Improving the assessment of risk factors relevant to potential carcinogenicity of gene therapies: a consensus paper.

Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organised by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This paper describes the consensus reached amongst delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the non-clinical toxicological assessment of GT products.

Regulatory Considerations for Genome-Edited T-cell Therapies.

Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.

Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results.

Recurrent glioblastoma (rGBM) remains a major unmet medical need, with a median overall survival of less than 1 year. Here we report the first six patients with rGBM treated in a phase 1 trial of intrathecally delivered bivalent chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2). The study's primary endpoints were safety and determination of the maximum tolerated dose. Secondary endpoints reported in this interim analysis include the frequency of manufacturing failures and objective radiographic response (ORR) according to modified Response Assessment in Neuro-Oncology criteria. All six patients had progressive, multifocal disease at the time of treatment. In both dose level 1 (1 ×107 cells; n = 3) and dose level 2 (2.5 × 107 cells; n = 3), administration of CART-EGFR-IL13Rα2 cells was associated with early-onset neurotoxicity, most consistent with immune effector cell-associated neurotoxicity syndrome (ICANS), and managed with high-dose dexamethasone and anakinra (anti-IL1R). One patient in dose level 2 experienced a dose-limiting toxicity (grade 3 anorexia, generalized muscle weakness and fatigue). Reductions in enhancement and tumor size at early magnetic resonance imaging timepoints were observed in all six patients; however, none met criteria for ORR. In exploratory endpoint analyses, substantial CAR T cell abundance and cytokine release in the cerebrospinal fluid were detected in all six patients. Taken together, these first-in-human data demonstrate the preliminary safety and bioactivity of CART-EGFR-IL13Rα2 cells in rGBM. An encouraging early efficacy signal was also detected and requires confirmation with additional patients and longer follow-up time. ClinicalTrials.gov identifier: NCT05168423 .

Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.

Chimeric antigen receptor (CAR) T cells demonstrate remarkable success in treating hematological malignancies, but their effectiveness in non-hematopoietic cancers remains limited. This study proposes enhancing CAR T cell function and localization in solid tumors by modifying the epigenome governing tissue-residency adaptation and early memory differentiation. We identify that a key factor in human tissue-resident memory CAR T cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, transforming growth factor ß (TGF-ß), which enforces a core program of both "stemness" and sustained tissue residency by mediating chromatin remodeling and concurrent transcriptional changes. This approach leads to a practical and clinically actionable in vitro production method for engineering peripheral blood T cells into a large number of "stem-like" CAR-TRM cells resistant to tumor-associated dysfunction, possessing an enhanced ability to accumulate in situ and rapidly eliminate cancer cells for more effective immunotherapy.

BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]-expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

Clinical practice: chimeric antigen receptor (CAR) T cells: a major breakthrough in the battle against cancer.

Chimeric antigen receptor (CAR) T cell therapy has come of age, offering a potentially curative option for patients who are refractory to standard anti-cancer treatments. The success of CAR T cell therapy in the setting of acute lymphoblastic leukemia and specific types of B cell lymphoma led to rapid regulatory approvals of CD19-directed CAR T cells, first in the United States and subsequently across the globe. Despite these major milestones in the field of immuno-oncology, growing experience with CAR T cells has also highlighted the major limitations of this strategy, namely challenges associated with manufacturing a bespoke patient-specific product, intrinsic immune cell defects leading to poor CAR T cell function as well as persistence, and/or tumor cell resistance resulting from loss or modulation of the targeted antigen. In addition, both on- and off-tumor immunotoxicities and the financial burden inherent in conventional cellular biomanufacturing often hamper the success of CAR T cell-based treatment approaches. Herein, we provide an overview of the opportunities and challenges related to the first form of gene transfer therapy to gain commercial approval in the United States. Ongoing advances in the areas of genetic engineering, precision genome editing, toxicity mitigation methods and cell manufacturing will improve the efficacy and safety of CAR T cells for hematologic malignancies and expand the use of this novel class of therapeutics to reach solid tumors.

CRISPR-engineered T cells in patients with refractory cancer.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

CRISPR/Cas9-based genome editing in the era of CAR T cell immunotherapy.

The advent of engineered T cells as a form of immunotherapy marks the beginning of a new era in medicine, providing a transformative way to combat complex diseases such as cancer. Following FDA approval of CAR T cells directed against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma, CAR T cells are poised to enter mainstream oncology. Despite this success, a number of patients are unable to receive this therapy due to inadequate T cell numbers or rapid disease progression. Furthermore, lack of response to CAR T cell treatment is due in some cases to intrinsic autologous T cell defects and/or the inability of these cells to function optimally in a strongly immunosuppressive tumor microenvironment. We describe recent efforts to overcome these limitations using CRISPR/Cas9 technology, with the goal of enhancing potency and increasing the availability of CAR-based therapies. We further discuss issues related to the efficiency/scalability of CRISPR/Cas9-mediated genome editing in CAR T cells and safety considerations. By combining the tools of synthetic biology such as CARs and CRISPR/Cas9, we have an unprecedented opportunity to optimally program T cells and improve adoptive immunotherapy for most, if not all future patients.

Retroviral and Lentiviral Safety Analysis of Gene-Modified T Cell Products and Infused HIV and Oncology Patients.

Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring.

Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat.

The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.

The Toll-like receptor 1/2 agonists Pam(3) CSK(4) and human ß-defensin-3 differentially induce interleukin-10 and nuclear factor-κB signalling patterns in human monocytes.

Human ß-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1ß, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-κB pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-κB signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes.

Dominant negative mutant cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat.

Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.