# US 10,371,695 B2 · Identifying compounds modifying a cellular phenotype

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- Number: US 10,371,695 B2
- Document ID: US10371695B2
- Status: Granted
- Kind: B2
- Application number: 15/321,888
- Filed: 2015-07-01
- Published: 2017-05-11
- Granted: 2019-08-06
- Earliest claimed: 2014-07-02
- Assignees: 2cureX ApS
- Applicants: 2cureX ApS
- Inventors: Grith Hagel; Ole Thastrup
- Web page: https://patentgrep.com/patent/US10371695B2

## CPC

- G01N 33/5011: {for testing antineoplastic activity}
- A61K 31/4745: condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines (yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48)
- A61K 31/513: having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
- A61K 31/519: ortho- or peri-condensed with heterocyclic rings
- A61K 31/555: containing heavy metals, e.g. hemin, hematin, melarsoprol
- A61K 31/704: attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin {(digitoxin A61K31/7048)}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- 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)

## Abstract

The present invention relates to a method and tools for extracting information on a compounds influence on a cellular phenotype. The method of the invention may be used as a very efficient procedure for testing the efficacy or resistance of single drugs or combinations of drugs on cells from individual patients. Thus, the methods may be useful for predicting efficacy of a drug on a given patient. The methods are also useful for testing of compounds for toxicity, identifying drug targets for known or novel compounds.

## Claims

### Claim 1

A method of identifying a compound or a combination of compounds that modify a least one cellular phenotype, said method comprising the steps of:
i) providing a library containing a plurality of library members, herein each library member is a compound or a combinations of compounds;
ii) providing a suspension of cells which may acquire said cellular phenotype;
iii) providing a cell-compatible support, wherein the support reversible can change between a sol state and a gel-state;
iv) providing an array containing a plurality of spaces;
v) adding said support in sol-state to the spaces of said array;
vi) adding library members to the spaces of said array, wherein at least two different library members are added to two different spaces,
wherein steps v) and vi) may be performed simultaneously or sequentially in any order,
vii) bringing the support to the eel-state;
viii) contacting the spaces of said array with the suspension of cells; and
ix) bringing the support into the sol-state,
wherein steps viii) and ix) may be performed simultaneously or sequentially in any order; and
x) bringing the support to the gel-state thereby entrapping cells in the support; and
xi) incubating the array under conditions allowing maintenance and/or growth of the cells,
xii) detecting the cellular phenotype in the cells,
xiii) identifying library members modifying the cellular phenotype,
thereby identifying a compound or a combination of compounds modifying said cellular phenotype.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said support is a temperature reversible gel.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said support is a sol-gel with a sol-gel transition temperature in the range of 0 to 35° C.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the support is a hydrogel selected from the group consisting of gelatinous gels and, copolymers, wherein the copolymer for example may be pluronic lecithin organogels or alginate hydrogels.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the array is a container comprising a plurality of compartments, which are separated by physical barriers.

### Claim 6 (depends on 5)

The method according to claim 5, wherein steps v) and vi) comprises adding the support and the library members to said compartments or wells, wherein at least two different library members are added to two different compartments or wells.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the cells are cells resected from a human being.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the cells are cancer cells provided at least in part in the form of spheroids and the cellular phenotype is in vitro growth of said spheroids.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the cell are primary cancer cells.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the cellular phenotype is selected from the group consisting of cell proliferation and cell death.

### Claim 11 (depends on 1)

The method according to claim 1, wherein one or more library me are drugs) useful in cancer treatment or a combination of drugs useful in cancer treatment.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the method comprises the steps of:
i) providing an array comprising a plurality of spaces, wherein each space comprises a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state; and
wherein at least 2 spaces further comprises different library members; and
wherein each library member is a compound or a combination of compounds, and
wherein the support is in the gel-state
ii) providing a suspension of cells which may acquire said cellular phenotype;
iii) contacting the spaces of said array with the suspension of cells; and
iv) bringing the support into the sol-state
wherein steps iii) and iv) may be performed simultaneously or sequentially in any order; and
v) bringing the support to the gel-state thereby entrapping cells in the support; and
vi) incubating the array under conditions allowing maintenance and/or growth of the cells,
vii) detecting the cellular phenotype in the cells,
viii) identifying library members modifying the cellular phenotype,
thereby identifying a compound or a combination of compounds modifying said cellular phenotype.

### Claim 13 (depends on 1)

A method of treatment of a clinical condition characterized by at least one cellular phenotype in an individual in need thereof, said method comprising the steps of:
i) obtaining cells associated with said clinical condition from an individual suffering from said clinical condition;
ii) identifying a compound or a combination of compounds modifying said at least one cellular phenotype characterizing said clinical condition by the method according to claim 1,
iii) administering a therapeutically effective amount of said compound or combination of compounds to said individual,
thereby treating said clinical condition.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the cells are causative of said clinical condition.

### Claim 15 (depends on 13)

The method according to claim 13, wherein the clinical condition is cancer.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the cellular phenotype is in vitro cancer cell proliferation.

### Claim 17 (depends on 15)

The method according to claim 15, wherein one or more library members are drugs useful in cancer treatment or a combination of drugs useful in cancer treatment.

### Claim 18 (depends on 15)

The method according to claim 15, wherein the method is a method for predicting the efficacy of treatment of a cancer in an individual in need thereof, the method comprising the steps of:
i) providing an array comprising a plurality of spaces, wherein each space comprises a cell-compatible support, wherein the support reversible can change between a sol-state and a gel-state; and
wherein at least 2 spaces further comprises different library members; and
wherein each library member is a drug or a combination of drugs useful in the treatment of cancer, and
wherein the support is in the gel-state
ii) providing a suspension of cancer cells or cancer cells in spheroids obtained from said individual;
iii) bringing the support to the gel-state;
iv) contacting the spaces of said array with the suspension of cells; and
v) bringing the support into the sol-state
wherein steps iv) and v) may be performed simultaneously or sequentially in any order;
vi) bringing the support to the gel-state thereby entrapping cells in the support; and
vii) incubating the array under conditions allowing growth of the cells
viii) detecting cancer cell proliferation and/or growth of cancer cell spheroids,
ix) identifying library members inhibiting proliferation of cancer cells and/or growth of cancer cell spheroids
wherein library members inhibiting proliferation of cancer cells and/or growth of cancer cell spheroids are predicted to be effective in treatment of cancer in said individual.