In vitro methods for inhibiting HHV-6 infection

Abstract

The present disclosure relates to methods for preventing, controlling and / or inhibiting rare events of foreign viral infections or viral replication / expansion following reactivation of latent viral infections during cell culture during the manufacture of cell-based pharmaceutical products, including CAR T cell pharmaceutical products. The invention also provides an in-vitro cell culture model of HHV-6 latent infection and a preparation method thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 398,364, filed on August 16, 2022, and U.S. Provisional Application No. 63 / 510,025, filed on June 23, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on August 14, 2023, named AT-055-03WO_ST26.xml, and is 37,605 bytes in size. Technical Field

[0005] The present disclosure relates to methods for preventing and / or inhibiting infection by foreign viruses and for preventing and / or inhibiting the rare event of infection reactivation following reactivation of a latent viral infection, in the manufacture of cell-based pharmaceutical products, including chimeric antigen receptor (CAR) T cell pharmaceutical products, during long-term cell culture, such as during long-term culture of primary cells with a latent viral infection. Background Art

[0006] Human herpesvirus 6 (HHV-6) usually infects young people. HHV-6 can be divided into two closely related but different subtypes, HHV-6A and HHV-6B. Both subtypes can establish lytic infection and latent infection, and both subtypes can infect human T cells. HHV-6B accounts for the vast majority of infections in the United States, the United Kingdom and Japan, while HHV-6A infection is mainly limited to sub-Saharan Africa. HHV-6B is the pathogen of infantile roseola and is associated with neurological diseases such as encephalitis. The epidemiology and symptoms associated with HHV-6A are still poorly defined. The specific triggers for the reactivation of the two HHV-6 strains are still unclear. The lack of HHV-6 latent infection and reactivation of cell culture models has hindered such research in this field. For therapies based on living cells, such as stem cell transplantation therapy, and therapies based on autologous and allogeneic CAR T cells, although rare, foreign viral infections may occur during cell culture in the manufacturing process. In addition, the reactivation of latently infected viruses (such as HHV-6) may also occur, but it is extremely rare. In allogeneic cell-based therapies, healthy donors are screened for transmissible and infectious diseases to minimize the risk in downstream manufacturing processes. In addition, as part of the quality control process, the reactivation of exogenous virus infection and endogenous or endemic viruses during cell culture during the manufacturing process can be detected by strict screening assays to ensure drug safety. Any batch of drugs that do not meet safety standards will be rejected.

[0007] Therefore, methods to prevent and control rare events of infection by adventitious viruses or post-reactivation infection are needed during the manufacturing of cell-based therapies to ensure drug safety and minimize waste. Cell culture models for HHV-6 latent infection and reactivation also need to be established. Summary of the invention

[0008] Provided herein are methods for preparing engineered immune cells, or methods for inhibiting and / or controlling rare events of exogenous viral infection and / or viral reactivation during cell culture in the manufacturing process of cell-based therapies, including CAR T cell therapies.

[0009] In one aspect, provided herein is a method for preventing, controlling or inhibiting HHV-6 replication after reactivation of immune cells isolated in cell culture, the method comprising contacting immune cells with antiviral agents in a culture medium. In certain embodiments, the antiviral agent is interferon (IFN), such as IFNα, ganciclovir, cidofovir or foscarnet or its salt. In another aspect, provided herein is a method for preventing, controlling or inhibiting HHV-6 replication, preventing, controlling or inhibiting HHV-6 transcriptional activation, preventing, controlling or inhibiting HHV-6 infection, or preventing, controlling or inhibiting HHV-6 replication or infection after reactivation in the process of preparing engineered immune cells, the method comprising the following steps: culturing immune cells, engineering the immune cells and contacting the immune cells with antiviral agents in a culture medium. In certain embodiments, the antiviral agent is IFNα, foscarnet, ganciclovir or cidofovir.

[0010] In some embodiments, HHV-6 is human HHV-6A. In certain embodiments, HHV-6 is human HHV-6B.

[0011] In some embodiments, the step of contacting the immune cells with the antiviral agent is performed by adding the antiviral agent to the culture medium. In certain embodiments, the antiviral agent is added to the culture medium on the 4th to the 10th day, the 5th to the 10th day, the 6th to the 10th day, the 7th to the 10th day, the 8th to the 10th day, or the 9th to the 10th day in the process of preparing engineered immune cells. In some embodiments, the antiviral agent is added to the cell culture medium after the cells have been cultured for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or at least 9 days. In some embodiments, the immune cells are contacted with the antiviral agent for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the immune cells are PBMCs. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are activated on the 1st day of the process of preparing engineered immune cells, optionally after the immune cells have been thawed. In some embodiments, the immune cells are activated by contacting the immune cells with anti-CD3 and anti-CD28 antibodies. In some embodiments, the antiviral agent is added to the cell culture medium 3 days, 4 days, 5 days, 6 days, 7 days or 8 days after the immune cells are activated. In some embodiments, the antiviral agent is IFN. In some embodiments, IFN is type I IFN or type III IFN. In some embodiments, IFN is IFNα. In some embodiments, IFN (e.g., IFNα) is added to the culture medium at a concentration of about 0.1ng / ml to about 100ng / ml, about 0.1ng / ml to about 10ng / ml or about 0.1ng / ml to about 1ng / ml. In some embodiments, the antiviral agent is foscarnet or its salt. In some embodiments, foscarnet or its salt is added to the culture medium at a concentration of about 8 μM to about 20 μM, about 8 μM to about 15 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, or about 15 μM. In some embodiments, the antiviral agent is ganciclovir or its salt. In some embodiments, ganciclovir or its salt is added to the culture medium at a concentration of about 25 μM to about 35 μM, about 25 μM to about 30 μM, about 27 μM to about 30 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, or about 35 μM. In some embodiments, the antiviral agent is cidofovir or its salt. In some embodiments, cidofovir or a salt thereof is added to the culture medium at a concentration of about 14 μM to about 25 μM, about 14 μM to about 20 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, or about 20 μM.

[0012] In some embodiments, the immune cell is a T cell, PBMC, IPSC or NK cell. In some embodiments, the immune cell is obtained from a patient or from a healthy donor. In some embodiments, the immune cell carries a latently infected HHV-6 genome or is latently infected by HHV-6.

[0013] In some embodiments, the immune cells exhibit reduced levels of HHV-6 replication, HHV-6 transcriptional activation, HHV-6 infection, or HHV-6 replication or infection after reactivation compared to control immune cells not exposed to the antiviral agent. In some embodiments, the immune cells exhibit the same level of combined Tcm and Tscm compared to control immune cells not exposed to the antiviral agent. In some embodiments, the method further includes a step of detecting or measuring HHV-6 DNA levels, RNA levels, or protein levels during the preparation of engineered immune cells. In some embodiments, after the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day, 14th day, 15th day, 16th day, 17th day, 18th day, 19th day, 20th day or 20th day of the process of preparing engineered immune cells, HHV-6 DNA levels, RNA levels and / or protein levels are measured. In some embodiments, at the beginning and / or at the end of the process of preparing engineered immune cells, HHV-6 DNA levels, RNA levels and / or protein levels are measured. In some embodiments, the HHV-6 DNA levels, RNA levels and / or protein levels measured at the beginning of the process of preparing engineered immune cells and at the end of the process of preparing engineered immune cells are equal. In some embodiments, the HHV-6 DNA level, RNA level and / or protein level measured from about the 1st day to about the 5th day of the process of preparing engineered immune cells are equivalent to the level measured from about the 15th day to about the 20th day of the process of preparing engineered immune cells. In some embodiments, the method also includes a step of detecting HHV-6 RNA levels in the process of preparing engineered immune cells, wherein the HHV-6 RNA level is undetectable. In some embodiments, HHV-6 DNA levels, RNA levels or protein levels are determined by PCR, qPCR, RT-PCR, RT-qPCR, ELISA, immunofluorescence assay or flow cytometry.

[0014] In some embodiments, the antiviral agent is IFN, and preferably the IFN is IFNα. In certain embodiments, the IFN is human IFNα.

[0015] Also provided herein is a method for preparing engineered immune cells, the method comprising the following steps: (a) culturing immune cells; (b) engineering the immune cells; and (c) contacting the immune cells with an antiviral agent such as interferon (IFN) in a culture medium, wherein the immune cells contain latent HHV-6 infection or are latently infected with HHV-6. In some embodiments, the step of contacting the immune cells with the antiviral agent is performed by adding the antiviral agent to the culture medium. In some embodiments, the antiviral agent is added to the culture medium on the 4th to 10th day, the 5th to 10th day, the 6th to 10th day, the 7th to 10th day, the 8th to 10th day, and the 9th to 10th day after culturing the immune cells. In some embodiments, the antiviral agent is added to the cell culture medium after the cells have been cultured for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or at least 9 days. In some embodiments, the immune cells are activated on the first day of the process of preparing engineered immune cells, optionally after the immune cells have been thawed. In some embodiments, the immune cells are activated by contacting the immune cells with anti-CD3 and anti-CD28 antibodies. In some embodiments, the antiviral agent is added to the cell culture medium 3 days, 4 days, 5 days, 6 days, 7 days or 8 days after the immune cells are activated. In some embodiments, the immune cells are contacted with the antiviral agent for about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In some embodiments, the antiviral agent is IFN, such as type I IFN or type III IFN, foscarnet, ganciclovir or cidofovir or its salt. In some embodiments, IFN is human IFNα. In some embodiments, the antiviral agent is or comprises IFN, which is added to the culture medium at a concentration of about 0.1 ng / ml to about 100 ng / ml, about 0.1 ng / ml to about 10 ng / ml or about 0.1 ng / ml to about 1 ng / ml. In some embodiments, IFN is IFNα. In certain embodiments, IFN is human IFNα. In some embodiments, the antiviral agent is or comprises foscarnet or a salt thereof. In some embodiments, foscarnet or a salt thereof is added to the culture medium at a concentration of about 8 μM to about 20 μM, about 8 μM to about 15 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM or about 15 μM. In some embodiments, the antiviral agent is or comprises ganciclovir or a salt thereof. In some embodiments, ganciclovir or a salt thereof is added to the culture medium at a concentration of about 25 μM to about 35 μM, about 25 μM to about 30 μM, about 27 μM to about 30 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM or about 35 μM.In some embodiments, the immune cells are T cells, PBMCs, iPSCs or NK cells. In some embodiments, the immune cells are obtained from patients or healthy donors.

[0016] In some embodiments, the method further comprises a step of detecting or measuring HHV-6 DNA level, RNA level or protein level after the step of contacting the immune cell with the antiviral agent. In some embodiments, the method further comprises a step of detecting or measuring HHV-6 DNA level before contacting the immune cell with the antiviral agent, wherein the HHV-6 DNA level measured after contacting the immune cell with the antiviral agent is equivalent to the HHV-6 DNA level measured before the step of contacting the immune cell with the antiviral agent.

[0017] In some embodiments, the step of engineering the immune cell includes introducing an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) into the immune cell. In some embodiments, the engineered immune cell is a CAR T cell. In some embodiments, the exogenous polynucleotide is introduced into the immune cell by lentiviral transduction or by adenovirus-associated virus transduction. In some embodiments, the step of engineering the immune cell also includes modifying one or two TCRα loci to reduce or eliminate the expression or activity of the TCRα gene.

[0018] In another aspect, provided herein are engineered immune cells or engineered immune cell populations produced by the methods described herein.

[0019] In another aspect, the present disclosure provides a method for generating an in vitro cell culture model of HHV-6 latent infection and reactivation, the method comprising the steps of: (a) infecting human lymphocytes with HHV-6; (b) culturing the infected cells for about 12 to about 19 days or until logarithmic growth of the virus is observed; and (c) serially passage the cells to maintain about 0.2-0.8×10 6 cells / cm 2The cell density of about 30 days to about 60 days, thereby establishing latent HHV-6 infection in the cell. In some embodiments, the method also includes detecting HHV-6 viral DNA or detecting HHV-6 viral RNA and / or HHV-6 protein after the cell is continuously passaged in step (c), wherein a constant level of HHV-6 DNA or the absence of detectable RNA and / or protein indicates latent infection. In some embodiments, the latent HHV-6 infection in the cell can be reactivated by a stimulant. In some embodiments, the stimulant is sodium butyrate and / or PMA (phorbol-12-myristic acid-13-acetate). In some embodiments, the method also includes the step of detecting the expression of one or more viral transcripts, wherein the one or more viral transcripts are detected to indicate viral reactivation.

[0020] In a related aspect, the present disclosure also provides an in vitro cell culture model for HHV-6 latent infection and reactivation produced by the methods described herein, wherein no more than 10 ng of extracted genomic DNA per 500 ng can be detected in the cells by qPCR. 4 The invention relates to a method for detecting the HHV-6 viral genome equivalent of a copy, and / or failing to detect HHV-6 transcripts in the cells by RT-qPCR. In some embodiments, active HHV-6 infection can be in cells, optionally induced or reactivated by a stimulus. In some embodiments, the stimulus comprises sodium butyrate and PMA. In some embodiments, active HHV-6 infection can be demonstrated by detecting one or more HHV-6 transcripts associated with viral function or structural genes, optionally detected by RT-PCR or RT-qPCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A shows a bar graph of HHV-6 viral DNA levels in selected CAR T drug products, and Figure 1B The lower panel shows immunofluorescence staining images of HHV-6 p41 protein levels with or without IFNα treatment. Figure 1B The upper panel shows the DAPI staining image of the cell nucleus. Figure 1C The data in show longitudinal analysis of HHV-6U31 DNA levels in two batches of CAR T cell cultures.

[0022] Figure 2A Depicted is the experimental design used to establish a cell culture model of HHV-6 latent infection. Figure 2BShown are U31 or U65-66 DNA levels in cells during the first 19 days after infection (left graph), and DNA levels in continuously cultured cells after two months of serial passage (time point 1) and after another week (time point 2) (right graph). DNA levels were measured by qPCR. Figure 2C Shown by using The absence of viral transcripts during the systematically determined continuous cell culture period. Figure 2D-Figure 2E Shown are the increased viral DNA levels detected upon reactivation ( Figure 2D ) and increased viral transcripts ( Figure 2E ). IE: viral immediate early gene; IE-E: viral immediate early-early gene; E: viral early gene; L: viral late gene. Figure 2F Shown are the increased viral transcripts detected over time following reactivation inducing treatment (Tx).

[0023] Figure 3A-3B Reactivation was shown to result in increased viral DNA levels in a cell culture latency model ( Figure 3A ), and increased IFNα added before or after reactivation suppressed viral DNA expansion ( Figure 3B ).

[0024] Figure 4A Depicted is the experimental design for testing the effect of IFNα on HHV-6 infection in a small-scale allogeneic CAR T generation process using cells from two donors. Figure 4B The fold exchange of viral genes U79, U90 and U100 RNA determined by RT-qPCR in the presence or absence of IFNα and with or without HHV-6 infection from day 11 to day 18 (i.e., 3 to 10 days after infection) is shown. The viral protein p41 and viral gene U31 DNA levels were measured under the same conditions and the results are shown in Figure 4C and Figure 4D middle.

[0025] Figure 5A The data in examined the cell expansion fold and CAR+ percentage from day 8 to day 18 (i.e., days 0-10 after HHV-6 infection) of the small-scale CAR T generation process. Figure 5B The cellular phenotypes of CAR+ cells at day 18 (i.e., 10 days after HHV-6 infection) under different treatment conditions were examined.

[0026] Figure 6A-6B The results in Figure 2 show that under different IFNα treatment conditions, in samples collected at different days during the process, HHV-6U100 transcripts ( Fig. 6A ) or U79 transcript ( Figure 6B) relative fold change. Figure 7A-7B is the same experiment repeated in cells from different donors.

[0027] Figure 8 Depicted are the results of apoptosis analysis of iCiHHV6 cells treated with different antiviral agents at day 3 or day 15 after antiviral treatment. Fig. 9 The data in show HHV-6 levels as determined by U65 gene copies analyzed by qPCR. DETAILED DESCRIPTION

[0028] In one aspect, the present disclosure provides a method for preventing, controlling or inhibiting HHV-6 infection or replication after HHV-6 reactivation of immune cells isolated in cell culture, the method comprising the step of contacting the immune cells with an antiviral agent (e.g., IFN) in a culture medium.

[0029] In a specific aspect, provided herein is a method for preventing, controlling or inhibiting HHV-6 amplification, replication, infection and / or transcriptional activation, the method comprising contacting an immune cell with an antiviral agent (e.g., IFN). One application is the manufacturing process of engineered immune cells (such as CAR T cells). By preventing rare events of HHV-6 amplification or replication, such as after virus reactivation during long-term cell culture during manufacturing, the rejection rate of clinical-grade batches can be further reduced.

[0030] As used herein, the terms "a" and "an" are used to indicate one or more. For example, reference to a "cell", "immune cell" or "antibody" means "one or more cells", "one or more immune cells" or "one or more antibodies".

[0031] Type I and type III interferons (IFNs) are a large class of proteins that jointly regulate the activity of the human immune system. IFN responses constitute the main first line of defense against viruses. Innate immune sensors recognize and activate type I and type III IFN responses to viral infections. Human type I IFNs include IFNα, IFNβ, IFNε, IFNκ, IFNω, and type III IFNs include IFNλ. IFN is an antiviral agent that activates immune cells, including leukocytes and T cells, as well as NK cells, and regulates the function of the immune system. IFN is released by virus-infected cells and exerts its function by binding to cell surface receptors and activating many interferon-stimulated genes (ISGs), which have the ability to interfere with every step of viral replication. See Park and Iwasaki, 2020, Cell Host & Microbe, 27: 870-878.

[0032] However, it has not been demonstrated that IFN can be used as a culture medium additive that can block HHV-6 infection, replication, infection after virus reactivation or virus replication / amplification, and / or transcriptional activation during the culture or manufacture of immune cells, or during the culture or manufacture of engineered immune cells. In certain embodiments, the immune cell or the engineered immune cell is an autologous CAR T cell. In certain embodiments, the immune cell or the engineered immune cell is an allogeneic CAR T cell.

[0033] In some embodiments, the IFN is a type I IFN. In certain embodiments, the IFN is IFNα or IFNβ. In some embodiments, the IFN is added to the culture medium at the following concentrations: about 0.01 ng / ml to about 100 ng / ml, about 0.01 ng / ml to about 10 ng / ml, about 0.01 ng / ml to about 1 ng / ml, about 0.01 ng / ml to about 0.1 ng / ml, about 0.1 ng / ml to about 100 ng / ml, about 0.1 ng / ml to about 10 ng / ml, about 0.1 ng / ml to about 1 ng / ml, about 0.1 ng / ml to about 0.9 ng / ml. ml, about 0.1 ng / ml to about 0.8 ng / ml, about 0.1 ng / ml to about 0.7 ng / ml, about 0.1 ng / ml to about 0.6 ng / ml, about 0.1 ng / ml to about 0.5 ng / ml, about 0.1 ng / ml, about 0.2 ng / ml, about 0.3 ng / ml, about 0.4 ng / ml, about 0.5 ng / ml, about 0.6 ng / ml, about 0.7 ng / ml, about 0.8 ng / ml, about 0.9 ng / ml or about 1 ng / ml. In certain embodiments, the IFN is IFNα. In certain embodiments, the IFN is human IFNα.

[0034] Although no formal treatment for HHV-6 has yet been developed, several existing antiviral agents, including foscarnet, ganciclovir, and cidofovir, have been shown to have in vitro efficacy against HHV-6. See hhv-6foundation.org / research / hhv-6-antiviral-drug-resistance. However, it has not been demonstrated that they can be used as culture medium additives that can block HHV-6 infection, replication, infection after viral reactivation, or viral replication / amplification, and / or transcriptional activation during the culture or manufacture of immune cells, or during the culture or manufacture of engineered immune cells. In some embodiments, the antiviral agent is foscarnet or a salt thereof. In some embodiments, foscarnet or a salt thereof is added to the culture medium at a concentration of about 8 μM to about 20 μM, about 8 μM to about 15 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, or about 15 μM. In some embodiments, the antiviral agent is ganciclovir or a salt thereof. In some embodiments, ganciclovir or a salt thereof is added to the culture medium at a concentration of about 25 μM to about 35 μM, about 25 μM to about 30 μM, about 27 μM to about 30 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, or about 35 μM.

[0035] The present disclosure provides a method for preventing or controlling HHV-6 replication of isolated immune cells or engineered immune cells. In some embodiments, HHV-6 replication can be the result of viral transmission after viral infection, latent viral infection reactivation (latency). In some embodiments, the method disclosed herein prevents the development of active infection. In some embodiments, the method disclosed herein prevents the progression (or spread) of viral infection after latent viral infection reactivation (or viral latency). In some embodiments, the method disclosed herein prevents the development of reactivated latent viral infection into active infection. Therefore, the method disclosed herein inhibits HHV-6 replication of isolated immune cells or engineered immune cells. In some embodiments, the method disclosed herein inhibits HHV-6 active infection of isolated immune cells or engineered immune cells. In certain embodiments, immune cells or engineered immune cells are autologous CAR T cells or allogeneic CART cells.

[0036] As used herein, the term "viral replication" or "viral amplification" may refer to active viral infection, as opposed to latent viral infection. Such active infection may or may not result in virion production and / or lytic infection. For endemic viruses that can establish latent infection in cells, an increase in viral DNA levels compared to latent infection levels, and / or detection or increase of one or more viral RNA transcripts associated with functional or structural genes compared to latent infection levels, can indicate active infection, and therefore indicate viral replication or viral amplification. Therefore, the present disclosure provides a method for preventing, controlling or inhibiting HHV-6 replication or amplification after viral reactivation. Viral DNA levels and viral RNA transcript levels can be detected by methods known in the art and / or described herein. In some embodiments, the virus is HHV-6.

[0037] In some embodiments, HHV-6 replication can be determined by detecting the presence or increase of viral DNA, as measured by methods including but not limited to PCR, qPCR, bulk DNA sequencing, or single cell DNA sequencing. In some embodiments, HHV-6 replication can be determined by detecting the presence or increase of viral RNA transcripts, as measured by methods including but not limited to RT-PCR, RT-qPCR, Batch RNA sequencing or single cell RNA sequencing method measured.In some embodiments, HHV-6 replication can be determined by detecting the presence or increase of viral protein expression, such as measured by including but not limited to flow cytometry, immunofluorescence staining or other immunological methods.In some embodiments, HHV-6 replication can be determined by detecting and / or quantifying the cytopathic effect (CPE) caused by viral replication.In some embodiments, immune cells or engineered immune cells can be co-cultured with a reporter cell line sensitive to HHV-6 infection, or the reporter cell line can be exposed to the cell culture supernatant of immune cells or engineered immune cells, and the HHV-6 infection of the reporter cell line can be detected and / or quantitatively reported according to methods known in the art and / or described herein to determine HHV-6 replication.

[0038] In another aspect, the disclosure provides a method for preventing or inhibiting HHV-6 transcriptional activation. In some embodiments, HHV-6 transcriptional activation can be the activation of one or more HHV-6 immediate early genes, early genes and / or late gene transcription. In some embodiments, HHV-6 transcriptional activation indicates viral reactivation. In some embodiments, HHV-6 transcriptional activation indicates productive or active viral infection. In some embodiments, HHV-6 transcriptional activation can be determined by detecting the presence or increase of viral RNA transcripts, as measured by methods including but not limited to RT-PCR or RT-qPCR.

[0039] In a related aspect, the present disclosure provides methods of preventing, controlling or inhibiting HHV-6 replication.

[0040] In addition, the present invention provides a method for preventing, controlling or inhibiting HHV-6 replication, preventing, controlling or inhibiting HHV-6 transcriptional activation, preventing, controlling or inhibiting HHV-6 infection, or preventing, controlling or inhibiting HHV-6 replication or infection after reactivation in the process of preparing engineered immune cells, the method comprising the following steps: culturing immune cells, engineering the immune cells and contacting the immune cells with IFN in a culture medium. Exogenous viruses, such as HHV-6, infect young people and establish endemic latent infections in the general population. The present disclosure provides a favorable method for preventing, controlling and / or inhibiting viral replication in the process of producing cell-based therapies, in vitro or in vitro cell culture, after such endemic latent infections are reactivated, but reactivation events are extremely rare. Cell-based therapy can be an autologous cell-based therapy derived from a patient's immune cells, engineered or obtained. Cell-based therapy can be a therapy of genetically allogeneic cells derived from, obtained or engineered from immune cells of healthy volunteers.

[0041] In some embodiments, the cell is an engineered immune cell. In some embodiments, the engineered immune cell is a CAR T cell. In some embodiments, the engineered immune cell is a CAR NK cell. In some embodiments, the engineered immune cell is an allogeneic or autologous immune cell. In some embodiments, the engineered immune cell is an autologous or allogeneic CAR T cell.

[0042] Immune cells

[0043] Cells suitable for use with the methods and / or reagents described herein include immune cells.

[0044] Prior to in vitro manipulation or genetic modification (e.g., as described herein), cells (e.g., immune cells) for the methods described herein can be obtained from subjects. Cells can be obtained from multiple non-limiting sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, stem cells or iPSC-derived immune cells, tissues from infection sites, ascites, pleural effusions, spleen tissue, and tumors. In some embodiments, any number of available and known T cell lines of technicians in the field can be used. In some embodiments, cells can be derived from healthy donors, from patients diagnosed with cancer, or from patients diagnosed with infection. In some embodiments, cells can be part of a mixed cell population presenting different phenotypic characteristics.

[0045] In some embodiments, the immune cells are autologous immune cells obtained from the subject who will ultimately receive the engineered immune cells. In some embodiments, the immune cells are allogeneic immune cells obtained from a donor who is a different individual than the subject who will receive the engineered immune cells.

[0046] In some embodiments, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, stem cells or iPSC-derived T cells, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, any number of techniques known to technicians can be used, such as FICOLL TM Isolate and obtain T cells from a volume of blood collected from a subject.

[0047] Cells can be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction, and the plasma fraction is placed in an appropriate buffer or culture medium for subsequent processing.

[0048] Using methods as described herein, PBMC can be directly used for the genetic modification of immune cells (such as CAR or TCR). In certain embodiments, after separating PBMC, T lymphocytes can be further separated, and cytotoxic and helper T lymphocytes can be sorted into primary, memory and effector T cell subsets before or after genetic modification and / or amplification.

[0049] In certain embodiments, for example, using PERCOLL TMGradient centrifugation, by lysing red blood cells and removing monocytes, separates T cells from PBMC. T cells, such as CCR7+, CD95+, CD122, CD27+, CD69+, CD127+, CD28+, CD3+, CD4+, CD8+, CD25+, CD62L+, CD45RA+ and CD45RO+T cells can be further separated by positive or negative selection techniques known in the art. For example, the enrichment of T cell colonies by negative selection can be achieved by a combination of antibodies for surface markers unique to cells selected for negative selection. One of the methods used herein is to sort cells and / or select by negative magnetic immunoadhesion or flow cytometry, which uses a monoclonal antibody mixture for cell surface markers present on cells selected for negative selection. For example, in order to enrich for CD4+ cells by negative selection, a monoclonal antibody mixture generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0050] In some embodiments, the T cell population is enriched for CD4+ cells.

[0051] In some embodiments, the T cell population is enriched for CD8+ cells.

[0052] In some embodiments, the T cell population is enriched for CD4+ and CD8+ cells.

[0053] In some embodiments, CD8+ cells are further sorted into primary, central memory, and effector cells by identifying cell surface antigens associated with each of these cell types. In some embodiments, the expression of phenotypic markers of primary T cells includes CD45RA+, CD95-, IL2Rβ-, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of stem cell memory T cells includes CD45RA+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers of effector memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7-, and CD62L-. In some embodiments, the expression of phenotypic markers of T effector cells includes CD45RA+, CD95+, IL2Rβ+, CCR7-, and CD62L-. Thus, CD4+ and / or CD8+ T helper cells can be sorted into naive, stem cell memory, central memory, effector memory, and T effector cells by identifying cell populations that have cell surface antigens.

[0054] It should be understood that PBMCs may further comprise other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying coding sequences of chimeric receptors as disclosed herein may be introduced into populations of human donor T cells, NK cells or NKT cells. Standard procedures are used to cryopreserve CAR-expressing T cells for storage and / or preparation for human subjects. In one embodiment, in vitro transduction, culture and / or amplification of T cells are performed in the absence of non-human animal-derived products, such as fetal bovine serum (fetal calf serum / fetalbovine serum). In various embodiments, cryopreservation medium may include, for example CS2, CS5 or CS10 or other medium containing DMSO, or medium without DMSO.

[0055] Engineered immune cells

[0056] Provided herein are engineered immune cells (e.g., CAR-T cells) expressing the CARs of the present disclosure.

[0057] In some embodiments, the engineered immune cells include a CAR population, each CAR including an extracellular antigen binding domain. In some embodiments, the engineered immune cells include a CAR population, each CAR including a different extracellular antigen binding domain. In some embodiments, the immune cells include a CAR population, each CAR including the same extracellular antigen binding domain.

[0058] The engineered immune cells can be allogeneic or autologous.

[0059] As used herein, "autologous" means that the cell, cell line or cell population used to treat a patient originates from the patient.

[0060] As used herein, "allogeneic" means that the cells, cell lines or cell populations used to treat a patient are not derived from the patient, but are derived from a donor. In some embodiments, the donor is a healthy donor.

[0061] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, or tumor infiltrating lymphocytes (TIL)), NK cells, NK-T cells, TCR expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the cells may be derived from a group consisting of CD4+T lymphocytes and CD8+T lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are αβT cells. In some exemplary embodiments, the engineered immune cells are γδT cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some embodiments, the engineered immune cells are human cells.

[0062] In some embodiments, the engineered immune cells can be derived from, for example but not limited to, stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells. Stem cells can be CD34+ or CD34-.

[0063] In some embodiments, the cell is obtained from peripheral blood or prepared therefrom. In some embodiments, the cell is obtained from peripheral blood mononuclear cells (PBMC) or prepared therefrom. In some embodiments, the cell is obtained from bone marrow or prepared therefrom. In some embodiments, the cell is obtained from umbilical cord blood or prepared therefrom. In some embodiments, the cell is a human cell. In some embodiments, a method including but not limited to the following is used to transfect or transduce cells by a nucleic acid vector: electroporation, sonoporation, biolistics (e.g., gene gun), transfection, lipofection, polymer transfection, nanoparticles, viral transduction or viral transfection (e.g., retrovirus, slow virus, AAV) or polymer complex (polyplex). In some embodiments, the cell is a T cell that has been reprogrammed from a non-T cell. In some embodiments, the cell is a T cell that has been reprogrammed from a T cell.

[0064] Binder

[0065] In embodiments, the disclosed methods include the use of antibodies or antigen binding agents (eg, including antigen binding domains or including antibodies or fragments thereof). As discussed below, in various embodiments, the engineered immune cells may further include a binding agent.

[0066] As used herein, the term "antibody" refers to a polypeptide comprising canonical immunoglobulin sequence elements sufficient to confer specific binding to a specific target antigen. As known in the art, a complete antibody produced in nature is a tetrameric reagent of approximately 150 kD, comprising two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD), which are associated with each other into a structure generally referred to as a "Y-shape". Each heavy chain comprises at least four domains (each about 110 amino acids in length) - these four domains are the amino-terminal variable (VH) domain (located at the end of the Y structure), followed by three constant domains: CHI, CH2, and carboxyl-terminal CH3 (located at the base of the stem of the Y). The short region is called a "switch" that connects the heavy chain variable region to the constant region. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides in the complete antibody to each other. Each light chain comprises two domains - the two domains are an amino-terminal variable (VL) domain followed by a carboxyl-terminal constant (CL) domain. Those skilled in the art are very familiar with antibody structure and sequence elements, recognize the "variable" and "constant" regions in the provided sequences, and understand that there can be some flexibility in defining the "borders" between these domains, so that different presentations of the same antibody chain sequence can, for example, mean that such borders at a certain position are offset by one or a few residues relative to different presentations of the same antibody chain sequence.

[0067] The complete antibody tetramer contains two heavy chain-light chain dimers, in which the heavy chain and the light chain are connected to each other by a single disulfide bond; the other two disulfide bonds connect the heavy chain hinge region to each other, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also glycosylated, usually on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold", which is formed by stacking two β folds (e.g., 3, 4 or 5 chain folds) in a compressed antiparallel β barrel. Each variable domain contains three hypervariable loops called "complement determining regions" (CDR1, CDR2 and CDR3) and four slightly unchanged "framework" regions (FR1, FR2, FR3 and FR4). When the natural antibody is folded, the FR region forms a β fold that provides a structural framework for the domain, and the CDR loop regions of the heavy and light chains are gathered together in three-dimensional space, thereby producing a single hypervariable antigen binding site located at the end of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells (including, for example, effector cells that mediate cytotoxicity). As is known in the art, the affinity and / or other binding properties of the Fc region for Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or employed in accordance with the present invention comprise a glycosylated Fc domain, including Fc domains having modified or engineered such glycosylation.

[0068] For the purposes of this disclosure, in certain embodiments, any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequences present in natural antibodies may be referred to and / or used as an "antibody", whether such polypeptide is produced naturally (e.g., by an organism reacting with an antigen) or produced by recombinant engineered, chemically synthesized, or other artificial systems or methods. In some embodiments, the antibodies are polyclonal; in some embodiments, the antibodies are monoclonal. In some embodiments, the antibodies have constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0069] In addition, as used herein, the term "antibody" may refer to any of the constructs or forms known or developed in the art for utilizing antibody structural and functional characteristics in alternative presentation. For example, in some embodiments, the form of the antibody employed in the methods of the present disclosure is selected from, but not limited to: a complete IgA, IgG, IgE, or IgM antibody; a bispecific or multispecific antibody (e.g., , etc.); antibody fragments, such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments and isolated CDRs or collections thereof; single chain variable fragments (scFV); polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); camelid antibodies (also referred to herein as nanobodies or VHH); shark antibodies, masked antibodies (e.g., ); Small modular immunopharmaceuticals (SMIPs) TM ); single-chain or tandem bivalent antibodies VHH; microantibodies; Ankyrin repeat protein or DART;TCR-like antibody; microprotein; and In some embodiments, the antibody may lack covalent modifications (e.g., attachment of polysaccharides) that are present in nature. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of polysaccharides), payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other side groups (e.g., polyethylene glycol, etc.).

[0070] As used herein, the term "antibody agent" generally refers to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex comprising an immunoglobulin structural element sufficient to confer specific binding. Exemplary antibody reagents include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that have the characteristics of mouse, rabbit, primate or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In multiple embodiments, the term "antibody agent" is used to refer to one or more of the constructs or forms known or developed in the art for utilizing antibody structure and functional characteristics in alternative presentation. For example, the form of the antibody agent used according to the present invention is selected from, but is not limited to: complete IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., ); Small modular immunopharmaceuticals (SMIPs) TM ); single-chain or tandem bivalent antibodies VHH; microantibodies; Ankyrin repeat protein or DART;TCR-like antibody; microprotein; and

[0071] The antibody or antibody agent for carrying out the method of the present disclosure can be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In certain embodiments, the antigen-binding molecule is selected from the group consisting of the following: scFv, Fab, Fab', Fv, F(ab')2, dAb and any combination thereof.

[0072] Antibodies and antibody agents include antibody fragments. Antibody fragments include a portion of a complete antibody, such as the antigen binding or variable region of a complete antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, bivalent antibodies, single domain antibodies, linear antibodies, multispecific antibodies formed by antibody fragment antibodies and scFv fragments and other fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibodies), single chain, Fab, Fa, F(ab')2 fragments and scFv. Antibodies can be complete antibodies, or immunoglobulins, or antibody fragments. Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli (E. coli), Chinese hamster ovary (CHO) cells or phages), as known in the art.

[0073] In some embodiments, the antibody or antibody agent may be a chimeric antibody (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species. In one example, a chimeric antibody may include a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody may be a "class-switched" antibody in which the class or subclass has been changed from a parent antibody. A chimeric antibody comprises an antigen-binding fragment thereof.

[0074] In some embodiments, the chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, Front. Biosci., 13:1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029). -10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et al., Methods 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer 83:252-260 (2000). Humanized antibodies are chimeric antibodies comprising amino acid residues from non-human hypervariable regions and amino acid residues from human framework regions. In certain embodiments, a humanized antibody will include substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the framework regions (FRs) correspond to those of a human antibody. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.

[0075] In some embodiments, the antibodies or antibody agents provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art (see, for example, van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol, 20: 450-459 (2008)). Human antibodies can be antibodies whose amino acid sequences correspond to those produced by humans or human cells or derived from antibodies of non-human origin using human antibody repertoires or other human antibody encoding sequences. This definition of human antibodies explicitly excludes humanized antibodies including non-human antigen binding residues. Human antibodies can be prepared using methods well known in the art.

[0076] Chimeric Antigen Receptor

[0077] As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to a target antigen, CAR can activate immune cells to attack and destroy cells carrying the antigen (e.g., cancer cells). CAR can also have co-stimulatory or signaling domains to increase its effectiveness. See Krause et al., Journal of Experimental Medicine (J. Exp. Med.), Vol. 188, No. 4, 1998 (619–626); Finney et al., Journal of Immunology (Journal of Immunology) Immunology, 1998, 161:2791–2797; Song et al., Blood, 119:696-706 (2012); Kalos et al., Sci. Transl. Med., 3:95 (2011); Porter et al., N. Engl. J. Med., 365:725-33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol., 56:59–83 (2016); U.S. Pat. Nos. 7,741,465 and 6,319,494.

[0078] The chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain that specifically binds to a target.

[0079] In some embodiments, the antigen-specific CAR further comprises a safety switch and / or one or more monoclonal antibody-specific epitopes.

[0080] i. Antigen binding domain

[0081] As discussed above, the CAR described herein includes an antigen binding domain. As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease.

[0082] In some embodiments, the antigen binding domain comprises a variable heavy chain, a variable light chain and / or one or more CDRs as described herein. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising a light chain CDR (CDR1, CDR2 and CDR3) and a heavy chain CDR (CDR1, CDR2 and CDR3).

[0083] An antigen binding domain is said to be "selective" when it binds one target more tightly or with higher affinity than it binds a second target.

[0084] The antigen binding domain of CAR selectively targets cancer antigens. In some embodiments, the cancer antigen is selected from EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, Muc3, Muc3, Muc16, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52 or CD34. In some embodiments, the CAR comprises an antigen binding domain that targets EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34.

[0085] In some embodiments, the cancer antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2) and Wilms tumor protein (WT-1).

[0086] Variants of antigen binding domains (e.g., variants of CDRs, VH and / or VL) are also within the scope of the present disclosure, e.g., variable light chains and / or variable heavy chains, each of which has at least 70%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-97%, 97%-99% or greater than 99% identity to the amino acid sequence of the antigen binding domain sequence. In some cases, such molecules comprise at least one heavy chain and one light chain, while in other cases, the variant forms contain two variable light chains and two variable heavy chains (or sub-portions thereof). Technicians will be able to use well-known techniques to determine suitable variants of antigen binding domains as shown herein. In certain embodiments, those skilled in the art can identify suitable regions of the molecule by targeting regions that are considered to be unimportant for activity, which regions can be changed without destroying activity.

[0087] In some embodiments, the polypeptide structure of the antigen binding domain is based on antibodies, including but not limited to monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the antigen binding domain comprises or consists of an avimer.

[0088] In some embodiments, the antigen binding domain is a scFv.In some embodiments, the antigen selective CAR includes a leader sequence or a signal peptide.

[0089] In other embodiments, the present disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the present disclosure relates to isolated polynucleotides encoding CAR. Also provided herein are vectors including polynucleotides and methods for preparing them.

[0090] In other embodiments, the present disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the present disclosure relates to isolated polynucleotides encoding CAR. Also provided herein are vectors including polynucleotides and methods for preparing them.

[0091] In some embodiments, CAR immune cells (e.g., CAR-T cells) may form a component of a cell population produced by implementing the method of the present disclosure, and the CAR immune cells include polynucleotides encoding safety switch polypeptides (e.g., RQR8 or rituximab mimetic epitopes). See, for example, WO2013153391A, which is hereby incorporated by reference in its entirety. In CAR immune cells (e.g., CAR-T cells) comprising polynucleotides, safety switch polypeptides may be expressed on the surface of CAR immune cells (e.g., CAR-T cells).

[0092] ii. Hinge domain

[0093] The extracellular domain of the CAR disclosed herein may include a "hinge" domain (or hinge region). The term generally refers to any such polypeptide having the function of connecting the transmembrane domain in CAR to the extracellular antigen-binding domain in CAR. Specifically, the hinge domain can be used to provide greater flexibility and accessibility for the extracellular antigen-binding domain.

[0094] The hinge domain may include up to 300 amino acids - in some embodiments 10 to 100 amino acids or in some embodiments, 25 to 50 amino acids. The hinge domain may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, CD28, 4-1BB or IgG (especially, the hinge region of IgG; it should be understood that the hinge region may contain members of the immunoglobulin family, such as some or all of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or their fragments), or from all or part of the constant region of the heavy chain of an antibody. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In some embodiments, the hinge domain is a part of a human CD8 alpha chain (e.g., NP_001139345.1). In other embodiments, the hinge and transmembrane domain include a part of a human CD8 alpha chain. In some embodiments, the hinge domain of the CAR described herein includes a subsequence of CD8α, IgG1, IgG4, PD-1 or FcγRIIIα, in particular a hinge region of any one of CD8α, IgG1, IgG4, PD-1 or FcγRIIIα. In some embodiments, the hinge domain includes a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1 or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein includes scFv, a CD8α human hinge and a transmembrane domain, a CD3ζ signaling domain and a 4-1BB signaling domain.

[0095] iii. Transmembrane domain

[0096] The CAR of the present disclosure is designed to have a transmembrane domain fused to the extracellular domain of CAR. It can be similarly fused to the intracellular domain of CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex. In some embodiments, a short linker can form a bond between any one or some of the extracellular, transmembrane and intracellular domains of CAR.

[0097] Suitable transmembrane domains of the CARs disclosed herein have the ability to: (a) express on the surface of immune cells, such as, but not limited to, lymphocytes, such as T helper cells (T h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg) cells or natural killer (NK) cells, and / or (b) interact with the extracellular antigen binding domain and the intracellular signaling domain to direct the cellular response of the immune cell against the target cell.

[0098] The transmembrane domain may be derived from a natural or synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0099] The transmembrane region particularly used in the present disclosure may be derived from (including, or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0100] As a non-limiting example, the transmembrane region can be derived from a T cell receptor, or a part of a T cell receptor, such as α, β, γ or δ, a polypeptide constituting a CD3 complex, an IL-2 receptor p55 (α chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor, especially Fcγ receptor III or CD protein. Alternatively, the transmembrane domain can be synthetic and can mainly include hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8 α chain (e.g., NP_001139345.1).

[0101] In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8α transmembrane domain.

[0102] In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain.

[0103] iv. Intracellular domain

[0104] The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide at least one activation of the normal effector function of the immune cell including the CAR. For example, the effector function of T cells can refer to cell lysis activity or helper cell activity, including the secretion of cytokines.

[0105] In some embodiments, the activating intracellular signaling domain for use in a CAR may be, for example, but not limited to, cytoplasmic sequences of T cell receptors and co-receptors that act coordinately to initiate signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0106] It is understood that suitable (e.g., activation) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, D3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0107] The intracellular domain of the CAR of the present disclosure may also be incorporated into a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) in addition to the above-mentioned activation domain to increase its effectiveness. The costimulatory domain may provide a signal other than the primary signal provided by the activation molecule as described herein.

[0108] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure may be derived from (or correspond to) for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 1a / CD18)), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNFR, integrin, signaling lymphocyte activation molecules, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD4 9D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITG B2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or a fragment or combination thereof. It should be understood that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0109] In some embodiments, the intracellular / cytoplasmic domain of CAR can be designed to include the 4-1BB / CD137 domain itself or any other desired intracellular domain combination suitable for the situation of CAR of the present disclosure. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI reference sequence: NM_001561.5.

[0110] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD28 domain itself or in combination with any other desired intracellular domain suitable for the context of the CAR of the present disclosure. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0111] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD3 zeta domain by itself or in combination with any other desired intracellular domain suitable for the context of the CAR of the present disclosure.

[0112] For example, the intracellular domain of CAR can include a portion of a CD3 ζ chain and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR disclosed herein can be connected to each other randomly or in a specified order. In some embodiments, the intracellular domain is designed to include an activation domain of CD3 ζ and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to include an activation domain of CD3 ζ and a signaling domain of 4-1BB.

[0113] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure includes the domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of the CAR of the present disclosure includes a portion of a co-stimulatory molecule selected from the group consisting of a fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0114] Genetic modification of CAR T cells

[0115] Also provided herein are engineered immune cells and populations of engineered immune cells expressing CAR (e.g., CAR-T cells or CAR+ cells), both of which are depleted of cells expressing endogenous TCRs.

[0116] In some embodiments, the engineered immune cells include CAR T cells, each CAR T cell includes an extracellular antigen binding domain and the expression of endogenous TCR is reduced or eliminated. In some embodiments, the engineered immune cell population includes a CAR T cell population, each CAR T cell includes two or more different extracellular antigen binding domains and the expression of endogenous TCR is reduced or eliminated. In some embodiments, the immune cells include a CAR population, each CAR T cell includes the same extracellular antigen binding domain and the expression of endogenous TCR is reduced or eliminated.

[0117] In some embodiments, the engineered immune cells according to the present disclosure include a destroyed or inactivated gene selected from the group consisting of CD52, DLL3, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα and TCRβ, and / or express CAR, multi-chain CAR and / or pTα transgene. In some embodiments, the isolated cells include polynucleotides encoding polypeptides including multi-chain CARs. In some embodiments, the isolated cells according to the present disclosure comprise two disrupted or inactivated genes selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, DLL3 and CD52, DLL3 and TCRα, DLL3 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ , TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or expression of CAR (comprising multi-chain CAR and / or pTα transgene). In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cell an endonuclease that can selectively inactivate the gene by selective DNA cutting. In some embodiments, the endonuclease can be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a large range of nucleases, a transcription activator-like effector nuclease (TALE-nuclease or ) or CRISPR (e.g., Cas9 or Cas12) endonucleases.

[0118] In some embodiments, the TCR is rendered nonfunctional in a cell according to the present disclosure by destroying or inactivating the TCRα gene and / or the TCRβ gene. In some embodiments, the TCR is rendered nonfunctional in a cell according to the present disclosure by destroying or inactivating the TCRα constant region (TRAC locus). In some embodiments, a method for obtaining a modified cell derived from an individual is provided, wherein the cell can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway that are easily obtained by this method are encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in patients in need; therefore, within the scope of the present disclosure is a method for treating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in patients in need, the method comprising treating the patient by administering an effective amount of modified cells to the patient, the modified cells comprising a destroyed or inactivated TCRα and / or TCRβ gene.

[0119] The present disclosure provides a method for determining the purity of an engineered immune cell colony lacking or having reduced endogenous TCR expression. In some embodiments, the engineered immune cell includes less than 5.0%, less than 4.0%, less than 3.0% TCR+ cells, less than 2.0% TCR+ cells, less than 1.0% TCR+ cells, less than 0.9% TCR+ cells, less than 0.8% TCR+ cells, less than 0.7% TCR+ cells, less than 0.6% TCR+ cells, less than 0.5% TCR+ cells, less than 0.4% TCR+ cells, less than 0.3% TCR+ cells, less than 0.2% TCR+ cells or less than 0.1% TCR+ cells. This colony can be the product of the disclosed method.

[0120] In some embodiments, the engineered immune cells according to the present disclosure may include one or more destroyed or inactivated genes. In some embodiments, the genes of the target antigen (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3 or CD34, CD70) may be In order to be knocked out to introduce a CAR targeting the same antigen (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3 or CD34, CD70 CAR), thereby avoiding induction of CAR activation. As described herein, in some embodiments, the engineered immune cell according to the present disclosure includes a destroyed or inactivated gene, the gene is selected from the group consisting of the following: MHC1 (β2M), MHC2 (CIITA), EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, tight junction protein -18.2, Muc17, FAPα, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3 or CD34, CD70, TCRα and TCRβ, and / or express CAR or multi-chain CAR. In some embodiments, the cell includes multi-chain CAR. In some embodiments, the isolated cells comprise two disrupted or inactivated genes selected from the group consisting of CD52 and TCRα, CDR52 and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, MHC-1 and TCRα, MHC-1 and TCRβ, MHC2 and TCRα, MHC2 and TCRβ, and / or express CAR or multi-chain CAR.

[0121] The engineered immune cells can be allogeneic or autologous.

[0122] In some embodiments, engineered immune cells or engineered immune cell colonies include T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor infiltrating lymphocytes (TIL)), NK cells, NK-T cells, TCR expressing cells, dendritic cells, killer dendritic cells, mast cells or B cells, and express CAR. In some embodiments, T cells can be derived from a group consisting of CD4+T lymphocytes, CD8+T lymphocytes, or a group including a combination of CD4+ and CD8+T cells.

[0123] In some embodiments, the engineered immune cells or engineered immune cell populations produced using the disclosed methods can be derived from, for example, but not limited to, stem cells. The stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0124] In some embodiments, the engineered immune cells or immune cell colonies produced using the disclosed method are obtained from peripheral blood or prepared therefrom. In some embodiments, the engineered immune cells are obtained from peripheral blood mononuclear cells (PBMC) or prepared therefrom. In some embodiments, the engineered immune cells are obtained from bone marrow or prepared therefrom. In some embodiments, the engineered immune cells are obtained from umbilical cord blood or prepared therefrom. In some embodiments, the cells are human cells. In some embodiments, cells are transfected or transduced by a nucleic acid vector using a method selected from the group consisting of the following: electroporation, sonoporation, biolistics (e.g., gene gun), lipofection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, slow virus, AAV) or polymer complex (polyplex).

[0125] In some embodiments, the engineered immune cells expressing antigen-specific CARs on the cell surface membrane include percentages greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% stem cell memory and central memory cells.

[0126] In some embodiments, the engineered immune cells expressing an antigen-specific CAR on the cell surface membrane include percentages of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70% stem cell memory and central memory cells.

[0127] In some embodiments, engineered immune cells expressing antigen-specific CARs on their surface membranes are enriched for T cells. CM and / or T SCM cells, such that the engineered immune cells include at least about 60%, 65%, 70%, 75% or 80% of the combined T CM and T SCM In some embodiments, engineered immune cells expressing antigen-specific CARs at their cell surface membranes are enriched for T cells. CM Cells and / or T SCM cells, such that the engineered immune cells include at least about 70% of the combined T CM Cells and T SCM In some embodiments, engineered immune cells expressing antigen-specific CARs at their cell surface membranes are enriched for T cells. CM Cells and / or T SCM cells, such that the engineered immune cells include at least about 75% of the combined T CM Cells and / or T SCM cell.

[0128] In some embodiments, the engineered immune cell is an inflammatory T lymphocyte expressing CAR. In some embodiments, the engineered immune cell is a cytotoxic T lymphocyte expressing CAR. In some embodiments, the engineered immune cell is a regulatory T lymphocyte expressing CAR. In some embodiments, the engineered immune cell is an auxiliary T lymphocyte expressing CAR.

[0129] In some embodiments, immune cells are engineered to resist one or more chemotherapy drugs. The chemotherapy drug may be, for example, a purine nucleotide analog (PNA), so that immune cells are suitable for cancer treatment combined with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, in separate or combined forms. PNA is metabolized into single, two, and triphosphate PNAs by deoxycytidine kinase (dCK). Its triphosphate form competes with ATP for DNA synthesis, serves as a pro-apoptotic agent, and is an effective inhibitor of the ribonucleotide reductase (RNR) involved in the production of trinucleotides.

[0130] In some embodiments, the isolated cells or cell lines of the present disclosure may include pTalpha or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRalpha gene.

[0131] The present disclosure also provides an engineered immune cell comprising any of the CAR polynucleotides described herein. In some embodiments, CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker, which provides identification and / or selection of cells receiving the vector.

[0132] After the polynucleotide encoding the CAR polypeptide is introduced into the cell, the CAR polypeptide can be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. The method of introducing a polynucleotide construct into a cell is known in the art. In some embodiments, a stable transformation method (e.g., using a lentiviral vector) can be used to integrate a polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct and a polynucleotide construct that is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to transduction, microinjection, electroporation, or particle bombardment. Polynucleotides can be included in a vector, such as a plasmid vector or a viral vector.

[0133] In some embodiments, an isolated nucleic acid is provided, comprising a promoter operably linked to a first polynucleotide encoding an antigen binding domain, at least one co-stimulatory molecule and an activation domain. In some embodiments, the nucleic acid construct is contained in a viral vector. In some embodiments, the viral vector is selected from the group consisting of: a retroviral vector, a murine leukemia virus vector, a SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector and a vaccinia virus vector. In some embodiments, the nucleic acid is contained in a plasmid.

[0134] In some embodiments, the isolated nucleic acid construct is contained in a viral vector and is introduced into the genome of the engineered immune cell by random integration (e.g., lentiviral or retroviral mediated random integration). In some embodiments, the isolated nucleic acid construct is contained in a viral vector or a non-viral vector and is introduced into the genome of the engineered immune cell by site-specific integration of homologous recombination (e.g., adenovirus mediated site-specific integration).

[0135] Preparation of engineered immune cells

[0136] Many known techniques can be used to prepare polynucleotides, polypeptides, vectors, antigen binding domains, immune cells, compositions, etc. according to the present disclosure.

[0137] Prior to in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. Cells expressing CAR can be derived from allogeneic or autologous sources and can be knocked out of endogenous TCR as described herein.

[0138] a. Source Materials

[0139] In some embodiments, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, any number of techniques known to the skilled person can be used, such as FICOLL TM Isolate and obtain T cells from a volume of blood collected from a subject.

[0140] Cells can be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and then placed in an appropriate buffer or culture medium for subsequent processing.

[0141] In some embodiments, for example, using PERCOLL TM Centrifugation performed by gradient, by lysing red blood cells and exhausting monocytes, separates T cells from PBMC. Specific subpopulations of T cells, (such as CD28+, CD4+, CD45RA- and CD45RO+T cells or CD28+, CD4+, CDS+, CD45RA-, CD45RO+ and CD62L+T cells) can be further separated by positive or negative selection techniques known in the art. For example, the enrichment of T cell colonies by negative selection can be achieved with a combination of antibodies for surface markers unique to cells selected for negative selection. One of the methods used herein is to sort and / or select cells by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies for cell surface markers present on cells selected for negative selection. For example, in order to enrich for CD4+ cells by negative selection, a mixture of monoclonal antibodies generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to separate the cell colonies of interest for use in the present disclosure.

[0142] Using methods as described herein, PBMC can be directly used for the genetic modification of immune cells (such as CAR or TCR).In certain embodiments, after separating PBMC, T lymphocytes can be further separated, and cytotoxicity and helper T lymphocytes can be sorted as primary, memory and effector T cell subsets before or after genetic modification and / or amplification.In some embodiments, CD8+ cells are further sorted as primary, stem cell memory, central memory and effector cells by identifying the cell surface antigens related to each of these CD8+ cell types.In some embodiments, the expression of the phenotypic markers of central memory T cells includes CD27, CD45RA, CD45RO, CD62L, CCR7, CD28, CD3 and CD127 and is negative for granzyme B.In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+T cells.In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

[0143] b. Stem cell-derived immune cells

[0144] In some embodiments, immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSC, mesenchymal cells, iPS cells and other types of stem cells can be cultivated into immortal cell lines or directly isolated from patients. Various methods for isolating, producing and / or cultivating stem cells are known in the art and can be used to practice the present disclosure.

[0145] In some embodiments, immune cells are induced pluripotent stem cells (iPSC) derived from reprogrammed T cells. In some embodiments, the source material can be an induced pluripotent stem cell (iPSC) derived from T cells or non-T cells. In some embodiments, the immune cells are iPSC-derived T cells. In some embodiments, the immune cells are iPSC-derived NK cells. The source material can be embryonic stem cells. The source material can be a B cell, or any other cell from peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0146] c. Genetic modification of isolated cells

[0147] Immune cells, such as T cells, can be genetically modified using known methods after isolation, or the immune cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro prior to genetic modification. In some embodiments, isolated immune cells can be genetically modified by mixing the cells with a volume of anti-CD3 / CD28 nanomatrix (e.g., TransAct TM ) is activated by contact.

[0148] In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and / or CD52. In some embodiments, gene editing techniques (e.g., CRISPR / Cas9, CRISPR / Cas12a, zinc finger nucleases (ZFNs), TALENs, MegaTAL nucleases, large-range nucleases) are used to genetically modify cells to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells (such as T cells) are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding CAR), followed by in vitro activation and / or amplification.

[0149] Certain methods for making the constructs and engineered immune cells of the present disclosure are described in PCT Application No. PCT / US15 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0150] It should be understood that PBMC may further include other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying the coding sequence of chimeric receptors as disclosed herein may be introduced into populations of human donor T cells, NK cells or NKT cells. Flow cytometry can be used to sort T cells carrying successful transduction of expression vectors to separate CD3 positive T cells, and then further proliferate to increase the number of these CAR expression T cells except for cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used to cryopreserve T cells expressing CAR, to store and / or prepare for human subjects. In one embodiment, in the absence of non-human animal derived products, such as fetal bovine serum (fetal calf serum / fetal bovine serum), T cells are transduced in vitro, cultured and / or amplified.

[0151] In order to clone a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow replication of the vector itself, and thereby amplify copies of the polynucleotide contained therein. The cloning vector may contain sequence components, generally including but not limited to a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, a replication origin may be selected to promote autonomous replication of the vector in a host cell.

[0152] In some embodiments, the disclosure provides a host cell containing the separation of the vector provided herein. The host cell containing the vector can be used for the expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include but are not limited to prokaryotic cells, fungal cells, yeast cells or higher eukaryotic cells, such as mammalian cells, particularly human cells.

[0153] The vector can be introduced into the host cell using any suitable method known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation, gene gun method, receptor mediated gene delivery, delivery mediated by polylysine, histone, chitosan and peptide. Standard methods for transfection and transformation of cells for expressing the vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used for a donor population of genetically modified immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a certain proportion of engineered cells express more than one different CAR.

[0154] In one embodiment, the present disclosure provides a method for storing genetically engineered cells expressing CAR or TCR. This involves cryopreservation of immune cells so that the cells remain alive after thawing. A portion of the immune cells expressing CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignant tumors. When necessary, cryopreserved transformed immune cells can be thawed, grown, and expanded to obtain more such cells.

[0155] In some embodiments, cells are formulated by first collecting the cells from their culture medium, and then washing and concentrating the cells in a culture medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic medium formulation, typically saline Normosol TM R (Abbott) or Plasma-Lyte TMA (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0156] d. Allogeneic CAR T cells

[0157] The process of manufacturing allogeneic CAR T therapy involves harvesting peripheral blood mononuclear cells (PBMC) from healthy, selected, screened and tested donors. Then, the cells are engineered to express CAR, which recognizes certain cell surface proteins expressed in blood or solid tumors. Allogeneic T cells are gene-edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can be knocked out so that the CAR T product is resistant to anti-CD52 antibody therapy. Therefore, anti-CD52 antibody therapy can be used to suppress the host immune system, and CAR T is allowed to remain implanted to achieve a complete therapeutic effect. Engineered T cells are then subjected to a purification step, and are ultimately cryopreserved in vials for delivery to patients.

[0158] e. Autologous CAR T cells

[0159] Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting the patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express CARs that recognize targets expressed on the cell surface of one or more specific cancer cells and kill cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

[0160] Control of foreign virus infection

[0161] HHV-6 can infect immune cells, including T cells, and establish latent infection (or latency). In in vitro cell culture, after a period of time, viral reactivation can be observed, but the event is rare. As disclosed herein, after any local latent virus reactivation present in source immune cells obtained from, for example, patients or healthy individuals, one or more exogenous cell culture additives or agents can be added during cell culture in the manufacturing process of cell-based therapy to prevent, control or inhibit viral replication, or prevent, control or inhibit viral transmission. In some embodiments, the source immune cells are T cells, PBMCs, NK cells, or iPSCs. In some embodiments, the exogenous agent can be IFN. In some embodiments, viral replication is HHV-6 viral replication. In some embodiments, the endemic latent virus is HHV-6.

[0162] In some embodiments, the exogenous agent is a type I IFN or a type III IFN. In certain embodiments, the IFN is IFNα or IFNβ. In some embodiments, the IFN is added to the culture medium at a concentration of about 0.01 ng / ml to about 100 ng / ml, about 0.01 ng / ml to about 10 ng / ml, about 0.01 ng / ml to about 1 ng / ml, about 0.01 ng / ml to about 0.1 ng / ml, about 0.1 ng / ml to about 100 ng / ml, about 0.1 ng / ml to about 10 ng / ml, about 0.1 ng / ml to about 1 ng / ml, about 0.1 ng / ml to about 0.9 ng / ml. ml, about 0.1 ng / ml to about 0.8 ng / ml, about 0.1 ng / ml to about 0.7 ng / ml, about 0.1 ng / ml to about 0.6 ng / ml, or about 0.1 ng / ml to about 0.5 ng / ml, about 0.1 ng / ml, about 0.2 ng / ml, about 0.3 ng / ml, about 0.4 ng / ml, about 0.5 ng / ml, about 0.6 ng / ml, about 0.7 ng / ml, about 0.8 ng / ml, about 0.9 ng / ml, or about 1 ng / ml. In certain embodiments, IFN is human IFNα. An exemplary human IFNα sequence is shown below (NCBI Accession No. NP_000596.2, IFNα2a):

[0163] MALTFALLVA LLVLSCKSSC SVG CDLPQTH SLGSRRTLML LAQMRRISLF SCLKDRHDFGFPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTETPLMKEDSIL AVRKYFQRIT LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE (SEQ ID NO: 19, indicated signal peptide is underlined)

[0164] An exemplary human IFNα sequence without a signal peptide is shown below:

[0165] CDLPQTH SLGSRRTLML LAQMRRISLF SCLKDRHDFG FPQEEFGNQF QKAETIPVLHEMIQQIFNLF STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRITLYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE(SEQ ID NO:20)

[0166] In some embodiments, IFNα comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, with or without a signal peptide. In some embodiments, IFNα comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

[0167] In some embodiments, the antiviral agent is foscarnet or its salt. In some embodiments, foscarnet or its salt (e.g., sodium salt) is added to the culture medium at a concentration of about 8 μM to about 20 μM, about 8 μM to about 15 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM or about 15 μM. In some embodiments, the antiviral agent is ganciclovir or its salt. In some embodiments, ganciclovir or its salt is added to the culture medium at a concentration of about 25 μM to about 35 μM, about 25 μM to about 30 μM, about 27 μM to about 30 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM or about 35 μM. In some embodiments, the antiviral agent is cidofovir or its salt. In some embodiments, cidofovir or a salt thereof is added to the culture medium at a concentration of about 14 μM to about 25 μM, about 14 μM to about 20 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, or about 20 μM.

[0168] The structure of phosphonoformic acid is shown below (sodium salt CAS number 63585-09-1):

[0169]

[0170] The structure of ganciclovir is shown below (CAS number 82410-32-0):

[0171]

[0172] The structure of cidofovir is shown below (CAS No. 113852-37-2):

[0173]

[0174] The one or more exogenous additives or agents can be added to the culture medium at different time points of the cell culture during the manufacturing process. In some embodiments, the additive is added at day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, or more than day 21 of the manufacturing process. In some embodiments, the additive is added at day 1 to day 5, day 6 to day 10, day 10 to day 15, day 15 to day 20, or after day 20 of the manufacturing process. In some embodiments, the additive is added on day 5 to 10, day 6 to 10, day 7 to 10, day 8 to 10, day 9 to 10, day 6 to 11, day 7 to 11, day 8 to 11, day 9 to 11, day 10 to 11, day 5 to 9, day 6 to 9, day 7 to 9, or day 8 to 9 of the manufacturing process. In some embodiments, the additive is added on day 9 to 15, day 10 to 15, day 11 to 15, day 12 to 15, day 13 to 15, day 14 to 15, day 9 to 13, day 9 to 12, day 9 to 11, day 9 to 10, day 10 to 13, day 10 to 14, or day 10 to 15 of the manufacturing process. In some embodiments, the additive is added on day 6 to 11, day 7 to 11, or day 8 to 11 of the manufacturing process. In some embodiments, the cells are contacted with the IFN in culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0175] Pharmaceutical composition

[0176] In some embodiments, cells are formulated by first collecting the cells from their culture medium, and then washing and concentrating the cells in a culture medium and container system (containing a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic medium formulation, typically physiological saline Normosol TM R (Abbott) or Plasma-Lyte TM A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0177] In embodiments, the desired therapeutic amount of cells in the composition is typically at least 2 cells (e.g., at least 1 CD8+ central or stem cell memory T cell and at least 1 CD4+ helper T cell subset; or two or more CD8+ central or stem cell memory T cells; or two or more CD4+ helper T cell subsets) or more typically greater than 10 2 cells, and up to and including 10 6 cells, up to and including 10 7 , 10 8 or 10 9 cells, and can be more than 10 10 The number of cells will depend on the intended use of the composition and the type of cells contained therein. The desired cell density is generally greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 Clinically relevant numbers of immune cells can be assigned to cells that accumulate to or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some aspects of the present disclosure, particularly since all infused cells will be redirected to a specific target antigen, administration may be performed over a period of about 10 5 / kg or about 10 6 / kg (10 per patient 6 -10 11 ) range. CAR therapy can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient being treated.

[0178] The CAR expressing cell colony of the present disclosure can be administered alone or in the form of a pharmaceutical composition combined with a diluent and / or with other components (such as IL-2 or other cytokines or cell colonies). The pharmaceutical composition of the present disclosure may include CAR expression or TCR expression cell colonies, such as T cells described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The present disclosure composition is preferably formulated for intravenous administration.

[0179] Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides that may serve as solvents or suspension media; polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0180] Treatment

[0181] The present disclosure includes methods for treating or preventing a patient's disease (e.g., cancer), the methods comprising administering an effective amount of CAR T cells or engineered immune cells including CAR disclosed herein to a patient in need. In some embodiments, an effective amount of CAR T cells or engineered immune cells have been analyzed for various properties according to the methods described in the present disclosure. In some embodiments, various properties, such as efficacy or multifunctionality, have been analyzed for CAR T cell drug products for therapeutic use according to the methods described in the present disclosure. In some embodiments, CAR T cells are TCR-CAR T cells, and various properties have been analyzed for CART drug products for therapeutic use according to the methods described in the present disclosure, such as the number or percentage of remaining TCR+CAR T cells and / or efficacy or multifunctionality.

[0182] Provided is a method for treating a disease or disorder including cancer. In some embodiments, the disclosure relates to generating a T cell-mediated immune response in a subject, comprising administering an effective amount of an engineered immune cell of the present application to the subject. In some embodiments, the T cell-mediated immune response is directed to a target cell or cell. In some embodiments, the engineered immune cell includes a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the disclosure includes a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one separated antigen binding domain described herein to a subject in need. In some aspects, the disclosure includes a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one immune cell to a subject in need, wherein the immune cell includes at least one chimeric antigen receptor, a T cell receptor, and / or a separated antigen binding domain as described herein. The immune cells containing CAR disclosed herein can be used to treat malignant tumors involving abnormal expression of biomarkers. In some embodiments, the CAR-containing immune cells of the present disclosure can be used to treat small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, scattered neuroendocrine tumors in the pancreas, bladder and prostate, testicular cancer and lung adenocarcinoma with neuroendocrine features. In an exemplary embodiment, CAR-containing immune cells (such as CAR-T cells of the present disclosure) are used to treat small cell lung cancer.

[0183] Also provided are methods for reducing tumor size in a subject comprising administering to the subject an engineered cell of the disclosure, wherein the cell comprises a chimeric antigen receptor comprising an antigen binding domain and binds to an antigen on the tumor.

[0184] In some embodiments, the subject suffers from a solid tumor or a hematological malignancy, such as a lymphoma or a leukemia. In some embodiments, the engineered cells are delivered to a tumor bed. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo.

[0185] A therapeutic agent, for example, a "therapeutically effective amount", "effective dose", "effective amount", or "therapeutically effective dose" of an engineered CART cell is any amount that, when used alone or in combination with another therapeutic agent, protects the subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disorders or disabilities caused by disease pain. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by analyzing the activity of an agent in an in vitro assay.

[0186] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as those with a formally diagnosed condition, those without a formally recognized condition, those under medical observation, those at risk of developing the condition, and the like.

[0187] The terms "treat" and "treatment" encompass therapeutic treatment, prophylactic treatment, and applications in which a subject is treated to reduce the risk that the condition or other risk factors will develop. Treatment does not require a complete cure of the condition and encompasses embodiments in which symptoms or potential risk factors are reduced. The term "prevention" does not require 100% elimination of the likelihood of an event occurring. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0188] The desired therapeutic amount of cells in the composition is generally at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset), or more usually greater than 10 2 cells, and up to 10 6 , up to and including 10 8 or 10 9 cells, and can exceed 10 10 The number of cells will depend on the intended use of the composition and the type of cells contained therein. The desired cell density is generally greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 108 cells / ml or greater. Clinically relevant numbers of immune cells can be assigned to cells with a cumulative count equal to or greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12In some aspects of the present disclosure, particularly since all infused cells will be redirected to a specific target antigen, 10 6 / kg(10 6 -10 11 The CAR therapy may be administered multiple times at doses within these ranges. The cells may be autologous, allogeneic, or xenogeneic to the patient being treated.

[0189] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 5 cells / kg, about 2X 10 5 cells / kg, about 3X 10 5 cells / kg, about 4X 10 5 cells / kg, about 5X 10 5 cells / kg, about 6X 10 5 cells / kg, about 7X 10 5 cells / kg, about 8X 10 5 cells / kg, about 9X 10 5 cells / kg, 2X 10 6 cells / kg, about 3X 10 6 cells / kg, about 4X 10 6 cells / kg, about 5X 10 6 cells / kg, about 6X 10 6 cells / kg, about 7X 10 6 cells / kg, about 8X 10 6 cells / kg, about 9X10 6 cells / kg, about 1X 10 7 cells / kg, about 2X 10 7 cells / kg, about 3X 10 7 cells / kg, about 4X 10 7 cells / kg, about 5X 10 7 cells / kg, about 6X 10 7 cells / kg, about 7X 10 7 cells / kg, about 8X 10 7 cells / kg or about 9X 10 7 cells / kg.

[0190] In some embodiments, the target dose of CAR+ / CAR-T+ / TCR+ cells is 1×10 6 -2×10 8 cells / kg, for example 2×106 In some embodiments, the dosage of the present invention is within the range of 10 cells / kg. It should be understood that administration above and below this range may be suitable for certain subjects, and appropriate administration levels may be determined by a healthcare provider as needed. In addition, multiple doses of cells may be provided according to the present disclosure.

[0191] In some aspects, the disclosure includes pharmaceutical compositions comprising at least one antigen binding domain as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0192] The CAR expressing cell colony of the present disclosure can be administered alone or in the form of a pharmaceutical composition combined with a diluent and / or with other components (such as IL-2 or other cytokines or cell colonies). The pharmaceutical composition of the present disclosure may include CAR expression or TCR expression cell colonies, such as T cells described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The present disclosure composition is preferably formulated for intravenous administration.

[0193] Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides that may serve as solvents or suspension media; polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0194] In some embodiments, when applied to a patient, an engineered immune cell expressing any one of the antigen-specific CARs described herein on the cell surface can reduce, kill or lyse the patient's endogenous antigen-expressing cells. In one embodiment, the reduction or lysis percentage of the cell of the antigen-expressing endogenous cell or cell line of any one of the engineered immune cells expressing antigens described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In one embodiment, the reduction or lysis percentage of the endogenous cells expressing antigen or the cell line expressing antigen produced by the engineered immune cells expressing antigen-specific CAR is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75% or about 25% to about 60%. In one embodiment, the cell expressing endogenous antigen is the bone marrow cell expressing endogenous antigen.

[0195] In one embodiment, the reduction or percentage of target cells (e.g., antigen-expressing cell lines) generated by engineered immune cells expressing an antigen-specific CAR of the present disclosure on the cell surface membrane can be measured using the assays disclosed herein.

[0196] The method may also include administering one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is a lymphocyte depletion (preconditioning) chemotherapeutic agent. For example, a method of conditioning a patient in need of T cell therapy comprises administering to the patient a prescribed beneficial dose of cyclophosphamide (between 200 mg / m 2 / day and 2000mg / m 2 / day, about 100 mg / m 2 / day and about 2000mg / m 2 / day; for example, about 100 mg / m 2 / day, about 200mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, about 900mg / m 2 / day, about 1000mg / m 2 / day, about 1500mg / m 2 / day or about 2000mg / m 2 / day) and the specified dose of fludarabine (between 20 mg / m 2 / day and 900mg / m 2 / day, about 10mg / m 2 / day and about 900mg / m 2 / day; for example, about 10 mg / m 2 / day, about 20mg / m 2 / day, about 30mg / m 2 / day, about 40mg / m 2 / day, about 40mg / m 2 / day, about 50mg / m 2 / day, about 60mg / m 2 / day, about 70mg / m 2 / day, about 80mg / m 2 / day, about 90mg / m 2 / day, about 100 mg / m 2 / day, about 500mg / m 2 / day or about 900 mg / m 2 A preferred dosing regimen involves treating a patient comprising administering to the patient about 300 mg / m daily prior to administering to the patient a therapeutically effective amount of engineered T cells. 2 / day of cyclophosphamide and about 30 mg / m 2 / day of fludarabine for three days.

[0197] In some embodiments, lymphocyte depletion further comprises administration of a CD52 antibody. In some embodiments, the CD52 antibody is alemtuzumab. In some embodiments, the CD52 antibody is administered IV at a dose of about 13-30 mg / day.

[0198] In other embodiments, the antigen binding domain, transduced (or otherwise engineered) cells, and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.

[0199] In some embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered with any number of chemotherapeutic agents, which can be administered in any order. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trishydroxymethylmelamine; nitrogen mustards, such as chlorambucil, naphthyl mustard, chlorphosphamide, estramustine, ifosfamide, dichloromethane, dichloromethane oxide hydrochloride, melphalan, novemb ichin), cholesterol, phenylacetic acid mustard, prednisone mustard, trolofosamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclarubicin, actinomycin, ostralimycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carminomycin, chromomycin, dactinomycin, daunomycin, detorubicin, 6-diaza-5-oxo-L-leuprocin ammonia, doxorubicin, epirubicin, esorubicin, idarucizumab, mexilomycin, mitomycin, mycophenolic acid, nogamycin, olivetomycin, peplomycin, sphaminomycin, puromycin, quinamycin, rodorubicin, streptozocin, streptozotocin, tuberculocidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dinotin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, Labine, 6-mercaptopurine, thiabendazole, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as captestosterone, drostanolone propionate, cyclothiodine, melastane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; acetoglucosalactone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestabuc; bisantrene; idatrox; defuvalamide; colchicine; diazocone; efamixin; elinium acetate; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; niteracrine;pentostatin; phenamet; pirarubicin; podophyllic acid; 2-acetylhydrazine; procarbazine; Razoxane; sizoran; spirogermanium; tricosporin; triazoline; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannochloridone; dibromomannitol; dibromodulanol; piperobroman; metoclotrine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel ( Rhne-Poulenc Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; mitoxantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as Targretin TM (Beselotin), Panretin TM (Yali Cuitonin);ONTAK TM(denileukin); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents were also administered when appropriate, including but not limited to CHOP, cyclophosphamide Doxorubicin (hydroxydoxorubicin), vincristine and prednisone.

[0200] In some embodiments, the chemotherapeutic agent is administered at the same time or within a week after the engineered cell, polypeptide or nucleic acid is administered. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months or 1 week to 12 months after the engineered cell, polypeptide or nucleic acid is administered. In other embodiments, the chemotherapeutic agent is administered at least 1 month before the cell, polypeptide or nucleic acid is administered. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0201] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, additional therapeutic agents that may be useful include PD-1 inhibitors, such as nivolumab. Pembrolizumab Pembrolizumab, pidilizumab, and atezolizumab

[0202]

[0203] Additional therapeutic agents suitable for combination with the present disclosure include, but are not limited to: ibrutinib Ofatumumab Rituximab Bevacizumab Trastuzumab Trastuzumab emtansine Imatinib Cetuximab ( Panitumumab Catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib ib), pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entecavir entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurine, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib nib), alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin-toxin conjugates, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitors (palbociclib).

[0204] In some embodiments, the composition including the immune cells containing CAR can be administered together with the treatment regimen to prevent cytokine release syndrome (CRS) or neurotoxicity. The treatment regimen for preventing cytokine release syndrome (CRS) or neurotoxicity can include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (eg, L-NIL or 1400W). In other embodiments, the composition including the immune cells containing CAR can be administered together with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, tramadol with propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed to cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept, ), Adalimumab and infliximab Chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular) and minocycline.

[0205] In certain embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, β and γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines.

[0206] Cell culture model of HHV-6 latent infection and reactivation

[0207] Cell lines in which at least part of the HHV-6 genome is integrated into the host cell genome have been reported for use in studying HHV-6 integration. However, no evidence of viral reactivation or expression of the HHV-6 gene spectrum after reactivation has been shown in these cell lines. Therefore, the utility of these cell lines for studying HHV-6 reactivation has not been demonstrated.

[0208] Therefore, in another aspect, an in vitro cell culture model of HHV-6 latent infection and reactivation is provided herein. In a related aspect, the present disclosure provides a method for establishing an in vitro cell culture model of HHV-6 latent infection and reactivation. In some embodiments, the cell is a human lymphocyte. In some embodiments, the cell culture model can be derived or produced by using a human lymphocyte cell line, and the human lymphocyte cell line includes but is not limited to CEM, Molt-3, Jhan or HSB2 cells. In some embodiments, the method comprises the following steps: infecting human lymphocytes with HHV-6; and continuously passage the infected cells to produce a cell culture model or cell line of HHV-6 latent infection and reactivation. In some embodiments, human lymphocytes are infected with HHV-6, and the multiplicity of infection (moi) is no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1 or no more than about 0.5moi. Infected cells are maintained in cell culture for a period of time before serial passage. In some embodiments, infected cells are maintained in cell culture and monitored until the virus reaches logarithmic growth in the cell. In some embodiments, infected cells are maintained in cell culture for about 12 to about 19 days before serial passage. In some embodiments, infected cells are maintained in cell culture before serial passage until the virus reaches logarithmic growth in the culture. In some embodiments, a logarithmic scale of 10 viral DNA increments detected by qPCR indicates logarithmic growth.

[0209] In some embodiments, the infected cells are continuously passaged in cell culture for a period of time. In some embodiments, the cells are continuously passaged in cell culture to dilute the cell density and / or avoid fusion of the cell culture. In some embodiments, the cells are continuously passaged in cell culture to maintain a cell density in the cell culture of no more than about 1×10 6 cells / cm 2 , no more than about 0.9×10 6 cells / cm 2 , not more than about 0.8×10 6 cells / cm 2 , not more than about 0.7×10 6 cells / cm 2 , not more than about 0.6×10 6 cells / cm 2 , not more than about 0.5×10 6 cells / cm 2 , not more than about 0.4×10 6 cells / cm2 , not more than about 0.3×10 6 cells / cm 2 , not more than about 0.2×10 6 cells / cm 2 or not more than about 0.1×10 6 cells / cm 2 , or about 0.2×10 6 cells / cm 2 About 0.9×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.7×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , or about 0.2×10 6 cells / cm 2 About 0.5×10 6 cells / cm 2 . In some embodiments, the cells are continuously passaged for about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, about 60 days, about 65 days, about 70 days, about 75 days, or about 80 days. In some embodiments, the method further comprises the step of detecting HHV-6 viral DNA or viral RNA, wherein a constant level of HHV-6 DNA or the absence of detectable HHV-6 RNA indicates HHV-6 latent infection.

[0210] In some embodiments, the HHV-6 latent infection cell culture model comprises about 10 2 to about 10 3 , about 10 2 to about 10 4 or about 10 3 to about 10 4 The HHV-6 genome equivalent contained no more than 10 copies per 500 ng of extracted genomic DNA. 2 No more than about 10 3 or not more than about 10 4In some embodiments, the viral genome equivalent is calculated by extrapolation by comparing the DNA amount of the viral gene detected in the sample with the standard curve established based on the predetermined amount of the DNA of the viral gene in the plasmid. In some embodiments, the viral gene is present in a copy of the viral genome (such as U31 or U65-66).

[0211] In some embodiments, HHV-6 latent infection cell culture model includes carrying detectable HHV-6 DNA as measured by methods known in the art and / or described herein, and as measured by methods known in the art and / or described herein, cells that do not show detectable HHV-6 RNA transcripts. In some embodiments, HHV-6 genome copy number is determined by, for example, qPCR detection of U31 or U65-66 viral DNA. In some embodiments, HHV-6 latent infection and reactivation cell culture models include cells that do not show detectable HHV-6 transcripts as determined by, for example, RT-qPCR.

[0212] In some embodiments, the cell culture model can induce HHV-6 immediate early gene, early gene and / or late gene expression after induction reactivation.In some embodiments, the cell culture model can induce one or more HHV-6 genes U95, U86, U39, U54, U14, U79-2, U24 or U46 to express.In certain embodiments, latent HHV-6 infection can be reactivated by sodium butyrate and PMA inducing cell culture model.

[0213] In vitro cell culture models allow induction of latent HHV-6 infection in a controlled manner, and can be used for a variety of applications. For example, using this cell culture model, the time and degree of HHV-6 reactivation can be controlled, and can be used to understand HHV-6 biology and carry out antiviral drug screening. Therefore, in related aspects, screening can affect HHV-6 replication, latent and / or reactivated medicaments, inhibitors or compounds, the method comprising contacting medicaments, inhibitors or compounds with cell culture models and analyzing the step of the impact of viral latent infection, reactivation, replication and / or lysis or active infection. In another aspect, the method for determining the optimal amount of medicaments, inhibitors or compounds for suppressing HHV-6 reactivation is disclosed herein, the method comprising contacting cells with different amounts of medicaments, inhibitors or compounds and analyzing the step of the impact of viral latent infection, reactivation, replication and / or lysis or active infection. In related aspects, disclosed herein is a method for determining the optimal time for applying an agent, inhibitor or compound for inhibiting HHV-6 reactivation, the method comprising contacting a cell with an agent, inhibitor or compound and analyzing the effect of latent viral infection, reactivation, replication and / or lysis or active infection. In some embodiments, the agent comprises CAR T cells or CAR T cells specific to HHV-6. In some embodiments, the degree of impact or impact can indicate the effectiveness of CAR T cells specific to HHV-6. In some embodiments, by methods described herein or known in the art, the impact is a change in the level of viral DNA replication, and / or a change in the level of viral RNA transcription, and / or a change in the level of protein expression.

[0214] All references cited herein, including patents, patent applications, articles, textbooks, etc., and references cited herein, to the extent they have not already been cited, are hereby incorporated by reference in their entirety.

[0215] The following examples are for illustrative purposes only. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description.

[0216] Example

[0217] Example 1 IFNα treatment effectively reduces HHV-6 viral load in CAR T cells

[0218] The drug product (DP) used for the experiment was selected at the end of the cell engineering process based on the screening results of HHV-6 and other foreign substances. Batch #1 is one of the few rare DPs with "positive" HHV-6 levels and is selected for the following study (called "HHV-6 high" group), while batch #2 is selected as a "low" level counterpart. Take out the DP bottle from LN2, thaw, and divide it into 4 flasks on day 0 (D0) respectively. We first tested the effects of type I IFN (such as IFNα) on the replication and infectivity of HHV-6. Flasks received complete culture medium (X-vivo+5% human serum (HS)+supplements) or complete culture medium with the addition of IFNα (100ng / mL, 1ng / mL and 0.01ng / mL of human IFN-α2a, Miltenyi). Cells were cultured for another 7 days (D1-D7), and cells were collected from all conditions every day for freezing. The culture medium was changed every three days, and IFNα was added or not to the corresponding flasks.

[0219] After cell culture, DNA was extracted from the collected samples and analyzed by qPCR for the HHV-6 marker U31 as described in Materials and Methods. Figure 1A The data in show all eight conditions (two batches of DP, each batch has 3 IFN α concentrations and untreated controls) from D1 to D7 HHV-6 viral DNA relative to the fold change of its D0 counterpart. The 1st batch in the control group without IFN α treatment shows a significant increase of HHV-6 viral DNA from D1 to D6, while "HHV-6 low" DP (the 2nd batch) shows a much smaller increase in the same period. For two DP batches, compared with the control, low doses of IFN α cause a decrease in viral load; Under higher doses of IFN α, the reduction is even more obvious.

[0220] To test whether HHV-6 protein expression was affected by IFNα treatment, we performed immunofluorescence on cells collected from batch 1 (“HHV-6 high”) DP culture D4 using an antibody specific for HHV-6p41, an HHV-6 early gene product. Figure 1B As shown, with the increase of IFNα concentration, we observed fewer HHV-6p41 positive cells in culture. For the second batch ("HHV-6 low") culture, even without IFNα treatment, only a few HHV-6p41 positive cells were observed at D4, and no matter what concentration was used, IFNα treatment resulted in no detectable HHV-6p41 positive cells (data not shown). Therefore, IFNα not only effectively reduces HHV-6 viral load, but also reduces viral protein expression in engineered CAR T cells.

[0221] Samples from two rare HHV-6-positive DPs (“HHV-6 high” or “HHV-6 low”) were retrospectively analyzed longitudinally to detect viral DNA levels during cell culture during the manufacturing process. By qPCR, both DP samples showed very low or undetectable U31 DNA levels at early time points in cell culture ( Figure 1C ). An increase in U31 DNA levels at later time points was observed in both the "HHV-6 high" sample (Batch #1) and the "HHV-6 low" sample (Batch #3).

[0222] Based on these results, we next developed an experimental model to study how IFNs can be used to suppress the reactivation of latent viruses such as HHV-6 in cell culture of engineered immune cells such as CAR T cells.

[0223] Example 2 Establishment of HHV-6 Latent Infection and Reactivation Cell Culture Model

[0224] As far as we know, there is currently no cell culture model suitable for HHV-6 latency and reactivation. Cell lines with at least part of the HHV-6 genome integrated into the chromosome have been reported. However, the utility of these cell lines for studying latent viral infection, viral reactivation and its control has not been demonstrated. See, for example, Gulve et al. 2017, Chromosomal integration of HHV-6A during non-productive viral infection. Nature, DOI: 10.1038 / s41598-017-00658-y. We established an in vitro cell culture model to facilitate the study of pathways for controlling viral reactivation. A latent infection model of human herpes virus 6B (strain Z-29) was established using human lymphoid CEM cells. CEM cells (2×10 7cells) and incubated at 37°C for 4 hours. The infected CEM cells were then washed and resuspended in appropriate culture medium over the course of 19 days. Cells were collected on days 1-6, 10, and 19 for qPCR analysis. To establish latent infection in the cells, on day 19, after logarithmic growth of the virus was observed, the infected CEM cells were divided 1 in 3 by volume and kept in culture for approximately 2 months, divided 1 in 3 every three days, and kept at a low cell density in the culture dishes until HHV-6B detection by qPCR remained constant. Genomic DNA was extracted from the collected samples and analyzed for viral infection using primer probes for detection of U31 and U65-66 viral genes. Viral adsorption was observed until day 5 after infection, and an increase in viral genomic DNA (vgDNA) was observed on days 10 and 19, indicating logarithmic growth of the virus ( Figure 2B , left panel). U31 (circles) and U65-66 (squares) Ct values ​​are reported on the left Y-axis, and viral genome equivalents based on U31 (triangles) and U65-66 (inverted triangles) are reported on the right Y-axis ( Figure 2B , left panel). The dashed lines represent the baseline of latent infection used, determined by U31 DNA (lower dashed line) or U65-66 DNA (upper dashed line). qPCR analysis of serial cultures of HHV-6B-infected CEM (CEM-HHV6) showed no increase in Ct values ​​after one week of culture, indicating that we had established latent infection ( Figure 2B , right). In the CEM-HHV6 model, by using The HHV-6B latency was confirmed by analyzing the expression of HHV-6B viral transcripts using the nanoString system (nanoString). Figure 2C The expression of 97 viral gene transcripts was analyzed from total RNA extracted from CEM (negative control) and CEM-HHV6 cells. Although constant viral DNA levels were detected in the cells ( Figure 2B , right), but no detectable viral transcripts were observed in continuous cultures ( Figure 2C ), which indicates viral latency.

[0225] We next tested whether we could induce viral reactivation in a latent cell culture model. CEM-HHV6 cells (1 × 10 cells) were incubated at 37°C with 100 ng / mL PMA (phorbol-12-myristate-13-acetate, catalog number 500582, Millipore-Sigma) and 1 mM butyrate sodium salt (catalog number 19-137, Millipore-Sigma) at a final concentration of 100 ng / mL. 7) for 2 days. On day 2, cells were washed and resuspended in RPMI + 5% FBS and cultured for 18 days. The medium was supplemented as needed. CEM-HHV6 cells were collected on days 5, 8, 12, 14, 16 and 19 after reactivation. HHV-6B viral reactivation was assessed by qPCR analysis of vgDNA of HHV-6B targeting U31 and U65-66 genes. Untreated CEM-HHV6 cells were used as negative control. Figure 2D As shown, compared with untreated CEM-HHV6 cells, vgDNA levels increased significantly starting from day 5 after reactivation and continued to increase exponentially until the last time point collected. HHV-6B reactivation was further assessed by systematic analysis of viral gene expression. Several viral RNA transcripts, including the immediate early gene U19 and the early gene U69, were detectable as early as day 5 after treatment ( Figure 2E ) and continues to increase over time ( Figure 2F Virus cytopathic effect (CPE) was also observed in reactivated cultures (data not shown). Figure 2E The expression profile of several immediate early (IE), immediate early early (IE-E), early I, and late (L) genes is shown, similar to the expression profile at the beginning of active infection after reactivation, Figure 2F The progress to active infection is shown.These results are consistent with the increase of HHV-6B vgDNA, and indicate the reactivation of latent HHV-6B virus in CEM-HHV6 cell model.CEM-HHV6 cells remain stable after at least four or five passages and after cell bank.Thawed cells from cell bank are maintained in culture for at least 30 days, and the phenotype (data not shown) of HHV-6 latent infection determined by qPCR can be maintained.

[0226] Example 3: IFNα effectively suppresses HHV-6 replication in the CEM-HHV6 cell reactivation model

[0227] use Figure 2A-2F The CEM-HHV6 reactivation model described in 2014 tested the effects of IFNα in a cell culture model similar to the one performed in DP ( Figure 1A). CEM-HHV6 cells were initially cultured in bulk prior to the start of the experiment. One day prior to the start of the experiment (D-1), an aliquot of CEM-HHV6 cells was removed and split prior to treatment with one of three IFNα concentrations (100 ng / mL, 1 ng / mL, and 0.01 ng / mL), which constituted a "pretreatment" of IFNα. Cells were collected from the start of the experiment (D0) and continued to be collected every few days throughout the process. On D3, Figure 2D The reactivation treatment (RA) described in was applied to an aliquot of cells that had undergone pretreatment, and an aliquot of cells that had not undergone pretreatment. From each of these samples, cells that had not undergone reactivation (NRA) were retained for growth. At D4, cells from all groups (RA / NRA and pretreatment / no pretreatment) were further subdivided, and some cells were treated with IFNα after reactivation ("post-treatment").

[0228] The results of this extensive series of treatments generated a wealth of data supporting the conclusions of the experiments performed with the CAR T DPs described above. All cells underwent DNA extraction followed by HHV-6 qPCR as described in Materials and Methods. In this cell culture model, HHV-6 reactivation was demonstrated by an increase in viral DNA after treatment with the reactivating agents sodium butyrate and PMA ( Figure 3A ). Data for IFNα-treated samples ( Figure 3B ) showed that with increasing doses of IFNα, the level of viral growth decreased, and at 100 ng / mL, there was almost no difference between NRA and RA samples, indicating that IFNα was counteracting the effects of RA treatment and suppressing viral growth even during induced reactivation events.

[0229] Example 4 IFNα reduces HHV-6 infectivity during small-scale CAR T production

[0230] Based on the available data, HHV-6 reactivation from PBMCs is an extremely rare event in CAR T manufacturing. To investigate the feasibility of using IFNα to control HHV-6 infectivity, we designed an experiment to test the effect of IFNα by intentionally introducing active HHV-6 infection during the manufacturing process. A schematic diagram of the experimental design is shown in Figure 4AAs shown. In brief, PBMCs from two different donors were thawed from frozen storage and cultured in complete medium (5% HS+X-vivo+ supplements) for activation and LVV transduction. After cell electroporation with TALEN mRNA, T cells were infected with HHV-6B (strain Z-29) at MOI=0.4 for 3 hours and then transferred to a G-Rex bioreactor for expansion for 10 days (Day 8 to Day 18). After 3 hours of HHV-6 infection at D8, IFNα was added to complete medium at different concentrations (100ng / mL, 10ng / mL, 1ng / mL and 0.1ng / mL) and kept in culture until the end of the expansion period. Cell samples were collected at D11, D13, D15 and D18, 3 days, 5 days, 7 days and 10 days after infection, to be determined by RT-qPCR ( Figure 4B ), flow cytometry ( Figure 4C ) and qPCR assay ( Figure 4D ) to determine HHV-6 viral infectivity. Descriptions of RT-qPCR assays, flow cytometry, and genomic DNA qPCR assays are shown in the Materials and Methods. Based on the RNA expression levels of HHV-6 viral genes U79, U90, and U100, two donors showed HHV-6 infection ( Figure 4B ). In the absence of any IFNα treatment, in donor #1, HHV-6 RNA levels increased from D11 to D18, i.e., day 3 to day 10 after infection, while RNA levels decreased from D11 to D18, even with treatment with the lowest concentration of IFNα. 10ng / mLIFNα can keep HHV-6 viral RNA below detectable levels at D18. In addition, after IFNα treatment, similar dose-dependent reductions in HHV-6 viral replication were seen in both donor #1 and donor #2, with donor #2 PBMCs first showing lower HHV-6 infectivity than donor #1. Flow cytometry data showed that even the lowest concentration of tested IFNα (0.1ng / mL) effectively reduced HHV-6 viral antigen p41 expression at D18 in both donors ( Figure 4C In addition, a dose-dependent inhibition of HHV-6U31 DNA levels was observed with IFNα treatment ( Figure 4D). By process D18, the lowest amount of tested IFNα (0.1 ng / mL) was able to reduce HHV-6 DNA levels by 16 to 38 times. Results from active infection experiments showed that in donor PBMC-engineered CAR T cells during small-scale manufacturing, IFNα added after HHV-6 infection effectively blocked HHV-6 infectivity, blocked viral spread, and prevented HHV-6 replication and amplification, as demonstrated by viral gene expression and viral DNA replication. IFNα treatment may have the same effect on patient PBMC-derived CAR T cells.

[0231] Example 5 Titration of IFNα dose for CAR T production

[0232] At certain IFNα doses, IFNα treatment can inhibit the proliferation of CAR-T cells (data not shown). In order to minimize the effect of IFNα on CAR-T cell proliferation and / or phenotype, we titrated the amount of IFNα to find the lowest effective treatment dose with minimal negative impact on CAR T expansion and phenotype in the HHV-6 infection model as described in Example 4. Figure 5A As shown in Figure 2, adding IFNα ranging from 0.1 ng / mL to 10 ng / mL to the culture from day 8 to day 18, i.e., day 0 to day 10 after infection, did not significantly affect the fold expansion of CAR T cells, however, the percentage of CAR+ cells increased in the group treated with higher doses of IFNα ( Figure 5A ). Compared with the group without HHV-6 infection and IFNα treatment, CART cells treated with IFNα also showed a younger phenotype (%CAR Tcm +%CAR Tscm cells) ( Figure 5B ). Treatment with 0.1 ng / mL to 1 ng / mL IFNα had less effect on %CAR+, CAR T cell expansion, and CAR-T cell phenotype compared with the higher concentration groups.

[0233] Example 6 Timing of adding IFNα to cell culture

[0234] The above data show that IFNα effectively inhibits HHV-6 infection of engineered CAR T cells and does not significantly affect CAR T cell expansion and cell phenotype. HHV-6 reactivation in primary PBMCs may occur in manufacturing processes with long-term cell culture, but this is an extremely rare event. We expect to understand whether the addition of antiviral agents, such as IFNα, at specific time points in the manufacturing process can prevent rare reactivation events. In order to simulate rare reactivation events, PBMC-derived CAR T cells were infected with exogenous HHV-6 at an MOI of 0.4 on day 8 of the small-scale manufacturing process. Cells were treated with IFNα at a concentration of 1 ng / μL or 10 ng / μL on day 0 (before activation), day 1 (activation), day 4 or day 6, or were not treated throughout the process. All cultures were infected with HHV-6 on day 8, regardless of whether the cells were pretreated with IFNα or not. A summary of the treatment conditions is shown in Table 1 below:

[0235] Table 1

[0236]

[0237]

[0238] The effect of IFNα on HHV-6 infection was analyzed in cells from two different donors. The viral gene U100 ( Fig. 6A and Fig. 7A ) and U79( Figure 6B and Figure 7B The y-axis represents the relative fold change of each value compared to endogenous GAPDH and untreated donor RNA (day 0 before IFNα treatment). Fig. 6A Results in Figure 5 show that the addition of IFNα on day 6, two days before infection (experimental conditions C7 and C8), resulted in the greatest reduction in U100 transcript levels compared to samples without IFNα pretreatment (C9), as well as the lowest levels of viral load determined by qPCR (data not shown). Fig. 6A The C7 and C8 samples shown also had the lowest fold changes for the U100 transcript. In this particular experiment, for unknown reasons, Fig. 6A The C5 and C6 samples shown in had fewer cells available for analysis. When transcripts of another viral gene, U79, were analyzed, all samples C5-C8 showed a decrease in transcripts ( Figure 6B The same experiment was repeated in cells from a different donor #3 ( Fig. 7A and Figure 7B ). Figure 6A-6B and Figure 7A-7B The data generally show IFN α dose-dependent responses, but some donor-specific variability has also been observed. These data show that, at the time point close to HHV-6 infection (being the alternative time or approximate time of HHV-6 reactivation in this experiment), such as carrying out IFN α processing on the 4th or 6th day of cell culture after activation on the 1st day, the HHV-6 reactivation in the manufacturing process can be most effectively controlled.

[0239] Viral DNA replication was also analyzed by qPCR of DNA of viral gene U31, and the results showed that the dose of IFNa added closer to infection (alternative time of reactivation) had the strongest suppressive effect on HHV-6 infection (data not shown).

[0240] Example 7 Effects of additional antiviral agents on PBMCs with integrated HHV-6 viral DNA

[0241] We next used PBMCs (iciHHV6) with integrated HHV-6 genomes to simulate persistent low-level HHV-6 infection and examine the effects of other known antiviral agents on controlling HHV-6 reactivation. Ganciclovir, cidofovir, and foscarnet are all small molecule inhibitors of HHV viral DNA polymerase. Artesunate is a small molecule antiviral agent targeting HHV immediate early genes. All four agents have been used clinically and show in vitro anti-HHV-6 activity. See, for example, hhv-6foundation.org / research / hhv-6-antiviral-drug-resistance; Reyman et al., 1995, Antiviral Research (Antiviral Res) .28:343-57; and Vittayawacharin et al., 2023, Transplant Cell Therapy (Transplant Cell Ther) .29:397. Cells were cultured in medium supplemented with X-vivo for three days and then divided into four subcultures. Different antiviral agents were added on day 4 at concentrations shown in Table 2. Afterwards, cells were cultured for another 15 days, with medium replaced twice a week, each time containing a fresh dose of the same antiviral agent at the same initial concentration. Cell sampling was performed at different days after the first addition of the antiviral agent. The effect of the antiviral agents on cell viability was analyzed using the Cell Apoptosis Screening Kit (Thermo Catalog No. V13242).

[0242] Table 2

[0243]

[0244] Figure 8The data in show that the tested concentrations of foscarnet and ganciclovir do not negatively affect cell health. On the other hand, artesunate and (to a lesser extent) cidofovir induce higher levels of apoptosis at the tested concentrations compared to other antiviral agents. Next, HHV-6 replication was analyzed by qPCR detection of copies of the HHV-6 gene U65. Fig. 9 The data in show that foscarnet is able to maintain HHV-6 levels at around its starting level, i.e., prevent any further HHV-6 expansion. Ganciclovir showed an initial decrease in viral load, followed by an increase at a later time point, indicating that concentrations above 25 μM may be more effective. In contrast, cells treated with artesunate and cidofovir showed increased HHV-6 levels during culture, indicating that the concentration of the tested drugs was insufficient to effectively suppress HHV-6 expansion. The results indicate that in a cell culture model, foscarnet is the most effective antiviral agent for controlling HHV6, followed by ganciclovir, which may be cidofovir when used at higher concentrations.

[0245] Materials and methods

[0246] DNA qPCR

[0247] Genomic DNA was extracted from cells and the DNA level of HHV-6 was determined by quantitative PCR (qPCR). The ThermoFisher Scientific TaqMan TM The experiments were performed using Rapid Virus 1-Step Master Mix (Catalog No. 4444434). The primers and probes for these genes are shown in Table 3.

[0248] Table 3 qPCR primer / probe set sequences

[0249]

[0250]

[0251] RNA RT-qPCR

[0252] Total RNA was extracted from cells and reverse transcription qPCR (RT-qPCR) analysis of U79, U90, U100 and GAPDH genes was used to determine HHV-6 infectivity. Primers and probes for these genes are shown in Table 4.

[0253] Table 4 RT-qPCR primer / probe set sequences

[0254]

[0255]

[0256] FACS

[0257] HHV6 infectivity was determined by intracellular staining using 1 μg / mL of an antibody against the HHV-6 p41 protein, and FACS analysis was performed to determine the percentage of cells expressing viral proteins.

[0258] %CAR+ and CAR- T cell phenotypes were determined by staining cells for the following markers: CD45, CD5, CD4, CD8, CAR. To identify memory T cell phenotypes, CD45RA and CD62L markers were used, e.g. eff (CD45RA+CD62L-), T em (CD45RA-CD62L-), T cm (CD45RA-CD62L+) and T scm (CD45RA+CD62L+) etc.

[0259] Immunofluorescence

[0260] Cells were fixed with 4% paraformaldehyde and permeabilized using 0.5% saponin, then incubated with HHV6 p41 antibody conjugated with 0.2 μg / mL Alexa- 488. Cells were stained with DAPI and then imaged at 20x magnification.

Claims

1. A method for preventing or inhibiting HHV-6 replication, preventing or inhibiting HHV-6 transcriptional activation, preventing or inhibiting HHV-6 infection, or preventing or inhibiting HHV-6 replication or infection after reactivation in the process of preparing engineered immune cells, the method comprising the following steps: Immune cells are cultured, engineered, and contacted with an antiviral agent in culture medium, optionally wherein the immune cells are suspected of having a latent HHV-6 infection.

2. The method of claim 1, wherein the step of contacting the immune cells with the antiviral agent is performed by adding the antiviral agent to the culture medium.

3. The method of claim 2, wherein the antiviral agent is added to the culture medium on day 4 to day 10, day 5 to day 10, day 6 to day 10, day 7 to day 10, day 8 to day 10, or day 9 to day 10 of the process of preparing the engineered immune cells.

4. The method of claim 2 or 3, wherein the antiviral agent is added to the cell culture medium after the cells have been cultured for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or at least 9 days.

5. The method of any one of claims 2-4, wherein the antiviral agent is added to the cell culture medium 3 days, 4 days, 5 days, 6 days, 7 days or 8 days after the immune cells have been activated, optionally, the immune cells are activated by anti-CD3 antibodies and anti-CD28 antibodies.

6. The method of any 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.

7. The method of any one of the preceding claims, wherein the antiviral agent is interferon (IFN), foscarnet, or ganciclovir.

8. The method of any one of the preceding claims, wherein the antiviral agent is a type I IFN.

9. The method of any one of the preceding claims, wherein the antiviral agent is a type III IFN.

10. 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.

11. 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.

12. 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.

13. 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.

14. The method of any one of the preceding claims, wherein the engineered immune cell is a T cell, PBMC, IPSC or NK cell.

15. The method of any of the preceding claims, wherein the engineered immune cells exhibit reduced levels of HHV-6 replication, HHV-6 transcriptional activation, HHV-6 infection, or HHV-6 replication or infection after reactivation compared to control engineered immune cells not exposed to the antiviral agent.

16. The method of any of the preceding claims, wherein the engineered immune cells express equivalent levels of combined Tcm and Tscm compared to control engineered immune cells not contacted with the antiviral agent.

17. The method according to any one of the preceding claims, further comprising a step of detecting or measuring HHV-6 DNA levels, RNA levels or protein levels during the preparation of the engineered immune cells.

18. A method as described in any of the preceding claims, further comprising a step of detecting or measuring the HHV-6 DNA level on the first day of the process of preparing the engineered immune cells, wherein the HHV-6 DNA level measured during the process of preparing the engineered immune cells is equivalent to the HHV-6 DNA level measured on the first day of the process of preparing the engineered immune cells.

19. The method of any one of the preceding claims, further comprising the step of detecting HHV-6 RNA levels during the preparation of the engineered immune cells.

20. The method of any one of claims 17-19, wherein the HHV-6 DNA level, RNA level or protein level is determined by PCR, qPCR, RT-PCR, RT-qPCR, ELISA, immunofluorescence assay or flow cytometry.

21. The method of any of the preceding claims, wherein the step of engineering the immune cell comprises introducing into the immune cell an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR).

22. The method of claim 21, wherein the exogenous polynucleotide is introduced into the immune cell by lentiviral transduction or by adeno-associated viral transduction.

23. The method of any of the preceding claims, wherein the step of engineering the immune cells further comprises modifying one or both TCR alpha loci to reduce or eliminate expression or activity of the TCR alpha gene.

24. The method of any one of claims 1-22, wherein the immune cells are obtained from a patient.

25. The method of any one of claims 1-23, wherein the immune cells are obtained from a healthy donor.

26. The method of any one of the preceding claims, wherein the immune cell carries a latent infection with HHV-6.

27. The method of any of the preceding claims, wherein the antiviral agent is human IFNα.

28. An engineered immune cell produced by the method of any one of claims 1-27.

29. A population of engineered immune cells as claimed in claim 28.

30. A method for generating an in vitro cell culture model of HHV-6 latent infection and reactivation, the method comprising the following steps (a) Infecting human lymphocytes with HHV-6; (b) culturing the infected cells for about 12 days to about 19 days; and (c) The cells are continuously passaged to maintain approximately 0.2-0.8×10 6 cells / cm 2 The cells are cultured at a cell density of about 30 to about 60 days, thereby establishing a latent HHV-6 infection in the cells.

31. The method of claim 30, further comprising detecting HHV-6 viral DNA or detecting HHV-6 viral RNA after the cells are continuously passaged in step (c), wherein a constant level of HHV-6 DNA or the absence of detectable RNA indicates a latent infection.

32. The method of claim 30 or 31, wherein the latent HHV-6 infection in the cell can be reactivated by a stimulus.

33. The method of claim 32, wherein the stimulant is sodium butyrate or PMA.

34. An in vitro cell culture model for HHV-6 latent infection produced by the method of claim 30, wherein no more than 10 DNA per 500 ng extracted can be detected in the cells by qPCR. 4 The present invention relates to the production of 100 copies of HHV-6 viral genome equivalents in the cells, and / or no HHV-6 transcripts can be detected in the cells by RT-qPCR.

35. The in vitro cell culture model of claim 34, wherein active HHV-6 infection can be induced or reactivated in the cells.

36. The in vitro cell culture model of claim 34 or 35, wherein the active HHV-6 infection can be induced or reactivated by contacting the cells with sodium butyrate and PMA.

37. The in vitro cell culture model of claim 35, wherein the active HHV-6 infection is demonstrated by detecting one or more HHV-6 transcripts via RT-PCR or RT-qPCR.

38. An in vitro cell culture model for HHV-6 latent infection, wherein no more than 10 4 The method comprises the steps of: generating a plurality of copies of HHV-6 viral genome equivalents in the cells, and / or failing to detect HHV-6 transcripts in the cells by RT-qPCR, wherein the cells are human lymphocytes.

39. The in vitro cell culture model of claim 38, wherein active HHV-6 infection can be induced or reactivated in the cells.

40. The in vitro cell culture model of claim 39, wherein the active HHV-6 infection can be induced or reactivated by contacting the cells with sodium butyrate and PMA.

41. The in vitro cell culture model of claim 39 or 40, wherein active HHV-6 infection is demonstrated by detecting HHV-6 transcripts by RT-PCR or RT-qPCR.

42. A method for preparing an engineered immune cell, the method comprising the following steps: (a) culturing immune cells; (b) engineering the immune cell; and (c) contacting the immune cell with an antiviral agent in culture medium, wherein the immune cell comprises a latent HHV-6 infection.

43. The method of claim 42, wherein the step of contacting the immune cells with the antiviral agent is performed by adding the antiviral agent to the culture medium.

44. The method of claim 42 or 43, wherein the antiviral agent is added to the culture medium on day 4 to day 10, day 5 to day 10, day 6 to day 10, day 7 to day 10, day 8 to day 10, or day 9 to day 10 after culturing the immune cells.

45. The method of any one of claims 42-44, wherein the antiviral agent is added to the cell culture medium after the cells have been cultured for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or at least 9 days.

46. ​​The method of any one of claims 42-45, wherein the antiviral agent is added to the cell culture medium 3 days, 4 days, 5 days, 6 days, 7 days or 8 days after the immune cells have been activated, optionally, the immune cells are activated by anti-CD3 antibodies and anti-CD28 antibodies.

47. The method of any one of claims 42-46, wherein the immune cells are contacted with the antiviral agent for about 2 days, about 3 days, about 4 days, or about 5 days.

48. The method of any one of claims 42-47, wherein the antiviral agent is a type I IFN or a type III IFN.

49. The method of any one of claims 42-48, wherein the antiviral agent is IFN, and 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.

50. The method of any one of claims 42-49, wherein the IFN is added to the culture medium at a concentration of about 0.1 ng / ml to about 1 ng / ml.

51. The method of any one of claims 42-50, 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.

52. The method of any one of claims 42-51, 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.

53. The method of any one of claims 42-52, wherein the immune cell is a T cell, PBMC, iPSC, or NK cell.

54. The method of any one of claims 42-53, further comprising a step of detecting or measuring HHV-6 DNA levels, RNA levels, or protein levels after the step of contacting the immune cells with the antiviral agent.

55. A method as claimed in claim 54, comprising a step of detecting or measuring the level of HHV-6 DNA before contacting the immune cells with the antiviral agent, wherein the level of HHV-6 DNA measured after contacting the immune cells with the IFN is equivalent to the level of HHV-6 DNA measured before the step of contacting the immune cells with the antiviral agent.

56. The method of claim 54, comprising the step of detecting HHV-6 RNA levels, wherein the HHV-6 RNA levels are undetectable.

57. The method of any one of claims 54-56, wherein the HHV-6 DNA level, RNA level or protein level is determined by PCR, qPCR, RT-PCR, RT-qPCR, ELISA, immunofluorescence assay or flow cytometry.

58. The method of any one of claims 42-57, wherein the step of engineering the immune cells comprises introducing into the immune cells an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR).

59. The method of claim 58, wherein the exogenous polynucleotide is introduced into the immune cell by lentiviral transduction or by adeno-associated viral transduction.

60. The method of any one of claims 42-59, wherein the step of engineering the immune cell further comprises modifying one or two TCR alpha loci to reduce or eliminate expression or activity of the TCR alpha gene.

61. The method of any one of claims 42-59, wherein the immune cells are obtained from a patient.

62. The method of any one of claims 38-60, wherein the immune cells are obtained from a healthy donor.

63. The method of any one of claims 48-62, wherein the IFN is human IFNα.

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