# US 2021/0330804 A1 · Cell penetrating conjugates and methods of use thereof

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- Number: US 2021/0330804 A1
- Document ID: US20210330804A1
- Status: Published application
- Kind: A1
- Application number: 17/184,237
- Filed: 2021-02-24
- Published: 2021-10-28
- Earliest claimed: 2013-08-29
- Assignees: City of Hope
- Applicants: City of Hope
- Inventors: Andreas HERRMANN; Hua YU; Piotr Marek SWIDERSKI; John Ernest SHIVELY; Lin LI
- Web page: https://patentgrep.com/patent/US20210330804A1

## CPC

- A61K 47/6843: {the antibody targeting a material from animals or humans}
- A61K 39/44: Antibodies bound to carriers
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61K 47/6807: {the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense}
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 16/084: Papillomaviridae (F); Polyomaviridae (F), e.g. SV40, BK virus or JC virus
- C07K 16/18: against material from animals or humans
- 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)
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 2310/315: Phosphorothioates
- C12N 2310/3513: Protein; Peptide
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are cell penetrating conjugates. The conjugates include a non-cell penetrating protein attached to a phosphorothioate nucleic acid or phosphorothioate polymer backbone, wherein the phosphorothioate nucleic acid or phosphorothioate polymer backbone enhances intracellular delivery of the non-cell penetrating protein. Also provided are compositions and kits comprising the cojugates.

## Claims

### Claim 1

A protein covalently attached to one or more phosphorothioate nucleic acids, wherein the protein has a molecular weight of greater than 25 kDa, wherein the protein binds an intracellular target, and wherein the protein is non-cell penetrating in the absence of said one or more phosphorothioate nucleic acids.

### Claim at position 2

-7. (canceled)

### Claim 8 (depends on 1)

The protein of claim 1, wherein each phosphorothioate nucleic acid is independently attached to a lysine, arginine, cysteine, or histidine of the protein.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

The protein of claim 1, wherein each phosphorothioate nucleic acid is independently 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length.

### Claim 11

(canceled)

### Claim 12

(canceled)

### Claim 13 (depends on 1)

The protein of claim 1, wherein the protein has a molecular weight of 25 to 750 kD.

### Claim 14 (depends on 1)

The protein of claim 1, wherein the protein is an antibody.

### Claim at position 10

-19. (canceled)

### Claim 20 (depends on 1)

The protein of claim 1, wherein the intracellular target is a target of a disease selected from the group consisting of autoimmune disease, inflammatory disease, metabolic disorder, developmental disorder, cardiovascular disease, liver disease, intestinal disease, infectious disease, endocrine disease, neurological disorder, and cancer.

### Claim 21 (depends on 20)

The protein of claim 20, wherein the intracellular target is a signaling molecule or transcription factor.

### Claim 22

(canceled)

### Claim 23

(canceled)

### Claim 24 (depends on 1)

The protein of claim 1, wherein the intracellular target is selected from the group consisting of STAT3, exportin 7, Her2, and Src.

### Claim at position 16

-27. (canceled)

### Claim 28 (depends on 1)

The protein of claim 1, made by contacting an unattached protein with an unattached phosphorothioate nucleic acid and allowing the unattached phosphorothioate nucleic acid to covalently bind to an amino acid of the unattached protein thereby attaching said protein and said phosphorothioate nucleic acid.

### Claim 29 (depends on 28)

The conjugated protein of claim 28, wherein the phosphorothioate nucleic acid comprises a covalent reactive moiety.

### Claim 30 (depends on 29)

The protein of claim 29, wherein the covalent reactive moiety is reactive with a lysine, arginine, cysteine or histidine of the protein.

### Claim at position 20

-35. (canceled)

### Claim 36 (depends on 1)

A cell comprising the protein of claim 1.

### Claim 37 (depends on 1)

A pharmaceutical composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.

### Claim 38 (depends on 37)

The pharmaceutical composition of claim 37, further comprising a second protein attached to one or more phosphorothioate nucleic acids.

### Claim 39

(canceled)

### Claim 40

(canceled)

### Claim 41 (depends on 38)

The pharmaceutical composition of claim 38, wherein the second protein binds a second intracellular target.

### Claim 42

(canceled)

### Claim 43 (depends on 1, 37)

A kit comprising the protein of claim 1 or the pharmaceutical composition of claim 37 and instructions for use.

### Claim 44 (depends on 43)

The kit of claim 43, further comprising a second protein attached to one or more phosphorothioate nucleic acids.

### Claim at position 30

-50. (canceled)

### Claim 51 (depends on 1)

A method of delivering a protein into a cell comprising contacting the cell with the protein of claim 1.

### Claim 52

(canceled)

### Claim 53

(canceled)

### Claim 54 (depends on 1)

A method of treating a disease in a subject comprising administering to the subject an effective amount of the protein of claim 1 wherein administration of the protein treats the disease in the subject.

### Claim 55 (depends on 54)

The method of claim 54, further comprising administering to the subject a second protein attached to one or more phosphorothioate nucleic acids.

### Claim at position 36

-61. (canceled)

### Claim 62 (depends on 54)

The method of claim 54, wherein the disease is selected from the group consisting of autoimmune disease, developmental disorder, inflammatory disease, metabolic disorder, cardiovascular disease, liver disease, intestinal disease, infectious disease, endocrine disease, neurological disorder, and cancer.

### Claim at position 38

-67. (canceled)