Method for inhibiting adventitious viral infection
OwnerThomas Charles PERTEL, Ren SONG +5inventors
InventorsThomas Charles PERTEL, Ren SONG, Diego A. VARGAS-INCHAUSTEGUI, Garima YAGNIK, Houman DEHGHANI, Wenjing LI, Jose PENA
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Abstract
The instant disclosure relates to methods for preventing, controlling and/or inhibiting the rare events of adventitious viral infection or viral replication/amplification after reactivation of latent viral infection during cell culture in the manufacturing process of cell-based drug products, including CAR T cell drug products. Also provided are in vitro cell culture models for HHV-6 latent infection and methods of making the same.
Claims63
A method of preventing or inhibiting HHV-6 replication, preventing or inhibiting HHV-6 transcription activation, preventing or inhibiting HHV-6 infection, or preventing or inhibiting HHV-6 replication or infection after reactivation in a process of making engineered immune cells, the method comprising the steps of culturing immune cells, engineering the immune cells, and contacting the immune cells with an antiviral agent in a culture medium, optionally wherein the immune cells are suspected to have latent HHV-6 infection.
The method of any one of the preceding claims, wherein the immune cells are contacted with the antiviral agent for about 2 days, about 3 days, about 4 days, or about 5 days.
The method of any one of the preceding claims, wherein the antiviral agent is interferon (IFN), foscarnet or ganciclovir.
The method of any one of the preceding claims, wherein the antiviral agent is a type I IFN.
The method of any one of the preceding claims, wherein the antiviral agent is a type III IFN.
The method of any one of the preceding claims, wherein the antiviral agent is IFN, and wherein the IFN is added to the culture medium at a concentration of about 0.1 ng/ml to about 100 ng/ml or about 0.1 ng/ml to about 10 ng/ml.
The method of any one of the preceding claims, wherein the antiviral agent is IFN, and wherein the IFN is added to the culture medium at a concentration of about 0.1 ng/ml to about 1 ng/ml.
The method of any one of the preceding claims, wherein the antiviral agent is foscarnet, and wherein foscarnet is added to the culture medium at a concentration of about 8 μM to about 15 μM.
The method of any one of the preceding claims, wherein the antiviral agent is ganciclovir, and wherein the ganciclovir is added to the culture medium at a concentration of 25 μM to about 35 μM.
The method of any one of the preceding claims, wherein the engineered immune cells are T cells, PBMCs, IPSCs or NK cells.
The method of any one of the preceding claims, wherein the engineered immune cells exhibit reduced levels of HHV-6 replication, HHV-6 transcription activation, HHV-6 infection, or HHV-6 replication or infection after reactivation, as compared to control engineered immune cells without being contacted with the antiviral agent.
The method of any one of the preceding claims, wherein the engineered immune cells exhibited comparable levels of combined Tcm and Tscm as compared to control engineered immune cells without being contacted with the antiviral agent.
The method of any one of the preceding claims, further comprising the step of detecting or measuring HHV-6 DNA levels, RNA levels or protein levels during the process of making the engineered immune cells.
The method of any one of the preceding claims, further comprising a step of detecting or measuring HHV-6 DNA levels on day one of the process of making the engineered immune cells, wherein the HHV-6 DNA levels measured during the process of making the engineered immune cells are comparable to the HHV-6 DNA levels measured on day one of the process of making the engineered immune cells.
The method of any one of the preceding claims, further comprising the step of detecting HHV-6 RNA levels during the process of making the engineered immune cells.
The method any one of the preceding claims, wherein the step of engineering the immune cells comprises introducing to the immune cells an exogenous polynucleotide that encodes a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR).
The method of any one of the preceding claims, wherein the step of engineering the immune cells further comprises modifying one or both TCRα genetic loci to reduce or eliminate the expression or activity of the TCRα gene.
The method of any one of the preceding claims, wherein the immune cells harbor latent infection of HHV-6.
The method of any one of the preceding claims, wherein the antiviral agent is human IFNα.
A method for generating an in vitro cell culture model of HHV-6 latent infection and reactivation comprising the steps of
- (a) infecting human lymphoid cells with HHV-6;
- (b) culturing the infected cells for about 12 to about 19 days;
- and
- (c) serially passaging the cells to maintain a cell density of about 0.2-0.8×106 cells per cm2 for about 30 days to about 60 days, thereby establishing latent HHV-6 infection in the cells.
An in vitro cell culture model for HHV-6 latent infection, wherein no more than 104 copies of HHV-6 viral genome equivalents per 500 ng extracted DNA can be detected by qPCR in the cells and/or no HHV-6 transcript can be detected by RT-qPCR in the cells, wherein the cells are human lymphoid cells.
A method of making engineered immune cells comprising the steps of (a) culturing immune cells;
- (b) engineering the immune cells;
- and (c) contacting the immune cells with an antiviral agent in a culture medium, wherein the immune cells comprise latent HHV-6 infection.
Dependencies are inferred from the claim text.