# US 5,702,890 A · Inhibitors of alternative alleles of genes as a basis for cancer therapeutic agents

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- Number: US 5,702,890 A
- Document ID: US5702890A
- Status: Granted
- Kind: A
- Application number: 08/379,680
- Filed: 1995-04-04
- Granted: 1997-12-30
- Earliest claimed: 1993-07-26
- Assignees: K.O. Technology, Inc.
- Inventors: David E. Housman
- Web page: https://patentgrep.com/patent/US5702890A

## CPC

- C12N 9/0028: {with NAD or NADP as acceptor (1.5.1)}
- A61P 35/00: Antineoplastic agents
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/70582: {CD71}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2881: {against CD71}
- C07K 16/40: against enzymes
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2319/00: Fusion polypeptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

This invention is directed to a therapeutic strategy, involving (1) identification of alternative alleles of genes coding for proteins vital for cell viability or cell growth and the loss of one of these alleles in cancer cells due to loss of heterozygosity (LOH) and (2) the development of inhibitors with high specificity for the single remaining alternative allele of the vital gene retained by the cancer cell after LOH. The inhibitors of this invention are specific for one alternative allele of a gene that codes for a protein vital to cell viability or cell growth. The targeted gene has two alternative alleles in which the inhibitors of this invention block only the one alternative allele still present in the cancer cells. Exposure to the inhibitor inhibits or kills cancer cells which have undergone LOH. Protein is still capable of being expressed in the normal cells exposed to the inhibitor by the unblocked alternative allele. This differential effect of the inhibitor on cancer cells and normal cells accounts for the high therapeutic index of the inhibitors of this invention when used as antineoplastic agents.

## Claims

### Claim 1

A method for identifying an inhibitor specific for an allele of a gene essential for cell viability or growth, wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of said gene;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor inhibits expression of at least one but less than all of said alleles or reduces the level of activity of a product of at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles is indicative that said potential inhibitor is an allele specific inhibitor.

### Claim 2 (depends on 1)

The method of claim 1,
wherein said potential allele specific inhibitor is a ribozyme, said method further comprising the steps of:
(a) constructing a ribozyme able to specifically cleave a ribonucleic acid target sequence, wherein said at least one but less than all of said alleles comprise said target sequence for said ribozyme and the remainder of said alleles do not comprise said target sequence for said ribozyme;
(b) contacting RNA from one of said alleles with said ribozyme; and
(c) determining whether said ribozyme specifically cleaves said RNA.

### Claim 3 (depends on 1)

The method of claim 1, wherein said potential allele specific inhibitor is an oligonucleotide having a sequence which is perfectly complementary to at least one but less than all of said alleles or to an RNA molecule transcribed from at least one but less than all of said alleles, further comprising the steps of:
(a) constructing said oligonucleotide;
(b) contacting said alleles or RNA transcribed from said alleles; and
(c) determining whether expression of said at least one but less than all of said alleles is reduced.

### Claim 4 (depends on 1)

The method of claim 1, wherein said potential allele specific inhibitor is an oligonucleotide, further comprising the steps of:
(a) constructing said oligonucleotide;
(b) contacting said alleles or RNA transcribed from said alleles; and
(c) determining whether said oligonucleotide inactivates at least one but less than all of said alleles.

### Claim 5 (depends on 1)

The method of claim 1, wherein said potential allele specific inhibitor is a low molecular weight compound.

### Claim 6

An inhibitor specific for an allele of a gene coding for a product vital for cell viability or cell growth, wherein said gene has at least two alternative alleles and wherein said inhibitor inhibits an activity of said product from at least one but less than all of said alternative alleles.

### Claim 7 (depends on 6)

The inhibitor of claim 6, wherein said product is an RNA.

### Claim 8 (depends on 6)

The inhibitor of claim 6, wherein said product is a protein.

### Claim 9 (depends on 6)

The inhibitor of claim 6, wherein said gene is the human transferrin receptor gene.

### Claim 10 (depends on 6)

The inhibitor of claim 6, wherein said gene is the human dihydrofolate reductase gene.

### Claim 11 (depends on 6)

The inhibitor of claim 6, wherein said inhibitor is an antibody or antibody fragment.

### Claim 12 (depends on 6)

The inhibitor of claim 6, wherein said inhibitor is an oligopeptide.

### Claim 13 (depends on 6)

The inhibitor of claim 6, wherein said inhibitor is an oligonucleotide.

### Claim 14 (depends on 6)

The inhibitor of claim 6, wherein said inhibitor is a ribozyme.

### Claim 15 (depends on 6)

The inhibitor of claim 6, wherein said inhibitor is a low molecular weight compound.

### Claim 16 (depends on 9)

The inhibitor of claim 9, wherein said at least one but less than all of said alternative alleles of said gene has a G at the first position of codon 142 (nucleotide 687).

### Claim 17 (depends on 9)

The inhibitor of claim 9, wherein said at least one but less than all of said alternative alleles of said gene has an A at the first position of codon 142 (nucleotide 687).

### Claim 18 (depends on 9)

The inhibitor of claim 9, wherein said at least one but less than all of said alternative alleles of said gene has an A at the third position of codon 708 (nucleotide 2387).

### Claim 19 (depends on 9)

The inhibitor of claim 9, wherein said at least one but less than all of said alternative alleles of said gene has a G at the third position of codon 708 (nucleotide 2387).

### Claim 20 (depends on 10)

The inhibitor of claim 10, wherein said at least one but less than all of said alternative alleles of said gene has an A at nucleotide 721.

### Claim 21 (depends on 10)

The inhibitor of claim 10, wherein said at least one but less than all of said alternative alleles of said gene has a T at nucleotide 721.

### Claim 22 (depends on 10)

The inhibitor of claim 10, wherein said at least one but less than all of said alternative alleles of said gene has a T at nucleotide 829.

### Claim 23 (depends on 10)

The inhibitor of claim 10, wherein said at least one but less than all of said alternative alleles of said gene has a C at nucleotide 829.

### Claim 24

A method for producing an inhibitor against at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a target gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell;
(b) screening to identify an inhibitor which inhibits at least one but less than all of said alternative allelic forms present in said cancer cell; and
(c) synthesizing said inhibitor