WO1999023259A1

WO1999023259A1 - Antisense compounds to insulin-like growth factor-1 receptor

Info

Publication number: WO1999023259A1
Application number: PCT/US1998/023418
Authority: WO
Inventors: Gerald Zon
Original assignee: Inex Pharmaceuticals Corp
Current assignee: Inex Pharmaceuticals Corp
Priority date: 1997-11-04
Filing date: 1998-11-03
Publication date: 1999-05-14
Anticipated expiration: 2000-05-04
Also published as: AU1302899A

Abstract

A method and compounds are provided for inhitibing the expression of insulin-like growth factor-1 receptor in tumor cells. An important aspect of the invention is the discovery that the antisense compounds of the invention are strong inducers of prefential apoptosis in tumor cells. Antisense compounds of the invention are directed to codons 4 through 9 of the messenger RNA encoding human insulin-like growth factor-1 receptor.

Description

ANTISENSE COMPOUNDS TO INSULIN-LIKE GROWTH FACTOR- 1 RECEPTOR

The invention relates generally to oligonucleotides and their use as therapeutic agents, and more particularly, to the use of oligonucleotide antisense compounds directed to insulin-like growth factor- 1 receptor (IGF-1R) for treating certain cancers.

Background The insulin-like growth factor- 1 receptor is a tyrosine kinase receptor with a 70% homology to the insulin receptor that plays a major role in cellular proliferation and maintenance. In various experimental settings, it has been shown to be necessary for optimal cell growth, to be required for establishment and maintenance of a transformed phenotype, and to confer a protective effect against apoptotic cell death, e.g. Ullrich et al, EMBO J., 5: 2503-2512 (1986); Baserga, Cell, 79: 927-930 (1994); Baserga, Cancer

Research, 55: 249-252 (1995); Resnicoff et al, Cancer Research, 55: 2463-2469 and 3739- 3741 (1995); and the like.

As a result of these observations, it has been suggested that drugs or methods designed to block IGF-1R action may lead to therapies for a variety of cancers, including ovarian carcinoma, gliobastoma, and lung carcinoma, e.g. Ambrose et al, J. Cell. Physiol, 159: 92-100 (1994); Resnicoff et al, Laboratory Investigation, 69: 756-760 (1993); and Baserga et al, International patent application PCT/US94/03314. In particular, it has been suggested that antisense compounds directed to messenger RNA (mRNA) encoding IGF- 1R may lead to new anti-cancer drugs by down-regulating the expression of IGF-1R in tumor cells.

While the antisense approach is theoretically compelling in that one can readily identify candidate compounds once a gene sequence of the protein sought to be down regulated is available, practice has shown that antisense compounds directed to different sites on the same gene can lead to dramatically different results in assays designed to measure a biological end point, such as cell growth, specific protein synthesis, or the like, e.g. Zalewski et al, International patent application PCT/US 94/00264; .and Bennett et al, Adv. Pharmacol., 28: 1-43 (1994). Consequently, the antisense approach reduces but does not eliminate the need to screen large numbers of compounds to find drug candidates.

In view of the above, the identification of antisense compounds to human IGF-1R having in vivo anti-tumor effects would lead to the development of compounds for treating a wide range of currently intractable cancers.

Summary of the Invention An objective of the invention is to provide methods and compounds for killing tumor cells while at the same time leaving normal cell populations unaffected.

Another objective of the invention is to provide methods for treating cancer, particularly ovarian carcinomas and glioblastomas.

A further objective of the invention is to provide an agent for inducing apoptosis in cells having an anchorage-independent growth phenotype.

Yet another objective of the invention is to provide antisense compounds capable of inhibiting the expression of IGF-1R in tumor cells. The invention accomplishes these and other objectives by providing methods and compounds for inhibiting the expression of IGF-1R in tumor cells which, in turn, induces apoptosis. In particular, the invention comprises antisense compounds specific for codons 4 to 9, inclusive, of human IGF-1R. Preferably, antisense compounds of the invention are oligonucleotides consisting of from 12 to 18 nucleotides. More preferably, antisense compounds of the invention consist of 5'-CCCTCCTCCGGAGCC-3' (SEQ ID NO: 1) or S'-GGACCCTCCTCCGGAGCCX (SEQ ID NO: 2). Still more preferably, the intemucleosidic linkages of antisense compound 5'-CCCTCCTCCGGAGCC-3' (SEQ ID NO: 1) consist solely of N3'- P5' phosphoramidate linkages, and the intemucleosidic linkages of antisense compound 5'-GGACCCTCCTCCGGAGCC-3' (SEQ ID NO: 2) consist solely of phosphorothioate linkages. Most preferably, antisense compound 5'- CCCTCCTCCGGAGCC-3' (SEQ ID NO: 1) has a 3' terminal hydroxyl group.

Definitions As used herein the term "codon" refers to a nucleotide triplet of a messenger RNA that is translated into an amino acid during protein synthesis in accordance with the genetic code. Codons are numbered relative to the "start" codon which is taken as 1. As used herein, "N3'-»P5' phosphoramidate" refers to an intemucleosidic linkage of the form:

3'-NH-P(=X)(OR>)-O- 5^*

wherein the 3' -and 5' refer to the carbon atoms of the sugar moieties of consecutive nucleosides which are connected by way of the linkage, and wherein R¹ is hydrogen or a phosphate protecting group, and X is a chalcogen, preferably oxygen or sulfur. More particularly, when R¹ is a phosphate protecting group it is hydrogen, methyl, or β- cyanoethyl.

As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and B.aker, DNA Replication, 2nd Ed. (Freeman, San Fr.ancisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g. stability, specificity, or the like.

The term "oligonucleotide" as used herein includes linear oligomers of natural or . modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, α- anomeric forms thereof, poly-amide nucleic acids, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units. Whenever .an oligonucleotide is represented by a sequence of letters, such as "ATGUCCTG," it will be understood that the nucleotides are in 5X3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes thymidine, and "U" denotes deoxyuridine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoramidate, and the like, as more fully described below.

As used herein, the term "antisense compound" or the term "antisense oligonucleotide" means an oligonucleotide designed to prevent or inhibit the expression of a gene by specifically hybridizing to its mRNA.

As used herein, the term "apoptosis" refers to the phenomena of programmed cell death or suicide which characterized by a variety of molecular, physiological, and morphological changes in a cell, including chromatin condensation, internucleosomal DNA cleavage (resulting in the hallmark DNA laddering), cytoskeletal disruption, cell shrinkage, and membrane blebbing. Indigenous cells undergoing the process typically do not trigger an inflammatory response in an organism.

Detailed Description of the Invention

The invention is directed to antisense compounds specific for the region of human IGF-1R bound by codons 4 to 9, inclusive. Antisense compounds directed to this region have been discovered to be significantly more effective in inducing apoptosis in tumor cells than other .antisense compounds. Antisense compounds of the invention comprise .any polymeric compound capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer- to-nucleoside interactions, such as Watson-Crick type of base pairing. Antisense compounds of the invention may also contain pendent groups or moieties, either as part of or separate from the basic repeat unit of the polymer, to enhance specificity, nuclease resistance, delivery, or other properties related to efficacy, e.g. cholesterol moieties, duplex intercalators such as acridine, poly-L-lysine, "end capping" with one or more nuclease- resisant linkage groups such as phosphorothioate, and the like.

Preferably, nuclease resistance is conferred on the antisense compounds of the invention by providing nuclease-resistant intemucleosidic linkages. Many such linkages are known in the art and are described in many reviews, e.g. Uhlmann and Peyman,

Chemical Reviews, 90: 543-584 (1990); Mesmaeker et al, Current Opinion in Structural Biology, 5: 343-355 (1995); Crooke et al, Exp. Opin. Ther. Patents 6: 855-870 (1996); Matteucci, Perspectives in Drug Discovery and Design, 4: 1-16 (1996); and the like. Preferably, antisense compounds of the invention have intemucleosidic linkages selected from the group consisting of phosphodiester, phosphorothioate, or phosphoramidate, particularly, N3'— »P5' phosphoramidate. Methods of synthesizing these latter compounds are fully described in the following references: Zon and Geiser, Anti-Cancer Drug Design, 6: 539-568 (1991); Stec et al, U.S. patent 5,151,510; Hirschbein, U.S. patent 5,166,387; Antisense compounds of the invention may also include nucleoside modifications, such as 2'-O-alkyl-substituted ribonucleosides, Altmann et al, Biochemical Society Transactions, 24: 630-637 (1996); 5-propynyl modificated pyrimidines, Froehler et al, Tetrahedron Lett., 33: 5307-5310 (1992); Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460; Koster et al, U.S. patent 4,725,677; Caruthers et al, U.S. patents 4,415,732; 4,458,066; and 4,973,679; Hirschbein et al, International application PCT US96/10418; Gryaznov et al, U.S. patent 5,631,135; and the like. Preferably, antisense compounds of the invention are synthesized by conventional means on commercially available automated DNA synthesizers, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer.

The length of the oligonucleotide moieties is sufficiently large to ensure that specific binding will take place only at the desired target polynucleotide and not at other fortuitous sites, as explained in many references, e.g. Rosenberg et al, International application PCT US92/05305; or Szostak et al, Meth. Enzymol. 68: 419-429 (1979). The upper range of the length is determined by several factors, including the inconvenience and expense of synthesizing and purifying oligomers greater than about 30-40 nucleotides in length, the greater tolerance of longer oligonucleotides for mismatches than shorter oligonucleotides, whether modifications to enhance binding or specificity are present, whether duplex or triplex binding is desired, the enhancement or reduction in duplex or triplex stability imparted by a particular analog, and tfye like. Preferably, antisense compounds consisting solely of N3'-»P5' phosphoramidate linkages have lengths in the range of about 12 to 15 nucleotides.

The antisense oligonucleotides of the invention are employed as one or more components of pharmaceutical compositions. Components of pharmaceutical compositions of the invention depend on several factors, including the nature of the disease or condition being treated, the location of disease lesions, the mode of drug delivery .and/or administration contemplated, the latter of which can include in vivo administration by way of a catheter into a target lesion or organ, topical application, intranasal administration, administration by implanted or transdermal sustained release systems, and the like.

Pharmaceutical compositions of the invention include a pharmaceutical carrier that may contain a variety of components that provide a variety of functions, including regulation of drug concentration, regulation of solubility, chemical stabilization, regulation of viscosity, absorption enhancement, regulation of pH, and the like. For example, in water soluble formulations the pharmaceutical composition preferably includes a buffer such as a phosphate buffer, or other organic acid salt, preferably at a pH of between about 7 and 8. For formulations containing weakly soluble antisense compounds, microemulsions may be employed, for example by using a nonionic surfactant such as Tween 80 in an amount of 0.04-0.05% (w/v), to increase solubility. Other components may include antioxidants, such as ascorbic acid, hydrophilic polymers, such as, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, dextrins, chelating agents, such as EDTA, and like components well known to those in the pharmaceutical sciences, e.g. Remington's Pharmaceutical Science, latest edition (Mack Publishing Company, Easton, PA).

Antisense compounds of the invention include the pharmaceutically acceptable salts thereof, including those of alkaline earths, e.g. sodium or magnesium, ammonium or N 4⁺, wherein X is C- -C4 alkyl. Other pharmaceutically acceptable salts include org-anic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic, and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, and benzenesulfonic; and inorganic acids such as hydrochloric, sulfuric, phosphoric, and sulfamic acids. Pharmaceutically acceptable salts of a compound having a hydroxyl group include the anion of such compound in combination with a suitable cation such as Na⁺, NH4⁺, or the like.

Sustained release systems suitable for use with the pharmaceutical compositons of the invention include semi-permeable polymer matrices in the form of films, microcapsules, or the like, comprising polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2_:hydroxyethyl methacrylate), and like materials, e.g. Rosenberg et al, International application PCT/US92/05305. Sustained release systems also include liposomally entrapped antisense compounds, e.g. as described in Liposome Technology, Vol. LI, Incorporation of Drugs, Proteins, an Genetic Material (CRC Press).

An effective amount of IGF-1R antisense oligonucleotide for particular applications depends on several factors, including the chemical nature of the antisense oligonucleotide, the disorder being treated, the method of administration, the stability of the complex between the antisense compound and its target, and the like. Preferably, an effective amount will provide a concentration of IGF-1R antisense oligonucleotide of between about 1 to 100 μM at the target polynucleotide; and more preferably, an effective amount will provide a concentration of antisense oligonucleotide of between about 1 to 10 μM at the target polynucleotide.

For each of the disorders described above, criteria for selecting patients for treatment and means for assessing therapeutic endpoints are well known in the art concerning the specific disorders.

Antisense Compound Selection Antisense compounds of the invention are selected on the basis of an in vivo diffusion chamber assay for apoptosis described in Resnicoff et al, Cancer Research 55: 2463-2469 (1995). The sequence of human IGF-1R is disclosed in Baserga et al, U.S. patent 5,643,788, which patent is incorporated by reference. Briefly, diffusion chambers are constructed from 14-mm Lucite rings with 0.1 μm pore-sized hydrophilic Durapore membranes (Millipore, Bedford, MA). The chambers are sterilized with ethylene oxide prior to use. Cells of a test population are preincubated with an antisense compound for 24 hr. at a concentration of from 1 to 120 μg/ml, after which 5x10⁵ cells are placed into the chambers. The chambers are then inserted into the subcutaneous tissue of rats (e.g., strain BD-LX, Chiles River Laboratories, Wilmington, MA) or mice (e.g., strain C57B 1/6) under .anesthesia, e.g. Halothane, or the like. Alternately, instead of pre incubation with antisense compound, cells are placed into a diffusion chamber, inserted in the subcutaneous tissue of a test animal, and the antisense compound is injected into the subcutaneous tissue adjacent to the chamber, e.g. 0.2 mg in 0.15 ml. Preferably, either sense or random sequence oligonucleotides are used as controls.

Cells recovered from the diffusion chambers (at various time points, but usually after 24 hr.) are counted in a hemocytometer and are also stained with trypan blue. Results are expressed as the percentage of cells recovered with respect to the size of the starting inoculation of cells. Apoptotic cells are detected either by the method of Sell et al, Cancer Research, 55: 303-306 (1995), or by the method of Gorczyca et al, Cancer Research, 53: 3186-3192 (1993). Briefly, by the method of Sell et al, cells are washed with phosphate buffered saline (PBS) and fixed by the addition of 70% ice-cold ethanol. After 10 min. the cells are washed with PBS -and treated with RNase A (75 units/ml) for 30 min. at 37°C, washed again in PBS, and resuspended in PBS containing 15 μg/ml propidium iodide. Cells are then analyzed by flow cytometry, e.g. Coulter Epics Profile U (Coulter Electronics, Inc., Hialeah, FL), or like instrument.

The effect of IGF-1R antisense compounds were determined in several tumor cell lines using the diffusion chamber assay with the following results:

Table I

Percent

Cell line Compound recovery Comments

C6¹ LR5328 3.4 SEQ ID NO: 1 (a idate with 3'-OH); preincubation with 1 μg/ml

C6 LR5377 42. SEQ ID NO: 3 (amidate)

C6 LR5406 180. control

C6 LR5407 174. control

C6 LR4437 1.05 SEQ ID NO: 2; 5 mg/ml s.c. inj. adjacent to chamber

C6 LR4437 0.8 SEQ ID NO: 2; 10 mg/ml s.c. inj. adjacent to chamber

Caov-3² LR6028 10.5 SEQ ID NO: 4 (amidate with 3'-NH₂); s.c. inj. 0.2 mg in 0.15 ml

Caov-3 LR6028 4.6 SEQ ID NO: 4 (amidate with 3'-NH₂); s.c. inj. 0.25 mg in 0.1 ml

Caov-3 LR6028 3.8 SEQ ID NO: 4 (amidate with 3'-NH₂); s.c. inj. 0.50 mg in 0.1 ml

Caov-3 LR6028 2.4 SEQ ID NO: 4 (amidate with 3'-NH₂); s.c. inj. 0.75 mg in 0.1 ml

Caov-3 LR6029 312.5 SEQ ID NO: 5 (control)

Caov-3 LR6030 190. control

HEY³ LR5328 0.3. SEQ ED NO: 1 (3'-OH); preincubation with 1 μg/ l

HEY LR4437 6.2 SEQ ID NO: 2 (thioate); preincubation with 1 μg/ml

C6 LR6001 48. SEQ ID NO: 6

C6 LR6002 41. SEQ ID NO: 7

C6 LR6003 31. SEQ ID NO: 8

C6 LR6004 28. SEQ ID NO: 9

C6 LR6005 42. SEQ ED NO: 10

C6 LR6006 34. SEQ ID NO: 11

1 ) Glial tumor cell line available from American Type Culture Collection (ATCC) under accession number

CCL 107.

2) Ovary adenocarcinoma cell line available from ATCC under accession number HTB 75.

3) Ovary adenocarcinoma cell line available from M.D. Anderson Cancer Center (Houston, TX).

Toxicity of IGF-1R Antisense Compound LR4437

The acute toxicity of repeated intravenous injections of LR4437 was determined in CD-I mice (Charles River Laboratories, Portage, Michigan). Animals were administered LR4437 on days 1, 3, 5, 8, 10, .and 12 via intravenous injection into a tail vein. On day 13 animals were sacrificed and subjected to a complete gross necropsy. Table LI summarizes the experimental set-up. Table H

Toxicity analysis included observations of the hematological states, standard clinical chemistry, histology, -and weight .and moφhology of org.ans of all test animals. There were no apparent test article-related clinical observations for male and female mice administered LR4437 by repeated dose up to 300 mg kg.

Claims

I claim:

1. A method of inducing apoptosis in a tumor cell, the method comprising contacting said tumor cell with an antisense oligonucleotide directed against an insulin- like growth factor- 1 receptor gene product in an amount effective to inhibit translation of said insulin-like growth factor- 1 receptor gene product in said tumor cell, the insulin-like growth factor- 1 receptor gene product having an AUG translation initiation codon, and the antisense oligonucleotide being complementary to a portion of an 18 nucleotide sequence of the insulin-like growth factor- 1 receptor gene product extending from four to nine codons downstream of the AUG translation initiation codon.

2. The method of claim 1 wherein said antisense oligonucleotide consists of from 12 to 18 nucleotides.

3. The method of claim 2 wherein said antisense oligonucleotide comprises a sequence of nucleosides and inter-nucleoside linkages, wherein the inter-nucleoside linkages are selected from the group consisting of phosphodiester, phosphorothioate, and N3'-»P5' phosphoramidate.

4. The method of claim 3 wherein said antisense oligonucleotide has the sequence of SEQ LD NO: 1.

5. The method of claim 4 wherein one or more of said inter-nucleoside linkages of said antisense oligonucleotide are N3XP5' phosphoramidate.

6. The method of claim 4 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are N3'- P5' phosphoramidate.

7. The method of claim 5 wherein said antisense oligonucleotide has a 3' terminal hydroxyl group.

8. The method of claim 3 wherein said antisense oligonucleotide has the sequence of SEQ LD NO: 2.

9. The method of claim 8 wherein one or more of said inter-nucleoside linkages of said antisense oligonucleotide are phosphorothioate.

10. The method of claim 9 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are phosphorothioate.

11. An antisense oligonucleotide for inducing apoptosis in a tumor cell, the antisense oligonucleotide having a nucleotide sequence complementary to a portion of an 18-nucleotide region of an insulin-like growth factor- 1 receptor gene product, the

18-nucleotide region extending from four to nine codons downstream of an AUG translation initiation codon of the insulin-like growth factor- 1 receptor gene product.

12. The antisense oligonucleotide of claim 11 consisting of from 12 to 18 nucleotides.

13. The antisense oligonucleotide of claim 12 comprising a sequence of nucleosides and inter-nucleoside linkages, wherein the inter-nucleoside linkages are selected from the group consisting of phosphodiester, phosphorothioate, and N3'— >P5' phosphoramidate.

14. The antisense oligonucleotide of claim 13 having the sequence of SEQ LD NO: 1.

15. The antisense oligonucleotide of claim 14 wherein one or more of said inter- nucleoside linkages of said antisense oligonucleotide are N3XP5' phosphoramidate.

16. The antisense oligonucleotide of claim 15 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are N3XP5' phosphoramidate.

17. The antisense oligonucleotide of claim 16 wherein said antisense oligonucleotide has a 3' terminal hydroxyl group.

18. The antisense oligonucleotide of claim 13 wherein said antisense oligonucleotide has the sequence of SEQ LD NO: 2.

19. The antisense oligonucleotide of claim 18 wherein one or more of said inter- nucleoside linkages of said antisense oligonucleotide are phosphorothioate.

20. The antisense oligonucleotide of claim 19 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are phosphorothioate.

21. A liposomal composition comprising: a) a liposome; and b) an antisense oligonucleotide for inducing apoptosis in a tumor cell, said antisense oligonucleotide having a nucleotide sequence complementary to a portion of an 18- nucleotide region of an insulin-like growth factor- 1 receptor gene product, said 18-nucleotide region extending from four to nine codons downstream of an AUG translation initiation codon of said insulin- like growth factor- 1 receptor gene product, and said antisense oligonucleotide not being greater than about 40 nucleotides in length.

22. The liposomal composition of claim 21 wherein said antisense oligonucleotide comprises from about 12 to about 18 nucleotides.

23. The liposomal composition of claim 22 wherein said antisense oligonucleotide comprises a sequence of nucleosides and inter-nucleoside linkages, wherein said inter- nucleoside linkages are selected from the group consisting of phosphodiester, phosphorothioate, and N3XP5' phosphoramidate.

24. The liposomal composition of claim 23 wherein said antisense oligonucleotide has the sequence of SEQ ID NO: 1.

25. The liposomal composition of claim 24 wherein one or more of said inter- nucleoside linkages of said antisense oligonucleotide are N3'-»P5' phosphoramidate.

26. The liposomal composition of claim 24 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are N3'-»P5' phosphoramidate.

27. The liposomal composition of claim 25 wherein said antisense oligonucleotide has a 3' terminal hydro xyl group.

28. The liposomal composition of claim 23 wherein said antisense oligonucleotide has the sequence of SEQ ID NO: 2.

29. The liposomal composition of claim 24 wherein all of said inter-nucleoside linkages of said antisense oligonucleotide are N3XP5' phosphodiester linkages.

30. A method for inducing apoptosis in a tumor cell, the method comprising: administering to a host containing said tumor cell, a liposome that comprises an antisense compound for inducing apoptosis in said tumor cell, said antisense compound having a nucleotide sequence complementary to a portion of an 18-nucleotide region of an insulin-like growth factor- 1 receptor gene product, said 18-nucleotide region extending from four to nine codons downstream of an AUG translation initiation codon of the insulin-like growth factor- 1 receptor gene product, said antisense compound not being greater than about 40 nucleotides in length, or a pharmaceutically acceptable salt thereof.

31. A method for inducing apoptosis in a tumor cell according to claim 30, wherein said antisense oligonucleotide has the sequence of SEQ ID NO: 2.

32. A method for inducing apoptosis in a tumor comprising:

(a) incubating in vitro a tumor cell with an antisense compound having a nucleotide sequence complementary to a portion of an 18-nucleotide region of an insulin-like growth factor- 1 receptor gene product, said 18-nucleotide region extending from four to nine codons downstream of an AUG translation initiation codon of the insulin- like growth factor- 1 receptor gene product, wherein the cultured cell remains viable, thereby producing a tumor cell culture;

(b) culturing said tumor cell culture in a diffusion chamber thereby producing a tumor cell-containing chamber, and

(c) inserting said tumor cell-containing chamber into a mammal for a therapeutically effective time thereby inducing apoptosis of said tumor in said mammal.

33. A method for inducing apoptosis in a tumor according to claim 32, wherein said antisense oligonucleotide has the sequence of SEQ ID NO: 2. SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: Gerald Zon (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: 11

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Stephen C. Macevicz, Lynx Therapeutics, Inc .

(B) STREET: 3832 Bay Center Place

(C) CITY: Hayward

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94545

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 inch diskette

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: Windows 95

(D) SOFTWARE: Microsoft Word 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Stephen C. Macevicz

(B) REGISTRATION NUMBER: 30,385

(C) REFERENCE/DOCKET NUMBER: LYNX-041

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (510) 670-9365

(B) TELEFAX: (510) 670-9302

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCCTCCTCCG GAGCC 15 (2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GGACCCTCCT CCGGAGCC 18

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCGGAGCCAG ACTTC 15

(2) INFORMATION FOR SEQ ID NO : 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STR7ANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCCTCCTCCG GAGCC 15

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CCCGAATCCT CCGCC 15

(2) INFORMATION FOR SEQ ID NO : 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGAGCCAGAC TTCAT 15

(2) INFORMATION FOR SEQ ID NO : 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TCCTTTTATT TGGGA 15

(2) INFORMATION FOR SEQ ID NO : 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AGACTTCATT CCTTT 15

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCACAGCGAG GTCGG 15

(2) INFORMATION FOR SEQ ID NO: 10

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 10: CGGAGACTTC ATTCC 15

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGAGCCCCCA CAGCG 15