R)-

6-(3 -isobutyrylamino-phenyl)-4- [( 1 -phenyl-ethyl-amino)-7H-pyrrolo [2,3 -d] -pyrimidine, (R)-6-(4-isobutyrylamino-phenyl)-4 [( 1 -phenyl-ethyl)-amino] -7H-pyrτolo [2,3 d]- pyrimidine, (R)-6-(4-pivaloylamino-phenyl)-4[(l-phenyl-ethyl)-amino]-7H- pyrrolo[2,3- d] -pyrimidine, or (R)-6-(3-pivaloylamino-phenyl)-4[(l -phenyl-ethyl)-amino]-7H- pyrrolo[2,3-d]-pyrimidine.

[0132] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (WO 1997/27199). In one embodiment the compound is 4- (2,3-dihydroindol-l-yl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimi dine, 4-(6-Chloro-2,3- dihydroindol-l-yl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidine, 4-(6-bromo-2,3- dihydroindol-l-yl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidine, 4-(6-methyl-2,3- dihydroindol-l-yl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidine, or 4-(l, 2,3,4- tetrahydroquinolin-l-yl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrim idine. [0133] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (WO 1998/07726). In one embodiment the compound is 4-(3- chloroanilino)-6-[(4-methoxybenzylamino)-methyl]-7H-pyrrolo[ 2,3-d]pyrimidine, 4-(3- chloroanilino)-6- [(4-hydroxybenzylamino)methyl] -7H-pyrrolo [2,3 -d]pyrimidine, 4-(3 - Chloroanilino)-7Hpyrrolo[2,3-d]pyrimidine-6-carbaldehyde O-methyl oxime, 4-(3- chloroanilino)-6(moφholin-4-yl-carbonyl)-7H-pyrrolo[2,3-d]p yrimidine, or 4-(3- chloroanilino)-6-[(4-methylpiperazin-l-yl)carbonyl]-7H-pyrro lo[2,3-d]pyrimidine. [0134] In a further embodiment the EGFR inhibitor is selected from compounds described in Altaian (US Patent 6,051,557). In one embodiment the compound is 5-(4- methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl-amine, 5-(3-benzyloxy-phenyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl-amine, 5-(3-fluoro-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl-amine, 5-(4-fluoro-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl-amine, or 5-(3-chloro- phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl-amine.

[0135] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (WOl 996/31510). In one embodiment the the compound is 4- (3-chloro-phenylamino)-3-(4-pivaloylamino-phenyl)-l H-pyrazolo[3,4-d]pyrimidine, 4- (3-chloro-phenylamino)-3-(3-pivaloylamino-phenyl)-l H-pyrazolo[3,4-d]pyrimidine, 3- (4-acetylamino-phenyl)-4-(3-chloro-phenylamino)-l H-pyrazolo[3,4-d]pyrimidine, or 3- (3 -acetylamino-phenyl)-4-(3 -chloro-phenylamino)- 1 H-pyrazolo [3 ,4-d]pyrimidine.

[0136] In a further embodiment the EGFR inhibitor is selected from compounds described in Bold et al. (WO1998/14449). In one embodiment the compound is as 4-(3- chlorophenylamino)-3 -(3 ,4-dimethoxybenzylamino)- 1 H-pyrazolo [3 ,4-d]pyrimidine, 4- (3 -chlorophenylamino)-3 (3 -hydroxy-4-methoxybenzylamino)- 1 H-pyrazolo [3 ,4- d]pyrimidine, 4-(3-chlorophenylamino)-3(4-hydroxy-3,5-dimethoxybenzylamino )-l H- pyrazolo [3 ,4-d]pyrimidine, 4-(3 -chlorophenylamino)-3 (2,3 - methylenedioxybenzylamino)-lH-pyrazolo[3,4-d]pyrimidine, or 3-(3- chlorobenzylamino)-4-(3-chlorophenylamino)-l H-pyrazolo[3,4-d]pyrimidine. [0137] In a further embodiment the EGFR inhibitor is selected from compounds described in Bold et al. (WOl 998/14450). In one embodiment the compound is as 4-(3- chloro-phenylamino)-3 -(pyrid-2-ylcarbonylamino)- 1 H-pyrazolo [3 ,4-d]pyrimidine, 4-(3 - chloro-phenylamino)-3-(2,3-methylenedioxy-benzoylamino)-lH-p yrazolo[3,4- djpyrimidine, rac-3-([N-benzyloxycarbonyl-valyl]-amino)-4-(3-chlorophenyla mino)- 1 H-pyrazolo [3 ,4-d]pyrimidine, rac-3 -((N-benzyloxycarbonyl-alanyl)-amino)-4-(3 - chloro-phenylamino)-l H-pyrazolo[3,4-d]pyrimidine, or rac-4-(3-chloro-phenylamino)- 3-((N-ethoxycarbonyl-valyl)-amino)- 1 H-pyrazolo[3 ,4-d]pyrimidine. [0138] In a further embodiment the EGFR inhibitor is selected from compounds described in Zimmermann (WO 1995/09847). In one embodiment the compound is N- [3-(l,l,2,2-tetrafluoro-ethoxy)-phenyl]-4-[2-(2-aminoethylam ino)-5-pyridyl]-2- pyrimidineamine, N- [3 -( 1 , 1 ,2,2-tetrafluoro-ethoxy)-phenyl] -4- [2- { 2-(4- formylpiperazinyl)ethylamino}-5-pyridyl]-2-pyrimidineamine, N-[3-(l, 1 ,2,2-tetrafluoro- ethoxy)-phenyl]-4-(2-carbamoyl-4-pyridyl)-2-pyrimidineamine, N-[3(l , 1 ,2,2- tetrafluoro-ethoxy)-phenyl] -4-(2-carboxy-4-pyridyl)-2-pyrimidineamine, or N- [3 - (1,1 ,2,2-tetrafluoro-ethoxy)-phenyl] -4-[ 1 -(2-phthalimido-ethyl)-3 - 1 H-indolyl] -2- pyrimidineamin.

[0139] In a further embodiment the EGFR inhibitor is selected from compounds described in Davis et al. (WO 1997/19065). In one embodiment the compound is 4-(4- cyanophenyl)-N-(3,4,5-trimethoxyphenyl)-2-pyrimidineamine, 4-(4- ethoxycarbonylphenyl)-N-(3 ,4,5-trimethoxyphenyl)-2-pyrimidineamine, 4-( 1 -naphthyl)- N-(3,4,5-trimethoxyphenyl)-2-pyrimidineamine, 4-(4,5-dimethylthiazol-2-yl)-N-(3,4,5- trimethoxyphenyl)-2-pyrimidineamine, or 4-(4-Methoxyphenyl)-N-(3,4,5- trimethoxyphenyl)-2-pyrimidinearnine.

[0140] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (WO1998/17662). In one embodiment the compound is 3-(3- bromophenyl)-5-hydroxy-7-methoxy-4-quinolone, 3-(3, 4-dichlorophenyl)-5-hydroxy-7- methoxy-4-quinolone, 3 -(3 -hydroxyphenyl)-5-hydroxy-7-methoxy-4-quinolone, 3 -(3 - chlorophenyl)-5-hydroxy-6,7-dimethoxy-4-quinolone, or 3-(3-chlorophenyl)-5-hydroxy-

6,7-methylenedioxy-4-quinolone.

[0141] In a further embodiment the EGFR inhibitor is selected from compounds described in Carter et al. (WO1999/35146). In one embodiment the compound is (4-(4- fluorobenzyloxy)-phenyl)-(6-(5-((2-methanesulphonyl-ethylami no)methyl)-furan-2-yl)- pyrido[3,4-d]pyrimidin-4-yl)-amine, (4-(3-fluorobenzyloxy)-phenyl)-(6-(5-((2- methanesulphonyl-ethylamino)methyl)-furan-2-yl)-pyrido[3,4d] pyrimidin-4-yl)-amine,

(4-benzenesulphonyl-phenyl)-(6-(5-((2-methanesulphonyl-et hylamino)-methyl)furan-2- yl)-pyrido[3,4-d]pyrimidin-4-yl)-amine, (4-benzyloxy-phenyl)-(6-(3-((2- methanesulphonyl-ethylamino)-methyl)-phenyl)-pyrido [3 ,4d]pyrimidin-4-yl)-amine, or

(4-benzyloxyphenyl)-(6-(5-((2-methanesulphonyl-ethylamino )-methyl)-furan-2-yl)- quinazolin-4-yl)-amine.

[0142] In a further embodiment the EGFR inhibitor is selected from compounds described in Cockerill et al. (WO 1999/35132). In one embodiment the compound is (1- benzyl-lH-indazol-5-yl)-(6-(2-(methanesulphonyl)-ethylaminom ethyl)-quinazolin-4yl) amine, 2-(4-(l-benzyl-lH-indazol-5-ylamino) quinazolin-6-yl)methylamino) acetic acid,

2-(4-(l -benzyl- lH-indazol-5-ylamino) quinazolin-6-yl)methylamino) acetamide, 2-(N-

(4-( 1 -benzyl- 1 H-indazol-5-ylamino) quinazolin-6-yl)methyl)-N-methylamino) acetamide, (2R)-I -(4-(I -benzyl- 1 H-indazol-5-ylamino) quinazolin-6- ylmethyl)pyrrolidine-2-carboxylic acid t-butyl ester.

[0143] In a further embodiment the EGFR inhibitor is selected from compounds described in Tang (WO1999/07701). In one embodiment the compound is 1 ,2-dimethyl-

6,7-bis(4-bromo-phenyl)imidazo[4,5-g]quinoxaline, l,2,9-trimethyl-6,7-bis(4- bromophenyl)-imidazol[4,5-g]quinoxaline, 2-amino-6,7-bis(4-bromophenyl)-l,9- dimethyl-imidazol[4,5-g]quinoxaline, or 6,7-Bis(4-bromophenyl)-2-Methylamino-l,9- dimethylimidazol-[4,5-g]quinoxaline.

[0144] In a further embodiment the EGFR inhibitor is selected from compounds described in Spada et al. (WO 1992/20642). In one embodiment the compound is 6-

(thien-3-yl)-l,8-naphthyridin-2(l H)-one, 3-(thien-3-yl)-6,7-dimethylquinoline, 6-(4- methoxyphenyl)- 1 ,8-naphthyridin-2(l H)-one, 5,6-dimethoxy-2-(2- phenylethenyl)benzothiazole, or 3 -( 1 -cyclopent- 1 -enyl)-6,7-dimethoxyquinoline. [0145] In a further embodiment the EGFR inhibitor is selected from compounds described in Barker et al. (CA2071087). In one embodiment the compound is 4-(3'- bromoanilino) quinazoline, 4-(3'-iodoanilino) quinazoline, 6-chloro-4-(3'-chloroanilino) quinazoline, 6-bromo-4-(3'-methylanilino) quinazoline, or 4-(3'-nitroanilino) quinazoline.

[0146] In a further embodiment the EGFR inhibitor is selected from compounds described in Barker (CA2086968). In one embodiment the compound is 6-(2- chloroacetamido)-4-(3 '-methylanilino) quinazoline, 6-dimethylamino-4-(3 '- methylanilino) quinazoline, 6-(2-bromoethoxy)-4-(3 '-methylanilino) quinazoline, 6-(2- methoxyethoxy)-4-(3 '-methylanilino) quinazoline, or 6-(2-dimethylaminoethoxy)-4-(3'- methylanilino) quinazoline.

[0147] In a further embodiment the EGFR inhibitor is selected from compounds described in Arnold et al. (EP837063). In one embodiment the compound is 4-(6- Chloro-2,3-dihydro-indol- 1 -yl)-6-phenyl-quinazoline, 7-methoxy-6-phenyl-3H- quinazolin-4-one, 4-(6-Chloro-2,3-dihydro-indol- 1 -yl)-7-methoxy-6-phenyl-quinazoline, { 6- [3 -(benzyl-methyl-amino)-prop- 1 -ynyl] -quinazolin-4-yl } -( 1 H-indol-5 -yl)-amine, or (3-Ethynyl-phenyl)-(6-pyridin-2-ylethynyl-quinazolin-4-yl)-a mine. [0148] In a further embodiment the EGFR inhibitor is selected from compounds described in Traxler et al. (US 5,686,457). In one embodiment the compound is 4-(m- fluoroanilino)-5,6-tetramethylene-7H-pyrrolo[2,3-d]pyrimidin e, 4-(m-methylanilino)- 5,6-tetramethylene-7H-pyrrolo[2,3-d]pyrimidine, 4-(m-methoxyanilino)-5,6- tetramethylene-7H-pyrrolo[2,3-d]pyrimidine, 4-(m-chloroanilino)-5,6-diphenyl-7H- pyrrolo[2,3-d]pyrimidine, or 4-(m-bromoanilino)-5,6-diphenyl-7H-pyrrolo[2,3- d]pyrimidine.

[0149] In a further embodiment the EGFR inhibitor is selected from compounds described in Schnur et al. (US 5,747,498). In one embodiment the compound is 4-(3- ethynyl-phenylamino)-6-(2-methoxy-ethoxy)-quinazolin-7-ol, 4-(3-ethynyl- phenylamino)-7-(2-methoxy-ethoxy)-quinazolin-6-ol, 1 - {2-[4-(3-ethynyl-phenylamino)- 6-(2-methoxy-ethoxy)-quinazolin-7-yloxy] -ethyl}- lH-pyridin-4-one, 1 -{2-[4-(3-

ethynyl-phenylamino)-7-(2-methoxy-ethoxy)-quinazolin-6-yl oxy]-ethyl}-lH-pyridin-4- one, or (3-ethynyl-phenyl)-(6-methoxy-quinazolin-4-yl)-amine. [0150] In a further embodiment the EGFR inhibitor is selected from compounds described in Chen et al. (US 5,789,427). In one embodiment the compound is (E)-2- aminothiocarbonyl-3-(3,5-diisoproyl-4-hydroxyphenyl) acrylonitrile, (E)-3-(3,5- diisopropyl-4-hydroxyphenyl-2-[(pyrid-2-yl)sulfonyl]acryloni trile, (E)-2- cyanomethylsulfonyl-3-(3,5-diisopropyl-4-hydroxvyphenyl) acrylonitrile), (E)-3-(3,5- diisopropyl-4-hydroxy-phenyl)-2-(phenylsulfonyl) acrylonitrile)or (E)-3-(3 ,5- diisopropyl-4-hydroxyphenyl)-2-[(4-trifluoromethyl)-phenylam ino carbonyl]acrylonitrile.

[0151] In a further embodiment the EGFR inhibitor is selected from compounds described in Tang et al. (US 5,650,415). In one embodiment the compound is - ethoxycarbonyl-4-(2-trifluoromethylbenzylamino)-6-trifluorom ethylquinoline, 3- ethoxycarbonyl-4-(3 -trifluoromethylbenzylamino)-6-trifluoromethylquinoline, 3 - ethoxycarbonyl-4-(4-trifluoromethylbenzylamino)-6-trifluorom ethylquinoline, 3- ethoxycarbonyl-4-(2-trifluoromethylphenylamino)-6-trifluorom ethylquinoline, 3- ethoxycarbonyl-4-(3-trifluoromethylphenylamino)-6-trifluorom ethylquinoline, or 3- ethoxycarbonyl-4-(4-trifluoromethylphenylamino)-6-trifluorom ethylquinoline. [0152] In a further embodiment the EGFR inhibitor is selected from compounds described in Himmelsbach et al. (CA2243994). In one embodiment the compound is 4- [(3-chloro-4-fluoro-phenyl) amino]-7-[(l-methyl-4piperidinyl) amino]-pyrido[4,3- d]pyrimidine, 4-[(3-chloro-4-fluoro-phenyl) amino]-7-[4-(4-piperidinyl)piperidin-l-yl]- pyrido[4,3-d]pyrimidine, 4-[(3-chloro-4-fluoro-phenyl) amino]-7-[(l-tert- butyloxycarbonyl-4-piperidinyl) amino]pyrido[4,3-d]pyrimidine, 4-[(3-chloro-4-fluoro- phenyl) amino]-7-[[trans-4(methoxycarbonyl)cyclohexyl)]-amino]pyrido [4,3- djpyrimidine, or 4-[(3-chloro-4-fluoro-phenyl) amino]-6-(4-amino-l-piperidinyl)- pyrido[3,4-d]pyrimidine.

[0153] Additional non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in Traxler (Exp. Opin. Ther. Patents (UK) 1998, 8, 1599-1625) and those described in Al-Obeidi et al. (Oncogene 2000, 19, 5690-5701). [0154] In one embodiment, the EGFR inhibitor is canertinib, PD169540, PD158780, AG1478, PD153035, CGP-59326, PKI-166, EKI-B569, or GW-572016.

[0155] In one embodiment the EGFR inhibitor is N-[-4-[(3-chloro-4- fluorophenyl)amino] -7- [3-(4-morpholinyl)propoxy] -6-quinazolinyl] -2-propenamide dihydrochloride (known as CI-1033 or PD183805 or Canertinib; Pfizer) (Smaill, J Med. Chem. 1999, 42, 1803-1815; Smaill, J Med Chem. 2000, 43, 1380-1397; Slichenmyer, Semin. Oncol. 2001, 28, 80-85. In one embodiment the EGFR inhibitor has the following structure:

[0156] In one embodiment the EGFR inhibitor is 4-[(3-bromophenyl)-amino]-6- (methylamino)-pyrido[3,4-d]pyrimidine (known as PDl 58780 (Pfizer)) (Rewcastle, J. Med. Chem. 1998, 41, 742-51 ; Cunnick, J. Biol. Chem. 1998, 273, 14468-14475). In one embodiment the EGFR inhibitor has the following structure:

[0157] In one embodiment the EGFR inhibitor is 4-(3-chloroanilino)-6,7- dimethoxyquinazoline (known as AG-1478; University of California) (Ward, Biochem. Pharmacol. 1994, 48, 659-666; US 5,457,105; EP0566266). In one embodiment the EGFR inhibitor has the following structure:

[0158] In one embodiment the EGFR inhibitor is the 4-[(3-bromophenyl)amino]-6,7- dimethoxyquinazoline hydrochloride (known as PD 153035; Bridges, J. Med. Chem. 1996, 39, 267-276; US 5,457,105; EP0566266). In one embodiment the EGFR inhibitor has the following structure:

[0159] In one embodiment the EGFR inhibitor is the CGP-59326 (Novartis) (Traxler, J. Med. Chem. 1996, 39, 2285-2292). In one embodiment the EGFR inhibitor has the following structure:

[0160] In one embodiment the EGFR inhibitor is 4-(R)-phenethylamino-6- (hydroxyl)-phenyl-7H-pyrrolo[2,3-d]-pyrimidine (known as PKI-166 (Novartis); Traxler, Clin. Cancer Res. 1999, 5, 3750s). In one embodiment the EGFR inhibitor has the following structure:

[0161] In one embodiment the EGFR inhibitor is EKI-569 (Wyeth; Torrance, Nat. Med. 2000, 6, 974-975). In one embodiment the EGFR inhibitor has the following structure:

[0162] In one embodiment the EGFR inhibitor is GW-2016 (also known as GW- 572016 or lapatinib ditosylate; GSK; Kim, IDrugs 2003, 6, 886-93). In one embodiment the EGFR inhibitor that has the following structure:

6.6 Pharmaceutical compositions

[0163] In one embodiment the small molecule inhibitor may be provided as a pharmaceutically acceptable salt deemed suitable by one of skill in the art. (See, Berge et al., J Pharm. Sci. 1977, 66, 1-19; and "Handbook of Pharmaceutical Salts, Properties, and Use," Stahl and Wermuth, Ed.; Wiley- VCH and VHCA, Zurich, 2002). [0164] Suitable acids for use in the preparation of pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(15)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D- glucuronic acid, L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic

acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL- mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene- 1,5- disulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L- pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

[0165] In certain embodiments, the small molecule inhibitor may also be provided as a prodrug, which is a functional derivative of the compound and is readily convertible into the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have enhanced solubility in pharmaceutical compositions over the parent compound. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. See Harper, Progress in Drug Research 1962, 4, 221-294; Morozowich et al. in "Design of Biopharmaceutical Properties through Prodrugs and Analogs," Roche Ed., APHA Acad. Pharm. Sci. 1977; "Bioreversible Carriers in Drug in Drug Design, Theory and Application," Roche Ed., APHA Acad. Pharm. Sci. 1987; "Design of Prodrugs," Bundgaard, Elsevier, 1985; Wang et al., Curr. Pharm. Design 1999, 5, 265-287; Pauletti et al., Adv. Drug. Delivery Rev. 1997, 27, 235-256; Mizen et al., Pharm. Biotech. 1998, 11, 345-365; Gaignault et al., Pr act. Med. Chem. 1996, 671-696; Asgharaejad in "Transport Processes in Pharmaceutical Systems," Amidon et al., Ed., Marcell Dekker, 185-218, 2000; Balant et al., Eur. J. Drug Metab. Pharmacokinet. 1990, 75, 143-53; Balimane and Sinko, Adv. Drug Delivery Rev. 1999, 39, 183-209; Browne, Clin. Neuropharmacol. 1997, 20, 1-12; Bundgaard, Arch. Pharm. Chem. 1979, 86, 1-39; Bundgaard, Controlled Drug Delivery 1987, 17, 179-96; Bundgaard, Adv. Drug Delivery Rev. 1992, 8, 1-38; Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130; Fleisher et al., Methods Enzymol. 1985, 112, 360-381 ; Farquhar et al., J. Pharm. Sci. 1983, 72, 324-325; Freeman et al., J. Chem. Soc, Chem. Commun. 1991, 875-877; Friis and Bundgaard, Eur. J. Pharm. Sci. 1996, 4, 49-59; Gangwar et al., Des. Biopharm. Prop. Prodrugs Analogs, 1977, 409-421 ; Nathwani and Wood, Drugs 1993, 45, 866-94; Sinhababu and Thakker, Adv. Drug

Delivery Rev. 1996, 19, 241-273; Stella et al., Drugs 1985, 29, 455-73; Tan et al., Adv. Drug Delivery Rev. 1999, 39, 117-151 ; Taylor, Adv. Drug Delivery Rev. 1996, 19, 131- 148; Valentino and Borchardt, Drug Discovery Today 1997, 2, 148-155; Wiebe and Knaus, Adv. Drug Delivery Rev. 1999, 39, 63-80; and Waller et al., Br. J. Clin. Pharmac. 1989, 28, 497-507; each of which is incorporated by reference herein in its entirety.

[0166] In one embodiment the small molecule inhibitor, a pharmaceutically acceptable salt, solvate, or prodrug thereof, can be administered in any form deemed useful by the practitioners of skill in the art. In certain embodiments, the small molecule inhibitor is administered in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients or diluents.

[0167] The small molecule inhibitor provided herein may be administered alone, or in combination with one or more other compounds provided herein, one or more other active ingredients. The pharmaceutical compositions that comprise a small molecule inhibitor provided herein may be formulated in various dosage forms for oral, parenteral, and topical administration. The pharmaceutical compositions may also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art {see, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Deliver Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, NY, 2003; Vol. 126).

[0168] In one embodiment, the pharmaceutical compositions are provided in a dosage form for oral administration, which comprise a small molecule inhibitor provided herein or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more pharmaceutically acceptable excipients or carriers.

[0169] In another embodiment, the pharmaceutical compositions are provided in a dosage form for parenteral administration, which comprise a small molecule inhibitor provided herein or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more pharmaceutically acceptable excipients or carriers.

[0170] In yet another embodiment, the pharmaceutical compositions are provided in a dosage form for topical administration, which comprise a small molecule inhibitor provided herein or a pharmaceutically acceptable salt, solvate, or prodrug thereof; and one or more pharmaceutically acceptable excipients or carriers. [0171] The pharmaceutical compositions provided herein may be provided in unit- dosage forms or multiple-dosage forms. Unit-dosage forms, as used herein, refer to physically discrete units suitable for administration to human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the active ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of unit- dosage forms include ampoules, syringes, and individually packaged tablets and capsules. Unit-dosage forms may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of multiple- dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. [0172] The pharmaceutical compositions provided herein may be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. [0173] In certain embodiments, the pharmaceutical composition provided herein is provided as a solid dosage form for oral administration.

[0174] In certain embodiments, the pharmaceutical composition provided herein is provided as a film-coated tablet.

6.7 RNA Molecules as Regulators

[0175] In certain embodiments, the inhibitor of the EGFR/ERK pathway is an RNA interference (RNAi) agent.

[0176] In certain embodiments, the RNAi agent is selected from the group consisting of siRNA, shRNA, and miRNA.

[0177] In one embodiment the inhibitor of the EGFR/ERK pathway is an RNAi agent selected from the group consisting of siRN A, shRNA, and miRNA.

[0178] In one embodiment the RNAi agent is an siRNA.

[0179] In one embodiment the RNAi agent is an shRNA.

[0180] In one embodiment the RNAi agent is an miRNA.

[0181] In one aspect provided herein are RNAi agents as inhibitors of the

EGFR/ERK pathway and methods of their use, wherein the RNAi agents target a gene associated with the EGFR/ERK pathway. In one embodiment said gene targeted by said

RNAi agent is egfr, erB-l, erB-2, erB-3, erB-4, PKC, SPK, RAS, Raf-1, MEK, a homolog or an isoform thereof. Exemplary mRNA sequences of transcripts of the egfr gene and its human homologue are listed as SEQ ID. 2, 4, 6, 8, 10, 12, 14, 16, 18, and

20.

[0182] In one aspect of the invention the RNAi agent targets a portion of a transcript of a gene in the EGFR/ERK signaling pathway. In some embodiments, the gene is egfr, also known as erbB-l or her I. In some embodiments, the gene is an egfr homologue such as erbB-2, erbB-3, and erbB-4. In some embodiments, the gene is encoding PKC,

SPK, Ras, Raf-1, or MEK in the EGFR/ERK signaling pathway.

[0183] In some embodiments, an RNAi agent of the present invention produces a corresponding RNAi-inducing molecule (e.g., an siRNA) that varies from 19 nt to 21 nt in length. In some embodiments, the resulting RNAi-inducing molecule is less than 19 nt in length. In some embodiments, the resulting RNAi-inducing molecule is 19 nt, 21 nt, 23 nt, 25 nt or 27 nt in length. In some embodiments, the resulting RNAi-inducing molecule is more than 21 nt, 23 nt, 25 nt or 27 nt in length. Because a functional siRNA duplex has a two-nucleotide overhang at one end of each of the sense and antisense strands, each of the inverted repeats form the corresponding siRNAs accordingly can contain from 21 nt to 23 nt.

[0184] In some embodiments, an RNAi agent of the present invention is a synthetic siRNA molecule. In some embodiments, an RNAi agent of the present invention includes an in vitro or in vivo transcription system (e.g., as an shRNA system) that produces RNAi-inducing molecules targeting, for example, a transcript of egfr. An in

vivo transcription system may be delivered to target tissue, organ, or sub cell type by a delivery system such as those developed at the Nanosystems Biology Cancer Center at the California Institute of Technology.

[0185] In some embodiments, for example, longer inverted repeats may be of use to construct the shRNAs core sequences that ultimately produce RNAi inducing molecules targeting a portion of a transcript of egfr. For example, Kim et al, have used as long as

27 nt to construct double stranded RNAs (dsRNAs) for gene silencing purpose. See,

Kim, et al. , 2005, "Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy," Nature Biotechnology 23:222-226, which is hereby incorporated by reference herein in its entirety.

[0186] In one embodiment, the ALK inhibitor is an RNAi agent selected from the group consisting of siRNA, shRNA, and miRNA.

[0187] In one embodiment, the RNAi agent inhibits the alk gene, or a homolog or an iso-form thereof.

[0188] In one embodiment, the RNAi agent is an siRNA.

[0189] In one embodiment, the RNAi agent is an shRNA.

[0190] In one embodiment, the RNAi agent is an miRNA.

[0191] In one embodiment, the RNAi agent is a siRNA of 19, 21 , 23, 25, 27 or more nucleotides.

[0192] In one embodiment, the EGFR inhibitor is a ribozyme or an antisense oligonucleotide targeting the egfr gene, a homolog, or an iso-form thereof

[0193] The sequence of the siRNA molecules of the instant disclosure may be defined by random screening methods or by using commercially or non-commercially available siRNA design software tools based on the nucleic acid coding sequence of a target gene, including but not limited to, for example, the siRNA design tools provided by companies such as siRNA Target Finder from Ambion, Inc. of Applied Biosystems,

Dharmacon, Invitrogen's BLOCK-iT RNAi Designer, Qiagen, siRNA Target Finder from Genscript, RNAi Design from IDT, RNAi Explorer from Gene Link; or siRNA design tools provided by any research institutions such as the siRNA Design Software from the University of Hong Kong, siDirect from the University of Tokyo, SiRNA at

Whitehead from the Whitehead Institute for Biomedical Research, Sirna-SiRNA Design from the Wadsworth Bioinformatics Center, or siSearch tool from the Stockholm

Bioinformatics Center. It will be understood by one skilled in the art that any method for siRNA sequence design, using algorithm developed from experimental results or from theoretical calculation, may be used to design sequence motif for siRNA. [0194] RNAi agents can be delivered by any method apparent to those skilled in the art. Two exemplary methods for triggering RNAi can be used in vivo: delivery of siRNAs, and delivery of plasmid and viral vectors that express a short hairpin RNA (shRNA) that is subsequently processed into active siRNA. See, for example, Reich et al., MoI Vis. 9:210-216 (2003); Soutschek et al, Nature 432:173-178 (2004); Urban- Klein et al, Gene Ther., 12(5):461-466 (2005); Li et al, Cell Cycle 5(18): 2103-9 (2006). Rubinson et al., Nat. Genet., 33, 401-406 (2003); and Stewart et al, RNA, 9: 493-501 (2003); each of which is hereby incorporated by reference herein in its entirety. [0195] Some embodiments of the present disclosure utilize one or more many of the existing delivery systems used for gene therapy are suitable for delivery RNAi agents of the present disclosure, including a viral delivery system, a retrovirus delivery system, a lentivirus delivery system, an adenovirus delivery system, and other viral delivery system using such as Adeno-associated viruses and insect Baculovirus. Delivery of RNAi can proceed with a wide variety of agents, such as: injection of pure, unmodified siRNA, chemically stabilized or modified RNA, encapsulating the siRNA in microparticles or liposomes, and binding siRNA to cationic or other particulate carriers. In additional embodiments, delivery systems based on liposomes, nanoparticles (e.g. , polyethylenimine: PEI), and magnets can also be used to deliver RNAi agents to a target organism, including a human subject for treating a subject suffering from substance-related disorders.

[0196] Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g., see U.S. Pat. Nos. 6,566,135; 6,566,131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321; and 5,981,732).

[0197] In one embodiment of the disclosure, the inhibitor is a ribozyme molecule that catalyzes specific cleavage of an RNA transcript of the egfr gene, or a homolog or iso-form thereof. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified,

short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0198] Both antisense oligonucleotides and ribozymes useful as modulators of a gene, such as the egfr gene or a homolog or iso-form thereof, can be prepared by known methods such as chemical syntheses, in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. In some embodiments, modifications to the nucleic acid modulators of the disclosure can be introduced in order to increase intracellular stability and/or half life. Moreover, antisense oligonucleotides can be delivered in vivo alone or in association with a vector. Additional details and examples for the methods of synthesis and delivery can be found, for example, in International Application by INSERM (Institut National de La Sante et de La Recherche Medicale), published as WO 2008/053270 A2; United States Patent Publication No. US 2005/0038049; and United States Patent Publication No. 2008/0176846; each of which is hereby incorporated by reference in its entirety.

6.8 Antibodies as Therapeutics

[0199] Some aspects of the present invention provide antibodies, to EGFR, a homologue of EGFR, another protein in the EGFR/ERK signaling pathway, or a fragment thereof, as therapeutic agents for treating a subject suffering from alcohol abuse, alcohol withdrawal or other forms of related substance abuse. Such antibodies include, but are not limited to, polyclonal, monoclonal, single chain monoclonal, recombinant, chimeric, humanized, mammalian, or human antibodies. [0200] In some embodiments, the present invention provides a method for the treatment or prevention of alcohol abuse or alcohol withdrawal in a subject having the disorder using monoclonal antibodies. The method provides monoclonal antibodies that are based on amino acid sequences of functional fragments and variants of egfr that comprise an antigenic determinant {i.e., a portion of a polypeptide that can be recognized by an antibody) or which are otherwise functionally active, as well as nucleic

acids encoding the foregoing. In some embodiments, egfr functional activity encompasses one or more known functional activities associated with a modulation of ethanol sedation in a subject; antigenicity (the ability to be bound by an antibody to a protein consisting of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19); immunogenicity (the ability to induce the production of an antibody that binds at a portion of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19), and so forth. In some embodiments, the subject is a mouse, a rat or a human. [0201] In some embodiments, the monoclonal antibodies comprise amino acid sequences having functionally inconsequential amino acid substitutions, and thus have amino acid sequences which differ from that of an antibody raised by the EGFR protein, a protein transcribed based on the native egfr sequence. Substitutions can be introduced by mutation into eg/r-encoding nucleic acid sequences that result in alterations in the amino acid sequences of the encoded egfr but do not alter egfr function. The substituted versions of the EGFR protein are used to generated the monoclonal antibodies of the present invention. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in egfr encoding sequences. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of egfr without altering myosin light chain kinase biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the egfr polypeptides of the invention are predicted to be particularly unsuitable for alteration. Amino acids for which conservative substitutions can be made are well known in the art. [0202] In one embodiment, the present invention includes a monoclonal antibody targeting an amino acid sequence having at least 70% identity to a portion of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. In some embodiments, the target amino acid sequence has at least 75%, 80%, 85%, 90%, or 95% identity to a portion of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. In a particular embodiment, the target amino acid sequence comprises a portion of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. In some embodiments, the portion of the amino acid sequence comprises 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 75

or more, 100 or more, 150 or more, 200 or more, 300 or more, 500 or more amino acid residues.

[0203] In one embodiment, the present invention includes a monoclonal antibody targeting a protein or a polypeptide product of the gene egfr, also known as erbB-l or herl . In some embodiments, the protein or polypeptide product is from an egfr homologue such as erbB-2, erbB-3, and erbB-A. In some embodiments, the antibody targets a protein or a poly peptide product of another gene in the EGFR/ERK signaling pathway PKC, SPK, Ras, Raf-1, or MEK. In some embodiments, the antibody targets the HB-EGF protein or a peptide product thereof.

[0204] For preparation of monoclonal antibodies directed toward EGFR, a homologue of EGFR, another protein in the EGFR/ERK signaling pathway, or a fragment thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, monoclonal antibodies may be prepared by the hybridoma technique originally developed by Kohler and Milstein, 1975, Nature 256:495-497, as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunol. Today 4:72), or the EBV- hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0205] Techniques for the production of single chain antibodies, as described in U.S. Patent 4,946,778, can also be adapted to produce single chain antibodies specific to EGFR. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al, 1988, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for EGFR. Antibody fragments that contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab'), fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab'), fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments. [0206] Techniques developed for the production of "chimeric" antibodies (Morrison et al, 1984, Proc. Natl. Acad. ScL U.S.A. 81 :6851-6855; Neuberger et al, 1984, Nature 312:604-608; Takeda et al, 1985, Nature 314:452-454) can also be used. For example,

nucleic acid sequences encoding a mouse antibody molecule specific to EGFR are spliced to nucleic acid sequences encoding a human antibody molecule. [0207] In addition, techniques have been developed for the production of humanized antibodies, and such humanized antibodies to EGFR, a homologue of EGFR, other proteins in the EGFR/ERK signaling pathway, or a fragment thereof are within the scope of the present invention. See, e.g., Queen, U.S. Patent No. 5,585,089 and Winter, U.S. Patent No. 5,225,539. An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs). The extent of the framework region and CDRs has been precisely defined. See Sequences of Proteins of Immunological Interest, Kabat, E. et al., 1983, U.S. Department of Health and Human Services. Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

[0208] Human antibodies may be used and can be obtained by using human hybridomas (Cote et al, 1983, Proc. Natl. Acad. ScL U.S.A., 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).

[0209] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or RIBA (recombinant immunoblot assay). For example, to select antibodies which recognize a specific domain of EGFR, one may assay generated hybridomas for a product which binds to a EGFR fragment containing such domain. For selection of an antibody that specifically binds a first EGFR homologue but which does not specifically bind a second, different EGFR homologue, one can select on the basis of positive binding to the first EGFR homologue and a lack of binding to the second EGFR homologue.

[0210] Antibodies specific to a domain of EGFR or a homologue thereof are also provided. The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the EGFR of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.

[0211] In some embodiments, a monoclonal antibody of the present invention targets a variant of EGFR, a homologue of EGFR, another protein in the EGFR/ERK signaling pathway, or a fragment thereof. Useful conservative substitutions are shown in Table 1 , "Preferred Substitutions." Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. If such substitutions result in a change in biological activity, then more substantial changes, indicated in Table 2 as exemplary are introduced and the products screened for egfr polypeptide biological activity.

[0212] Non-conservative substitutions that effect: (1) the structure of the polypeptide backbone, such as a β-sheet or α-helical conformation; (2) the charge; (3) hydrophobicity; or (4) the bulk of the side chain of the target site, can modify egfr polypeptide function or immunological identity. Residues are divided into groups based on common side-chain properties as denoted in Table 2. Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.

[0213] The variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see Carter, 1986, Biochem. J. 237: 1-7; Zoller and Smith, 1987, Methods Enzymol. 154:329-50), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985, Gene 34:315-323) or other known techniques can be performed on cloned eg/r-encoding DNA to produce egfr variant DNA (Ausubel et al., 2001, Current Protocols In Molecular Biology, John Wiley and Sons, New York (current edition); Sambrook et al, Molecular Cloning, A Laboratory Manual, 3d. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0214] In certain embodiments, monoclonal antibodies used in the present invention target egfr mutants, homologous thereof, or derivatives having an amino acid substitution with a non-classical amino acid or chemical amino acid analog. Non- classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α -amino isόbutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Na- methyl amino acids, and amino acid analogs in general.

[0215] Although most of the methods and systems described herein are provided by way of illustration using the EGFR protein as an example, they are generally applicable to other proteins in the EGFR/ERK pathway, including PKC, SPK, Ras, Raf, MEK. The exemplary embodiments provided herein should not be construed as any limitation of the scope of the present invention.

[0216] In accordance with the present invention, known antibodies against EGFR, previously developed for treatment of cancer, can be used as a therapeutic agent for treating a subject suffering from alcohol abuse, alcohol withdrawal or other forms of related substance abuse. In some embodiments, these previously developed antibodies may be used for prevention of substance abuse such as alcohol abuse, alcohol withdrawal or other forms of related substance abuse. These antibodies include but are not limited to Cetuximab (ImC lone Systems Inc., New York City, New York) and Panitumumab (Amgen Inc., Thousand Oaks, CA).

6.8.1 Cetuximab

[0217] The U.S. Food and Drug Administration (FDA) has approved Cetuximab (marketed under the name Erbitux®) for treating patients with advanced colorectal cancer that has spread to other parts of the body. Cetuximab is the first monoclonal antibody approved to treat this type of cancer and is indicated as a combination treatment to be given intravenously with irinotecan, another drug approved to fight colorectal cancer, or alone if patients cannot tolerate irinotecan.

[0218] Cetuximab is a chimeric monoclonal antibody, an epidermal growth factor receptor (EGFR) inhibitor, given by intravenous injection for treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab is believed to operate by binding to the extracellular domain of the EGFR of all cells that express EGFR, which includes the subset "cancer cells," preventing ligand binding and activation of the receptor. This blocks the downstream signaling of EGFR resulting in impaired cell growth and proliferation. Cetuximab has also been shown to mediate antibody dependent cellular cytotoxicity (ADCC). Clinically, Cetuximab has been used to treat colon cancer or head and neck cancer. In some embodiments, Cetuximab is given concurrently with the chemotherapy drug irinotecan (Camptosar), a form of chemotherapy that blocks the effect of DNA topoisomerase I, resulting in fatal damage to the DNA of affected cells.

6.8.2 Panitumumab

[0219] In the present invention, Panitumumab, another known human monoclonal antibody may be used as a therapeutic agent for treating a subject suffering from alcohol abuse, alcohol withdrawal or other forms of related substance abuse. [0220] Panitumumab (ABX-EGF) is the first fully human monoclonal antibody specific to the EGF receptor (manufactured by Amgen and marketed as Vectibix®). The compound works by binding to the extracellular domain of the EGFR (epidermal growth factor receptor) preventing its activation. This in turn, results in halting of the cascade of intracellular signals dependent on this receptor. Panitumumab is a recombinant, human IgG2 kappa monoclonal antibody that binds specifically to the human Epidermal Growth Factor Receptor (EGFR). Overexpression of EGFR is detected in many human cancers, including those of the colon and rectum. When Panitumumab binds to EGFR, it competitively inhibits the binding of ligands for EGFR which results in inhibition of cell growth, induction of apoptosis, decreased proinflammatory cytokine and vascular growth factor production.

[0221] Panitumumab is specifically indicated for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan- containing chemotherapy regimens.

[0222] Panitumumab is produced by immunization of transgenic mice (xenomouse) that are able to produce human immunoglobulin light and heavy chains. After immunization of these animals a specific clone of B cells that produced an antibody against EGFR was selected and immortalized in Chinese hamster ovary (CHO) cells. These cells are then used for the full scale manufacture of the antibody. [0223] Panitumumab is delivered intravenous and has a half time around 7.5 days, ranging from 4 to 11 days. Panitumumab is supplied as a sterile, colorless, preservative- free solution containing 20 mg/mL designed for intravenous infusion. The recommended initial dose of the drug is 6 mg/kg administered over 60 minutes as an intravenous infusion every 14 days. If safety or tolerability concerns arise, dosage may be reduced by 50%. Doses higher than 1000 mg should be infused over 90 minutes. [0224] Additional information regarding Panitumumab is found in, for example, Wainberg and Hecht, 2006, "Panitumumab in colon cancer: a review and summary of ongoing trials," Expert opinion on biological therapy, Nov; 6(11): 1229-35; Saif and Cohenuram, 2006, "Role of panitumumab in the management of metastatic colorectal cancer," Clinical Colorectal Cancer, 6(2): 118-24; Gibson et al., 2006, "Randomized phase III trial results of panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, in metastatic colorectal cancer," Clinical colorectal cancer, 6(1):29-31 ; and Tyagi, 2005, "Recent results and ongoing trials with panitumumab (ABX-EGF), a fully human anti-epidermal growth factor receptor antibody, in metastatic colorectal cancer," Clinical colorectal cancer, 5(l):21-3, each of which is hereby incorporated herein by reference in its entirety.

6.8.3 Additional Antibodies and Polypeptides

[0225] Although they both target the EGFR, panitumumab (IgG2) and cetuximab (IgGl) differ in their isotype and they might differ in their mechanism of action. Monoclonal antibodies of the IgGl isotype may activate the complement pathway and mediate ADCC (antibody-dependent cellular cytotoxicity) better than their IgG2 counterparts, hence although they have not been documented, differences in the responses in treatments with these two antibodies might be expected. Other monoclonals in clinical development are zalutumumab, nimotuzumab, and matuzumab.

[0226] Antibodies targeting other molecules in the EGFR/ERK pathway can be also used in the present invention including Trastuzumab, which is a humanized monoclonal antibody that acts on the HER2/neu (erbB2) receptor. [0227] Additionally, polypeptides based on the sequences of any of the aforementioned protein sequences may be used to alter the signal transduction activity of the EGFR pathway. A polypeptide of the invention can be synthesized through methods of in vitro peptide synthesis that are known in the art; or they can be synthesized using a vector-based expression system by a plasmid encoding the sequence of the polypeptide. It will be understood by one skilled in the art that, peptide sequences that have the desired activity may be easily identified through standard random screening methods.

6.9 Methods for delivery/administration

[0228] The invention provides compositions, including pharmaceutical compositions, containing a polypeptide or a polynucleotide encoding such a polypeptide of the invention. The polypeptide can be synthesized using standard peptide synthesis methods. The compositions optionally contain other components, including, for example, a storage solution, such as a suitable buffer, e.g., a physiological buffer. In a preferred embodiment, the composition is a pharmaceutical composition and the other component is a pharmaceutically acceptable carrier, such as are described in Remington's Pharmaceutical Sciences, 16th editions, Osol, ed., 1980. [0229] A pharmaceutically acceptable carrier suitable for use in the invention is nontoxic to cells, tissues, or subjects at the dosages employed, and can include a buffer (such as a phosphate buffer, citrate buffer, and buffers made from other organic acids), an antioxidant (e.g., ascorbic acid), a low-molecular weight (less than about 10 residues) peptide, a polypeptide (such as serum albumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (such as glycine, glutamine, asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide, and/or other carbohydrates (including glucose, mannose, and dextrins), a chelating agent (e.g., ethylenediaminetetratacetic acid [EDTA]), a sugar alcohol (such as mannitol and sorbitol), a salt-forming counterion (e.g., sodium), and/or an anionic surfactant (such as Tween™, Pluronics™, or PEG). In one embodiment, the pharmaceutically acceptable carrier is an aqueous pH-buffered solution.

[0230] Preferred embodiments include sustained-release pharmaceutical compositions. An exemplary sustained-release composition has a semipermeable matrix of a solid hydrophobic polymer to which the polypeptide is attached or in which the polypeptide is encapsulated. Examples of suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L-glutamic acid and T-ethyl-L-glutamase, non- degradable ethylene-vinylacetate, a degradable lactic acid-glycolic acid copolymer, and poly-D-(-)-3-hydroxybutyric acid. Such matrices are in the form of shaped articles, such as films, or microcapsules.

[0231] Exemplary sustained release compositions include polypeptides attached, typically via ε-amino groups, to a polyalkylene glycol (e.g., polyethylene glycol [PEG]). Attachment of PEG to proteins is a well-known means of reducing immunogenicity and extending in vivo half-life; see, e.g., Abuchowski et al., 1977, J Biol. Chem. 252:358286. Any conventional "pegylation" method can be employed, provided the "pegylated" variant retains the desired function(s).

[0232] In another embodiment, a sustained-release composition includes a liposomally entrapped polypeptide. Liposomes are small vesicles composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing polypeptides are prepared by known methods, such as, for example, those described in Epstein, et al, 1985, PNAS USA 82:3688-92, and Hwang, et al., 1980 PNAS USA, 77:4030-34. Ordinarily the liposomes in such preparations are often small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the specific percentage being adjusted to provide the optimal therapy. Useful liposomes can be generated by the reverse-phase evaporation method, using a lipid composition including, for example, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). If desired, liposomes are extruded through filters of defined pore size to yield liposomes of a particular diameter.

[0233] Moreover, methods for delivering polypeptides and peptides are known and widely practiced in the art, including a peptide-based delivery system, a polymer-based delivery system, a liposome-based delivery system, an immunosome-based delivery system, a virosome-based delivery system. For example, Morris et al. demonstrated that

simply incubating cells with short amphipathic peptides in the presence of the green fluorescent protein (GFP), a 238 amino acid protein, resulted in endocytosis of GFP in the intact cells. See Morris et al, 2001, "A Peptide Carrier for the Delivery of Biologically Active Proteins into Mammalian Cells," Nat Biotechnol. 19(12): 1 173-1176. Also, liposome-based delivery methods were shown to be successful in delivering both polypeptides and polynucleotides; for example, protease inhibitor hirudin was delivered using liposome coated with dextran. See Mumper and Hoffman, 2000, "The Stabilization and Release of Hirudin from Liposomes or Lipid- Assemblies Coated with Hydrophobically Modified Dextran," AAPS PharmSciTech. 1(1):E3. Liposomes conjugated with antibody molecules and PEG were successfully delivered to target tumor cells in mouse, including lung cancer cells (which are at a relatively accessible locale of the organism) and to solid tumor tissues (which are in relatively less accessible locales of the organism). The PEG-liposomes used ranged from 60 to 400 nanometers in diameters, which can obviously accommodate large size proteins. See Maruyama, 2002, "PEG-immunoliposome," Biosci Rep. 22(2):251-266. Additional examples of immunoliposomes that are suitable for targeted delivery of proteins into cells in vivo, can be found in Park, 2002, "Tumor-directed targeting of liposomes," Biosci Rep. 22(2):267-281; Mastrobattista et al, 2002, "Targeted Liposomes for Delivery of Protein- Based Drugs into the Cytoplasm of Tumor Cells," J Liposome Res. 12(1 -2): 57-65; and Ulrich, 2002, "Biophysical Aspects of Using Liposomes as Delivery Vehicles," Biosci Rep. 22(2): 129- 150. Additionally, polymer-based delivery methods, infusion methods and injections methods were also known and used in the art. Examples can be found at Idea and Uekamab, 1999, "Cyclodextrins in Peptide and Protein Delivery," Advanced Drug Delivery 36(l):101-123; Kanamasa et al., 2001, "tPA via Infusion Catheters Followed by Continuous IV Infusion for 3 Days Prevents Intimal Hyperplasia after Balloon Injury," Angiology 52(12):819-825; and Bremer et al, 1997, "Protein Delivery with Infusion Pumps," Pharm Biotechnol 10:239-254. Each of the above-mentioned references is hereby incorporated herein by reference in its entirety. [0234] Additionally, methods for transporting polynucleotides into organisms are known in the art. It will also be understood by one skilled in the art that most methods for delivering polypeptides into target cells may be also useful for delivering polynucleotide sequence encoding such polypeptides. For example, liposome-based

delivery methods, polymer-based delivery methods, and infusion and injection methods, as set forth in prior discussion, are suitable for protein delivery as well as for gene delivery. Examples of such applications can be found at Abe et al , 2002, "Intra-arterial Delivery of p53 -containing Adenoviral Vector into Experimental Brain Tumors," Cancer Gene Ther. 9(3):228-235; Wright et al, 2001, "In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo," Gene Ther. 8(24): 1833- 1839; Duguid et al, 1998, "A Physicochemical Approach for Predicting the Effectiveness of Peptide-Based Gene Delivery Systems for Use in Plasmid-Based Gene Therapy," Biophys J. 74(6):2802-2814; and Xu et al, 2002, "Systemic Tumor-targeted Gene Delivery by Anti-transferrin Receptor scFv-immunoliposomes," MoI Cancer Ther. l(5):337-346. In particular, Mastrobattista et al. demonstrated that virosomes (liposome containing virus particles) can be coated with PEG and antibodies and used for delivering polypeptides and polynucleotides. In additional, traditional virus-based delivery system, dentrimer-based system can all be used for delivering polynucleotides of the invention. Each of the above-mentioned references is hereby incorporated herein by reference in its entirety.

[0235] It will be understood by one skilled in the art that any of the suitable methods mentioned hereinabove may be combined with any knowledge or technology that is known in the art in delivering the composition of the present invention, including but not limited to small peptides, small inorganic compounds, small organic compounds, siRNAs, proteins, and antibodies, or a combination thereof.

7. EXAMPLES

7.1 Example 1: The effect of TARCEVA ^® on ethanol and sucrose self- administration.

[0236] The present example demonstrates the effect of TARCEVA ^® on ethanol and sucrose self-administration. Different groups of animals will be trained to self administer either 10% ethanol or 5% sucrose. Rats are housed individually and food is always available in the home cage.

[0237] Ethanol and sucrose self-administration:

Before the beginning of operant self-administration training, rats are exposed to a 10% ethanol (10E) solution as the only liquid source in their home cages for 3 days. SeIf-

administration training sessions are initiated by the extension of two levers. Each response at the active lever(s) is reinforced by 0.1 ml of solution paired with the illumination of a white cue light above the lever for 5 sec and a 5 sec tone. Responses at the inactive lever are recorded as a measure of nonspecific behavioral activation but have no programmed consequences. Subjects trained to lever-press for oral ethanol for the self-administration and reinstatement procedures undergo a brief period (21 hour) of water restriction during the initial training of the operant response (5 days maximum). Previous studies (Arolfo et al, 2004, Alcohol Clin Exp Res, 28(9), 1308; Nie et al, 2003, Psychopharmacology (Berl) 168 (1-2), 222) have shown that similar water restriction schedules before the beginning of operant self administration does not compromise animals' health. Hughes et al. (Hughes et al., 1994, Laboratory animal science 44 (2), 135) showed that rats deprived of fluid for 21 hr/day for 3 months did not differ from ad libitum controls with respect to weight loss, organ and tissue appearance at necropsy, hematologic examination or clinical chemical analysis. [0238] Rats quickly learn to consume much, if not all, of their daily fluid needs in a short, restricted period (reviewed by Evans, 1990, Neurotoxicology and Teratology, 12(5): 531). Rats are monitored daily for weight loss and signs of dehydration. After initial overnight water restriction, rats are placed in operant chambers for a 14 hr overnight session on an FRl schedule (one reward for every lever press) with 10% sucrose (10S) as the reinforcer and both levers active. Animals are kept on 1 hr water restriction for the next 5 days during which they receive one 45 min session per day on an FRl schedule with 1OS as the reinforcer and one active lever. They are then given free access to water in their home cages for the remainder of the experiment and are trained for 2-3 more sessions with 1OS as the reinforcer. The next day, sessions are shortened to 30 minutes and the ratio of responding is increased to FR3. Operant self- administration is initiated according to the sucrose fading technique as described above (Samson, 1986, Alcohol Clin. Exp. Res. 10, 436-442) with minor modifications. Ten percent ethanol (10E) is then be added to the 10% sucrose solution (10S/ 10E) and the rats receive 3-4 sessions of this solution. Over the next 8-10 sessions, the sucrose concentration is gradually decreased (i.e., 5, 2, and finally 0%) after which the rats have 20-22 sessions with 1OE only until a stable baseline consumption is achieved (no more than 20% variation between sessions over 3 consecutive days, in excess of 0.3g/kg). A

separate group of animals is trained to self administer 5% sucrose, this group of animals will serve as a control group. Following approximately two weeks of daily 30 min self- administration sessions with 10% ethanol or 5% sucrose as the reinforcer, animals are injected with either vehicle or a dose of TARCEV A ^® (1-80 mg/kg i.p.) immediately prior to the self-administration session to determine the drug effects on 10% ethanol or 5% sucrose self-administration. Each group of animal receives only one drug, but receives multiple doses of the same drug over a period of 4 weeks in random order, with one injection per week.

[0239] Operant Self Administration of 10% Ethanol.

This protocol is the same as described for 10% ethanol self-administration. Once a stable baseline consumption is achieved (no more than 20% variation between sessions over 3 consecutive days, in excess of 0.3g/kg), rats are food deprived to begin nicotine self-administration training.

[0240] Nose-poke Training. In order to motivate animals to learn a second behavior (nose-poking) they require a short period of food restriction in the home cage so that food delivery can be used as a reward for learning the nose poke behavior. Rats are food restricted (animals have access to 2Og of pellet food per day in the home cage) for 1 week and if any animal falls below 90% of their basal weight food pellets are returned. The general health and well being of each animal is monitored daily and any signs of distress such as changes in behavior, coat condition, eye color and individual body weights are measured. If there are any signs of distress food pellets are returned. During the week of food restriction the animals are trained to nose-poke for food rewards in daily sessions in the operant behavior chambers. Food rewards are predicated on animal responses. A response in the left nose-poke hole results in immediate delivery of a food pellet paired with the illumination of a white, cue light directly above the nose-poke hole for 5 sec. Food pellets (45 mg, Bioserve, San Diego, CA, USA) are delivered to a food trough situated between the two nose-poke holes. A response on the right nose-poke hole is recorded but elicits no programmed consequence. Levers previously associated with ethanol delivery is not extended during nose-poke, food training sessions. Water is available ad libitum in the home cage. [0241] Drug Tests. The effects of a single injection of one dose of TARCEV A ^® are tested during self administration of ethanol. TARCEV A ^® or vehicle is administered 15

min prior to the test sessions. Drug dose test order is based on a within subjects Latin square design.

[0242] Loss of Righting Reflex and Blood Ethanol Concentrations in Rats. After isolation on a surgery table, Long Evans rats were injected intraperitoneally with ethanol at the dose of 3.6 g/kg (20% vol/vol). When the animal appeared to be sedated, it was laid on its back. The duration of the loss of righting reflex (LORR) was evaluated by measuring the time interval between the loss and the recovery of the righting reflex. Animals were judged to have regained their righting reflex when they could right themselves three times within 30 sec after being placed on their backs. To examine the effects of erlotinib on ethanol-induced sedation, drug (40 mg/kg) or vehicle were administered 30 min before the administration of the ethanol. To examine the effects of erlotinib (40mg/kg i.p) on blood ethanol clearance, blood samples were collected from the lateral tail vein of the sedated animals at 30, 60, 120, 240, and 480 min following ethanol injection. Blood ethanol concentrations were determined using gas chromatography methods adapted from Doyon et α/., 2003.

7.2 Example 2: The effect of TARCEV A ^® on reinstatement of drug- seeking.

[0243] The present example measures the effect of TARCEV A ^® on reinstatement of drug-seeking. Following the testing of TARCEVA on operant self-administration as described above, animals undergo extinction procedures, in which pressing of the active lever produces no reinforcers and no visual/audio stimuli. This signals to the animal that ethanol is no longer available, and the animal stops pressing the active lever when placed into the operant chamber. Then one of the following reinstatement paradigm is used.

[0244] Context-induced reinstatement: Subjects are trained to self-administer solutions of either ethanol (4.5% or 10%, v/v), sucrose (5%, g/v), beer (4.5% ethanol v/v) or near beer on a continuous fixed-ratio- 1 (FRl) schedule of reinforcement (FR-I), as previously described. The experiment consists of three phases: acquisition/maintenance in either the first or the second context (random and counterbalanced assignment, referred to as context A), extinction in the other context (referred to as context B) and reinstatement in the acquisition/maintenance context (A)

and the extinction, context (B). Animals are tested for operant response reinstatement with no reward availability in the acquisition context (context A) the day after the last extinction session, 2 and 3 weeks later. Animals are also tested in the extinction context (context B) 15 days after the last extinction session (Burattini et al. , 2006, Neuroscience 139(3): 877). Food and water are available ad libitum in their home cages. Animals are injected with either vehicle or a dose of TARCEV A ^® prior to the reinstatement session. As before, each group of animal receives only one drug but receives multiple doses of the same drug over a period of 4 weeks in random order, with one injection per week. All reinstatement sessions are conducted once per week.

[0245] Extinction of Ethanol Self Administration. Rats continue with daily self- administration sessions but without reinforcers available. During extinction sessions all visual (light stimuli) and auditory (tone) cues are absent. Extinction sessions continue until animals reach a predetermined set of criteria pertaining to nose poke and lever press responses.

[0246] Reinstatement of Ethanol Self Administration. On reinstatement test days animals are placed in the chambers and are presented with contingent cues previously associated with ethanol and nicotine delivery. Animal response activity is recorded but no rewards are delivered. Reinstatement sessions occur once weekly with extinction sessions in between test days.

[0247] Drug Tests. The effects of a single injection of one dose of TARCEV A ^® is tested during two phases of the experiment: (1) extinction and (2) reinstatement. TARCEV A ^® or vehicle is administered 15 min prior to the test sessions. Drug dose test order is based on a within subjects Latin square design.

7.3 Example 3: The effect of T ARCE V A ^® on ethanol sensitivity including loss of righting reflex and blood ethanol clearance.

[0248] The present example measures the effect of TARCEV A ^® on ethanol sensitivity including loss of righting reflex and blood ethanol clearance. [0249] Loss of Righting Reflex (LORRI (C57BL/6J): This procedure provides an objective measure of the sedating effects of ethanol by examining the length of time mice lay "unconscious" (i.e., unable to right themselves after being placed on their backs) after receiving a high dose of ethanol. These experiments determine if the

candidate drugs enhance or reduce the sedating effects of high doses of ethanol. Mice are housed with free access to food and water at all times. Animals are administered vehicle or a dose of TARCEV A ^® (1-80 mg.kg i.p) ten minutes prior to injection of ethanol (3.2 or 3.6 g/kg i.p.). After the ethanol injection, mice are placed on their backs, and the time taken to lose the righting reflex as well as to regain the righting reflex are measured. Because repeated administration of ethanol at high doses can result in acute tolerance to the loss of righting reflex, the analysis of multiple doses of candidate drugs, or multiple doses of ethanol, is not performed within the same animal. [0250] Blood Ethanol Clearance (Long Evans and Wistar rats): Any drug that reduces voluntary ethanol consumption or sedation could perhaps do so by altering the metabolism or clearance of ethanol from the bloodstream. To determine if TARCEV A ^® alter blood ethanol clearance, animals are housed with free access to food and water and are administered either vehicle or the most effective dose of TARCEV A ^® in altering ethanol consumption or sedation (derived from experiments above) 10 min prior to the administration of ethanol (3.6 g/kg). Then, at 10, 30, 90, 180 and 270 min following the ethanol injection, a small nick is made in the lateral portion of the base of the tail of each rat, and approximately 20 ul of blood is collected into a heparinized capillary tube and later analyzed for ethanol content. Each animal is then euthanized immediately following the last blood collection time point by CO2 inhalation.

7.4 Example 4: Erlotinib selectively decreases ethanol consumption and seeking

[0251] The role of erlotinib in modulating ethanol seeking and consumption using two different drinking paradigms has been evaluated. This experiment shows that Erlotinib can diminish or inhibit an addiction related behavior in rats suffering from alcohol dependence. The experiment also shows that Erlotinib ameliorates the effects of alcohol dependence in rats as well as alleviates the withdrawal symptoms in rats suffering from alcohol dependence. [0252] RESULTS

[0253] The effect of erlotinib on ethanol consumption and reward in rats using the continuous-access-two-bottle-choice drinking paradigm was measured {see Materials and Methods). This paradigm differs significantly from the operant self-administration

paradigm as the rats consume the reward on a voluntary basis; the reward is freely available and its delivery is neither contingent upon specific behaviors (lever pressing) nor associated with discrete cues (light/tone). When the rats had maintained a stable baseline consumption of 10% ethanol for 8 weeks, erlotinib (5, 20 and 40 mg/kg i.p.), given 30 minutes before access to 10% ethanol, significantly decreased ethanol consumption for up to 24 hours. There was an overall main effect of erlotinib on ethanol consumption at 30 minutes [F(3,l 1)=4.5; PO.009, Fig. 3A], at 16 hours [F(3,l 1)=7.7; PO.0005; Fig. 3B] and at 24 hours [F(3,l l)=7.0; P<.0009; not shown] compared to vehicle. Post hoc group differences are indicated in Figs. 3A&B. Erlotinib selectively decreased ethanol consumption and did not have an overall effect on water consumption [F(3,l I)=O.6, n.s., not shown] at any of the time points examined on sucrose consumption after 24 hours (Fig. 5).

[0254] Furthermore, the amount of ethanol consumed between 24 to 48 hours following the erlotinib administration did not differ from ethanol consumption after vehicle treatment [F(3,l l)=2.05; n.s., not shown]. This shows that a rebound increase in ethanol consumption was not observed following erlotinib treatment. The main caveat with the continuous-access-two-bottle-choice paradigm is that a sucrose fading technique is required to train the rats to consume 10% ethanol and that when this is achieved the animals consume only low to moderate quantities of ethanol (0.5 g/kg/30 minutes or 2.2g/kg/24 hours).

[0255] Therefore the effect of erlotinib on ethanol mediated behaviors using an operant self-administration model of drinking and reward seeking in rats was examined. In this model, the delivery of the ethanol (10%) reward was contingent upon a visual (light) and auditory (3 second tone) cue. In addition, the rats were trained to selectively press an active lever three times to receive the ethanol reward {see Materials and Methods). No reward was received if the rats pressed the inactive lever and the event was merely recorded as a measure of nonspecific behavioral activity. When the rats had maintained a stable level of responding over approximately 70 sessions (approximately four months of ethanol exposure), erlotinib (20, 40 or 80 mg/kg i.p.) was administered 120 minutes prior to the session. Erlotinib treatment had an overall main effect on operant self-administration of 10% ethanol [F(3,l l)=6.0, PO.002] and post hoc analysis

revealed that 80 mg/kg significantly inhibited operant self-administration of 10% ethanol compared to vehicle (Fig. 4 ).

[0256] The present study shows that erlotinib reduces ethanol consumption and seeking in rats and did not inhibit water consumption. [0257] MATERIAL AND METHODS [0258] Animals and Housing

[0259] Adult, male Wistar and Long Evans rats (Harlan Indianapolis, IN), were individually housed in ventilated Plexiglas cages. The rats were given time to acclimatize to the individual housing conditions and handling before the start of the experiments. All rats were housed in a climate controlled room on a 12 hour reversed light-dark cycle (lights off at 10 a.m.). Food and water were available ad libitum, except for short periods during initial training in the operant self-administration paradigm, as outlined below. All procedures were pre-approved by the Gallo Center Institutional Animal Care and Use Committee and were in accordance with NIH guidelines for the Humane Care and Use of Laboratory Animals. [0260] Operant Self-Administration [0261] Apparatus

[0262] Testing was conducted in standard operant conditioning chambers (Coulbourn Instruments, Allentown, PA) enclosed in ventilated, sound-attenuating cubicles. Each chamber housed two retractable levers on the right wall with a liquid dipper system placed centrally between them. A house light was present on the wall opposite the levers and remained on at all times during the operant session. Stimulus lights were present above each lever. An apparatus to emit a tone under specific operant conditions was also present. Upon correct (active) lever press(es) the stimulus light above the active (right) lever was illuminated for 3 seconds and was accompanied by a 3 second tone to reinforce availability of reward in the dipper receptacle. The dipper port was illuminated for 10 seconds whilst the dipper cup was available. Stimulus, fluid delivery and operant responses were all controlled and recorded by a computer (Coulbourn Instruments) using Graphic State 2.0 software. [0263] Operant Self-Administration Training

[0264] Prior to beginning the operant self-administration training, twelve male Wistar rats were exposed to 10% ethanol as the only liquid source in their home cages

for four days. The mean body weight was 253 ± 3 g at the start of training. Rats were then fluid restricted for 22 hours before being placed in the operant chambers for a 14 hour overnight session. During the overnight session, the rats were rewarded with a reinforcer of 0.1 ml of a solution consisting of 10% w/v sucrose after a single lever press (FRl protocol of reinforcement). During a session on the FRl protocol, only the right (active) lever was available for the rat to press to facilitate learning. The start of a session was signaled by illumination of the house light. In addition to the reinforcer, both a visual (light) and auditory (3 second tone) stimuli cue were presented after a press on the active lever. Following the overnight session, the rats were trained daily for 45 minutes on the FRl protocol and the rats were restricted to 2 hours water access after the behavioral session. Once stable responding levels were established, rats were given free access to water in the home cage and continued on an FRl schedule for three to four additional sessions. Subsequently, training sessions were reduced to 30 minutes and the FR3 protocol of reinforcement was introduced (i.e. 3 active lever presses required for 0.1 ml reward). A second, inactive lever was also introduced at this time. Presses on the inactive lever yielded no programmed consequence and were recorded as a measure of nonspecific behavioral activity. Once a baseline level of pressing was established, 10% ethanol was added to the 10% sucrose solution for the ethanol group. Over the next eight to ten sessions, the sucrose concentration was gradually decreased (5%, 3%, 1.5%) until the rats responded on an FR3 schedule for 10% v/v ethanol without any sucrose (Samson, 1986, Alcohol Clin. Exp. Res. 10, 436-42). Rats were kept on the FR3 protocol with 10% v/v ethanol as the reinforcer for approximately 70 sessions or four months prior to drug testing. The mean body weights were 580 ± 13 g at the first erlotinib test session.

[0265] Feeding EGFR inhibitors to Drosophila

[0266] The protocol for feeding erlotinib (Tarceva®) to flies was derived from a previously published method (Bainton et al., 2000). Flies of the genotype w Berlin were collected at 0-2 days post-eclosion, 25 males per large food vial. The following day, flies were transferred to large vials without food, each lined with a strip of Whatman 3 MM paper (8 x 2.5 cm) and containing 1 ml of 5% sucrose, 2% yeast, and 0.02 ml of 40 mg/ml Tarceva® (obtained from OSI Pharmaceuticals, Melville, NY) freshly dissolved in 25% cyclodextrin/0.9% saline (vehicle); final drug concentration in food was 0.8

mg/ml. Gefitinib feeding was carried out as for erlotinib, on Whatman 3 MM paper containing 1 ml of 5% sucrose, 2% yeast, and 0.01 ml of 50 mg/ml gefitinib (Iressa®) (Biaffin GmbH & Co KG, Kassel, Germany [www.proteinkinase.de]) freshly dissolved in DMSO (vehicle); final concentration of gefitinib was 0.5 mg/ml. Control vials contained an equivalent volume of vehicle without drug. Vials were capped with large buzz plugs which were dampened with water to prevent dehydration. Flies were returned to the 25 ⁰ C incubator and were kept on drug for 40-41 hours until they were assayed for sedation.

[0267] Two-Bottle-Choice Drinking Paradigms

[0268] All fluids were presented in 100-ml graduated glass cylinders with stainless steel drinking spouts inserted through two grommets in front of the cage 15 minutes after the light went out in the reversed light-dark cycle room. The placement of the ethanol bottle was alternated daily to control for side preferences. Bottles were weighed 30 minutes, 6 hours and 24 hours after the fluids were presented and measurements were taken to the nearest gram. The weight of each rat was measured daily in order to calculate the gram per kilogram ethanol intake (g/kg) ethanol and sucrose intake. [0269] Continuous-Access-Two-Bottle-Choice Drinking Paradigm [0270] After the acclimatization period, rats were divided into two groups, one received 10% ethanol continuously and the other was given continuous access to 5% sucrose. After the acclimatization period twelve Long Evans rats (249 ± 4 g) were given access to a bottle containing a solution of 10% (v/v) ethanol and 10% (w/v) sucrose and a separate water bottle. Over the next 12 days, the sucrose concentration was gradually decreased (i.e. from 10% to 5, 2% and 0% sucrose) until rats had continuous access to one bottle of 10% v/v ethanol and one bottle of water. Drug administrations began after the rats had maintained stable baseline drinking levels (2.2 ± 0.2 g/kg/24 hours) of the 10% v/v ethanol solution for 8 weeks (10 weeks of ethanol consumption including the sucrose fading period). The mean body weight was 448 ± 10 g at the first erlotinib test session. The eleven Long Evans rats that were assigned to the sucrose group received a bottle of 5% sucrose and had ad libitum access to water and the sucrose solution for the remainder of the experiment. Drug administrations began after the rats had maintained stable baseline drinking levels (24.5 ± 0.7 g/kg/24 hours) for 2 weeks. The mean body weight was 263 ± 4 g at the first erlotinib test session.

[0271] Drugs and Treatment Schedules

[0272] Ethanol and sucrose solutions were prepared in tap water from 95% (v/v) ethanol (Gold Shield Chemical Ac, Hay ward, CA, USA) and (w/v) sucrose (Fisher Scientific, NJ, USA), respectively. Erlotinib was generously provided by OSI Pharmaceuticals. All rats in the acute erlotinib experimental groups (operant self- administration, and continuous-access two bottle choice) received each of the 4 treatments (vehicle, 20, 40 and 80 mg/kg for the operant group and vehicle 5, 20, and 40 mg/kg for the two bottle group). Within each treatment group, each injection was given seven days apart using a Latin square design, and thus each rat served as its own control. Erlotinib was dissolved in 25% cyclodextrin in saline and administered as a i.p injection, in a volume of 1 ml/kg, 120 minutes before the start of the operant self-administration session or 30 minutes before ethanol and water bottles were presented. All drug solutions were prepared immediately before each injection. [0273] Statistics

[0274] Statistical analysis was performed using GraphPadPrism software and data was analyzed by repeated measures ANOVA followed Newman-Keuls Post hoc analysis was used when a significant overall main effect was found (PO.05).

7.5 Example 5: The effect of TARCEVA on ethanol sensitivity in flies.

[0275] The present example demonstrates the effect of TARCEV A ^® on ethanol sensitivity in flies.

[0276] Flies show increased sensitivity (Fig. 6) to ethanol sedation after being fed the EGFR inhibitor TARCEV A ^® (erlotinib; OSI Pharmaceuticals; Genentech).

T ARCEV A ^® at a final concentration of 0.8 mg/ml was fed continuously to adult male w; wt Berlin flies in a solution of 5% sucrose, 2% yeast for 40-41 hr (Bainton et α/., 2000,

Curr Biol 10 (4): 187), immediately before flies were assayed for sedation. For sedation assays, each sample consisted of 20-25 flies, and the ethanol/air relative flow rate was

100/50 E/A (sedation assays performed as per A. Rothenfluh et al, 2006, Cell 127 (1):

199; *, P=0.006 based on one-way ANOVA of values for ST50, the time at which 50% of the flies in each sample were sedated; N=8).

[0277] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were

specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

7.6 EXAMPLE 6: Comparison of Effects of TARCEVA® in Rats

[0278] The protocol for feeding erlotinib (Tarceva®) to flies was derived from a previously published method (Bainton et al., 2000). Flies of the genotype w Berlin were collected at 0-2 days post-eclosion, 25 males per large food vial. The following day, flies were transferred to large vials without food, each lined with a strip of Whatman 3 MM paper (8 x 2.5 cm) and containing 1 ml of 5% sucrose, 2% yeast, and 0.02 ml of 40 mg/ml Tarceva® (obtained from OSI Pharmaceuticals, Melville, NY) freshly dissolved in 25% cyclodextrin/0.9% saline (vehicle); final drug concentration in food was 0.8 mg/ml.

[0279] Control vials contained an equivalent volume of vehicle without drug. Vials were capped with large buzz plugs which were dampened with water to prevent dehydration. Flies were returned to the 25° C incubator and were kept on drug for 40-41 hours until they were assayed for sedation.

[0280] Referring to FIGS. 7A-7F, samples of 25 flies each were treated identically to when they were assayed in the ethanol LOR assay, and were fed a sucrose/yeast mixture containing either erlotinib (0.8 mg/ml) dissolved in vehicle or vehicle alone for 40 or 41 hr. (A) Flies fed erlotinib (Tarceva®) did not show altered ethanol pharmacokinetics. Flies were exposed to ethanol vapor (100/50 E/ A) for 30 min in the tracking apparatus and ethanol levels were measured in extracts of erlotinib (Tarceva®)- and vehicle-fed flies. One-way ANOVA revealed no significant effect of erlotinib (p=0.\ 1; F _{U 5} =2.85; n=8). (B) Blue dye (erioglaucine, Sigma 861 146, 0.2%) was included in food for both erlotinib (Tarceva®)- and vehicle-fed flies. Feeding behavior of flies was quantified at the time of assay by comparing total gut volumes in drug- and vehicle-fed flies, as assessed by dye uptake measured from fly extracts (absorbance units at 630 run, AU). No significant effect of erlotinib (Tarceva®) on feeding was detected

by one way ANOVA (p-0.5; Fi ₁ H=O^; n=6). (C) Effect of erlotinib on locomotor behavior of flies was quantified in the tracking apparatus. Average locomotor velocities of flies pre-fed either erlotinib (Tarceva®) or vehicle were measured in air alone and during early times of exposure to ethanol vapor/humidified air (100/50 E/A; gray bar), before flies had begun to sedate. No effect of erlotinib (Tarceva®) on fly velocity was seen in the absence of ethanol, while a modest increase in velocities of drug-fed flies was seen during ethanol exposure; while consistent (data not shown), this effect was not significant by two-way repeated measures ANOVA (p=0λ3; F _lj7 =3.02; n=4). (D, E) The effect of erlotinib (Tarceva®) on the latency and duration of loss of righting reflex (LORR) was measured in adult Long Evans rats. Vehicle or erlotinib (40 mg/kg i.p) was administered 30 mins prior to ethanol (3.6 g/kg i.p). Both the latency and the duration of the LORR were measured (see Methods) and the data, analyzed by Student's unpaired t- test, revealed that erlotinib (Tarceva®) did not effect either (D) the latency to LORR (p=0.665), n=6 or (E) the duration of the LORR 0?=0.295), n=6). (F) The effect of erlotinib (Tarceva®) (40mg/kg i.p) on blood ethanol clearance was measured in adult Long-Evans rats. Blood samples were collected from the lateral tail vein at 30, 60, 120, 240, and 480 min following ethanol injection (3.6 g/kg i.p). One-way ANOVA revealed that erlotinib did not effect blood ethanol clearance at any time point (/?>0.05), n=5. [0281] Loss of Righting Reflex and Blood Ethanol Concentrations in Rats [0282] After isolation on a surgery table, Long Evans rats were injected intraperitoneally with ethanol at the dose of 3.6 g/kg (20% vol/vol). When the animal appeared to be sedated, it was laid on its back. The duration of the loss of righting reflex (LORR) was evaluated by measuring the time interval between the loss and the recovery of the righting reflex. Animals were judged to have regained their righting reflex when they could right themselves three times within 30 sec after being placed on their backs. To examine the effects of erlotinib on ethanol-induced sedation, drug (40 mg/kg) or vehicle were administered 30 min before the administration of the ethanol. To examine the effects of erlotinib (40rng/kg i.p) on blood ethanol clearance, blood samples were collected from the lateral tail vein of the sedated animals at 30, 60, 120, 240, and 480 min following ethanol injection. Blood ethanol concentrations were determined using gas chromatography methods adapted from Doyon et al. (2003).

[0283] The EGFR/ERK pathway has an evolutionarily-conserved role in regulating ethanol behaviors in adult flies and mammals. Administration of a well-studied EGFR inhibitors, erlotinib (Tarceva®) to adult flies resulted in enhanced sensitivity in the LOR assay. While our results do not rule out a developmental role for the EGFR/ERK pathway, they do demonstrate that this pathway functions in the adult fly to regulate the sedative effects of ethanol. Strikingly, treatment of adult rats with erlotinib significantly decreased ethanol preference in a two-bottle free choice paradigm. This effect appears to be ethanol-specific, as preference for a second rewarding substrate, sucrose, was not affected by erlotinib administration. Together, these data reveal a potentially conserved role for the EGFR pathway in regulating ethanol behaviors in both flies and rodents, and offer a possible therapeutic avenue for the treatment of alcoholism in humans.

7.7 EXAMPLE 7: Pharmacological Inhibition of the EGFR Pathway in

Drosophila

[0284] To determine whether inhibition of EGFR signaling specifically during adulthood would alter sensitivity to ethanol-induced sedation, we pharmacologically inhibited the EGFR in adult flies using the well-characterized inhibitor erlotinib (Tarceva®). This small molecule drug is orally bioavailable and a selective inhibitor of the tyrosine kinase activity of the EGFR that acts by preventing receptor autophosphorylation and activation (Ciardiello et al., 2004). Adult flies were fed a sucrose/yeast mixture containing erlotinib for a period of ~40 hours, then tested in the ethanol LOR assay. Erlotinib-fed flies were more sensitive to the sedating effects of ethanol compared to flies fed vehicle alone (Fig. 6A, B). This enhanced sensitivity does not appear to be due to altered ethanol pharmcokinetics, as ethanol absorption was normal (Fig. 7A), nor due to non-specific effects on fly fitness, as erlotinib did not alter total food intake (Fig. 7B), baseline locomotor velocity, nor the startle response to the smell of ethanol (Fig. 7C). A second orally active, specific and potent inhibitor of EGFR tyrosine kinase activity is the drug gefitinib (Iressa®) (see Ranson and Wardell, 2004, "Gefitinib, a novel, orally administered agent for the treatment of cancer," J Clin Pharm Ther 29: 95-103; Ono and Kuwano, 2006, "Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR- targeting drugs," CHn Cancer Res 12, 7242-7251; and Dutta and Maity, 2007, "Cellular

responses to EGFR inhibitors and their relevance to cancer therapy," Cancer Lett 254, 165-177. Similar to erlotinib, adult flies fed gefitinib for -40 h exhibited increased sensitivity to ethanol-induced sedation (Figure 6C, D). These data indicate that inhibition of the EGFR pathway specifically during adulthood is sufficient to elicit enhanced ethanol sensitivity in Drosophila.

7.8 EXAMPLE 8: Acute Pharmacological Inhibition of the EGFR

Reduces Ethanol Consumption in Rats

[0285] Having demonstrated an effect of pharmacological inhibition of the EGFR on ethanol-induced behaviors in Drosophila, we next asked whether acute administration of erlotinib might alter ethanol-induced behaviors in mammals. The effects and pharmacokinetics of erlotinib have been well characterized in mammals including humans (Broniscer et al., 2007), and appropriate doses have been determined for oral administration in rodents (Carey et al., 2006). We evaluated the effects of erlotinib on 10% ethanol and 5% sucrose consumption in rats using the continuous-access two- bottle-choice drinking paradigm. Once rats had achieved a stable baseline consumption of 10% ethanol or 5% sucrose, erlotinib (5, 20 and 40 mg/kg i.p.) was administered 30 minutes prior to access to either ethanol or sucrose. Erlotinib (Tarceva®)significantly decreased ethanol consumption for up to 24 hours in a dose-dependent manner (Fig. 5A). Interestingly, the effect of the drug was selective for ethanol, as it had no effect on 5% sucrose consumption (Fig. 5B). There was no overall effect on water consumption (Fig. 5C), indicating a reduction in ethanol preference rather than simply an alteration in overall fluid consumption. Finally, the amount of ethanol consumed between 24 to 48 hours following erlotinib administration did not differ from ethanol consumption after vehicle treatment (data not shown); thus, erlotinib (Tarceva®) did not cause a rebound increase in ethanol consumption. In summary, our data demonstrate that acute inhibition of EGFR pathway signaling during adulthood is sufficient to significantly decrease ethanol consumption and preference in rats.

[0286] Finally, we demonstrate that pharmacological inhibition of EGFR signaling during adulthood causes increased sensitivity to ethanol-induced sedation in flies, and decreased ethanol preference in a two-bottle choice assay in rats.

7.9 Genetic Interactions with The EGFR/ERK Pathway

[0287] Proteins in the EGFR/ERK pathway interact extensives among themselves and with other proteins. It is therefore possible to target one of these proteins to achieve desired effects on the EGFR/ERK pathway. Based on our observations that enhanced EGFR signaling and loss of hppy function both lead to resistance to the sedative effects of ethanol, while reduced EGFR signaling and hppy overexpression lead to the opposite effect, enhanced sensitivity, we reasoned that hppy may function as an inhibitor of the EGFR pathway. In order to test this hypothesis, we resorted to the fly eye, where the developmental role of EGFR signaling has been thoroughly studied (reviewed in Dominguez et al., 1998, Current Biology 8:1039-1048). Specifically, we tested whether overexpression of hppy-RB could enhance or suppress the rough eye phenotypes induced by expression of EGFR pathway components using the GMR-GAL4 driver, which drives expression in developing retinal cells (Moses and Rubin, 1991, Genes and Development 5:583-593). Expression of UAS-hppy™ ¹ under the control 0ϊGMR-GAL4 resulted in an essentially wild-type eye phenotype (compare Fig. 1OA and 10B). Overexpressing the EGFR using UAS-Egfr™ ⁷ resulted in a very strong rough eye phenotype with prominent blistering in the dorsal anterior section of the eye (Fig. 10C). A rough eye phenotype was also observed when expressing a constitutively active form of the MAPK rolled, using the UAS-rl ^ACτ transgene, under the control of GMR-GAL4 (Fig. 10E). We found that hppy overexpression was able to suppress the rough eye and blistering phenotypes induced by overexpression of the EGFR (Fig. 10D). In contrast, expression of hppy did not affect the rough eye phenotype caused by expression of activated rolled (Figs. 1OE, 10F). Having shown that hppy expression could suppress the rough eye phenotype induced by EGFR pathway activation, we asked whether hppy expression could enhance the rough eye phenotype caused by the expression of the transcription factor YAN, which acts downstream of rolled to inhibit the transcription of EGFR pathway activated genes (O'Neill et al., 1994, Cell 78:137-147). Indeed, while expression of wild-type yan, using the UAS-yan ^wτ transgene, produced an overall normal looking eye with an orderly arrangement of ommatidia (Fig. 10G), the combined expression of hppy and yan under GMR-GAL4 control produced a severe rough and "glossy" eye phenotype in which large swaths of ommatidia were absent (Fig. 10H), showing that hppy and yan act synergistically to inhibit downstream targets of the EGFR pathway. Taken together, our

data implicates a role for hppy as an inhibitor of EGFR signaling, acting downstream of the EGFR, but upstream of the MAPK rolled.

[0288] Interestingly, when expressing various EGFR pathway components with the

GMR-GAL4 driver, we observed that expression of either the dominant negative form of the EGFR (UAS-Egfr ^m ) or an activated form of yan (UAS-yan ^ACτ ), resulted in reduced viability. Interestingly, co-expression of hppy (UAS-hppy ^RBl ) potently enhanced the lethality of both GMR-GAL4 driven UAS-Egfr™ and UAS-yan ^ACτ . Taken together, these data provide evidence that hppy modulates the EGFR pathway in its requirement for normal eye development and viability, and are consistent with hppy functioning as an inhibitor to the pathway, acting downstream of the EGFR and upstream of the ERK

Rolled.

[0289] To determine whether hppy interacts with the EGFR pathway in the context of ethanol-induced behaviors, we tested the LOR sensitivity of flies expressing the UAS- egfr™ ^Ai transgene pan-and hppy ^11'51 neuronally (under the control of elav-GAL4 ^cl5S ) in the homozygous hppy ^KG5531 mutant backgrounds. In contrast to the enhanced ethanol sensitivity seen in flies expressing the UAS-egfr ^RNM transgene pan-neuronally, the hppy ^KG5537 and the hppy ^11'51 mutants completely suppressed the sedation sensitivity observed in flies expressing UAS-egfr ^mAl . This finding is consistent with our model in which hppy functions as an inhibitor to the EGFR pathway, functioning downstream of the EGFR to regulate ethanol-induced sedation..

[0290] Thus, hppy can modulate EGFR signaling in a manner that is consistent with it acting as an inhibitor of the pathway, operating downstream of the EGFR but upstream of the ERK rolled.

[0291] Discussion

[0292] Happyhour regulates ethanol-induced sedation and EGFR signaling in

Drosophila

[0293] Utilizing a forward genetic approach to search for Drosophila mutants displaying altered responses to ethanol, we identified and characterized two P-element mutants in the CG7097 /happyhour (hppy) gene region. We found that decreased hppy expression resulted in decreased sensitivity to the sedative effects of ethanol, as measured in a modified LOR assay, whereas neuronal overexpression of hppy caused the opposite effect. Through in situ hybridization and quantitative RT-PCR, we found

evidence for hppy expression in adult brains, and behavioral rescue experiments demonstrated that neuronal expression of hppy was sufficient to rescue the hppy sedation resistance phenotype. In addition, we found that complete rescue could be achieved by restoring expression of specifically the hppy-RB transcript, suggesting functional redundancy between the hppy-RB and hppy-RA transcripts or that the hppy-RA transcript may not be required for normal ethanol-induced sedation behaviors. [0294] Like its mammalian homologs, the GCK-I subfamily of Ste20 family kinases, both hppy transcripts are predicted to encode proteins that bear N-terminal serine/threonine kinase domains and C-terminal regulatory domains known as citron homology domains. In vitro studies of these homologs of hppy, including germinal center kinase (GCK) (Pombo et al., 1995), GCK-like kinase (GLK; Diener et al, 1997, Proc Natl Acad Sci USA 94:9687-9692), kinase homologous to SPS1/STE20 (KHS; Tung and Blenis, 1997, Oncogene 14:653-659), and hematopoietic progenitor kinase (HPKl; Kiefer et al., 1996, Embo J 15:7013-7025), have revealed that these GCK-I subfamily members specifically activate the JNK signaling cascade, but not the ERK or p38 MAPK pathways. HPKl (Hu et al, 1996, Genes Dev 10:2251-2264) and GLK (Diener et al., 1997) have both been shown to phosphorylate MAP3Ks in the JNK signaling pathway, positioning these GCK-I subfamily kinases as MAP4Ks, situated upstream of the traditional three-tiered MAPK signaling cascade, but downstream of membrane signaling elements. While more distantly related Ste20 group kinases, such as those belonging to the GCK-VIII subfamily of thousand and one (TAO) kinases, have been shown to act as MAP3 kinases, activating both the JNK and the p38 stress- activated MAP kinase cascades (Hutchison et al, 1998, J Biol Chem 273:28625-28632 and Yustein et ah, 2000, Oncogene 19:710-718), until this study there have not been any reports of GCK kinases having a modulatory role on ERK signaling. [0295] In this study we present evidence to show that HPPY, a presumed MAP4K in the GCK-I subfamily of Ste20 kinases, can indeed modulate ERK signaling in a manner that is consistent with it acting as an inhibitor of ERK signaling functioning upstream of ERK itself but downstream of the EGFR. We find that HPPY can enhance and suppress the rough eye phenotypes brought about by EGFR/ERK perturbations as well as enhance the semi-lethality induced by expression of EGFR/ERK pathway inhibitors. In addition, decreasing levels of hppy completely suppressed the enhanced ethanol sensitivity

brought about by neuronal EGFR downregulation. These findings are consistent with our hypothesis that hppy is epistatic to egfr, and that HPPY acts through the EGFR/ERK pathway to modulate ethanol-induced sedation in Drosophila. [0296] An in vitro study of another GCK-I subfamily kinase, HPKl, offers up an intriguing possibility (Anafi et al., 1997, J Biol Chem 272:27804-2781 1) of biochemical mechanism through which hppy inhibits EGFR/ERK signaling. In this study, the authors found that HPKl physically associates with the EGFR adaptor protein Grb2 both in a yeast two-hybrid assay as well as in transfected mammalian cells. EGF stimulation recruits the Grb2/HPK1 complex to the autophosphorylated EGFR. This recruitment then leads to the tyrosine phosphorylation of HPKl, although the functional consequences of this phosphorylation are yet unknown. It would be interesting to determine whether such an association might exist between HPPY and members of the EGFR/ERK signaling cascade, and what consequences this association may have on the signaling of this MAP kinase pathway.

[0297] From our experiments we cannot rule out a role for hppy in regulating JNK signaling, although the lack of effect of JNK signaling perturbation on behavioral sensitivity to ethanol strongly suggests that hppy does not mediate its effects on ethanol- induced sedation through the JNK pathway. We also found that hppy mutant flies do not respond differently from controls when exposed to a variety of stress stimuli known to activate the JNK and p38 pathways, including oxidative stress, heat stress, and starvation (data not shown), further supporting the hypothesis that hppy is not involved in transducing signals through these stress-activated MAPK pathways. Indeed, in vitro studies in HeIa cells support this hypothesis, demonstrating a lack of involvement of hppy in mediating JNK activation in response to stress stimuli such as osmotic stress and the protein synthesis inhibitor anisomycin (Findlay et al, 2007). Instead, Findlay et al. offer evidence that hppy can act as a nutrient sensor of amino acids and can stimulate phosphorylation of S6 kinase (S6K) through the mammalian target of rapamycin (mTOR) signaling pathway. The authors show that overexpression of wild-type hppy, but not a kinase-inactive mutant, can induce this S6K phosphorylation, demonstrating that hppy can in fact function as a kinase (Findlay et al, 2007). Interestingly, the mTOR signaling pathway, a key regulator of cell growth, is itself intimately regulated by inputs from the ERK signaling pathway (Sarbassov et al, 2005, Curr Opin Cell Biol 17:596-

603), suggesting the attractive possibility that hppy may exert its stimulatory role on the mTOR pathway via its effects on EGFR/ERK pathway signaling. [0298] The EGFR/ERK pathway mediates ethanol-induced sedation in Drosophila [0299] The ERK signaling cascade has traditionally been studied for its roles in regulating a variety of developmental processes, including cell division, survival, and differentiation (reviewed in Chen et ah, 2001; Pearson et ah, 2001). More recent research, however, has revealed that the ERK pathway also plays important roles in mediating synaptic plasticity in post-mitotic neurons (Sweatt, 2004) (Mazzucchelli and Brambilla, 2000). For instance, establishment of long-term potentiation requires the activation of ERK, and inhibition of ERK signaling has been shown to disrupt both hippocampal- and amygdala-dependent learning (Brambilla et al., 1997, Nature 390:281-286.; Atkins et al, 1998, Nat Neurosci 1 :602-609; and Selcher et α/., 1999, Learn Mem 6:478-490). In addition, the EGFR/ERK pathway has been implicated in the regulation of circadian rhythms via its activation by transforming growth factor-α in the suprachiasmatic nucleus (Hao and Schwaber, 2006, Brain Research 1088:45-48). Recently, an interesting literature has documented the inhibitory effects of ethanol on the EGFR/ERK pathway in neurons. Ethanol administration inhibits EGFR and ERK phosphorylation both in neuronal cell cultures (Kalluri and Ticku, 2003, Neurochem Res 28:765-769; Chandler and Sutton, 2005; Ma et al., 2005) as well as in mouse and rat brains (Kalluri and Ticku, 2002, Eur J Pharmacol 451 :51 -54; and Sanna et al., 2002, Brain Res 948:186-191).

[0300] The present invention provides a previously undocumented role for the EGFR/ERK pathway in mediating the behavioral responses to ethanol in Drosophila. Neuronal manipulations that activate the EGFR/ERK pathway result in decreased sensitivity to the sedative effects of ethanol, whereas inhibition of the pathway results in increased sensitivity to ethanol-induced sedation. These effects were seen through manipulations of various components of the EGFR/ERK pathway, including ERK itself, the MAP3K (dRaf), the EGFR, the EGF receptor ligand Spitz, and the enzyme, Rhomboid- 1, that processes Spitz into its active form. In contrast, we find no evidence for the other two major MAPK pathways, the JNK and p38 pathways, in mediating the sedative response to ethanol, suggesting a specific role for the ERK pathway. The EGFR/ERK pathway joins other growth factor pathways, such as the insulin, glial cell

line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) pathways, as regulators of the behavioral response to ethanol (Janak et al., 2006, Alcohol Clin Exp Res 30:214-221).

[0301] The pathways through which the EGFR/ERK cascade detects ethanol signals and how it might transduce those signals into a behavioral response remain unknown. The ERK cascade is activated through a variety of sources, including growth factors, serum, and ligands for heterotrimeric G protein-coupled receptors (reviewed in Chen et al., 2001). Interestingly, recent studies have implicated a role for both GABA _A and NMDA receptors in ethanol-mediated inhibition of ERK in vitro, suggesting that ethanol may influence ERK activation state via signaling through these receptors (Kalluri and Ticku, 2002, 2003). The targets of ERK signaling are multitudinous, and our knowledge of the substrates of ERK signaling is ever expanding. In addition to phosphorylating and activating various transcription factors, ERK also targets various protein kinases, voltage-gated ion channels, and various second messenger systems such as cytosolic phospholipase A2 (Atkins et al, 1998; Chen et al, 2001). One alluring target of ERK signaling is the phosphodiesterase (PDE) 4D3, a cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase which negatively regulates cAMP/PKA signaling by degrading intracellular cAMP (Chen et al, 2001). The cAMP signaling pathway, in turn, has been shown to regulate the ERK pathway, with inhibition of ERK signaling being induced by C-RAF phosphorylation by PKA (Dumaz and Marais, 2005, Febs J 272:3491-3504). Various studies in both Drosophila and mammals have demonstrated a role for the cAMP/PKA signaling cascade in mediating the behavioral responses to ethanol (Moore et al 1998; Park et al, 2000; Thiele et al, 2000; Mass et al, 2005), raising the exciting possibility that "crosstalk" between these two conserved pathways may be integral to regulating ethanol induced sedation behaviors.

7.10 Effect of Expression of EGFR in Different Cell Types on Ethanol- Induced Sedation

[0302] In this study we present evidence to show that HPPY, a presumed MAP4K in the GCK-I subfamily of Ste20 kinases, can indeed modulate ERK signaling in a manner that is consistent with it acting as an inhibitor of ERK signaling functioning upstream of ERK itself but downstream of the EGFR. We find that HPPY can enhance and suppress

the rough eye phenotypes brought about by EGFR/ERK perturbations as well as enhance the semi-lethality induced by expression of EGFR/ERK pathway inhibitors. In addition, decreasing levels of hppy completely suppressed the enhanced ethanol sensitivity brought about by neuronal EGFR downregulation. These findings are consistent with our hypothesis that hppy is epistatic to egfr, and that HPPY acts through the EGFR/ERK pathway to modulate ethanol-induced sedation in Drosophila.

[0303] P[GAL4], UAS-egfr™ ⁷ , and P[GAL4] + UAS-egfr ^m flies were tested in the booz-o-mat sedation assay and ST50 values were calculated for each genotype (see Methods). One-way ANOVA of the ST50 values followed by post-hoc Newman-Keuls testing were performed. P[GAL4] + lines with ST50 values that were significantly higher than both the P[GAL4] alone and the UAS-egft^ alone were considered to be resistant (n=8 for each genotype, ns= not significant). IPCs = Insulin producing cells; DA neurons = Dopaminergic neurons; 5HT neurons = Serotonergic neurons; AL = Antennal lobe; AMC = Antenno-mechanosensory center; SEG = Subesophageal ganglion; CC = Central complex; LPC = Lateral protocerebrum; MB = Mushroom Body; DGI = Dorsal giant interneurons; EB = Ellipsoid body; LN _V neurons = Ventral lateral neurons; OL = Optic lobes.

[0304] The observation that EGFR activation specifically in either insulin-producing cells (IPCs) or dopaminergic cells affects ethanol sensitivity is consistent with previous studies implicating both the IPCs (Corl et al., 2005) and dopaminergic systems (Bainton et al., 2000) in the behavioral response to ethanol in Drosophila, and suggests that the EGFR/ERK pathway may interact with the insulin and dopamine signaling pathways to control drug responses. Equally interesting is our observation that EGFR activation in many other brain regions had no effect on ethanol-induced sedation, brain regions that include those previously shown to play a role in ethanol-related behaviors, such as the EB (Urizar et al., 2007) and the cells defined by the 201 Y GAL4 line (Rodan et al., 2002). Thus, the EGFR pathway appears to play a role in only a subset of brain regions that regulate the flies' response to ethanol.

7.11 Perturbation of EGFR Signaling in Neuronal Cells Alters Ethanol Sensitivity

[0305] Since pan-neuronal activation of the EGFR pathway resulted in increased resistance to ethanol-induced sedation, specific cells/brain regions responsible for this phenotype were identified, a small-scale screen was conducted, driving egfr expression using 15 pre-selected GAL4 driver lines whose expression patterns had been characterized as shown in the abobe Table. It was found that expression of a wild-type EGFR transgene (UAS-egfr™ ⁷ ) in such brain regions as the mushroom body, ellipsoid body, or the ventral lateral neurons had no measurable effect on ethanol-induced sedation (See the Table above). Egfr expression using a muscle driver, MHC-GAL4, also produced no effect, while driving egfr expression using a glial driver, repo-GAL4, resulted in lethality (See the Table above and data not shown). In contrast, strongly increased resistance to ethanol-induced sedation was observed when driving egfr expression in insulin-producing cells (IPCs) using the dilp2-GAL4 driver (Rulifson et al., 2002) (Fig. 10A) or in dopaminergic cells using the TH-GAL4 driver (Friggi-Grelin et al., 2003) (Fig. 10D). More modest resistance was observed when expressing egfr with Ddc-GAL4, which drives expression in most dopaminergic and serotonergic neurons (Li et al., 2000). Importantly, expression of GAL4 and gross morphology of cells was unaffected by egfr expression driven by dilp2-GAL4, TH-GAL4, or Ddc-GAL4 {e.g., compare FIGs. 1OB and 1OC, FIGs. 1OE and 1OF, and data not shown). In summary, these data show that perturbation of the EGFR pathway in discrete subsets of CNS neurons is sufficient to induce marked resistance to ethanol-induced sedation. [0306] The remaining MAPK signaling pathway, the ERK pathway, was tested for its potential role in regulating ethanol sensitivity. We asked if perturbations of the ERK pathway, specifically the EGFR pathway, had an effect on ethanol-induced sedation by driving expression of various EGFR pathway transgenes using the pan-neuronal drivers elav-GAL4 ^cl55 or elav-GAL4 ^3E1 . We also attempted to drive transgene expression with the ubiquitous driver Tub-GAL4, but found that this resulted in lethality in all cases except when driving the expression of a secreted form of the EGFR ligand encoded by the spitz (spi) gene, a condition that produced viable and healthy flies. [0307] Manipulations that enhanced EGFR signaling at several levels in the pathway potently increased resistance to ethanol-induced sedation (Fig. 11). Increasing

expression of secreted Spitz, by driving expression of a UAS-spi transgene using either Tub-GAL4 or elav-GAL4 ^ci55 , strongly increased resistance to ethanol-induced sedation (Fig. 1 IA and data not shown). Marked resistance was also produced by driving neuronal expression of a wild-type EGFR transgene (UAS-egfr ^{m '} , Fig. 1 IB), a gain-of-function Raf MAP3K {UAS-dRaβ ^ov , data not shown), or a constitutively active form of the ERK rolled (rl) (UAS-rl ^ACτ , Fig. HC).

[0308] Conversely, inhibiting EGFR signaling resulted in the opposite effect, decreased resistance to ethanol-induced sedation. For example, a mutation in rhomboid- 1 (rho-1), which encodes an enzyme that activates EGFR signaling through proteolysis of the ligand Spitz (Lee et al., 2001), displayed enhanced sensitivity to ethanol-induced sedation. In this mutant, which harbors a P element insertion in the promoter region of rho-1, mRNA levels were reduced to about 30% of wild-type as measured by QPCR. [0309] Finally, to determine if neuronal inhibition of EGFR signaling would result in increased sensitivity to the sedating effects of ethanol, we utilized a line expressing an RNAi transgene that targets the egfr, UAS-egfr™ ^k \ which strongly reduces egfr transcript levels as determined by QPCR (Fig. 8B). Indeed, expression of using the pan-neuronal driver e/αv-GλZ¥ ^cl55 resulted in increased sensitivity to ethanol- induced sedation (Fig. 8A). Taken together, our data strongly support a role for the EGFR/ERK pathway in regulating ethanol-induced sedation in Drosophila, where inhibition of the pathway leads to enhanced sensitivity to the sedating effects of ethanol, while its activation leads to the opposite phenotype, increased resistance to sedation.

SEQUENCE LISTING

SEQ ID NO.: 1 (Locus: NP_005219; epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), isoform a precursor [Homo sapiens])

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEV VLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQI IRGNMYYENSYALA VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDF QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGC TGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFK NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF ENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVI ISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVV ALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGS GAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGI CLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA RNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSY GVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPK FRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQ QGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTED SIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLN TVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRV APQSSEFIGA

SEQ ID NO.: 2 (Locus: NM_005228; epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), mRNA transcript sequence, EGFR isoform a precursor [Homo sapiens])

CCCCGGCGCAGCGCGGCCGCAGCAGCCTCCGCCCCCCGCACGGTGTGAGCGCCCGAC GCG GCCGAGGCGGCCGGAGTCCCGAGCTAGCCCCGGCGGCCGCCGCCGCCCAGACCGGACGAC AGGCCACCTCGTCGGCGTCCGCCCGAGTCCCCGCCTCGCCGCCAACGCCACAACCACCGC GCACGGCCCCCTGACTCCGTCCAGTATTGATCGGGAGAGCCGGAGCGAGCTCTTCGGGGA GCAGCGATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTC

TGCCCGGCGAGTCGGGCTCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAG CTC ACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACTGT GAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTC TTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGA ATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCC TTAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGA AATTTACAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAAC GTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATG GACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGC TGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAG TGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCA GGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCC ACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGAT GTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAAT TATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATG GAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAAC GGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACAC TTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGT GACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTA AAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCAT GCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT GCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGAT GGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAA AAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGC TGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCG GAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAAC TGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCAC CTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACG AATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTG GTGGTGGCCCTGGGGATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACG CTGCGGAGGCTGCTGCAGGAGAGGGAGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCT CCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTG GGCTCCGGTGCGTTCGGCACGGTGTATAAGGGACTCTGGATCCCAGAAGGTGAGAAAGTT

AAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAG GAA ATCCTCGATGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTG GGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTC CTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGT GTGCAGATCGCAAAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTG GCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTG GCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTGCCTATC AAGTGGATGGCATTGGAATCAATTTTACACAGAATCTATACCCACCAGAGTGATGTCTGG AGCTACGGGGTGACCGTTTGGGAGTTGATGACCTTTGGATCCAAGCCATATGACGGAATC CCTGCCAGCGAGATCTCCTCCATCCTGGAGAAAGGAGAACGCCTCCCTCAGCCACCCATA TGTACCATCGATGTCTACATGATCATGGTCAAGTGCTGGATGATAGACGCAGATAGTCGC CCAAAGTTCCGTGAGTTGATCATCGAATTCTCCAAAATGGCCCGAGACCCCCAGCGCTAC CTTGTCATTCAGGGGGATGAAAGAATGCATTTGCCAAGTCCTACAGACTCCAACTTCTAC CGTGCCCTGATGGATGAAGAAGACATGGACGACGTGGTGGATGCCGACGAGTACCTCATC CCACAGCAGGGCTTCTTCAGCAGCCCCTCCACGTCACGGACTCCCCTCCTGAGCTCTCTG AGTGCAACCAGCAACAATTCCACCGTGGCTTGCATTGATAGAAATGGGCTGCAAAGCTGT CCCATCAAGGAAGACAGCTTCTTGCAGCGATACAGCTCAGACCCCACAGGCGCCTTGACT GAGGACAGCATAGACGACACCTTCCTCCCAGTGCCTGAATACATAAACCAGTCCGTTCCC AAAAGGCCCGCTGGCTCTGTGCAGAATCCTGTCTATCACAATCAGCCTCTGAACCCCGCG CCCAGCAGAGACCCACACTACCAGGACCCCCACAGCACTGCAGTGGGCAACCCCGAGTAT CTCAACACTGTCCAGCCCACCTGTGTCAACAGCACATTCGACAGCCCTGCCCACTGGGCC CAGAAAGGCAGCCACCAAATTAGCCTGGACAACCCTGACTACCAGCAGGACTTCTTTCCC AAGGAAGCCAAGCCAAATGGCATCTTTAAGGGCTCCACAGCTGAAAATGCAGAATACCTA AGGGTCGCGCCACAAAGCAGTGAATTTATTGGAGCATGACCACGGAGGATAGTATGAGCC CTAAAAATCCAGACTCTTTCGATACCCAGGACCAAGCCACAGCAGGTCCTCCATCCCAAC AGCCATGCCCGCATTAGCTCTTAGACCCACAGACTGGTTTTGCAACGTTTACACCGACTA GCCAGGAAGTACTTCCACCTCGGGCACATTTTGGGAAGTTGCATTCCTTTGTCTTCAAAC TGTGAAGCATTTACAGAAACGCATCCAGCAAGAATATTGTCCCTTTGAGCAGAAATTTAT CTTTCAAAGAGGTATATTTGAAAAAAAAAAAAAGTATATGTGAGGATTTTTATTGATTGG GGATCTTGGAGTTTTTCATTGTCGCTATTGATTTTTACTTCAATGGGCTCTTCCAACAAG GAAGAAGCTTGCTGGTAGCACTTGCTACCCTGAGTTCATCCAGGCCCAACTGTGAGCAAG GAGCACAAGCCACAAGTCTTCCAGAGGATGCTTGATTCCAGTGGTTCTGCTTCAAGGCTT CCACTGCAAAACACTAAAGATCCAAGAAGGCCTTCATGGCCCCAGCAGGCCGGATCGGTA CTGTATCAAGTCATGGCAGGTACAGTAGGATAAGCCACTCTGTCCCTTCCTGGGCAAAGA AGAAACGGAGGGGATGGAATTCTTCCTTAGACTTACTTTTGTAAAAATGTCCCCACGGTA CTTACTCCCCACTGATGGACCAGTGGTTTCCAGTCATGAGCGTTAGACTGACTTGTTTGT

CTTCCATTCCATTGTTTTGAAACTCAGTATGCTGCCCCTGTCTTGCTGTCATGAAAT CAG CAAGAGAGGATGACACATCAAATAATAACTCGGATTCCAGCCCACATTGGATTCATCAGC ATTTGGACCAATAGCCCACAGCTGAGAATGTGGAATACCTAAGGATAGCACCGCTTTTGT TCTCGCAAAAACGTATCTCCTAATTTGAGGCTCAGATGAAATGCATCAGGTCCTTTGGGG CATAGATCAGAAGACTACAAAAATGAAGCTGCTCTGAAATCTCCTTTAGCCATCACCCCA ACCCCCCAAAATTAGTTTGTGTTACTTATGGAAGATAGTTTTCTCCTTTTACTTCACTTC AAAAGCTTTTTACTCAAAGAGTATATGTTCCCTCCAGGTCAGCTGCCCCCAAACCCCCTC CTTACGCTTTGTCACACAAAAAGTGTCTCTGCCTTGAGTCATCTATTCAAGCACTTACAG CTCTGGCCACAACAGGGCATTTTACAGGTGCGAATGACAGTAGCATTATGAGTAGTGTGG AATTCAGGTAGTAAATATGAAACTAGGGTTTGAAATTGATAATGCTTTCACAACATTTGC AGATGTTTTAGAAGGAAAAAAGTTCCTTCCTAAAATAATTTCTCTACAATTGGAAGATTG GAAGATTCAGCTAGTTAGGAGCCCACCTTTTTTCCTAATCTGTGTGTGCCCTGTAACCTG ACTGGTTAACAGCAGTCCTTTGTAAACAGTGTTTTAAACTCTCCTAGTCAATATCCACCC CATCCAATTTATCAAGGAAGAAATGGTTCAGAAAATATTTTCAGCCTACAGTTATGTTCA GTCACACACACATACAAAATGTTCCTTTTGCTTTTAAAGTAATTTTTGACTCCCAGATCA GTCAGAGCCCCTACAGCATTGTTAAGAAAGTATTTGATTTTTGTCTCAATGAAAATAAAA CTATATTCATTTCCACTCTAAAAAAAAAAAAAAAΆA

SEQ ID NO.:3 (Locus: NP 958439; epidermal growth factor receptor isoform b precursor [Homo sapiens])

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEV VLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALA VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDF QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGC TGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFK NCTSI SGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF ENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGS

SEQ ID NO.:4 (Locus: NM_201282; epidermal growth factor receptor isoform b precursor [Homo sapiens] mRNA transcript sequence)

CCCCGGCGCAGCGCGGCCGCAGCAGCCTCCGCCCCCCGCACGGTGTGAGCGCCCGAC GCG

GCCGAGGCGGCCGGAGTCCCGAGCTAGCCCCGGCGGCCGCCGCCGCCCAGACCGGAC GAC AGGCCACCTCGTCGGCGTCCGCCCGAGTCCCCGCCTCGCCGCCAACGCCACAACCACCGC GCACGGCCCCCTGACTCCGTCCAGTATTGATCGGGAGAGCCGGAGCGAGCTCTTCGGGGA GCAGCGATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTC TGCCCGGCGAGTCGGGCTCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTC ACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACTGT GAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTC TTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGA ATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCC TTAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGA AATTTACAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAAC GTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATG GACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGC TGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAG TGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCA GGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCC ACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGAT GTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAAT TATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATG GAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAAC GGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACAC TTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGT GACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTA AAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCAT GCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT GCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGAT GGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAA AAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGC TGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCG GAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAAC TGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCAC CTGTGCCATCCAAACTGCACCTACGGGTCCTAATAAATCTTCACTGTCTGACTTTAGTCT CCTGACACTATTCATTTGA

SEQ ID NO.:5 (Locus: NP 958440; epidermal growth factor receptor isoform c precursor [Homo sapiens])

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEV VLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALA VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDF QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGC TGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFK NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGLS

SEQ ID NO.: 6 (Locus: NM 201283; epidermal growth factor receptor isoform c precursor [Homo sapiens] mRNA transcript sequence)

CCCCGGCGCAGCGCGGCCGCAGCAGCCTCCGCCCCCCGCACGGTGTGAGCGCCCGAC GCG GCCGAGGCGGCCGGAGTCCCGAGCTAGCCCCGGCGGCCGCCGCCGCCCAGACCGGACGAC AGGCCACCTCGTCGGCGTCCGCCCGAGTCCCCGCCTCGCCGCCAACGCCACAACCACCGC GCACGGCCCCCTGACTCCGTCCAGTATTGATCGGGAGAGCCGGAGCGAGCTCTTCGGGGA GCAGCGATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTC TGCCCGGCGAGTCGGGCTCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTC ACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACTGT GAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTC TTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGA ATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCC TTAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGA AATTTACAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAAC GTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATG GACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGC TGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAG TGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCA GGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCC ACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGAT GTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAAT TATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATG GAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAAC GGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACAC

TTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGG GGT GACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTA AAGGAAATCACAGGTTTGAGCTGAATTATCACATGAATATAAATGGGAAATCAGTGTTTT AGAGAGAGAACTTTTCGACATATTTCCTGTTCCCTTGGAATAAAAACATTTCTTCTGAAA TTTTACCGTTAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.: 7 (Locus: NP_958441; epidermal growth factor receptor isoform d precursor [Homo sapiens])

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEV VLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALA VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDF QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGC TGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFK NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF ENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGPGNESLKAMLFCLFKLSSCNQSNDGSVSHQSGS PAAQESCLGWIPSLLPSEFQLGWGGCSHLHAWPSASVIITASSCH

SEQ ID NO.: 8 (Locus: NM_201284; epidermal growth factor receptor isoform d precursor [Homo sapiens] mRNA transcript sequence)

CCCCGGCGCAGCGCGGCCGCAGCAGCCTCCGCCCCCCGCACGGTGTGAGCGCCCGAC GCG GCCGAGGCGGCCGGAGTCCCGAGCTAGCCCCGGCGGCCGCCGCCGCCCAGACCGGACGAC AGGCCACCTCGTCGGCGTCCGCCCGAGTCCCCGCCTCGCCGCCAACGCCACAACCACCGC GCACGGCCCCCTGACTCCGTCCAGTATTGATCGGGAGAGCCGGAGCGAGCTCTTCGGGGA GCAGCGATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTC TGCCCGGCGAGTCGGGCTCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTC ACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACTGT GAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTC TTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGA ATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCC TTAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGA AATTTACAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAAC

GTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCG ATG GACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGC TGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAG TGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCA GGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCC ACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGAT GTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAAT TATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATG GAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAAC GGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACAC TTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGT GACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTA AAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCAT GCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT GCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGAT GGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAA AAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGC TGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCG GAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAAC TGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCAC CTGTGCCATCCAAACTGCACCTACGGGCCAGGAAATGAGAGTCTCAAAGCCATGTTATTC TGCCTTTTTAAACTATCATCCTGTAATCAAAGTAATGATGGCAGCGTGTCCCACCAGAGC GGGAGCCCAGCTGCTCAGGAGTCATGCTTAGGATGGATCCCTTCTCTTCTGCCGTCAGAG TTTCAGCTGGGTTGGGGTGGATGCAGCCACCTCCATGCCTGGCCTTCTGCATCTGTGATC ATCACGGCCTCCTCCTGCCACTGAGCCTCATGCCTTCACGTGTCTGTTCCCCCCGCTTTT CCTTTCTGCCACCCCTGCACGTGGGCCGCCAGGTTCCCAAGAGTATCCTACCCATTTCCT TCCTTCCACTCCCTTTGCCAGTGCCTCTCACCCCAACTAGTAGCTAACCATCACCCCCAG GACTGACCTCTTCCTCCTCGCTGCCAGATGATTGTTCAAAGCACAGAATTTGTCAGAAAC CTGCAGGGACTCCATGCTGCCAGCCTTCTCCGTAATTAGCATGGCCCCAGTCCATGCTTC TAGCCTTGGTTCCTTCTGCCCCTCTGTTTGAAATTCTAGAGCCAGCTGTGGGACAATTAT CTGTGTCAAAAGCCAGATGTGAAAACATCTCAATAACAAACTGGCTGCTTTGTTCAATGC TAGAACAACGCCTGTCACAGAGTAGAAACTCAAAAATATTTGCTGAGTGAATGAACAAAT GAATAAATGCATAATAAATAATTAACCACCAATCCAACATCCAGA

SEQ ID NO.: 9 (Locus: NP_001036064; v-erb-a erythroblastic leukemia viral oncogene homolog 4 isoform JM-a/CVT-2 precursor [Homo sapiens])

MKPATGLWVWVSLLVAAGTVQPSDSQSVCAGTENKLSSLSDLEQQYRALRKYYENCE VVM GNLEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYLPLENLRIIRGTKLYEDRYALAIF LNYRKDGNFGLQELGLKNLTEILNGGVYVDQNKFLCYADTIHWQDIVRNPWPSNLTLVST NGSSGCGRCHKSCTGRCWGPTENHCQTLTRTVCAEQCDGRCYGPYVSDCCHRECAGGCSG PKDTDCFACMNFNDSGACVTQCPQTFVYNPTTFQLEHNFNAKYTYGAFCVKKCPHNFVVD SSSCVRACPSSKMEVEENGIKMCKPCTDICPKACDGIGTGSLMSAQTVDSSNIDKFINCT KINGNLIFLVTGIHGDPYNAIEAIDPEKLNVFRTVREITGFLNIQSWPPNMTDFSVFSNL VTIGGRVLYSGLSLLILKQQGITSLQFQSLKEISAGNIYITDNSNLCYYHTINWTTLFST INQRIVIRDNRKAENCTAEGMVCNHLCSSDGCWGPGPDQCLSCRRFSRGRICIESCNLYD GEFREFENGSICVECDPQCEKMEDGLLTCHGPGPDNCTKCSHFKDGPNCVEKCPDGLQGA NSFIFKYADPDRECHPCHPNCTQGCNGPTSHDCIYYPWTGHSTLPQHARTPLIAAGVIGG LFILVIVGLTFAVYVRRKSIKKKRALRRFLETELVEPLTPSGTAPNQAQLRILKETELKR VKVLGSGAFGTVYKGIWVPEGETVKIPVAIKILNETTGPKANVEFMDEALIMASMDHPHL VRLLGVCLSPTIQLVTQLMPHGCLLEYVHEHKDNIGSQLLLNWCVQIAKGMMYLEERRLV

HRDLAARNVLVKSPNHVKITDFGLARLLEGDEKEYNADGGKMPIKWMALECIHYRKF THQ SDVWSYGVTIWELMTFGGKPYDGIPTREI PDLLEKGERLPQPPICTIDVYMVMVKCWMID ADSRPKFKELAAEFSRMARDPQRYLVIQGDDRMKLPSPNDSKFFQNLLDEEDLEDMMDAE EYLVPQAFNIPPPIYTSRARIDSNRNQFVYRDGGFAAEQGVSVPYRAPTSTIPEAPVAQG ATAEI FDDSCCNGTLRKPVAPHVQEDSSTQRYSADPTVFAPERSPRGELDEEGYMTPMRD KPKQEYLNPVEENPFVSRRKNGDLQALDNPEYHNASNGPPKAEDEYVNEPLYLNTFANTL GKAEYLKNNILSMPEKAKKAFDNPDYWNHSLPPRSTLQHPDYLQEYSTKYFYKQNGRIRP IVAENPEYLSEFSLKPGTVLPPPPYRHRNTVV

SEQ ID NO.: 10 (Locus: NM_001042599; Homo sapiens v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) (ERBB4), transcript variant JM-a/CVT-2, mRNA transcript sequence)

CACGCGCGCCCGGCTGGGGGATCTCCTCCGCGTGCCCGAAAGGGGGATATGCCATTT GGA CATGTAATTGTCAGCACGGGATCTGAGACTTCCAAAAAATGAAGCCGGCGACAGGACTTT GGGTCTGGGTGAGCCTTCTCGTGGCGGCGGGGACCGTCCAGCCCAGCGATTCTCAGTCAG TGTGTGCAGGAACGGAGAATAAACTGAGCTCTCTCTCTGACCTGGAACAGCAGTACCGAG CCTTGCGCAAGTACTATGAAAACTGTGAGGTTGTCATGGGCAACCTGGAGATAACCAGCA TTGAGCACAACCGGGACCTCTCCTTCCTGCGGTCTGTTCGAGAAGTCACAGGCTACGTGT

TAGTGGCTCTTAATCAGTTTCGTTACCTGCCTCTGGAGAATTTACGCATTATTCGTG GGA CAAAACTTTATGAGGATCGATATGCCTTGGCAATATTTTTAAACTACAGAAAAGATGGAA ACTTTGGACTTCAAGAACTTGGATTAAAGAACTTGACAGAAATCCTAAATGGTGGAGTCT ATGTAGACCAGAACAAATTCCTTTGTTATGCAGACACCATTCATTGGCAAGATATTGTTC GGAACCCATGGCCTTCCAACTTGACTCTTGTGTCAACAAATGGTAGTTCAGGATGTGGAC GTTGCCATAAGTCCTGTACTGGCCGTTGCTGGGGACCCACAGAAAATCATTGCCAGACTT TGACAAGGACGGTGTGTGCAGAACAATGTGACGGCAGATGCTACGGACCTTACGTCAGTG ACTGCTGCCATCGAGAATGTGCTGGAGGCTGCTCAGGACCTAAGGACACAGACTGCTTTG CCTGCATGAATTTCAATGACAGTGGAGCATGTGTTACTCAGTGTCCCCAAACCTTTGTCT ACAATCCAACCACCTTTCAACTGGAGCACAATTTCAATGCAAAGTACACATATGGAGCAT TCTGTGTCAAGAAATGTCCACATAACTTTGTGGTAGATTCCAGTTCTTGTGTGCGTGCCT GCCCTAGTTCCAAGATGGAAGTAGAAGAAAATGGGATTAAAATGTGTAAACCTTGCACTG ACATTTGCCCAAAAGCTTGTGATGGCATTGGCACAGGATCATTGATGTCAGCTCAGACTG TGGATTCCAGTAACATTGACAAATTCATAAACTGTACCAAGATCAATGGGAATTTGATCT TTCTAGTCACTGGTATTCATGGGGACCCTTACAATGCAATTGAAGCCATAGACCCAGAGA AACTGAACGTCTTTCGGACAGTCAGAGAGATAACAGGTTTCCTGAACATACAGTCATGGC CACCAAACATGACTGACTTCAGTGTTTTTTCTAACCTGGTGACCATTGGTGGAAGAGTAC TCTATAGTGGCCTGTCCTTGCTTATCCTCAAGCAACAGGGCATCACCTCTCTACAGTTCC AGTCCCTGAAGGAAATCAGCGCAGGAAACATCTATATTACTGACAACAGCAACCTGTGTT ATTATCATACCATTAACTGGACAACACTCTTCAGCACAATCAACCAGAGAATAGTAATCC GGGACAACAGAAAAGCTGAAAATTGTACTGCTGAAGGAATGGTGTGCAACCATCTGTGTT CCAGTGATGGCTGTTGGGGACCTGGGCCAGACCAATGTCTGTCGTGTCGCCGCTTCAGTA GAGGAAGGATCTGCATAGAGTCTTGTAACCTCTATGATGGTGAATTTCGGGAGTTTGAGA ATGGCTCCATCTGTGTGGAGTGTGACCCCCAGTGTGAGAAGATGGAAGATGGCCTCCTCA CATGCCATGGACCGGGTCCTGACAACTGTACAAAGTGCTCTCATTTTAAAGATGGCCCAA ACTGTGTGGAAAAATGTCCAGATGGCTTACAGGGGGCAAACAGTTTCATTTTCAAGTATG CTGATCCAGATCGGGAGTGCCACCCATGCCATCCAAACTGCACCCAAGGGTGTAACGGTC CCACTAGTCATGACTGCATTTACTACCCATGGACGGGCCATTCCACTTTACCACAACATG CTAGAACTCCCCTGATTGCAGCTGGAGTAATTGGTGGGCTCTTCATTCTGGTCATTGTGG GTCTGACATTTGCTGTTTATGTTAGAAGGAAGAGCATCAAAAAGAAAAGAGCCTTGAGAA GATTCTTGGAAACAGAGTTGGTGGAACCATTAACTCCCAGTGGCACAGCACCCAATCAAG CTCAACTTCGTATTTTGAAAGAAACTGAGCTGAAGAGGGTAAAAGTCCTTGGCTCAGGTG CTTTTGGAACGGTTTATAAAGGTATTTGGGTACCTGAAGGAGAAACTGTGAAGATTCCTG TGGCTATTAAGATTCTTAATGAGACAACTGGTCCCAAGGCAAATGTGGAGTTCATGGATG AAGCTCTGATCATGGCAAGTATGGATCATCCACACCTAGTCCGGTTGCTGGGTGTGTGTC TGAGCCCAACCATCCAGCTGGTTACTCAACTTATGCCCCATGGCTGCCTGTTGGAGTATG

TCCACGAGCACAAGGATAACATTGGATCACAACTGCTGCTTAACTGGTGTGTCCAGA TAG CTAAGGGAATGATGTACCTGGAAGAAAGACGACTCGTTCATCGGGATTTGGCAGCCCGTA ATGTCTTAGTGAAATCTCCAAACCATGTGAAAATCACAGATTTTGGGCTAGCCAGACTCT TGGAAGGAGATGAAAAAGAGTACAATGCTGATGGAGGAAAGATGCCAATTAAATGGATGG CTCTGGAGTGTATACATTACAGGAAATTCACCCATCAGAGTGACGTTTGGAGCTATGGAG TTACTATATGGGAACTGATGACCTTTGGAGGAAAACCCTATGATGGAATTCCAACGCGAG AAATCCCTGATTTATTAGAGAAAGGAGAACGTTTGCCTCAGCCTCCCATCTGCACTATTG ACGTTTACATGGTCATGGTCAAATGTTGGATGATTGATGCTGACAGTAGACCTAAATTTA AGGAACTGGCTGCTGAGTTTTCAAGGATGGCTCGAGACCCTCAAAGATACCTAGTTATTC AGGGTGATGATCGTATGAAGCTTCCCAGTCCAAATGACAGCAAGTTCTTTCAGAATCTCT TGGATGAAGAGGATTTGGAAGATATGATGGATGCTGAGGAGTACTTGGTCCCTCAGGCTT TCAACATCCCACCTCCCATCTATACTTCCAGAGCAAGAATTGACTCGAATAGGAACCAGT TTGTATACCGAGATGGAGGTTTTGCTGCTGAACAAGGAGTGTCTGTGCCCTACAGAGCCC CAACTAGCACAATTCCAGAAGCTCCTGTGGCACAGGGTGCTACTGCTGAGATTTTTGATG ACTCCTGCTGTAATGGCACCCTACGCAAGCCAGTGGCACCCCATGTCCAAGAGGACAGTA GCACCCAGAGGTACAGTGCTGACCCCACCGTGTTTGCCCCAGAACGGAGCCCACGAGGAG AGCTGGATGAGGAAGGTTACATGACTCCTATGCGAGACAAACCCAAACAAGAATACCTGA ATCCAGTGGAGGAGAACCCTTTTGTTTCTCGGAGAAAAAATGGAGACCTTCAAGCATTGG ATAATCCCGAATATCACAATGCATCCAATGGTCCACCCAAGGCCGAGGATGAGTATGTGA ATGAGCCACTGTACCTCAACACCTTTGCCAACACCTTGGGAAAAGCTGAGTACCTGAAGA ACAACATACTGTCAATGCCAGAGAAGGCCAAGAAAGCGTTTGACAACCCTGACTACTGGA ACCACAGCCTGCCACCTCGGAGCACCCTTCAGCACCCAGACTACCTGCAGGAGTACAGCA CAAAATATTTTTATAAACAGAATGGGCGGATCCGGCCTATTGTGGCAGAGAATCCTGAAT ACCTCTCTGAGTTCTCCCTGAAGCCAGGCACTGTGCTGCCGCCTCCACCTTACAGACACC GGAATACTGTGGTGTAAGCTCAGTTGTGGTTTTTTAGGTGGAGAGACACACCTGCTCCAA TTTCCCCACCCCCCTCTCTTTCTCTGGTGGTCTTCCTTCTACCCCAAGGCCAGTAGTTTT GACACTTCCCAGTGGAAGATACAGAGATGCAATGATAGTTATGTGCTTACCTAACTTGAA CATTAGAGGGAAAGACTGAAAGAGAAAGATAGGAGGAACCACAATGTTTCTTCATTTCTC TGCATGGGTTGGTCAGGAGAATGAAACAGCTAGAGAAGGACCAGAAAATGTAAGGCAATG

CTGCCTACTATCAAACTAGCTGTCACTTTTTTTCTTTTTCTTTTTCTTTCTTTGTTT CTT TCTTCCTCTTCTTTTTTTTTTTTTTTTTTAAAGCAGATGGTTGAAACACCCATGCTATCT GTTCCTATCTGCAGGAACTGATGTGTGCATATTTAGCATCCCTGGAAATCATAATAAAGT TTCCATTAGAACAAAAGAATAACATTTTCTATAACATATGATGGTGTCTGAAATTGAGAA TCCAGTTTCTTTCCCCAGCAGTTTCTGTCCTAGCAAGTAAGAATGGCCAACTCAACTTTC ATAATTTAAAAATCTCCATTAAAGTTATAACTAGTAATTATGTTTTCAACACTTTTTGGT TTTTTTCATTTTGTTTTGCTCTGACCGATTCCTTTATATTTGCTCCCCTATTTTTGGCTT

TAATTTCTAATTGCAAAGATGTTTACATCAAAGCTTCTTCACAGAATTTAAGCAAGA AAT ATTTTAATATAGTGAAATGGCCACTACTTTAAGTATACAATCTTTAAAATAAGAAAGGGA GGCTAATATTTTTCATGCTATCAAATTATCTTCACCCTCATCCTTTACATTTTTCAACAT TTTTTTTTCTCCATAAATGACACTACTTGATAGGCCGTTGGTTGTCTGAAGAGTAGAAGG GAAACTAAGAGACAGTTCTCTGTGGTTCAGGAAAACTACTGATACTTTCAGGGGTGGCCC AATGAGGGAATCCATTGAACTGGAAGAAACACACTGGATTGGGTATGTCTACCTGGCAGA TACTCAGAAATGTAGTTTGCACTTAAGCTGTAATTTTATTTGTTCTTTTTCTGAACTCCA TTTTGGATTTTGAATCAAGCAATATGGAAGCAACCAGCAAATTAACTAATTTAAGTACAT TTTTAAAAAAAGAGCTAAGATAAAGACTGTGGAAATGCCAAACCAAGCAAATTAGGAACC TTGCAACGGTATCCAGGGACTATGATGAGAGGCCAGCACATTATCTTCATATGTCACCTT TGCTACGCAAGGAAATTTGTTCAGTTCGTATACTTCGTAAGAAGGAATGCGAGTAAGGAT TGGCTTGAATTCCATGGAATTTCTAGTATGAGACTATTTATATGAAGTAGAAGGTAACTC TTTGCACATAAATTGGTATAATAAAAAGAAAAACACAAACATTCAAAGCTTAGGGATAGG TCCTTGGGTCAAAAGTTGTAAATAAATGTGAAACATCTTCTCATGCAATTATTTTATTAT CCAACACACTAATCTTTTGATACTTTATATAATTCCCTTTCTTCATATACTGCATCCAGT ACTAGAACCATCATTATTATGTATCATTTTGAAAGAATACCTGATGAGATGAAGGATGAG AACAAATGACAGAGATGAGTCTCCAAGTAAAGGGGGCCTCACATCAATAATTAGGAAACT TAGATATAAGTCGCCCTTTTCTGAAAATTCTACCCCAAGTCATTTAGATTTTTAAAAAAT ATTTCTAATGTTAAAATATTGGGACCAAATTAGAATCAATAGTATAAGATTAATTAATTA GAGTAAAAATATCTATTAAGGCAGAGAAAGTTTAGAGAAAAAAATCCAAAGAAATTTGTG TTTCTTCCTATTCTGAACAAGTAAATCCATCCATCCATCCATCCAAACCTCCTTTATCTA ACTGTGTCTACTAAAAGCACCATGTTTTGTGGGGAACACTCAGATAAATGGAATATCATC CTCAACTTCAAAATTCTATGATCTAGGAGATTTAATTAAAATGACATTTTAATTTTTCTA TGCGTTCCAACAATCAGATTGCATAGTCTCTTTTGTGAATAGCTGTCATATAATCAGTTG TACTGTAAGATATCTCCTTTAAACTCATTTGGGATATAAGTTAAACATCCTTCAAATTGT TGATGTTGACAAACAGGATAATTTCAATAATATTATTCAAACATAAACTGGTCTAGGAGA ATATTGCATCACTGACTAATTAGCCTATCTAGAGTCTAACTTCACCATTAAACCAAAAGC AGATGGTGGTCCTTGGCCAAGAATATTGGAGACATTGGAGTTGGTTTTTTTCTAAGCTAT AAGAAGTGAGGCGAGCTGAAAAAGTATGGTAGAGCAGGAGAAGGGTTTGTGAGATTCCTT CTAGTGAAGTTCACCCTCAAACTTTTCAGGGGTAAAGACACAGAGTGATTCAGGGGCCAC AATCTAATAGCTCAGGGCTCTCCTATCCATTCAGAGAAGTCTCTAGGAAAAGGGATCTCA TATCAGTACTTATGAAAAATTGAATATAAGCCTCCCTTTCTAAATAAATCTGCATCGAGT

CATCACAGCCCTCTTTTTGGATACTATACCTTGATTTTTTTTTTCTGATTTACAATA TGC ATATGGTTTCTACTGGGCTATAGAAAGCAGAATCACTCATTTTGGAGAAGGAAAAAATGA ATAGTTAAAACAAACTTTTAACTGTTAAGGTAACAGAAATGTATTTAGTGAATGTCTCTT TCCTCCTAAGAACACAAGACTTCTACATGTTGGGTAATACCTAGAGATGCATGTAGGAAT

AATCCAAAATGACCCAAATGCTTTATAATAGCACCACTTTATAATTCTTTTGAATGA TTT CTGTAGTATATAATTGACTTCAGTTGTTTGAGTGTTTTTTGTTTTATTTTTGTCCCCCCT GGGAAAACATATTTCAGCATGTATAAGAGGGAGAAAAAAAGTTTCATTCCTTCCAGAGAA TAACTTATTTAGTCCAGTAGGGTAGAATTTTAAAATGTCAGTTAAAGTCTTCAAAGTGCT TGGGGGGATATCAGATTCCAGAGGCCAATTGTAGCAATTGAAATTTGCAGAATCAATTAT GTAAATCTGAGACAAATTAGTATTAAAATTACACGGAGTATATTTTTTAAATCACCCAAC TTTGTAGATTATACCTATTTTGGGCAGGTATGGAAAAATTTTGCAGTTAAATGATTGCCT AAAGAAAGTGGTAAACAGGTGAGGAAAGATGGCCTCTGATCTAGGATAGATCCAGAACCA CAAAGCATCTGCACCACAAAAGGTGTTAGACTACCAAGCAGCTCCTGGTTTTCTGCATAG TATTAGTAGCACAGCTTAGGATGAGAATCCTTTCTCCAGTAACATTCTTAAAATAGCATG AAAAACAACGCAAAACTCAAATTTCTATTAAAACACACAAACTAAAATCAAGTGATTCTT TTTTGTAGATTAGGGAGAAGGACTGAATATCTAATTTAAGAGAAGGAATAGTGTTTAAGT GTTATAGTGTGTGAGCTAATACCTTCTAAAGGAAAGACATGGCATGAAGATTGTGCATAC TTACAATGCTAAGGAAAAATCAAGAAAAGGACTGTGTGAGGCTCTGCTACTAGATGAAGT TGGAAGGACTATTAATGTGCTTCTTGAAGTATCAAAAATGAAAAGAAAATTAAAATTGTT TAAGCCTGACAGGGAAGGATGTAAATACAAGTTTTTCTAGAGCTCTCTAACCTTTATTTC AAAACTGGAATTATTCATCCATCTGTAATTGTTGATAATTTAACTAGTATATGTAGTTCA TAAGGTAATAGAAAAGGTGATCATGAAAGCATGTATATAACTGGACAGAACCACGATAAT GCTATAAGATGTAGATTTAGTTAGGTTATCAGATGTTAAATGATTTTAATATTATTAAAT AAATCAAACTAGAAAACTAACCACAAGTATAATGTAACAAAGTTAAATGCAGGATATAAA AATGTAGGATGGATTTTGCATAGTAAAAAGATAAGTTTGCCATTTAAAATTGTTGTTTGT TGGGTTTAGCTGAAAGTAGGCATATATGGTTCCACTTGGGAAAACTTGCTTTAAAGCATT ACAATGAACAATTTTTTCTCATTCTCTTATTCCTTTATCACTTTTTAAATGTAAAGAAAA TTGTATTTATTTATTTTTTTAAATAAACACCACCTTGCAGAATTTAATAGGCAAACATGT TACATATGACTAAGTAAGGGTCTTCAAGATGAAGTAAAGAAAATGTAAATGTTCTATTAC CTTATGCAGAGACAAAAAAAAAAAGGAGTGGTGTCATTTAGCTAGCAAACAAACAAAATA CAGTTAATTGGTGATATGTCCTTTCTTTTCTCACTATGCCCTCTTGCCTCCAAAAATGAC AACAAAGAATCACAATTTTTCTGATAAATAAATGCTAAACCAAGCGTTTCAAACTATTGC ATTGCCATTCTTTTGGACTTTAGTTATTAGAATGATGATTGTTATAGGGCAAATGAGAAA TCCATGTGCATCAGCTTCTAGTTGTTAAAAAAACCAGATAAATTAACTTCTACTGTATAC TGTGGGCAGAGGATCCTAGAGCTGATCCTACAACATCAGCTTCTAGTTGTTAAAAAAAAA AAAAGAAACAGATAAATTAACTTCTACTGTATATACTGTGGGCAGAGGATCTTACTGTGC CTCTGTTTGTGTACATGGACTTCGGTGTGTATCAGTTTGAAGGACAGCCTTGCCCCATGT AAACATATAAATGCAGATTGGTATCGCCTGGTTGCTATTTGCTTAAGAACAAATATTATA CAGATGAGATCAGGCATAATTTTAAAAGATCATTATCAGTGGAGACCTCATTATTACTGA TATTACAATGGGGCCAGTTTTTATACTTCTGGGTAGAATTAATAAAATTTTTCTGATCCC

AGAGATCTGAGTTCTCTCTGCAGTTGGAAACAAGAAGCTGTTGTGGGCATTGTGTCG GGC CAGGGGCCCTTGTGTTTGTGTGGGCAAATATCTTTTAGCAGTGTGAGCTGCTTTTTTCTT TTCATTAAAAGTCTCTCTAAAATAATAGAAATTTCAGATACTCGGTTCAAGTCTCACTGA TTTTGTAGAGGTCCAAAAATGTAGGATCTGTCACTTTTGCAGGCCCCTGCCTCACCTAAT TCCTGGCCAGGTGACATTTTGGGCAGAAGTAAATGCTTCTATAGTCACAAGCTAAAATGA CTCTAAGCCCCAATTTCACGGGGGGTATTCACATGCTTCCTCTGGAAAATACTCTTTGAC AGTCAGCTTTGCAAGTAAGTGATTACCTTGTTAGGAATCAAAGAAAAATGTATTTCTCTC TGACCTTTAGAGGAAAATAGAATCCTTCCCTTTTTTGCCCATTGACACAACTGGCACTGC TCTCTTCCCTTTCTACCACCCTGGTTCAAAGTAGTCCCCCGATGCTGTCCTGTTCCTTTC TTAAGCCATAGTGGATCTCTGAGATCCTACACCCCACTTTGTGAAACACTGACTTCATCT TTGCCCTCGAATGCCTGATTTTTTCATAAGAGATTCTAGCAATTTGGACACTGTTTAAGT GAACTATCAAACTACCGCATAGAGAATATTTAAGCTATTAAAATTATGGTTTCCCATGAA GATCAATTCTCTGTGTCCTTCCCTATAGGAATTTGAGACGAGTTAGCCCTGTGATGAATC TTGAAACTCACATATGTCCACATACACTTGGTAGAACTTCGATTTAATCTTTACATAAAA GCTGTACATATAACCAAGAAGTTATTTTTGCCAGTAAATTAACTTATTTGCTTTATTCAT CTTATTTGGTTCCTAATCGTAAATATTTTGTAGCTGCTGTAAATTTTTTTCTCCCAAATG AGGAGTCTTATTATCATAAAGGTAAAGGCTATTCAGCTTTGATAACCACCTGCAATTCTT TTTTGGATCATTCATCCATCTAACAAATACATAATGAGGACAGTTCATGTTAATGAAAAT CCATGTTGTTTAATAGAATGCCATCCTTTACCTACTTTTGCTCTTTATGGACGTTTTTCT TTTCATGCTCTAGTGAGCTTTCCCTATATCATGAGAAGTGGTTATATTTGTGCAAATATA CAAATATAGGAAAACAAAGATTCATACCTGTAGGCAATAGTCTAACTTGTCCAAACCACT TTGCCTTTACTGCTATTTTTATCCCCAATGCGTAGATATTTCCCCCAGGCCTATAGCCTT TGTGAAGGAAAGCAAATCATACCTCCTGTATATTGACACGAATCTGGTTTTCAAATGTCA TTTCCAGATTTTTTAGTTAATTGGGGGTTGTCCTTTTCCCTTAATGTGAGAGTCATTTTC CTGTATATTTCTGGATCTCTCAGGGGCTGGGAGGGGGGAGTGAGGGGACTACAACCATAG CACTCCAAGAACCCTTTTGGGATTACTCCAGTAATCAACTACGAAAGTTATTTTCTAAAT GTAGATATGTAAGGTGTTCTTTTAAAGTAAGGTACTTTGAAATATGTAGCATAAACTGGT ACTGCTGTTAAATGGGTCGATTATTAAACGGAGCAGCTGTGTGAGGGCAGCTAACTTTGA ATGCCTGTCTCCCTGGCTGGTGTGTCTCCTTCTCATGTTGAGAGCACCAGGGATTGCGTG GCTGCATGCTGAAACCGCATTTTCCCATGGTGTATGACTAGTTCATCTCTTTCTTGAGCA CCATTACAAGAAGATCAAATGAAAATGAGATCAATGTGGAAGACAATTCATAGCACAAAA AAAGTCATCTTAAATCTACTCTCAAACATTCATCTTATACATGCATCAAAGTAATTTACT GACATCAGTTTGGGTGAGAGAGGGAGTCACTTTACTGAAAAGGCAGAGGCTTAAGGTGTA TACATTTGTACTCACTTCCTTATTTTCTTAACTTGTAAGCAGAAAACAAGCCCTCTCTCT TGTGAAGTATCTTCAAAGGATTGGGGTGCAAAAATACCTTGCTGGTAAGCCATCAATGTT TTATTTAAATCCCTGCATTCAAAGTTAGCTGCCTTTTTGAAATAAACAAACAAAAAATAC

TACTGTATGTTTGAAAATGTGAATAGTATTTTTATAGCTTGTTAAAGACATGGCTAG TTG CATTTGTAAATAAGTATAATGTTGCTTTGATTTTCTTTTGTGGACATCTTTATTTGGAAC ATAATTGTCTTTAGGGTTGATTTGTATATAAGTAATTGGCCTGTGATTGTTTCTTTTTTG GTTGGAAGTTATCATTTTGACATTACTTGTGATTCTGTGTTCAGCACTATTGTGATGTGT TCAACCTCTGCACTCGCTTACACAATAGGATATGCCAATTGTGTGTGGTGTAATGTTATT TTGATTTTTTTCCATGTTATTGATGAAGGATCATGCACCTAACACATACTAACTTTTTTA ATGTTAGGCATATTTTTAGTATACTTTCTCTTATTCTTTCTTCTCCTCCAACCTTTTACC CATCCTCCTTCCTTTCCCTCATTCCTGTTGTTATTTGAGAATGAGGGAGAAACAGTATTT TACATTTATGTAATTAGGCTTTTCCGTTAGTTCTCAAGGATCCTCTTTTGGCTCTTGGGA AAGAATTGTACCTGTACAAGGCAATTATAGAATGCGAACTGCTTTGCCTCATTCCATACT GATCATCCCAGCTGAACAATTTGAAAACTGTTCTGCCTTTTTGTTACATGAATCTGTCAG AAATATATTTTTAATTTAATATAAATGAAATTCAATAAAATATGAAACAAACGTTAAAAA AAAAAAAAAAAAA

SEQ ID NO.: 11 (Locus: NP_005226; v-erb-a erythroblastic leukemia viral oncogene homolog 4 isoform JM-a/CVT-1 precursor [Homo sapiens]

MKPATGLWVWVSLLVAAGTVQPSDSQSVCAGTENKLSSLSDLEQQYRALRKYYENCEWM GNLEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYLPLENLRIIRGTKLYEDRYALAIF LNYRKDGNFGLQELGLKNLTEILNGGVYVDQNKFLCYADTIHWQDIVRNPWPSNLTLVST NGSSGCGRCHKSCTGRCWGPTENHCQTLTRTVCAEQCDGRCYGPYVSDCCHRECAGGCSG PKDTDCFACMNFNDSGACVTQCPQTFVYNPTTFQLEHNFNAKYTYGAFCVKKCPHNFVVD SSSCVRACPSSKMEVEENGIKMCKPCTDICPKACDGIGTGSLMSAQTVDSSNIDKFINCT KINGNLIFLVTGIHGDPYNAIEAIDPEKLNVFRTVREITGFLNIQSWPPNMTDFSVFSNL VTIGGRVLYSGLSLLILKQQGITSLQFQSLKEISAGNIYITDNSNLCYYHTINWTTLFST INQRIVIRDNRKAENCTAEGMVCNHLCSSDGCWGPGPDQCLSCRRFSRGRICIESCNLYD GEFREFENGSICVECDPQ ^' CEKMEDGLLTCHGPGPDNCTKCSHFKDGPNCVEKCPDGLQGA NSFIFKYADPDRECHPCHPNCTQGCNGPTSHDCIYYPWTGHSTLPQHARTPLIAAGVIGG LFILVIVGLTFAVYVRRKSIKKKRALRRFLETELVEPLTPSGTAPNQAQLRILKETELKR VKVLGSGAFGTVYKGIWVPEGETVKIPVAIKILNETTGPKANVEFMDEALIMASMDHPHL VRLLGVCLSPTIQLVTQLMPHGCLLEYVHEHKDNIGSQLLLNWCVQIAKGMMYLEERRLV HRDLAARNVLVKSPNHVKITDFGLARLLEGDEKEYNADGGKMPIKWMALECIHYRKFTHQ SDVWSYGVTIWELMTFGGKPYDGI PTREIPDLLEKGERLPQPPICTIDVYMVMVKCWMID ADSRPKFKELAAEFSRMARDPQRYLVIQGDDRMKLPSPNDSKFFQNLLDEEDLEDMMDAE EYLVPQAFNIPPPIYTSRARIDSNRSEIGHSPPPAYTPMSGNQFVYRDGGFAAEQGVSVP YRAPTSTIPEAPVAQGATAEIFDDSCCNGTLRKPVAPHVQEDSSTQRYSADPTVFAPERS

PRGELDEEGYMTPMRDKPKQEYLNPVEENPFVSRRKNGDLQALDNPEYHNASNGPPK AED EYVNEPLYLNTFANTLGKAEYLKNNILSMPEKAKKAFDNPDYWNHSLPPRSTLQHPDYLQ EYSTKYFYKQNGRIRPIVAENPEYLSEFSLKPGTVLPPPPYRHRNTVV

SEQ ID NO.: 12 (Locus: NM_005235; v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) (ERBB4), transcript variant JM-a/CVT-1, mRNA [Homo sapiens]

CACGCGCGCCCGGCTGGGGGATCTCCTCCGCGTGCCCGAAAGGGGGATATGCCATTT GGA CATGTAATTGTCAGCACGGGATCTGAGACTTCCAAAAAATGAAGCCGGCGACAGGACTTT GGGTCTGGGTGAGCCTTCTCGTGGCGGCGGGGACCGTCCAGCCCAGCGATTCTCAGTCAG TGTGTGCAGGAACGGAGAATAAACTGAGCTCTCTCTCTGACCTGGAACAGCAGTACCGAG CCTTGCGCAAGTACTATGAAAACTGTGAGGTTGTCATGGGCAACCTGGAGATAACCAGCA TTGAGCACAACCGGGACCTCTCCTTCCTGCGGTCTGTTCGAGAAGTCACAGGCTACGTGT TAGTGGCTCTTAATCAGTTTCGTTACCTGCCTCTGGAGAATTTACGCATTATTCGTGGGA CAAAACTTTATGAGGATCGATATGCCTTGGCAATATTTTTAAACTACAGAAAAGATGGAA ACTTTGGACTTCAAGAACTTGGATTAAAGAACTTGACAGAAATCCTAAATGGTGGAGTCT ATGTAGACCAGAACAAATTCCTTTGTTATGCAGACACCATTCATTGGCAAGATATTGTTC GGAACCCATGGCCTTCCAACTTGACTCTTGTGTCAACAAATGGTAGTTCAGGATGTGGAC GTTGCCATAAGTCCTGTACTGGCCGTTGCTGGGGACCCACAGAAAATCATTGCCAGACTT TGACAAGGACGGTGTGTGCAGAACAATGTGACGGCAGATGCTACGGACCTTACGTCAGTG ACTGCTGCCATCGAGAATGTGCTGGAGGCTGCTCAGGACCTAAGGACACAGACTGCTTTG CCTGCATGAATTTCAATGACAGTGGAGCATGTGTTACTCAGTGTCCCCAAACCTTTGTCT ACAATCCAACCACCTTTCAACTGGAGCACAATTTCAATGCAAAGTACACATATGGAGCAT TCTGTGTCAAGAAATGTCCACATAACTTTGTGGTAGATTCCAGTTCTTGTGTGCGTGCCT GCCCTAGTTCCAAGATGGAAGTAGAAGAAAATGGGATTAAAATGTGTAAACCTTGCACTG ACATTTGCCCAAAAGCTTGTGATGGCATTGGCACAGGATCATTGATGTCAGCTCAGACTG TGGATTCCAGTAACATTGACAAATTCATAAACTGTACCAAGATCAATGGGAATTTGATCT TTCTAGTCACTGGTATTCATGGGGACCCTTACAATGCAATTGAAGCCATAGACCCAGAGA AACTGAACGTCTTTCGGACAGTCAGAGAGATAACAGGTTTCCTGAACATACAGTCATGGC CACCAAACATGACTGACTTCAGTGTTTTTTCTAACCTGGTGACCATTGGTGGAAGAGTAC TCTATAGTGGCCTGTCCTTGCTTATCCTCAAGCAACAGGGCATCACCTCTCTACAGTTCC AGTCCCTGAAGGAAATCAGCGCAGGAAACATCTATATTACTGACAACAGCAACCTGTGTT ATTATCATACCATTAACTGGACAACACTCTTCAGCACAATCAACCAGAGAATAGTAATCC GGGACAACAGAAAAGCTGAAAATTGTACTGCTGAAGGAATGGTGTGCAACCATCTGTGTT CCAGTGATGGCTGTTGGGGACCTGGGCCAGACCAATGTCTGTCGTGTCGCCGCTTCAGTA GAGGAAGGATCTGCATAGAGTCTTGTAACCTCTATGATGGTGAATTTCGGGAGTTTGAGA

ATGGCTCCATCTGTGTGGAGTGTGACCCCCAGTGTGAGAAGATGGAAGATGGCCTCC TCA CATGCCATGGACCGGGTCCTGACAACTGTACAAAGTGCTCTCATTTTAAAGATGGCCCAA ACTGTGTGGAAAAATGTCCAGATGGCTTACAGGGGGCAAACAGTTTCATTTTCAAGTATG CTGATCCAGATCGGGAGTGCCACCCATGCCATCCAAACTGCACCCAAGGGTGTAACGGTC CCACTAGTCATGACTGCATTTACTACCCATGGACGGGCCATTCCACTTTACCACAACATG CTAGAACTCCCCTGATTGCAGCTGGAGTAATTGGTGGGCTCTTCATTCTGGTCATTGTGG GTCTGACATTTGCTGTTTATGTTAGAAGGAAGAGCATCAAAAAGAAAAGAGCCTTGAGAA GATTCTTGGAAACAGAGTTGGTGGAACCATTAACTCCCAGTGGCACAGCACCCAATCAAG CTCAACTTCGTATTTTGAAAGAAACTGAGCTGAAGAGGGTAAAAGTCCTTGGCTCAGGTG CTTTTGGAACGGTTTATAAAGGTATTTGGGTACCTGAAGGAGAAACTGTGAAGATTCCTG TGGCTATTAAGATTCTTAATGAGACAACTGGTCCCAAGGCAAATGTGGAGTTCATGGATG AAGCTCTGATCATGGCAAGTATGGATCATCCACACCTAGTCCGGTTGCTGGGTGTGTGTC TGAGCCCAACCATCCAGCTGGTTACTCAACTTATGCCCCATGGCTGCCTGTTGGAGTATG TCCACGAGCACAAGGATAACATTGGATCACAACTGCTGCTTAACTGGTGTGTCCAGATAG CTAAGGGAATGATGTACCTGGAAGAAAGACGACTCGTTCATCGGGATTTGGCAGCCCGTA ATGTCTTAGTGAAATCTCCAAACCATGTGAAAATCACAGATTTTGGGCTAGCCAGACTCT TGGAAGGAGATGAAAAAGAGTACAATGCTGATGGAGGAAAGATGCCAATTAAATGGATGG CTCTGGAGTGTATACATTACAGGAAATTCACCCATCAGAGTGACGTTTGGAGCTATGGAG TTACTATATGGGAACTGATGACCTTTGGAGGAAAACCCTATGATGGAATTCCAACGCGAG AAATCCCTGATTTATTAGAGAAAGGAGAACGTTTGCCTCAGCCTCCCATCTGCACTATTG ACGTTTACATGGTCATGGTCAAATGTTGGATGATTGATGCTGACAGTAGACCTAAATTTA AGGAACTGGCTGCTGAGTTTTCAAGGATGGCTCGAGACCCTCAAAGATACCTAGTTATTC AGGGTGATGATCGTATGAAGCTTCCCAGTCCAAATGACAGCAAGTTCTTTCAGAATCTCT TGGATGAAGAGGATTTGGAAGATATGATGGATGCTGAGGAGTACTTGGTCCCTCAGGCTT TCAACATCCCACCTCCCATCTATACTTCCAGAGCAAGAATTGACTCGAATAGGAGTGAAA TTGGACACAGCCCTCCTCCTGCCTACACCCCCATGTCAGGAAACCAGTTTGTATACCGAG ATGGAGGTTTTGCTGCTGAACAAGGAGTGTCTGTGCCCTACAGAGCCCCAACTAGCACAA TTCCAGAAGCTCCTGTGGCACAGGGTGCTACTGCTGAGATTTTTGATGACTCCTGCTGTA ATGGCACCCTACGCAAGCCAGTGGCACCCCATGTCCAAGAGGACAGTAGCACCCAGAGGT ACAGTGCTGACCCCACCGTGTTTGCCCCAGAACGGAGCCCACGAGGAGAGCTGGATGAGG AAGGTTACATGACTCCTATGCGAGACAAACCCAAACAAGAATACCTGAATCCAGTGGAGG AGAACCCTTTTGTTTCTCGGAGAAAAAATGGAGACCTTCAAGCATTGGATAATCCCGAAT ATCACAATGCATCCAATGGTCCACCCAAGGCCGAGGATGAGTATGTGAATGAGCCACTGT ACCTCAACACCTTTGCCAACACCTTGGGAAAAGCTGAGTACCTGAAGAACAACATACTGT CAATGCCAGAGAAGGCCAAGAAAGCGTTTGACAACCCTGACTACTGGAACCACAGCCTGC CACCTCGGAGCACCCTTCAGCACCCAGACTACCTGCAGGAGTACAGCACAAAATATTTTT

ATAAACAGAATGGGCGGATCCGGCCTATTGTGGCAGAGAATCCTGAATACCTCTCTG AGT TCTCCCTGAAGCCAGGCACTGTGCTGCCGCCTCCACCTTACAGACACCGGAATACTGTGG TGTAAGCTCAGTTGTGGTTTTTTAGGTGGAGAGACACACCTGCTCCAATTTCCCCACCCC CCTCTCTTTCTCTGGTGGTCTTCCTTCTACCCCAAGGCCAGTAGTTTTGACACTTCCCAG TGGAAGATACAGAGATGCAATGATAGTTATGTGCTTACCTAACTTGAACATTAGAGGGAA AGACTGAAAGAGAAAGATAGGAGGAACCACAATGTTTCTTCATTTCTCTGCATGGGTTGG TCAGGAGAATGAAACAGCTAGAGAAGGACCAGAAAATGTAAGGCAATGCTGCCTACTATC AAACTAGCTGTCACTTTTTTTCTTTTTCTTTTTCTTTCTTTGTTTCTTTCTTCCTCTTCT

_{TTTTTTTTTTTTTTTTTA} Jy^ _GCAGA ^

AGGAACTGATGTGTGCATATTTAGCATCCCTGGAAATCATAATAAAGTTTCCATTAG AAC AAAAGAATAACATTTTCTATAACATATGATGGTGTCTGAAATTGAGAATCCAGTTTCTTT CCCCAGCAGTTTCTGTCCTAGCAAGTAAGAATGGCCAACTCAACTTTCATAATTTAAAAA TCTCCATTAAAGTTATAACTAGTAATTATGTTTTCAACACTTTTTGGTTTTTTTCATTTT GTTTTGCTCTGACCGATTCCTTTATATTTGCTCCCCTATTTTTGGCTTTAATTTCTAATT GCAAAGATGTTTACATCAAAGCTTCTTCACAGAATTTAAGCAAGAAATATTTTAATATAG TGAAATGGCCACTACTTTAAGTATACAATCTTTAAAATAAGAAAGGGAGGCTAATATTTT TCATGCTATCAAATTATCTTCACCCTCATCCTTTACATTTTTCAACATTTTTTTTTCTCC ATAAATGACACTACTTGATAGGCCGTTGGTTGTCTGAAGAGTAGAAGGGAAACTAAGAGA CAGTTCTCTGTGGTTCAGGAAAACTACTGATACTTTCAGGGGTGGCCCAATGAGGGAATC CATTGAACTGGAAGAAACACACTGGATTGGGTATGTCTACCTGGCAGATACTCAGAAATG TAGTTTGCACTTAAGCTGTAATTTTATTTGTTCTTTTTCTGAACTCCATTTTGGATTTTG AATCAAGCAATATGGAAGCAACCAGCAAATTAACTAATTTAAGTACATTTTTAAAAAAAG AGCTAAGATAAAGACTGTGGAAATGCCAAACCAAGCAAATTAGGAACCTTGCAACGGTAT CCAGGGACTATGATGAGAGGCCAGCACATTATCTTCATATGTCACCTTTGCTACGCAAGG AAATTTGTTCAGTTCGTATACTTCGTAAGAAGGAATGCGAGTAAGGATTGGCTTGAATTC CATGGAATTTCTAGTATGAGACTATTTATATGAAGTAGAAGGTAACTCTTTGCACATAAA TTGGTATAATAAAAAGAAAAACACAAACATTCAAAGCTTAGGGATAGGTCCTTGGGTCAA AAGTTGTAAATAAATGTGAAACATCTTCTCATGCAATTATTTTATTATCCAACACACTAA TCTTTTGATACTTTATATAATTCCCTTTCTTCATATACTGCATCCAGTACTAGAACCATC ATTATTATGTATCATTTTGAAAGAATACCTGATGAGATGAAGGATGAGAACAAATGACAG AGATGAGTCTCCAAGTAAAGGGGGCCTCACATCAATAATTAGGAAACTTAGATATAAGTC GCCCTTTTCTGAAAATTCTACCCCAAGTCATTTAGATTTTTAAAAAATATTTCTAATGTT AAAATATTGGGACCAAATTAGAATCAATAGTATAAGATTAATTAATTAGAGTAAAAATAT CTATTAAGGCAGAGAAAGTTTAGAGAAAAAAATCCAAAGAAATTTGTGTTTCTTCCTATT CTGAACAAGTAAATCCATCCATCCATCCATCCAAACCTCCTTTATCTAACTGTGTCTACT AAAAGCACCATGTTTTGTGGGGAACACTCAGATAAATGGAATATCATCCTCAACTTCAAA

ATTCTATGATCTAGGAGATTTAATTAAAATGACATTTTAATTTTTCTATGCGTTCCA ACA ATCAGATTGCATAGTCTCTTTTGTGAATAGCTGTCATATAATCAGTTGTACTGTAAGATA TCTCCTTTAAACTCATTTGGGATATAAGTTAAACATCCTTCAAATTGTTGATGTTGACAA ACAGGATAATTTCAATAATATTATTCAAACATAAACTGGTCTAGGAGAATATTGCATCAC TGACTAATTAGCCTATCTAGAGTCTAACTTCACCATTAAACCAAAAGCAGATGGTGGTCC TTGGCCAAGAATATTGGAGACATTGGAGTTGGTTTTTTTCTAAGCTATAAGAAGTGAGGC GAGCTGAAAAAGTATGGTAGAGCAGGAGAAGGGTTTGTGAGATTCCTTCTAGTGAAGTTC ACCCTCAAACTTTTCAGGGGTAAAGACACAGAGTGATTCAGGGGCCACAATCTAATAGCT CAGGGCTCTCCTATCCATTCAGAGAAGTCTCTAGGAAAAGGGATCTCATATCAGTACTTA TGAAAAATTGAATATAAGCCTCCCTTTCTAAATAAATCTGCATCGAGTCATCACAGCCCT CTTTTTGGATACTATACCTTGATTTTTTTTTTCTGATTTACAATATGCATATGGTTTCTA CTGGGCTATAGAAAGCAGAATCACTCATTTTGGAGAAGGAAAAAATGAATAGTTAAAACA AACTTTTAACTGTTAAGGTAACAGAAATGTATTTAGTGAATGTCTCTTTCCTCCTAAGAA CACAAGACTTCTACATGTTGGGTAATACCTAGAGATGCATGTAGGAATAATCCAAAATGA CCCAAATGCTTTATAATAGCACCACTTTATAATTCTTTTGAATGATTTCTGTAGTATATA ATTGACTTCAGTTGTTTGAGTGTTTTTTGTTTTATTTTTGTCCCCCCTGGGAAAACATAT TTCAGCATGTATAAGAGGGAGAAAAAAAGTTTCATTCCTTCCAGAGAATAACTTATTTAG TCCAGTAGGGTAGAATTTTAAAATGTCAGTTAAAGTCTTCAAAGTGCTTGGGGGGATATC AGATTCCAGAGGCCAATTGTAGCAATTGAAATTTGCAGAATCAATTATGTAAATCTGAGA CAAATTAGTATTAAAATTACACGGAGTATATTTTTTAAATCACCCAACTTTGTAGATTAT ACCTATTTTGGGCAGGTATGGAAAAATTTTGCAGTTAAATGATTGCCTAAAGAAAGTGGT AAACAGGTGAGGAAAGATGGCCTCTGATCTAGGATAGATCCAGAACCACAAAGCATCTGC ACCACAAAAGGTGTTAGACTACCAAGCAGCTCCTGGTTTTCTGCATAGTATTAGTAGCAC AGCTTAGGATGAGAATCCTTTCTCCAGTAACATTCTTAAAATAGCATGAAAAACAACGCA AAACTCAAATTTCTATTAAAACACACAAACTAAAATCAAGTGATTCTTTTTTGTAGATTA GGGAGAAGGACTGAATATCTAATTTAAGAGAAGGAATAGTGTTTAAGTGTTATAGTGTGT GAGCTAATACCTTCTAAAGGAAAGACATGGCATGAAGATTGTGCATACTTACAATGCTAA GGAAAAATCAAGAAAAGGACTGTGTGAGGCTCTGCTACTAGATGAAGTTGGAAGGACTAT TAATGTGCTTCTTGAAGTATCAAAAATGAAAAGAAAATTAAAATTGTTTAAGCCTGACAG GGAAGGATGTAAATACAAGTTTTTCTAGAGCTCTCTAACCTTTATTTCAAAACTGGAATT ATTCATCCATCTGTAATTGTTGATAATTTAACTAGTATATGTAGTTCATAAGGTAATAGA AAAGGTGATCATGAAAGCATGTATATAACTGGACAGAACCACGATAATGCTATAAGATGT AGATTTAGTTAGGTTATCAGATGTTAAATGATTTTAATATTATTAAATAAATCAAACTAG AAAACTAACCACAAGTATAATGTAACAAAGTTAAATGCAGGATATAAAAATGTAGGATGG ATTTTGCATAGTAAAAAGATAAGTTTGCCATTTAAAATTGTTGTTTGTTGGGTTTAGCTG AAAGTAGGCATATATGGTTCCACTTGGGAAAACTTGCTTTAAAGCATTACAATGAACAAT

TTTTTCTCATTCTCTTATTCCTTTATCACTTTTTAAATGTAAAGAAAATTGTATTTA TTT ATTTTTTTAAATAAACACCACCTTGCAGAATTTAATAGGCAAACATGTTACATATGACTA AGTAAGGGTCTTCAAGATGAAGTAAAGAAAATGTAAATGTTCTATTACCTTATGCAGAGA CAAAAAAAAAAAGGAGTGGTGTCATTTAGCTAGCAAACAAACAAAATACAGTTAATTGGT GATATGTCCTTTCTTTTCTCACTATGCCCTCTTGCCTCCAAAAATGACAACAAAGAATCA CAATTTTTCTGATAAATAAATGCTAAACCAAGCGTTTCAAACTATTGCATTGCCATTCTT TTGGACTTTAGTTATTAGAATGATGATTGTTATAGGGCAAATGAGAAATCCATGTGCATC AGCTTCTAGTTGTTAAAAAAACCAGATAAATTAACTTCTACTGTATACTGTGGGCAGAGG ATCCTAGAGCTGATCCTACAACATCAGCTTCTAGTTGTTAAAAAAAAAAAAAGAAACAGA TAAATTAACTTCTACTGTATATACTGTGGGCAGAGGATCTTACTGTGCCTCTGTTTGTGT ACATGGACTTCGGTGTGTATCAGTTTGAAGGACAGCCTTGCCCCATGTAAACATATAAAT GCAGATTGGTATCGCCTGGTTGCTATTTGCTTAAGAACAAATATTATACAGATGAGATCA GGCATAATTTTAAAAGATCATTATCAGTGGAGACCTCATTATTACTGATATTACAATGGG GCCAGTTTTTATACTTCTGGGTAGAATTAATAAAATTTTTCTGATCCCAGAGATCTGAGT TCTCTCTGCAGTTGGAAACAAGAAGCTGTTGTGGGCATTGTGTCGGGCCAGGGGCCCTTG TGTTTGTGTGGGCAAATATCTTTTAGCAGTGTGAGCTGCTTTTTTCTTTTCATTAAAAGT CTCTCTAAAATAATAGAAATTTCAGATACTCGGTTCAAGTCTCACTGATTTTGTAGAGGT CCAAAAATGTAGGATCTGTCACTTTTGCAGGCCCCTGCCTCACCTAATTCCTGGCCAGGT GACATTTTGGGCAGAAGTAAATGCTTCTATAGTCACAAGCTAAAATGACTCTAAGCCCCA ATTTCACGGGGGGTATTCACATGCTTCCTCTGGAAAATACTCTTTGACAGTCAGCTTTGC AAGTAAGTGATTACCTTGTTAGGAATCAAAGAAAAATGTATTTCTCTCTGACCTTTAGAG GAAAATAGAATCCTTCCCTTTTTTGCCCATTGACACAACTGGCACTGCTCTCTTCCCTTT CTACCACCCTGGTTCAAAGTAGTCCCCCGATGCTGTCCTGTTCCTTTCTTAAGCCATAGT GGATCTCTGAGATCCTACACCCCACTTTGTGAAACACTGACTTCATCTTTGCCCTCGAAT GCCTGATTTTTTCATAAGAGATTCTAGCAATTTGGACACTGTTTAAGTGAACTATCAAAC TACCGCATAGAGAATATTTAAGCTATTAAAATTATGGTTTCCCATGAAGATCAATTCTCT GTGTCCTTCCCTATAGGAATTTGAGACGAGTTAGCCCTGTGATGAATCTTGAAACTCACA TATGTCCACATACACTTGGTAGAACTTCGATTTAATCTTTACATAAAAGCTGTACATATA ACCAAGAAGTTATTTTTGCCAGTAAATTAACTTATTTGCTTTATTCATCTTATTTGGTTC CTAATCGTAAATATTTTGTAGCTGCTGTAAATTTTTTTCTCCCAAATGAGGAGTCTTATT ATCATAAAGGTAAAGGCTATTCAGCTTTGATAACCACCTGCAATTCTTTTTTGGATCATT CATCCATCTAACAAATACATAATGAGGACAGTTCATGTTAATGAAAATCCATGTTGTTTA ATAGAATGCCATCCTTTACCTACTTTTGCTCTTTATGGACGTTTTTCTTTTCATGCTCTA GTGAGCTTTCCCTATATCATGAGAAGTGGTTATATTTGTGCAAATATACAAATATAGGAA AACAAAGATTCATACCTGTAGGCAATAGTCTAACTTGTCCAAACCACTTTGCCTTTACTG CTATTTTTATCCCCAATGCGTAGATATTTCCCCCAGGCCTATAGCCTTTGTGAAGGAAAG

CAAATCATACCTCCTGTATATTGACACGAATCTGGTTTTCAAATGTCATTTCCAGAT TTT TTAGTTAATTGGGGGTTGTCCTTTTCCCTTAATGTGAGAGTCATTTTCCTGTATATTTCT GGATCTCTCAGGGGCTGGGAGGGGGGAGTGAGGGGACTACAACCATAGCACTCCAAGAAC CCTTTTGGGATTACTCCAGTAATCAACTACGAAAGTTATTTTCTAAATGTAGATATGTAA GGTGTTCTTTTAAAGTAAGGTACTTTGAAATATGTAGCATAAACTGGTACTGCTGTTAAA TGGGTCGATTATTAAACGGAGCAGCTGTGTGAGGGCAGCTAACTTTGAATGCCTGTCTCC CTGGCTGGTGTGTCTCCTTCTCATGTTGAGAGCACCAGGGATTGCGTGGCTGCATGCTGA AACCGCATTTTCCCATGGTGTATGACTAGTTCATCTCTTTCTTGAGCACCATTACAAGAA GATCAAATGAAAATGAGATCAATGTGGAAGACAATTCATAGCACAAAAAAAGTCATCTTA AATCTACTCTCAAACATTCATCTTATACATGCATCAAAGTAATTTACTGACATCAGTTTG GGTGAGAGAGGGAGTCACTTTACTGAAAAGGCAGAGGCTTAAGGTGTATACATTTGTACT CACTTCCTTATTTTCTTAACTTGTAAGCAGAAAACAAGCCCTCTCTCTTGTGAAGTATCT TCAAAGGATTGGGGTGCAAAAATACCTTGCTGGTAAGCCATCAATGTTTTATTTAAATCC CTGCATTCAAAGTTAGCTGCCTTTTTGAAATAAACAAACAAAAAATACTACTGTATGTTT GAAAATGTGAATAGTATTTTTATAGCTTGTTAAAGACATGGCTAGTTGCATTTGTAAATA AGTATAATGTTGCTTTGATTTTCTTTTGTGGACATCTTTATTTGGAACATAATTGTCTTT AGGGTTGATTTGTATATAAGTAATTGGCCTGTGATTGTTTCTTTTTTGGTTGGAAGTTAT CATTTTGACATTACTTGTGATTCTGTGTTCAGCACTATTGTGATGTGTTCAACCTCTGCA CTCGCTTACACAATAGGATATGCCAATTGTGTGTGGTGTAATGTTATTTTGATTTTTTTC CATGTTATTGATGAAGGATCATGCACCTAACACATACTAACTTTTTTAATGTTAGGCATA TTTTTAGTATACTTTCTCTTATTCTTTCTTCTCCTCCAACCTTTTACCCATCCTCCTTCC TTTCCCTCATTCCTGTTGTTATTTGAGAATGAGGGAGAAACAGTATTTTACATTTATGTA ATTAGGCTTTTCCGTTAGTTCTCAAGGATCCTCTTTTGGCTCTTGGGAAAGAATTGTACC TGTACAAGGCAATTATAGAATGCGAACTGCTTTGCCTCATTCCATACTGATCATCCCAGC TGAACAATTTGAAAACTGTTCTGCCTTTTTGTTACATGAATCTGTCAGAAATATATTTTT AATTTAATATAAATGAAATTCAATAAAATATGAAACAAACGTTAAAAAAAAAAAAAAAAA A

SEQ ID NO.: 13 (Locus: NP_004439; erbB-2 isoform a [Homo sapiens]

MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQ GNL ELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNG DPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDI FHKNNQLA LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQC AAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSAN

IQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPD SLP DLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTV PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQEC VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVG ILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETEL RKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSP YVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVR LVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM IDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDA EEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEG AGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYV NQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

SEQ ID NO: 14 (Locus: NM_004448; erbB-2 isoform a mRNA transcript sequence [Homo sapiens], also known as Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), transcript variant 1 , mRNA)

GGAGGAGGTGGAGGAGGAGGGCTGCTTGAGGAAGTATAAGAATGAAGTTGTGAAGCT GAG ATTCCCCTCCATTGGGACCGGAGAAACCAGGGGAGCCCCCCGGGCAGCCGCGCGCCCCTT CCCACGGGGCCCTTTACTGCGCCGCGCGCCCGGCCCCCACCCCTCGCAGCACCCCGCGCC CCGCGCCCTCCCAGCCGGGTCCAGCCGGAGCCATGGGGCCGGAGCCGCAGTGAGCACCAT GGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCGGAGCCGC GAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGAC CCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGA ACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCA GGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGAT TGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGA CCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCA GCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCT CTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCT CACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGG CTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGC

CGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTG TGC TGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAG TGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTC CATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTA CAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGA GGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGT GTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATAT CCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTT TGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGA GACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGA CCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTA CTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACT GGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCC CTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGA GGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTG GGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAGTGCGT GGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGTGAATGCCAGGCACTGTTT GCCGTGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGC TGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCC CAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGG CGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGG CTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCATCTCTGCGGTGGTTGGCAT TCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCGACGGCAGCAGAA GATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGAC ACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAG GAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCC TGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCC CAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATA TGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTAT GCCCγATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGG A CCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCT CGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAAT TACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGG GGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCA CCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAA ACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCT

GCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGAT GAT γGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCA G GGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGA CAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGA GGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGG CATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACT AGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGC TGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCT CCCCACACATGACCCCAGCCCTCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCC CTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAA CCAGCCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGCCCTCTGCCTGCTGCCCG ACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGT CAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGACACCCCAGGG AGGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAGCCCAGCCTTCGACAACCTCTA TTACTGGGACCAGGACCCACCAGAGCGGGGGGCTCCACCCAGCACCTTCAAAGGGACACC TACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGAACCAGAAGGCCAAG TCCGCAGAAGCCCTGATGTGTCCTCAGGGAGCAGGGAAGGCCTGACTTCTGCTGGCATCA AGAGGTGGGAGGGCCCTCCGACCACTTCCAGGGGAACCTGCCATGCCAGGAACCTGTCCT AAGGAACCTTCCTTCCTGCTTGAGTTCCCAGATGGCTGGAAGGGGTCCAGCCTCGTTGGA AGAGGAACAGCACTGGGGAGTCTTTGTGGATTCTGAGGCCCTGCCCAATGAGACTCTAGG GTCCAGTGGATGCCACAGCCCAGCTTGGCCCTTTCCTTCCAGATCCTGGGTACTGAAAGC CTTAGGGAAGCTGGCCTGAGAGGGGAAGCGGCCCTAAGGGAGTGTCTAAGAACAAAAGCG ACCCATTCAGAGACTGTCCCTGAAACCTAGTACTGCCCCCCATGAGGAAGGAACAGCAAT GGTGTCAGTATCCAGGCTTTGTACAGAGTGCTTTTCTGTTTAGTTTTTACTTTTTTTGTT TTGTTTTTTTAAAGATGAAATAAAGACCCAGGGGGAGAATGGGTGTTGTATGGGGAGGCA AGTGTGGGGGGTCCTTCTCCACACCCACTTTGTCCATTTGCAAATATATTTTGGAAAACA

GCTA

SEQ ID NO: 15 (Locus: NP_001005862; erbB-2 isoform b [Homo sapiens])

MKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIA HNQ VRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSE DCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPA LVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQR CEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTA

PLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQG LGI SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLA CHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQ NGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINC THSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRL LQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHV RENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLL DIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPARE IPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQ NEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQ RYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERP KTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPER GAPPSTFKGTPTAENPEYLGLDVPV

SEQ ID NO.: 16 (Locus: NMJ)01005862; erbB-2 isoform b mRNA transcript [Homo sapiens], also known as Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), transcript variant 2, mRNA)

GTTCCCGGATTTTTGTGGGCGCCTGCCCCGCCCCTCGTCCCCCTGCTGTGTCCATAT ATC GAGGCGATAGGGTTAAGGGAAGGCGGACGCCTGATGGGTTAATGAGCAAACTGAAGTGTT TTCCATGATCTTTTTTGAGTCGCAATTGAAGTACCACCTCCCGAGGGTGATTGCTTCCCC ATGCGGGGTAGAACCTTTGCTGTCCTGTTCACCACTCTACCTCCAGCACAGAATTTGGCT TATGCCTACTCAATGTGAAGATGATGAGGATGAAAACCTTTGTGATGATCCACTTCCACT TAATGAATGGTGGCAAAGCAAAGCTATATTCAAGACCACATGCAAAGCTACTCCCTGAGC AAAGAGTCACAGATAAAACGGGGGCACCAGTAGAATGGCCAGGACAAACGCAGTGCAGCA CAGAGACTCAGACCCTGGCAGCCATGCCTGCGCAGGCAGTGATGAGAGTGACATGTACTG TTGTGGACATGCACAAAAGTGAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGC CAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCA GGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATAT CCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCA GAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCT AGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCT GCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCG

GAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAA CCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCC GATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCG CACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCA TGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCA CTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGA CACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGAC TGCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCT GCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCC CTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTAC CAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCT GCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCT CCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGA CAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCA CAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTC ACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGT GCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGC CAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCG AGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGG CCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGTGAATGC CAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTT TGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGT GGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCC AGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCT GGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCATCTCTGC GGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCG ACGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGT GGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGA GACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGG CATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGA AAACACATCCCCCAAAGC ^' CAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGT GGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGT GACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCT GGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGA GGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAA CCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTA

CCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCG CCG GCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGAC TTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAA GGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAA ATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTC CCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGC CAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCT GGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCC GGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGG GGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACC CTCCGAAGGGGCTGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGGG GCTGCAAAGCCTCCCCACACATGACCCCAGCCCTCTACAGCGGTACAGTGAGGACCCCAC AGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCC TGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGCCCTCT GCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAA GAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTT GACACCCCAGGGAGGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAGCCCAGCCTT CGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGGCTCCACCCAGCACCTT CAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGAAC CAGAAGGCCAAGTCCGCAGAAGCCCTGATGTGTCCTCAGGGAGCAGGGAAGGCCTGACTT CTGCTGGCATCAAGAGGTGGGAGGGCCCTCCGACCACTTCCAGGGGAACCTGCCATGCCA GGAACCTGTCCTAAGGAACCTTCCTTCCTGCTTGAGTTCCCAGATGGCTGGAAGGGGTCC AGCCTCGTTGGAAGAGGAACAGCACTGGGGAGTCTTTGTGGATTCTGAGGCCCTGCCCAA TGAGACTCTAGGGTCCAGTGGATGCCACAGCCCAGCTTGGCCCTTTCCTTCCAGATCCTG GGTACTGAAAGCCTTAGGGAAGCTGGCCTGAGAGGGGAAGCGGCCCTAAGGGAGTGTCTA AGAACAAAAGCGACCCATTCAGAGACTGTCCCTGAAACCTAGTACTGCCCCCCATGAGGA AGGAACAGCAATGGTGTCAGTATCCAGGCTTTGTACAGAGTGCTTTTCTGTTTAGTTTTT ACTTTTTTTGTTTTGTTTTTTTAAAGATGAAATAAAGACCCAGGGGGAGAATGGGTGTTG TATGGGGAGGCAAGTGTGGGGGGTCCTTCTCCACACCCACTTTGTCCATTTGCAAATATA TTTTGGAAAACAGCTA

SEQ ID NO.: 17 (Locus:NP_001973; erbB-3 isoform 1 precursor [Homo sapiens])

MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCE VVM GNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVM LNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTI DWRDIVRDRDAEIVVKDNG

RSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSG PQD TDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTS CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGN LDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGG RSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEE RLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEP REFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPI YKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLG GTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVF GTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPG SSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNV LLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVT VWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTI DVYMVMVKCWMIDENIRPTFKE LANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEED NLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPR PVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLL TPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYM NRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDY EYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATD SAFDNPDYWHSRLFPKANAQRT

SEQ ID NO.: 18 (Locus: NM_001982.2; erbB-3 isoform 1 precursor [Homo sapiens], also known as Homo sapiensv-eτb-b 2 erythroblastic leukemia viral oncogene homolog 3 (avian) (ERBB3), transcript variant 1, mRNA)

ACACACACACACCCCTCCCCTGCCATCCCTCCCCGGACTCCGGCTCCGGCTCCGATT GCA ATTTGCAACCTCCGCTGCCGTCGCCGCAGCAGCCACCAATTCGCCAGCGGTTCAGGTGGC TCTTGCCTCGATGTCCTAGCCTAGGGGCCCCCGGGCCGGACTTGGCTGGGCTCCCTTCAC CCTCTGCGGAGTCATGAGGGCGAACGACGCTCTGCAGGTGCTGGGCTTGCTTTTCAGCCT GGCCCGGGGCTCCGAGGTGGGCAACTCTCAGGCAGTGTGTCCTGGGACTCTGAATGGCCT GAGTGTGACCGGCGATGCTGAGAACCAATACCAGACACTGTACAAGCTCTACGAGAGGTG TGAGGTGGTGATGGGGAACCTTGAGATTGTGCTCACGGGACACAATGCCGACCTCTCCTT CCTGCAGTGGATTCGAGAAGTGACAGGCTATGTCCTCGTGGCCATGAATGAATTCTCTAC TCTACCATTGCCCAACCTCCGCGTGGTGCGAGGGACCCAGGTCTACGATGGGAAGTTTGC CATCTTCGTCATGTTGAACTATAACACCAACTCCAGCCACGCTCTGCGCCAGCTCCGCTT GACTCAGCTCACCGAGATTCTGTCAGGGGGTGTTTATATTGAGAAGAACGATAAGCTTTG

TCACATGGACACAATTGACTGGAGGGACATCGTGAGGGACCGAGATGCTGAGATAGT GGT GAAGGACAATGGCAGAAGCTGTCCCCCCTGTCATGAGGTTTGCAAGGGGCGATGCTGGGG TCCTGGATCAGAAGACTGCCAGACATTGACCAAGACCATCTGTGCTCCTCAGTGTAATGG TCACTGCTTTGGGCCCAACCCCAACCAGTGCTGCCATGATGAGTGTGCCGGGGGCTGCTC AGGCCCTCAGGACACAGACTGCTTTGCCTGCCGGCACTTCAATGACAGTGGAGCCTGTGT ACCTCGCTGTCCACAGCCTCTTGTCTACAACAAGCTAACTTTCCAGCTGGAACCCAATCC CCACACCAAGTATCAGTATGGAGGAGTTTGTGTAGCCAGCTGTCCCCATAACTTTGTGGT GGATCAAACATCCTGTGTCAGGGCCTGTCCTCCTGACAAGATGGAAGTAGATAAAAATGG GCTCAAGATGTGTGAGCCTTGTGGGGGACTATGTCCCAAAGCCTGTGAGGGAACAGGCTC TGGGAGCCGCTTCCAGACTGTGGACTCGAGCAACATTGATGGATTTGTGAACTGCACCAA GATCCTGGGCAACCTGGACTTTCTGATCACCGGCCTCAATGGAGACCCCTGGCACAAGAT CCCTGCCCTGGACCCAGAGAAGCTCAATGTCTTCCGGACAGTACGGGAGATCACAGGTTA CCTGAACATCCAGTCCTGGCCGCCCCACATGCACAACTTCAGTGTTTTTTCCAATTTGAC AACCATTGGAGGCAGAAGCCTCTACAACCGGGGCTTCTCATTGTTGATCATGAAGAACTT GAATGTCACATCTCTGGGCTTCCGATCCCTGAAGGAAATTAGTGCTGGGCGTATCTATAT AAGTGCCAATAGGCAGCTCTGCTACCACCACTCTTTGAACTGGACCAAGGTGCTTCGGGG GCCTACGGAAGAGCGACTAGACATCAAGCATAATCGGCCGCGCAGAGACTGCGTGGCAGA GGGCAAAGTGTGTGACCCACTGTGCTCCTCTGGGGGATGCTGGGGCCCAGGCCCTGGTCA GTGCTTGTCCTGTCGAAATTATAGCCGAGGAGGTGTCTGTGTGACCCACTGCAACTTTCT GAATGGGGAGCCTCGAGAATTTGCCCATGAGGCCGAATGCTTCTCCTGCCACCCGGAATG CCAACCCATGGAGGGCACTGCCACATGCAATGGCTCGGGCTCTGATACTTGTGCTCAATG TGCCCATTTTCGAGATGGGCCCCACTGTGTGAGCAGCTGCCCCCATGGAGTCCTAGGTGC CAAGGGCCCAATCTACAAGTACCCAGATGTTCAGAATGAATGTCGGCCCTGCCATGAGAA CTGCACCCAGGGGTGTAAAGGACCAGAGCTTCAAGACTGTTTAGGACAAACACTGGTGCT GATCGGCAAAACCCATCTGACAATGGCTTTGACAGTGATAGCAGGATTGGTAGTGATTTT CATGATGCTGGGCGGCACTTTTCTCTACTGGCGTGGGCGCCGGATTCAGAATAAAAGGGC TATGAGGCGATACTTGGAACGGGGTGAGAGCATAGAGCCTCTGGACCCCAGTGAGAAGGC TAACAAAGTCTTGGCCAGAATCTTCAAAGAGACAGAGCTAAGGAAGCTTAAAGTGCTTGG CTCGGGTGTCTTTGGAACTGTGCACAAAGGAGTGTGGATCCCTGAGGGTGAATCAATCAA GATTCCAGTCTGCATTAAAGTCATTGAGGACAAGAGTGGACGGCAGAGTTTTCAAGCTGT GACAGATCATATGCTGGCCATTGGCAGCCTGGACCATGCCCACATTGTAAGGCTGCTGGG ACTATGCCCAGGGTCATCTCTGCAGCTTGTCACTCAATATTTGCCTCTGGGTTCTCTGCT GGATCATGTGAGACAACACCGGGGGGCACTGGGGCCACAGCTGCTGCTCAACTGGGGAGT ACAAATTGCCAAGGGAATGTACTACCTTGAGGAACATGGTATGGTGCATAGAAACCTGGC TGCCCGAAACGTGCTACTCAAGTCACCCAGTCAGGTTCAGGTGGCAGATTTTGGTGTGGC TGACCTGCTGCCTCCTGATGATAAGCAGCTGCTATACAGTGAGGCCAAGACTCCAATTAA

GTGGATGGCCCTTGAGAGTATCCACTTTGGGAAATACACACACCAGAGTGATGTCTG GAG CTATGGTGTGACAGTTTGGGAGTTGATGACCTTCGGGGCAGAGCCCTATGCAGGGCTACG ATTGGCTGAAGTACCAGACCTGCTAGAGAAGGGGGAGCGGTTGGCACAGCCCCAGATCTG CACAATTGATGTCTACATGGTGATGGTCAAGTGTTGGATGATTGATGAGAACATTCGCCC AACCTTTAAAGAACTAGCCAATGAGTTCACCAGGATGGCCCGAGACCCACCACGGTATCT GGTCATAAAGAGAGAGAGTGGGCCTGGAATAGCCCCTGGGCCAGAGCCCCATGGTCTGAC AAACAAGAAGCTAGAGGAAGTAGAGCTGGAGCCAGAACTAGACCTAGACCTAGACTTGGA AGCAGAGGAGGACAACCTGGCAACCACCACACTGGGCTCCGCCCTCAGCCTACCAGTTGG AACACTTAATCGGCCACGTGGGAGCCAGAGCCTTTTAAGTCCATCATCTGGATACATGCC CATGAACCAGGGTAATCTTGGGGAGTCTTGCCAGGAGTCTGCAGTTTCTGGGAGCAGTGA ACGGTGCCCCCGTCCAGTCTCTCTACACCCAATGCCACGGGGATGCCTGGCATCAGAGTC ATCAGAGGGGCATGTAACAGGCTCTGAGGCTGAGCTCCAGGAGAAAGTGTCAATGTGTAG GAGCCGGAGCAGGAGCCGGAGCCCACGGCCACGCGGAGATAGCGCCTACCATTCCCAGCG CCACAGTCTGCTGACTCCTGTTACCCCACTCTCCCCACCCGGGTTAGAGGAAGAGGATGT CAACGGTTATGTCATGCCAGATACACACCTCAAAGGTACTCCCTCCTCCCGGGAAGGCAC CCTTTCTTCAGTGGGTCTCAGTTCTGTCCTGGGTACTGAAGAAGAAGATGAAGATGAGGA GTATGAATACATGAACCGGAGGAGAAGGCACAGTCCACCTCATCCCCCTAGGCCAAGTTC CCTTGAGGAGCTGGGTTATGAGTACATGGATGTGGGGTCAGACCTCAGTGCCTCTCTGGG CAGCACACAGAGTTGCCCACTCCACCCTGTACCCATCATGCCCACTGCAGGCACAACTCC AGATGAAGACTATGAATATATGAATCGGCAACGAGATGGAGGTGGTCCTGGGGGTGATTA TGCAGCCATGGGGGCCTGCCCAGCATCTGAGCAAGGGTATGAAGAGATGAGAGCTTTTCA GGGGCCTGGACATCAGGCCCCCCATGTCCATTATGCCCGCCTAAAAACTCTACGTAGCTT AGAGGCTACAGACTCTGCCTTTGATAACCCTGATTACTGGCATAGCAGGCTTTTCCCCAA GGCTAATGCCCAGAGAACGTAACTCCTGCTCCCTGTGGCACTCAGGGAGCATTTAATGGC AGCTAGTGCCTTTAGAGGGTACCGTCTTCTCCCTATTCCCTCTCTCTCCCAGGTCCCAGC CCCTTTTCCCCAGTCCCAGACAATTCCATTCAATCTTTGGAGGCTTTTAAACATTTTGAC ACAAAATTCTTATGGTATGTAGCCAGCTGTGCACTTTCTTCTCTTTCCCAACCCCAGGAA AGGTTTTCCTTATTTTGTGTGCTTTCCCAGTCCCATTCCTCAGCTTCTTCACAGGCACTC CTGGAGATATGAAGGATTACTCTCCATATCCCTTCCTCTCAGGCTCTTGACTACTTGGAA CTAGGCTCTTATGTGTGCCTTTGTTTCCCATCAGACTGTCAAGAAGAGGAAAGGGAGGAA ACCTAGCAGAGGAAAGTGTAATTTTGGTTTATGACTCTTAACCCCCTAGAAAGACAGAAG CTTAAAATCTGTGAAGAAAGAGGTTAGGAGTAGATATTGATTACTATCATAATTCAGCAC TTAACTATGAGCCAGGCATCATACTAAACTTCACCTACATTATCTCACTTAGTCCTTTAT CATCCTTAAAACAATTCTGTGACATACATATTATCTCATTTTACACAAAGGGAAGTCGGG CATGGTGGCTCATGCCTGTAATCTCAGCACTTTGGGAGGCTGAGGCAGAAGGATTACCTG AGGCAAGGAGTTTGAGACCAGCTTAGCCAACATAGTAAGACCCCCATCTCTTTAAAAAAA

AAAAAAAAAAAAAAAAAAAAACTTTAGAACTGGGTGCAGTGGCTCATGCCTGTAATC CCA GCCAGCACTTTGGGAGGCTGAGATGGGAAGATCACTTGAGCCCAGAATTAGAGATAAGCC TATGGAAACATAGCAAGACACTGTCTCTACAGGGGAAAAAAAAAAAAGAAACTGAGCCTT AAAGAGATGAAATAAATTAAGCAGTAGATCCAGGATGCAAAATCCTCCCAATTCCTGTGC ATGTGCTCTTATTGTAAGGTGCCAAGAAAAACTGATTTAAGTTACAGCCCTTGTTTAAGG GGCACTGTTTCTTGTTTTTGCACTGAATCAAGTCTAACCCCAACAGCCACATCCTCCTAT ACCTAGACATCTCATCTCAGGAAGTGGTGGTGGGGGTAGTCAGAAGGAAAAATAACTGGA CATCTTTGTGTAAACCATAATCCACATGTGCCGTAAATGATCTTCACTCCTTATCCGAGG GCAAATTCACAAGGATCCCCAAGATCCACTTTTAGAAGCCATTCTCATCCA

SEQ ID NO.: 19 (Locus: NP_001005915; erbB-3 isoform s precursor [Homo sapiens])

MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCE VVM GNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVM LNYNTNSSHALRQLRLTQLTGQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTL

AAV

SEQ ID NO.: 20 (Locus: NM_001005915; erbB-3 isoform s precursor mRNA transcript sequence [Homo sapiens], also known as Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) (ERBB3), transcript variant s, mRNA).

ACACACACACACCCCTCCCCTGCCATCCCTCCCCGGACTCCGGCTCCGGCTCCGATT GCA ATTTGCAACCTCCGCTGCCGTCGCCGCAGCAGCCACCAATTCGCCAGCGGTTCAGGTGGC TCTTGCCTCGATGTCCTAGCCTAGGGGCCCCCGGGCCGGACTTGGCTGGGCTCCCTTCAC CCTCTGCGGAGTCATGAGGGCGAACGACGCTCTGCAGGTGCTGGGCTTGCTTTTCAGCCT GGCCCGGGGCTCCGAGGTGGGCAACTCTCAGGCAGTGTGTCCTGGGACTCTGAATGGCCT GAGTGTGACCGGCGATGCTGAGAACCAATACCAGACACTGTACAAGCTCTACGAGAGGTG TGAGGTGGTGATGGGGAACCTTGAGATTGTGCTCACGGGACACAATGCCGACCTCTCCTT CCTGCAGTGGATTCGAGAAGTGACAGGCTATGTCCTCGTGGCCATGAATGAATTCTCTAC TCTACCATTGCCCAACCTCCGCGTGGTGCGAGGGACCCAGGTCTACGATGGGAAGTTTGC CATCTTCGTCATGTTGAACTATAACACCAACTCCAGCCACGCTCTGCGCCAGCTCCGCTT GACTCAGCTCACCGGTCAGTTCCCGATGGTTCCTTCTGGCCTCACCCCTCAGCCAGCCCA AGACTGGTACCTCCTTGATGATGACCCAAGACTGCTCACTCTAAGTGCCTCTTCCAAGGT GCCTGTCACCTTGGCCGCTGTCTAAAGGTCCATTGCTCCCTAAGCAATAGAGGGCCCCCA GTAGGGGGAGCTAGGGGCATCTGCTCCAGGGAAAGGAACCCTGTGTCCTTGTGGGGCTGG AGTCAGAGCTGGATCTGTTAACCGTTTTTCTAATTTCAAAGTACAGTGTACCGGAGGCCA

GGCCTGATGGCTTACACCTGTAATCCCAGCATTTTGGGAGGCCAAGGAGGGCAGATC ACT TGAGATCAGGAGTTTGAGACCAGCCTGGCCAACATGGCGAAACCCTGTCTCTACTAAAAA TACAAAAAAATAAAATAAAATAAAAAATTA