Production Of Chimeric Antigen Receptor Research Articles

NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness

AbstractAdoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22, interleukin-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors.

Custom CARs: Leveraging the Adaptability of Allogeneic CAR Therapies to Address Current Challenges in Relapsed/Refractory DLBCL.

Cellular therapies have transformed the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL), which typically does not respond well to salvage chemotherapy. Recently, approximately 40% of r/r DLBCL patients across three different trials achieved a complete remission at 1 year after receiving treatment with autologous chimeric antigen receptor (CAR) T cells (auto-CARs). These successes have prompted studies of auto-CARs in second-line settings, in which axicabtagene ciloleucel and lisocabtagene maraleucel both showed improved event-free survival over autologous hematopoietic cell transplantation (AHCT). While encouraging, this data also highlights that 60% of patients relapse or progress following treatment with auto-CARs. Individual disease characteristics and logistical challenges of cell engineering also limit patients’ eligibility for auto-CARs. Allogeneic CAR T cells (allo-CARs) may address some of these limitations as they may mitigate delays associated with auto-CARs, thereby reducing the need for bridging chemotherapies and increasing availability of cellular products for patients with aggressive lymphomas. By being sourced from healthy donors who have never been exposed to cytotoxic chemotherapy, allo-CARs can be created from T cells with better fitness. Allo-CARs made from specific cellular subsets (e.g., stem cell memory or naïve/early memory T cells) may also have increased efficacy and long-term persistence. Additionally, allo-CARs have been successfully created from other cell types, including natural killer cells, gamma-delta T-cells and induced pluripotent stem cells. These cell types can be engineered to target viral antigens, enabling precision targeting of virally driven DLBCL. As allogeneic donor cells can be banked and cryopreserved in batches, they can be made more readily available, potentially reducing logistical hurdles and costs compared to engineering auto-CARs. This may ultimately create a more sustainable platform for cell therapies. Challenges with allo-CARs that will need to be addressed include graft versus host disease, alloimmunization, potentially decreased persistence relative to auto-CARs, and antigen escape. In short, the adaptability of allo-CARs makes them ideal for treating patients with r/r DLBCL who have progressed through standard chemotherapy, AHCT, or auto-CARs. Here, we review the published literature on patients with r/r DLBCL treated with allogeneic CAR products manufactured from various cell types as well as forthcoming allogeneic CAR technologies.

Haematology laboratory parameters to assess efficacy of CD19-, CD22-, CD33-, and CD123-directed chimeric antigen receptor T-cell therapy in haematological malignancies.

Chimeric antigen receptor (CAR) T cell products are available to treat relapsed/refractory B-lymphoblastic leukaemia/lymphoma (B-ALL), diffuse large B-cell lymphoma, mantle-cell lymphoma, and myeloma. CAR products vary by their target epitope and constituent molecules. Hence, there are no common laboratory assays to assess CAR T cell expansion in the clinical setting. We investigated the utility of common haematology laboratory parameters to measure CAR T cell expansion and response. Archived CellaVision images, absolute lymphocyte counts, and Sysmex CPD parameters spanning 1month after CD19-CAR, UCAR19, CD22-CAR, CD33-CAR, and UCAR123 therapy were compared against donor lymphocyte infused control patients. Additionally, CellaVision images gathered during acute EBV infection were analysed. CellaVision images revealed a distinct sequence of three lymphocyte morphologies, common among CD19-CAR, CD22-CAR and UCAR19. This lymphocyte sequence was notably absent in CAR T cell non-responders and stem-cell transplantation controls, but shared some features seen during acute EBV infection. CD19-CAR engraftment kinetics monitored by quantitative PCR show an expansion and persistence phase and mirror CD19-CAR ALC kinetics. We show other novel CAR T cell therapies (UCAR19, CD22-CAR, CD33-CAR and UCAR123) display similar ALC expansion in responders and diminished ALC expansion in non-responders. Furthermore, the CPD parameter LY_WY fluorescence increased within the first week after CD19-CAR infusion, preceding the peak absolute lymphocyte count (ALC) by 3.7 days. Autologous and allogeneic CAR T cell therapy produce unique changes in common haematology laboratory parameters and could be a useful surrogate to follow CAR T-cell expansion after infusion.

Characterizing piggyBat—a transposase for genetic modification of T cells

Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.

Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti-Leukemia Efficacy of CAR-T Cells via SOCE-Calcineurin-NFAT and Glycolysis Pathways.

Chimeric antigen receptor (CAR) T cells are potent agents for recognizing and eliminating tumors, and have achieved remarkable success in the treatment of patients with refractory leukemia and lymphoma. However, dysfunction of T cells, including exhaustion, is an inevitable obstacle for persistent curative effects. Here, the authors initially found that calcium signaling is hyperactivated via sustained tonic signaling in CAR‐T cells. Next, it is revealed that the store‐operated calcium entry (SOCE) inhibitor BTP‐2, but not the calcium chelator BAPTA‐AM, markedly diminishes CAR‐T cell exhaustion and terminal differentiation of CAR‐T cells in both tonic signaling and tumor antigen exposure models. Furthermore, BTP‐2 pretreated CAR‐T cells show improved antitumor potency and prolonged survival in vivo. Mechanistically, transcriptome and metabolite analyses reveal that treatment with BTP‐2 significantly downregulate SOCE‐calcineurin‐nuclear factor of activated T‐cells (NFAT) and glycolysis pathways. Together, the results indicate that modulating the SOCE‐calcineurin‐NFAT pathway in CAR‐T cells renders them resistant to exhaustion, thereby yielding CAR products with enhanced antitumor potency.

Anti-BCMA CAR-T Cell Therapy in Relapsed/Refractory Multiple Myeloma Patients With Extramedullary Disease: A Single Center Analysis of Two Clinical Trials

BackgroundThe prognosis of relapsed/refractory multiple myeloma (RRMM) patients with the extramedullary disease was significantly poor. Extramedullary multiple myeloma (EMM) patients gained limited benefits from traditional drugs. Anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy seems to be a promising approach to treat RRMM patients. However, very few clinical studies are designed for EMM. Our study aimed to compare and assess the safety, efficacy, and pharmacokinetics of anti-BCMA CAR-T cell therapy in EMM and non-EMM.MethodsThe results from published anti-BCMA CAR-T clinical trials, in which raw data of EMM patients were available, were reviewed and summarized. Two trials conducted in our clinical centers were analyzed and presented with detailed data.ResultsAccording to published anti-BCMA CAR-T clinical trials, the ORR of EMM ranged from 57% to 100%, with the complete remission (CR) rate of 29% to 60%. Between February 22, 2017, and September 26, 2019, a total of 61 subjects (EMM 25; non-EMM 36) received anti-BCMA CAR-T cell infusion. The data-cutoff date was April 1, 2021. There were no statistical differences between EMM and non-EMM groups in adverse events (AEs), including cytokine release syndrome (CRS). The most common AEs of grade ≥ 3 in both groups were hematologic toxicities. There was no significant difference in the objective response rate (ORR) and ≥ complete remission (CR) rate between both groups. However, the ≥ CR rate of the EMM group was lower than the non-EMM group receiving the fully human anti-BCMA CAR-T cell therapy (p = 0.026). The median progression-free survival (PFS) for EMM and the non-EMM group was 121 days and 361 days, respectively (p = 0.001). The median overall survival (OS) for EMM and the non-EMM group was 248 days and 1024 days, respectively (p = 0.005). The Cmax and AUC0-28d for EMM group were lower than non-EMM group (Cmax, p = 0.016; AUC0-28d, p = 0.016). Extramedullary disease was an independent prognostic risk factor for PFS (hazard ratio, 2.576; 95% CI, 1.343 to 4.941; p = 0.004) and OS (hazard ratio, 2.312; 95% CI, 1.165 to 4.592; p = 0.017) in RRMM patients receiving anti-BCMA CAR-T cell therapy.ConclusionsBased on our results, EMM patients could benefit from the two anti-BCMA CAR products, although they had a shorter PFS and OS compared with non-EMM patients.Clinical Trial Registration http://www.chictr.org.cn, identifier ChiCTR-OPC-16009113 and ChiCTR1800018137.

Scalable Manufacturing of CAR T cells for Cancer Immunotherapy.

As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development.This review provides a critical appraisal of these technologies that can be leveraged to scale and optimize the production of next generation CAR T cells.

PI3K Inhibition with Idelalisib for Optimized Production of Chimeric Antigen Receptor T Cells

Introduction: Genetically modified T cells expressing chimeric antigen receptors (CARTs) are currently among the most encouraging treatment approaches in cancer immunotherapy. Efficacy and persistence of adoptively transferred CARTs are linked to the proportion of naïve-like T cells (TN) and stem cell memory-like (TSCM) T cells defined by stem cell-like properties such as a high proliferative and self-renewal capacity. Optimization of CART production is important to shift the balance towards these subsets. The PI3K/mTOR/AKT pathway is one of the main pathways involved in T cell differentiation. In the current study we evaluated the inhibition of PI3K by the clinically approved small molecule idelalisib during culturing of CARTs in order to increase less differentiated T cell subpopulations in the final cell product.Methods: Human peripheral blood mononuclear cells (PBMCs) of ten healthy donors were transduced with a 3rd generation retroviral CD19-specific CART vector containing CD28 and 4-1BB as costimulatory domains (kindly provided by Malcolm Brenner, Baylor College of Medicine, Houston). PBMCs were activated with anti-CD3/anti-CD28 and stimulated with IL-7/IL-15. Cultivation was performed with or without 1µM idelalisib. TN was defined as CD45RA+CCR7+, central memory-like (TCM) as CD45RA-CCR7+, effector memory-like (TEM) as CD45RA-CCR7- and effector-like (TEff) as CD45RA+CCR7- T cells. Detailed analysis of T cell subpopulations, cytokine secretion and expression of exhaustion and homing markers was performed using flow cytometry over 17 days.Results: Viability (94±3% vs 87±9%; p=0.006) and transduction efficiency (65±13% vs 60±12%; p=0.001) of CARTs were significantly higher in presence of idelalisib at day 14. Both groups showed similar evolution of CD3+/CD4+ cells (48%±13% vs 46±13%, p=0.1) and CD3+/CD8+ cells (50%±13% vs 51±13%, p=0.2) at day 14. In addition, the TN subset of CD3+ (31±11% vs 23±9%, p<0.001), CD3+/CD8+ (36±15% vs 27±12%, p<0.001) and CD3+/CD4+ (25±11% vs 19±10%, p=0.003) CARTs was significantly increased with idelalisib. Cultivation with the inhibitor reduced the expression of the exhaustion marker Tim-3 (66±10% vs 82±5%, p=0.003) and raised CD62L expression (84±9% vs 70±9%, p<0.001) of CD3+ CARTs at day 14. Both groups showed low PD-1 expression (4±3% vs 6±3%; p=0.003) after 14 days of cultivation. At day 10 of culture CARTs propagated with idelalisib showed significantly lower CD4+ T helper 1 cell (20±10% vs 23±8%; p=0.05) and higher CD4+ T helper 2 cell (72±9% vs 67±7%; p=0.004) counts. Intracellular cytokine staining at day 15 after co-culture with the CD19+ Daudi cell line showed significantly lower TNF-α (47±7% vs 58±6%; p<0.001) and IFN-γ production (33±13% vs 40±13%; p<0.001) in presence of idelalisib at CD3+ CARTs. This effect was reversible and similar cytokine production for TNF-α (64±7%) and IFN-γ (40±13%) could be achieved when CARTs were rested for one day without idelalisib.Conclusions: PI3K inhibition during CART production shifted the T cells into subpopulations that allow higher persistence, long-term engraftment and sustained antitumor activity. CARTs showed a less exhausted phenotype and a higher capacity to migrate into lymph nodes. Evaluation of the effect of idelalisib for CART cultivation in patient-derived donor cells is currently ongoing. The identification of the optimal culturing condition of CARTs will further increase the benefit of this promising treatment approach in cancer immunotherapy. DisclosuresDreger:medac: Other; medac: Other: Travel grants; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; medac: Other; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; medac: Other; medac: Other; medac: Other: Travel grants; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Jansen: Consultancy; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Gilead: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Riemser: Consultancy, Research Funding; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Riemser: Consultancy, Research Funding; Riemser: Consultancy, Research Funding; Jansen: Consultancy; Riemser: Consultancy, Research Funding; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Other: Travel grants, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; Jansen: Consultancy; Gilead: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; medac: Other: Travel grants; medac: Other: Travel grants.

Co-Transplantation of CAR-Gene Engineered Precursor T Cells Leads to Suppressed T Cell Development and Increased NK Cell-Based CAR-Mediated Anti-Leukemic Activity

BACKGROUND: Production of chimeric antigen receptor (CAR) -engineered T cells is currently done for each patient individually and relies on a patient-derived T cell product of heterogeneous quality. On target/off tumor effects represent a major obstacle for a broader application of CARs. Broader application of this powerful therapeutic tool is hampered by limited production capacity and the non-desired sequelae from CAR-derived on target/off tumor effects. Here we investigated whether precursor T cells (preTs) engineered to express a clinically relevant CAR can potentially address some of these limitations.METHODS: Murine LSK cells were transduced with a 2nd generation murine CAR (CD28CD3zeta configuration) against CD19 in a lentiviral backbone and consecutively differentiated into preTs using the OP9-DL1 feeder layer system. CAR-engineered preTs (8 x 106) of MHC matched (B6/NCrl) or mismatched origin (B10.A) were co-transplanted with syngeneic T-cell-depleted bone marrow into irradiated B6 recipients. Mice were challenged with a lethal dose of CD19+ murine leukemia cells 20 days after adoptive transfer and assessed for B cell recovery and survival. For in vivo NK cell depletion studies, the anti-NK1.1 antibody (clone: PK136) was used. Sorted CAR preTs previous to co-transfer and after in vivo maturation were analyzed by microarray technology.RESULTS: Afterco-transplantation of in vitro generated CAR-engineered preTs up to 60% (p<0.001) and 100% (p<0.0001) leukemia free survival was achieved in fully MHC-mismatched or MHC-matched recipients respectively (Figure 1A). No graft-versus-host disease was induced. Anti-leukemic effects entirely relied on the in vivo development of CAR-expressing natural killer (NK) cells since NK cell depletion after hematopoietic stem cell transplantation (HCT) resulted in a complete abrogation of anti-leukemic effects (Figure 1B, p<0.001). As shown by gene expressing profiling enforced CAR-expression on preTs fosters transcriptional key factors being associated with NK cell development such as Nfil3, Id2, and Eomes. CAR-expression on co-transplanted preTs blocked thymic seeding completely and prevented further T-cell development in vivo. In contrast, CAR-expression favored NK cell development with strong anti-leukemic effects when compared to signaling-defective CAR preTs early after adoptive transfer of engineered preTs (Figure 1C, p<0.001). CAR-bearing NK-cells persisted for up to 60 days post-transplant allowing for complete B-cell recovery thereafter.CONCLUSION: Co-transplantation of CAR-engineered preTs is of high clinical relevance since they can be prefabricated and frozen as a “off the shelf product”. Limited persistence of their NK-progenies after HCT makes them an attractive platform for CARs when on target/off tumor effects are of specific concern.Figure 1: Co-transplantation of mCD19 CAR-engineered preTs provides strong anti-leukemia effects that are mediated by preT-derived CAR-expressing NK cells. (A) Irradiated B6/NCrl recipients were transplanted with either 3 x 106 T cell depleted bone marrow cells (TCDBM) only (n= 10) or additionally with 8 x 106 of either syngeneic (n= 10) or allogeneic CAR-transduced preTs (n= 10). Mice were challenged with 1.2 x 106 C1498-CD19-GFP cells on day 20 after transplantation (day 0) and followed for survival (B) Mice co-transplanted with CAR-preTs were treated i.p. weekly either with a depleting anti-NK1.1 antibody (200µg) or with PBS for control (n= 10, respectively). All mice received 1.2 x 106 C1498-CD19-GFP cells on day 20 after transplantation and survival was assessed after leukemia challenge. (C) Progenies of co-transplanted CAR-, ΔCAR- (CAR defective for signaling) or Ctrl-transduced preTs were analyzed within the BM of transplanted mice by flow cytometry 14 days after transplantation. Numbers of NK1.1+NKp46+ cells within the gate are depicted (CAR, ΔCAR, Ctrl, n=4 respectively). (A, B) Survival curves were analyzed with Mantel-Cox (log-rank) test and (C) with one-way ANOVA followed by Turkey's post-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. [Display omitted] DisclosuresSauer:Neovii: Research Funding.

Photoporation with Biodegradable Polydopamine Nanosensitizers Enables Safe and Efficient Delivery of mRNA in Human T Cells

AbstractSafe and efficient production of chimeric antigen receptor (CAR)‐T cells is of crucial importance for cell‐based cancer immunotherapy. Physical transfection methods have quickly gained in importance in the context of transfecting T‐cells, since they are readily compatible with different cell types and a broad variety of cargo molecules. In particular, nanoparticle‐sensitized photoporation has been introduced in recent years as a gentle yet efficient method to transiently permeabilize cells, allowing subsequent entry of external cargo molecules into the cells. Gold nanoparticles (AuNPs) have been used the most as photothermal sensitizers because they can easily form laser‐induced vapor nanobubbles, a photothermal phenomenon that is shown to be particularly efficient for permeabilizing cells. However, as AuNPs are not biodegradable, clinical translation is hampered. Here, for the first time, the possibility to form laser‐induced vapor nanobubbles from biocompatible polymeric nanoparticles is reported. Compared to electroporation, the most used physical transfection method for T cells, 2.5 times more living mRNA transfected human T cells are obtained via photoporation sensitized by polydopamine nanoparticles. This shows that photoporation is a viable approach for efficiently producing therapeutic engineered T‐cells at a throughput easily exceeding 105 cells per second.

Robust CAR-T memory formation and function via hematopoietic stem cell delivery

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

Allogeneic CAR T Cells: An Alternative to Overcome Challenges of CAR T Cell Therapy in Glioblastoma.

Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the major breakthroughs in cancer immunotherapy in the last decade. Outstanding results in hematological malignancies and encouraging pre-clinical anti-tumor activity against a wide range of solid tumors have made CAR T cells one of the most promising fields for cancer therapies. CAR T cell therapy is currently being investigated in solid tumors including glioblastoma (GBM), a tumor for which survival has only modestly improved over the past decades. CAR T cells targeting EGFRvIII, Her2, or IL-13Rα2 have been tested in GBM, but the first clinical trials have shown modest results, potentially due to GBM heterogeneity and to the presence of an immunosuppressive microenvironment. Until now, the use of autologous T cells to manufacture CAR products has been the norm, but this approach has several disadvantages regarding production time, cost, manufacturing delay and dependence on functional fitness of patient T cells, often reduced by the disease or previous therapies. Universal “off-the-shelf,” or allogeneic, CAR T cells is an alternative that can potentially overcome these issues, and allow for multiple modifications and CAR combinations to target multiple tumor antigens and avoid tumor escape. Advances in genome editing tools, especially via CRISPR/Cas9, might allow overcoming the two main limitations of allogeneic CAR T cells product, i.e., graft-vs.-host disease and host allorejection. Here, we will discuss how allogeneic CAR T cells could allow for multivalent approaches and alteration of the tumor microenvironment, potentially allowing the development of next generation therapies for the treatment of patients with GBM.

CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy

The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor β (FRβ) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRβ+ TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRβ+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing

The majority of CD19-directed CAR T cell products are manufactured using an autologous process. Although using a patient's leukapheresis reduces the risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect production of chimeric antigen receptor (CAR) T cells, such as myeloid cells. In this work, the effect of monocytes (CD14) on the level of activation, growth, and transduction efficiency was monitored across well plate and culture bag platforms using healthy donor leukapheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T cell starting material. Two activation reagents were tested in well plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant effect on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to unsorted leukapheresis. Cytotoxicity assays revealed that CAR T cell products produced from donor material depleted of monocytes and isolated T cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T cells produced using depleted starting material displayed a more rested and naive state.

Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures

ABSTRACT Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single-cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen-specific stimulation. Analysis of 53,191 single-cell transcriptomes showed APRIL-based CAR products to contain several subpopulations of cells, with cellular composition reproducible from donor to donor, and all major cellular subsets compatible with CAR expression. Only 50% of CAR-expressing cells displayed transcriptional changes upon CAR-specific antigen exposure. The resulting molecular signature for CAR T-cell activation provides a rich resource for future dissection of underlying mechanisms. Targeted data interrogation also revealed that a small proportion of antigen-responding CAR-expressing cells displayed an exhaustion signature, with both known markers and genes not previously associated with T-cell exhaustion. Comprehensive single-cell transcriptomic analysis thus represents a powerful way to guide the assessment and optimization of clinical-grade CAR-T-cells, and inform future research into the underlying molecular processes.

Paving the Way Towards Universal Chimeric Antigen Receptor Therapy in Cancer Treatment: Current Landscape and Progress.

Chimeric antigen receptor (CAR) therapy has been proved effective in a stream of clinical trials, especially in hematologic malignancies. However, current CAR therapy is highly personalized as cells used are derived from patients themselves, which can be costly, time-consuming, and sometimes fails to achieve optimal therapeutic results due to poor quality/quantity of patient-derived cells. On the contrary, universal CAR therapy, which is based on healthy individuals’ cells, circumvents several limitations of current autologous CAR therapy. To achieve the universality of CAR therapy, the allogeneic cell transplantation related issues, such as graft-versus-host disease (GVHD) and host-versus-graft activities (HVGA), must be addressed. In this review, we focus on current progress regarding GVHD and HVGA in the universal CAR therapy, followed by a universal CAR design that may be applied to allogeneic cells and a summary of key clinical trials in this field. This review may provide valuable insights into the future design of universal CAR products.

IMMU-07. IMMUNE EFFECTOR CELL ASSOCIATED NEUROTOXICITY (ICANS) AMONG PEDIATRIC AND AYA PATIENTS: MD ANDERSON CANCER CENTER EXPERIENCE

INTRODUCTIONImmune effector cell associated neurotoxicity (ICANS) and cytokine release syndrome (CRS) are potentially life-threatening complications associated with immune effector cell (IEC) therapies. We characterize ICANS in pediatric and adult young adolescent (AYA) patients receiving IEC therapy at our institution.METHODSWe reviewed clinical characteristics and severity (based on ASTCT Consensus Criteria) in pediatric and AYA patients with IEC products from 2018–2019 at MDACC.RESULTSNine patients, median age 15.5 (range: 3–25) years received chimeric antigen receptor (CART) IEC therapy. Four (44%) developed ICANS within median of 8 (range: 3–27) days of CAR T cell infusion and median 6 (range: 2–7) days after CRS. Primary diagnoses were pre-B cell acute lymphoblastic leukemia (8) and mediastinal large B-cell lymphoma (1). Median CRS and ICANS severity grade was 2 (range 1–4). Symptoms included altered mental status (AMS) (5), seizure (1), aphasia (2), impaired ability to write a standard sentence (4). Neuroimaging did not correlate to ICANS symptoms or severity. EEG was performed in 3 and 1 had background slowing correlating with aphasia. CSF was obtained in two revealing lymphocytosis. All received prophylactic anti-epileptic medication and tocilizumab for concomitant CRS. Three received steroids.CONCLUSIONICANS may present in almost half of pediatric patients within one week of receiving CART products associated with CRS. CAR-T trafficking into the CSF may explain pleocytosis in the CSF. Prospective studies may clarify. Impaired ability to write a standard sentence and the Cornell Assessment of Pediatric Delirium (CAPD) may be early indicators of ICANS in pediatric/AYA patients.

IMMU-52. IMMUNE EFFECTOR CELL ASSOCIATED NEUROTOXICITY (ICANS) AMONG PEDIATRIC AND AYA PATIENTS: MD ANDERSON CANCER CENTER EXPERIENCE

Abstract INTRODUCTION Immune effector cell associated neurotoxicity (ICANS) and cytokine release syndrome (CRS) are potentially life-threatening complications associated with immune effector cell (IEC) therapies. We characterize ICANS in pediatric and adult young adolescent (AYA) patients receiving IEC therapy at our institution. METHODS We reviewed clinical characteristics and severity (based on ASTCT Consensus Criteria) in pediatric and AYA patients who received IEC products from 2018–2019 at MDACC. RESULTS Nine patients, median age 15.5 (range: 3–25) years received chimeric antigen receptor (CART) IEC therapy. Four (44%) developed ICANS within median of 8 (range: 3–27) days of CAR T cell infusion and median 6 (range: 2–7) days after CRS. Primary diagnoses were pre-B cell acute lymphoblastic leukemia (8) and mediastinal large B-cell lymphoma (1). Median CRS and ICANS severity grade was 2 (range 1–4). Symptoms included altered mental status (AMS) (5), seizure (1), aphasia (2), impaired ability to write a standard sentence (4). Neuroimaging did not correlate to ICANS symptoms or severity. EEG was performed in 3 and 1 had background slowing correlating with aphasia. CSF was obtained in two revealing lymphocytosis. All received prophylactic anti-epileptic medication and tocilizumab for concomitant CRS. Three received steroids. CONCLUSION ICANS may present in almost half of pediatric patients within one week of receiving CART products associated with CRS. CAR-T trafficking into the CSF may explain pleocytosis in the CSF. Prospective studies may clarify. Impaired ability to write a standard sentence and the Cornell Assessment of Pediatric Delirium (CAPD) may be early indicators of ICANS in pediatric/AYA patients.

Immune Effector Cell Associated Neurotoxicity (ICANS) Among Pediatric and AYA Patients: MD Anderson Cancer Center Experience

Introduction Immune effector cell associated neurotoxicity (ICANS) and cytokine release syndrome (CRS) are unique potentially life-threatening complications associated with immune effector cell (IEC) therapies. ICANS presents with varying symptoms ranging from confusion to seizure and cerebral edema. There is no consensus on the precise pathophysiology related to ICANS. The incidence of this toxicity may vary based on the specific product, underlying diagnosis and host factors. Here, we characterize ICANS in pediatric and adult young adolescent (AYA) patients receiving IEC therapy at our institution. Methods We reviewed clinical characteristics, neurological severity and outcomes in pediatric and AYA patients who received IEC standard of care products from 2018-2019 at MDACC. Severity was retrospectively assessed based on ASTCT Consensus Criteria. Results Nine patients, median age 15.5 (range: 3-25) years received standard of care, chimeric antigen receptor (CART) IEC therapy. The primary diagnoses were pre-B cell acute lymphoblastic leukemia (ALL) (n=8) and primary mediastinal large B-cell lymphoma (n=1). Of these patients, 4 (44%) developed ICANS within a median of 8 (range: 3-27) days of CAR T cell infusion. ICANS was associated with CRS in all affected patients, occurring within a median of 6 (range: 2-7) days after CRS. Median CRS and ICANS severity grade was 2 (range 1-4) in all 9 patients. Symptoms included altered mental status (AMS) (5), seizure (1), aphasia (2), impaired ability to write a standard sentence (4). Neuroimaging did not correlate to ICANS symptoms or severity. EEG was obtained in 3 affected patients but only correlated to background slowing in one with aphasia. Lumbar puncture was performed in 2 patients with ICANS and was remarkable for lymphocytosis. All patients had received prophylactic anti-epileptic medication. All patients received tocilizumab for concomitant CRS and 3 received steroids. Conclusion Almost half of pediatric patients receiving standard of care CART products may experience ICANS within one week CAR T cell infusion that is associated with CRS. Pleocytosis in the CSF may be due to CAR-T trafficking into the CSF and prospective studies may clarify its significance. Impaired ability to write a standard sentence and the Cornell Assessment of Pediatric Delirium (CAPD) may be early indicators of ICANS in pediatric/AYA patients.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.