# US 12,005,121 B2 · Compositions and methods for delivery of nucleic acids to cells

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- Number: US 12,005,121 B2
- Document ID: US12005121B2
- Status: Granted
- Kind: B2
- Application number: 17/823,492
- Filed: 2022-08-30
- Published: 2023-02-02
- Granted: 2024-06-11
- Earliest claimed: 2019-08-30
- Assignees: Yale University
- Applicants: Yale University
- Inventors: Elias Quijano; Peter Glazer
- Web page: https://patentgrep.com/patent/US12005121B2

## CPC

- 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}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 48/0025: {wherein the non-active part clearly interacts with the delivered nucleic acid}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 35/00: Antineoplastic agents
- C07K 14/4747: {Apoptosis related proteins}
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/77: Internalization into the cell
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/3513: Protein; Peptide
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- 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

Compositions and methods of use thereof for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 3E10 monoclonal antibody or an antigen binding, cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof, and (b) a nucleic acid cargo including, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex.

## Claims

### Claim 1

A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a non-covalent complex of (a) an antibody or antigen-binding fragment thereof, and (b) a therapeutic polynucleotide,
wherein the antibody or antigen-binding fragment thereof comprises VH complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and VL CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26;
and wherein the cancer comprises one or more cells that express a functional ENT2 transporter.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to SEQ ID NO:2 and a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to SEQ ID NO:7 or 8.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the VH has the amino acid sequence of SEQ ID NO:2 and the VL has the amino acid sequence of SEQ ID NO:7 or 8.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 and a VL having an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 53-55.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the VH has an amino acid sequence of SEQ ID NO: 6 and the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 53-55.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monovalent, divalent, or multivalent single chain variable fragment (scFv).

### Claim 8 (depends on 1)

The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the bispecific antibody comprises:
a first heavy chain or antigen-binding fragment thereof comprising CDRs having the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and a first light chain or antigen binding fragment thereof comprising CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; and
a second heavy chain, or antigen-binding fragment thereof, and a second light chain, or antigen binding fragment thereof, that associate to specifically bind a target cell-type, tissue, or organ.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the therapeutic polynucleotide is a polynucleotide inhibitor of protein translation.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the polynucleotide inhibitor of protein translation is an antisense oligonucleotide.

### Claim 12 (depends on 10)

The method according to claim 10, wherein the polynucleotide inhibitor of protein translation is selected from the group consisting of an siRNA, an miRNA, a shRNA, and an external guide sequence (EGS).

### Claim 13 (depends on 10)

The method according to claim 10, wherein the polynucleotide inhibitor targets mRNA encoding an oncogenic protein.

### Claim 14 (depends on 10)

The method according to claim 10, wherein the composition comprises:
a first complex of (a) an antibody or antigen-binding fragment thereof comprising VH CDRs having the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and VL CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and (b) a first polynucleotide inhibitor of protein translation; and
a second complex of (a) an antibody or antigen-binding fragment thereof comprising VH CDRs having the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and VL CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and (b) a second polynucleotide inhibitor of protein translation;
wherein the first polynucleotide inhibitor of protein translation and the second inhibitor of protein translation are different inhibitors of protein translation.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the therapeutic polynucleotide is an mRNA or plasmid.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the mRNA encodes a polypeptide ligand for a receptor of an immune cell.

### Claim 17 (depends on 16)

The method according to claim 16, wherein the polypeptide ligand stimulates the immune system of the subject.

### Claim 18 (depends on 15)

The method according to claim 15, wherein the mRNA encodes an antigen.

### Claim 19 (depends on 18)

The method according to claim 18, wherein the antigen is a viral antigen.

### Claim 20 (depends on 1)

The method according to claim 1, wherein the cancer is selected from the group consisting of a pancreatic cancer, a skin cancer, a brain cancer, a colorectal cancer, and a breast cancer.

### Claim 21 (depends on 1)

The method according to claim 1, wherein the cancer is selected from the group consisting of a vascular cancer, multiple myeloma, an adenocarcinoma, a sarcoma, a bone cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a kidney cancer, a liver cancer, a lung cancer, a nasopharangeal cancer, a prostate cancer, a stomach cancer, and a uterine cancer.

### Claim 22 (depends on 1)

The method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient.