United States Patent [19] Huang

US006069231A

6,069,231 May 30, 2000

[11] Patent Number:

[45] Date of Patent:

[54] PR DOMAIN PEPTIDES

[75] Inventor: Shi Huang, San Diego, Calif.

[73] Assignee: La Jolla Cancer Research Foundation, La Jolla, Calif.

[21] Appl. No.: 08/516,859

[22] Filed: Aug. 18, 1995

Related US. Application Data

[63] Continuation-in-part of application No. 08/399,411, Mar. 6, 1995, Pat. No. 5,831,008, which is a continuation-in-part of application No. 08/292,683, Aug. 18, 1994, abandoned.

[51] Int. Cl.7 .......................... .. C06K 7/06; C06K 14/435

[52] US. Cl. ......................... .. 530/327; 530/300; 530/350 [58] Field of Search ................................... .. 530/300, 324,

530/350

[56] References Cited

PUBLICATIONS

George et al. Macromolecular Sequencing & Synthesis Selected Methods & Applications, pp. 127—149, 1988. Bourne et al., “The GTPase superfamily: conserved struc ture and molecular mechanism.” Nature, 349:117—127

(1991). Boyd et al., “A region in the C—terminus of adenovirus 2/5 Ela protein is required for association With a cellular phosphoprotein and important for the negative modulation of T24—ras mediated transformation, tumorigenesis and metastasis.” EMBO. J. 12:469—478 (1993). Van Cherington et al., “Separation of simian virus 40 large T antigen transforming and origin—binding functions from the ability to block differentiation.” Mol. Cell. Biol., 8:1380—1384 (1988). DeCaprio et al., “SV40 large tumor antigen forms a speci?c complex With the product of the retinoblastoma susceptibil ity gene.” Cell, 54:275—283 (1988). Defeo—Jones et al., “Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product.” Nature, 352:251—254 (1991). DoWdy et al., “Physical interaction of the retinoblastoma protein With human D cyclins.” Cell 73:499—511 (1993). Durfee et al., “The retinoblastoma protein associates With the protein phosphatase type 1 catalytic subunit.” Genes Dev., 7:555—569 (1993). Dyson et al., “Adenovirus E1A makes tWo distinct contacts With the retinoblastoma protein.” J. Virol., 66:4606—4611 (1992). EWen et al., “Functional interactions of the retinoblastoma protein With mammalian D—type cyclins.” Cell, 73:487—497 (1993). Ford et al., “Nuclear protein With sequence homology to translation initiation factor eIF—4A.” Nature, 332:736—738

(1988). HarloW et al., “Association of adenovirus early region 1A proteins With cellular polypeptides.” Mol. Cell. Biol., 6:1579—1589 (1986).

Hirling et al., “RNA helicase activity associated With the human p68 protein.” Nature, 339:562—564 (1989). Hu et al., “The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations.” EMBO J., 9:1147—1155

(1990). Huang et al., “TWo distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.” EMBO J., 9:1815—1822 (1990). Huang et al., “A cellular protein that competes With SV40 T antigen for binding to the retinoblastoma gene product.” Nature, 350:160—162 (1991). Huang et al., “The retinoblastoma protein region required for interaction With the E2F transcription factor includes the T/ElAbinding and carboXy—terminal sequences.” DNA Cell Biol., 11:539—548 (1992). Kaelin, Jr. et al., “De?nition of the minimal simian virus 40 large TAntigen and adenovirus E1A—binding domain in the retinoblastoma gene product.” Mol. Cell. Biol., 10:3761—3769 (1990). Keller and Maniatis, “Identi?cation and characterization of a novel repressor of [3—interferon gene expression.” Genes Dev., 5:868—879 (1991). Kimelman et al., “Ela regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related.” J. Virol, 53:399—409 (1985). M. KoZak, “An analysis of 5‘ noncoding sequences from 699 vertebrate messenger RN .” Nucl. Acids Res. 15:8125—8148 (1987). Krieg and Melton, “Functional messenger RNAs are pro duced by SP6 in vitro transcription of cloned cDNAs.” Nucl. Acids Res., 12:7057—7070 (1984). Lane and Hoef?er, “SV40 large T shares an antigenic determinant With a cellular protein of molecular Weight 68,000.” Nature, 288:167—170 (1980). Lillie et al., “Functional domains of adenovirus type 5 Ela proteins.” Cell, 50:1091—1100 (1987). LudloW et al., “SV40 large T antigen binds preferentially to an under phosphorylated member of the retinoblastoma susceptibility gene product family.” Cell, 56:57—65 (1989). Mihara et al., “Cell cycle—dependent regulation of phospho rylation of the human retinoblastoma gene product.” Sci ence, 246:1300—1303 (1989). E. Moran, “A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus ElAproducts.” Nature, 334:168—170 (1988).

(List continued on neXt page.)

Primary Examiner—Lila Feisee Assistant Examiner—Christine Saoud Attorney, Agent, or Firm—Campbell & Flores LLP

[57] ABSTRACT

The present invention provides PR domain peptides, Which are about 100 to about 120 amino acids in length and contain three highly conserved sequences of about 10 to about 12 amino acids, separated by less conserved sequences of about 20 to about 35 amino acids.

18 Claims, 26 Drawing Sheets

6,069,231 Page 2

OTHER PUBLICATIONS

Moran and Matthews, “Multiple functional domains in the adenovirus E1A gene.” Cell, 48:177—178 (1987). J .R. Nevins, “E2F: a link betWeen the Rb tumor suppressor protein and viral oncoproteins.” Science, 258:424—429 (1992). PaWson and Gish, “SH2 and SH3 domains: from structure to function.” Cell 71:359—362 (1992). Qin et al., “Identi?cation of a groWth suppression domain Within the retinoblastoma gene product.” Genes Dev., 6:953—964 (1992). Quinlan et al., “GroWth factor induction by the adenovirus type 5 E1A 12S protein is required for immortiliZation of primary epithelial cells.” Mol. Cell. Biol., 8:3191—3203 (1988). Quinlan and Douglas, “ImmortiliZation of primary epithelial cells requires ?rst— and second—exon functions of adenovi rus type 5 12S.” J. Virol, 66:2020—2030 (1992). Ren et al., “Identi?cation of a ten—amino acid proline—rich SH3 binding site.” Science, 259:1157—1161 (1993). Saraste et al., “The P—loop—a common motif in ATP— and GTP—binding proteins.” Trends. Biochem. Sci., 15 :430—434 (1990). Scheffner et al., “RNA unWinding activity of SV40 large T antigen.” Cell 57:955—963 (1989). Smith and Ziff, “The amino—terminal region of the aden ovirus serotype 5 E1a protein performs tWo separate func tions When expressed in primary baby rat kidney cells.” Mol. Cell. Biol, 8:3882—3890 (1988). Subramanian et al., “Enhanced ras oncogene mediated cell transformation and tumorigenesis by adenovirus 2 mutants lacking the C—terminal region of E1a protein.” Oncogene, 4:415—420 (1989). Templeton et al., “Nonfunctional mutants of the retinoblas toma protein are characteriZed by defects in phosphoryla tion, viral oncoprotein association, and nuclear tethering.” Proc. Natl. Acad. Sci. USA, 88:3033—3037 (1991). Wang et al., “Identi?cation of speci?c adenovirus E1A N—terminal residues critical to the binding of cellular pro teins and the control of cell groWth.” J. Virol, 67:476—488 (1993). RA. Weinberg, “Tumor suppressor genes.” Science, 254:1138—1146 (1991). Welch and Wang, “A C—terminal protein—binding domain in the retinoblastoma protein regulates nuclear c—Abl tyrosine kinase in the cell cycle.” Cell, 75:779—790 (1993). Whyte et al., “Association betWeen an oncogene and an anti—oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product.” Nature 334:124—129 (1988). Whyte et al., “Cellular targets for transformation by the adenovirus E1A proteins.” Cell, 56:67—75 (1989). Walker et al., “Distantly related sequences in the ot— and [3—subunits of ATP synthase, myosin, kinases and other ATP—requiring enZymes and a common nucleotide binding fold.” Embo J., 1(8):945—951 (1982). Chen et al., “Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation.” Cell, 58:1193—1198 (1989). DeCaprio et al., “The product of the retinoblastoma suscep tibility gene has properties of a cell cycle regulatory ele ment.” Cell, 58:1085—1095 (1989). Iggo and Lane, “Nuclear protein p68 is an RNA—dependent ATPase.” EMBO J., 8(6):1827—1831 (1989).

Moran et al., “Identi?cation of separate domains in the adenovirus E1A gene for immortiliZation activity and the activation of virus early genes.” Mol. and Cell. Biol., 6(10):3470—3480 (1986). Buchkovich et al., “The retinoblastoma protein is phospho rylated during speci?c phases of the cell cycle.” Cell, 58:1097—1105 (1989). Chen et al., “Identi?cation of a Human Homologue of Yeast Nuc2 Which Interacts With the Retinoblastoma Protein in a Speci?c Manner.” Cell Growth & Di?rer 6:199—210 (1995). BartholomeW and Ihle, “Retroviral insertions 90 kilobases proximal to the Evi—1 myeloid transforming gene activate transcription from the normal promoter.” Mol. Cell. Biol., 11(4):1820—1828 (1991). Morishita et al., “Expression of the Evi—1 Zinc ?nger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony—stimulating factor.” Mol. Cell. Biol, 12(1):183—189 (1992). Buyse et al., “The retinoblastoma protein binds to RIZ, a Zinc—?nger protein that shares an epitope With the adenovi rus E1Aprotein.”Proc. NatLAcad. Sci. USA, 92:4467—4471

(1995). Kreider et al., “Loss of erythropoietin responsiveness in erythroid progenitors due to expression of the Evi—1 myeloid—transforming gene.” Proc. Natl. Acad. Sci. USA, 90:6454—6458 (1993). Miyoshi et al., “t(8;21)breakpoints on chromosome 21 in acute myeloid leukemia are clustered Within a limited region of a single gene, AML1.” Proc. Natl. Acad. Sci. USA, 88:10431—10434 (1991). Nucifora et al., “Consistent intergenic splicing and produc tion of multiple transcripts betWeen AML1 at 21q22 and unrelated genes at 3q26 in (3;21) (q26;q22)translocations.” Proc. Natl. Acad. Sci. USA, 91:4004—4008 (1994). Morishita et al., “Unique expression of the human Evi—1 gene in an endometrial carcinoma cell line: sequence of cDNAs and structure of alternatively spliced transcripts.” Oncogene, 5:963—971 (1990). Lee et al., “Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation.” Genes & Development, 8:2008—2021 (1994). Garriga et al., “Migrations of the caenorhaba'itis elegans HSNs are regulated by egl—43, a gene encoding tWo Zinc ?nger proteins.” Genes & Development, 7:2097—2109 (1993). Turner et al., “Blimp—1, a novel Zinc ?nger—containing protein that can drive the maturation of B lymphocytes into immunoglobulin—secreting cells.” Cell, 77:297—306 (1994). Weinberg Robert A., “The retinoblastoma protein and cell cycle control.” Cell, 81:323—330 (1995). Morishita et al., “EVI—1 Zinc ?nger protein Works as a transcriptional activator via binding to a consensus sequence of GACAAGATAAGATAAN 1128 CTCATCTTC.” Onco gene, 10:1961—1967 (1995). Mitani et al., “Generation of the AML1—EVI—1 fusion gene in the t(3;21) (q26;q22) causes blastic crisis in chronic myelocytic leukemia.” EMBO J., 13(3):504—510 (1994). Rechsteiner Martin, “Regulation of enZyme levels by pro teolysis: the role of pest regions.” Aa'v. Enzyme. Regal, 27:135—151 (1988). Huang Shi, “Blimp—1 is the murine homolog of the human transcriptional repressor PRDI—BF1.” Cell, 78:9 (1994). Morishita et al., “Retroviral activation of a novel gene encoding a Zinc ?nger protein in IL—3—dependent myeloid leukemia cell lines.” Cell, 54:831—840 (1988).

6,069,231 Page 3

Morishita et al., “The Evi—1 Zinc ?nger myeloid transform- Perkins et al., “Patterns of Evi—1 expression in embryonic ing gene is normally expressed in the kidney and in devel- and adult tissues Suggest that Evi_1 plays an important

regulatory role in mouse development.” Development, oping oocytes.” Oncogene, 5:1419—1423 (1990). 111;479_487 (1991)_

U.S. Patent May 30, 2000 Sheet 1 0f 26 6,069,231

cow.“ com." cum." com“ one." 0mm omh cow omw own oow oNH

FE0440<442<<F248008F8088r004085000<<9609r0009r9600<200 00000 0U0<0U00040UOU0 000 8000 00UUOUO0U0£<0< 4.000 mvm mom mmv 00H mm mm

U.S. Patent May 30, 2000 Sheet 4 0f 26 6,069,231

GAA'I'TCCCGG CTCACTGAAG CTTGGCACGT GCGCTCTGGA

ATATCTGAAT GATCTCAGTA CAATGAAGGA GTGCCTTTTC

CCT'I‘TCTACC CTGCCTCCTT GAAGCATGCA TTAGAGTCGT

T (SEQ ID NO: 94)

FIG. 1B

U.S. Patent May 30, 2000 Sheet 7 0f 26 6,069,231

m .GE

msusnlmzm $5 60 who to H ._. K. ._..

I80 _ _ E \\\0 , _ i wot EQQETCN 3&5 ~._ wEmETcN m< mm

U.S. Patent May 30, 2000 Sheet 8 0f 26 6,069,231

As‘ ‘2* [*1

010 50501050 5001M?)

5 6

FIG. 4 1234

7 $51M 5 i. :2 Egg 3mg 3 .. 5.15 m

PPL

B30"

12 3 4 5

FIG. 5A FIG. 5B

U.S. Patent May 30, 2000 Sheet 9 0f 26 6,069,231

Em 1mm?

nmirci .m _. my NEIHWG ?wmwlm _. my NE

w pHb

FIG. 6

U.S. Patent May 30, 2000 Sheet 10 0f 26 6,069,231

+211“

wwmhww wwwkmw m “THEM. hmmu

FIG. 7A FIG. 75 FIG. 7C

U.S. Patent May 30, 2000 Sheet 11 0f 26 6,069,231

U.S. Patent May 30, 2000 Sheet 12 0f 26 6,069,231

03% 00m ovm ooo omv own o:

U.S. Patent May 30, 2000 Sheet 13 0f 26 6,069,231

ommm owhm ovum ONmN oovm ommm owHm ovoN oNmH oomH

U.S. Patent May 30, 2000 Sheet 14 0f 26 6,069,231

cmmv oomv omoq owmm ovmm omhm Doom omvm comm ovmm ooom

U.S. Patent May 30, 2000 Sheet 15 0f 26 6,069,231

02% 03m oumm oovm 33 0w 7... owom ommq 003 cm: 82 avg.

U.S. Patent May 30, 2000 Sheet 16 0f 26 6,069,231

1. Clone SY

G GAG TGG GGG CCA GTC ACC CGG AGC CTT CAG CGC

E W G P V T R S L Q R

AGC ACC AAG CAG GAG CTG AAG GAc TTG CAG (95)

s T K Q E. L K D L Q (102)

2. Clone lY

GGG GCC GGC GAA ACA GCG GCG GCG GCG GCG GCC C'I'C

G A E E T A A A A A A L

GGT GCT CTG AGG CTG GGc CGG CGG GCG CGG (96)

G A L R L G R R A R (103)

— Number in parentheses is SEQ ID NO: .

FIG. 9B

U.S. Patent May 30, 2000 Sheet 17 0f 26 6,069,231

GCIYACYXGCIAGCCTCACAAGCA'IGAAGHIITGKXCIGITCCTMCCIA SO A'IGA'IOCCXTI‘ TI‘IGICCCGI‘ AAA‘IGTIAAC ACICA'IGAAG CA'IACIXICGS lOO mmcmmmmmm 150 AACUIAAACCICXIEXAGICACCIWOXKBIGAGCTCIGGSAAGT 200 GIGFITGAGGCCITGZXIEICACICCITTTTTCIIATGIGRAATGIQTKE 250 WWWAAACIGTICCAATCAIGAAA 300 AWTGATTTIGIYAAAGIGCXRTTICCIKHCAGIGGIWICI'ICA 350 AGALEACACIITCIGIATCIGCUUGIGAAGAGAATEAACAA‘IYXAAAGIGT 400 GAAGRQIGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 450 AAAAAAAAAAAAAGQXImCCGC 474

FIG. 9C