A multiparametric CAR T cell potency assay that employs real-time cellular impedance and live cell imaging

Development of a Regulated, Optimized Mesothelin Chimeric Antigen Receptor (CAR) for the Treatment of Mesothelin Positive Cancers

APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy

A Novel Method for Molecular Enumeration of Circulating Tisa-Cel and Axi-Cel in Lymphoma Patients

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T-cells. In order to overcome a lot-to-lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum-free media [Prime-XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T-Cell Expansion Xeno-Free Medium, Takara Bio™ (TB) and TCM GMP-Prototype, CellGenix™ (CG)] to the standard CAR T-cell medium containing FBS (RCF). After 12 days of CD19.CAR T-cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T-cells was evaluated using intracellular staining, a chromium release assay and a long-term co-culture assay. Expansion and viability did not differ between the CAR T-cells generated in serum-free media compared to the standard FBS-containing medium. The CG CAR T-cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF-α+ CAR T-cells than the CAR T-cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T-cells generated with serum-free media exhibited a higher cytotoxicity than the CAR T-cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T-cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte-activation gene 3 and T-cell immunoglobulin and mucin-domain containing-3. The CAR T-cells cultured in FF had a higher percentage of central memory CAR T-cells (40.0 vs. 14.3%, P=0.0470) than the CAR T-cells cultured with FBS, whereas the CAR T-cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T-cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum-free media. Given the advantages of freedom from bovine material and consistent quality, serum-free media hold promise for the future development of the field of GMP manufacturing of CAR T-cells.

Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

Chimeric Antigen Receptor (CAR) immunotherapy has transformed cancer therapeutics and has shown great success in treating hematopoietic malignancies. To date, autologous CAR-T-cell therapy has been the main clinical approach. However, autologous CAR-T cells have several major limitations including high-cost and complexity of production, donor-to-donor variability, and potentially severe toxicities. Allogenic NK cell-based therapies have also been shown to mount potent responses against hematopoietic malignancies (i.e., AML). However, unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as cytokine release syndrome, neurotoxicity, or graft-vs-host-disease. Therefore, this approach provides an important platform for allogeneic CAR-enabled therapies. Several groups have demonstrated the clinical efficacy of allogeneic CAR-expressing NK cells that utilize CARs developed for T-cells. However, recent studies using NK cell-based activating receptor signaling modules such as NKG2D and 2B4 as CAR co-stimulatory domains proved increased anti-tumor activity compared to T-cell CARs. The goal of this study was to develop next-generation NK cell-optimized CARs to improve targeting and potency of our iPSC-derived NK cell therapies. To achieve this, we developed a platform to comprehensively screen immune cell signaling modules in iPSC-derived NK cells. First, we constructed a library of 44 novel CAR constructs containing signaling modules from diverse NK cell specific signaling receptors. Next, we developed a viral-based CAR expression protocol in mature iPSC-derived NK cells that yields high CAR expression (>75% CAR+) while maintaining high viability (>90%). We then screened for CAR activity using two co-culture target cell killing assays (eSight impedance assay and caspase 3/7 killing assay) and two NK cell-resistant CD19+ target cell lines (Raji and SupB15). Our screen identified 7 novel CAR signaling modules that performed better than both a second-generation T-cell CAR (CD28-CD28-zeta) and 3 previously described NK cell-CARs (NKG2D-2B4-zeta, CD28-OX40-zeta, and CD28-OX40L-zeta). These results were consistent across both functional assays and both target cell lines tested adding support to our findings. Overall, we have successfully developed an efficient CAR screening platform in iPSC-derived NK cells and have identified NK optimized CARs that enable increased potency of NK cells compared to current CAR solutions. The most potent CARs identified in our screen are being engineered into CISH-knockout iPSC-derived NK cells which were previously developed by us that demonstrate improved in vivo anti-tumor activity, in vivo persistence, metabolic fitness, polyfunctionality and resistance to cell exhaustion to create next-generation CAR-NK cell-based immunotherapies. Citation Format: Lee Swanson, Gabrielle Allen, Kenyon Lyon, Wael Tadros, Max Shabla, Dan S. Kaufman, Huang Zhu. Development of an iPSC-derived NK cell screening platform for discovery of NK cell optimized Chimeric Antigen Receptors (CARs) for next-generation CAR-NK cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 554.

Biomarker correlates of clinical outcomes from a global Phase 1b study of JNJ-90014496, CD20/CD19 bi-specific chimeric antigen receptor (CAR) T-cell therapy for patients with large B-cell lymphoma (LBCL)

The first chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T-cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T-cells eliminate all B-lineage cells, including nonmalignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections. Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T-cells and subsequent testing of the CAR T-cells in various in vitro assay with targeT-cells demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in targeT-cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T-cells, and our tests confirmed this. To improve IGK CAR T-cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T-cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs, hence enabling T-cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T-cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún Melkorka Maggadóttir, Gunnar Kvalheim, Erlend Bremertun Smeland, June Helen Myklebust, Else Marit Inderberg, Sébastien Wälchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A035.

Carmod, an Open-Label, Multicenter, Proof of Concept, Phase II Trial, Investigating the Safety and Efficacy of Golcadomide in Post-CART for Patients with Relapsed/Refractory High-Risk Aggressive Large B-Cell Lymphoma

Chimeric antigen receptor (CAR) T cell therapy has proven to be highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. These efforts face multiple challenges, including off-tumor target expression and checkpoint inhibition of CAR T cell activity in the tumor microenvironment. CAR T cells are much more potent compared to antibody therapeutics, therefore there is a need for more stringent CAR T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. In this study we employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model, which expresses both BCMA and ROR1. The anti-ROR1 CAR T cells used in this study recognized both the mouse and human proteins whereas the anti-BCMA CAR T cells recognized only the human protein. RNA ISH revealed that BCMA was only expressed in the xenograft tumor and in no mouse organs, while ROR1 was found to be expressed in the xenograft tumor as well as at low levels in mouse lung and liver. Duplex RNA ISH assay with probes targeting the CAR 3’ UTR and either IFNG or GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from vehicle, anti-ROR1 CAR T cell, or anti-BCMA CAR T cell treated mice. Activated anti-BCMA CAR T cells expressing GZMB and IFNG were found only in the xenograft tumor, where BCMA was expressed. In contrast, activated anti-ROR1 CAR T cells were found almost exclusively in mouse lung and liver with very few anti-ROR1 CAR T cells being found in the xenograft tumor, consistent with the previously observed pulmonary and hepatic toxicity of anti-ROR1 CAR T cells that was not predicted by IHC analysis of ROR1 protein presumably due to lack of antibody sensitivity. Lastly, we employed a multiplex ISH-IHC approach to confirm the presence of activated anti-BCMA CAR T cells in the xenograft tumor through simultaneous detection of the BCMA CAR 3’ UTR, IFNG, GZMB, and CD3, validating the anti-tumor activity of anti-BCMA CAR T cells. These data thus demonstrate how the RNAscope ISH assay can be utilized for CAR T efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR T cell pharmacodynamics and activation in tumor models. This technology has equal utility in understanding both CAR T and TCR-T cell activity in patient tumors. Citation Format: Helly Pimentel, Helen Jarnagin, Hailing Zong, Courtney Todorov, Kenneth Ganley, Fay Eng, Kevin Friedman, Molly Perkins, Shannon Grande, Courtney M. Anderson, Bingqing Zhang, Christopher Bunker, James B. Rottman, Xiao-Jun Ma. Preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2324.

Introduction: Despite significant progress in the chimeric antigen receptor (CAR)-T cell therapy of B cell malignancies, the 5-year progression-free survival rate in large B cell lymphoma following CD19-targeting CAR-T cell treatments remains below 50%. While some treatment failures were attributed to CD19 antigen loss, immunogenicity of murine single-chain fragment variable (scFv) elements has been implicated in relapse and resistance to CAR-T cell re-treatment. In this study, we evaluated fully human CD20 and CD19 dual-targeting CAR constructs (h20.19 CARs) combining human scFvs and distinct co-stimulatory domains and architectures, to mitigate tumor antigen escape, reduce immunogenicity, and improve CAR functionality. Methods: The h20.19 CAR constructs were assembled from fully human scFvs against lymphoma antigens CD19 and CD20, with hinge, transmembrane and co-stimulatory domains derived from 4-1BB, CD28, OX40 or ICOS. Tandem second generation (T8/8BBz), tandem third generation (T28/28BBz, T8/28BBz), and bicistronic (D28z/BBz, DBBz/28z, DOXz/ICz) CARs were evaluated. CAR T cells were derived from healthy donors’ CD4+ and CD8+ T cells by lentiviral transduction, and CAR expression was evaluated by flow cytometry. CAR-mediated target cell killing was assessed by co-culture assays, and cytokine elaboration by ELISA. In vivo CAR-T cell function was evaluated in a Raji lymphoma NSG xenograft model using low CAR-T dose (2x106/mouse). Tumor-free survivors were rechallenged with Raji cells to compare CAR-T cell potency and persistence. Results: The hCD20.19 CAR-T cells lysed CD19+CD20+ tumor lines Raji, NALM-6 and REH, and single-antigen lines 293T CD19 and 293T CD20, but not the parental CD19-CD20- 293T cells, indicating antigen-specific efficacy. Cytokines TNFα, IL2 and IFNγ were strongly upregulated in response to Raji cells. During repeat co-culture with Raji cells, the third-generation tandem hCD20.19 CARs and the bicistronic CARs showed greater cytotoxicity than the second-generation CAR T8/8BBz. In vivo, all third-generation tandem and bicistronic CARs induced rapid and durable Raji tumor rejection by study day 7 post CAR-T, with no apparent toxicity. Upon long rest and rechallenge with Raji cells on study day 50, all h20.19 CARs attenuated tumor progression as compared to age-matched untreated mice cohort. CARs T8/28BBz and D28z/BBz afforded the least protection from Raji rechallenge, whereas CAR DOXz/ICz mediated rapid tumor rejection, but mice met euthanasia criteria by study day 71 due to weight loss. The tandem CAR T28/28BBz and bicistronic CAR DBBz/28z, demonstrated tumor rejection, persistence and safety after rechallenge, up to study day 86. Conclusions: In summary, the fully human dual-targeting h20.19 CARs exhibit robust anti-tumor function and persistence in vitro and in vivo, mediate long-term remissions and resist tumor rechallenge. Citation Format: Peirong Hu, Brittany Steimle, Ngoc Tran, Pradyot Dash, Dina Schneider. Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo [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 3198.

A Novel Protease-Mediated Chimeric Antigen Receptor (CAR): “Double-Arm” CAR-T Cell System Improves Target Specificity of CAR-T Cell Therapy

Chimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy. We propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells. We designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion. We detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells. We developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.

Figure 1 Type-2 function CAR T cell cluster is associated with 8-year ultra-long remission. (A) UMAP clustering of CDI 9-specific stimulated CAR T cells from the Discovery Cohort patients and...

Phase I CD19 and CD22 targeting Chimeric Antigen Receptor (CAR) T cell trials have shown tremendous results against acute lymphoid leukemia (ALL). Across these trials there were variations in the types of costimulatory molecules included in the CAR constructs and this lead to the discovery that CAR T cells with CD28 result in earlier potency and activation with decreased persistence, while CARs with 4-1BB show increased expansion and persistence. There is great interest in translating these results in ALL trials into other diseases such as acute myeloid leukemia (AML). It is unclear which costimulatory domain in a CAR will be most effective in treating AML. To look at how co-stimulatory domains impact CAR functionality in AML, we developed CD33 targeting CD28 and 4-1BB CARs. In vitro testing of the constructs revealed that CD33 CD28 CAR consistently produced more IL2 and Interferon-gamma than CD33 4-1BB across multiple AML cell lines. To translate these findings in vivo, xenograft models were injected with Molm 14 AML cells and treated with either CD33 CD28 or CD33 4-1BB CAR T cells. By bioluminescence imaging, CD33 CD28 treated mice had no detectable disease while CD33 4-1BB treated mice were ridden with leukemia. Combined, the in vitro and in vivo results suggest that the co-stimulatory domain does play a critical role in CAR T cell functionality and may improve CAR potency. To confirm the presence of AML in mice detected by bioluminescence, flow cytometry was performed on tissues from mock and CD33 4-1BB treated mice. No leukemia was found in the bone marrow of mock T cell treated mice. In contrast, CD33 4-1BB treated animals were clear of any leukemia in the bone marrow, suggesting the presence of extra medullary disease (EMD). The development of EMD in the less potent CD33 4-1BB CAR treated mice suggests that CAR immune pressure may be potent enough to clear primary sites of leukemia such as the bone marrow, but unable to eliminate disease in secondary tissues that AML can seed. This is not surprising since treatment of AML with chemotherapy often leads to the development of extramedullary disease in the form of chloromas. To further investigate the effects of these two factors, we moved onto another AML model, THP1, that regularly presents with EMD even in the absence of CAR pressure. With CD33 CD28 against THP1, there was clearance in compartments bone marrow, however CD33 CD28 CAR was not able to prevent the development of EMD. These experiments suggest that although the CD28 costimulatory domain is more potent than 4-1BB in Molm14, the potency of CD28 is still not able to overcome EMD in all models. Using further studies of different AML models, we will continue to tease apart the contribution the impact of immune pressure from CARs, the natural progression of AML models, and effect of CAR potency on leukemia distribution. Citation Format: Lila Yang, Samiksha Tarun, Christopher D. Chien, Mark E. Kohler, Haiying Qin, Terry J. Fry. Analysis of CAR 41-BB versus CD28 co-stimulatory domains exposes emergence of extramedullary disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1534.