AbstractQuantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3–5 h and then migrate to the liver and spleen for up to 2–3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.
BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).
Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control
Abstract Background Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. Methods Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. Results After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. Conclusion We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.
BackgroundDespite remarkable response rates mediated by anti-CD19 chimeric antigen receptor (CAR) T cells in selected B cell malignancies, CAR T cell therapy still lacks efficacy in the vast majority of tumors. A substantial limiting factor of CAR T cell function is the immunosuppressive tumor microenvironment. Among other mechanisms, the accumulation of adenosine within the tumor can contribute to disease progression by suppressing anti-tumor immune responses. Adenosine 2a- and 2b-receptor (A2A and A2B)-mediated cAMP build-up suppresses T cell effector functions. In the present study we hypothesize, that combination therapy with the selective A2A/A2B dual antagonist AB928 (etrumadenant) enhances CAR T cell efficacy.Materials and MethodsSecond generation murine (anti-EPCAM) and human (anti-MSLN) CAR constructs, containing intracellular CD28 and CD3ζ domains, were fused via overlap extension PCR cloning. Murine or human T cells were retrovirally transduced to stably express the CAR constructs. A2A/A2B signaling in CAR T cells was analyzed by phospho-specific flow cytometry of CREB (pS133)/ATF-1 (pS63). CAR T cell activation was quantified by flow cytometry and enzyme-linked immunosorbent assay (ELISA) of IFN-γ, IL-2 and TNF-α. CAR T cell proliferation was assessed by flow cytometry. CAR T cell cytotoxicity was assessed by impedance based real-time cell analysis.ResultsAB928 protected murine CAR T cells from cAMP response element-binding protein (CREB) phosphorylation in the presence of stable adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA). NECA inhibited antigen-dependent CAR T cell cytokine secretion in response to four murine tumor cell lines. CAR T cell-mediated tumor cell lysis as well as proliferation were decreased in the presence of NECA or adenosine. Importantly, AB928 fully restored CAR T cell cytotoxicity, proliferation, and cytokine secretion in a dose dependent manner. Further, AB928 also restored antigen dependent cytokine secretion of human CAR T cells in the presence of NECA.ConclusionsHere we used the A2A/A2B dual antagonist AB928 to overcome adenosine-mediated suppression of CAR T cells. We found that AB928 enhanced important CAR T cell effector functions in the presence of the adenosine analogue, suggesting that combination therapy with AB928 may improve CAR T cell efficacy. This study was limited to in vitro experiments. To confirm the relevance of our findings, this combination therapy must be further investigated in an in vivo setting.Disclosure InformationM. Seifert: None. M. Benmebarek : None. B. Cadilha : None. J. Jobst: None. J. Dörr: None. T. Lorenzini: None. D. Dhoqina: None. J. Zhang: None. J. Zhang: None. U. Schindler: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Amgen Inc., Arcus Biosciences. Other; Significant; Arcus Biosciences. S. Endres: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Biosciences.
Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.
Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.
Abstract Hodgkin lymphoma (HL) generally carries a good prognosis. However, 10-15% of patients relapse or are refractory to first-line therapy. These patients have a poor prognosis and would benefit from innovative approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor redirected T cells (CART19, CTL019) for refractory B cell malignancies. Despite the B-cell origin of the malignant Hodgkin Reed-Sternberg (HRS) cells, B-cell antigens, in particular CD19, are typically not expressed in HL. We sought to define a HL-associated cell membrane antigen that could be targeted by CAR T cells. Given the relative paucity of the malignant cells and the importance of the immunosuppressive tumor microenvironment in HL, the ideal target would be expressed on neoplastic cells as well as on infiltrating immune cells in order to provide robust stimulation of the CAR T cells. Immunohistochemistry for novel HL targets on 10 patient samples revealed that 5/10 patients expressed CD123 on the HRS cells. CD123 was also seen on immune cells of the microenvironment in most samples. CD123 is the α chain of the receptor for interleukin-3 (IL-3), an important cytokine in hematopoietic growth and differentiation that has been previously shown to promote HL cell line growth (Aldinucci et al, Leuk & Lymph, 2005). As primary HL is non-engraftable in mice we turned to immortalized HL cell lines and confirmed that CD123 is expressed by flow cytometry and Q-PCR in four different HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). To determine the role of IL-3 signaling in HL we engrafted NOD-SCID-γ-chain KO mice that overexpress human cytokines including IL-3 (NSG-S mice) with the luciferase-expressing HDLM-2 cell line. After i.v. injection, the neoplastic cells progressively formed disseminated soft tissue masses. Serial injections of a neutralizing anti-IL3 antibody slowed the growth of tumor, suggesting that CD123 may be a particularly relevant target in HL. We therefore sought to investigate the utility of anti-CD123 CAR T cells (CART123) for the treatment of HL. We have recently described the activity of CART123 in human acute myeloid leukemia (Gill et al, Blood, 2014). Our construct is a 2nd generation CAR, comprising 4-1BB co-stimulatory and CD3-ζ chain signaling domains with an anti-CD123 scFv. In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells (Table 1). Moreover, long-term co-culture (20 days) of CART123 with HDLM-2 cells at a 1:1 ratio led to T cell proliferation and complete elimination of HL cells by day 4. To confirm these in vitro data, we developed a rigorous in vivo model injecting 1 million luciferase+ HDLM-2 cells i.v. on day 0. Serial bioluminescent imaging (BLI) demonstrated low level of tumor on day 7, which was followed by gradual increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day 43 when the tumor burden was 20-fold higher than baseline, mice were treated with 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 6 months (Figure 1 and 2). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In summary, we show for the first time that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. We have previously demonstrated that CART123 lead to myelosuppression, suggesting that our findings could be translated to treat patients with refractory HL with a combined CART123 and rescue autologous bone marrow transplantation. Table 1 In vitro activity of CART123 compared to untransduced control T cells (UTD) against a HL cell line (HDLM-2). IN VITRO EXPERIMENT CART123* UTD CD107a Degranulation (4 hrs, E:T = 1:5) 59.3% 2.69% Specific Killing (24 hrs) E:T = 2:1 57% 5% E:T = 0.25:1 27% 1% Proliferating cells (CFSE based) (5 days, E:T = 1:1) 96.4% 20% Cytokine production (24 hrs, E:T = 1:1) (Luminex, MFI) INF-γ 38,265 42 IL-2 85,604 0 TNF-α 10,684 55 MIP-1β 40,038 111 IL-6 16,425 110 GM-CSF 99,915 285 *All P values are <0.05 when compared to UTD Figure 2 Figure 2. Disclosures Ruella: Novartis: Research Funding. Kenderian:Novartis: Research Funding. Shestova:Novartis: Research Funding. Chen:Novartis: Research Funding. Scholler:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Gill:Novartis: Research Funding.
BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-HCT) is a standard treatment for relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However ~30-40% of patients (pts) still relapse after HCT. We report a cohort of 20 r/rB-ALL pts, who relapsed after HCT, and enrolled in the CAR2.0 study receiving one or two types of CAR-T cells targeting various B-ALL antigens. METHOD: Pts with r/r B-ALL who relapsed after allo-HCT and did not have significant active comorbiditeis, were enrolled in the study. The target antigens were determined based on immunostaining of each pt's leukemia cells, and CAR-T infusions included a single, or a combination of CAR-Ts targeting the following antigens: CD19, CD22, CD123 and CD38. T cells were collected from pts (N=4) or their allogeneic donors (N=16) and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine lymphodepleting therapy before infusion of 0.2-5.8x106 CAR-T/kg per infusion. In addition to disease response, we carefully monitored the quality of apheresis cells, efficiency of gene transfer, T cell proliferation rate, CAR-T infusion dose, and the CAR-T copy number in peripheral blood. RESULTS: Among the 20 enrolled pts, 11 were <18 years of age, and 7 were BCR- ABL (P190) positive. Before CAR-T treatment, 7 pts had ≤grade 2 active graft-versus-host disease (GVHD), and 13 pts received chemotherapy or targeted therapy after their relapse post HCT. Six pts had extramedullary relapse and 2 of them also had bone marrow relapse. The tumor burden in bone marrow ranged from minimal residual disease (MRD) negative to 66% of blasts, based on flow cytometry before CAR-T therapy. Five pts had >10% blasts in bone marrow, 8 pts had <3% blasts, and 7 pts had MRD negative bone marrow (summarized in the Table below). Based on the GVHD history, chimerism state and the available T-cell sources, 16 pts used allogeneic HCT donor T-cells for CAR-T preparation. All pts were full donor chimeras prior to CAR-T infusion, except one pt who had 41% donor cells in bone marrow. Eleven pts received a single CD19 CAR-T infusion, with a mean dose of 1.6x106 CAR-T/kg, and ten achieved an MRD remission and one had progressive disease (PD) within 60 days by flow cytometry. The remaining 9 pts received 2 CAR-Ts (CD19 plus CD22, CD123 or CD38 CAR-Ts) given on the same day, and resulted in 8 CR and 1 PD within 60 days. After CAR-T infusion, no cytokine release syndrome (CRS) was observed in 8 pts, and 12 pts experienced CRS of grade 1, which was consistent with the previously described low toxicity profile of the 4SCAR design. Acute GVHD ≤ grade 2 developed in 5 pts within one month following CAR-T cell infusion but all responded well to supportive care and/or cyclosporine infusion. The 2 pts who developed PD after CAR-T infusion included the one with 41% donor chimerism and had grade 2 GVHD and active infections before CAR-T infusion. The other pt with PD following CAR-T had severe bone marrow suppression, low leukocyte count, infections and was transfusion dependent before enrollment. This emphasizes the need for controlling comorbidities before infusion of CAR-T cells. In summary, total 18 patients (90%) achieved negative MRD remission within 2 months of therapy with acceptable CRS. Four pts relapsed (after being in remission for 3 months) and 14 pts are in continued remission, 6 of which for > 1 year. None of these 20 pts received a second HCT after CAR-T infusion. GVHD developed in 5/16 (31%) pts after donor source CAR-T cell infusion within one month, but all responded well to treatment. CONCLUSION: This study focuses on CAR-T cell therapy following relapse after HCT. While the expanded study is ongoing, we present results of the first 20 pts. Use of donor-derived or recipient-derived CAR-T products in pts who relapsed after allo-HCT is well tolerated and it may prolong life expectancy of these pts while maintaining good quality of life. Table Disclosures No relevant conflicts of interest to declare.
BackgroundCAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).Materials and MethodsOverexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.ResultsOverexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.ConclusionsOverexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.Disclosure InformationA. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.
