Specific Chimeric Antigen Receptor Research Articles

Abstract 2907: Development of a CAR-NK cellular platform for cancer immunotherapy

Abstract Although Chimeric Antigen Receptor (CAR)-T cell therapy has proven to be successful in treating multiple hematological malignancies, their use is limited due to the potential life-threatening adverse events, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). CAR natural killer (CAR-NK) cells are rapidly emerging as a promising alternative cellular therapeutic with improved efficacy and reduced adverse effects. Moreover, due to the intrinsic characteristics of NK cells, whereby they lack antigen-specific cell surface receptors, CAR-NK cells offer the additional advantage of being an “off-the-shelf” product, thus satisfying the requirement for large-scale production for cancer immunotherapy, giving it the potential to reach more patients at a reduced cost. Here we outline the method to generate, expand and enrich human primary CD19 CAR-NK cells, and demonstrate their efficacy in-vitro against CD19-expressing target cancer cells. Human primary NK cells were isolated from healthy donors and transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV, as an empty vector transduced control. Transduction efficiency greater than 10% or 20% was achieved following transduction and expansion over a 7-day period in response to CD19 CAR LV or GFP LV respectively. Following sufficient expansion, cytotoxicity of the CD19 CAR-NK cells was tested in-vitro in a Tumor Killing Assay (TKA) against CD19+ (Raji) or CD19- (SKOV-3) target cells. As expected, CD19 CAR-NK cells displayed increased cytotoxicity towards CD19+ target cells when compared to non-transduced or GFP transduced controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting specific CD19 CAR mediated killing. Moreover, CD19 CAR-NK cells showed increased degranulation and IFNꝩ secretion when compared to both non-transduced and GFP-transduced controls, suggesting increased NK cell activation and cytotoxicity. Additionally, when degranulation of CD19 CAR positive and CD19 CAR negative NK populations was directly compared, the level of NK cell degranulation was analogous between the two populations, suggesting that CD19 CAR NK cells can enhance the cytotoxic activity of neighboring non-transduced cells, resulting in enhanced killing of CD19+ target cells by the total NK population present. Taken together these data demonstrate the generation, expansion and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This culture system provides the scientific community a platform to screen novel CAR-constructs and to test novel immunomodulators which could enhance NK cell expansion or functional potential to further develop new, more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Ravindra Mode, Kaitlin Mitchell, David Cobeta-Lopez, Sabrina de Munnik, Rene McLaughlin, Gemma Moiset, Louise Brackenbury, Lauren Schewitz-Bowers, Robert Nunan. Development of a CAR-NK cellular platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2907.

Abstract 1780: TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements

Abstract The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1780.

Abstract 909: Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission

Abstract Despite high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), approximately half of the patients relapse within the first year, representing an urgent question to address in the next stage of cellular immunotherapy. To interrogate the molecular determinants of ultra-long CAR T persistence, we sequenced one million pre-infusion CAR T cells from 82 pediatric ALL patients and 6 healthy donors and obtained high quality multi-omics data from ~700k single cells at the basal level or upon antigen specific stimulation. We identified that elevated type-2 functionality in both CD4+ and CD8+ subsets was associated with five patients maintaining a median B-cell aplasia duration of 8.4 years (BCA-L group), which was independently validated using both intracellular flow cytometry and multiplexed secretomic assay. Further network analysis revealed several pathways promoting the activation of the Th2 master regulator GATA3 in BCA-L cells. Through ligand-receptor interaction analysis, type-2 cytokines were found to regulate a specific cluster of Tim-3+ terminal effector cells showing overactivation of cytotoxicity, impaired immune function, elevated exhaustion signature and diminished proliferative potentiality, and CAR T cells from BCA-L patients showed reduced dysfunctional hallmarks. In addition, activated BCA-L cells maintained a significantly higher level of oxidative phosphorylation metabolism that is likely associated with their observed central memory phenotype, whereas CAR T cells from patients who developed primary resistance or relapse exhibited imbalanced functional and metabolic programs. Specifically, CAR T products from initial non-responders showed hyperactive glycolysis metabolism, and the CD19-negative relapsed patients (RL- group) had significantly attenuated gene module expression of a broad spectrum of metabolic categories, including major energy-producing metabolisms. By direct comparison with BCA-L cells, our analysis identified a strikingly impaired mTOR signaling in RL- CAR T cells, along with reduced proliferation activities and a systematic deficiency of pathways orchestrating immune response. Although the mechanism is still unclear, our hypothesis is that the impaired mTOR signaling could only endow blunted functional and metabolic response in RL- cells, thereby conferring more opportunities to leukemic cells to escape the attack. In summary, our large-scale single-cell multi-omics dataset provides valuable insights for dissecting mechanistic basis of CAR T cell longevity in mediating 8-year leukemia remission, highlighting several therapeutic strategies to edit specific CAR T functionality to ameliorate clinical outcome, particularly boosting the type-2 immunity to mitigate dysfunction thereby extending persistence. Citation Format: Zhiliang Bai, J. Joseph Melenhorst, Stephan A. Grupp, Rong Fan. Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 909.

Abstract 906: Development of a repeated challenge potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract The development of immunotherapies relies on the use of in vitro potency assays - which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells - to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, each representing an important aspect driving clinical efficacy of the cellular product. Importantly, adoptive cell therapies must exhibit potent tumor killing over a prolonged period in vivo. Here, we describe an in vitro potency assay that quantifies immune cell-mediated cytotoxicity in response to repeated challenges with target cells. The repeated challenge assay was performed with two separate protocols, each providing a unique assessment of the effector cell function. In the first method, SKOV3 target cells (5k cells/well) were seeded into a multiwell microplate with embedded electrodes in the substrate. Electrical impedance measurements from the electrodes in each well detected the attachment and proliferation of the target cells. HER-2 specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells at a 1:1 effector to target ratio. Cytolysis of the target cells was calculated by comparing treated wells to no treatment control wells. Additional SKOV3 target cells (5k per well) were added every 24 hours for the repeated challenge conditions, with total tumor cell killing computed from the area under the impedance curve. The repeated challenge condition reached complete killing after the 6th tumor cell challenge (144 hours after first effector addition), whereas the single challenge condition reached complete killing within 96 hours of effector addition. Flow cytometry analysis revealed that the effector cells proliferated significantly more for the repeated challenge condition (4.7 fold increase) than the single challenge condition (1.5 fold increase). In the second method, the CAR T cells were removed after each challenge, counted, and added to a fresh plate of SKOV3 target cells at a 1:1 effector to target cell ratio. Here, the E:T ratio was kept constant for each challenge, allowing a direct comparison of the killing kinetics (KT50) for each subsequent challenge. The KT50 decreased from the first challenge (19.9 hr) to the fourth challenge (83.5 hr). Flow cytometry analysis revealed a reduction in CD8+ cells for the repeated challenge condition, indicating a change in the make-up of the immune cell population. The paradigms described above facilitate a label-free, in vitro workflow to assess potency of immune effector cells across repeated challenges, with the opportunity to incorporate additional assays, like flow cytometry or cytokine release, after each successive challenge. Citation Format: Denise Sullivan, Stacie Chvatal, Daniel Millard. Development of a repeated challenge potency assay for evaluation of immune cell-mediated cytotoxicity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 906.

Abstract 3199: Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells

Abstract The measurement of chimeric antigen receptor (CAR) expression is critical for the development, qualification and quantitation of CAR-T cells for clinical applications. There is a comprehensive range of tools commercially available for specifically measuring expression of the CAR on CD19-targeting CAR-T cells. However, with the rapidly expanding repertoire of CAR-T cells in development targeting a diverse range of tumour antigens, there is a paucity of readily implementable assays that specifically measure CAR expression, which is essential for translation into the clinic. We have developed several CARs targeting the cancer stem cell marker LGR5, one of which (CNA3103) is the lead candidate in an upcoming clinical trial in advanced metastatic colorectal cancer. Previously, the level of CNA3103 expression in this system has been determined indirectly by measuring expression of tEGFR on the CAR-T cell surface, taking advantage of co-expression of the CAR and tEGFR upon transduction with our lentiviral construct. tEGFR expression was found over many CAR-T cell manufacturing batches to report transduction efficiency reproducibly with high sensitivity. However, to specifically quantify expression of the CNA3103, an assay utilising an Fc-tagged recombinant human LGR5 protein, in combination with an anti-human IgG1 Fc-specific secondary antibody was developed. A number of optimisation steps including titration of multiple batches of rhLGR5-Fc, different anti-human IgG1 Fc-specific antibodies and a range of incubation parameters were tested. This resulted in development of an assay that yielded very similar reproducibility and sensitivity to that previously observed with the indirect tEGFR surrogate assay. Overall, the findings highlight the utility of using CAR-targeting recombinant proteins for evaluating CAR expression on CAR-T cells, providing a general flow cytometry-based staining strategy that may be adapted for assessing a diverse repertoire of CARs. Citation Format: Timona Tyllis, Caitlin Abbott, Dylan McPeake, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Emma Thompson, Lih Tan, Allison Cowin, Claudine Bonder, Timothy Sadlon, Simon Barry, Shaun McColl. Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3199.

A Case Study of Chimeric Antigen Receptor T Cell Function: Donor Therapeutic Differences in Activity and Modulation with Verteporfin.

Chimeric antigen receptor (CAR) T cells have recently been demonstrated to extract and express cognate tumor antigens through trogocytosis. This process may contribute to tumor antigen escape, T cell exhaustion, and fratricide, which plays a central role in CAR dysfunction. We sought to evaluate the importance of this effect in epidermal growth factor receptor variant III (EGFRvIII) specific CAR T cells targeting glioma. EGFRvIII-specific CAR T cells were generated from various donors and analyzed for cytotoxicity, trogocytosis, and in vivo therapeutic activity against intracranial glioma. Tumor autophagy resulting from CAR T cell activity was evaluated in combination with an autophagy inducer (verteporfin) or inhibitor (bafilomycin A1). CAR T cell products derived from different donors induced markedly divergent levels of trogocytosis of tumor antigen as well as PD-L1 upon engaging target tumor cells correlating with variability in efficacy in mice. Pharmacological facilitation of CAR induced-autophagy with verteporfin inhibits trogocytic expression of tumor antigen on CARs and increases CAR persistence and efficacy in mice. These data propose CAR-induced autophagy as a mechanism counteracting CAR-induced trogocytosis and provide a new strategy to innovate high-performance CARs through pharmacological facilitation of T cell-induced tumor death.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR Tcell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

SSTR2 as an anatomical imaging marker and a safety switch to monitor and manage CAR T cell toxicity

The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells.

Establishing Immunocompetent Leukemia Models to Investigate the Impact of CAR T Cells on the Immune Microenvironment and Bone Marrow Niche

Background Chimeric antigen receptor (CAR) T cells need to overcome limited expansion and persistence within the highly immunosuppressive tumor microenvironment (TME) to successfully eliminate acute myeloid leukemia (AML) in the clinic. CD123 has been identified as a promising target for AML because of its high expression on leukemia stem cells and AML blasts, with a clear increased differential expression when compared to healthy hematopoietic progenitors (HPCs). To date, preclinical evaluation of CD123-CAR T cells have used human xenograft models, which lack a functional immune system. This cross-species evaluation of CAR T cells inadequately addresses interactions between CAR T cells and the immune microenvironment, a limitation of many CAR T cell therapy studies. We have generated immunocompetent murine leukemia models to evaluate murine CD123 (mCD123)-specific CAR T cells and thus investigate the impact of the AML TME on CAR T cell dysfunction. Methods and Results We used biopanning of a murine phage display library on recombinant mCD123 to select a panel of mCD123-specific single chain variable fragments (scFvs). 4 scFvs, encoding 'top binders', were subcloned into a MSGV1 retroviral vector encoding a CD28.z-CAR to generate 4 mCD123-CARs. Second-generation mCD123-CAR T cells were generated by retroviral transduction of activated splenocytes. Only one mCD123-CAR construct was expressed on the cell surface as judged by flow cytometric analysis using F(ab')2 (55.8±11.5% N=7) or recombinant mCD123 (59.0±12.1% N=6) for detection, and T cells expressing this CAR were selected for further analysis. To test the antigen specificity of mCD123-CAR T cells, we expressed full-length mCD123 on 2 murine leukemia cell lines: C1498, a spontaneous myelomonocytic AML cell line, and a Arf-null, BCR-ABL1 preB-ALL. In addition, we analyzed a panel of virally induced NUP98::KDM5A AML generated by retroviral transduction of murine HPCs and determined all samples naturally expressed mCD123 antigen at different densities (range 38-77% N=4). We co-cultured mCD123-CAR in the presence of mCD123+ leukemia cell lines, with mB7-H3-CAR, SP6-CAR, or non-transduced T cells serving as controls. mCD123-CAR T cells recognized CD123+ targets, as evidenced by significantly increased cytokine secretion and cytolytic activity in comparison to controls (N=6, p<0.01). Antitumor activity was maintained in repeat stimulation assays for up to 5 stimulations. To determine in vivo toxicity of mCD123-CAR T cells, non-tumor bearing C57BL/6 mice were given 200 mg/kg cyclophosphamide (Cy) and one effector T cell infusion generated from transgenic luciferase mice (N=5) and monitored by bioluminescence imaging for 10 days. Bone marrow and spleen were harvested 10 days after injection for flow cytometry evaluation and colony formation unit (CFU) readouts. We detected no significant differences in toxicities between mCD123-CAR and control T cells. We next evaluated the anti-leukemia activity of mCD123-CAR T cells. C57BL/6 mice were injected with preB-ALLArf-/CD123+ cells on day 0 and received 100 mg/kg of Cy on day 2. On day 4, they were injected with 10x106 mCD123-CAR T cells. Mice receiving tumor only, tumor + Cy, or SP6-CAR served as controls. mCD123-CAR T cells had significant anti-leukemia activity resulting in a prolonged survival advantage in comparison to controls (N=5 per group, p<0.001). We focused on delineating the TME in our established models and preliminary analysis of comprehensive flow cytometry and single cell transcriptomic data on bone marrow. Our findings revealed that the TME of our established immune competent leukemia CAR T cell therapy models recapitulate human disease, having an increase in dying tumor cells within the bone marrow of mice treated with mCD123-CAR, with a number of other populations (e.g. dendritic cells, regulatory T cells) approximating the tumor-only microenvironment profile. Conclusions We have developed functional mCD123-CAR T cells targeting AML and ALL and have established immunocompetent models to test them. mCD123-CAR T cells demonstrated potent anti-leukemic activity in vitro and have anti-leukemia activity with minimal to no toxicity in vivo. These findings will provide insight into the impact of the AML TME on CAR T cell functionality, and inform future strategies to improve CAR T cells in the immunosuppressive TME.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety.

This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.

496 Study of Affinity Tuning in AML-Targeted CAR-NK Cells

CD123 is overexpressed on AML blasts compared to healthy myeloid progenitor cells. There is opportunity to design anti-CD123 therapy to discriminate on antigen density. Our goal is to study affinity modification of the single chain variable fragment (scFv) in the context of chimeric antigen receptor (CAR)-modified NK cell activation and target killing. We hypothesize that lower affinity CAR-NK cells will demonstrate improved discrimination between tumor and healthy tissue. We designed NK-cell specific CARs to contain anti-CD123 scFvs binding distinct target epitopes (26292 or 7G3) fused to intracellular portions of NK-activating receptors 4-1BB.CD3Z and 2B4.CD3Z. We synthesized novel 26292 and known 7G3 variants to capture a spectrum of CD123 binding affinities (26292: Kd=15nM; variant Kd range 0.29-160nM; 7G3: Kd=8.75nM; variant Kd range 23.9-64.9nM). These scFv were generated using site-directed mutagenesis of the wild type sequences and subcloned into CAR backbones. Retroviral transduction of primary human NK cells was performed, and CAR expression was measured using FACS (range 23.9%-89.6% CAR+ NK cells). Raji target cells (CD123neg) were engineered and sorted to obtain model cell lines with stable low (Raji.Low) and high (Raji. High) CD123 expression, comparable to healthy cells (CD123.Low) and AML blasts (CD123.High). We tested CAR-NK cell activation and antigen-specific cytotoxicity using in vitro co-culture assays. We established a mouse model of disease using implantation of Raji cells engineered to express firefly Luciferase (Raji.ffLuc). Antitumor cytotoxicity of 26292 and 7G3-based CAR-NK cells with a range of binding affinities showed CAR-specific activation but did not differentiate on target antigen density. Similarly, no differences were seen in CAR-NK activation as measured by TNFa and IFNy secretion following coculture with CD123-positive cells. NSG mice implanted with Raji.ffLuc had median survival of 42 days without treatment. There are no significant differences across a broad range of variable affinity 26292 and 7G3-based anti-CD123 CAR-NK cells in effector cell activation or target killing when assayed short-term. These data support the value of additional long-term investigation of the immune synapse and CAR-NK antitumor activity in vivo. This work is supported by the St. Baldrick's Foundation Fellowship Award and the Johns-Hopkins-Allegheny Health Network Cancer Research Fund.

CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models.

Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.

Chondroitin sulfate proteoglycan 4 expression in chondrosarcoma: A potential target for antibody-based immunotherapy.

Chondrosarcoma is a common primary bone malignancy whose phenotype increases with its histologic grade. They are relatively resistant to chemotherapy and radiation therapy limiting curative options for disseminated disease. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface proteoglycan that is highly expressed across various human cancers, including chondrosarcoma, and has restricted distribution in healthy tissues, making it an attractive target for the antibody-based therapy. CSPG4 specific chimeric antigen receptor (CAR) T cell therapies have been shown to be effective in treating other cancers such as melanoma and triple negative breast cancer. The goal of this study was to assess the prevalence of CSPG4 in human chondrosarcoma and to assess the efficacy of CSPG4 specific CAR T cells in lysing chondrosarcoma cells in vitro. Using immunohistochemistry (IHC), we stained a tissue microarray containing primary conventional and dedifferentiated chondrosarcoma from 76 patients with CSPG4 specific monoclonal antibodies (mAbs). In addition, we incubated 2 chondrosarcoma cell lines with CSPG4-targeting CAR T cells and subsequently evaluated cell survival. Our results showed medium to high expression of CSPG4 in 29 of 41 (71%) conventional chondrosarcoma tumors and in 3 of 20 (15%) dedifferentiated chondrosarcoma tumors. CSPG4 expression showed a positive association with time to metastasis and survival in both subtypes. CSPG4 CAR T treated cell lines showed a lysis of respectively >80% and 70% demonstrating CSPG4-targeted CAR T cells effective in killing CSPG4-positive chondrosarcoma tumors.

Intracavity generation of glioma stem cell-specific CAR macrophages primes locoregional immunity for postoperative glioblastoma therapy.

Glioblastoma multiforme (GBM) remains incurable despite aggressive implementation of multimodal treatments after surgical debulking. Almost all patients with GBM relapse within a narrow margin around the initial resected lesion due to postsurgery residual glioma stem cells (GSCs). Tracking and eradicating postsurgery residual GSCs is critical for preventing postoperative relapse of this devastating disease, yet effective strategies remain elusive. Here, we report a cavity-injectable nanoporter-hydrogel superstructure that creates GSC-specific chimeric antigen receptor (CAR) macrophages/microglia (MΦs) surrounding the cavity to prevent GBM relapse. Specifically, we demonstrate that the CAR gene-laden nanoporter in the hydrogel can introduce GSC-targeted CAR genes into MΦ nuclei after intracavity delivery to generate CAR-MΦs in mouse models of GBM. These CAR-MΦs were able to seek and engulf GSCs and clear residual GSCs by stimulating an adaptive antitumor immune response in the tumor microenvironment and prevented postoperative glioma relapse by inducing long-term antitumor immunity in mice. In an orthotopic patient-derived glioblastoma humanized mouse model, the combined treatment with nanoporter-hydrogel superstructure and CD47 antibody increased the frequency of positive immune responding cells and suppressed the negative immune regulating cells, conferring a robust tumoricidal immunity surrounding the postsurgical cavity and inhibiting postoperative glioblastoma relapse. Therefore, our work establishes a locoregional treatment strategy for priming cancer stem cell-specific tumoricidal immunity with broad application in patients suffering from recurrent malignancies.

Chimeric antigen receptor T-cell therapy in adults: management of toxicities and implications for critical care

Chimeric antigen receptor (CAR) T-cell therapy is potentially life-changing in patients with haematological malignancies refractory to multiple lines of chemoimmunotherapy and stem cell transplantation. This adoptive cell immunotherapy involves harvesting and engineering patients' own T lymphocytes to express specific antitumour CARs, thereby directing them to target tumour cells. The CAR also has an intracellular, co-stimulatory domain causing in vivo CAR T-cell expansion and persistence. Chimeric antigen receptor T-cells destroy cancer cells but may also induce life-threatening, reversible toxicities that may require support from the critical care team.

Chimeric Antigen Receptor (CAR) Regulatory T-Cells in Solid Organ Transplantation.

Solid organ transplantation is the treatment of choice for various end-stage diseases, but requires the continuous need for immunosuppression to prevent allograft rejection. This comes with serious side effects including increased infection rates and development of malignancies. Thus, there is a clinical need to promote transplantation tolerance to prevent organ rejection with minimal or no immunosuppressive treatment. Polyclonal regulatory T-cells (Tregs) are a potential tool to induce transplantation tolerance, but lack specificity and therefore require administration of high doses. Redirecting Tregs towards mismatched donor HLA molecules by modifying these cells with chimeric antigen receptors (CAR) would render Tregs far more effective at preventing allograft rejection. Several studies on HLA-A2 specific CAR Tregs have demonstrated that these cells are highly antigen-specific and show a superior homing capacity to HLA-A2+ allografts compared to polyclonal Tregs. HLA-A2 CAR Tregs have been shown to prolong survival of HLA-A2+ allografts in several pre-clinical humanized mouse models. Although promising, concerns about safety and stability need to be addressed. In this review the current research, obstacles of CAR Treg therapy, and its potential future in solid organ transplantation will be discussed.

Glioblastoma, an opportunity T cell trafficking could bring for the treatment.

Infiltrating into the vital structure of the brain, located in the inaccessible anatomical region, and having molecular heterogeneity, glioblastoma (GBM) -with no doubt- is one of the deadliest cancers. Using the blood and brain barrier (BBB), GBM makes a shield to restrict the reach of chemotherapeutic agents to the tumor site and evolves a unique microenvironment to furnish all the essentials for cancer cells survival to conceal neoplastic cells from immunosurveillance. 99 papers which met the criteria of eligibility were included in this review by consensus. The included articles were classified based on their design and level of evidence. Given this characteristic, immunotherapies for a while enjoyed unprecedented attention as a solution for GBM treatment; however, it did not take long before the enthusiasm for their application was muted. It became apparent that cancer cells intelligently find a way to manipulate the anti-tumor responses of agents by attracting immunosuppressive lymphocytes into the brain using the lymphatic vessels. This event makes GBM a good model for immunotherapy resistance. However, the presence of lymphatic vessels has fired up an idea of the adoptive attraction of effector T lymphocytes to the tumor milieu. This was when engineering and cloning technologies, which have given life to one of the recent treatment strategies using artificial T cells named chimeric antigen receptors (CAR) T-cells, came to action to design specific CAR T-cells for the treatment of GBM. The present review summarizes the recent advances in CAR T-cell-based treatments in GBM and discusses why this approach could be positioned as a pillar of the next-generation of immunotherapies for this type of brain tumor.

Abstract 128: A Chimeric Antigen Receptor Targeting MDA-LDL Activates Regulatory T Cells In The Presence Of Human Atherosclerotic Plaque

Introduction: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to reduce the risk of MI and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a Chimeric Antigen Receptor (CAR) targeting malonaldehyde-modified LDL (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. Oxidized-LDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane and CD28/CD3z cytoplasmic domains. CD4 + CD25 + CD127 low/- Tregs were purified from human blood via FACS and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy (CEA). Autologous ox-CAR-Tregs were analyzed for activation after ex-vivo co-culture with CEA samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising ox-CAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared to controls, based on CD71 expression, cytokine expression and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using IHC. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex-vivo co-culture with atherosclerotic plaque. Conclusion: An optimized CAR targeting MDA-LDL activates Tregs when cultured with human atherosclerotic plaque ex-vivo.