Compositions and methods for evaluating potency of listeria-based immunotherapeutics
AssigneeAdvaxis, Inc.
InventorsAnu WALLECHA, Mike GRACE
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Abstract
Methods and compositions are provided for assessing antigen presentation and potency of Listeria-based immunotherapeutics in inducing an immune response.
Claims23
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.
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.
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.
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.
The method of any preceding claim, wherein the ratio of APCs to T cells in step (b) is 1:1 to 4:1.
The method of any preceding claim, wherein the number of APCs in step (b) is about 5000 to about 40,000.
The method of any preceding claim, wherein the APCs are co-cultured with the T cells for about 18-24 hours.
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.
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.
The method of any preceding claim, wherein the disease-associated antigenic peptide is a tumor-associated antigen.
The method of any preceding claim, wherein the recombinant Listeria-based immunotherapeutic is a Listeria monocytogenes strain.
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.
Dependencies are inferred from the claim text.