Modeling predicts differences in CAR T cell signaling due to biological variability

In recent decades, chimeric antigen receptors (CARs) have been successfully used to generate engineered T cells capable of recognizing and eliminating cancer cells. The structure of CARs frequently includes costimulatory domains, which enhance the T cell response upon antigen encounter. However, it is not fully known how the CAR co-stimulatory domains influence T cell activation in the presence of biological variability. In this work, we used mathematical modeling to elucidate how the inclusion of one such co-stimulatory molecule, CD28, impacts the response of a population of engineered T cells under different sources of variability. Particularly, our simulations demonstrate that CD28-bearing CARs mediate a faster and more consistent population response under both target antigen variability and kinetic rate variability. We identify kinetic parameters that have the most impact on mediating cell activation. Finally, based on our findings, we propose that enhancing the catalytic activity of lymphocyte-specific protein tyrosine kinase (LCK) can result in drastically reduced and more consistent response times among heterogeneous CAR T cell populations.

A Comprehensive Review of Recent Advancements in Cancer Immunotherapy and Generation of CAR T Cell by CRISPR-Cas9

The mechanisms involved in immune responses to cancer have been extensively studied for several decades, and considerable attention has been paid to harnessing the immune system’s therapeutic potential. Cancer immunotherapy has established itself as a promising new treatment option for a variety of cancer types. Various strategies including cancer vaccines, monoclonal antibodies (mAbs), adoptive T-cell cancer therapy and CAR T-cell therapy have gained prominence through immunotherapy. However, the full potential of cancer immunotherapy remains to be accomplished. In spite of having startling aspects, cancer immunotherapies have some difficulties including the inability to effectively target cancer antigens and the abnormalities in patients’ responses. With the advancement in technology, this system has changed the genome-based immunotherapy process in the human body including the generation of engineered T cells. Due to its high specificity, CRISPR-Cas9 has become a simple and flexible genome editing tool to target nearly any genomic locus. Recently, the CD19-mediated CAR T-cell (chimeric antigen receptor T cell) therapy has opened a new avenue for the treatment of human cancer, though low efficiency is a major drawback of this process. Thus, increasing the efficiency of the CAR T cell (engineered T cells that induce the chimeric antigen receptor) by using CRISPR-Cas9 technology could be a better weapon to fight against cancer. In this review, we have broadly focused on recent immunotherapeutic techniques against cancer and the use of CRISPR-Cas9 technology for the modification of the T cell, which can specifically recognize cancer cells and be used as immune-therapeutics against cancer.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered TCR-T cells against hepatocellular carcinoma

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Genetic ablation of PRDM1 in antitumor T cells enhances therapeutic efficacy of adoptive immunotherapy

Adoptive cancer immunotherapy can induce objective clinical efficacy in patients with advanced cancer; however, a sustained response is achieved in a minority of cases. The persistence of infused T cells is an essential determinant of a durable therapeutic response. Antitumor T cells undergo a genome-wide remodeling of the epigenetic architecture upon repeated antigen encounters, which inevitably induces progressive T-cell differentiation and the loss of longevity. In this study, we identified PR domain zinc finger protein 1 (PRDM1) i.e., Blimp-1, as a key epigenetic gene associated with terminal T-cell differentiation. The genetic knockout of PRDM1 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) supported the maintenance of an early memory phenotype and polyfunctional cytokine secretion in repeatedly stimulated chimeric antigen receptor (CAR)-engineered T cells. PRDM1 disruption promoted the expansion of less differentiated memory CAR-T cells in vivo, which enhanced T-cell persistence and improved therapeutic efficacy in multiple tumor models. Mechanistically, PRDM1-ablated T cells displayed enhanced chromatin accessibility of the genes that regulate memory formation, thereby leading to the acquisition of gene expression profiles representative of early memory T cells. PRDM1 knockout also facilitated maintaining an early memory phenotype and cytokine polyfunctionality in T-cell receptor-engineered T cells as well as tumor-infiltrating lymphocytes. In other words, targeting PRDM1 enabled the generation of superior antitumor T cells, which is potentially applicable to a wide range of adoptive cancer immunotherapies.

Product Characteristics and Multi-Arm Clinical Trial Design for TSC-100 and TSC-101, TCR-T Cells That Target Leukemia Following Hematopoietic Cell Transplantation

Background: While adoptive cell therapies such as CAR-T therapies have transformed the treatment of lymphoid malignancies by targeting lineage-specific antigens, they have yet to demonstrate safety and efficacy against myeloid malignancies. T cells expressing T cell Receptors (TCRs) for the HLA-A*02:01-restricted minor histocompatibility antigens HA-1 and HA-2 have been observed to clonally expand after hematopoietic cell transplantation (HCT) in donor-recipient pairs mismatched for these antigens. These expanded T cells clones are associated with significantly lower relapse rates (Marijt et al. Proc. Natl. Acad. Sci. 2003; Spierings et al. Biol. Blood Marrow Transplant. 2013) indicating a specific graft versus leukemia effect. Engineered T cells expressing an HA-1-targeting TCR have demonstrated safety and preliminary anti-leukemic activity in patients with relapsed leukemia after HCT (Krakow et al. ASH 2020). We have developed engineered TCR-T cell products, TSC-100 and TSC-101, that target HA-1 and HA-2 respectively for the treatment of leukemias after HCT and present their product characteristics and the clinical trial design here. Methods and Results: To minimize potential safety risks, process variability, and costs associated with lentiviruses, our proprietary T-Integrate manufacturing platform uses a transposon/ transposase system delivered into pan T cells. This enables the introduction of larger vectors with an increased number of functional elements. Our transposon vector encodes both the α and β chains of the TCR under a strong promoter. We find high levels of cell-surface TCR that suppresses endogenous TCRs, thereby minimizing non-specific alloreactivity and potential graft versus host disease (GvHD). In mixed lymphocyte reactions, we find undetectable to minimal alloreactivity of the engineered T cell product compared with non-engineered T cells. The vector also encodes the α and β chains of CD8 ensuring that both CD8+ and CD4+ T cells in the product acquire cytotoxicity and we demonstrate efficient killing of target cells by both T cell types. The vector includes a short peptide tag that is recognized by a GMP-grade antibody, enabling efficient purification of engineered T cells during manufacturing along with the ability to track these T cells in patients. The manufacturing process generates more than 10 billion cells with an estimated vein-to-vein time of ~3 weeks, including product release testing. We routinely find high product purity exceeding 90% engineered T cells, high cytotoxicity in vitro and vector copy numbers <5 copies/cell, ensuring low risks of oncogenicity. The planned clinical trial design is a multi-arm Phase 1/2 trial that includes a control arm for safety and early efficacy comparisons. Patients with AML, ALL and MDS planned for HCT are eligible. Since disease burden is lowest soon after HCT with lower risks of acquired resistance, TCR-T treatment will begin shortly after HCT to prevent disease relapse. Because relapse rates are far higher with reduced intensity conditioning (RIC), only RIC-eligible patients are included. To ensure that all patient-donor pairs are mismatched for the minor antigens, only patients eligible for haploidentical transplantation will be included which enables HLA mismatches. Assignment to treatment or control arms is determined by HLA type as well as the minor antigen status as determined by a PCR-based central lab assay. Since both HA-1 and HA-2 are presented on HLA-A*02:01, all patients with HLA-A*02:01 are eligible for TCR-T treatment. HA-1-positive patients are assigned to the TSC-100 treatment and HA-2 positive patients are assigned to the TSC-101 arm. Donors for these patients are required to be mismatched at either HLA-A*02:01 or the minor antigens. HLA-A*02:01 negative patients will be assigned to the control arm. Analysis of CIBMTR datasets found that this HLA-based 'biological randomization' did not affect outcomes. Dosing of TCR-T cells begins upon count recovery after RIC-HCT and up to three doses will be administered every 40 days if there is no high-grade toxicity. Early readouts of biological activity include monitoring for minimal residual disease and kinetics of donor chimerism. Recruitment begins in Q1 2022 and after the recommended Phase 2 dose has been identified, the study will transition to a Phase 2 study to assess relapse rates of TSC-100- and TSC-101-treated patients versus the control arm. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.

163 Improved anti-tumor activity of next-generation TCR-engineered T cells through CD8 co-expression

BackgroundSuccessful targeting of solid tumors with TCR-engineered T cells (TCR-T) will require eliciting of antigen-specific, multi-dimensional, sustained anti-tumor immune response by infused T cells while overcoming the suppressive tumor microenvironment. First-generation TCR-T approaches have demonstrated clinical efficacy in some solid cancers. However, effective treatment across several solid tumor indications may require engineered T cells with enhanced anti-tumor activity. Here, we show pre-clinical data from one of the engineering approaches currently being developed for next-generation ACTengine® TCR-T product candidates. We evaluated the impact of co-expression of different CD8 co-receptors on functionality of CD4+ and CD8+ T cells genetically modified with an HLA class I-restricted TCR and determined the depth and durability of anti-tumor response in vitro.MethodsHere, we used a PRAME-specific TCR currently being tested in the ACTengine® IMA203 clinical trial. T cells expressing either the TCR alone or co-expressing the TCR and CD8α homodimer (TCR.CD8α) or CD8αβ heterodimer (TCR.CD8αβ) were characterized for transgene expression, antigen-recognition, and functional efficacy in vitro. Comprehensive evaluation of CD4+ T cells expressing TCR.CD8α or TCR.CD8αβ was performed focusing on cytotoxic potential and the breadth of cytokine response against target-positive tumor cell lines.ResultsIntroduction of CD8α or CD8αβ enabled detection of transgenic TCR on the surface of CD4+ T cells via HLA multimer-guided flow cytometry otherwise lacking in the TCR only transduced T cells. Co-expression of either form of CD8 co-receptor endowed CD4+ T cells with the ability to recognize and kill target positive tumor cells; however, genetic modification with TCR.CD8αβ led to more pronounced CD4+ T cell activation as compared to TCR.CD8α. Most distinct differences were observed in the breadth and magnitude of cytokine responses, less in cytotoxic activity against tumor cells. T cells expressing TCR.CD8αβ showed superior induction of Th1 cytokines e.g. IFNγ, TNFα, IL-2, GM-CSF in vitro upon antigen stimulation as compared to TCR.CD8α-T cells. Additionally, TCR.CD8αβ T cells demonstrated more efficient engagement with antigen-presenting cells and consequently, modulation of cytokine response than TCR.CD8α-T cells.ConclusionsOur findings illustrate that engaging CD4+ T cells via CD8 co-expression potentiates anti-tumor activity of HLA class I restricted TCR-T cells in vitro. The pleiotropic effects mediated by activated CD4+ T cells including acquired cytotoxicity may potentially improve outcomes in solid tumor patients when applied clinically. In addition, the differential functional profile of TCR-T cells co-expressing either CD8α or CD8αβ suggests that optimizing the type of co-receptor is relevant to maximize anti-tumor response.

561 Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

BackgroundOver 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for therapy innovation. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (Msln) contributes to the malignant and invasive phenotype in ovarian cancer, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors.MethodsThe ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill murine ovarian tumor cells in vitro and in vivo. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies administered alone or in combination, ultimately allowing targeting up to three inhibitory receptors simultaneously. Single cell RNA sequencing was used to profile the impact of combination checkpoint blockade on both the engineered T cells and the tumor microenvironment.ResultsIn a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limited engineered T cell persistence and ability to kill cancer cells. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased anti-tumor function by intratumoral TCRMsln T cells. Single cell RNA-sequencing revealed distinct transcriptome changes in engineered T cells and the TME following triple blockade compared to single- and double-agent treatment. Moreover, combining adoptive immunotherapy with triple checkpoint blockade prolonged survival in the cohort of treated tumor-bearing mice, relative to TCRMsln with or without anti-PD1, or double-agent treatments.ConclusionsInhibitory receptor/ligand interactions within the tumor microenvironment can dramatically reduce T cell function, suggesting tumor cells may evade T cell responses by upregulating the ligands for PD-1, Tim-3 and Lag-3. In a model of advanced ovarian cancer, triple checkpoint blockade significantly improved the function of transferred engineered T cells and improved outcomes in mice in a setting in which single checkpoint blockade had no significant activity. The results suggest that T cell therapy with triple blockade, which can ultimately be more safely pursed in a cell intrinsic form through T cell genetic engineering, may overcome barriers to achieving therapeutic efficacy in patients.Ethics ApprovalThe Institutional Animal Care and Use Committees of the University of Washington and the Fred Hutchinson Cancer Research Center approved all animal studies.

CAR T Cell Therapy’s Potential for Pediatric Brain Tumors

Malignant central nervous system tumors are the leading cause of cancer death in children. Progress in high-throughput molecular techniques has increased the molecular understanding of these tumors, but the outcomes are still poor. Even when efficacious, surgery, radiation, and chemotherapy cause neurologic and neurocognitive morbidity. Adoptive cell therapy with autologous CD19 chimeric antigen receptor T cells (CAR T) has demonstrated remarkable remission rates in patients with relapsed refractory B cell malignancies. Unfortunately, tumor heterogeneity, the identification of appropriate target antigens, and location in a growing brain behind the blood–brain barrier within a specific suppressive immune microenvironment restrict the efficacy of this strategy in pediatric neuro-oncology. In addition, the vulnerability of the brain to unrepairable tissue damage raises important safety concerns. Recent preclinical findings, however, have provided a strong rationale for clinical trials of this approach in patients. Here, we examine the most important challenges associated with the development of CAR T cell immunotherapy and further present the latest preclinical strategies intending to optimize genetically engineered T cells’ efficiency and safety in the field of pediatric neuro-oncology.