# US 11,999,973 B2 · Method for producing recombinant oncolytic adenovirus with modified E1a cancer-specific promoter

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- Number: US 11,999,973 B2
- Document ID: US11999973B2
- Status: Granted
- Kind: B2
- Application number: 16/604,194
- Filed: 2018-04-10
- Published: 2020-01-30
- Granted: 2024-06-04
- Earliest claimed: 2017-04-10
- Assignees: EpicentRx, Inc.
- Applicants: EpicentRx, Inc.
- Inventors: Tony R. Reid; Bryan T. Oronsky; Christopher Larson
- Web page: https://patentgrep.com/patent/US11999973B2

## CPC

- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2710/10321: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/10332: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/10352: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to a method for producing a recombinant virus, e.g., a recombinant oncolytic adenovirus, using an A549 host cell.

## Claims

### Claim 1

A method for producing a recombinant oncolytic adenovirus comprising:
(a) infecting an A549 host cell with the recombinant oncolytic adenovirus to produce an infected A549 host cell or introducing a nucleic acid comprising a nucleotide sequence encoding the recombinant oncolytic adenovirus into an A549 host cell; and
(b) suspension culturing the A549 host cell in a serum-free medium, under conditions to permit production of the recombinant oncolytic adenovirus,
wherein the recombinant oncolytic adenovirus comprises an E1a gene comprising an E1a protein coding region operably linked to a modified regulatory sequence comprising (i) deletion of a functional Pea3 binding site, (ii) deletion of a functional E2F binding site, (iii) deletion of a functional TATA box, (iv) deletion of a functional CAAT box, (v) deletion of the nucleotides corresponding to the nucleotides from 195-244 of the Ad5 genome (SEQ ID NO: 1), or (vi) has two, three, or four of (i), (ii), (iii), and (iv).

### Claim 2 (depends on 1)

The method of claim 1, wherein the A549 host cell is a SF-BMAdR 281 A549 cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the A549 host cell is cultured for at least 3 days.

### Claim 4 (depends on 1)

The method of claim 1, further comprising, after step (b), the step of purifying the recombinant oncolytic adenovirus.

### Claim 5 (depends on 4)

The method of claim 4, wherein the step of purifying the recombinant oncolytic adenovirus comprises lysing the A549 host cell.

### Claim 6 (depends on 4)

The method of claim 4, wherein the step of purifying the recombinant oncolytic adenovirus comprises nuclease treatment.

### Claim 7 (depends on 4)

The method of claim 4, wherein the step of purifying the recombinant oncolytic adenovirus comprises ion exchange chromatography.

### Claim 8 (depends on 7)

The method of claim 7, wherein the step of purifying the recombinant oncolytic adenovirus comprises: (i) lysing the infected A549 host cell to produce a cell lysate;
(ii) treating the cell lysate with nuclease to produce a treated cell lysate; and (iii) purifying the recombinant virus from the treated cell lysate by ion exchange chromatography.

### Claim 9 (depends on 1)

The method of claim 1, wherein the method results in at least 10× more recombinant oncolytic adenovirus compared to a similar method that comprises, in step (a), introducing a nucleic acid comprising a nucleotide sequence encoding a recombinant oncolytic virus into a HEK293 host, and, in step (b), suspension culturing the HEK293 host cell in a serum-free medium, under conditions to permit production of the recombinant oncolytic adenovirus.

### Claim 10 (depends on 1)

The method of claim 1, wherein the method results in at least 10× more recombinant oncolytic adenovirus compared to a similar method that comprises, in step (b), adherent culturing the A549 host cell in a serum-free medium, under conditions to permit replication of the recombinant oncolytic adenovirus.

### Claim 11 (depends on 1)

The method of claim 1, wherein the method results in at least 10× more recombinant oncolytic adenovirus compared to a similar method that comprises, in step (b), suspension culturing the A549 host cell in a serum-containing medium, under conditions to permit replication of the recombinant oncolytic adenovirus.

### Claim 12 (depends on 1)

The method of claim 1, wherein the E1a promoter has the deletion of a functional Pea3 binding site.

### Claim 13 (depends on 1)

The method of claim 1, wherein the E1a promoter has the deletion of a functional TATA box.

### Claim 14 (depends on 1)

The method of claim 1, wherein the recombinant oncolytic adenovirus comprises a polynucleotide deletion that results in a virus sequence comprising the junction sequence corresponding to CTAGGACTG (SEQ ID NO: 3), AGTGCCCG (SEQ ID NO: 8) and/or TATTCCCG (SEQ ID NO: 9) of the Ad5 genome (SEQ ID NO: 1).

### Claim 15 (depends on 1)

The method of claim 1, wherein the E1a promoter has the deletion of a functional CAAT box.

### Claim 16 (depends on 1)

The method of claim 1, wherein the recombinant oncolytic adenovirus comprises a nucleotide sequence encoding a transgene.

### Claim 17 (depends on 16)

The method of claim 16, wherein the nucleotide sequence is inserted into an E1b-19K insertion site, and wherein the E1b-19K insertion site is located between the start site of E1b-19K and the stop site of E1b-19K.

### Claim 18 (depends on 16)

The method of claim 16, wherein the transgene is not operably linked to an exogenous promoter sequence and/or the recombinant virus selectively expresses the transgene in a hyperproliferative cell.

### Claim 19 (depends on 16)

The method of claim 16, wherein the transgene encodes a polypeptide selected from CD80, CD137L, IL-23, IL-23A/p19, p40, IL-27, IL-27A/p28, IL-27B/EBI3, ICAM-1, a TGF-β Trap, TGF-β, CD19, CD20, IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, CD154, CD86, BORIS/CTCFL, FGF, IL-24, MAGE, NY-ESO-1, acetylcholine, interferon-gamma, DKK1/Wnt, p53, thymidine kinase, an anti-PD-1 antibody heavy chain or light chain, and an anti-PD-L1 antibody heavy chain or light chain.

### Claim 20 (depends on 1)

The method of claim 1, wherein the recombinant virus selectively replicates in a hyperproliferative cell.

### Claim 21 (depends on 1)

The method of claim 1, wherein the Pea3 binding site is selected from Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and Pea3 V.

### Claim 22 (depends on 1)

The method of claim 1, wherein the E2F binding site is selected from E2F I and E2F II.

### Claim 23 (depends on 1)

The method of claim 1, wherein the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5).