c-Jun Overexpressing CAR-T Cells are Exhaustion-Resistant and Mediate Enhanced Antitumor Activity

SUMMARYCAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

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Synthetic Chimeric Antigen Receptors (CARs) Rapidly Induce Exhaustion and Augmented Glycolytic Metabolism In Human T Cells and Implicate Persistent CD28 Signaling As a Driver Of Exhaustion In Human T Cells

Blood ◽

10.1182/blood.v122.21.192.192 ◽

2013 ◽

Vol 122 (21) ◽

pp. 192-192

Author(s):

Adrienne H. Long ◽

Rimas J. Orentas ◽

Crystal L. Mackall

Keyword(s):

T Cells ◽

Antitumor Efficacy ◽

Antigen Receptors ◽

Antitumor Effects ◽

Car T Cells ◽

Human T Cells ◽

Car T ◽

In Vitro Expansion

Abstract Introduction Chimeric antigen receptors (CARs) provide a promising new approach for the adoptive immunotherapy of cancer. Though impressive antitumor activity has been observed with some CAR T cells, other CAR T cells demonstrate poor antitumor efficacy in vivo despite high cytolytic capacity in vitro due to poor expansion and persistence. Whether exhaustion of CAR T cells mirrors exhaustion that occurs naturally in chronically stimulated human T cells has not yet been studied. Here, we report that expression of select CD28 containing CARs in normal human T cells rapidly induces an exhausted state characterized by high PD-1 expression, poor persistence and poor antitumor efficacy, whereas other CARs do not induce this phenotype. Results Human T cells were expanded with anti-CD3/CD28 beads, and then transduced with a second-generation (CD28-CD3ζ) disialoganglioside 2 (GD2) specific CAR or a second-generation (CD28-CD3ζ) CD19 specific CAR. By day 7 of in vitro expansion, GD2 CAR T cells developed a metabolism more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced controls. Neither CAR population was exposed to antigen during this expansion period. Using a Seahorse Extracellular Flux Analyzer, the ratio of glycolysis to oxidative phosphorylation rates (ECAR:OCR ratio) of GD2 CAR T cells was found to be double that of CD19 CAR T cells or controls on day 7. The highly glycolytic metabolism of GD2 CAR T cells was associated with an exhausted phenotype. GD2 CAR T cells expressed higher levels of PD-1, TIM-3 and LAG-3, and transcription repressor BLIMP-1, compared to CD19 CAR T cells or untransduced controls. Additionally, GD2 CAR T cells were poor cytokine producers, generating <10x lower levels of IL2, TNFα and IFNγ than CD19 CAR T cells upon in vitro co-incubation with a GD2+CD19+ osteosarcoma line (143B-CD19), despite maintaining comparable in vitro cytolytic ability. GD2 CAR T cells showed poor in vitro expansion and increased rates of apoptosis compared to controls. GD2 CAR T cells also did not persist and did not mediate antitumor effects against GD2+CD19+ tumors in a murine xenograft model in vivo, whereas CD19 CAR T cells completely eradicated CD19+ tumors and persisted in both the spleen and tumor compartments. To rule out the possibility that diminished cytokine production and in vivo efficacy was related to antigen specific effects, T cells were co-transduced with both the GD2 and CD19 CARs. Though single-transduced CD19 CAR T cells show no signs of an altered metabolism or exhaustion and have strong antitumor efficacy, CD19 CAR T cells co-transduced with the GD2 CAR demonstrate an exhausted phenotype and diminished antitumor efficacy similar to that of single-transduced GD2 CAR T cells. Thus, expression of the GD2 CAR confers a dominant exhausted phenotype in T cells, and prevents otherwise efficacious CARs from mediating strong antitumor effects. We hypothesized that chronic signaling of CD3ζ and CD28 via the GD2 CAR results in exhaustion. Interestingly, however, we did not identify GD2 expression in the culture system. Point mutations in the CAR antigen-binding site, though abrogating GD2 binding, did not prevent the development of exhaustion. Thus, we postulate that constitutive receptor signaling may occur via interactions between the framework regions of the CAR receptors. Importantly however, substitution of 4-1BB for the CD28 domain in the GD2 CAR substantially diminished PD-1 expression, one of the hallmark features of exhausted T cells. Conclusions We report that expression of a CD28 containing GD2 CAR induces both an altered metabolism and an exhausted state in human T cells, resulting in poor in vivo persistence and antitumor efficacy. We hypothesize that tonic signaling through the GD2 CAR induces this phenotype and have identified the CD28 domain as an important component contributing to this phenotype. Rapid induction of exhaustion mediated via a synthetic receptor provides a novel model system to identify mechanistic factors required for this phenotype in human T cells. Work is currently underway to molecularly define the basis for the exhaustion of GD2 CAR T cells and to probe a potential role for altered T cell metabolism as a contributor to T cell exhaustion in human T cells. Disclosures: No relevant conflicts of interest to declare.

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Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-113438 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2205-2205 ◽

Cited By ~ 3

Author(s):

Elisa De Togni ◽

Miriam Y Kim ◽

Matt L Cooper ◽

Julie Ritchey ◽

Julie O'Neal ◽

...

Keyword(s):

T Cells ◽

Cell Line ◽

Myeloid Leukemia ◽

Cytotoxic Effects ◽

Car T Cells ◽

Human T Cells ◽

Car T ◽

Acute Myeloid

Abstract Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

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CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo

Blood ◽

10.1182/blood-2011-03-344275 ◽

2012 ◽

Vol 119 (3) ◽

pp. 696-706 ◽

Cited By ~ 180

Author(s):

De-Gang Song ◽

Qunrui Ye ◽

Mathilde Poussin ◽

Gretchen M. Harms ◽

Mariangela Figini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Car T Cells ◽

Human T Cell ◽

Human T Cells ◽

Car T ◽

Cd27 Costimulation

AbstractThe costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-XL protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB–costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

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Interleukin 21 Enhances Survival and Expansion of CAR T Cells Via Inhibition of Their Terminal Differentiation during Interaction with Tumor Target Cells

Blood ◽

10.1182/blood-2018-99-116294 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4545-4545 ◽

Cited By ~ 2

Author(s):

Martin Štach ◽

Jan Musil ◽

Petr Cetkovsky ◽

Pavel Otahal

Keyword(s):

T Cells ◽

Conflicts Of Interest ◽

Terminal Differentiation ◽

Target Cells ◽

Antitumor Effects ◽

Tumor Target ◽

Color Flow ◽

Car T Cells ◽

Car T

Abstract Background: Efficiency of CAR T cell based therapies against cancer is often limited by a poor survival of CAR T following recognition of tumor target cells. Interaction of CAR T with target cells induces their rapid differentiation into late memory subtypes (Teff) which lack expression of CD27, CD28, CD62L and CCR7. Although these terminally differentiated T cells are highly cytotoxic, their in vivo engraftment capacity is lower which thus reduces their in vivo survival and enables only temporary antitumor effects. It is generally believed that CAR T cells with early memory phenotypes (Tscm + Tcm) would provide stronger antitumor effects due to better survival in vivo. Recently, we have developed a transposon-based protocol of clinical-grade CAR19 T manufacture (Otahal et al, Cytotherapy 2018) which uses a combination of cytokines IL-4, IL-7 and IL-21 which strongly enhance the generation of CAR T cells with Tscm/Tcm phenotypes. Methods: We have thoroughly studied the effects of IL-21 on the survival, differentiation status and the expression of major immunoinhibitory receptors using CAR T cells specific to antigens CD19 and PSMA. After the co-culture of CAR T with their tumor target cells, the phenotypes were analyzed by multi-color flow cytometry, together with the assessment of effector functions and proliferation. We have compared the outcomes of signaling initiated by IL-21 on the fate of CAR T during this co-culture with the effects initiated by IL-2. Results: We have found out that IL-21 is a strong regulator of CAR T memory differentiation initiated by recognition of tumor target cells. IL-21 supported expansion of CAR T with Tscm/Tcm phenotypes and inhibited their terminal differentiation into CD45RA+/- CD62L neg, CD27 neg, CD28 neg late memory subtypes (i.e. Teff and Tem). Additionally, IL-21 suppressed up-regulation of inhibitory receptors PD-1 and TIGIT by CAR T cells. Both IL-21 and IL-2 were indispensable to maintain proliferation of CAR T following their activation via the recognition of tumor target cells however, IL-2 induced a rapid differentiation of CAR T into late memory subtypes and resulted in significantly lower expansion than CAR T cells co-cultivated with tumor cells in the presence of IL-21. Conclusions: Our data strongly suggest that the in vivo functions of CAR T cells can be significantly boosted by omitting the use of IL-2 during production because IL-2 drives CAR T towards their terminal effector differentiation state that reduces their ability to form long-lived memory cells. We are currently developing CAR T with transgenically expressed IL-21 and we are preparing a clinical testing of CAR T manufactured according to this protocol in patients diagnosed with relapsed-refractory B-ALL and B-NHL. Supported by grants NV15-34498A and Primus/MED/34, MH CZ - DRO (Institute of hematology and blood transfusion, IN - 00023736) and by gifts from Heřmanský foundation. Disclosures No relevant conflicts of interest to declare.

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Engineering of CD19-Specific Chimeric Antigen Receptor T Cells with the Integrin CD103 Results in Augmented Therapeutic Efficacy Against Human Lymphoma in a Preclinical Model

Blood ◽

10.1182/blood-2018-99-117354 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2050-2050

Author(s):

Hongxing Sun ◽

Shan He ◽

Lijun Meng ◽

Ying Wang ◽

Hanghang Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chimeric Antigen Receptor ◽

Preclinical Model ◽

Car T Cells ◽

Nsg Mice ◽

Human T Cells ◽

Car T ◽

Human Lymphoma

Abstract Whether tumor-reactive T cells can infiltrate into the tumor to execute effector function is essential for controlling tumor growth. CD103 is an integrin protein (αE) that binds integrin β7 to form the heterodimeric integrin complex αEβ7. CD103 is important for T cell retention in peripheral tissues by interacting with E-cadherin and a promising prognosis biomarker for assessment of tumor-reactive T cells infiltrating in the tumor from various types of cancer, such as lung cancer, ovarian cancer and cervical cancers. However, CD103 is not expressed on the surface of circulating peripheral blood T cells that are genetically modified to express a chimeric antigen receptor (CAR) for adoptive T cell therapy. Whether CD103 expression on the surface of tumor-reactive CAR T cells is functionally important for their anti-tumor activity has not been previously determined. Using a preclinical model of human lymphoma expressing E-cadherin, we demonstrate that engineering of CD19-specific human CAR T cells with CD103 significantly improves their therapeutic effects on eliminating pre-established human lymphoma in immune deficient NSG mice (NOD.scid.Il2Rγcnull). We synthesized a codon optimized CD19-specific CAR containing 4-1BB and CD3zeta intracellular signaling domains (named CD19-BBz-CAR), cloned it into lentiviral vector and infected human T cells. As expected, the resultant human CD19-BBz-CAR T cells possessed potent capacity to cure human B cell leukemia in NSG mice that had been intravenously inoculated with Raji leukemic/lymphoma cells. Notably, while approximately 10% of non-CAR T cells produced high levels of CD103 from these NSG mice, CD19-BBz-CAR T cells failed to upregulate CD103, suggesting that the expression of CD19-BBz-CAR inhibits the induction of CD103 in vivo. Ex vivo assay confirmed that CD19-BBz-CAR caused dose-dependent decrease of CD103 expression in human T cells cultured in the presence of TGF-β1. This effect was mediated by the expression of costimulatory molecule 41BB, which is known essential for sustaining CD19-BBz-CAR T cells in vivo. To circumvent the repression effect of 41BB on induction of CD103, we incorporated the gene encoding integrin αE into the CAR structure to generate CD103-CD19-BBz-CAR T cells. Intriguingly, as compared to conventional CD19-BBz-CAR T cells, CD103-CD19-BBz-CAR T cells expressed high levels of CD62L and CD45RA, which resemble less differentiated T cells, produced higher levels of IL-2, which is crucial for promoting T cell expansion and function, and underwent greater expansion in cultures. Upon adoptive transfer into NSG mice that had subcutaneous human Raji lymphoma, CD103-engineering of CD19-BBz-CAR T cells dramatically decreased the distal metastasis of lymphoma, increased the infiltration of CAR T cells into the solid lymphoma, and improved the in vivo persistence of tumor-reactive CAR T cells. As a result, transfer of CD103-CD19-BBz-CAR T cells significantly increased overall survival rate of lymphoma mice compared to conventional CD19-BBz-CAR T cells (40% versus 10%, p<0.05). Our findings suggest that engineering tumor-reactive T cell with CD103 may represent a novel strategy to improve their anti-tumor efficacy. Moreover, this newly established CD103-CAR structure may have broad implication in the solid tumor treatment. Disclosures Barta: Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

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ALL Xenografts Reveal the Importance of Anti-CD19-Chimeric Antigen Receptor Cell Dose, Cell Persistence and Surprising Antitumor Activity of CD4+ Anti-CD19-CAR T Cells in Eradicating Pediatric Acute Lymphocytic Leukemia In Vivo

Blood ◽

10.1182/blood.v118.21.574.574 ◽

2011 ◽

Vol 118 (21) ◽

pp. 574-574

Author(s):

Daniel W. Lee ◽

James N Kochenderfer ◽

Rimas J Orentas ◽

Elizabeth G. Gardner ◽

Crystal L Mackall

Keyword(s):

T Cells ◽

T Cell ◽

Lymphocytic Leukemia ◽

51Cr Release ◽

Antitumor Effects ◽

Car T Cells ◽

Cell Dose ◽

Car T ◽

Release Assay

Abstract Abstract 574 Relapsed and refractory pediatric acute lymphocytic leukemia (ALL) remains a difficult therapeutic challenge and accounts for a sizable number of cancer-related deaths in children. Chimeric antigen receptors (CAR) are genetically engineered molecules that combine antibody specificity for a target antigen with the potent cytotoxic potential of activated T cells. CAR based T cell immunotherapy is currently under study in several clinical trials and encouraging early response data is beginning to emerge. However, studies in pediatric malignancies are still lacking. We have studied the efficacy of a second-generation CAR containing the T-cell receptor zeta signaling subunit, the signaling domain of CD28, and a single chain variable fragment directed against CD19. CD19 is present on nearly 100% of pediatric ALL blasts and normal B cells but not on hematopoietic stem cells. Anti-CD19-CAR T cells are generated using retroviral transduction of T cells activated with K562 based artificial antigen presenting cells expressing the high-affinity Fc-receptor (CD64) loaded with anti-CD3 and the co-stimulatory molecule, 41BB-ligand. Transduction efficiency, as measured by flow cytometry, averaged 50–80%. Unselected anti-CD19-CAR T-cells specifically and robustly killed four CD19+ ALL but not CD19– target cell lines (45-60% lysis at E:T ratios as low as 2.5:1 in 4h 51Cr release assay) and produced significant levels of IFNg, TNFa, and IL-2 when encountering CD19+ ALL cell lines but not CD19– cells. In xenograft models, 3 × 10^6 unselected anti-CD19-CAR T-cells injected via tail vein eliminated engrafted ALL (NALM6 cell line stably expressing both GFP and firefly luciferase, NALM6-GL) within 48 hours in 5/5 immunodeficient NOG mice (Figure 1) whereas all animals that received 3 × 10^6 activated but untransfected T-cells required sacrifice within 18 days. Nearly two months after T cell infusion, CD19-CAR T cells bearing an effector memory phenotype could still be detected in peripheral blood. When lower T cell doses were used, antitumor effects were less potent and were associated with a lack of persistence of anti-CD19-CAR T cells in vivo. CD4+ T cells are not classically cytotoxic and normally require Class II presentation of target peptides for recognition. However, since CAR based recognition is MHC independent, we hypothesized that both CD4+ and CD8+ anti-CD19-CAR T-cells may mediate cytotoxicity. To test this, CD4+ and CD8+ T-cells from the same donor were negatively selected using immunomagnetic beads, then activated and transduced as before. A 4-hour 51Cr release assay demonstrated cytotoxicity from CD8+ CAR T-cells similar to unselected cells but no appreciable cytotoxicity from CD4+ CAR T cells. However, in NOG xenografts, 1 × 10^6 CD4+ CD19-CAR T cells produced complete responses in 4/5 mice by Day 10 as compared to 5/5 complete responses in animals receiving 1 × 10^6 CD8+ CD19-CAR T cells. While antitumor effects mediated by CD8+ T cells were rapid and nearly complete within 3 days, antitumor effects of CD4+ T cells were slower, peaking at approximately day 14. Together, these results demonstrate notable differences in efficacy of anti-CD19-CAR based therapy with small changes in cell dose, unexpected activity of CD4+ anti-CD19-CAR based T cells in xenografts which is not reflected by activity in short term killing assays, and evidence that cure of mice is associated with persistence of anti-CD19-CAR modified T-cells in this model. These results inform clinical development of this emerging therapy by emphasizing the importance of cell dose, cell persistence and the novel observation that CD4+ anti-CAR T cells may be important effectors in mediating antitumor effects of this therapy. Disclosures: No relevant conflicts of interest to declare.

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112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0112 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A124-A124

Author(s):

Letizia Giardino ◽

Ryan Gilbreth ◽

Cui Chen ◽

Erin Sult ◽

Noel Monks ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Single Chain ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Animal Care ◽

Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

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Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model

Scientific Reports ◽

10.1038/s41598-021-94490-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Naomi S. Sta Maria ◽

Leslie A. Khawli ◽

Vyshnavi Pachipulusu ◽

Sharon W. Lin ◽

Long Zheng ◽

...

Keyword(s):

T Cells ◽

Real Time ◽

Pet Imaging ◽

Dose Group ◽

Low Dose ◽

High Dose ◽

Car T Cells ◽

Nsg Mice ◽

Car T

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.

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98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0098 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A109-A109

Author(s):

Jiangyue Liu ◽

Xianhui Chen ◽

Jason Karlen ◽

Alfonso Brito ◽

Tiffany Jheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Next Generation ◽

Phase 3 ◽

T Cell Therapy ◽

Cell Therapies ◽

Car T Cells ◽

Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

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114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0114 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A126-A126

Author(s):

John Goulding ◽

Mochtar Pribadi ◽

Robert Blum ◽

Wen-I Yeh ◽

Yijia Pan ◽

...

Keyword(s):

T Cells ◽

Cancer Immunotherapy ◽

Tumor Immunity ◽

Immune Cell ◽

Nk Cell ◽

Tumor Escape ◽

Car T Cells ◽

Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

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