198. CD4+ IL13Rα2-Specific CAR T Cells Exhibit Potent Effector Function Against Glioblastoma

BackgroundAdoptive cell transfer of chimeric antigen receptor (CAR) modified T cells has demonstrated great therapeutic success against certain hematological malignancies. However, a substantial number of patients experienced relapse at some point after treatment with the underlying mechanisms not fully understood. Emerging data suggest that the undesired clinical outcome is related to different aspects, which include: the tumor heterogeneity, the tumor microenvironment, as well as intrinsic characteristics of the CAR T cells. In this work, we aimed to understand the diversity of CAR T cells generated from different donors, using multiparameter in vitro characterization.MethodsLeukapheresis from healthy donors were collected to generate CAR T cells using the GMP-compliant CliniMACS Prodigy® platform, enabling an automated and closed engineering of CAR T cells in a highly reproducible manner. We performed an in-depth characterization of the resulting CAR T cells by exploring differences in the immunophenotype, cell fitness and effector function of the freshly prepared as compared to frozen CAR T cell samples. Specifically, we designed several flow cytometry panels for the extensive characterization of immunophenotypes of interest such as: proliferative capacity, differentiation, activation and exhaustion. Cell fitness status was determined by the rate at which cells undergo apoptosis following stress. Finally, effector function was determined by the ability of the activated CAR T cells to secrete proinflammatory cytokines including IFN-g, TNF-a and IL-2. The associations between these different parameters were analyzed using comprehensive statistical approaches.ResultsWith our established workflow, over 20 healthy-donor derived CAR T cells were generated and characterized. We have observed donor-dependent variations and responses for most of the explored parameters. In general, the freezing and thawing process negatively affected cell fitness and effector function of the CAR T cells and resulted in altered immunophenotypes. Additionally, correlations between certain immunophenotypes and cell fitness/effector function were identified.ConclusionsCollectively, we established a workflow for multiparameter characterization of CAR T cells and assessed the intrinsic variability of CAR T cells for both research and clinical application.

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Abstract 602: A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function

10.1158/1538-7445.am2017-602 ◽

2017 ◽

Author(s):

Benjamin Boyerinas ◽

Sara Miller ◽

Ryan Murray ◽

Stacie Seidel ◽

Geoffrey Parsons ◽

...

Keyword(s):

T Cells ◽

Immune Suppression ◽

Effector Function ◽

Signal Conversion ◽

Car T Cells ◽

Car T

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Effects of Prior Exposure to Tec Kinase(BTK/ITK) Inhibitors on Kte-X19 Products

Blood ◽

10.1182/blood-2021-146941 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3849-3849

Author(s):

Irene Scarfò ◽

Kathleen M.E. Gallagher ◽

Mark B. Leick ◽

Michael C. Kann ◽

Justin Budka ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Stock Options ◽

Effector Function ◽

Prior Exposure ◽

Publicly Traded ◽

Car T Cells ◽

Car T ◽

Post Infusion ◽

Privately Held

Abstract Introduction: Frequent and durable responses were recently reported in relapsed or refractory (R/R) mantle cell lymphoma (MCL) patients treated with KTE-X19, an autologous CD19-targeted chimeric antigen receptor-engineered T-cell (CAR-T) product (Wang et al. N Engl J Med. 2020). Most patients enrolled had received at least one line of Tec kinase inhibitor prior to KTE-X19 manufacturing, either in the form of ibrutinib, a Bruton's tyrosine kinase (BTK) and Inducible T cell kinase (ITK) inhibitor, or acalabrutinib, a more selective BTK inhibitor. Pharmacokinetic expansion of KTE-X19 was higher in ibrutinib-treated patients relative to acalabrutinib-treated patients. We previously showed that prolonged exposure to ibrutinib enhanced T cell effector function and proliferation in patients with CLL (Fraietta et al, Blood, 2016). To assess the impact of Tec kinase inhibitor on KTE-X19 products and downstream clinical outcomes, we examined the phenotype, transcriptional profile and cytokine production of KTE-X19 infusion products and post-infusion lymphocytes from patients with R/R MCL treated on the Zuma-2 study. Study Design and Methods: We evaluated biospecimens from MCL patients who enrolled on the Zuma-2 clinical trial (NCT02601313) and who were previously treated with ibrutinib (n=14) or acalabrutinib (n=6). Samples analyzed consisted of KTE-X19 CAR T products and peripheral blood mononuclear cells (PBMC) collected 7 days after infusion. Lymphocytes were assessed for CAR expression, T cell phenotype, transcriptional profile and cytokine production. In addition, CAR T cell phenotypes and cytokines were profiled following co-culture of KTE-X19 with CD19 + Toledo cells (DLBCL). Results: Flow cytometric analysis of KTE-X19 demonstrated similar distributions of CD4+ and CD8+ T cells and comparable frequencies of central and effector memory populations in the CAR+ T cells derived from patients with prior exposure to ibrutinib vs. acalabrutinib. T helper subset analysis trended towards enrichment of Th1/Th17 populations within the CAR+ CD4+ cells of the ibrutinib cohort. This finding was further supported by transcriptional profiling of sorted CAR+ T cells from infusion products, where Th1/Th17, Jak/STAT and activation-related genes were enriched in the cohort with prior ibrutinib exposure. In addition, the Th1 phenotype was more frequent in PBMC of ibrutinib-exposed patients (8/14) compared to acalabrutinib-exposed patients (1/4). Interestingly, a shift from a central memory-dominant product towards an effector memory phenotype was observed in peripheral CD4+ and CD8+ CAR T cells in the ibrutinib cohort, whereas acalabrutinib post-infusion CAR T cells maintained a central memory phenotype. In vitro stimulation of KTE-X19 CAR-T infusion products with tumor cells resulted in a significant enrichment of the Th1 population in patients who had received ibrutinib compared to those that received acalabrutinib (p=0.0058). Following stimulation, CAR-T cells from the acalabrutinib cohort produced higher levels of Th2 cytokines, including IL-4, IL-5, and IL-13 as well as GM-CSF compared to the ibrutinib cohort. Conclusions: Analysis of KTE-X19 infusion products and day 7 post-infusion PBMC demonstrated that CAR T cells from patients with prior ibrutinib exposure have a Th1 predominant phenotype, suggesting that ibrutinib but not acalabrutinib promotes Th1 differentiation and effector function, potentially through the inhibition of ITK. Furthermore, our data suggest that inhibition of non-BTK targets such as ITK may play a role in driving a Th17 phenotype. Prior exposure to ibrutinib may increase CAR T cell effector function to a greater extent than exposure to acalabrutinib to enhance clinical outcome in patients with MCL. Disclosures Budka: Kite Pharma: Current Employment. Sowrirajan: Kite Pharma: Current Employment. Nguyen: Kite Pharma: Current Employment. Shen: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties; Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

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CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus

Cancers ◽

10.3390/cancers13051050 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1050

Author(s):

Ana Textor ◽

Laura Grunewald ◽

Kathleen Anders ◽

Anika Klaus ◽

Silke Schwiebert ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Effector Function ◽

Human Neuroblastoma ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Cell Effector Function ◽

Cell Effector

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

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Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

10.1101/2020.11.07.372334 ◽

2020 ◽

Author(s):

Young-Ho Lee ◽

Hyeong Ji Lee ◽

Hyung Cheol Kim ◽

Yujean Lee ◽

Su Kyung Nam ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Immune Checkpoint ◽

Therapeutic Option ◽

B Cell Lymphoma ◽

Effector Function ◽

Antitumor Effects ◽

Car T Cells ◽

Car T ◽

Checkpoint Receptors

AbstractCD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations—PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4 and PD-1/TIGIT—and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD-1 single downregulation, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT–downregulated CAR T cells generated from diffuse large B-cell lymphoma patient-derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint-mediated resistance in CAR T therapy.

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Loss of TET2 Uncouples Proliferative and Effector Functions in CAR T Cells

Blood ◽

10.1182/blood-2020-142957 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-1

Author(s):

Nayan Jain ◽

Zeguo Zhao ◽

Archana S. Iyer ◽

Michael Lopez ◽

Judith Feucht ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Stress Test ◽

Effector Function ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Anti Tumor Activity ◽

Central Memory Phenotype

Chimeric antigen receptor (CAR) T cells have opened a new paradigm for the treatment of leukemia and lymphoma. Their production, however, is laborious, requiring tens of millions of CAR T cells per infusion. This constraint could be significantly alleviated if safe and more efficacious T cells could be generated. In a patient with chronic lymphocytic leukemia, treated with anti-CD19 CAR T cells, a recent report described the emergence of a single T cell clone that at its expansion peak accounted for 94% of circulating CAR T cells, coinciding with the development of cytokine release syndrome and tumor regression (Fraietta et. al. Nature 2018). Insertional mutagenesis in this T cell had disrupted an allele of TET2, an epigenetic regulator mediating the oxidation of 5-methylcytosine. The other allele appeared to bear an inherited hypomorphic variant, resulting in the near complete loss of TET2 function in this clone. To understand the mechanisms accounting for this chance clinical finding, we investigated the effect of TET2 loss in human T cells engineered to express different chimeric receptors. Using CRISPR/Cas9, we edited TET2 in T cells engineered to express a CD19-specific second-generation CAR encompassing the costimulatory domain of either CD28 or 4-1BB (Rv-1928z and Rv-19BBz). TET2 disruption enhanced the in vivo anti-tumor activity of Rv-19BBz but not Rv-1928z CAR T cells tested under stress test conditions using limiting CAR T cell doses (as previously described in a human B cell acute lymphoblastic leukemia (B-ALL) NALM6 model, Zhao et. al. Cancer Cell 2015). Since Rv-1928z induces potent effector differentiation but limited persistence compared to Rv-19BBz, we hypothesized that loss of TET2 could amplify the expansion and persistence of 4-1BB-costimulated T cells but not override the differentiation program imparted by Rv-1928z. To test this hypothesis, we utilized two orthogonal approaches known to limit exhaustion and increase persistence of CD28-costimulated CAR T cells, Rv-1928z co-expressed with 4-1BB ligand (Rv-1928z-41BBL) and 1928z driven by the TRAC promoter (TRAC-1928z). Disruption of TET2 enhanced the anti-tumor efficacy of both these CAR T cells and promoted acquisition of a central memory phenotype. However, over time (50-200 days), TET2-edited TRAC-1928z and Rv-1928z-41BBL attained a hyper-proliferative phenotype ultimately requiring euthanasia due to splenomegaly and extensive CAR T cell accumulation in various organs. Post-mortem analysis found no evidence of NALM6 in these mice. This was in contrast to stress test studies with Rv-1928z and Rv-19BBz where most mice succumbed to NALM6 progression. These observations established an essential role for CAR signaling in determining the phenotypic outcome of TET2 loss in T cells. To examine the long-term effects of TET2 disruption in the context of all 4 receptors, we treated human B-ALL bearing mice with curative doses of all 4 CAR T cells and followed them for up to 200 days. We found that all 4 CAR expressing TET2-edited T cells could eventually attain a hyper-proliferative phenotype, but with varying frequency depending on the CAR design (Rv-1928z-41BBL and TRAC-1928z > Rv-19BBz > Rv-1928z). To assess their effector function, NALM6-bearing mice were infused with adoptively transferred hyper-proliferative TET2-edited CAR T cells. Strikingly, these T cells were unable to elicit any tumor control, despite their maintaining a central memory phenotype as assessed by flow cytometry. This loss of effector function was observed for all 4 CAR T cell types, suggesting a discrepancy between function and flow cytometric phenotype. Transcriptional, methylation and genome accessibility studies revealed a unique T cell state wherein the proliferative program is uncoupled from effector response. We identified a unique transcriptional and epigenetic signature that is manifested in a loss of effector function while maintaining robust proliferation. This state stands in contrast to the classically described T cell exhaustion state where loss of effector function is preceded by loss of proliferative ability. TET2 disruption thus promotes a CAR T cell proliferative program that depends on the CAR design but does not in itself enhance anti-tumor activity. Disclosures Sadelain: Atara: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Minerva: Other: Biotechnologies , Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding.

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