Generation Chimeric Antigen Receptor Research Articles

GD2 targeting CAR T cells for neuroblastoma

Treatment of neuroblastoma is a significant clinical unmet need in paediatric oncology epitomised by high-risk disease in which relapse is common and outcomes for children with relapse or primary refractory disease are typically poor, with 4-year progression-free survival for relapse/refractory disease of 6%. Immunotherapy targeting disialoganglioside GD2 using monoclonal antibodies (mAb) has become a component of standard of care treatment in neuroblastoma following published studies that have demonstrated clinical activity and survival benefit associated with this treatment. Hence a number of research groups have developed and clinically evaluated chimeric antigen receptor gene modified T cells (CAR-T cells) targeting GD2 in patients with relapsed and refractory neuroblastoma. Preclinical and clinical results using a range of receptor technologies and immune effectors have demonstrated the basic safety and feasibility of this approach, progressing into clinical data exhibiting promise for sustained patient benefit.

Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering.

ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient's own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).

Long-term Remissions Following CD20-directed Chimeric Antigen Receptor Adoptive T cell Therapy.

Chimeric antigen receptor (CAR) T cell therapy produces high response rates in refractory B-cell non-Hodgkin lymphoma (NHL), but long-term data are minimal to date. Here, we present long-term follow-up of a pilot trial testing a CD20-targeting 3rd generation CAR in patients with relapsed B-cell lymphomas following cyclophosphamide-only lymphodepletion. Two of the 3 patients in the trial, with mantle cell lymphoma and follicular lymphoma, had remissions lasting more than 7 years, though they ultimately relapsed. The absence of B cell aplasia in both patients suggested a lack of functional CAR T cell persistence, leading to the hypothesis that endogenous immune responses were responsible for these long remissions. Correlative immunologic analyses supported this hypothesis, with evidence of new humoral and cellular anti-tumor immune responses proximal to clinical response time points. Collectively, our results suggest that CAR T cell therapy may facilitate epitope spreading and endogenous immune response formation in lymphomas.

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

Chimeric antigen receptor-natural killer cell therapy: current advancements and strategies to overcome challenges.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is a novel immunotherapy targeting cancer cells via the generation of chimeric antigen receptors on NK cells which recognize specific cancer antigens. CAR-NK cell therapy is gaining attention nowadays owing to the ability of CAR-NK cells to release potent cytotoxicity against cancer cells without side effects such as cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GvHD). CAR-NK cells do not require antigen priming, thus enabling them to be used as "off-the-shelf" therapy. Nonetheless, CAR-NK cell therapy still possesses several challenges in eliminating cancer cells which reside in hypoxic and immunosuppressive tumor microenvironment. Therefore, this review is envisioned to explore the current advancements and limitations of CAR-NK cell therapy as well as discuss strategies to overcome the challenges faced by CAR-NK cell therapy. This review also aims to dissect the current status of clinical trials on CAR-NK cells and future recommendations for improving the effectiveness and safety of CAR-NK cell therapy.

Magnetic CAR T cell purification using an anti-G4S linker antibody

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients' own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Abstract 44: A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells

Abstract Chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies, while promising and efficacious, can also result in a variety of adverse effects. Of note, on-target, off-tumor toxicity is one adverse effect that continues to limit the safety and efficacy of CAR-T cells and has even led to life-threatening or fatal outcomes in patients (Morgan et al. 2010.). To address this, we have developed a unique, codon-based approach by replacing strategically selected codons within CAR non-antigen binding domains with their rare synonymous isoforms. Hoekema et al. (1987) reported the ability of synonymous rare codons to alter gene and protein expression in other model systems. In CAR-T cells, we aim to use this novel, codon-based approach to fine tune and modulate CAR-T cell expression and function to elicit full on-tumor activity while lessening off-tumor toxicity. In our approach, we replaced specific endogenous codons within the non-antigen binding domains of Hu8F4-CAR, a TCR-like 2nd generation CAR developed by our group which targets the leukemia-associated antigen PR1/HLA-A2 (Ma et al. 2016.). Briefly, we created variants of Hu8F4-CAR containing rare codon substitutions in two subtypes: intracellular-based (type 1) and combined extracellular- and intracellular-based (type 2). Each variant subtype (type 1 or 2) had either between 6 and 30 or 27 and 57 rare codons introduced, respectively. Expression profiling and functional assessment of the Hu8F4-CAR variants was assessed in the Jurkat mutant cell line (J.RT3-T3.5) or human primary healthy peripheral blood mononuclear cells (PBMCs) that were enriched for T cells, respectively. Expression profiling results show that surface expression is reduced in J.RT3-T3.5 cells when assessed by flow cytometry by 1-1.5-fold compared to unmodified Hu8F4-CAR. When evaluated by immunoblotting, novel lower molecular weight bands emerge only in rare codon-modified variants, suggesting that rare codons may have altered translational or post-translational processing leading to truncated CAR protein isoforms (30, 45, or 60kDa vs 73kDa for unmodified Hu8F4-CAR), which may contribute to reduced cell surface CAR expression. Furthermore, we have conducted in vitro functional testing of the rare codon-modified variants and show reduced activity against non-malignant off-tumor targets that express PR1 and/or HLA-A2 including healthy donor PBMCs, mobilized hematopoietic cells, and bone marrow. Importantly, in addition to reduced off-tumor activity against healthy myeloid cells, rare codon-modified variants preserve their activity against on-tumor target cells, including the U937-A2+ cell line and primary acute myeloid leukemia. Altogether, these results suggest that our approach can modulate CAR expression and functionality creating a novel methodology to reduce the likelihood of off-tumor adverse effects typically associated with CAR therapies. Citation Format: Rolando A. Vedia, Sijie Lu, Anne V. Philips, Anna Sergeeva, Lisa S. St John, Karen Clise-Dwyer, Gheath Alatrash, Jeffrey J. Molldrem. A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 44.

Genome editing approaches for universal chimeric antigen receptor T cells

Autologous chimeric antigen receptor (CAR) T cell therapy has revolutionised the management of certain B-cell malignancies. However, as bespoke therapies, challenges include complex manufacturing logistics and risks ranging from suboptimal harvests to inadvertent transduction and masking of blast populations. Premanufactured, ready -to -use allogeneic CAR T cells could mitigate some of these hurdles if barriers created by HLA (Human leukocyte antigen) mismatching can be addressed. Genome editing to disrupt TCRαβ (T-cell receptor αβ) expression has been shown to be effective in addressing alloreactivity and avoiding graft versus host disease (GVHD). Platforms including transcription activator-like effector nucleases (TALENs), homing endonucleases and clustered regularly interspersed short palindromic repeats (CRISPR) / Cas9 have allowed multiplex editing of TCR genes in combination with CD52, the target antigen of alemtuzumab, as a strategy to evade lymphodepletion used to prevent host v graft rejection effects. Alternative approaches have targeted pathways to prevent HLA expression on donor T cells, and have also allowed targeted insertion of CAR genes, including placing transgene expression under the control of endogenous transcriptional machinery. These tools have rapidly progressed to clinical trials, and applications have extended beyond B-cell malignancies, showing promising early results in other settings, including relapsed/refractory(r/r) T-cell leukaemia. Short term immunological effects and toxicities have been generally manageable, and long-term monitoring is ongoing to help build confidence in safety over time.

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer.

As chimeric antigen receptor (CAR) T cell therapy continues to gain attention as a valuable treatment option against different cancers, strategies to improve its potency and decrease the side effects associated with this therapy have become increasingly relevant. Herein, we report an alternative CAR design that incorporates transmembrane domains with the ability to recruit endogenous signaling molecules, eliminating the need for stimulatory signals within the CAR structure. These endogenous signaling molecule activating (ESMA) CARs triggered robust cytotoxic activity and proliferation of the T cells when directed against the triple-negative breast cancer (TNBC) cell line MDA-MB-231 while exhibiting reduced cytokine secretion and exhaustion marker expression compared to their cognate standard second generation CARs. In a NOD SCID Gamma (NSG) MDA-MB-231 xenograft mouse model, the lead candidate maintained longitudinal therapeutic efficacy and an enhanced T cell memory phenotype. Profound tumor infiltration by activated T cells repressed tumor growth, further manifesting the proliferative capacity of the ESMA CAR T cell therapy. Consequently, ESMA CAR T cells entail promising features for improved clinical outcome as a solid tumor treatment option.

Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.

Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.

And-Gate CAR T-Cells to Improve Tumour Specificity and Targeting of Low-Expression Antigens in Multiple Myeloma

Whilst chimeric antigen receptor (CAR) T cells have shown remarkable success, further novel targets are required. However, our previous cell surface proteomics work in multiple myeloma (MM) showed that target choice is highly limited by an absence of truly tumour-specific antigens, and often very low on-tumour expression of target proteins in some primary samples. One approach to improve tumour specificity is to use combinatorial antigen recognition, or “AND-gates”, whereby a CAR T cell must recognise two different antigens for full activation and target cell elimination. It has also been reported that this approach might overcome low target expression. We therefore decided to explore the potential for AND-gate targeting in MM and identify a suitable target pairing. To determine a comprehensive list of viable AND-gate combinations for MM, we first integrated our MM proteomics data with a normal tissue proteomics dataset. From 777 extracellular proteins expressed in primary MM, we identified a total of 287,890 possible pairwise combinations. We next excluded any pairs with overlapping expression in vital, healthy tissues, reducing potential combinations to 664. Additional filtering steps to remove difficult-to-target proteins and combinations where either target was expressed on T cells - thus avoiding T cell fratricide - resulted in a final list of 92 AND-gate targets. From our prioritised candidates, we selected TNFRSF8 and TMPRSS11E as an exemplar pairing. By mass spectrometry, both TNFRSF8 and TMPRSS11E were expressed across all 8 profiled primary MM samples at comparable levels to existing CAR T cell targets, such as GPRC5D, and had no predicted overlapping expression in normal tissue. To test our AND-gate combination, we designed a ‘split-CAR’ system in which TNFRSF8 was targeted by a 1 st generation CAR with an intracellular signalling domain, but without a co-stimulatory receptor (CAR-only construct), and TMPRSS11E was targeted by a construct with a chimeric co-stimulatory receptor (CCR), but lacking an intracellular signalling domain (CCR-only construct). Constructs were initially validated in a Jurkat cell line model of T cell activation. Jurkats were transformed with CCR-only, CAR-only, or both constructs, as well as with full 2 nd generation CARs targeting TNFRSF8 or TMPRSS11E. These cells were co-cultured with the TNFRSF8+TMPRSS11E+ human MM cell line XG1 or the TNFRSF8+TMPRSS11E- erythroleukemic cell line K562. As anticipated, neither the CCR-only nor the CAR-only Jurkats were activated, with no production of IFNγ or IL2 ( Fig. 1A). Whilst Jurkats expressing the full 2 nd generation TMPRSS11E CAR were activated in co-culture with XG1, Jurkats expressing the TNFRSF8 2 nd generation CAR were not, despite expression of TNFRSF8 on XG1. Jurkats co-expressing both components of the split-CAR were markedly activated when co-cultured with TNFRSF8+TMPRSS11E+ XG1 cells (but not with TNFRSF8+TMPRSS11E- K562 cells), as demonstrated by a large increase in expression of CD69, IFNγ, and IL2 ( Fig. 1A). Thus, the split-CAR system showed a very high degree of specificity, requiring both antigens to be co-expressed on the target cell. Moreover, it was activated even when full 2 nd generation CARs against TNFRSF8 were not. Next, we sought to investigate the cytolytic activity of our split-CAR constructs in donor T cells. In a cytotoxicity assay, T cells expressing the CAR-only or CCR-only construct did not exhibit significant killing of XG1 cells ( Fig. 1B). In parallel with the Jurkat reporter assays, only 2 nd generation TMPRSS11E CAR T cells, and not 2 nd generation TNFRSF8 CAR T cells, were cytotoxic to XG1. Finally, donor T cells expressing both components of the split-CAR demonstrated potent killing of dual-positive XG1 cells ( Fig. 1B), but not single-positive K562 cells. We have thus developed an analytical pipeline for the discovery of AND-gate CAR T cell targets for MM, also applicable to other tumours. This approach led to the development of an extremely potent, but highly selective, split-CAR targeting TNFRSF8 and TMPRSS11E. Importantly, our split-CAR targeted very low levels of TNFRSF8 antigen that were insufficient to trigger activation of a 2 nd generation TNFRSF8 CAR-T. In summary, our work suggests that AND-gate targeting can increase not just specificity, but potency, even against very low expression tumour targets in MM. Link to preprint https://doi.org/10.1101/2023.04.04.535580

Restoration of LAT Signaling in CD33-Directed CAR T Cells Improves Potency and Persistence Against Acute Myeloid Leukemia

Despite the development of novel therapies, the majority of patients with acute myeloid leukemia (AML) will not achieve long-term remissions. Patients with relapsed/refractory (r/r) AML have particularly poor outcomes, as conventional chemotherapy regimens are often unable to elicit long-term remissions. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative in eligible patients, however, bridging therapies which can induce deep remissions prior to HSCT are needed. Second generation chimeric antigen receptor (CAR) T cells targeting lineage-restricted antigens, such as CD19, have induced remissions in up to 80 - 90% of pediatric patients with r/r B-lineage acute lymphoblastic leukemia (ALL), however, post-CAR relapses are common and only ~50% of treated patients achieve durable remissions. Similar strategies targeting myeloid-restricted antigens with 2 nd generation CAR T cells in AML have not replicated the success seen in ALL. As clinical experience has highlighted the impact of CAR T cell potency, persistence, and antigen-sensitivity to long-term efficacy in ALL, strategies to boost these properties in myeloid-directed CAR T cells are necessary to extend the clinical benefit of this therapy to patients with AML. Utilizing a global phosphoproteomics screen, we recently identified that inefficient phosphorylation of the Linker for activation of T cells (LAT) and the subsequent decrease in the downstream signaling pathways results in suboptimal CAR T cell activation. To overcome inefficient LAT activation, we developed a novel bicistronic CAR platform, consisting of a 2 nd generation CAR along with an Adjunctive LAT-Activating CAR (ALA-CART) which improved sensitivity to low-antigen leukemia cells, increased in vivo potency, and enhanced persistence in xenograft models of ALL. We hypothesized that the restoration of LAT signaling via the ALA-CART platform would also improve potency and persistence in pre-clinical models of AML. We found that while CD33 ALA-CART cells responded to CD33+ AML cells in vitro, they were less cytotoxic and produced lower levels of cytokines than CD33-28z or CD33-BBz 2 ndgeneration CAR T cells. However, CD33 ALA-CART cells demonstrated increased potency in xenograft models, eradicating AML cells at much lower doses than CD33-28z and CD33-BBz CAR T cells (Fig. 1A, p<0.0001). This in vivo potency correlated with an enrichment of T stem cell memory cells in the ALA-CART product, resulting in increased persistence of CD33 ALA-CART cells after leukemia clearance (Fig. 1B, p=0.0139). The ALA-CART platform was associated with increased mitochondrial mass and function, suggesting that the increased signaling through LAT results in an enhanced capacity for oxidative phosphorylation, potentially contributing to the increased persistence of CD33 ALA-CART cells in vivo. Thus, the ALA-CART platform, designed to restore LAT signaling in CAR T cells, demonstrates that CAR molecules can be rationally designed to enhance both in vivo potency and persistence, and represents a promising strategy for the development for more effective AML-directed CAR T cells.

Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells. With a 5-year overall survival of approx. 30%, the prognosis of patients with acute myeloid leukemia remains poor, in particular for those with relapse or refractory disease. The adoptive transfer of T-cells genetically modified to express a chimeric antigen receptor (CAR) have shown impressive remission rates in patients with B-cell lymphoid malignancies. In contrast, CAR T-cell therapy for AML has induced durable remission only in a minority of patients so far, highlighting the need for strategies to improve anti-tumor efficacy and persistence of AML-specific CAR T-cell products. Adapting an established high throughput screening platform, we aimed to identify compounds with the potential to enhance CAR T-cell mediated cytotoxicity through interaction with tumor and/or CAR T-cells. Methods: We established a high throughput platform to determine the cytotoxicity of four different AML-specific CAR T-cell products targeting CD33 (CD33.CD28.41BB.zeta), CD70 (CD27.zeta), CLL1 (CLL1.CD28.zeta) and EMR2 (also known as ADGRE2; EMR2.41BB.zeta) in the presence of 32 compounds at five different concentrations in a 10,000-fold concentration range. CAR T-cells were generated by transduction of activated T-cells derived from peripheral blood mononuclear cells (PBMCs) of three healthy donors with a gamma retroviral vector containing the different CAR constructs. On day 11 after activation, CAR T-cells and non-CAR-transduced control T-cells were incubated with luciferase-expressing target cells at an effector-target ratio of 1:4 in 384-well plates. Viability of target cells cultured with CAR T-cells, control T-cells or in the absence of T-cells was calculated after 24 and 48 hours using a luciferase signal intensity assay. Combinatorial toxicities were calculated with the Bliss Independence model for synergistic effect and the highest single agent model for additive combination effect. Results: In quality analysis, the intensity of luminescence signals in control wells (tumor only, non-CAR-transduced control T-cells) of different plates was similar and principal component analysis (PCA) of samples derived from different days (batches) did not show significant differences ruling out relevant edge or batch effects that could negatively impact the screening results. Of all tested compounds birinapant, a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of apoptosis protein (IAP) family proteins and the hypomethylating agent (HMA) decitabine showed the strongest improvement of CAR T cell cytotoxicity in an additive but also in a synergistic manner. These effects were evident in CAR T-cell products derived from all different T cell donors without significant differences. Depending on the structural composition of the CAR construct, the strongest drug-induced improvement of CAR T-cell cytotoxicity was seen either after 24 or 48 hours. The third generation CAR T- cell construct (CD33.CD28.41BB.zeta) showed enhanced cytotoxicity already after 24 hours, whereas significant anti-leukemic efficacy of CD27.zeta, CLL1.CD28.zeta and EMR2.41BB.zeta CAR T-cells became evident at the later 48 hours timepoint. Importantly, co-treatment with birinapant and decitabine particularly enhanced the cytotoxicity of EMR2-specific CAR T-cells which exhibited the lowest baseline anti-tumor efficacy compared to all other CAR constructs highlighting the potential of distinct chemical compounds to improve the functionality of AML-specific CAR T-cells. Conclusion: Our data suggest that high throughput drug interaction screens are a reliable approach to investigate the impact of chemical compounds on the functionality of AML-specific CAR T-cell products. HMA and SMAC mimetics are promising candidates that may potentially enhance the cytotoxicity of CAR T-cells. Ongoing studies are evaluating the effect of HMAs and SMAC mimetics on CAR T-cell proliferation and anti-tumor efficacy against AML cell lines and primary AML patient samples upon serial antigen stimulation. Furthermore, in-depth proteogenomic characterization to identify molecular signatures of enhanced functionality are currently being performed.

Multinational Retrospective Analysis of Bridging Therapy Prior to Chimeric Antigen Receptor T Cells for Relapsed/Refractory Acute Lymphoblastic Leukemia in Children and Young Adults

Background: Chimeric antigen receptor (CAR) T cells targeting CD19 are a well-established treatment option for children and young adults suffering from relapsed and/or refractory B-lineage acute lymphoblastic leukemia. Nonetheless, there is still insufficient data about the proper management of bridging therapy between eligibility for therapy and administration of CAR T cells taking into consideration that most of the patients are heavily pretreated. Bridging therapy has been designed to achieve a low leukemia burden prior to CAR T cell infusion. However, systematic data of bridging therapy are still limited and the effect of bridging therapy on outcome, side effects and response to CAR T cell therapy is still poorly understood. With this retrospective, multinational, large-scale study, we strive to understand the impact of low- and high-intensity bridging regimens on a variety of outcome parameters in order to improve the basis for clinical decision making in bridging therapy prior to CAR T cell administration. Methods: Real-world data were collected from 83 patients receiving 88 CAR T cell therapies from twelve different sites in Germany, Austria and Switzerland. Data were collected anonymously via paper case report forms and subsequently analyzed. Performed treatments were classified into the categories 1) no systemic therapy, 2) low-intensity therapy and 3) high-intensity therapy. Bridging therapies were defined as high-intensity if at least one chemotherapeutic agent of the following was given: cyclophosphamide/ifosfamide, etoposide, anthracyclines or other agents with high toxicity potential (intravenous methotrexate, platinum-based antineoplastic drugs, thiotepa, high-dose cytarabine, fludarabine). Low-intensity bridging therapies comprised the administration of steroids, vincristine, low-dose cytarabine, PEG-asparaginase/Erwinia asparaginase and oral maintenance therapy (mercaptopurine, thioguanine, oral methotrexate, hydroxyurea). The administration of specific chemotherapeutic agents as well as immunotherapies and targeted therapies was assessed. CAR therapies comprised CD19 2 nd generation CAR T cell products from commercial and academic providers. We then analyzed the impact of different bridging regimens on several outcome parameters such as overall and disease-free survival, adverse events, tumor burden and performance status at defined time points (eligibility, leukapheresis if performed, lymphodepletion/CAR T cell infusion). Results: 33 of 88 treatments were classified as high-intensity and 34 as low-intensity bridging regimens. Prior to 13 CAR T cell administrations no systemic bridging therapy was given, 8 of 88 bridging regimens could not be stratified due to incomplete data (Figure 1). Between eligibility and apheresis, mostly low-intensity therapy or no systemic therapy was given. Within the period between apheresis and CAR T cell infusion, treatment diversified due to the heterogeneity of the cohort. Patient characteristics are listed in Table 1. Patients receiving a high-intensity bridging therapy had a significantly higher tumor burden at time point of eligibility defined by blasts in bone marrow and by measurement of minimal residual disease (MRD) compared to patients treated with a low-intensity or no systemic bridging therapy. Tumor burden within the two groups converged over the time of bridging therapy. However, at time of lymphodepletion, patients in the high-intensity group showed a significantly lower performance status indicated by Karnofsky/Lansky score than patients in the low-intensity/no systemic therapy group, reflecting the higher toxicity potential. Furthermore, these patients suffered significantly more often from bacterial adverse events and mucositis. Neither overall nor disease-free survival differed significantly between the two bridging regimen groups. Conclusion: In this retrospective cohort data, a high-intensity bridging therapy has not improved the outcome of CAR T cell therapy in terms of overall and disease-free survival. Yet high-intensity bridging therapy has caused more mucositis, bacterial adverse events and worsened the performance status. Our study suggests that a low-intensity bridging regimen may be preferred whenever tumor burden and disease kinetics allow this treatment strategy.

Targeting Normal Myelopoiesis Negatively Affects CAR T Cell Activity

Chimeric antigen receptor (CAR) T cells have not been successful for the treatment of acute myeloid leukemia (AML) to date, and the reason for this is not clear. While CAR T cells have been traditionally studied as independent agents targeting tumor, in reality they work in the context of the endogenous host immune system, which may influence outcomes of therapy. We hypothesized that targeting normal myelopoiesis is detrimental to CAR T cell activity, and this creates a unique obstacle to effective CAR T cell therapy for AML. We generated prototypical CAR T cells targeting B cell (CD19) and myeloid (CD33) antigens, using a standard 2 nd generation CAR design with 4-1BB and CD3ζ costimulatory domains. In parallel we created tumor cell lines expressing both targets by adding CD33 expression to a CD19+ B cell line (Ramos +33), and by adding CD19 expression to a CD33+ myeloid cell line (THP1 +19). For ease of tracking tumor cells were labeled with GFP, and CAR T cells were labeled with RFP. CD19-targeting CAR T cells (CART19) and CD33-targeting CAR T cells (CART33) exhibited similar anti-tumor activity in standard in vitro cytotoxicity assays and in vivo NSG mouse models. We then proceeded to add PBMCs to CAR T cell and tumor co-culture and measured tumor cell clearance and CAR T cell expansion using flow cytometry and the Incucyte Live-Cell analysis system. Addition of PBMCs enhanced the tumor clearance and CAR T cell expansion of both CART19 and CART33 in short-term co-culture assays (7-10 days). Fractionation of PBMCs revealed that CD33+ classical monocytes were able to replicate the effect of PBMCs, while the presence of CD19+ B cells did not have any effect. Monocytes are viable in vitro for less than 7 days. To assess the long-term effects of monocytes on CAR T cells, we performed serial replating assays, in which fresh tumor cells and monocytes were added to CAR T cells every 7 days. Ongoing monocyte support further improved CART19 expansion and tumor clearance. However, the continuous presence of monocytes was detrimental to CART33 activity upon serial replating, as evidenced by suboptimal T cell expansion and loss of tumor control. To study the effects of normal myelopoiesis on CAR T cell activity in vivo, NSGS mice (NOD/SCID/IL2Rg null mice with human SCF, IL-3 and GM-CSF expression) were injected with human cord blood CD34+ cells to engraft human hematopoiesis. Mice were then injected with THP1 +19 tumor cells, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from the same cord blood product as the CD34+ cells. Consistent with our in vitro data, CART19 and CART33 showed better anti-tumor activity in the presence of human hematopoiesis. Notably, while the anti-tumor activity of CART19 and CART33 was equivalent in non-humanized mice, CART19 had better long-term activity in humanized mice, as mice treated with CART33 had late tumor relapses. To directly compare the effects of myeloid cells as bystanders versus targets, we lentivirally transduced cord blood CD34+ cells with human CD19 so that they would be targets for CART19. We then differentiated these cells into myeloid progenitors (MP) by culturing in IMDM with 20% FBS and the following cytokines: SCF (100ng/ml), FLT3L (50ng/ml), IL-3 (10ng/ml), and GM-CSF (10ng/ml). After 10 days of culture >95% of MP expressed CD33, and were selected for >95% CD19 expression (Fig A). Addition of non-transduced MP to CAR T cell and Ramos +33 co-culture enhanced CART19-mediated tumor clearance without affecting CART19 expansion. However, the presence of MP CD19+ had a detrimental effect on both tumor control and expansion of CART19, confirming that CAR T cells are less effective when targeting myeloid cells. In contrast, both MP and MP CD19+ had similar effects on CART33 that mirrored the negative effects of MP CD19+ on CART19 (Fig B). To summarize, we show that normal myeloid cells have beneficial effects on CAR T cell function, and targeting these same myeloid cells is detrimental to CAR T cell activity. These findings provide insight into the reason for suboptimal CAR T cell activity in AML, and show that independent of target antigen and CAR construct, the nature of the cells being targeted can change the effectiveness of this therapy.

EXTH-08. IMMUNOTHERAPEUTIC TARGETING OF FIBROBLAST ACTIVATION PROTEIN (FAP) IN TREATMENT REFRACTORY GLIOBLASTOMA USING NOVEL CAR-T CELL THERAPY

Abstract Glioblastoma (GBM), the most common malignant primary adult brain tumor, is characterized by extensive cellular and genetic heterogeneity. The standard of care therapy and clinical trials have not been successful in improving patients’ survival which underscores an urgent need for developing new effective therapies. In silico and in vitro analysis have revealed high level of Fibroblast Activation Protein (FAP) expression in GBM tissue by multiple cell populations including endothelial cells, fibroblasts, stromal and tumor cells, but not normal brain tissue, making FAP a safe and suitable therapeutic target. As chimeric antigen receptor (CAR) expressing T cells have shown promising results against multiple cancers, we aimed to target FAP in GBM by FAP specific CAR-T cells generated using a novel GMP-ready FAP-CAR construct produced based on a virus-free CAR gene transfer using advanced sleeping beauty transposon technology and pre-clinically test their anti-tumor efficacy. Our data revealed that co-culturing CAR-T cells with FAP+ GBM cells (U87) resulted in increased surface expression of T-cell activation markers. Moreover, FAP CAR-T mediated cytotoxicity against FAP+ GBM cells was also observed in the co-culture of GBM and CAR-T cells. For further validation, the effect of FAP CAR-T cells will be tested on patient-derived GBM organoids. The in vivo efficacy of FAP CAR-T cells is currently under investigation and we anticipate that the treatment of GBM tumor-bearing mice with FAP CAR-Ts results in extended survival and tumor burden reduction. These rigorously obtained data show high level of efficacy for FAP-specific CAR-T cells against GBM suggesting that clinical development of this therapeutic modality can provide a novel therapeutic strategy for GBM patients. Therefore, upon successful completion of the preclinical study, we plan to conduct a phase I dose-escalation trial of autologous FAP-CAR T cells for patients with recurrent Glioblastoma.

Charting a killer course to the solid tumor: strategies to recruit and activate NK cells in the tumor microenvironment.

The ability to expand and activate natural Killer (NK) cells ex vivo has dramatically changed the landscape in the development of novel adoptive cell therapies for treating cancer over the last decade. NK cells have become a key player for cancer immunotherapy due to their innate ability to kill malignant cells while not harming healthy cells, allowing their potential use as an "off-the-shelf" product. Furthermore, recent advancements in NK cell genetic engineering methods have enabled the efficient generation of chimeric antigen receptor (CAR)-expressing NK cells that can exert both CAR-dependent and antigen-independent killing. Clinically, CAR-NK cells have shown promising efficacy and safety for treating CD19-expressing hematologic malignancies. While the number of pre-clinical studies using CAR-NK cells continues to expand, it is evident that solid tumors pose a unique challenge to NK cell-based adoptive cell therapies. Major barriers for efficacy include low NK cell trafficking and infiltration into solid tumor sites, low persistence, and immunosuppression by the harsh solid tumor microenvironment (TME). In this review we discuss the barriers posed by the solid tumor that prevent immune cell trafficking and NK cell effector functions. We then discuss promising strategies to enhance NK cell infiltration into solid tumor sites and activation within the TME. This includes NK cell-intrinsic and -extrinsic mechanisms such as NK cell engineering to resist TME-mediated inhibition and use of tumor-targeted agents such as oncolytic viruses expressing chemoattracting and activating payloads. We then discuss opportunities and challenges for using combination therapies to extend NK cell therapies for the treatment of solid tumors.

Efficacy and Safety of TLR2 Co-Stimulated CAR T Cells in CD19-Positive Lymphoid Malignancies with Central Nervous System Involvement

Background CD19 chimeric antigen receptor (CAR) T cell therapy showed remarkable remission rates in patients with refractory or relapsed (R/R) non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL). However, data on outcomes of CAR T cell therapy for patients with central nervous system (CNS) involvement are limited, as most of them have been excluded from most pivotal CAR-T studies because of concerns about treatment-related neurotoxicity. Here, we show results of a third generation of CD19 CAR T cells for patients with R/R B cell cancers with CNS involvement in a single-center cohort study. Methods A total of 21 patients with R/R B cell malignancies (including ALL and NHL) with CNS involvement were included in this study. Patients derived peripheral lymphocytes were collected through apheresis and lentivirally transduced with a 3 rd generation CAR containing CD28 and TLR2 co-stimulatory domains. The cells were expanded ex vivo and administered in a single infusion (1×10 6 or 2×10 6 CAR T cells per kilogram of body weight) after a conditioning regimen of cyclophosphamide and fludarabine. Results Of the 21 patients with CNS involvement, 11 patients with ALL and 10 with NHL were included in this study, with a data cutoff of July 2023. All 11 patients with ALL had both bone marrow (BM) and cerebrospinal fluid (CSF) involvement. Of the 10 patients with NHL, 5 of the 10 lymphoma patients had parenchyma involvement, 6 patients (6/10) had CSF infiltration and 1 had oculus sinister involvement; the majority of patients (8/10) were diagnosed with secondary CNS lymphoma, whereas 2 patient was primary CNS lymphoma. Of the 21 patients who were treated, the overall response rate (ORR) was 71% (15/21), with 73% (8/11) in ALL and 70% (7/10) in NHL. 67% of patients with ALL (7/11) had a complete remission in both BM and CSF, while 40% of NHL patients (4/10) had a complete response and 30% (3/10) received a partial response. The median overall survival (OS) of ALL was 14.3 months, with a median follow-up of 20.3 months, while the median OS of NHL was not reached with a median follow-up of 14.2 months. Median progression-free survival (PFS) of ALL and NHL were 11.1 months (95% CI, 0.7-29.9 months) and 4.8 months (95% CI, 1.0-15.3 months), respectively (P = 0.84). In the total CNS cohort, cytokine release syndrome (CRS) of any grade was seen in 20 patients (95%), grade 3 in 3 patients (14%). ICANS occurred in 9 patients (42.8%, including 5 ALL and 4 NHL patients), grade 3 in 5 patients (23.8%), and there was no grade 4 event. Median time to ICANS was 5 days (range, 2-10 days) after CAR-T infusion. All the CRS or ICANS were manageable. No significant difference in occurrence of and time to ICANS was seen between ALL and NHL patients (P = 0.22 and 0.96). CAR T cells were identified in CSF of patients following infusion, and the median time to peak CAR T cells in CSF of ALL and NHL were 6.5 days and 10 days post-infusion, respectively (P = 0.141). Median time to last documented follow-up of all patients (alive or dead) was 18.1 months (range 1.0-31.2 months). At the end point of observation, 5 of the 11 ALL patients and 5 of 10 NHL patients were still alive with sustained CR of CNS disease and systemic disease (Figure 1). Conclusion TLR2-costimulated CAR T cell therapy for patients with R/R B cell malignancies with CNS involvement is safe, feasible, and results in high response rates and durable remission in both ALL and NHL patients with CNS disease. The clinical trial was registered at www.clinicaltrials.gov as # NCT04605666.

CD19-CAR Cytokine Induced Killer Cells Armored with IL-18 Control Tumor Burden, Prolong Mouse Survival and Result in In Vivo Persistence of CAR-CIK Cells in a Model of B-Cell Acute Lymphoblastic Leukemia

Rationale: Cytokine induced killer cells (CIK) are a promising cancer immunotherapy. Recently CIK cells modified to express chimeric antigen receptors (CAR) target CD19 expressing hematological malignancies including B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging clinical trial results, insufficient CAR-CIK anti-tumor activity and persistence pose major obstacles towards improved efficacy in vivo. Therefore, we optimized our CAR-CIK platform by introducing two modifications to the CD19-CAR construct. The first modification “armored” CAR-CIK cells by inserting the IL-18 gene in a bicistronic DNA plasmid enabling simultaneous surface expression of the CAR protein and secretion of IL-18, which demonstrated improved CAR-T cell function. The second modification attenuated the CD28 cytoplasmic signaling domain of the CAR molecule to enhance anti-tumor activity and in vivo persistence since recent findings demonstrate that persistent or chronic CAR signaling can impair anti-tumor activity and in vivo persistence. Methods: CARCIK-1918 cells generated from donor-derived CIK cells are engineered to express CAR and IL-18 using the non-viral Sleeping Beauty (SB) transposon system. CIK cells were generated from donor PBMCs by the sequential addition of IFN-ϒ and stimulation with anti-CD3 antibody plus IL-2. After 2 days of stimulation CIK cells were co-electroporated with SB100X transposase mRNA and plasmid DNA containing a bicistronic transgene encoding both the anti-CD19 CAR and IL-18. This second-generation CAR contains a fusion of the CD28 transmembrane domain, intracellular CD28 and, TCR CD3ζ chain. In some instances, the cytoplasmic CD28 signaling domain is modified to attenuate CD28 signaling. This method of gene transfer results in stable surface expression of the CAR on CD3+ cells through a 17-day culture period. Day 17 cells are harvested, frozen and used for all subsequent studies. CARCIK-1918 cells are stimulated in vitro with CD19+ REH tumor cells and the phenotype of activated cells was characterized by multi-color flow cytometry. IL-18 secretion was determined by cytokine bead array analysis of supernatants collected post stimulation. CARCIK-1918 cells with or without the attenuated CD28 signaling domain were tested in vivo in a xenograft model of B-ALL, using NSG mice engrafted with CD19+ Raji tumor cells. Tumor burden was assessed weekly by bioluminescence imaging, the in vivo persistence of CARCIK-1918 cells was measured by flow cytometry, and survival was monitored throughout the study. Results: CARCIK-1918 cells express both IL-18 and CAR with or without attenuated CD28 signaling. Furthermore, stimulation of CARCIK-1918 cells with CD19+ REH tumor cells increases the frequency of CAR-CIK cells with a memory phenotype and concurrent upregulation of the high affinity IL-2 receptor, CD25, and the costimulatory receptor CD137. Expression of IL-18 leads to higher engraftment of CAR-CIK cells in tumor bearing NSG mice compared to unarmored controls. NSG mice engrafted with luciferase positive Raji cells demonstrated decreased bioluminescent signal following CARCIK-1918 treatment, consistent with CARCIK derived anti-leukemic activity. This anti-tumor activity corresponded to a statistically significant improvement in survival as mice treated with 2 nd generation CAR transgene CARCIK cells with or without attenuated CD28 signaling had a median survival of 37.5 (n=10; p=0.0001) and 38 (n=12; p =0.0001 days), respectively, compared to untreated mice that develop progressive highly disseminated tumor and succumb to disease by 3 weeks post tumor inoculation (n=11; median survival 18 days). Interestingly, mice treated with CARCIK-1918 cells with the CD28 attenuated signaling domain exhibited better tumor control and enhanced persistence in vivo, despite no significant improvement in survival compared to WT CD28 signaling CARCIK-1918 cells. Summary: This study demonstrates that administration of CAR-CIK cells armored with IL-18 results in a significant survival advantage for tumor challenged animals versus untreated animals. Furthermore, modifying the cytoplasmic CD28 domain to attenuate signaling, results in better control of tumor burden, and persistence of CARCIK-1918 cells in vivo. Our study is the first report of IL-18 armored CAR-CIK cells which may present a promising, novel strategy for the treatment of B-ALL.