METHODS OF USE

Field of the Invention

The present invention relates generally to the field ol cell physiology, and more

particularly, to programmed cell death, or apoptosis. The novel peptides. compositions and methods ol ^" the invention are useful for modulating apoptosis in cells.

Background of the Invention

The phenomenon of programmed cell death, or "apoptosis," is involved in and important to the normal course of a wide variety υf developmental processes, including immune and nervous system maturation. Apoptosis also plays a role in adult tissues having high cell

turnover rates (Ellis, R. E., et al., Ann. Rev. Cell. Biol. 7: 663-698 (1991); Oppenheim, R. W.,

Ann. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J., et ai, Ann. Rev. Immunol. 10: 267-293 ( 1992); Raff, M. C, Nature 356: 397-400 (1992)). In addition to its role in development,

apoptosis has heen implicated as an important cellular safeguard against tumorigenesis

(Williams, G. T., Cell 65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)). Under

certain conditions, cells die by apoptosis in response to high-level or deregulated expression of

oncogenes (Askew, D., et al, Oncogene 6: 1915-1922 (1991); Evan, G. I., et ai, Cell 69:

1 19- 128 (1992); Rao, L., et al, Proc. Natl. Acad Sci. USA 89: 7742-7746 ( 1992); Smcyne,

R. J., et al., Nature 363: 166-169 (1993); Tanaka, S., et al.. Cell 77: 829-83^ 1994); Wu, X.,

et ai , Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994)). Suppression of the apoptotic

program, by a variety of genetic lesions, may contribute to the development and progression υf malignancies. This is well illustrated by the frequent mutation of the p53 tumor suppressor gene in human tumors (Levinc, A. J., et al., Nature 351: 453-456 ( 1991)).

Other factors have been identified which appear tυ play roles in regulating apoptosis. One of these, the Insulin-Like Growth Factor-I Receptor (IGF-IR), is a member of the tyrosine kinase family of signal transducing molecules. The IGF-IR is activated by the ligands IGF-I, IGF-II and insulin at supra-physiological concentrations, and plays an important role in the development, growth, and survival of normal cells (LeRoith, D., et al.. Endocrine Revs: 16: 143- 163 ( 1995); Lowe, W.L., Jr. "Biological actions of the Insulin-like growth factor receptors," in LeRoith. D., Ed., Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press,

Boca Raton. Pub. ( 1991 ); Bascrga, R.. et al.. Cell Prolif. 27: 63-71 ( 1994)). Over-expression of the IGF-IR leads to the transformation of fibroblasts and conversely, IGF-IR null fibroblasts are refractory to transformation by a number of oncogenes (Sell, C, et al., Mol. Cell Biol. 14: 3604-3612 ( 1994)). There is considerable evidence for a role for the IGF-IR in the maintenance of tumor cells in vitro and in vivo. IGF-IR levels are elevated in tumors of lung (Kaiser, U., et al., J. Cancer Res. Clin Oncol. 119: 665-668 ( 1993); Moody, T.W. and Cuttitta, F., Life Sciences 52: 1 161-1 173 ( 1993)), breast (Pollak, M.N., et al., Cancer Lett. 38: 223-230 (1987); Foekens, J.A., et al.. Cancer Res. 49: 7002-7009 (1989) Cullen, K.I., et al., Cancer Res. 49: 7002-7009 (1990)) and colon (Remaole-Bennet, M. M., et al., J. Clin. Endocrinol. Metab. 75: 609-616

( 1992); Guo, Y.S., et ai, Gastroenterol. 102: 1 101- 1 108 ( 1992)). Increased levels υf IGF-I and/υr IGF-II expression have been associated with human tumors (McCauley, V.M., et ai, Cancer Res. 50: 251 1-2517 ( 1990); Bhatavdekar, J.M., et l., Neoplas a 41: 101- 103 ( 1994)). Many of these tumor cell types respond lo IGF-I with a proliferative signal in culture

(Nakanishi. Y., et al.. J. Clin. Invest. 82: 354-359 (1988); Freed, K.A. and Hcmngton, A.C., J. Mol. Endocrinol. 3: 509-514 ( 1989)), and autocrine or paracrine loops for proliferation in vivo have been postulated (LeRoith, D., et ai. Endocrine Revs. 16: 143- 163 ( 1995); Yee, D., et al., Mol. Endocrinol. 3: 509-514 (1989)). IGF-I protects from apoptosis induced by cytokine withdrawal in IL-3-depcndent hemopoietic cells (Rodriguez-Tarduchy, G., et ai, J. Immunol. 149: 535-540 (1992)), and from serum withdrawal in Rat-1/mycER cells (Harrington, E., et ai, EMBO J. 13: 3286-3295 ( 1994)). Of cytokines present in fetal bovine serum, including the mitogens EGF and PDGF, IGF-I proved to be the most potent in inhibition of myc-induced death in Rat-1 cells. The anti- apoptotic function of IGF-I was evident in the post-commitment stage of the cell cycle and also in cells blocked in cell cycle progression by cloposidc or thymidine.

The demonstration that c-myc driven fibroblasts arc dependent on IGF-I for their survival suggests that there is an impoπant role for the IGF-IR in the maintenance ol oncogene driven tumor cells by specifically inhibiting apoptosis, a role distinct from the better characterized proliferative effects. This would be similar to a role thought to be played by other anti-apoptotic genes such as bcl-2 in promoting tumor survival (McDonnell, T. J., et ai. Cell 57: 79-88 (1989); Hockcnberry, D. M., et ai, Nature 348: 334-336 (1990)). The protective effects of IGF-I are dependent upon receptor levels rather than on availability of the ligand (Resnicoff, M., et ai. Cancer Res. 55: 3739-3741 (1995a)). Support for an anti-apoptotic function of IGF-IR in the maintenance of tumor cells was also provided by a study using antisense oligonucleotides to the IGF-IR that identified a quantitative relationship between IGF- IR levels, the extent of apoptosis and the tumorigenic potential of a rat syngeneic tumor (Rescinoff, M., et ai, Cancer Res. 55: 3739-3741 (1995b)).

Fibroblasts from IGF-IR null mice have been used to demonstrate a requirement for the

IGF-IR in transformation, and also to map domains in the receptor essential for the proliferative and transformation function of the IGF-IR. Specifically, the C-terminal region of the IGF-IR is required for the transformation function. Receptors which are truncated at amino acid 1229 fail lo transform fibroblasts derived from IGF-IR null mice, but retain lull proliferative activity (Surmacz, E., et ai, Exp. Cell Res. 218: 370-380 ( 1995)). Within the C-terminal region, the transforming activity has been further localized to a domain between amino acids 1245 and 1294; substitution of the single tyrosine 1251 with phenylalanine impairs transformation function (Miura, M., et ai, J. Biol. Chem. 270: 22639-22644 (1995b)), substitution of the four serines ( 1280-1283) completely abolishes transformation (Li et ai, submitted), and substitution of histidine 1293 and lysine 1294 reduces transformation activity (Hongo et ai, submitted). All of the transformation-defective, truncated and point mutant receptors retain proliferative capacity. These studies indicate that two separate functions of the receptor, proliferation and transformation, are spatially distinct within the receptor and that transformation may need additional signals to those required for proliferation. Mutations at the ATP binding site in the kinase domain, at the tyrosine cluster in the kinase domain, or at tyrosine 950 (the major binding site for well defined substrates of the IGF-IR, IRS-1 and SHC) abolish both proliferation and transformation (Miura, M., et ai, Cancer Res. 55: 663-667 ( 1995a); Li, S., et ai, J. Biol. Chem. 269: 32558-32564 (1994); Gronbcrg, M., et ai, J. Biol. Chem. 268: 23435-23440 (1993)). As the preceeding discussion demonstrates, while recent studies have advanced the general understanding of the transformation and proliferative functions of the IGF-IR in vertebrate cells, the apparent anti-apoptotic function of the IGF-IR remains less well characterized. Elucidation of IGF-IR domains involved in the receptor's anti-apoptotic function would be of great value in the development of compositions which modulate the survival υf

certain cells, such as cancer cells. The ability to modulate the anti-apoptotic activity of the IGF- IR would alsυ allow the development of compositions and strategies for treating cells affected by diseases, such as neurodegenerative diseases, and by acute hypoxic injury, such in stroke, in which activation of the IGF-IR's anti-apoptotic function would be beneficial. Conversely, inactiviaiion of the anti-apoptotic function υf the IGF-IR in tumor cells would be a useful and specific treatment strategy. Accordingly, a need persists to identify the potential domain(s) in the IGF-IR responsible for its anti-apoptotic function.

Summary of the Invention

In a broad aspect, the present invention is directed to novel compositions comprising domains of the IGF-IR that regulate the survival or anti-apoptotic function of the receptor upon ligand binding. These novel compositions, collectively referred to herein as "Active Survival Domains," comprise peptide regions υf the IGF-IR, modulation of which enhances or diminishes anti-apoptotic response upon receptor activation. Modulation of the Active Survival Domains of the IGF-IR, as well as compositions which effect such modulation, form additional broad aspects of the invention.

In one embodiment, the present invention is directed to C-terminal deletion mutants of the IGF-IR. Vertebrate cells comprising the C-terminal deletion mutants of the invention exhibit enhanced anti-apoptotic response upon ligand binding to the receptor. C-terminal deletion mutants according to this aspect of the invention include, but are not limited to, deletion mutants I229d, 1245d and 1293d (i.e., mutant IGF-IR peptides truncated at amino acids F1229, R1245 and HI293, respectively), and their functional equivalents.

In another aspect, the invention is directed to isolated and purified C-terminal peptides,

which may alternatively be synthetically produced, compnsmg the last 108, 92 or 44 am o acids ot the IGF-IR, preterably compπsing IGF-IR cytoplasmic domain constructs designated MyCF, CF, MyCF-N, MyCF- id, MyCF-C. MyCF-29, MyCF-62, CF-N, CF- id, and CF-C, or constructs ot MyCF, CF and MyCF-N having mutations at Y 1250F/Y1251F, H 1293F/K1294R or SI 280- 1283 A, and to molecules that mimic and/υr lnterlcre with their structure and/or tuncuon, useful tor inducing or modulating the apoptotic state of a cell. Chemical compounds that disrupt the lunction of the Active Survival Domain have utility as apoptosis-mυdulating agents. Accυrd gly, in yet another aspect, the invention us directed to agents capable ol disrupting Active Survival Domain tuncuon Such agents include, but are not limited to, molecules that bind to ihc Active Survival Domain, molecules that lnterlcre with the interaction oi the Active Survival Domain with other peptide sequences derived trom cellular proteins (including, but not limited to, the 108, 92 and 44 amino acid C-lerminal peptides or other peptide sequences ot the IGF-IR itself), and molecules comprising the Active Survival Domain which is altered in some manner. The invention tuπhcr provides screening methods to identity molecules that modulate apoptosis by disrupting the lunction ot the Active Survival Domain by, tor example, intramolecular interaction with the IGF-IR, which accordingly comprise additional contemplated embodiments. In one aspect, such screening methods comprise competitive binding assays wherein the ability ot a putative modulating molecule to bind tυ truncated I229d, 1254d or 1293d deletion mutants ot the IGF-IR is measured in the presence ot a suitably labeled C- terminal peptide.

In another embodiment, the invention is directed to single or multiple point mutants of the IGF-IR. Vertebrate cells comprising the point mutated IGF-IR ol the invention exhibit altered anti-apop.υtic response upon ligand binding to the receptor. Point mutated IGF-IR

compositions according to this aspect ot the invention include, but are not limited to, the mutant

YI251F and the double mutants Y1250F/Y1251F and H1293F/KI294R, and their lunctional equivalents The phosphorylation state ot the point mutated IGF-IR compositions ot the invention compπses an additional aspect ot the invention, as does the modulation ot that state, which may be accomplished according to the invention through, tor example, inhibition ot the respective protein tyrosine kinase or phosphotyrosine phosphatase, by means ot which the anti- apoptotic signal υl the point mutated IGF-IR compositions ol the invention may be attected. Yet additional embodiments ot the invention comprise the use ot the point mutated IGF- IR compositions ol the invention as screening markers tor molecules which modulate apoptosis Such embodiments include, bul are not limited tυ, assays which measure the ability ot a putative apoptosis modulating molecule to compete with olher peptides and proneins (including, but not limited to, other peptide sequences ot the IGF-IR itself), which arc identilied to bind specitically to the pυint mutated IGF-IR compositions ot the invention, in order to modulate the apoptυlie stale ol a cell. In one specifically contemplated exemplary embodiment are provided assays in which the ability ot a putative apoptosis modulating molecule to bind to a point mutated receptor is reduced or lost or gained when measured against the same molecule's allimty tor the wild type (i.e., non-mutated) receptor

Molecules identified by means υt the screening assays ol the invention will be candidates as useful therapcuue drugs tor the in vivo, ex vivo or in vitro treatment ot target cells alone or in combination with suitable carriers and excipients. Such compositions and their use compπse additional embodiments ot the invention

In additional aspects, the present invention relates to products and processes involved in the cloning, preparation and expression ot C-terminal mutant IGF-IR compositions and point mutated IGF-IR compositions according to the invention (collectively, ' .autant IGF-IR

compositions"): antibodies with speciticity to these mutant IGF-IR compositions, and nucleotide sequences encoding these mutant IGF-IR compositions or poπions thereot Peptides compπsing the mulant IGF-IR compositions ot the invention are usetul tor producing monoclonal and polyclonal antibodies thereto Such anubodies, and fragments thereot, arc usetul tor detecting and isolating proteins compnsmg the mutant IGF-IR cυmpυsitions in biological specimens including, tor example, cells trom all human tissues including heart tissue, lung tissue, tumor cells, brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas, as well as tor modulating the apoptotic activity ol proteins comprising the mutant IGF-IR compositions, such as C-terminal tragments, in and trom such biological specimens, and constitute additional aspects ot the invention

In yet another aspect, the invention provides tor expression vectors containing genetic sequences, hosts translormed with such expression vectors, and methods tor producing the recombinant mulant IGF-IR compositions ot the invention

The present invention is lurther directed to methυds lor inducing or suppressing apoptosis in the cells and/or tissues of individuals sutteπng trom disorders characteπzed by inappropriate cell pro lcration or survival, or by mappropπate cell death, respectively Disorders characteπzed by inappropriate cell prolileration and/υr survival include, lor example, inflammatory conditions, cancer, including lymphomas, genotypic tumors, etc Disorders characteπzed by inappropπate cell death include, lor example, autoimmune diseases, acquired immunodeficiency disease (AIDS), cell death due to radiation therapy or chemotherapy, acute hypoxic |ury, etc.

The present invention also relates to methods tor detecting the presence ot the IGF-IR anti-apoptotic domain, as well as methods directed tυ the diagnosis ot disorders, which disorders are associated with an increased oi decreased level υt expression υl proteins

compnsmg the IGF-IR anti-apoptotic domain, as compared to the expected level ot expression ot such proteins in the normal cell population.

The present invention relates to the therapeutic use of peptides comprising the IGF-IR anti-apoptotic domain. The present invention also relates to memods tor modulating the apoptotic state ot a cell by administering peptides compπsing AcUve Survival Domain sequences or compounds that modulate the activity ot the Active Survival Domain to an individual sulleπng Irom a disorder characterized by inappropπatc cell proliteration or inappropπate cell death, in order to stabilize inappropriate cell proliferation (i.e., induce apoptosis) or stabilize inappropriate cell death (i.e., suppress apoptosis), respectively, and/or in either case to restore normal cell behavior

In another aspect, the present invention is directed to the surprising discovery that C- terminal amino acid peptides trom the IGF-IR, including the C-termial 108 amino acid peptide, arc cytotoxic tυ tumor cells. These peptides specifically inhibit and or kill cells which are dependent upon the IGF-IR C-terminus, i.e., cells which exhibit anchorage-independent growth and or apoptotic stimulus provided by transfection tn vitro or growth in a bioditlusion chamber in vivo. The cytotoxic C-terminal ammo acid peptides (and their tunctiυnal analυgs) of the invention are usetul as therapeutic agents and in screening assays tor other agents which modulate the anti-apoptotic tuncuon ol the IGF-IR.

In yet another aspect, the invention is directed to a method ot assaying IGF-IR anti- apoptotic tuncuon in IL-3-dependent cells, which method allows the demonstration ot the dissociation ot survival tuncuon trom mitogenic and transtorming lunctions in the receptor structure.

These and olher υb|ects and aspects of the invention will be apparent to those ol skill tro the description which follows

Description of the Figures

Figure 1.

Viability of FL5.12 cells stably transfected with IGF-IR compared with FL5.12/Bcl-2 cells. Cells were cultured at 5 xl0 ⁶ /mL in medium containing 5% FBS, 5% FBS + IGF-I, υr 5% FBS + IL-3, and the viability was monitored at timcpoints shown by trypan blue exclusion. Fig. IA shows FL5.12/neo cells and Fig. IB shows FL5.12 IGF-IR-I cells. FL5.12 cells/BCL-2 cells are compared with FL5.12/neo cells in Fig. IC for their viability in 5% FBS. Data pυints represent the mean and standard deviation υf cell viability derived from triplicate cultures. The expressiυn of wt IGF-IR υn FL5.12 cells as determined by indirect immunυfluorescencc staining with the Ab- 1 mAb directed to the human IGF-IR is shown in panel D; the thin line represents staining obtained with the negative control (no primary antibody) and the thick line rcprcsets staining with the Ab- 1 mAb.

Figure 2.

Proliferation of FL5.12 IGF-IR in the presence of IL-3 or IGF-I. Cells were seeded at 1 x lOVmL in 5% FBS, 5% FBS + IGF-I, or 5% FBS + IL-3. At the indicated timepoints an

aliquot was removed and the cell number determined.

Nucleotide and predicted amino acid sequence ot IGF-IR. Amino acids ot the proreceptor are numbered above starting at Glu 1, and are preceeded by a 30-residue signal sequence; nucleotides arc numbered tυ the right. Expenmentally determined peptide sequences are

underlined and numbered; potential N-lmked glycosylation sites are overhned; cysteine residues are shaded; the transmembrane domain is heavily underlined. The potential ATP binding site is indicated by asterisks over Gly 976, 978 and 981, and by an arrow over Lys 1003. The putative precursor processing site is boxed. From Ullrich, A., et ai, EMBO J 5(10): 2503- 2512 (1986).

Fig. 3B.

Schematic diagram ot the cDNA structure υt the IGF-IR showing cysteme-πch regions of a chain and kinase domain of B chain υf the dimer. Relative positions υf deletion mutants described herein are indicated. Alter Ullrich, A., et ai, EMBO J 5(10): 2503-2512 (1986).

Figure 4A.

Expression levels ol IGF-IR mutants alter stable translection in FL5.12 cells. Cells were assayed by indirect immunolluorescence tor binding ot the Ab- 1 monoclonal antibody directed against the human IGF-IR. The thin line represents staining obtained with the negative control (no primary antibody), and the bold line represents Ab- 1 binding. The name of each mutant expressed is indicated above the box.

Figure 4B

Survival curves lor FL5 12 cells expressing the K1003R mutant IGF-IR, the Y950F mutant, (the Yl 131, 1135, 1136 F mutant, the Y1250F/1251F mutant, the S1280- 1283A mutant, and the H 1293/F/K1294R mutant Cells were incubated in medium containing IL-3 lor 24 hours, washed extensively, and cultured at 5 x 10 ⁵ /mL in medium containing 5% FBS, 5% FBS +

IGF-, or 5% FBS + IL-3 Cell viability was monitored by trypan blue exclusion at 24, 48, and 72 hours alter replating, and the data are presented as % viability ol total cells plotted against

Figure 5

IGF-I protccuυn Irυm apoptosis induced by IL-3 withdrawal in FL5.12 cells expressing truncated IGF-IRs. Cells were incubated in medium containing IL-3 tor 24 hours, washed extensively, and cultured at 5 x lOVmL in medium containing 5% FBS, 5% FBS + IGF-, or 5% FBS + IL-3 Cell viability was monitored by trypan blue exclusion at 24, 48, and 72 hours, and the data are presented as % viability ot total cells plotted against time Each point represents the mean and standard deviation ol triplicate cultures Panel A, FL5 12 cells expressing 1229d IGF-IR mutant; panel B, 1245d IGF-IR mutant; and panel C, 1293d IGF-IR mutant.

Figure 6. IGF-IR cytoplasmic domain constructs. Constructs encoding the nucleoude sequences tor IGF- IR Iragments were generated by PCR amplification trom tull length IGF-IR sequences Each sequence was tused at the 3' end with the sequence lor the 7-ammo-acιd flag antigenic tag (F) A second version was made ot each construct that was moditied by lusing the tirst 16 am o acid sequence ot SRC to the 5' end as a signal tor myπstylauon (My) and membrane anchorage

(as described herein). MyBF encodes the entire IGF-IR cytoplasmic domain starting at am o acid 930 and extending to amino acid 1337 tused to My at the 5' end and F at the 3' end MyKCF is a construct encoding the IGF-IR kinase domain and the entire C-termmus starting at amino acid 972 extending to amino acid 1337 lused to My at the 5' end and lo F at the 3' end MyCF encodes the entire C-terminus ol the IGF-IR and starts al amino acid 1225 (in the kinase domain) and extends to amino acid 1337 tused to My at the 5' end and to F at the 3' end. CF encodes the entire C-terminus ot the IGF-IR and starts at amino acid I223(ιn the kinase domain) and is tused to F at the 3' end. MyKC20 encodes 20 amino acids trom the IGF- IR kinase domain starting at am o acid 1210 and extending to ammo acid 1229 lused to My at the 5' end and tυ F at the 3' end MyCF-N encodes ihe N-terminal portion ol the IGF-IR C- termmus and extends Irυm am o acids 1225- 1269 tused to My at the 5' end and tυ F at the 3' end. MyCF-mid encodes the middle region ol the IGF-IR C-terminus extending trom amino acids 1260-1307 tused to My at the 5' end and F at the 3' end. MyCF-C encodes the C- lermmal end ot the IGF-IR C-terminus extending trom amino acids 1301-1337 lused to My at the 5' end and to F at the 3' end. MyCF-29 encodes an IGF-IR C-terminal fragment trom amino acid 1231- 1259 tused tυ My at the 5' end and to F at the 3' end. MyCF-62 encodes an IGF-IR C-tcrminal tragment beginning at amino acid 1246 and extending to amino acid 1307 tused to My at the 5' end and to F at the 3' end. Also made were constructs ot CF-N, CF-mid and CF-C, as well as constructs ot MyCF, CF and MyCF-N which featured the mutations at Y1250F/Y1251F, H1293F/K1294R and/or SI 280- 1283 A.

Figure 7.

Transient expression ot IGF-IR C-tcrminus tragment MyCF into MCF-7 results in cytotoxicity.

MCF-7 cells were transiently transfected with a marker plasmid encoding β-galα iosidase, a

pcDNA3 control vector υr with the pcDNA3 plasmid containing the sequences for CF or MyCF by the lipofectamine methυd and incubated in the presence of IGF-I for 48 hυurs. Cells were stained with X gal at 48 hυurs and live and dead blue cells were counted by microscopic analysis. Panel A, total live and dead blue cell number for each plasmid transfected. Data represent the mean and standard deviation of live and dead cell number from triplicate transfections. Expression of CF and MyCF proteins in MCF-7 cells by transient transfection is shown by western blot analysis of immunoprecipitated proteins in panel B. The anti-flag monoclonal antibody was used for immunoprecipitation and western blots, and detection was by the ECL protocol.

Figure 8.

Transient transfection of CF, MyCF, and MyKCF is toxic to R+ cells. Cells were transfected with a marker plasmid encoding β-galactosidase, together with R+ a plasmid containing CF, MyCF, or MyKCF by the lipofectamine method, incubated wilh 10% FBS υr IGF-I (50 ng/mL) for 24 hours and stained with X gal. Live and dead blue cells were counted by microscopic analysis. Panel A shows R+ cells incubated in 10% FBS and panel B shows R+ cells incubated in IGF-I. Data represent mean and standard deviation of live and dead cells from triplicate transfections.

Figure 9.

Transient expression of MyCF, MyKC20, MyCF-N, MyCF-N (50/51), MyCF-mid, and MyCF-C in R ⁺ cells. Cells were transfected with a marker plasmid encoding the β-galactosidase gene, together with plasmid encoding the various IGF-IR constructs by lipofectamine, incubated in seriim free medium containing IGF-I (50ng/mL) tot 24 hours and stained with X-gal. Live and

dead blue cells were counted by microscopic analysis Data represent mean and standard deviation ot live and dead cells trom triplicate transactions

Figure 10 Modilied pGEX-2TK template vector encoding MyCF lused to GST and antennapedia sequence. The cloning region ot the P-GEX-2TK vector was moditied in the cloning region to contain the My (lirst 16 ammo acids trom SRC) sequence cloned in at the Nco-I restπcuon site, the flag tag (F) cloned in at the Sac-I and the antennapedia sequence (Ap) cloned in at the Xba I restπcuon site The IGF-IR C-terminal sequence encompassing am o acids 1223 to 1337 were cloned in between the KpN-I and Sac-I restπction sites This plasmid produces MyCF that is lused to GST al the N-tcrminus and to Ap at the C-tcrminus A linker region between GST and MyCF contains a ihrombin cleavage site and a protein kinase A site tor ^<2 P labeling ot the protein Any DNA sequence can be inserted into this vector tor fusion to GST, flag tag and Ap.

Figure I I.

IGF-IR bait constructs tor use in yeast two-hybπd system screening. IGF-IR constructs were cloned into the yeast two hybπd system bait plasmid pAS-2 to produce protems that are tused to the GAL-4 DNA binding domain and a hemagglutinin (HA) epitope tag. Y2Hwt is the pAS-2 plasmid encoding the entire IGF-IR cytoplasmic domain sequence encompassing ammo acids 931-1337 Y2H 950 is an identical construct except it contains the Y950F mutation Y2H CF is the pAS-2 plasmid encoding the IGF-IR C-terminus and extend from am o acid 1225 to 1337. Y2H-CF 50/51 is CF containing the Y1250F/YI251F mutation, Y2H CF 93/94 is CF containing the H1293F/K1294R mutation, and Y2H CF 1280-83 is CF containing the S1280- I280A mutation.

Detailed Description of the Invention

Technical and scientific lerms used herein have ihe meanings commonly understood by one of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies known to those υf skill in the art.

Publications and other materials setting forth such known methodologies to which reference is made are incoφorated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook, J., et ai, Molecular Cloning: A Laboratory Manual. 2d Ed., Cold Spring Harbor Laboratory Press, Planview, New York ( 1989); Kaufman, P. B., et ai, Eds., Handbook υf Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton ( 1995); McPherson, M. J., Ed., Directed Mutagenesis: A Practical Approach. IRL Press, Oxford (1991); Jones, J., Amino Acid and Peptide Synthesis. Oxford Science Publications, Oxford (1992); Austen, B. M. and Westwood, O. M. R., Protein Targetinu and Secretion, IRL Press, Oxford ( 1991). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention; however, preferred materials and/or methods are described. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted.

According to the invention, protein domains of the IGF-IR, termed herein "Active Survival Domains," have been identified which are involved in the anti-apoptotic function of the IGF-IR. IGF-IRs containing mutations at Y950, in the kinase domain, and in the C-terminus were expressed in the IL-3-dependent murine B cell line FL5.12. These cells have been extensively used as a model for cell death studies, because they rapidly undergo apoptosis upon IL 3 withdrawal. When transfected with anti-apoptotic genes such as hcI-2, apoptosis induced

by IL-3 withdrawal is inhibited (Hockcnberry et ai, 1990), υr when transfected with apoptosis- inducing genes such as bak, there is an acceleration of the onset υf apoptosis upon IL-3 deprivaUon. The isolation and characterization of bak is described in co-pending United States application Serial Number 08/321,071, filed 1 1 October 1994, which is a continuation-in-part of United States application Serial Number 08/287,427, filed 9 August 1994 (bak is rcfeπed tυ

therein as bcl-y), the disclosures of which are incorporated herein by reference.

The IL-3-dependcnt cell line FL5.12 provides an unambiguous system in which lo study the anti-apoptotic lunction of the IGF-IR and is prefeπed for these purposes. In contrast to tibroblast-likc cells, FL5.12 cells express very low numbers υf endogenous IGF-IR (< 1000/cell) thereby reducing the possibility ot cross-talk between endogenous receptors and those transfected into the cells. Cells transtcctcd with wild type ("wt") IGF-IR are protected from apoptosis by IGF-I in a manner analogous to the protection provided by over-expression of Bcl- 2 in these cells. The anti-apoptotic function of the IGF-IR was not accompanied by a significant mitogenic signal in FL5. I2 cells, since the cells did not proliferate in the presence of IGF-I.

A series of mutants of the IGF-IR, which had previously been analyzed for their proliferative and transforming function in IGF-IR null cells, was analyzed for the ability to protect from apoptosis induced by IL-3 withdrawal in FL5.12 cells. The kinase-inactivating mutation at K1003R provided a receptor which does not have anti-apoptotic function, in accordance with its loss of proliferation and transformation (Kato et ai, 1993). Tyrosine 950 in the IGF-IR has been shown to interact with IRS-1 by two-hybrid system analysis (O'Neill et ai, 1994, Gustafson et al., 1995) and the tyrosine cluster (1 131 , 1 135, 1 136) in the kinase domain is required to maximize mis interaction (Gustafson et ai, 1995). Mutants Y950F, and VI 131, 1135, 1 136F both retain protection from apoptosis, although neither of these mutants

has miiogenic υr translorm g lunction in R cells (Miura et ai, 1995a). This result indicates that the domam(s) required tor ihe proliierauon and iranslormation tuncuυns υt the IGF-IR are separate trom the domam(s) lcquired tυr the anti-apoptυtic tuncuon (rcleπed to herein as "Active Survival Domains"), and lurther suggests that this function is not mediated by IRS- 1 In the C-terminus υl the IGF-IR, the twυ mutations Y1251F and H1293F/K1294R abolished IGF-I-mediated protection trom IL-3 withdrawal, and comprise contemplated Ac ve Survival Domains as that term is used herein. Interesungly, these two regions are also required lor translormatiυn lunction, although both mutants retain proliierauon in R- cells (Miura et ai, 1995b, Hυngo et ai, 1996). The IGF-IR with mutations at the tour seπnes (1280- 1283) has proliteralivc tuncuon, but does not have translormmg ability (Li et ai, submitted) In FL5.12 cells, this mulant retains anti-apoptotic lunction These results suggest lhat there may be a heretotorc unrecognized degree of overlap in the transtorming and anti-apoptotic functions ot the IGF-IR receptor with regard to a requirement lor certain residues in the C-terminus. Inhibitiυn υt apoptυsis may thus be a component ol the translormatiυn υl cells, but additional signals are also required. Compaπson υl the anu-apoptotic and transtormatiυn lunction data tor these mutants (Table I) reveals that mhibi on ot apoptosis is essenUal tor transformation. Mutants lhat did not protect trom apoptosis were non-transtυrming

An unexpected and surprising lindmg by the present inventors is that the IGF-IRs that were truncated in the C-terminus retained anu-apoptotic lunction, although point mutations within the regions that were deleted abolished the anti-apoptotic function ot the full length receptor The truncated receptors appear to have enhanced anti-apoptotic tuncuon since significant protecuon trom IL-3 withdrawal was evident with low levels ot receptor expression, and in 5% FBS without exυgcnously added IGF-I The truncated receptors were sull responsive to IGF-I, and did not aμj/ear tυ be cυnstilulively phosphorylated by Western blot analysis υt

ικ

phosphotyrosine content. These truncated receptors do not have transforming function; they have proliferative function and this does not appear to be enhanced (Surmacz, et ai, 1995).

While not intending to be bound by any particular theory, this suggests two possibilities for the role of the C-terminus in IGF-IR anii-apoptotic function. The first is that the C-terminus provides a regulatory role on other parts of the receptor, and in the absence of this regulation the receptor has increased anti-apoptotic activity. The second possibility is that it contains within it a potentially negative signal for cell survival, or pro-apoptotic signal, that is suppressed by o er regions of the receptor. Such a negative signal would be analogous to an inactive IGF-IR without ligand stimulation that provides no anti-apoptotic lunction. This negative signal is also manifest in the presence of IGF-1 when mutations are present at Y 1251 or H1293/K 1294. The cytoloxicily detected with the transient transfections of C-terminal fragments provides further evidence for an inhibitory role of this region υf the IGF-IR on survival. Studies with stably expressed C-terminus fragments in ovarian carcinoma cells indicate that those Iragments are specifically inhibitory when the cells arc placed under conditions in which cells require the lunction of the IGF-IR C-terminus for anchorage independent growth or growth in a biodiffusion chamber in vivo.

In support of a regulatory role for the C-terminus of the IGF-IR in providing an anti- apoptotic signal, it has been noted during the course of the studies on transformation that the truncated receptors or C terminal mutants have hyper-phosphorylation of IRS- 1 and SHC. It has also been reported in protein interaction studies in yeast lhat C-terminal truncated IGF-IRs and those with mutations in the C-terminus had enhanced interaction with both SHC and IRS- 1 (Tartare-Deckert et ai, 1995).

The present inventors observed that mutant Y950F, which presumably can no longer interact with IRS- 1, did not abolish protection from apoptosis. These data indicate lhat it is

unlikely that the enhanced activity υt the truncated receptors is due to increased signaling through IRS- 1 or SHC Therefore, an alternauve site on the receptor in the kinase domain or juxta-membrane region must be propυsed as being essential lυr the anti-apoptotic lunction ol the receptor Such a region could be susceptible to regulation by the C-terminus ot the IGF-IR and compπses an Active Survival Domain according lo the invention. On the other hand, it is also possible that such a survival domain tuncuons by inhibiung pro-apoptotic signals present in the C-termmus.

Although not intending tυ be bound, the present inventors have been able to articulate a non-limiting hypothesis as to how the C-terminus lunctions to regulate the an ti -apoptotic tuncuon ol the IGF-IR. In the tull length IGF-IR, the C-lerminus might interact directly with the receptor to attenuate its tuncuon, or indirectly thrυugh interaciiυn with υthcr inhibitory proteins Such interactions could exist when the receptor is not active Binding ol the ligand IGF-I results in activation υt the kinase domain, and phosphorylation ot key residues in the kinase domain or in the C-terminus ot the receptor may release the inhibitory interactions. Such a model is also consistent with the cytotoxic effects ot C-terminal fragments when transfected into cells These molecules may be cytotoxic by virtue ot two possible mechanisms. They may interact with cndogenυus receptors as outlined above and block their anu-apoptotic function Alternatively, the C-tcrmmal Iragments may be able to induce apoptosis by competing lor proteins that normally interact with the C-terminus of endogenous IGF-IR and block the anti- apoptotic tuncuon. Data trom stable expression ot these molecules in ovarian carcinoma cells are consistent with the posited mechanism. Agents that mimic the effects ot the Y 125 IF or H1293F/K1294R mutauons, and agents that behave like the C-terminal Iragments ot the IGF-IR, then, are also useful tor therapeuuc intervention in tumor cells according to the present invention.

The anti-apoptotic function ol the IGF-IR requires domains that are not the same as those required lor the mitogenic or transtorming function ot the receptor This implies that there exists one or more specific domains required tor the anti-apoptotic signal, termed herein "Active Survival Domains," apparently susceptible to regulation by the C- terminus ot the receptor. Such regulatory interactions at the level ot the receptor provide a specific target tor therapeutic intervention in tumor cells.

As used herein, "apoptosis inducing agent" includes any biological, chemical, biochemical or physical means ot inducing a complete or partial apoptotic response in a target cell Target cells may be normal cells, or cells having aberrant growth or proliierauon, such as tumor cells. Most nucleated eukaryotic cells tested have shown the capacity to undergo apoptosis in response to appropriate stimuli, including non-mammalian cells such as avian and nematode

Examples ol apoptosis inducing agents include UV light, hyperthermia or heat shock, calcium, ATP, actinomycin D, A23187 Ca ²⁺ -Mg ²⁺ lonophorcs, cytochalasin B, cycloheximide, antι-CD3/T-cell receptor antibodies, epipodophyllotoxins, gliotoxm, glucocorticoids, lymphotoxins, RU486, TCDD, TGF-β 1, oxidative stress, viral infections, chemotherapeutic drugs, cold shock, gamma radiation, cisplatin, etoposide, temposides, DNA alkylating agents, macromolecular synthesis inhibitors, immunological agents such as natural killer cells, effector cells, lymphotoxins, K cells, T cells, and the like, and others, as descnbed tor example in Green, D. R. et ai, Apoptosis and Cancer, in Principles and Practice of Oncology Updates

Volume 8, J. B. Lippincott Company, January 1994 Number 1, and Gerschenson, L.E., et al

FASEB J. 6: 2450 - 2455 (1992).

The Active Survival Domain compositions of the present invention are used to idenuty known and putative apoptosis inducing agents, for example, in assays using the IGF-IR, Us

ligands including IGF-I, IGF-II and insulin, and receptor interacting proteins found in cells

Preferred agents according to the invention are molecules, including peptides and proteins, which bind υi otherwise interact with an Active Survival Domain and thereby may or may not affect the function ot the Active Survival Domain, releπed to herein as "Survival Domain Interacting Agents " As used herein, then, a "Survival Domain Interacting Agent" means a molecule that is recognized by a particular protein, which parucular protein is preferably a receptor protein, and most preferably, the IGF-IR The agent bound by υr reacting with the protein is called a "Survival Domain Interacting Agent," a term which is detinitionally meaningful only in terms ol its counterpart protein The term "Survival Domain Interacting Agent" does not imply any particular molecular size or other structural or compositional teature other than that the substance in question is capable ot binding υr otherwise interacting with the protein Also, a "Survival Domain Interacting Agent" may serve either as the natural ligand to which the protein binds or interacts, or as a tunctional analogue that may act as an agonist or antagυnist As described herein, transtected cells may be used as a model tor studying apoptosis.

For controlled investigation, mammalian cells lacking the IGF-IR may be transicctcd with an expression construct encoding the IGF-IR ot the invention Cells are produced lhat encode the protem that is otteπ tunctionally equivalent to the wild-type protein Thus, the binding properties ot interacting proteins may be analyzed, including naturally occurπng and syntheuc Survival Domain Interacting Agents. The transtected cells find particular use tor the identification ot agents having pharmaceutical efficacy. Transtected cells may be contacted with a putative drug agent, and the amount of apoptosis modulation determined, as compared to the control cells in the absence ol the putative drug. Agents identified according to the invention find a vaπety ot uses, including modulators ol apoptosis, inhibitors ot

neurodegenerative diseases, tumors, viral diseases, and identification ot tumor promoters

The present mvenuon also provides lor o er polypepudes compπsing Iragments of the protein ol the invention and polypeptides substantially homologous thereto The protein peptides ot the invention will generally exhibit at least about 80% homology with naturally occurπng sequences υl the IGF-IR, typically at least about 85% homology, and more usually at least about 97% homology. The length ot compaπson sequences will generally be at least about 16 amino acids residues, usually at least about 20 residues, more usually at least about

24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

The present mvenuon also includes fusion polypeptides between the IGF-IR, which may be iruncated, and other proteins For example, homologous polypeptides may be tused with omer proteins, υr other apopto.sis-modulating proteins, resulting in fusion proteins having mixed tunctionahues. Examples υl suitable proteins are members ot the Bcl-2 family ot proteins, Bak,

Bax and the like Similarly, fusions may be generated wim heterologυus prυteins, lυr example, the first 16 aminυ acids ol SRC. Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sultonauon, biotinylation, υr the addition ot other moieties, using methods known in the art. In some embodiments, the modificauon will be useful labelling reagents, or serve as purification targets, tor example, allinity ligands

Preferred according to the invention are IGF-IR cytoplasmic domain constructs designated

MyCF, CF, MyCF-N, MyCF-mid, MyCF-C, MyCF-29, MyCF-62, CF-N, CF-mid, and CF-C, and constructs ot MyCF, CF and MyCF-N having mutations at Y1250F/Y1251F,

H1293F/K1294R or S1280-1283A, as well as molecules that mimic and/or interfere with their structure and/or tuncuon

Fusion polypeptides will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods, as are generally described in Sambrook, et ai, supra:

Mernlield, J Amer. Chem Soc 85: 2149-2156 ( 1963) Merπtield, Science 232: 341-347

( 1986); and A erton, et ai, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press, Oxford ( 1989)

The nucleic acid compositions ot the mvenuon will generally be in RNA or DNA forms, mixed polymeric lυrms, or any synthetic nucleotide structure capable ot binding in a base- specitic manner to a complementary strand of nucleic acid An example ot a suitable synthetic nucleotide structure is described in Nielson, P.E., et ai, Science 254: 1497-1500 ( 1991) The described nucleic acid embodiment is typically derived from genomic DNA υr cDNA, prepared by synthesis, υr derived trom combinations thereof. The DNA compositions generally include the complete coding region encoding the IGF-IR, or Iragments thereot, e.g., compπsing at least

8 codons, usually at least 12 codons. or usually at least about 15 codυns, typically at least about 20 codons, more typically at least about 30 codons and preferably even more One or more introns may be present

The nucleic acids encoding the IGF-IR or Iragments thereot such as C-terminal fragments, may be used to prepare an expression construct for the IGF-IR. The expression construct normally compπses one or more DNA sequences encoding the IGF-IR operably linked and under the transcriptional control ot a native or other promoter. Usually the promoter will be a eukaryotic promoter tor expression in a mammalian cell. The transcπptional regulatory sequences will typically include a heterologous promoter or enhancer which is recognized by the host cell. The selection ot an appropriate promoter will depend on the host cell.

Convenient expression vectors are commercially available.

The compositions of the present invention have utility tor modulating the growth and differentiation ot cells through the apoptotic process. Modulation ol the apoptotic process includes dccelerauon ot thu <ate ot apoptosis in a populauon ot cells, or eliminauon ot the

cellular apoptouc response to apoptosis inducing agents. Modulation of the apoptotic process also includes induction or acceleration of apoptosis where it is desirable to increase the rate of cell death or to specifically target a population of cells. For example, the induction of apoptosis in tumor cells or in other cells showing increased proliferation and growth provides an effective therapy to decrease or abolish the growth of these cells. The compounds of the present invention also have utility in combatting drug resistance, which is a common problem with cuπent cancer treatments. Drug resistance may be a resistance to apoptosis in general, and thus, the proteins of the present invention may be used to decrease drug resistance. In this embodiment, the compounds of the invention may be used in conjunction with other anti- neoplastic agents. Mechanisms of drug resistance are described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, supra. In some embodiments, the compositions of the invention may be used to assay tissue injury and regeneration. A suitable model system for the assay of tissue injury is the thymus of dexamethasone-treated rats, as described in Schwartzman, R., et al., Endocrinol. 128(2): 1190-1197 (1991). The compositions of the present invention thus have utility for a variety of therapeutic indications, including as anti-viral, anti-microbial, or anti-parasitic agents, as anti-neoplastic agents for the treatment of tumors, including but not limited to tumors of the lung, breast, pancreas and liver, as well as for acute lymphoblastic or myeloid leukemia, chronic myeloid, myelogenous, granulocytic, or lymphatic leukemia, acquired immune deficiency syndrome (AIDS), neurodegenerative diseases, myelodysplatic syndrome, Hodgkin's lymphoma, malignant lymphomas such as non-Hodgkin's lymphoma, or Burkitt's lymphoma, neoplasms and the like. The quantities of active ingredient necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered. Thus, treatment dosages should be titra^u to

optimize safety and efficacy Typically, dosages used in vitro may provide usetul guidance in the amounts usetul lor in situ administration ot the acϋve ingredients. Animal testing ot effective doses tor treatment ot particular disorders will provide lurther predictive indicaUon ol human dosage. Vanous considerauons are descnbed, lor example, Goodman and Gilman's The Pharmacological Basis ot Therapeutics, 7th Ed., MacMillan Publishing Co,, New York ( 1985), and Remington's Pharmaceutical Sciences 18th Ed., Mack Publishing Co , Easton, Penn (1990) Methods tor administration are discussed therein, including oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, lontophoretic administration, and the like

By "lunctional equivalent" is meant a peptide possessing a biological activity or immunυlυgical characteristic substanually similar to thai ol a composition ot the mvenuon, and is intended to include "tragments", "vaπants", "analogs", "homologs", υr "chemical deπvatives" possessing such activity or charactenstic Functional equivalents ot a peptide comprising an Active Survival Domain, then, may not share an identical am o acid sequence, and conservative or non-conservative amino acid substitutions ot conventional or unconventional amino acids are possible

Reference herein to "conservative" amino acid substitution is intended to mean me mterchangeability ol ammo acid residues having similar side chains For example, glycine, alanine, valine, leucine and isoleucine make up a group ot ammo acids having aliphatic side chains; seπne and threonine are ammo acids having aliphauc-hydroxyl side chains; asparagine and glutamine are amino acids having amide-containing side chains; phenylalanine, tyrosine and tryptophan are amino acids having aromauc side chains; lysine, argmine and hisudme are ammo acids having basic side chains; and cysteme and methionine are ammo acids having sulfur- containing side chains. Interchanging one amino acid trom a given group with another amino acid from that same group would be considered a conservative substituuon Preferred

conservative substitution groups include asparagine-glutamine, alanine-valinc, lysine-argm e, phenylalanine- tyrosine and valine -leucine-isoleucine.

The biological activity ol an Active Survival Domain and its tuncuonal equivalents may be affected by the sub-cellular localization ot these compositions. Accordingly, in another preteπed embodiment υl the invention, the Active Survival Domain peptides ol the invention will have lused to their N-terminal end an appropnate sequence, which may preferably be the SRC sequence tor myπstylation (My) lor targeting to cell membranes. Other suitable means ot effecting sub-cellular localization, including the selection of suitable hydrophobic tails, may be employed by those of skill using known methods.

Agents capable oi modulating Active Survival Domain lunctions may include peptides compπsing an Active Survival Domain, as well as mutants ot an Active Survival Domain or of proteins comprising an Active Survival Dυmain. A "mutant" as used herein refers to a peptide having an am o acid sequence which differs trom that of the naturally occurring peptide υr protein by at least one ammo acid. Mutants may have the same biological and immunological activity as the naturally occurring Active Survival Domain or the naturally occuπing protein. However, the biological or immunological activity υf mutants may differ or be lacking. For example, an Active Survival Domain mutant may lack the biological activity which characteπzes naturally occurπng Active Survival Domain peptide, but may be usetul as an antigen for raising antibodies against an Active Survival Domain or tor the detection or purification of anubodies against an Active Survival Domain, or as an agonist (competitive or non-compeutive), antagontst, or partial agonist of the function of the naturally occurnng Active

Survival Domain peptide. Preferred mutants according to the present invention include "mutant

IGF-IR compositions" as defined herein.

Modulauon of Actι\ _^ Jurvival Domain mediated functions may be effected by agonists

υr antagonists ot Active Survival Domain peptides as well Screening ol peptide libraries, compound libraries and other information banks to identity agonists or antagonists ot the tuncuon ol proteins compπsing an Active Survival Domain is accomplished with assays tor detecting the ability ot potential agonists υr antagonists to inhibit or augment Active Survival Domain binding

For example, GST lusion proteins ot the IGF-IR C-termmus or fragments thereot are used lor screening inhibitors ot IGF-IR-mteracting proteins In such an assay, GSH-Agarose is used to immobilize a GST-tusion protein. Binding ol a radiolabeled interacting protein ( ^,S S- Met-labeled in vitro translated protein or ³² P-labeled-vιa protein kinase A-GST tusion protem, cleaved with thrombm, and punfied lrom GST) would be detected and/or measured by scintillation counting For large-scale, rapid-throughput screening, purified proteins are used in a 96-well plate tormat. Such an assay uses puπtied GST lusion proteins and punfied biotinylated proteins Biounylated proteins are produced in E. coli using PinPoint vectors (Promega) by methods known to those ol skill. Protems υt interest are lused lυ a segment ot the bioun carboxylase earner protein which is bioytinylated in an E. coli strain expressing the gene lor biotin ligase, birA The assay utilizes neutravidin-coated plates to which a purified biotinylated protein is bound Binding ot a GST lusion interacting protein is detected by ELISA using a GST MAb. Conversely, GST fusion proteins are immobilized on 96-well plates. Biotinylated interacting protein in this embodiment is detected using a fluorochrome-tagged Streptavidin (Pierce). For both types of assays, inhibitors ot the interaction would score as a decreased signal (ELISA or fluorescence).

Suitable labels lor use in screening assays according to me mvenuon include a detectable label such as an enzyme, radioactive isotope, fluorescent compound, chemiluminescent compound, or biolummescent compound. Those ot ordinary skill in the art will know of ,tiier

2X

suitable labels or will be able to ascertain such using routine expeπmentation Furthermore, the binding υl these labels to the peptides is accomplished using standard techniques known in the art

A high speed screen tor agents that bind directiy to the Active Survival Domain may

employ immobilized or "tagged" combinatorial hbraπes Agents that bind specifically to such libraries are candidates to be tested lor their capacity to alter Active Survival Domain tuncuon. Depending upon the nature ot the alterauon, such agents would be usetul tor suppressing aberrant apoptosis in degenerative disorders or following ischemic m|ury, or in initiating or enhancing the apoptotic cascade in abeπant survival or proliferative disorders such as cancer For example, purified GST fusions to IGF-IR C-terminal Iragments are usetul lor the detection ot cellular proteins lhat interact with specific regions υt the C-lcrminus In υne exemplary embodiment, these proteins are used to screen an λ-ZAP (Stratagene) expression library (Skolnik, E. Y., et al. Cell 65: 83-90 ( 1991)) The λ-ZAP library is plated on E. coli, and resulting plaques are transleπed to isopropyl- β-D-thiogalactυside (IPTG)-ιmpregnated nitrocellose filters to induce protein expression. Expressed proteins are probed lor IGF-IR C- terminus interaction usmg ^,2 P-radιolabelcd GST fusion proteins or unlabled GST lusion proteins capable ot detection by a MAb to GST Alternatively, GST fusions could be immobilized on Glutathione (GSH)-Agarose columns to capture interacting protems lrom cell lysates Cell lysates ot FL5.12 cells stabily expressing the IGF-IR, with and without IGF-1 sumulation, are passed over the column, followed by washing. Interacting proteins remaining bound to the column can be co-eluted with the GST fusion protem using tree GSH, or without the GST- lusion protein using a salt, pH, guanidine HCl, or detergent gradient Interacting proteins are analyzed by, lor example, SDS-PAGE and silver staining, or by lodmauon, SDS-PAGE, and autoradiography Idcntificauon ot an interacung protein is accomplished, tor example, by SDS-

PAGE, excision ot the band, and microsequencing ot the N-termmus 11 necessary, the gene encoding the protein ol interest is cloned by screening a cDNA library with a radiolabeled DNA probe corresponding to the N-terminal sequence

Another exemplary method ol identifying interacting proteins accordmg to me mvenuon is to measure an enzymatic activity, such as a kinase, phosphotase or protease activity, associated with and/or required lor the tuncuon ot the IGF-IR. Such proteins may not interact directly or tightly with the receptor and may be missed in the screens described above. It a phosphatase or kinase is directed towards mutant or wt versiυns ot the IGF-IR C-terminus, this activity may be detected in vitro by, tor example, lysates Irom FL5.12/IGF-IR cells and Iragments ot the IGF-IR that are substrates tor the kmase or phυsphatase activity. Accυrding to this aspect ol the invention, then, in vitio kinase assays are earned out by incubating FL5.12/IGF-IR cell lysates with CF protein or fragments thereot in the presence ot ³² P-labelled ATP in lOmM Tπs butter containing lOmM MnCL and luM cold ATP. Phosphatase activity is detected by incubating tractionatcd FL5.12/IGF-IR cell lysate with phosphυrylated IGF-IR or Iragments thereot that have been immunoprecipitatcd trom cells, and following the ability of d e cell lysate to de-phosphorylate IGF-IR. Idenuficauon υt enzymatic activity in cell lysates lhat is specific tor a region υl the IGF-IR allows the turther isolation, puπticauon and sequencing ot the protein responsible tor this activity by standard biochemical methods such as, tor example, those descnbed in "Protein puπticauon: Principles and Practice," bv Robert Scopes (Ed: C. Cantor, Springer Verlag, Heidelberg, 1982)

Antibodies against the Active Survival Domain peptides of the invention may be used to screen cDNA expression libraries tor identifying clones containing cDNA inserts encodmg structurally related, immunocrossreacuve proteins which may be members of the Active Survival Domain lamily ot .otems Screening ot cDNA and mRNA expression libranes is

known in the art Similarly, antibodies against Active Survival Domain peptides are used to idenufy or puπly immunocrossreacuve proteins related to this domain, or to detect or determine the amount ol prυteins containing the Active Survival Domain in a cell or cell population, tor example, in tissue or cells, such as tumor cells or lymphocytes, obtained Irom a patient. Known methods lor such measurements include immunoprecipitation ot cell extracts tollowed by PAGE, in situ detection by immunohistochemical methods, and ELISA methods, all of which are well known in the art.

Modulation ot apoptosis according to the invention includes methods employing specific antisense polynucleotides complimentary to all or part ot the nucleotide sequences encoding agents which modulate Active Survival Domain lunction as disclosed herein Such complimentary antisense polynucleotides may include nucleotide additions, deletions, substitutions and transpositions, providing that specific hybridization to the target sequence persists Soluble antisense RNA or DNA oligonucleotides which can hybndize specifically to mRNA species encoding agents according to the invention, and which prevent transcnption ot the mRNA species and/or translation ol the encoded polypeptide, are contemplated as complimentary antisense polynucleotides according to the invention Production ot proteins agents as contemplated herein is inhibited by antisense polynucleotides according to me invention, and such antisense polynucleotides may inhibit apoptosis, senescence and the like, and or reverse the transformed phenotype ot cells. A heterologous expression cassette may be used to produce antisense polynucleoudes in a transtectant or transgenic cell Anusense polynucleoudes also may be administered as soluble oligonucleotides to the external environment ot the target cell, such as the culture medium ot cells in vitro or the interstitial fluid (e.g., via the circulatory system) in vivo. Anusense polynucleotides and their use are known to tfiose ot skill, and are described, tor example, in Melton, D.A.. Ed., Antisense RsA

and DNA. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( 1988).

Active Survival Domain mimetic agents are υf use in the therapeutic treatment of cancer and viral disease. Peptidomimetics of an Active Survival Domain peptide are also provided by the present invention, and can act as drugs for the modulation of apoptosis by, for example, blocking the function of proteins, preferably the IGF-IR, comprising the respective

Active Survival Domain. Peptidomimetics are commonly understood in the pharmaceutical industry tυ include non-peptide drugs having properties analogous to those υf those of tiie mimicked peptide. The principles and practices of peptidomimetic design arc known in the art and are described, for example, in Fauchere J., Adv. Drug Res. 15: 29 (1986); and Evans, et ai,

J. Med. Chem. 30: 1229 ( 1987). Peptidomimetics which bear structural similarity to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Typically, such peptidomimetics have one or more peptide linkages optionally replaced by a linkage which may convert desirable properties such as resistance to chemical breakdown in vivo. Such linkages may include -CH ₂ NH-, -CH S-, -CH -CH ₂ -, -CH=CH-, -COCH ₂ -, - CH(OH)CH ₂ -, and -CH.SO-. Peptidomimetics may exhibit enhanced pharmacological properties (biological half life, absoφtion rates, etc.), different specificity, increased stability, production economies, lessened antigenicity and the like which makes their use as therapeutics particularly desirable.

It is possible to employ the invention for detection or determination of proteins (or antibodies specific thereto) comprising an Active Survival Domain, for example, in fractions from tissue/organ excisions, by means of immunochemical or other techniques in view of the antigenic properties thereof. This is useful, for example, in the performance of tissue biopsies and olher histochemical procedures known to those of skill, including but not limited tυ paraffin embedment for immunohistochemistry. Immunization of anii αls with peptides comprising an

Active Survival Domain alone or in conroncuon with adjuvants by known methods can produce antibodies specific lor the Active Survival Domain peptide Antiserum obtained by conventional procedures may be utilized lor this puφose For example, a mammal, such as a rabbit, may be immunized with a pep de comprising an Active Survival Domain, thereby inducing the tormauon ot polyclonal antibodies thereagainst Monoclonal antibodies also may be generated using known procedures Such anubodies can be used accordmg to the invention to detect the presence and amount ot peptides compπsing an Active Survival Domam

It will be appreciated by those ot skill that the precise chemical structure of peptides compπsing an Active Survival Domain will vary depending upon a number of factors. For example, a given protein may be obtained as an acidic or basic salt, or in neutral torm, since lonizable carboxyl and ammo groups are found in the molecule For the puφoses ot the invention, then, any torm ot pepude comprising an Active Survival Domain which retains the therapeutic or diagnostic acuvity ol the naturally occurring peptide is intended to be within the scope ot the present mvenuon. The Acuve Survival Domain peptides and other compositions ot the present mvenuon may be produced by recombinant DNA techniques known m the art. For example, nucleotide sequences encoding Active Survival Domain peptides ot the invention may be inserted into a suitable DNA vector, such as a plasmid, and the vector used to transform a suitable host. The recombinant Active Survival Domam peptide is produced in the host by expression The transformed host may be a prokaryotic or eukaryotic cell. Preferred nucleotide sequences for this puφose encoding an Active Survival Domain are 1229-1337, Y1251 and H1293/K1294,

as shown in Figure 3.

Polynucleotides encoding peptides comprising an Active Survival Domain may be genomic or cDNA, isolated trom clone libraries by conventional methods including

hybridization screening methods. Alternatively, synthetic polynucleotide sequences may be constructed by known chemical synthetic methods for the synthesis of oligonucleotides. Such synthetic methods are described, for example, in Blackburn, G.M. and Gait, M.J., Eds., Nucleic Acids in Chemistry and Biology, IRL Press, Oxford, England (1990), and it will be evident mat commercially available oligonucleotide synthesizers also may be used according to the manufacturer's instructions. One such manufacturer is Applied Bio Systems.

Polymerase chain reaction (PCR) using primers based on the nucleotide sequence data disclosed herein may be used to amplify DNA fragments from mRNA pools, cDNA clone libraries or genomic DNA. PCR nucleotide amplification methods are known in the art and are described, for example, in Erlich, H.A., Ed., PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, New York ( 1989); U.S. Patent No. 4,683,202; U.S. Patent No. 4,800,159; and U.S. Patent No. 4,683, 195. Various nucleotide deletions, additions and substitutions may be incoφorated into the polynucleotides of the invention as will be recognized by those of skill, whυ will also recognize that variation in die nucleotide sequence encoding Active Survival Domain peptides may occur as a result of, for example, allelic polymorphisms, minor sequencing errors, and the like. The polynucleotides encoding Active Survival Domain peptides of the invention may include short oligonucleotides which are useful, for example, as hybridization probes and PCR primers. The polynucleotide sequences of the invention also may comprise a portion of a larger polynucleotide and, rough polynucleotide linkage, they may be fused, in frame, with one or more polynucleotide sequences encoding different proteins. In this event, the expressed protein may comprise a fusion protein. Of course, the polynucleotide sequences of the invention may be used in the PCR method to delect the presence of mRNA encoding Active Survival Domain peptides in the diagnosis of ( seasc or in forensic analysis.

cDNAs encoding protems which interact with an Active Survival Domain (or proteins containing such a domain) can be iden iied by screening cDNA expression libranes, employing known methods Examples ot such metiiods include the yeast two-hybπd system (Chien, et ai, Proc. Natl Acad Sci. 88: 9578 (1991)), and the E colilBCCP interactive screening system (Germino, et ai, Proc Natl Acad. Sci 90: 1639 ( 1993)) Suitable cDNA libraries will include mammalian cDNA libranes, such as human, mouse or rat, which may contain cDNA produced Irom RNA and a single cell, tissue or organ type or developmental stage, as are know in the art

A nucleotide sequence encoding a protein or peptide comprising an Active Survival Domain may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini tor ligation, restriction enzyme digestion to provide appropriate termini, filling in ot cohesive ends as appropnate, alkaline phosphatase treatment to avoid undesirable joining, and hga on with appropriate hgases Techniques for such manipulations are disclosed, lor example, by Sambrook, et al , supra, and arc well known

The sequence ol amino acid residues in a protein or peptide compπsing an Active Survival Domain is designated herein either through the use ot their commonly employed three-letter designauons or by their single-letter designations A listing ol these three-letter and one-letter designauons may be found in textbooks such as Lehninger, A , Biochemistry. 2d Ed, Worth Publishers, New York, New York (1975). When the amino acid sequence is listed hoπzontally, me ammo terminus is intended to be on the left end whereas the carboxy terminus is intended to be at die nght end. The residues ot amino acids in a peptide may be separated by hyphens. Such hyphens arc intended solely to facilitate the presentation ot a sequence. The rational design ol Active Survival Domain mimei-w or binding molecules, based

on modeled (or expeπmentally determined) pepude structure, may be earned out by those ot skill, using known methods ot rational drug design. Therapeuuc or prophylactic methods tor treating pathological conditions such as autoimmune disease, neurodegenerative disease, cancer and the like, are accomplished by the administration ot an effective amount ot a therapeutic agent capable ol specifically altering Active Survival Domain function, thereby modulating the biological activity υt Active Survival Domain containing proteins and the apoptotic state in a patient

Truncated IGF-IR molecules comprising an Active Survival Domain, disclosed herein, as well as other small peptide derivatives that constitute a "minimal" Acuve Survival Domain, are demonstrated herein to retain the apoptosis modulating tuncuon exhibited by wild-type IGF- IR These molecules, or peptidomimetic derivatives, may protect against apoptosis in cells by providing the same biological signal produced by IGF-IR. Such agents compπse a novel class ot therapeutic agent capable ot altecting the apoptotic state of a target cell

Any mode ol administration which results in the delivery ot the therapeutic agents ot the invention across the cell membrane and into the target cell is contemplated as within the scope of me present invention. The site ot administration and cells will be selected by one ot ordinary skill in the art based upon an understanding ot the particular degenerative disorder being treated. In addition, die dosage, dosage frequency, and lengm ot course ot treatment can be determined and optimized by one ot ordinary skill in the art depending upon the particular degenerative disorder being treated. The particular mode ot administration can also be readily selected by one of ordinary skill in the art and can include, tor example, oral, intravenous, subcutaneous, intramuscular, etc., widi die requirement that the therapeuuc agent cross me cell membrane. Pnnciples ot pharmaceuueal dosage and drug delivery are known and are descnbed, tor- example, in Ansel, H. C. anu Popovich, N G., Pharmaceutical Dosage Forms and Dru ^g

Delivery Systems. 5th Ed. Lea & Febiger, Pub , Philadelphia, PA (1990) It is possible, tor example, to utilize liposomes to specifically deliver the agents ot the invention. Such liposomes can be produced so that tiiey contain additiυnal biυactive compounds and the like such as drugs, radioisotopes, lectins and toxins, which would act at the target site. Suitable agents for use according to the invention include Active Survival Domain peptides and mimetics, Iragments, lunctional equivalents and/or hybrids or mutants thereof, as well as mutants, and vectors containing cDNA encoding any ot the foregoing. Agents can be administered alone or in combination with and/or concuπently with other suitable drugs and/or courses ot therapy The agents ol the present invention are suitable lor the treatment υl degenerative disorders, including disorders characteπzed by inappropπatc cell proliferation or inappropπate cell death or some cases, both Inappropπate cell proliferation will include the statistically significant increase in cell number as compared to the proliierauon of that particular cell type m the normal population Also included are disorders whereby a cell is present and/or persists in an inappropπate location, e.g., the presence of fibroblasts in lung tissue after acute lung injury. For example, such cells include cancer cells which exhibit the properties ot invasion and metastasis and are highly anaplastic. Such cells include but are not limited to, cancer cells including, tor example, tumor cells. Inappropπate cell death will include a statistically significant decrease in cell number as compared to the presence ot that particular cell type in the normal population. Such underrepresentation may be due to a particular degenerauve disorder, including, lor example, AIDS (HIV), which results in the inappropriate death of T-cells, and autoimmune diseases which are characteπzed by inappropriate cell death. Autoimmune diseases are disorders caused by an immune response directed against self antigens. Such diseases are characterized by the presence ot circulating autoanti bodies oi

cell-mediated immunity against autoantigens in conjunction with inflammatory lesions caused by lmmunologically competent cells or immune complexes in tissues containing the autoantigens. Such diseases include systemic lupus erythematosus (SLE), rheumatoid arthnus. Standard reterence works setting forth the general pπnciples ol immunology include Stiles, D. P., and Ten, A. I., Basic and Clinical Immunology, 7th Ed., Appleton & Lange, Publ.,

Norwalk, CT ( 1991); and Abbas, A. K.. et ai, Cellular and Molecular Immunology. W. B. Saunders Co., Publ., Philadelphia, PA ( 1991).

The Active Survival Domain peptides, mimetics, agents and the like disclosed herein, as well as vectors comprising nucleotide sequences encoding them or their corresponding antisense sequences, and hosts compπsing such vectors, may be used in the manufacture ot medicaments tor the trcaiment ot diseases.

Cells and non-human transgenic animals having one or more tunctionally impaired alleles encoding a protein comprising an Active Survival Domain may be generated using homologous targeting constructs trom genomic clones ot proteins compπsing an Active Survival Domain. Methods tor the production of homologous targeting constructs are known and described, for example, in Bradley, et ai, Bio/Technology 10: 534 (1992); and Koh, et ai, Science 256: 1210 (1992). For example, "knock-out" mice may be generated which are homozygous or heterozygous tor an inactivated allele of the IGF-IR or other protein compnsmg an Active Survival Domain by use of homologous targeting. Such mice are useful as research subjects for the investigation ot disease and tor other uses. Methods ot producing chimenc targeted mice are known and are described, for example, in Robertson, E.J., Ed., Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Washington, D.C. ( 1987), which also describes the manipulation ot embryonic stem cells. In addition, transgenes for expressing polypeptides comprising an Active S. i zival Domain at high levels

or under the contrυl ot selected transcription control sequences may be constructed using the cDNA or genomic gene of a protein comprising an Active Survival Domain Transgenes so constructed can be introduced into cells and transgenic non-human animals by known methods. Such transgenic cells and transgenic non-human animals may be used as screens lor agents which modulate apoptosis.

The invention may be appreciated in certain aspects with reference to the following examples, ottered by way ot illustration, not by way ot limitation

EXAMPLES

Expression plasmids:

The generation υt pBPV IGF-IR containing the wt IGF-IR and all ot the mutants used in this study has been described (Kato et ai, 1993, Miura et ai, 1995, Li et al , 1994, Miura et al., 1995b, Li et ai, submitted, Hongυ et ai, submitted, Surmacz, et ai, 1995) These IGF- IR cDNA constructs were released from a shutdc vector SK-IGF-IR (IGF-IR cDNA cloned into pBluescπpt SK, (Stratagene, La Jolla, CA) by digestion with Sal I and Xba I, and sub-cloned into the Xhol and Xba I site ot pcDNA3 (Invitrogen, San Diego, CA). The numbcπng ot ammo acids in the IGF-IR is that proposed by Ullrich et ai, 1986

Transfection of FL5.12 cells with IGF-IR containing plasmids:

FL5.12 cells were maintained in Iscove's modified defmed medium (IMDM) supplemented with I mM L-glutamine, 10% fetal bovine serum, and 10% (vol/vol) conditioned medium from the IL-3-producmg cell line WEHI-3B. Cells (5x10*) were transiectcd with 20 μg DNA by electroporation (200 V, 960 μF) or by lipofectamine (Gibco/BRL, Life Technologies,

Inc., Grand Island, NY) using 400 ng DNA, for 3.5 hours. Cells were seeded at 1 xlϋV mL (2 mL well) in 24 well plates in IMDM/10%FBS supplemented with 10% WEHI CM. G418 (geneticin, Gibco/ BRL Life Technologies Inc.) was added 48 hr later to a final concentration of 1 mg/mL. Medium was replenished every 3 to 4 days and emerging drug resistant cells were screened for IGF-IR expression by indirect immunofluorescence. Some υf the cell lines were sub-clυned by limiting dilution in 96 well plates. The FL5.12/Bcl-2 cell line has been previously described in co-pending United States application Serial Number 08/321,071, filed 1 1 October 1994, which is a continuation-in-part of United States application Serial Number 08/287,427, filed 9 August 1994, the disclosures of which are incorporated herein by reference.

Indirect Immunofluorescence Assays:

Cells (2x 10 ^s ) were suspended in IMDM containing 25 mM Hepes and 10% human pooled AB serum in 96 well round bottom plates. Anti-IGF-IR mAb (Ab- l, Oncogene Sciences, Cambridge, MA), was added at a final concentration of 1 ug/mL in a final volume of 100 μL and incubated for 1 hr at 20°C. Cells were washed three times and exposed to tluorescein-labelled (Fab') ₂ Iragments of goat Ig to mouse IgG for 30 min. at 4°C. Cells were again washed twice, and the cell-associated fluorescence was quantified using a FACSCAN flow cytometer (Becton Dickinson, San Jose, CA).

Cell cycle Analysis:

DNA content of cells was quantified using propidium iodide staining with the cellular DNA flow cytometric analysis reagent set (Boehringer Mannheim, Indianapolis, IL). FL5.12 cell lines transfected with IGF-IR, neo, or Bcl-2 were incubated in IMDM/5% FBS in the presence or absence υf IGF-I or WEHI CM for the indicated time periods. For DNA staining, 10" cells

were removed, washed once with PBS and fixed in ice cold 70% EtOH tor 10 min. Cells were washed 2 x with PBS, resuspended in 1ml ot PBS and treated with RNAse tor 30 min. at 37°C. Cells were chilled on ice and propidium lυdide was added. Fluorescence was immediately quantified on the FACSCAN, and the data were analyzed using the CellFit sottware (Becton Dickinson).

Cell viability Assays:

Cells were plated at 3 x lOVmL in medium containing IL-3 lor 24 hr, washed 3 times in serum free medium and plated at 5 x lO ^*5 cells/mL in IMDM containing 5% FBS (2ml/well) in 24 well plates. IGF-I (50ng/mL) or IL-3 (WEHI CM, 10%) was added to triplicate cultures. At the indicated time points 200 μl aliquots were removed trom each well and viability was determined by cυunting live and dead cells after trypan blue staining. The percentage viable cells was calculated from the total number of cells per well and all data represent the mean of triplicate cultures lor each condition.

IGF-IR B chain fragment constructs and modifications:

A series ot nucleotide sequences encoding the cytoplasmic domain ot the IGF-IR or fragments thereof was constructed by PCR amplification from full length IGF-IR. Each sequence was fused at the 3' end with the sequence for the 7 amino acid flag antigenic tag (International Biotechnologies, Inc., New Haven, CT). In a second version, each sequence was also fused at the 5' end to the sequence encoding the first 16 amino acids of SRC tused to the N-termmus to serve as a site for yristylation (My) and potential membrane anchorage (Resh, 1994 Cell 76: 411). In addition, constructs encompassing the entire cytoplasmic domain of the IGF-IR B chain (MyBF), or which included the kinase domain and _* J-termιnus (MyKCF), were

made. A tragment ot similar size to CF deπved lrom the Immunoglobulin kappa light chain sequence (MyV4BKF) and a lragment dcnved from the IGF-IR kinase domain (MyKC20) were made as controls.

The vanous IGF-IR B chain constructs indicating the IGF-IR ammo acids included are shown in Figure 6 as the version including the My and flag tag sequences

CF and MyCF were also constructed with the Y 1250/ 1251 mutations, the H1293F/K1294R mutations, the S1280- 1283A mutations, and with the combined mutations at mutation at Y 1250F/ 1251 F and H 1293F/K 1294R The CF-N and MyCF-N constructs were also made with the Y1250F/1251F mutation All ot the C-termmal constructs were expressed in the eucaryotic expression vector pcDNA 3 and die retroviral pBabe vector lor transient, constituuve or inducible expression in cells, and lor in vitro translation υl the proteins They were also cloned into the prokaryotic Glutatiuone S-transterase gene fusion vector pGEX-2TK (Pharmacia, Uppsala, Sweden) and expressed and purified as GST tusiυn proteins Some υt these cυnstructs were also synthesised as peptides wim or without modifications at the tyrosmes tor icrυ- injection studies and protein interaction studies.

Transient transfection assays:

Two cell lines were used to test CF and MyCF constructs tor function by transient transfection assays: the breast carcinoma cell line MCF-7, and R+ cells, which are fibroblasts derived irom the IGF-IR null mouse and have been transtected with the human IGF-IR (Sell et ai, 1994 ). The transient transfections were performed as previously descnbed (Miura et al , 1993). Cells were plated in 24 well plates at 4 x IO ⁴ cells/well in Dulbecco's modified essenual medium (DMEM) containing 10% FBS lor 18 hours. The C-tcrmmal tragment expression plasmids (400ng) or the pcDNA3 '. uor alone were transtected along with a marker plasmid

( 160 ng) encoding β -galactosidase by the lipofectamine procedure (Gibco/BRL, Life

Technologies). Four hours after transfection, medium cυntaimng 10% FBS, or IGF-I (50ng/mL) was added to the cells and incubation was continued for 24 or 48 hr. Cells were then fixed and stained widi X-gal to detect β -galactosidase in cells that received plasmid. The number of blue cells was counted by microsocopic examination and scored as live (flat blue cells) or dead (round blue cells). Cell killing in this assay is also manifested by a reduction in the number of blue cells obtained (Chittenden et ai, 1995). All transfections were performed in triplicate and the data arc presented as the mean and standard deviation υf three cultures.

IGF-I inhibits apoptosis in FLS.12 cells stably transfected with a wt IGF-IR:

FL5.12 cells are dcπved Irom a muπnc B lymphoblastoma, and are dependent on IL-3 for proliferation and survival in culture. In order to test the ability of the IGF-I IGF-IR pair to inhibit apoptosis induced by IL-3 wididrawal, FL5.12 cells were transtected with a human IGF-IR containing plasmid under the control of the CMV enhancer/ promoter. Cells expressing IGF-IR were selected by indirect immunofluorescence staining with an anti-IGF-IR mAb (Ab-

1 ). Cells expressing different levels of IGF-IR were sorted by FACS analysis and cultured under normal cυnditions. Interestingly, after a week or so in culture the level of IGF-IR increased on the lower expressing cells until all clones expressed similar levels (Fig. 3). FL5.12/IGF-IR cells were then analyzed for their viability upon IL-3 withdrawal in the presence of medium containing 5% FBS, or 5% FBS + IGF-I. The neo-expressing cells died rapidly upon IL-3-withdrawal (Fig. IA), and IGF-I provided a minimal survival effect, presumably due to the low levels of endogenous IGF-IRs. The FL5.12/IGF-IR cells demonstrated greater viability in the presence of 5% FBS alone compared with neo cells and in die presence of IGF- I, these cells exhibited viability comparable lo those in the presence of IL-3 over the time

period of the assay (Fig. IB). The survival signal in FBS is probably provided by the IGF-I or IGF-II present in FBS. The IGF-I protective effect in FL5.12/IGF-IR cells was of a similar magnitude to the anti-apoptotic signal provided by Bcl-2 over-expression in FL5.12 cells upon IL-3 withdrawal. Bcl-2 protection from IL-3-withdrawaI is shown in Fig. IC where it is compared with FL5.12/neo cells.

IGF-I provides a survival signal, not a proliferative signal in FL5.12 cells.

We next investigated whether IGF-I was replacing IL-3 as a mitogen for FL5.12 cells or whether it provided an anti-apoptotic signal only. Proliferation of FL5.12/IGF-IR cells was measured by counting the total cell number in cultures seeded at I x 10 ^s cells /mL in the presence of the same concentrations of IGF-I or IL-3 as those used for survival assays. The cells in IGF-I did not demonstrate a significant increase in cell number in the presence of IGF-I compared with cells seeded in the presence of IL-3 (Fig. I D). In order to determine the cell cycle distribution of FL5.12/IGF-IR cells maintained in the above assay conditions, the DNA content was analyzed by propidium iodide (Fig. 2). The percentage of cells in each stage of the cell cycle is shown above the histogram peaks. At 24 hr of IL-3 withdrawal, FL5.12/IGF-IR cells in the presence of IGF-I demonstrate progression from S phase through to GVM phase, but there do not appear to be any new cells entering S phase (Fig. 2A). By 48 hours, 87% of the cells are situated in the G. G, phase, widi <10% of cells located in S or GVM phases (Fig. 2B). The FL5.12/IGF-IR cells at 48 hr have a similar distribution to that of the FL5. l2/Bcl-2 cells cultured without IL-3 at 48 hours (Fig. 2C). This is in contrast to the FL5.12/IGF-IR cells cultured in die presence of IL-3 at 48 hours, which are distributed diroughout all stages of the cell cycle. Altogether these two assays demonstrate tiiat the expression of IGF-IRs in FL5.12

cells results in the cells responding to IGF-I with an anti-apoptotic stimulus, but not with a mitogenic stimulus, when cell death is induced by IL-3 withdrawal

Expression of point mutants of the IGF-IR in FL5.12 cells and analysis of their ability to protect FL5.12 cells from IL-3 withdrawal.

We sought to determine it a particular domain ot the IGF-IR is responsible tor mediating me anu-apoptotic effects ol IGF-I We were particularly interested in determining whether the anu-apoptotic signal is mediated by regions ot the receptor lhat arc ditterent trom the regions previously shown to be required tor its translormmg or prohlerative functions The locauon ot the vanous mutations in the IGF-IR is depicted in Fig 3 Constructs ot die IGF-IR containing mutations were transtccicd into FL5 12 cells, and clones expressing these receptors were selected by indirect immunofluorescence analysis with the mAb Ab- I Cells expressing the mutant receptors at approximately equivalent levels, shown in Fig 4A, were selected to be analyzed in survival assays tor the ability ol IGF-I to inhibit apoptosis induced by IL-3 witiidrawal The viability ol the cultures was monitored over 72 hours in a manner analogous to that descnbed tor wt IGF-IR in Fig 1 The survival data lor all ot the IGF-IR mutant data are summarized in Table I, where they are compared with the published results tor the prohlerative and transforming function υt the mutants

Mutant Y950F (Miura et al , 1995a) was tested tor survival function in FL5.12 cells because tyrosine 950 is required to bind to IRS-1 and SHC (Gustafson et al , 1995, Tartare- Deckert, et ai, 1995). The Y950F expressing cells demonstrated survival in the presence ot IGF-I quite comparable to that ot FL5.12 cells expressing wt IGF-IRs (Fig 4B) This suggests that interaction with IRS- 1 or SHC via this tyrosine 950 is not required lor IGF-IR inhibition ot apoptosis

To assess the requirement for kinase domain function, two mutations in the kinase domain were tested; one at the ATP binding lysine residue, K1003R (Kato et ai, 1993), and another in the tyrosine cluster where the three tyrosines residues 1 131, 1 135, and 1 136 are changed to phenylalanine (Li et ai, 1994). FL5.12 cells expressing the receptor mutated at lysine K1003 had negligible IGF-I-mediated protection from IL-3 withdrawal (Fig. 4B). The tyrosine cluster mulant shows a good IGF-I protective effect at 48 hr with 65%> of the cells retaining viability cυmpared to 20% with the K1003R mutant However, at 72 hours this effect is much diminished to 18% viability. This mutant has been shown to have no proliferative or transforming potential in fibroblasts (Li et ai, 1994), but it clearly demonstrates anti-apoptotic function, although it is impaired when compared to that provided by wt or Y950F receptors.

Five mutants having changes in the C-terminus υf ihc IGF-IR were analyzed. Tyrosines

1250 and 1251 were mutated singly or together tυ phenylalanine (Miura et ai, 1995b). Cells expressing the Y1250F receptor demonstrated IGF-I-mediated protection from IL-3 withdrawal at levels similar to that provided by wt receptors (Table I). In contrast, cells expressing the Y1251F mutation or the Y1250F/1251F double mutation had much diminished IGF-I protection from IL-3-withdrawal, with the effect being more pronounced in die double mutant (Fig. 4B). This shows that Y1251 is required for the survival function.

A mutant derived by replacing all four serines at 1280- 1283 with alanines, previously shown to have no transforming function, provided a receptor which retained IGF-I-mediated survival (Fig. 4B). Mutant H1293F/K1294R failed to mediate a survival signal upon IL-3 withdrawal (Fig. 4B). This mutant replaces two amino acids that are situated at the beginning of an eight amino acid stretch of basic residues; a sequence that is not shared with the insulin receptor. The survival curve (Fig. 4B) indicates that there is approximately 30% survival rernainint; at 48 hours, which is diminished to zero at 72 hours. This suggests that these

residues contribute to IGF-IR-mediated inhibition of apoptosis. The last point mutant to be analyzed m the C-terminus was Y1316F. This mutant had an intermediate effect in that it retained protection from IL-3 withdrawal, but tiie degree ot protection was significantly reduced trom that ot wt receptors. Altogether, analysis ot the C-termmal mutants in FL5.12 cells suggests that domains required for inhibition ot apoptosis are partially overlapping with, but separable trom, those required for transformation.

Expression of truncation mutants of the IGF-IR in FL5.12 cells and analysis of their ability to protect from IL-3- withdrawal:

A seπes ot C-termmal truncation mutants ot the IGF-IR was also expressed in FL5.12 cells for the ability to protect FL5.12 cells trom IL-3 withdrawal. Mutants were truncated immediately below the kinase domain (1229d), 6 ammo acid residues in tront of the Y1251 residue (1245d), and immediately m front υt the H1293/K1294 residues (1293d). The truncated receptors 1229d and 1245d are expressed at lower levels than all of die other mutant or wt receptors (compare Fig. 5A with Fig. 5B). As discussed above, the level of IGF- 1 -mediated protection trom apoptosis is correlated with die levels ot receptor expression tor wt IGF-IR. Efforts to obtain higher levels of expression of the 1229d and 1245d mutants by transfection or sub-cloning were not successful. However, several clones of FL5.12 cells expressing each truncated receptor were anlayzed for IGF-I-mediated protection trom IL-3 withdrawal.

IL-3 withdrawal assays were performed as described above and the survival curves are shown in Fig. 5. All three of the truncated receptors showed IGF-I-medialed protection from IL-3- withdrawal. This was an unexpected finding in view ot the above results with the Y 125 IF and H1293F/K1294R point mutants, which are located witiiin the dc ied portion ot die I245d

and 1293d IGF-IRs, respectively, and appear to be required for inhibition of apoptosis (Fig. 5).

Interestingly, cells expressing the 1229d and 1245d mutants, characterized by very low levels υf IGF-IR compared widi cells expressing the wt or point mutants analyzed above, exhibit less cell death in 5% FBS alone and more IGF-I protection from IL-3 withdrawal than cells expressing receptor mutant Y1250F or the tyrosine cluster mutant. The truncated mutants 1229d and 1245d, therefore, appear to have enhanced anti-apoptotic function compared with wt IGF- IR.

Table I. Summary of mitogenic, transforming, and anti-apoptotic function of IGF-IR mutants.

Receptor Mitogenic ¹ Transforming* Aim- Apoptotic"

WT +++ +++ +++

Y950 F - +++ 1003 -

Y1 131 , 1 135, 1 136 F - ++

Y1250F +++ +-H- +++

YI251F +++ +/-

Y 1250/1251F -H-+

S 1280-1283 A +++

H 1293 F/ K 1294 R +++

Y 1316 F -H-+ +++ del. 1229 +++ ddeell.. 11224455 ++++++ - +++ del. 1293 ++ ++ +++

' data deπved from references (Coppola et al., 1994, Li el al., 1994, Surmacz et al., 1995, Miura el al., 1995 a and b, Hongo ct al., 1996, Li et al., submitted.) ^h dala summarized from IGF- 1 -mediated protection afforded by mutant IGF-IRs in IL-3 withdrawal assays with

FL5.12 cells.

4X

Fragments of the C-terminus of the IGF-IR are cytotoxic when transiently transfected into cells:

The results described above indicate that the IGF-IR when truncated in the C-terminus has enhanced anti-apoptotic function, suggesting that the C-terminus has a regulatory role or an inhibitory effect on IGF-I-mediated survival. To test this possibility further, DNA fragments of the C-terminus fused to the DNA encoding the 7 amino-acid flag tag were PCR amplified from the IGF-IR and cloned inio the pcDNA-3 expression plasmid for transfection into cells.

In order to facilitate a potential requirement of membrane anchorage for function a 15 amino acid sequence from SRC encoding die site for myristylation was added to the N-terminus of the CF fragment (MyCF). A third fragment included the kinase domain (MyKCF). These constructs were transiently transfected into MCF-7 cells and R+ cells along with a marker plasmid encoding β-galactosidase. At 24 hr and 48 hr post transfection die cells were stained with X-gal and die number of blue cells were counted by microscopic examination and scored as viable or dead. The data for MCF-7 cells cultured in the presence of IGF-I for 48 hr after transfection are shown in Fig. 7 and indicate that the MyCF fragment transfection results in 75% fewer viable cells than transfection with the pcDNA3 vector alone or transfection with the CF and

MyKCF-containing vectors. This is indicative of toxicity to the MCF-7 cells and further suggests that die C-terminal fragment needs to be membrane-anchored to elicit mis toxicity. To establish that these proteins were expressed in die cells, they were immunoprecipitated widi the anti-flag tag mAb from MCF-7 cells 24 hr after transfection and detected by Western blotting with the same antibody. CF and MyCF can be seen in Fig. 7B. Under these conditions the

MyKCF co-migrated with Ig at -50 KD and is not detectable. The results of transient transfection in R+ cell is shown in Fig. 8 where the cells were cultured in the presence of FBS or IGF-I at .. transfection. MyCF is toxic when the cells were cultured in the presence of eititer

FBS υr IGF-I, whereas CF is more toxic to cells cultured in the presence υt IGF-I This suggests that the CF tragment is specifically inhibitory to cells that are dependent on IGF-I tor survival. Altogether these data indicate that the C-terminus ot the IGF-IR has a direct cytotoxic effect on cells when it is transiently transtected into them Similar transient translection assays using MyKC20, MyCF-N, MyCF-mid, and MyCF-N compared wim the tull length MyCF molecule showed that the MyCF-N tragment is almost as toxic as the parent MyCF tragment By contrast, the MyCF-mid and MyCF-C fragments are much less toxic than the lull length MyCF molecule, and in this respect, are comparable in their toxicity to the MyKC20 tragment (Fig. 9). The MyCF-N tragment with the Y1250F/1251F mutation has a very slight reversal υt tυxicity compared with MyCF-N In transient translations with tull length MyCF containing the mutations at Y1250FY 1251F, H1293F/K1294R or S1280- 1283 A, partial reversion ol cytotoxicity was also observed Expeπments to prepare constructs which contain two or three of these mutations together are ongυing to confirm their expected inactivation ot killing lunction. There are several possibilities tor the mechanism ot action ot the C-termmal Iragments.

IGF-IRs that are truncated in the C-terminus have enhanced anti-apoptυtic function, suggesting that the C-termmus has a negaUve regulatory role in this lunction The C- terminus may recruit or interact with proteins (such as phosphatases) diat dampen the anti-apoptotic function ot the receptor. While not intending to be bound by any particular tiieory, over-expression of the C- terminal fragments in die cell could result in an increased recruitment or activation ot these negative regulatory molecules, and thereby inactivate the anti-apoptotic function of endogenous receptors. Otiier possibdities for die cytotoxic function of the C-terminal fragments include tiiat they could bind to and prevent positive anti-apoptotic signaling molecules trom interacting widi endogenous IGF-IRs.

Immunoprecipitation of die CF υr MyCF Iragments tiiat have been transiently transfected into R ⁺ cells followed by western blotting with anti-phosphotyrυsine antibody demonstrated dial these molecules are phosphorylated on tyrosine. However, it is not known at this point if the phosphorylation is required for tiieir action. Potential serine phosphorylation and effects these molecules may have υn the kinase activity, tyrosine or serine phosphorylation of endogenous

IGF-IRS in R+ cells is subject to ongoing investigation. In addition, experiments are underway to determine it ^' they interact with endogenous IGF-IRs or otiier proteins by immunoprecipitation

In order to facilitate die transport of the C-terminal proteins across cell membranes, they were fused to the third domain of the antennapedia homeodomain (Derossi, D. E al., 1994 J. Biol. Chem. 269: 1044). These constructs were made by modifying pGEX-2TK by inserting die

(My) (SRC sequence fragment), the Hag tag, and me Antennapedia (Ap) sequence into the cloning region. The sequences for the various IGF-IR fragments were then cloned in between the sequences for (My) and flag. The resulting plasmids produced C-terminal Iragments widi

GST fused to the N-terminus of My and Ap fused to the C-terminus of flag. A linker region between the GST and fused domains contains a thrombin cleavage site and a protein kinase A site for ^,2 P labeling of the constructs. Such a MyCFAp construct is depicted in Figure 10, and this template vector can be used by those of skill to insert any DNA sequence to conveniently produce modified proteins with or without My, flag or Ap.

Other non-limiting contemplated modifications of these proteins include the addition of different membrane localization sequences such as a CAAX sequence for farnesylation or the putative membrane localization sequence from the C-terminus of Bcl-2 or otiier members of this family. Tyrosine phosphorylation of GST-fusion proteins can be accomplished by expression in E. coli strain TKXl (Stratagene) which expresses an inducible protein tyrosine kinase derived from the ELK protein (Letwin, K., et al., 1988 Oncogene 3: 621). Such fusion proteins could

be linked to mυnoclυnal υr other antibodies for delivery to cells or for use as ligands on affinity columns to purify interacting proteins. GST fusion proteins of the peptides could also be used in affinity columns. The various purified proteins will also be useful for in vitro assays such as kinase and phosphatase assays to measure the function of interacting proteins or their effect on full length IGF-IR that has been immunoprccipitated from cells.

Production of Monoclonal Antibodies to IGF-IR C-terminus:

The IGF-IR C terminal fragment (CF) consisting of amino acids 1225 to 1337 fused to the flag tag at its C terminus was cloned into the prokaryotic GST Fusion vector resulting in expression of a protein consisting of glutathione S-transferase (GST) fused to the N-terminus of CF. GST-CF was purified from E. coli using GSH affinity chromatography. Eight week old CAFI/J mice were immunised intraperitoneally (IP) with 30ug of GST-CF in PBS, boosted IP on day 4 and day 6 with 50 ug or GST-CF, and boosted two more times intravenously with 100 ug of GST-CF υn days 13 and 17. On day 20 mouse spleen cells were fused with the myeloma cell line p3X63/AG8.653 and hybridomas were selected in HAT medium and sub¬ cloned by limiting dilution. Supernatants from surviving cells were tested in ELISA assays for reactivity widi GST or with GST-CF. Those not reacting with GST were further tested in western blots for reactivity with endogenous native IGF-IR immunoprecipitated from FL5.12/IGF-IR cells using the Ab- 1 monoclonal antibody. Hybridoma supernatants were also tested against cell lysates from F15.12/IGF-IR cells. Those found to react with a band at approximately 95KD (IGF-IR B subunit migration under PAGE reducing conditions) were selected for further sub-cloning. For these analyses a commercially available polyclonal antisera raised against a synthtetic peptide coπesponding to amino acids 1347-1366 (Santa Cruz Biotechnology, Santa Cruz, CA) was used in western blots as a positive control. The CF-

directed mAbs are also being tested in western blots of GST-CF protein, in immuprecipitations of native IGF-IR from cells or in vitro translated CF protein. The MyKC20 construct is being used a negative control for antibodies that cross-react with part of the IGF-IR kinase domain. A panel of these monoclonal antibodies is currently being assembled. Epitope mapping of the CF mAbs is being caπied out by testing the antibodies for reactivity with smaller fragments of CF (CF- N, CF-mid, CF-C, CF 29, and CF 62) and with mutant CF fragments (Y1250F/Y1251F, S1280-1283A, and H1293F/K1294R) by western blotting with these proteins purified as GST fusions or by western blotting on anti-flag tag immunoprecipitation of ese constructs expressed in cells by transient transfection. These analyses will determine which part of the IGF-IR C-terminus antibodies are reacting with and identify a monoclonal antibody reacting with an active survival domain.

Antibodies reacting wim specific region of CF are useful for CF detection in biochemical studies with CF and endogenous IGF-IR or the interacting proteins. These antibodies are also useful for immunohistochemical studies to detect IGF-IR or Active Survival Domains of IGF-IR in cells and tissue specimens, including frozen or paraffin-embedded tissue sections. Anti-CF mAbs are also being tested by micro-injection techniques for modulating the function of CF or endogenous IGF-IR in cells. Survival domain reactive mAbs could potentially inhibit or activate the survival of cells.

IGF-IR C-terminal fragments inhibit transformation in vitro and induce apoptosis in vivo when stably expressed in ovarian carcinoma cells.

The ovarian carcinoma cell line CaOV-3 was stably transfected with CF, MyCF, or MyKCF constructs. Growth in liquid culture was normal. Cells were plated as single cells in soft agar and colonies were counted after two weeks growth.

Table II. Colony formation in soft agar by CaOV-3 cells.

CaOV-3 iraπsicctants (clone #) No. of colonies (triplicate cultures ) vector 121 , 125, 1 17 CF #12 312, 318, 292 CF #13 22, 12, 17 CF # 18 2, 0, 0 MyCF # 9 12, 7, 1 1

MyCF # 12 1 , 0, 0 MyCF # 14 0, 0, ^' 0 MyKCF # 2 12, 9, 5 MyKCF # 3 10, 9, 7 MyKCF # 9 58, 50, 45

CaoV-3 cells expressing IGF-IR C-tcrminal fragments were inoculated (5 x 10 ^s ) in bio- diffusion chambers and the chambers were implanted in the sub-cutaneous tissue of rats (Rescinoff, M., et ai. Cancer Res. 55: 3739-3741 (1995b)). At 24 hr. the chambers were removed and the cells recovered and counted. The data are presented as a precentage of cells inocculated.

Table III. Survival of CaOV-3 cells in Bio-diffusion chambers in vivo.

CaOV-3 tra sicctaπt (clone #) Percent cells recovered vector 212.3

MyKCF #2 197.5 MyKCF # 3 185.0

MyKCF # 9 230.0

MyCF # 9 1.4

MyCF # 12 1.0

MyCF # 14 0.4

The mechanism of CF and MyCF inhibition of CaOV-3 cells is the subject of ongoing investigation. Although not intending to be bound by any particular theory, potential mechanisms could include specific inhibition of IGF-IR C-terminal signalling when cells arc cultured under conditions in which they a.. dependent upon these signals.

Identification of proteins that interact with IGF-IR C-terminus by yeast two-hybrid analysis: The IGF-IR cytoplasmic domain (starting at amino acid 931) and an identical construct

containing the mutation Y950F were cloned into the yeast two-hybrid bait vector pAS2 (O'Neill et al, 1994 Mol. Cell Biol. 14: 6433). Constructs cloned into diis vector are expressed as fusion proteins widi the GAL-4 DNA binding domain and a hemagglutinin (HA) epitope tag. The IGF-

IR cytoplasmic region with the mutated Y950 was used so as to exclude d e interaction of known substrates (IRS-1 and SHC) from interacting with the bait Both of these kinase domain- containing constructs could be phosphorylated when expressed in yeast as determined by immunoprecipitation with the anti-HA monoclonal antibody and western blotting with an antibody aga st phosphotyrosine. IGF-IR C-terminal Iragments (starting at amino acid 1225) fused to the flag tag antigenic epitope at their C-terminus (wt υr with the Y1250/1251F υr H1293F/K1294R mutations) were also cloned into die pAS-2 vector. All of the pAS-2 vector constructs are shown in Figure 1 1.

A B-cell library cloned into die pGAD GH vector containing the GAL 4 activation domain sequence was screened for interacting proteins widi die Y2H 950, and a HELA cell library was screened with the Y2H CF and Y2H CF 50/51 baits. This produced 21 HELA cell library- derived clones which interacted with Y2HCF and 31 HELA cell library-derived clones which interacted withY2HCF 50/51. Each of these clones was re-mated with vector, Y2H CF, or Y2H CF (50/51). In ongoing experiments, each of these clones is also being re-mated with Y2H CF (93/94),

Y2H (1280-83), Y2H wt and Y2H 950. Comparing the ability of the different interacting proteins obtained to interact with die different baits will provide further insight into the interacting domain of a bait. For example, if certain interacting proteins interact wilh die mutant constructs (Cf- 50/51, CF 1280-83 or CF 93/9) but not widi wt constructs, then this result would

map the site ot interaction to the mutated residues. Proteins that would interact with Y2H CF construct, but not with the Y2H wt construct, require an inactive kinase or dephosphorylation of key residues for tiieir interaction. On the other hand, further screening of die HELA library for proteins that interact with the Y2H wt bait but not with the Y2H CF baits, will allow the identification of interacting proteins which represent interacting proteins requiπng the kinase activity of the IGF-IR and possible autophosphorylation of residues in the C-terminus. Such genetic analysis with die different IGF-IR constructs of die invention will help to further define the site of interaction and die biological relevance of the interacting proteins to the function of Active Survival Domains or for the cytotoxicity of the C-tcrminal fragments.

Yeast DNA puπfied from interacting clones obtamed widi baits Y2H CF and Y2H CF 50/51 was translormcd into E. coli and plasmid DNA was prepared for sequencing. A summary of the homologous genes identified to date is shown in Table IV.

Table IV. IGF-IR C-terminus interacting protein gene homologues identified by yeast two-hybrid analysis.

Clone number Homology identified Genbank accession number

M'64 Human calmodulin-dependent protein gb/L 14778/HUMCALAA phosphatase subunit (PPP3CA or calcineuπn) gb/M29275/RATCNRA

(identical)

M68 Homo sapiens Uba 80 mRNA lor ubiquitin emb/X63237/HSUBA80R

(identical)

Human ubiquitin mRNA 3 ^* end gb M10939/HUMUBCP

M9 yx70h05.rl Homo sapiens cDNA clone gb/N31805

(identical)

M4 Homo sapiens cDNA clone yc22h03.sl gb/T63506

(identical)

M30 Human fetal brain cDNA 5' end Gen-25G1 1 dbj/D59384/HUM025Gl 1 B W"93 Homo sapiens cDNA clone yu 22 aOS.Rl gb H78414

(identical)

W45 Human Tumor antigen (L6) mRNA complete gb/M90657/HUML6A W22 yc()5h06.rl Homo sapiens cDNA clone gn/T64108

(partial)

W10 Homo sapiens mitochondrial DNA for loop emb/X89832/ F ;SLAS44 attachment sequence (identical)

W24 Human mRNA tor elongation factor 1 alpha emb/X03558/H86225 subunit (EF-1 ) (identical)

W31 Homo sapiens CpG DNA, clone 124b4 emb/Z59071/HS124B4R Human tctal brain cDNA 5' end GEN- 1 b| D53306 HUM 1 5D09B W37 Human ribosomal protein Ll 1 homologue gb/L05092 mRNA, 5'end (identical)

"clones originally lound to interact with Y2H CF 50/51 "clones originally lound to interact wilh Y2H CF

In ongυing expeπments, CF-interacting genes identified in Table IV with unknown function are being searched for homology to known sequences associated with kinase or phosphatase activity, protein interaction motifs such as SH2 or SH3 binding domains, sequences asociated with enzyme activity such as proteases, or homology to known signal transduction proteins. Genes with known function, such as calcineuπn, are being tested for their functional requirement lor survival domain activity This will include transtcr υl these genes into expression vectors lor inducible or constitutive expression in cells, followed by assays tor the effect of this expression on the anti-apoptotic function of the cells. These proteins are also being expressed in bactcna for in vitro biochemical assays such as modulation υl IGF-IR kinase activity υr dephosphorylation of key residues in the receptor. These studies should define the mechanism υt action of the active survival domain and its dependence υn a key interacting protem. From diese expeπments the inventors hope to design biochemical assays that will modulate the actvity of one or more key interacting proteιn(s) required tor IGF-IR survival function, or will modulate its interaction with or activity towards key residues in the IGF-IR.

All publications mentioned in this specification are herein incoφorated by reference, to the same extent as if each individual publication was specifically and individually indicated to be incoφorated by reference. It will be understood that the mvention is capable of further modifications and this application is intended to cover any variations, uses, or adoptions of the invention including such departures from the present disclosure as come within known or

customary practice within the art to which the invention pertains, and is intended to be limited only by the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: IMMUNOGEN, INC. and

THOMAS JEFFERSON UNIVERSITY

(ii) TITLE OF INVENTION: ACTIVE SURVIVAL DOMAINS OF IGF-IR

AND METHODS OF USE

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: HALE and DORR LLP

(B) STREET: 1455 Pennsylvania Avenue, N.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: USA

(F) ZIP: 20004

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOΞ

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT - TO BE ASSIGNED

(B) FILING DATE: 01-APR-1997

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/625,819

(B ) FILING DATE : 01 -APR- 1996

(C ) CLASSIFICATION :

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: WIXON, Henry N.

(B) REGISTRATION NUMBER: 32,073

(C) REFERENCE/DOCKET NUMBER: 104322.162

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 942-8459

(B) TELEFAX: (202) 942-8484

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4989 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 46..4149

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 136..4149

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : _{ττττττττττ} TTTTGAGAAA GGGAATTTCA TCCCAAATAA AAGGA ATG AAG TCT 54

Met Lys Ser -30

GGC TCC GGA GGA GGG TCC CCG ACC TCG CTG TGG GGG CTC CTG TTT CTC 1.02 Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu Leu Phe Leu -25 -20 -15

TCC GCC GCG CTC TCG CTC TGG CCG ACG AGT GGA GAA ATC TGC GGG CCA 150 Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu lie Cys Gly Pro -10 -5 1 5

GGC ATC GAC ATC CGC AAC GAC TAT CAG CAG CTG AAG CGC CTG GAG AAC 198 Gly lie Asp lie Arg Asn Asp Tyr Gin Gin Leu Lys Arg Leu Glu Asn 10 15 20

TGC ACG GTG ATC GAG GGC TAC CTC CAC ATC CTG CTC ATC TCC AAG GCC 246 Cys Thr Val lie Glu Gly Tyr Leu His lie Leu Leu lie Ser Lys Ala 25 30 35

GAG GAC TAC CGC AGC TAC CGC TTC CCC AAG CTC ACG GTC ATT ACC GAG 294 Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val lie Thr Glu 40 45 50

TAC TTG CTG CTG TTC CGA GTG GCT GGC CTC GAG AGC CTC GGA GAC CTC 342 Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu Gly Asp Leu 55 60 65

TTC CCC AAC CTC ACG GTC ATC CGC GGC TGG AAA CTC TTC TAC AAC TAC 390 Phe Pro Asn Leu Thr Val lie Arg Gly Trp Lys Leu Phe Tyr Asn Tyr 70 75 80 85

GCC CTG GTC ATC TTC GAG ATG ACC AAT CTC AAG GAT ATT GGG CTT TAC 438 Ala Leu Val lie Phe Glu Met Thr Asn Leu Lys Asp lie Gly Leu Tyr 90 95 100

AAC CTG AGG AAC ATT ACT CGG GGG GCC ATC AGG ATT GAG AAA AAT GCT 486 Asn Leu Arg Asn lie Thr Arg Gly Ala lie Arg lie Glu Lys Asn Ala 105 110 115

GAC CTC TGT TAC CTC TCC ACT GTG GAC TGG TCC CTG ATC CTG GAT GCG 534 Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu lie Leu Asp Ala 120 125 130

GTG TCC AAT AAC TAC ATT GTG GGG AAT AAG CCC CCA AAG GAA TGT GGG 582 Val Ser Asn Asn Tyr lie Val Gly Asn Lys Pro Pro Lys Glu Cys Gly 135 140 145

GAC CTG TGT CCA GGG ACC ATG GAG GAG AAG CCG ATG TGT GAG AAG ACC 630 Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys Glu Lys Thr 150 155 160 165

ACC ATC AAC AAT GAG TAC AAC TAC CGC TGC TGG ACC ACA AAC CGC TGC 678 Thr lie Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr Asn Arg Cys 170 175 180

CAG AAA ATG TGC CCA AGC ACG TGT GGG AAG CGG GCG TGC ACC GAG AAC 726 Gin Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys Thr Glu Asn 185 190 195

AAT GAG TGC TGC CAC CCC GAG TGC CTG C."' AGC TGC AGC GCG CCT GAC 774 As ^' n Glu Cys Cys His Pro Glu Cys Leu GJ./ Ser Cys Ser Ala Pro Asp 200 205 210

AAC GAC ACG GCC TGT GTA GCT TGC CGC CAC TAC TAC TAT GCC GGT GTC 822

Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr Ala Gly Val 215 220 225

TGT GTG CCT GCC TGC CCG CCC AAC ACC TAC AGG TTT GAG GGC TGG CGC 870 Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu Gly Trp Arg 230 235 240 245

TGT GTG GAC CGT GAC TTC TGC GCC AAC ATC CTC AGC GCC GAG AGC AGC 918 Cys Val Asp Arg Asp Phe Cys Ala Asn lie Leu Ser Ala Glu Ser Ser 250 255 260

GAC TCC GAG GGG TTT GTG ATC CAC GAC GGC GAG TGC ATG CAG GAG TGC 966 Asp Ser Glu Gly Phe Val lie His Asp Gly Glu Cys Met Gin Glu Cys 265 270 275

CCC TCG GGC TTC ATC CGC AAC GGC AGC CAG AGC ATG TAC TGC ATC CCT 1014 Pro Ser Gly Phe lie Arg Asn Gly Ser Gin Ser Met Tyr Cys lie Pro 280 285 290

TGT GAA GGT CCT TGC CCG AAG GTC TGT GAG GAA CAA AAG AAA ACA AAG 1062 Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Gin Lys Lys Thr Lys 295 300 305

ACC ATT GAT TCT GTT ACT TCT GCT CAG ATG CTC CAA GGA TGC ACC ATC 1110 Thr lie Asp Ser Val Thr Ser Ala Gin Met Leu Gin Gly Cys Thr lie 310 315 320 325

TTC AAG GGC AAT TTG CTC ATT AAC ATC CGA CGG GGG AAT AAC ATT GCT 1158 Phe Lys Gly Asn Leu Leu lie Asn lie Arg Arg Gly Asn Asn lie Ala 330 335 340

TCA GAG CTG GAG AAC TTC ATG GGG CTC ATC GAG GTG GTG ACG GGC TAC 1206 Ser Glu Leu Glu Asn Phe Met Gly Leu He Glu Val Val Thr Gly Tyr 345 350 355

GTG AAG ATC CGC CAT TCT CAT GCC TTG GTC TCC TTG TCC TTC CTA AAA 1254 Val Lys He Arg His Ser His Ala Leu Val Ser Leu Ser Phe Leu Lys 360 365 370

AAC CTT CGC CTC ATC CTA GGA GAG GAG CAG CTA GAA GGG AAT TAC TCC 1302 Asn Leu Arg Leu He Leu Gly Glu Glu Gin Leu Glu Gly Asn Tyr Ser 375 380 385

TTC TAC GTC CTC GAC AAC CAG AAC TTG CAG CAA CTG TGG GAC TGG GAC 1350 Phe Tyr Val Leu Asp Asn Gin Asn Leu Gin Gin Leu Trp Asp Trp Asp 390 395 400 405

CAC CGC AAC CTG ACC ATC AAA GCA GGG AAA ATG TAC TTT GCT TTC AAT 1398 His Arg Asn Leu Thr He Lys Ala Gly Lys Met Tyr Phe Ala Phe Asn 410 415 420

CCC AAA TTA TGT GTT TCC GAA ATT TAC CGC ATG GAG GAA GTG ACG GGG 1446 Pro Lys Leu Cys Val Ser Glu He Tyr Arg Met Glu Glu Val Thr Gly 425 430 435

ACT AAA GGG CGC CAA AGC AAA GGG GAC ATA AAC ACC AGG AAC AAC GGG 1494 Thr Lys Gly Arg Gin Ser Lys Gly Asp He Asn Thr Arg Asn Asn Gly 440 445 450

GAG AGA GCC TCC TGT GAA AGT GAC GTC CTG CAT TTC ACC TCC ACC ACC 1542 Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr Ser Thr Thr 455 460 465

ACG TCG AAG AΛI CGC ATC ATC ATA ACC TGG CAC CGG TAC CGG CCC CCT 1590 Thr Ser Lys Asn Arg He He He Thr Trp His Arg Tyr Arg Pro Pro 470 475 480 485

GAC TAC AGG GAT CTC ATC AGC TTC ACC GTT TAC TAC AAG GAA GCA CCC 1638 Asp Tyr Arg Asp Leu He Ser Phe Thr Val Tyr Tyr Lys Glu Ala Pro 490 495 500

TTT AAG AAT GTC ACA GAG TAT GAT GGG CAG GAT GCC TGC GGC TCC AAC 1686 Phe Lys Asn Val Thr Glu Tyr Asp Gly Gin Asp Ala Cys Gly Ser Asn 505 510 515

AGC TGG AAC ATG GTG GAC GTG GAC CTC CCG CCC AAC AAG GAC GTG GAG 1734 Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys Asp Val Glu 520 525 530

CCC GGC ATC TTA CTA CAT GGG CTG AAG CCC TGG ACT CAG TAC GCC GTT 1782 Pro Gly He Leu Leu His Gly Leu Lys Pro Trp Thr Gin Tyr Ala Val 535 540 545

TAC GTC AAG GCT GTG ACC CTC ACC ATG GTG GAG AAC GAC CAT ATC CGT 1830 Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu Asn Asp His He Arg 550 555 560 565

GGG GCC AAG AGT GAG ATC TTG TAC ATT CGC ACC AAT GCT TCA GTT CCT 1878 Gly Ala Lys Ser Glu He Leu Tyr He Arg Thr Asn Ala Ser Val Pro 570 575 580

TCC ATT CCC TTG GAC GTT CTT TCA GCA TCG AAC TCC TCT TCT CAG TTA 1926 Ser He Pro Leu Asp Val Leu Ser Ala Ser Asn Ser Ser Ser Gin Leu 585 590 595

ATC GTG AAG TGG AAC CCT CCC TCT CTG CCC AAC GGC AAC CTG AGT TAC 1974 He Val Lys Trp Asn Pro Pro Ser Leu Pro Asn Gly Asn Leu Ser Tyr 600 605 610

TAC ATT GTG CGC TGG CAG CGG CAG CCT CAG GAC GGC TAC CTT TAC CGG 2022 Tyr He Val Arg Trp Gin Arg Gin Pro Gin Asp Gly Tyr Leu Tyr Arg 615 620 625

CAC AAT TAC TGC TCC AAA GAC AAA ATC CCC ATC AGG AAG TAT GCC GAC 2070 His Asn Tyr Cys Ser Lys Asp Lys He Pro He Arg Lys Tyr Ala Asp 630 635 640 645

GGC ACC ATC GAC ATT GAG GAG GTC ACA GAG AAC CCC AAG ACT GAG GTG 2118 Gly Thr He Asp He Glu Glu Val Thr Glu Asn Pro Lys Thr Glu Val 650 655 660

TGT GGT GGG GAG AAA GGG CCT TGC TGC GCC TGC CCC AAA ACT GAA GCC 2166 Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys Thr Glu Ala 665 670 675

GAG AAG CAG GCC GAG AAG GAG GAG GCT GAA TAC CGC AAA GTC TTT GAG 2214 Glu Lys Gin Ala Glu Lys Glu Glu Ala Glu Tyr Arg Lys Val Phe Glu 680 685 690

AAT TTC CTG CAC AAC TCC ATC TTC GTG CCC AGA CCT GAA AGG AAG CGG 2262 Asn Phe Leu His Asn Ser He Phe Val Pro Arg Pro Glu Arg Lys Arg 695 700 705

AGA GAT GTC ATG CAA GTG GCC AAC ACC ACC ATG TCC AGC CGA AGC AGG 2310 Arg Asp Val Met Gin Val Ala Asn Thr Thr Met Ser Ser Arg Ser Arg 710 715 720 725

AAC ACC ACG GCC GCA GAC ACC TAC AAC ATC ACC GAC CCG GAA GAG CTG 2358 Asn Thr Thr Ala Ala Asp Thr Tyr Asn He Thr Asp Pro Glu Glu Leu 730 735 740

GAG ACA GAG TAC CCT TTC TTT GAG AGC AGA GTG GAT AAC AAG GAG AGA 2406 Glu Thr Glu Tyr Pro Phe Phe Glu Ser Arg Val Asp Asn Lys Glu Arg 745 750 755

ACT GTC ATT TCT AAC CTT CGG CCT TTC ACA TTG TAC CGC ATC GAT ATC 2454 Thr Val He Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg He Asp He 760 765 770

CAC AGC TGC AAC CAC GAG GCT GAG AAG CTG GGC TGC AGC GCC TCC AAC 2502 His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser Ala Ser Asn 775 780 785

TTC GTC TTT GCA AGG ACT ATG CCC GCA GAA GGA GCA GAT GAC ATT CCT 2550 Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly Ala Asp Asp He Pro 790 795 800 805

GGG CCA GTG ACC TGG GAG CCA AGG CCT GAA AAC TCC ATC TTT TTA AAG 2598 Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn Ser He Phe Leu Lys 810 815 820

TGG CCG GAA CCT GAG AAT CCC AAT GGA TTG ATT CTA ATG TAT GAA ATA 2646 Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu He Leu Met Tyr Glu He 825 830 835

AAA TAC GGA TCA CAA GTT GAG GAT CAG CGA GAA TGT GTG TCC AGA CAG 2694 Lys Tyr Gly Ser Gin Val Glu Asp Gin Arg Glu Cys Val Ser Arg Gin 840 845 850

GAA TAC AGG AAG TAT GGA GGG GCC AAG CTA AAC CGG CTA AAC CCG GGG 2742 Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu Asn Pro Gly 855 860 865

AAC TAC ACA GCC CGG ATT CAG GCC ACA TCT CTC TCT GGG AAT GGG TCG 2790 Asn Tyr Thr Ala Arg He Gin Ala Thr Ser Leu Ser Gly Asn Gly Ser 870 875 880 885

TGG ACA GAT CCT GTG TTC TTC TAT GTC CAG GCC AAA ACA GGA TAT GAA 2838 Trp Thr Asp Pro Val Phe Phe Tyr Val Gin Ala Lys Thr Gly Tyr Glu 890 895 900

AAC TTC ATC CAT CTG ATC ATC GCT CTG CCC GTC GCT GTC CTG TTG ATC 2886 Asn Phe He His Leu He He Ala Leu Pro Val Ala Val Leu Leu He 905 910 915

GTG GGA GGG TTG GTG ATT ATG CTG TAC GTC TTC CAT AGA AAG AGA AAT 2934 Val Gly Gly Leu Val He Met Leu Tyr Val Phe His Arg Lys Arg Asn 920 925 930

AAC AGC AGG CTG GGG AAT GGA GTG CTG TAT GCC TCT GTG AAC CCG GAG 2982 Asn Ser Arg Leu Gly Asn Gly Val Leu Tyr Ala Ser Val Asn Pro Glu 935 940 945

TAC TTC AGC GCT GCT GAT GTG TAC GTT CCT GAT GAG TGG GAG GTG GCT 3030 Tyr Phe Ser Ala Ala Asp Val Tyr Val Pro Asp Glu Trp Glu Val Ala 950 955 960 965

CGG GAG AAG ATC ACC ATG AGC CGG GAA CTT GGG CAG GGG TCG TTT GGG 3078 Arg Glu Lys He Thr Met Ser Arg Glu Leu Gly Gin Gly Ser Phe Gly 970 975 980

ATG GTC TAT GAA GGA GTT GCC AAG GGT GTG GTG AAA GAT GAA CCT GAA 3126 Met Val Tyr Glu Gly Val Ala Lys Gly Val Val Lys Asp Glu Pro Glu 985 990 995

ACC AGA GTG GCC ATT AAA ACA GTG AAC GAG GCC GCA AGC ATG CGT GAG 3174 Thr Arg Val Ala He Lys Thr Val Asn Glu Ala Ala Ser Met Arg Glu 1000 1005 1010

AGG ATT GAG TTT CTC AAC GAA GCT TCT GTG ATG AAG GAG TTC AAT TGT 3222 Arg He Glu Phe Leu Asn Glu Ala Ser Val Met Lys Glu Phe Asn Cys 1015 1020 1025

CAC CAT GTG GTG CGA TTG CTG GGT GTG GTG TCC CAA GGC CAG CCA ACA 3270 His His Val Val Arg Leu Leu Gly Val Val Ser Gin Gly Gin Pro Thr 1030 1035 1040 1045

CTG GTC ATC ATG GAA CTG ATG ACA CGG GGC GAT CTC AAA AGT TAT CTC 3318 Leu Val He Met Glu Leu Met Thr Arg Gly Asp Leu Lys Ser Tyr Leu 1050 1055 1060

CGG TCT CTG AGG CCA GAA ATG GAG AAT AAT CCA GTC CTA GCA CCT CCA 3366 Arg Ser Leu Arg Pro Glu Met Glu Asn Asn Pro Val Leu Ala Pro Pro 1065 1070 1075

AGC CTG AGC AAG ATG ATT CAG ATG GCC GGA GAG ATT GCA GAC GGC ATG 3414 Ser Leu Ser Lys Met He Gin Met Ala Gly Glu He Ala Asp Gly Met 1080 1085 1090

GCA TAC CTC AAC GCC AAT AAG TTC GTC CAC AGA GAC CTT GCT GCC CGG 3462 Ala Tyr Leu Asn Ala Asn Lys Phe Val His Arg Asp Leu Ala Ala Arg 1095 1100 1105

AAT TGC ATG GTA GCC GAA GAT TTC ACA GTC AAA ATC GGA GAT TTT GGT 3510 Asn Cys Met Val Ala Glu Asp Phe Thr Val Lys He Gly Asp Phe Gly 1110 1115 1120 1125

ATG ACG CGA GAT ATC TAT GAG ACA GAC TAT TAC CGG AAA GGA GGC AAA 3558 Met Thr Arg Asp He Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys 1130 1135 1140

GGG CTG CTG CCC GTG CGC TGG ATG TCT CCT GAG TCC CTC AAG GAT GGA 3606 Gly Leu Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu Lys Asp Gly 1145 1150 1155

GTC TTC ACC ACT TAC TCG GAC GTC TGG TCC TTC GGG GTC GTC CTC TGG 3654 Val Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp 1160 1165 1170

GAG ATC GCC ACA CTG GCC GAG CAG CCC TAC CAG GGC TTG TCC AAC GAG 3702 Glu He Ala Thr Leu Ala Glu Gin Pro Tyr Gin Gly Leu Ser Asn Glu 1175 1180 1185

CAA GTC CTT CGC TTC GTC ATG GAG GGC GGC CTT CTG GAC AAG CCA GAC 3750 Gin Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp Lys Pro Asp 1190 1195 1200 1205

AAC TGT CCT GAC ATG CTG TTT GAA CTG ATG CGC ATG TGC TGG CAG TAT 3798 Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys Trp Gin Tyr 1210 1215 1220

AAC CCC AAG ATG AGG CCT TCC TTC CTG GAG ATC ATC AGC AGC ATC AAA 3846 Asn Pro Lys Met Arg Pro Ser Phe Leu Glu He He Ser Ser He Lys 1225 1230. 1235

GAG GAG ATG GAG CCT GGC TTC CGG GAG GTC TCC TTC TAC TAC AGC GAG 3894 Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser Phe Tyr Tyr Ser Glu 1240 1245 1250

GAG AAC AAG CTG CCC GAG CCG GAG GAG CTG GAC CTG GAG CCA GAG AAC 3942 Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu Asp Leu Glu Pro Glu Asn 1255 1260 1265

ATG GAG AGC GTC CCC CTG GAC CCC TCG GCC TCC TCG TCC TCC CTG CCA 3990 Met Glu Ser Val Pro Leu Asp Pro Ser Ala Ser Ser Ser Ser Leu Pro 1270 1275 1280 1285

CTG CCC GAC AGA CAC TCA GGA CAC AAG GCC GAG AAC GGC CCC GGC CCT 4038 Leu Pro Asp Arg His Ser Gly His Lys Ala Glu Asn Gly Pro Gly Pro 1290 1295 1300

GGG GTG CTG GTC CTC CGC GCC AGC TTC GAC GAG AGA CAG CCT TAC GCC 4086 Gly Val Leu Val Leu Arg Ala Ser Phe Asp Glu Arg Gin Pro Tyr Ala 1305 1310 1315

CAC ATG AAC GGG GGC CGC AAG AAC GAG CGG GCC TTG CCG CTG CCC CAG 4134 His Met Asn Gly Gly Arg Lys Asn Glu Arg Ala Leu Pro Leu Pro Gin 1320 1325 1330

TCT TCG ACC TGC TGA TCCTTGGATC CTGAATCTGT GCAAACAGTA ACGTGTGCGC 4189 Ser Ser Thr Cys

1335

ACGCGCAGCG GGGTGGGGGG GGAGAGAGAG TTTTAACAAT CCATTCACAA GCCTCCTGTA 4249

CCTCAGTGGA TCTTCAGTTC TGCCCTTGCT GCCCGCGGGA GACAGCTTCT CTGCAGTAAA 4309

ACACATTTGG GATGTTCCTT TTTTCAATAT GCAAGCAGCT TTTTATTCCC TGCCCAAACC 4369

CTTAACTGAC ATGGGCCTTT AAGAACCTTA ATGACAACAC TTAATAGCAA CAGAGCACTT 4429

GAGAACCAGT CTCCTCACTC TGTCCCTGTC CTTCCCTGTT CTCCCTTTCT CTCTCCTCTC 4489

TGCTTCATAA CGGAAAAATA ATTGCCACAA GTCCAGCTGG GAAGCCCTTT TTATCAGTTT 4549

GAGGAAGTGG CTGTCCCTGT GGCCCCATCC AACCACTGTA CACACCCGCC TGACACCGTG 4609

GGTCATTACA AAAAAACACG TGGAGATGGA AATTTTTACC TTTATCTTTC ACCTTTCTAG 4669

GGACATGAAA TTTACAAAGG GCCATCGTTC ATCCAAGGCT GTTACCATTT TAACGCTGCC 4729

TAATTTTGCC AAAATCCTGA ACTTTCTCCC TCATCGGCCC GGCGCTGATT CCTCGTGTCC 4789

GGAGGCATGG GTGAGCATGG CAGCTGGTTG CTCCATTTGA GAGACACGCT GGCGACACAC 4849

TCCGTCCATC CGACTGCCCC TGCTGTGCTG CTCAAGGCCA CAGGCACACA GGTCTCATTG 4909

CTTCTGACTA GATTATTATT TGGGGGAACT GGACACAATA GGTCTTTCTC TCAGTGAAGG 4969

TGGGGAGAAG CTGAACCGGC 4989

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1337 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu -30 -25 -20 -15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu He -10 -5 1

Cys Gly Pro Gly He Asp He Arg Asn Asp Tyr Gin Gin Leu Lys Arg 5 10 15

Leu Glu Asn Cys Thr Val He Glu Gly Tyr Leu His He Leu Leu He 20 25 30

Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val 35 40 45 50

He Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu 55 60 65

Gly Asp Leu Phe Pro Asn Leu Thr Val He Arg Gly Trp Lys Leu Phe 70 75 80

Tyr Asn Tyr Ala Leu Val He Phe Glu Met Thr Asn Leu Lys Asp He 85 90 95

Gly Leu Tyr Asn Leu Arg Asn He Thr Arg Gly Ala He Arg He Glu 100 105 110

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu He 115 120 125 130

Leu Asp Ala Val Ser Asn Asn Tyr He Val Gly Asn Lys Pro Pro Lys 135 140 145

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys 150 155 160

Glu Lys Thr Thr He Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr 165 170 175

Asn Arg Cys Gin Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys 180 185 190

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser 195 200 205 210

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr 215 220 225

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu 230 235 240

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn He Leu Ser Ala 245 250 255

Glu Ser Ser Asp Ser Glu Gly Phe Val He His Asp Gly Glu Cys Met 260 265 270

Gin Glu Cys Pro Ser Gly Phe He Arg Asn Gly Ser Gin Ser Met Tyr 275 280 285 290

Cys He Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Gin Lys 295 300 305

Lys Thr Lys Thr He Asp Ser Val Thr Ser Ala Gin Met Leu Gin Gly 310 315 320

Cys Thr He Phe Lys Gly Asn Leu Leu He Asn He Arg Arg Gly Asn 325 330 335

Asn He Ala Ser Glu Leu Glu Asn Phe Met Gly Leu He Glu Val Val 340 345 350

Thr Gly Tyr Val Lys He Arg His Ser His Ala Leu Val Ser Leu Ser 355 360 365 370

Phe Leu Lys Asn Leu Arg Leu He Leu Gly Glu Glu Gin Leu Glu Gly 375 380 385

Asn- Tyr Ser Phe Tyr Val Leu Asp Asn Gin A_,n Leu Gin Gin Leu Trp 390 395 400

Asp Trp Asp His Arg Asn Leu Thr He Lys Ala Gly Lys Met Tyr Phe

405 410 415

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu He Tyr Arg Met Glu Glu 420 425 430

Val Thr Gly Thr Lys Gly Arg Gin Ser Lys Gly Asp He Asn Thr Arg 435 440 445 450

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr 455 460 465

Ser Thr Thr Thr Ser Lys Asn Arg He He He Thr Trp His Arg Tyr 470 475 480

Arg Pro Pro Asp Tyr Arg Asp Leu He Ser Phe Thr Val Tyr Tyr Lys 485 490 495

Glu Ala Pro Phe Lys Asn Val Thr Glu Tyr Asp Gly Gin Asp Ala Cys 500 505 510

Gly Ser Asn Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys 515 520 525 530

Asp Val Glu Pro Gly He Leu Leu His Gly Leu Lys Pro Trp Thr Gin 535 540 545

Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu Asn Asp 550 555 560

His He Arg Gly Ala Lys Ser Glu He Leu Tyr He Arg Thr Asn Ala 565 570 575

Ser Val Pro Ser He Pro Leu Asp Val Leu Ser Ala Ser Asn Ser Ser 580 585 590

Ser Gin Leu He Val Lys Trp Asn Pro Pro Ser Leu Pro Asn Gly Asn 595 600 605 610

Leu Ser Tyr Tyr He Val Arg Trp Gin Arg Gin Pro Gin Asp Gly Tyr 615 620 625

Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp Lys He Pro He Arg Lys 630 635 640

Tyr Ala Asp Gly Thr He Asp He Glu Glu Val Thr Glu Asn Pro Lys 645 650 655

Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys 660 665 670

Thr Glu Ala Glu Lys Gin Ala Glu Lys Glu Glu Ala Glu Tyr Arg Lys 675 680 685 690

Val Phe Glu Asn Phe Leu His Asn Ser He Phe Val Pro Arg Pro Glu 695 700 705

Arg Lys Arg Arg Asp Val Met Gin Val Ala Asn Thr Thr Met Ser Ser 710 715 720

Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr Tyr Asn He Thr Asp Pro 725 730 735

Glu Glu Leu Glu ~ ιτ Glu Tyr Pro Phe Phe Glu Ser Arg Val Asp Asn ^* 740 745 750

Lys Glu Arg Thr Val He Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg 755 760 765 770

He Asp He His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser 775 780 785

Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly Ala Asp 790 795 800

Asp He Pro Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn Ser He 805 810 815

Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu He Leu Met 820 825 830

Tyr Glu He Lys Tyr Gly Ser Gin Val Glu Asp Gin Arg Glu Cys Val 835 840 845 850

Ser Arg Gin Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu 855 860 865

Asn Pro Gly Asn Tyr Thr Ala Arg He Gin Ala Thr Ser Leu Ser Gly 870 875 880

Asn Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gin Ala Lys Thr 885 890 895

Gly Tyr Glu Asn Phe He His Leu He He Ala Leu Pro Val Ala Val 900 905 910

Leu Leu He Val Gly Gly Leu Val He Met Leu Tyr Val Phe His Arg 915 920 925 930

Lys Arg Asn Asn Ser Arg Leu Gly Asn Gly Val Leu Tyr Ala Ser Val 935 940 945

Asn Pro Glu Tyr Phe Ser Ala Ala Asp Val Tyr Val Pro Asp Glu Trp 950 955 960

Glu Val Ala Arg Glu Lys He Thr Met Ser Arg Glu Leu Gly Gin Gly 965 970 975

Ser Phe Gly Met Val Tyr Glu Gly Val Ala Lys Gly Val Val Lys Asp 980 985 990

Glu Pro Glu Thr Arg Val Ala He Lys Thr Val Asn Glu Ala Ala Ser 995 1000 1005 1010

Met Arg Glu Arg He Glu Phe Leu Asn Glu Ala Ser Val Met Lys Glu 1015 1020 1025

Phe Asn Cys His His Val Val Arg Leu Leu Gly Val Val Ser Gin Gly 1030 1035 1040

Gin Pro Thr Leu Val He Met Glu Leu Met Thr Arg Gly Asp Leu Lys 1045 1050 1055

Ser Tyr Leu Arg Ser Leu Arg Pro Glu Met Glu Asn Asn Pro Val Leu 1060 1065 1070

Ala Pro Pro Ser Leu Ser Lys Met He Gin Met Ala Gly Glu He Ala 1075 1080 1085 1090

Asp Gly Met Ala Tyr Leu Asn Ala Asn Lys Phe Val His Arg Asp Leu 1095 1100 1105

Ala Ala Arg Asn Cys Met Val Ala Glu Asp Phe Thr Val Lys He Gly 1110 1115 1120

Asp Phe Gly Met Thr Arg Asp He Tyr Glu Thr Asp Tyr Tyr Arg Lys

1125 1130 1135

Gly Gly Lys Gly Leu Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu 1140 1145 1150

Lys Asp Gly Val Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe Gly Val 1155 1160 1165 1170

Val Leu Trp Glu He Ala Thr Leu Ala Glu Gin Pro Tyr Gin Gly Leu 1175 1180 1185

Ser Asn Glu Gin Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp 1190 1195 1200

Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys 1205 1210 1215

Trp Gin Tyr Asn Pro Lys Met Arg Pro Ser Phe Leu Glu He He Ser 1220 1225 1230

Ser He Lys Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser Phe Tyr 1235 1240 1245 1250

Tyr Ser Glu Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu Asp Leu Glu 1255 1260 1265

Pro Glu Asn Met Glu Ser Val Pro Leu Asp Pro Ser Ala Ser Ser Ser 1270 1275 1280

Ser Leu Pro Leu Pro Asp Arg His Ser Gly His Lys Ala Glu Asn Gly 1285 1290 1295

Pro Gly Pro Gly Val Leu Val Leu Arg Ala Ser Phe Asp Glu Arg Gin 1300 1305 1310

Pro Tyr Ala His Met Asn Gly Gly Arg Lys Asn Glu Arg Ala Leu Pro 1315 1320 1325 1330

Leu Pro Gin Ser Ser Thr Cys 1335