# US 2021/0239681 A1 · Compositions and methods for evaluating potency of listeria-based immunotherapeutics

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- Number: US 2021/0239681 A1
- Document ID: US20210239681A1
- Status: Published application
- Kind: A1
- Application number: 17/049,004
- Filed: 2019-04-25
- Published: 2021-08-05
- Earliest claimed: 2018-04-27
- Assignees: Advaxis, Inc.
- Applicants: Advaxis, Inc.
- Inventors: Anu WALLECHA; Mike GRACE
- Web page: https://patentgrep.com/patent/US20210239681A1

## CPC

- G01N 33/505: {involving T-cells}
- G01N 33/5055: {involving macrophages}
- G01N 2333/195: from bacteria
- G01N 2333/555: Interferons [IFN]
- 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)

## Abstract

Methods and compositions are provided for assessing antigen presentation and potency of Listeria-based immunotherapeutics in inducing an immune response.

## Claims

### Claim 1

A method of assessing potency of a Listeria-based immunotherapeutic, comprising:
(a) infecting antigen presenting cells (APCs) with the recombinant Listeria-based immunotherapeutic to provide infected APCs, wherein the recombinant Listeria-based immunotherapeutic expresses a disease-associated antigenic peptide;
(b) co-culturing the infected APCs with a population of T cells enriched for T cells having reactivity to the disease-associated antigenic peptide; and
(c) determining a cytokine production profile of the T cells, wherein an increase in the cytokine production indicates expression of the disease-associated antigenic peptide in the infected APCs.

### Claim 2 (depends on 1)

The method of claim 1, wherein the APCs are THP-1 cells.

### Claim 3

The method of any preceding claim, wherein step (a) comprises infecting the APCs with the recombinant Listeria-based immunotherapeutic at a multiplicity of infection (MOI) of 1-200.

### Claim 4 (depends on 3)

The method of claim 3, wherein the APCs are infected with the recombinant Listeria-based immunotherapeutic at an MOI of about 1, about 2, about 5, about 10, about 20, about 100, or about 200.

### Claim 5

The method of any preceding claim, wherein infecting the APCs comprises incubating the APCs with the recombinant Listeria-based immunotherapeutic for 0.5-24 hours.

### Claim 6 (depends on 5)

The method of claim 5, wherein infecting the APCs comprises incubating the APCs with the recombinant Listeria-based immunotherapeutic for about 1 hour, about 2 hours, about 5 hours, or about 24 hours.

### Claim 7

The method of any preceding claim, wherein the APCs are washed and cultured for 18-24 hours prior to co-culture with the T cells.

### Claim 8

The method of any preceding claim, wherein the ratio of APCs to T cells in step (b) is 1:1 to 4:1.

### Claim 9

The method of any preceding claim, wherein the number of APCs in step (b) is about 5000 to about 40,000.

### Claim 10

The method of any preceding claim, wherein the APCs are co-cultured with the T cells for about 18-24 hours.

### Claim 11

The method of any preceding claim, wherein the APCs are co-cultured with the T cells in the presence of a protein secretion inhibitor, optionally wherein the protein secretion inhibitor is brefeldin A.

### Claim 12

The method of any preceding claim, wherein determining a cytokine expression profile of the T cells comprises measuring the level of interferon gamma (IFNγ) produced by the T cells.

### Claim 13 (depends on 12)

The method of claim 12, wherein determining a cytokine expression profile of the T cells comprises measuring the level of IFNγ produced by the T cells and secreted into a culture media.

### Claim 14 (depends on 12, 13)

The method of claim 12 or 13, wherein IFNγ is detected by enzyme-linked immunosorbent assay (ELISA).

### Claim 15

The method of any preceding claim, wherein the disease-associated antigenic peptide is a tumor-associated antigen.

### Claim 16

The method of any preceding claim, wherein the recombinant Listeria-based immunotherapeutic is a Listeria monocytogenes strain.

### Claim 17 (depends on 16)

The method of claim 16, wherein the Listeria monocytogenes comprises a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptide.

### Claim 18 (depends on 17)

The method of claim 17, wherein the PEST-containing peptide is listeriolysin O (LLO) or a fragment thereof, and the disease-associated antigenic peptide is a human papillomavirus (HPV) protein E7 or a fragment thereof.

### Claim 19 (depends on 17, 18)

The method of claim 17 or 18, wherein the recombinant Listeria-based immunotherapeutic is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

### Claim 20 (depends on 17)

The method of claim 17, wherein the recombinant Listeria-based immunotherapeutic is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of listeriolysin O (LLO).

### Claim 21 (depends on 16)

The method of claim 16 wherein the Listeria monocytogenes strain is ADXS11-001, and the T cell is an HPV-reactive T cell or an HPV-E7-reactive T cell.

### Claim 22

A method of assessing potency of a Listeria-based immunotherapeutic, comprising:
(a) infecting THP-1 cells with a recombinant Listeria-based immunotherapeutic at an MOI of 1-20 for 2 hours to provide infected THP-1 cells, wherein the recombinant Listeria-based immunotherapeutic comprises a live attenuated Listeria monocytogenes strain genetically modified to express a fusion protein of listeriolysin O (LLO) or a fragment thereof and a human papillomavirus (HPV) 16 protein E7 tumor antigen comprising HPV 16 protein E7 or a fragment thereof;
(b) washing the THP-1 cells and culturing the THP-1 cells for an additional 18-24 hours in the absence of gentamicin;
(c) co-culturing the infected THP-1 cells with T cells having reactivity to an HPV16 E7 antigenic peptide for 18-24 hours; and
(d) measuring interferon gamma (IFNγ) production, wherein an increase in IFNγ production indicates expression of the HPV 16 protein E7 tumor antigen or a fragment thereof in the infected THP-1 cells.

### Claim 23 (depends on 22)

The method of claim 22, wherein the HPV 16 E7 tumor antigen comprises SEQ ID NO: 101.