Chimeric Antigen Receptor Expression Research Articles

ATA3431: Allogeneic CD19/CD20 Bispecific CAR EBV T Cells for the Treatment of B-Cell Malignancies

BACKGROUND: Chimeric antigen receptor (CAR) therapy targeted against CD19 has yielded remarkable clinical responses in the treatment of relapsed/refractory B-cell malignancies, but about a third of patients ultimately relapse due to the loss or downregulation of CD19. Furthermore, technical and logistical challenges associated with autologous CAR T therapy limits access for a majority of eligible patients. To help address these challenges, we have developed an allogeneic bispecific tandem CAR directed against both CD19 and CD20 (ATA3431) built on our Epstein-Barr virus (EBV) T-cell platform. EBV T cells maintain expression of endogenous T-cell receptors that have inherently low alloreactivity due to their recognition of defined viral antigens. CAR EBV T cells have demonstrated a favorable safety profile with no associated evidence for graft-versus-host disease or high-grade cytokine release syndrome (Curran KJ, 2020). The bispecific tandem CAR design allows either CD19 or CD20 target antigen recognition to activate the CAR EBV T cell, with the goal of extending durability of response and limiting antigen escape. In clinical trials, autologous bispecific CAR T cells were shown to be efficacious and safe (Larson 2023, Shah 2020). Additionally, a 1XX mutated CD3ζ signaling domain and our optimized CAR EBV T-cell platform preserve a central memory phenotype, robust expansion, functional persistence, and potent antitumor activity of ATA3431. METHODS: We generated CD20/CD19 bispecific CAR EBV T cells containing a modified CD3ζ signaling domain 1XX using our EBV T-cell platform. As a benchmark comparator, we generated autologous CD20/CD19 bispecific CAR T cells using a 12-day process derived from OKT3/CD28-activated T cells. The final products were characterized via immunophenotyping and evaluated for in vitro and in vivo response to antigen expressing targets. RESULTS: CD20/CD19 bispecific CAR EBV T cells generated from multiple healthy donors demonstrated stable CAR expression, a high proportion of T-cell memory populations, and consistent HLA-independent killing of CD19 + and/or CD20 + tumor cells over repeated in vitrochallenges. ATA3431 mediated highly potent antitumor activity in an established disseminated tumor model of Burkitt's lymphoma and correlated with long-term in vivo persistence of CAR T cells without additional cytokine support. Moreover, ATA3431-treated animals showed superior tumor growth inhibition compared to animals treated with autologous CD20/CD19 bispecific benchmark CAR T cells. No treatment-related toxicities were observed in animals treated with ATA3431. CONCLUSIONS: Preclinical evaluations of ATA3431 show B-cell tumor growth inhibition without any treatment-related toxicities and thereby support advancing ATA3431 for clinical evaluation. Curran KJ, Sauter CS, Kernan CS, et al. Durable remission following “off-the-shelf” chimeric antigen receptor (CAR) T-cells in patients with relapse/refractory (R/R) B-cell malignancies. 2020 Transplantation & Cellular Therapy Meetings. Orlando. Larson SM, Walthers CM, Ji B, Ghafouri SN, et al. CD19/CD20 bispecific chimeric antigen receptor (CAR) in naive/memory T cells for the treatment of relapsed or refractory non-Hodgkin lymphoma. Cancer Discov. 2023 Mar 1;13(3):580-597. doi: 10.1158/2159-8290.CD-22-0964. PMID: 36416874; PMCID: PMC9992104. Shah NN, Johnson BD, Schneider D, et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial. Nat Med. 2020 Oct;26(10):1569-1575. doi: 10.1038/s41591-020-1081-3. Epub 2020 Oct 5. PMID: 33020647.

Dual Split-Signaling TIM3+CLEC12a Targeting CAR T-Cells with Optimized Signaling As a Safe Potential Therapy for Acute Myeloid Leukemia

Background: The success of chimeric antigen receptor (CAR) T-cell therapies targeting B cell maturation antigen (BCMA) in multiple myeloma and CD19 in B-cell malignancies has demonstrated the immense potential of this immunotherapeutic strategy. However, the translation of CAR T-cell therapy to acute myeloid leukemia (AML) patients has encountered challenges due to the absence of surface antigens specifically or preferentially expressed on AML cells. To overcome this hurdle, we explored the feasibility and safety of simultaneously targeting the AML-associated antigens CLEC12a and TIM3 using an AND-gate strategy in which the essential stimulatory and co-stimulatory T-cell activation signals are segregated into separate CARs. To improve the safety of our strategy we also attenuated signaling through the stimulatory anti-CLEC12a CAR via affinity-optimization or through targeted mutations in its immunoreceptor tyrosine-based activation motif (ITAM) regions. Methods: To identify the most optimal dual-split CAR T-cell configuration, we designed several chimeric antigen receptor constructs in which we targeted CLEC12a with stimulatory CARs (sCARs) with high (C hi) or low (C low) affinities. In addition, the high affinity C hi stimulatory CAR was mutated in the second and third ITAM regions (denoted as C hixx) to attenuate the CD3ζ stimulatory signal. TIM3 was targeted by a costimulatory CAR (cCAR) containing both CD28 and 41BB signaling domains. The dual-split CARs were generated using two separate retroviral constructs. After the assessment of the CAR expression and specific surface markers associated with memory phenotype and exhaustion, dual-split CAR T-cells were functionally tested using an isogenic cell line model for specific lysis against dual CLEC12a/TIM3 positive or single CLEC12a or TIM3 positive isogenic target cells as well as for dual antigen-specific cytokine secretion. The dual-split CAR T-cells were also tested for their efficiency to specifically eliminate leukemic stem/progenitor cells by the colony formatting unit (CFU) assay. Moreover, anti-AML activity and persistence of the dual-split CAR T-cells was assessed using an AML PDX mouse model in which human primary AML cells expressing TIM3 and CLEC12a were injected. Results: Using the isogenic cell line model, we revealed that the dual-split CAR T-cells with the combination of the high affinity CLEC12a sCAR and TIM3 cCAR (C hi+T) show antigen-specific cytokine secretion and cytotoxicity, and that this specificity was enhanced when the CLEC12a sCAR was attenuated through ITAM mutations (C hixx+T). In CFU assays, all tested CLEC12a/TIM3 dual-split CAR T-cell combinations exhibited potent inhibition (up to 95%) of leukemia blast viability and leukemia stem/progenitor self-renewal. In vivo, although AML did not engraft in all mice, the best-performing dual-split C hixx+T1 CAR T-cells displayed potent anti-leukemic activity, as they prevented engraftment in two additional mice compared to control groups (n=6 each group). Furthermore, in contrast to control groups dual-split CAR T-cells exhibited prolonged persistence in the blood up to 20 weeks after injection. Conclusion: We successfully generated highly specific dual-targeting CAR T-cells by splitting the T-cell activation signals into two simultaneously expressed CARs, and using a novel approach of attenuating the sCAR through mutations in ITAM regions. Overall, we show that dual-split CAR T cells targeting CLEC12a and TIM3, particularly the combination C hixx+T CAR T-cells, have specific and promising anti-AML activity. Our findings underscore the potential of innovative dual-split CAR T-cell therapies as a novel and effective immunotherapeutic approach for combating AML.

Lipid nanoparticles outperform electroporation in mRNA-based CAR T cell engineering

Engineered Tcells expressing chimeric antigen receptors (CARs) have been proven as efficacious therapies against selected hematological malignancies. However, the approved CAR Tcell therapeutics strictly rely on viral transduction, a time- and cost-intensive procedure with possible safety issues. Therefore, the direct transfer of invitro transcribed CAR-mRNA into Tcells is pursued as a promising strategy for CAR Tcell engineering. Electroporation (EP) is currently used as mRNA delivery method for the generation of CAR Tcells in clinical trials but achieving only poor anti-tumor responses. Here, lipid nanoparticles (LNPs) were examined for exvivo CAR-mRNA delivery and compared with EP. LNP-CAR Tcells showed a significantly prolonged efficacy invitro in comparison with EP-CAR Tcells as a result of extended CAR-mRNA persistence and CAR expression, attributed to a different delivery mechanism with less cytotoxicity and slower CAR Tcell proliferation. Moreover, CAR expression and invitro functionality of mRNA-LNP-derived CAR Tcells were comparable to stably transduced CAR Tcells but were less exhausted. These results show that LNPs outperform EP and underline the great potential of mRNA-LNP delivery for exvivo CAR Tcell modification as next-generation transient approach for clinical studies.

Enhanced antitumor efficacy, proliferative capacity, and alleviation of T cell exhaustion by fifth-generation chimeric antigen receptor T cells targeting B cell maturation antigen in multiple myeloma

Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) has been approved for treating multiple myeloma (MM). Some clinical studies reported suboptimal outcomes, including reduced cytotoxicity of CAR-T cells and tumor evasion through increased expression of programmed death-ligand 1 (PD-L1). To enhance CAR-T cell efficiency and overcome PD-L1-mediated T cell suppression, we developed anti-BCMA-CAR5-T cells equipped with three costimulatory domains and the ability to secrete anti-PD-L1 single-chain variable fragment (scFv) blockade molecules. Anti-BCMA-CAR4-T cells contained a fully human anti-BCMA scFv and three intracellular domains (CD28, 4–1BB, and CD27) joined with CD3ζ. Anti-BCMA-CAR5-T cells were generated by fusing anti-BCMA-CAR4 with anti-PD-L1 scFv. Both anti-BCMA-CAR4-T and anti-BCMA-CAR5-T cells demonstrated comparable antitumor activity against parental MM cells. However, at an effector-to-target ratio of 1:2, only anti-BCMA-CAR5-T cells maintained cytolytic activity against PD-L1 high MM cells, unlike anti-BCMA-CAR4 T cells. Anti-BCMA-CAR5-T cells were specifically activated by BCMA-expressing target cells, resulting in increased CAR-T cell proliferation, release of cytolytic mediators, and pro-inflammatory cytokines. Anti-BCMA-CAR5-T cells demonstrated specific cytotoxicity against BCMA-expressing target cells, leading to decreased target cell numbers, increased CAR-T cell numbers, and preserved CAR expression during antigenic re-stimulation. Interestingly, only anti-BCMA-CAR5-T cells showed reduced PD-1 receptor levels, which correlated with decreased PD-L1 expression on target cells. We successfully generated anti-BCMA-CAR5-T cells capable of secreting anti-PD-L1 scFv. These cells exhibited superior antitumor efficiency, proliferative capacity, and alleviated T-cell exhaustion against MM cells. Further investigation into the antitumor efficacy of anti-BCMA-CAR5-T cells is warranted in ex vivo and clinical research settings.

Unlocking the potential of Tregs: innovations in CAR technology.

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR. Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells. LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro. Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor T Cells: The Intersection of Stem Cells and Immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy is a promising cell-based immunotherapy applicable to various cancers. High cost of production, immune rejection, heterogeneity of cell product, limited cell source, limited expandability, and relatively long production time have created the need to achieve a universal allogeneic CAR-T cell product for "off-the-shelf" application. Since the innovation of induced pluripotent stem cells (iPSCs) by Yamanaka et al., extensive efforts have been made to prepare an unlimited cell source for regenerative medicine, that is, immunotherapy. In the autologous grafting approach, iPSCs prepare the desired cell source for generating autologous CAR-T cells through more accessible and available sources. In addition, generating iPSC-derived CAR-T cells is a promising approach to achieving a suitable source for producing an allogeneic CAR-T cell product. In brief, the first step is reprogramming somatic cells (accessible from peripheral blood, skin, etc.) to iPSCs. In the next step, CAR expression and T cell lineage differentiation should be applied in different arrangements. In addition, in an allogeneic manner, human leukocyte antigen/T cell receptor (TCR) deficiency should be applied in iPSC colonies. The allogeneic iPSC-derived CAR-T cell experiments showed that simultaneous performance of HLA/TCR deficiency, CAR expression, and T cell lineage differentiation could bring the production to the highest efficacy in generating allogeneic iPSC-derived CAR-T cells.

Deletion of the inhibitory co-receptor CTLA-4 enhances and invigorates chimeric antigen receptor T cells

Chimeric antigen receptor (CAR) Tcell therapy targeting CD19 has achieved tremendous success treating Bcell malignancies; however, some patients fail to respond due to poor autologous Tcell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR Tcell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR Tcells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the Tcell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of Tcells from patients with leukemia that previously failed CAR Tcell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient Tcells, providing a strategy for increasing patient responses to CAR Tcell therapy.

NK Cell Therapeutics for Hematologic Malignancies: from Potential to Fruition.

The current review focuses on the preclinical development and clinical advances of natural killer (NK) cell therapeutics for hematologic malignancies and offers perspective on the unmet challenges that will direct future discovery in the field. Approaches to improve or re-direct NK cell anti-tumor functions against hematologic malignancies have included transgenic expression of chimeric antigen receptors (CARs), administration of NK cell engagers including BiKEs and TriKEs that enhance antibody-dependent cellular cytotoxicity (ADCC) by co-engaging NK cell CD16 and antigens on tumors, incorporation of a non-cleavable CD16 that results in enhanced ADCC, use of induced memory-like NK cells alone or in combination with CARs, and blockade of NK immune checkpoints to enhance NK cytotoxicity. Recently reported and ongoing clinical trials support the feasibility and safety of these approaches. NK cell-based therapeutic strategies hold great promise as cost-effective, off-the-shelf cell therapies for patients with relapsed and refractory hematologic diseases.

Targeted Delivery of Chimeric Antigen Receptor into T Cells via CRISPR-Mediated Homology-Directed Repair with a Dual-AAV6 Transduction System.

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.

Anti-Cancer Potential of Transiently Transfected HER2-Specific Human Mixed CAR-T and NK Cell Populations in Experimental Models: Initial Studies on Fucosylated Chondroitin Sulfate Usage for Safer Treatment.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte "on-target off-tumor" reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect.

Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering.

The programmed cell death protein 1 (PD-1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD-1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD-1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)-mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co-encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co-delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD-1 to primary human T cells ex vivo and strong CAR expression and PD-1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancerimmunotherapies.

FOXO1 Inhibition Generates Potent Nonactivated CAR T Cells against Solid Tumors.

Chimeric antigen receptor (CAR) T cells have shown promising results in the treatment of B-cell malignancies. Despite the successes, challenges remain. One of them directly involves the CAR T-cell manufacturing process and especially the ex vivo activation phase. While this is required to allow infection and expansion, ex vivo activation dampens the antitumor potential of CAR T cells. Optimizing the nature of the T cells harboring the CAR is a strategy to address this obstacle and has the potential to improve CAR T-cell therapy, including for solid tumors. Here, we describe a protocol to create CAR T cells without ex vivo preactivation by inhibiting the transcription factor FOXO1 (CAR TAS cells). This approach made T cells directly permissive to lentiviral infection, allowing CAR expression, with enhanced antitumor functions. FOXO1 inhibition in primary T cells (TAS cells) correlated with acquisition of a stem cell memory phenotype, high levels of granzyme B, and increased production of TNFα. TAS cells displayed enhanced proliferative and cytotoxic capacities as well as improved migratory properties. In vivo experiments showed that CAR TAS cells were more efficient at controlling solid tumor growth than classical CAR T cells. The production of CAR TAS from patients' cells confirmed the feasibility of the protocol in clinic.

CAR-NK Cells Generated with mRNA-LNPs Kill Tumor Target Cells In Vitro and In Vivo.

Natural killer (NK) cells are cytotoxic lymphocytes that are critical for the innate immune system. Engineering NK cells with chimeric antigen receptors (CARs) allows CAR-NK cells to target tumor antigens more effectively. In this report, we present novel CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect NK cells expanded from primary PBMCs and to generate functional CAR-NK cells. CD19-CAR mRNA and BCMA-CAR mRNA were embedded into LNPs that resulted in 78% and 95% CAR expression in NK cells, respectively. BCMA-CAR-NK cells after transfection with CAR mRNA-LNPs killed multiple myeloma RPMI8226 and MM1S cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner in vitro. In addition, CD19-CAR-NK cells generated with CAR mRNA-LNPs killed Daudi and Nalm-6 cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner. Both BCMA-CAR-NK and CD19-CAR-NK cells showed significantly higher cytotoxicity, IFN-gamma, and Granzyme B secretion compared with normal NK cells. Moreover, CD19-CAR-NK cells significantly blocked Nalm-6 tumor growth in vivo. Thus, non-viral delivery of CAR mRNA-LNPs can be used to generate functional CAR-NK cells with high anti-tumor activity.

Nonviral technologies can pave the way for CAR-NK cell therapy.

Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf" product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor-natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality.

A phase 1, first-in-human (FIH) study of autologous macrophages engineered to express an anti-HER2 chimeric antigen receptor (CAR) in participants (pts) with HER2-overexpressing solid tumors.

TPS2666 Background: Macrophages are abundant in the solid tumor microenvironment (sTME) and can promote tumor growth (M2) or enhance anti-tumor immunity (M1). CAR expression can redirect macrophage function to selectively target and phagocytose antigen overexpressing cancer cells. CAR-M can reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and anti-tumor immunity. CT-0508 is comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies showed that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden, prolonged survival, and was safe. Notably, anti-HER2 CAR-M treatment led to activation of the sTME, with infiltration of CD8+ and CD4+ T cells, NK cells, dendritic cells, and increased activated CD8+ tumor infiltrating lymphocytes. In a pre-clinical anti-PD1 resistant solid tumor model, mice that received anti-HER2 CAR-M and anti-PD1 demonstrated improved tumor control, overall survival, and TME activation compared to single treatment alone, indicating synergy and capacity for CAR-M to sensitize solid tumors to checkpoint blockade. Methods: This Phase 1, First in Human study evaluates the safety, tolerability, cell manufacturing feasibility, trafficking, TME activation, and preliminary evidence of efficacy of investigational product CT-0508 in 18 participants (pts) with locally advanced (unresectable) /metastatic solid tumors overexpressing HER2. Pts previously treated with anti-HER2 therapies are eligible. Filgrastim mobilized autologous CD14+ monocytes are collected by apheresis, followed by manufacturing and cryopreservation. Group 1 pts (n = 9; enrollment complete) received fractionated doses of CT-0508 over Days 1, 3, and 5. Group 2 pts (n = 9) receive CT-0508 as a single infusion on D1. Additional cohorts include: CT-0508 co-administered with pembrolizumab and CT-0508 monotherapy administered intraperitoneally in pts with peritoneal predominant disease. Correlative assessments include pre- and post-treatment biopsies and blood samples for safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial information: NCT04660929 .

Targeting CD47 in lymphoid malignancies with CAR T cells.

2513 Background: Despite major therapeutic advances of chimeric antigen receptor (CAR) T cells directed against CD19 in relapsed and refractory non-Hodgkin lymphoma (NHL), two thirds of patients experience relapse within the first two years. Nearly one third of these relapses are associated with CD19 antigen-loss escape thus leveraging alternate CAR targets may improve outcomes. CD47, an anti-phagocytic “don’t eat me” receptor known to be overexpressed in NHL, is an ideal CAR target as it is potentially indispensable for tumor progression. Methods: CD47-CAR binding domains were engineered either as a camelid single variable heavy chain nanobody (CD47-VHH-CAR) or as the CD47 binding domain of cognate receptor SIRPα (SIRPα-CAR), each combined with a CD28 costimulatory domain and a CD3ζ activation domain with two mutated ITAMs (1XX). In vitro co-culture assays were performed with lymphoid mouse tumor cell lines BM185 and A20. For in vivo experiments, syngeneic BALB/c mice were sublethally irradiated for lymphodepletion then injected intravenously with 1x105 luciferase+ BM185 cells. Two days later, mice were injected with 2x106 CAR T cells, and bioluminescence imaging was performed to monitor tumor progression. For bio-localization experiments, CAR T cells were co-transduced with a separate vector encoding luciferase. Results: CD47-CAR T cell expansion in vitro was hindered due to fratricide, which can be rescued by CD47 CRISPR knockout. Both CD47-VHH-CAR and SIRPa-CAR T cells exhibited potent and specific in vitro lysis of BM185 and A20 cells. CD47-VHH-CAR T cells showed transient anti-leukemia activity, however, survival was not significantly prolonged. T cell bio-localization analysis revealed that, compared to CD19-CAR T cells, CD47-VHH-CAR T cells exhibited prolonged retention in the thorax with minimal bone marrow trafficking, suggesting CD47 antigen-specific pulmonary sequestration, and inferior expansion. SIRPα-CAR T cells engineered to secrete pro-inflammatory cytokine IL-18 significantly improved CAR T cell expansion, resulting in bone marrow trafficking and clearance of BM185 tumor. However, recipients of IL-18-secreting SIRPa-CAR T cells experienced lethal CAR T cell-related toxicity. To improve the safety of CD47-CAR T cells, we engineered an NFAT-responsive vector expressing the CD47-CAR modified with E. coli-derived dihydrofolate reductase drug destabilization domain to limit CD47-CAR expression to T cells exposed to the stabilizing antibiotic trimethoprim and receiving a licensing NFAT signal from a CD19-CAR. Conclusions: In vivo activity of CD47-targeting CAR T cells is limited by thoracic retention and limited expansion, which can be partially overcome by overexpression of IL-18, though enhanced toxicity precludes survival prolongation. Our findings suggest that strategies to spatiotemporally restrict CAR expression or activation will be necessary to achieve safe and efficacious CD47-CAR T cell activity.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of β2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

Chimeric antigen receptor (CAR) modified T cells can induce complete remissions in patients with advanced hematological malignancies. Nevertheless, the efficacy is mostly transient and remains so far poor in the treatment of solid tumors. Crucial barriers to long-term CAR T cell success encompass loss of functional capacities known as "exhaustion", among others. To extend CAR T cell functionality, we reduced interferon regulatory factor 4 (IRF4) levels in CAR T cells using a one-vector system encoding a specific short-hairpin (sh) RNA along with constitutive CAR expression. At baseline, CAR T cells with downregulated IRF4 showed equal cytotoxicity and cytokine release compared to conventional CAR T cells. However, under conditions of repetitive antigen encounter, IRF4low CAR T cells displayed enhanced functionality with superior cancer cell control in the long-term compared with conventional CAR T cells. Mechanistically, the downregulation of IRF4 in CAR T cells resulted in prolonged functional capacities and upregulation of CD27. Moreover, IRF4low CAR T cells were more sensitive to cancer cells with low levels of target antigen. Overall, IRF4 downregulation capacitates CAR T cells to recognize and respond to target cells with improved sensitivity and endurance.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

Adoptive cell therapy (ACT) utilizing γδ Tcells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific Tcell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ Tcells with CAR or secreted bispecific Tcell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ Tcells. Using a CD19-specific CAR, approximately 60% of γδ Tcells are modified after mRNA electroporation and these cells show potent anticancer activity invitro and invivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ Tcell cytotoxicity, both invitro and invivo, and promotes killing of target cells by modified and unmodified γδ Tcells. Taken together, we show that transient transfection of γδ Tcells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.