Chimeric Antigen Receptor Signaling Research Articles

Tonic 4-1BB signaling from chimeric antigen receptors (CARs) impairs expansion of T cells due to Fas-mediated apoptosis

Abstract High expression of CARs in T cells or the presence of CAR ligands in cell culture results in tonic CAR signaling that enhances differentiation and exhaustion of T cells and compromises anti-tumor activity. A recent study demonstrated tonic signaling negatively affected T cells expressing a CD28.zeta GD2-specific CAR, indicating 4-1BB costimulation may improve their function. However, whether 4-1BB is a uniformly optimal costimulatory signal for CAR-transduced T cells is unclear. We found that retroviral expression of several 2nd generation CARs with a 4-1BB domain (4-1BB.zeta) unexpectedly impaired T cell expansion. High expression of 4-1BB.zeta CD19 and kappa CARs led to upregulation of Fas and FasL and enhanced caspase 8-dependent apoptosis. Decreasing CAR expression in retro- or lentiviral systems normalized Fas levels and restored T cell expansion. However, a similar approach did not rescue expansion of 4-1BB.zeta CD5 CAR T cells, likely because the presence of the target antigen CD5 on T cells sustains high tonic signaling. 4-1BB.zeta CD5 CAR T cells recruit Fas and FasL to form stable fratricidal immunologic synapses. Disrupting TRAF2 binding site in the 4-1BB domain prevented Fas upregulation and restored T cell function but compromised costimulation. Therefore, we developed a regulated retroviral expression system that uses a small molecule to reversibly inhibit CAR expression, preventing tonic signaling and the resulting fratricide and differentiation. Withdrawing the inhibitor restores CAR expression and the anti-tumor function of T cells. Hence, we have described a mechanism by which chronic 4-1BB signaling may undermine CAR T cell function and provide a solution that curtails detrimental tonic CAR signaling.

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

Abstract B021: Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice

Abstract Background: The adoptive transfer of T cell engineered with chimeric antigen receptors (CARs) is currently considered a highly promising therapeutic option for treating so far incurable malignant diseases. In order to reduce adverse reactions associated with CAR engineered T cells, a flexible modular universal chimeric antigen receptor (UniCAR) platform is developed, which allows a precise control of CAR T cell reactivity to manage side effects while preserving efficacy. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CARs into two individual components. The UniCAR is structurally similar to second-generation CARs, however the single chain variable fragment (scFv) does not recognize a cell surface antigen but a short peptide motif derived from a human nuclear autoantigen. Antigen-specificity and crosslinking of UniCAR expressing T cells to target cells is provided by a separate targeting module (TM) compromising a scFv, binding to specific cell surface antigens, fused to the aforementioned peptide motif. Thus, T cells engineered to express UniCARs remain inactivated after infusion, since the UniCAR target is not available for binding under physiological conditions. Only in the presence of the TM, antigen specific activation of UniCAR T cells occurs, which allows for a more sophisticated regulation. Methods: We developed a novel radio-immune target module (TM), which consists of an anti-PSCA (prostate stem cell antigen) scFv for redirecting of UniCARs to PSCA expressing tumor cells. Three different tags are fused in a row to the C-Terminus of the scFv to functionalize the molecule for combined immune- and radiotherapy. First, a ten amino acid (aa) long tag mediates the crosslink to T cells genetically modified with the UniCAR. Further downstream, a peptide tag (Rx) for enhanced conjugation of radionuclides and finally, a 6xhistidine (His) tag are located. TM-Rx efficacy was evaluated for target cell killing in-vitro and compared to a TM without Rx-tag. In-vivo efficacy of the TM-Rx was also investigated in a high and low-tumor burden mouse model. Results: Rx-TM and TM equivalently kill PSCA-expressing tumor cells in-vitro in presence of UniCARs in an E:T (Effector:Target) ratio of 1:1 after 24 hours. UniCAR T cell engrafted NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice show no clinical signs of autoimmunity neither in presence nor absence of the TM. One week treatment with TM delays subcutaneous tumor growth and prolongs survival of tumor bearing, UniCAR T cell engrafted NSG mice. Longterm infiltration of tumor tissue by UniCARs could be observed over 16 weeks in the high and low-tumor burden ectopic carcinoma model. Conclusion: The UniCAR technology shows efficacy both in-vitro and in-vivo. Extension of the TM with an additional tag does not impair functionality of the UniCAR platform. Short-treatment prolongs survival of tumor bearing mice but does not lead to cure, therefore combination with other treatment modalities e.g. radiation oncology seems reasonable. Citation Format: Elham Pishali Bejestani, Malte von Bonin, Marc Cartellieri, Armin Ehninger, Mechthild Krause, Michael Baumann, Gerhard Ehninger, Michael Bachmann. Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B021.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival

ABSTRACTEpidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Unicar: A Novel Modular Retargeting Platform Technology for CAR T Cells

The adoptive transfer of T cells engineered with chimeric antigen receptors (CARs) is currently considered as a highly promising therapeutic option for treatment of otherwise incurable malignant diseases. CARs combine the cellular and humoral arm of the immune response by assembling a single-chain fragment variable (scFv) as binding moiety which provides the antigen-specificity and an activating immune receptor. It has been demonstrated both in vitro and in vivo, that CAR engrafted effector T cells mediate long-lasting anti-tumor responses. Despite encouraging clinical efficacy targeting CD19 in recent clinical trials, the appearance of potentially life-threatening adverse reactions and the lack of control mechanisms once initiated, prevent more widespread application of the CAR technology.To overcome limitations of conventional CAR T cells, a unique chimeric antigen receptor (UniCAR) technology was developed (Fig. 1) which allows precise control of CAR T cell reactivity, thus lowering the risk of side effects while preserving efficacy. Moreover, the UniCAR technology enables the retargeting of engrafted T cells against more than one antigen simultaneously or subsequently, thus reducing the risk for development of antigen-loss tumor variants under treatment. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CAR into two individual components. T cells are engineered to express a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein. Thus, T cells engineered to express UniCAR remain inactivated after re-infusion, since the UniCAR target is not available for binding under physiological conditions. The ultimate antigen-specificity of the system is provided separately by targeting modules (TMs) comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR binding domain. Here we provide first in vitro and in vivo prove of concept for this new approach. Antigen-specific redirection of T cells armed with the universal CAR in the presence of different targeting modules against various antigens (CD33, CD123, CD19, CD20, PSCA, PSMA,) was effective at femtomolar concentrations of the targeting module both. Taken together, the modular nature of UniCAR technology will allow retargeting of autologous, patient-derived T cells to several antigens under controlled pharmacological conditions and has the potential to become a highly effective treatment option for late stage cancer patients with reduced risks for side effects. [Display omitted] DisclosuresCartellieri:Cellex Patient Treatment GmbH: Employment. Loff:GEMoaB Monoclonals GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership; Cellex Patient Treatment GmbH: Equity Ownership. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.

Abstract B2-33: Computational model of chimeric antigen receptor-engineered T cells

Abstract Introduction. The purpose of this study is to create a predictive model that can be used to test hypotheses about chimeric antigen receptor (CAR) activation of T cells in order to optimize the development of these engineered proteins used for cancer immunotherapy. CARs are engineered proteins that contain an extracellular antibody-like region linked to a selection of intracellular signaling domains involved in T cell activation. CAR-engineered T cells have shown promise in treating B cell lymphoma, but they have not been successfully applied to other types of cancers. Additionally, there have been serious side effects when T cell activation goes awry. The specific mechanisms of the signaling and co-stimulatory domains on the CAR are not well understood, and therefore they are hard to control. Developing a computational model capable of comprehensively and quantitatively describing the biochemical reactions involved the CAR-mediated T cell signaling network will help guide the development and optimization of CAR-based cancer immunotherapy. Methods. We have constructed a model of T cell signaling mediated by CARs. Due to the complexity of the multiprotein interactions in this pathway, a rule-based model was written in BioNetGen and implemented in MatLab. The model consists of coupled, nonlinear differential equations, which can be solved to predict the concentrations of molecular species in CAR-mediated T cell signaling. In parallel, we have obtained signaling data using flow cytometry measurements to quantify the time required for ERK phosphorylation, indicating signaling through the MAPK pathway. These data are used to calibrate the model and estimate unknown parameter values. Results. Our computational model is comprised of 181 rules, encoding 1,989 reactions between 20 species. The initial parameter estimates are based on experimentally measured values and a model of T cell receptor signaling (Altan-Bonnet, et al., 2005). Importantly, our model expands upon previous works by modeling separate pathways for specific signaling domains that can be incorporated into the CAR and incorporating more detail into the large multi-protein complexes downstream of the CAR. Extending the model to these multi-protein complexes allows us to predict the levels of downstream proteins that correlate to specific responses of T cell activation, including cell proliferation and survival. We constructed two CAR proteins with different signaling domains (CD3ζ-only and CD3ζ+CD28), and stably expressed them in the Jurkat T cell line. We similarly expressed CD19 in the K562 cell line to be used as target cells. The CAR cells were stimulated with target cells and stained for intracellular flow cytometry analysis of doubly phosphorylated ERK. We performed a local sensitivity analysis to identify the effect of individual parameters on the model predictions. This analysis identified six parameters that most significantly influenced the model. These parameters were fit to ERK phosphorylation data, and we estimated the optimal parameter values required to match the experimental measurements using a nonlinear optimization procedure. Interestingly, the fitted model predicts that the time required for phosphorylation of ERK differs in CD3ζ-only and CD3ζ-CD28 CARs, matching our experimental measurements. The fitting procedure identified multiple parameter sets that fit the data equally well, indicating model robustness. We are currently collecting additional flow cytometry measurements to validate the model predictions. Conclusions. The model is able to accurately represent the effects of CAR signaling to provide insight into how different signaling domains control the strength and persistence of activated T cells. This understanding can be used to optimize CAR engineered T cells to target cancer cells in a manner that is both sensitive and specific. Citation Format: Jennifer Rohrs, Pin Wang, Stacey D. Finley. Computational model of chimeric antigen receptor-engineered T cells. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-33.

Abstract B12: Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptor

Abstract Recent success of adoptive transfer of T cells expressing chimeric antigen receptors (CARs) in patients with blood-borne malignancies has resulted in growing enthusiasm to apply the same approach to treat solid tumors. CAR-expressing T cells, however, will likely face many challenges that will limit their efficacy in solid tumors. One possible obstacle will be the intrinsic negative feedback system that prevent continuing T cell responses. The transcription factor Ikaros, which has been shown to suppress endogenous T effector functions through epigenetic remodeling of cytokine gene loci, may be one such negative regulator. Here we show that anti-mesothelin CAR (mesoCAR) T cells with reduced levels of Ikaros consistently mediate better in vitro cell killing and in vivo tumor regression than their wild-type counterparts. This increased anti-tumor activity was accompanied by increased persistence of mesoCAR T cells, as well as increased IFN-γ production. Enhanced cytolytic activity was also evidenced in mesoCAR T cells with reduced Ikaros, which could be partially explained by increased CD107a upregulation and Granzyme B production. Reduction in the Ikaros level in mesoCAR T cells did not affect T cell receptor and CAR signaling, but was able to render them more resistant to soluble inhibitory factors like TGF-β and adenosine. In summary, inhibition of the transcriptional repressor Ikaros in CAR T cells could be an attractive strategy to overcome immunosuppressive tumor microenvironment and enhance their tumoricidal capacity. Further clinical development is warranted. Citation Format: Shaun O'Brien, Rajan M. Thomas, Steven M. Albelda, Andrew D. Wells, Liang-Chuan S. Wang. Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptor. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B12.

Abstract 3142: Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptors

Abstract Recent success of adoptive transfer of T cells expressing chimeric antigen receptors (CARs) in patients with blood-borne malignancies has resulted in growing enthusiasm to apply the same approach to treat solid tumors. CAR-expressing T cells, however, will likely face many challenges that will limit their efficacy in solid tumors. One possible obstacles will be the intrinsic negative feedback systems that prevent continuing T cell responses. The transcription factor Ikaros which has been shown to suppress endogenous T effector functions through epigenetic remodeling of cytokine gene loci may be one such negative regulator. Here we show that anti-mesothelin CAR (mesoCAR) T cells with reduced levels of Ikaros consistently mediate better in vitro cell killing and in vivo tumor regression than their wild-type counterparts. This increased anti-tumor activity was accompanied by increased persistence of mesoCAR T cells, as well as increased IFN-g. Enhanced cytolytic activity was also evidenced in mesoCAR T cells with reduced Ikaros, which could be partially explained by increased CD107a upregulation and Granzyme B production. Reduction in the Ikaros level in mesoCAR T cells did not affect T cell receptor and CAR signaling, but was able to render them more resistant to soluble inhibitory factors like TGF-B and adenosine. In summary, inhibition of the transcriptional repressor Ikaros in CAR T cells could be an attractive strategy to overcome immunosuppressive tumor microenvironments and enhance their tumoricidal capacity. Further clinical development is warranted. Citation Format: Shaun O'Brien, Liang-Chuan Wang, Kheng Newick, Rajan M. Thomas, Andrew Wells, Steven M. Albelda. Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2015-3142

Abstract 4702: 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells

Abstract Published reports have demonstrated high response rates of CD19 chimeric antigen receptor (CAR) modified T cells against acute lymphoblastic leukemia. CARs incorporating either CD28 or 4-1BB costimulatory domains mediated significant anti-leukemic effects. However CD19-41BBz CAR T cells could be detected for months and induced prolonged B cell depletion, whereas most patients treated with CD19-28z CAR T cells had no detectable CAR+ T cells by day 28, and B cell recovery was routinely observed. We report that CAR T cells incorporating the CD28 versus 4-1BB costimulatory domains show differential susceptibilities to T cell exhaustion. Using the tonically signaling 14g2a-based GD2 CAR as a model system, we identify that GD2-28z CARs rapidly acquire an exhaustion phenotype, characterized by high level expression of PD1, TIM3 and LAG3, high expression of exhaustion related transcription factors Blimp1 and T-bet, poor proliferative capacity, poor cytokine production, and poor anti-tumor efficacy in vivo. Interestingly, second generation GD2-28z CARs induce greater exhaustion than first generation GD2-z CARs, implicating CD28 costimulation in the induction of exhaustion. In contrast, GD2-41BBz CARs express lower levels of PD1 and TIM3, show increased cytokine production, improved persistence in vivo, and improved anti-tumor efficacy against both osteosarcoma and neuroblastoma xenografts. Similarly, CD19-41BBz CARs have improved persistence and lower expression of exhaustion markers compared to CD19-28z CARs following leukemia challenge. Finally, CD22-BBz CARs show lower exhaustion marker expression, improved persistence, and improved anti-leukemia efficacy in immunodeficient mice compared to CD22-28z CARs. Gene expression experiments are underway to further delineate the mechanism by which 4-1BB signaling ameliorates exhaustion in the setting of chronic CAR signaling. Together, this data demonstrates that exhaustion is a critical factor limiting the efficacy of CAR expressing T cells. CD28 and 4-1BB have opposing effects on exhaustion in CAR T cells, with CD28 signaling augmenting the phenotype while 4-1BB signaling mitigating exhaustion. This data is the first to demonstrate differential effects of costimulatory pathways on T cell exhaustion, likely explains differential persistence observed in the clinical trials of CAR therapeutics, and provides important insights for CAR design for future clinical applications. Citation Format: Adrienne H. Long, Waleed M. Haso, Jillian P. Smith, Alec J. Walker, Terry J. Fry, Rimas J. Orentas, Crystal L. Mackall. 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2015-4702

Abstract 3140: Antigen-induced downmodulation is associated with diminished efficacy of a novel chimeric antigen receptor targeting anaplastic lymphoma kinase

Abstract CD19 directed Chimeric Antigen Receptor (CAR) T cell therapies have shown clinical responses against B cell malignancies. However, CARs targeting other tumor-associated antigens have been less successful. Factors limiting CAR efficacy are not well understood. Anaplastic lymphoma kinase (ALK) is a cell-surface protein overexpressed in a large fraction of neuroblastomas, the most common extracranial solid tumor of childhood. We constructed MSGV1.ALK.BBZ retroviral vectors using single-chain variable fragment (scFv) sequences from murine monoclonal antibodies targeting ALK and transduced these into human PBMCs. ALK-CAR T cells lysed ALK+ tumor lines, but produced limited amounts of IFN-g and IL-2 upon co-culture with ALK+ tumor cells compared to CD19-CARs co-cultured with CD19+ targets. In order to identify potential differences between ALK-CAR and CD19-CAR induced signaling we employed a reporter system in which GFP expression was directed by an NFAT-responsive promoter and could be visualized by flow cytometry. T cells were co-transduced with lentiviral NFAT-GFP and MSGV1.ALK.BBZ or MSGV1.CD19.BBZ. Transduced T cells were co-cultured with tumor lines expressing ALK or CD19 antigen. GFP expression was induced within 4h of antigen exposure in double transduced T cells and persisted for at least 24h. The magnitude of GFP expression correlated positively with antigen density on tumor targets. Additionally, >50% of GFP+ cells retained CD19-CAR surface expression after antigen encounter. However, we observed drastically reduced surface ALK-CAR expression on GFP+ T cells after encounter with tumor targets. Upon further examination, we noticed that ALK-CARs were down-modulated from the T cell surface within 1h of exposure to antigen, and remained internalized for at least 24h. The magnitude of NFAT translocation in ALK-CAR T cells, as measured by GFP intensity, was also markedly lower than the magnitude of NFAT translocation in CD19-CAR T cells. These results suggest that limited numbers of CARs remaining on the T cell surface after initial antigen encounter may contribute to diminished ALK-CAR T cell efficacy, possibly by preventing temporal summation of repeated CAR signaling. Work is underway to prevent CAR down-modulation after antigen encounter, and to evaluate the impact of increased surface CAR retention on CAR functionality. In summary, optimal design of new CAR therapeutics requires a better understanding of essential factors limiting CAR efficacy. Through evaluation of a novel CAR targeting ALK, we have identified CAR down-modulation as one factor that may influence CAR-T efficacy, and may be amenable to modulation in order to improve CAR functionality. Citation Format: Alec J. Walker, Ling Zhang, Adrienne H. Long, Rimas J. Orentas, Crystal L. Mackall. Antigen-induced downmodulation is associated with diminished efficacy of a novel chimeric antigen receptor targeting anaplastic lymphoma kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2015-3140

4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic anti-tumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We discovered that tonic CAR CD3ζ phosphorylation, triggered by antigen-independent clustering of CAR scFvs, can induce early exhaustion of CAR T cells that limits anti-tumor efficacy. Such activation is present to varying degrees in all CARs studied, with the exception of the highly effective CD19 CAR. We further identify that CD28 costimulation augments, while 4-1BB costimulation ameliorates, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the dramatic anti-tumor effects of CD19 CARs and for the observations that CD19.BBz CAR T cells are more persistent than CD19.28z CAR T cells in clinical trials.

Abstract 2943: 14g2a-based GD2-specific chimeric antigen receptors (CARs) constitutively signal, leading to rapidly induced T-cell exhaustion and poor antitumor efficacy in vivo

Abstract Chimeric antigen receptors (CARs) provide a promising new approach for generating T cells for the adoptive immunotherapy of cancer. In both pre-clinical and clinical studies, we have observed potent anti-tumor effects using CD19 CAR T cells (CART) to treat B cell malignancies. However, we observe less success when targeting solid tumors with GD2 CART. We hypothesized that the observed differences could be due to a more hostile microenvironment in solid tumors, and/or variable CAR potency. To address these issues, we created a CD19+GD2+ osteosarcoma (143B-CD19). This double positive solid tumor line allowed us to directly compare the in vivo efficacy of the GD2 and CD19 CARs, normalized for tumor type. In vitro 51Cr release demonstrated that CD19 and GD2 CART were equally active against 143B-CD19. However, in vivo models showed a significant difference in anti-tumor efficacy. NSG mice were injected with 143B-CD19 and treated with control, GD2 or CD19 CART 14d later. GD2 CART failed to persist in mice and did not inhibit tumor outgrowth. However, CD19 CART did persist and cleared the mice of all CD19+ solid tumor disease. Therefore, we conclude that the solid tumor microenvironment is not a barrier to effective CART therapies, and hypothesize that poor GD2 CAR potency leads to poor in vivo efficacy. Further comparison of GD2 and CD19 CART demonstrated that excessive, rather than insufficient, signaling by the GD2 CAR contributes to the poor in vivo efficacy. GD2 CART have increased expression of activation makers (CD25, CD127, CD137) and increased size compared to CD19 CART following in vitro expansion. Despite this increased activation, GD2 CART expand poorly, have high rates of apoptosis, produce IL2, TNFa and IFNg poorly, and express high levels of PD1, TIM3, LAG3, and BLIMP1. This phenotype is consistent with T cell exhaustion, a state of poor effector function due to chronic antigen stimulation. This phenotype is prevented by knocking out GD2 CAR signaling through point mutations in the CD3 zeta and CD28 domains of the receptor, implicating tonic signaling through the CAR during in vitro expansion as the source of T cell exhaustion. Interestingly, we did not identify GD2 expression in the culture system. Point mutations in the CAR antigen-binding site, though blocking GD2 binding, did not prevent exhaustion. Thus, we postulate that constitutive receptor signaling may occur via interactions between the framework regions of the CAR, leading to exhaustion. This exhausted phenotype was recapitulated with CD19 CART when expanded in the presence of a crosslinking anti-idiotype antibody, suggesting that over-activation via other CARs also has the potential to limit efficacy. Ongoing experiments focus on elucidating the molecular pathways that link CAR signaling and induction of T cell exhaustion, as well as identifying the structural features of the GD2 CAR that lead to excessive signaling. Citation Format: Adrienne Long, Rimas J. Orentas, Crystal L. Mackall. 14g2a-based GD2-specific chimeric antigen receptors (CARs) constitutively signal, leading to rapidly induced T-cell exhaustion and poor antitumor efficacy in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2014-2943

Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases.

Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs.

Abstract IA22: Advanced T cell engineering for cancer immunotherapy.

Abstract Targeted eradication of cancer by adoptive transfer of antigen specific T cells is the subject of intensive clinical exploration. While many of the key conceptual and technical barriers that have previously limited translation to human clinical trials have been overcome, as reflected by the growing number of FDA IND-supported studies in progress, significant refinement is needed to evolve this therapeutic modality for reproducibly effective and safe applications in the oncology clinic. The approach of genetically engineering T cells for cancer adoptive therapy is now at the forefront of translational research activities in this field. Our group, as well as others, has advanced technologies to target tumor cells using T cells modified to express chimeric antigen receptors (CARs). These engineering strategies, when applied to the genetic modification of central memory T cells (TCM) endowed with the capacity to engraft and repopulate functional memory T niches, is the basis of an unprecedented opportunity to advance cancer immunotherapy applications beyond CML and melanoma. Accordingly, we have combined our development of third generation self-inactivating (SIN) lentiviral vectors that direct the expression of tumor targeting CARs with cGMP compliant methodologies to isolate, transduce, and expand TCM-derived effector cells (TE(CM)) for the purpose of conducting clinical trials, initially for patients with CD19+ B cell malignancies (leukemias and lymphomas). Recent CD19-specific CAR T cell trials involving patients with CLL and ALL have revealed unprecedented anti-tumor potency, however, tumor regressions have been accompanied by significant life-threatening toxicities and subsequent ongoing B cell aplasia. The anti-tumor potency of current iterations of second generation CD19-specific CAR T cells therefore needs to be matched with strategies to 1.) regulate the functional outputs mediated by CAR signaling, and, 2.) effect the timely ablation of CAR+ T cells to allow for B cell reconstitution. To date, CAR T cell clinical trials have involved vector systems that utilize constitutive promoters. The field clearly needs regulated CAR expression systems capable of responding in real time to clinician-prescribed small molecule inputs to better manage the complex interplay of clinical parameters related to the tempo/extent of tumor regression and side effects, including on-target collateral ablation of healthy cells, for example non-neoplastic B cells, and toxicities resulting from T cell cytokine production (cytokine storm) and tissue inflammation. The Jensen laboratory is focused on developing clinically useful small drug regulated transgene transcriptional regulatory systems and/or drug regulated riboswitches that modulate the stability of transgene mRNA to affect changes in transgene expression levels. The current status of our development of these technologies will be presented. The development of T cell ablation mechanisms that are both highly effective and triggered by commercially available FDA-licensed drugs will be increasingly important as long term engraftment becomes the normative outcome following adoptive transfer of CAR redirected T cells. Ablation of CAR expressing T cells is a significant issue given the observed long-term persistence of CD19-specific T cells, their ongoing stimulation by nascent B cell precursors from bone marrow precursors, and their capacity to mediate ongoing B cell aplasia. Routine clinical use of suicide technologies has been hampered by the immunogenicity of xenogeneic suicide constructs such as HSV-TK, and the lack of commercially available pharmaceutical grade triggering drugs such as those that trigger dimerization based caspase suicide transgenes. Our group has therefore endeavored to design alternate suicide strategies based on the engineering of human EGFR to derive an inert truncated type I transmembrane-extracellular segment of human EGFR, designated EGFRt, that retains the binding epitope of cetuximab. Our studies support the utility of EGFRt when co-expressed with CAR's as a selection, tacking, and suicide construct. Current IND supported clinical trials are employing co-expressed EGFRt and will examine the suicide utility of this construct in patients. Citation Format: Michael Jensen. Advanced T cell engineering for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA22.

Abstract A3: Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases.

Abstract The use of chimeric antigen receptor (CAR)-redirected CD8+ T cells in the treatment of malignancy is a novel strategy that has shown tremendous promise; however, it remains likely this strategy could be enhanced when combined with additional approaches. We hypothesized that deletion of signaling mediators that negatively regulate TCR signal transduction might enhance CAR signaling and function, since CARs utilize the same machinery that transduces TCR signals. We tested CAR activity and function in T cells that lacked one or both lymphocyte-specific isoforms of diacylglycerol kinase (dgk), proteins that metabolize the second messenger DAG and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells, and identify dgks as a potential target for improving the clinical potential of CARs. Citation Format: Matthew Riese, Liang-Chuan Wang, Anjana Ranganathan, Gary Koretzky, Steven Albelda. Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A3.

Effect of Rituximab On the Activity of T Cells Expressing CD20-Specific Chimeric Antigen Receptors

AbstractAbstract 4222 BACKGROUND:Adoptive cellular therapy using autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) has emerged as a promising therapy for lymphoma. Clinical trials for lymphoid malignancies to date have primarily targeted either the CD19 or CD20 antigens. While CD20 has a more established track record as an immunotherapy target, one potential drawback of targeting CD20 with CAR+ T cells is the theoretical possibility that residual levels of circulating anti-CD20 antibodies (Ab) from prior chemoimmunotherapy regimens could partially or completely block CAR-antigen interactions. This could negatively impact the efficacy of CD20-targeted CAR+ T cells. However, previous data from our group and others indicate that CD20 CAR+T cell function is only partially blocked by anti-CD20 Ab, and T cell function in the setting of anti-CD3 × anti-CD20 bispecific Ab is not blocked by rituximab (R) levels of up to 100 μg/ml. Collectively, these data suggest that a very low number of available CD20 binding sites may be sufficient to trigger CAR signaling and T cell activation. METHODS:We tested the effect of different levels of R on in vitro function of polyclonal T cells from healthy donors negatively selected by MACS, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 1st or 3rdgeneration (αCD20-ζ or αCD20-CD28–41BB-ζ) anti-CD20 CARs. T cells were re-stimulated 1 week after initial activation by co-culture with antigen presenting cells (APCs) that had been pre-incubated for 30 minutes with varying concentrations of R (ranging from 0 to 800 μg/ml). APCs were K562 cells transduced to express CD80 with or without CD20 (denoted “K80” and “K80-20”), or Ramos lymphoma cells. Proliferation, cytokine secretion, and cytotoxicity were then assessed as discussed below. RESULTS:We first used flow cytometry to test whether varying concentrations of R blocked binding of the Leu16 Ab and, as expected, found a dose-dependent blockade of CD20 on each cell line, with 50 μg/ml and 200 μg/ml causing near-complete blockade of K80-20 and Ramos cells, respectively. However, despite this apparent blockade, proliferation was largely unimpaired in CFSE-labeled 1st or 3rd generation CAR+ T cells cultured with K80-20 or Ramos cells pre-incubated with R concentrations of up to 400 μg/ml. We concurrently measured cytokine secretion of these T cells using Luminex assays and found that IL-2 and IFN-γ secretion decreased with increasing R levels, but 50–85% of baseline levels were still achieved at R concentrations of up to 100 μg/ml. Cytotoxicity against K80-20 and Ramos target cells in standard 51Cr-release assays by 1st and 3rd generation CAR+ T cells was largely preserved at low R concentrations, and 50–75% of cytolytic activity was retained at 100 μg/ml. Nonspecific proliferation, cytokine secretion, and cytotoxicity were excluded in these experiments by using CAR+ T cells incubated with K80 cells lacking CD20 expression, or T cells transduced with an empty vector as negative controls. Mouse xenograft experiments are currently ongoing to test the effect of serum R levels on the in vivo anti-tumor efficacy of CD20-CAR T cells. CONCLUSIONS:These in vitro results suggest that despite apparent blockade of the CD20 antigen, CAR+ T cells targeting CD20 retain significant activity in the presence of R concentrations of up to 100 ug/ml. Patients receiving 2–3 cycles of R-chemotherapy have serum R trough levels in the range of 30–70 μg/ml. We therefore predict that residual serum R levels will not present a significant impediment to CD20-targeted adoptive T cell therapy given after salvage R-chemotherapy. Disclosures:Jensen:ZetaRx: Equity Ownership, Patents & Royalties. Maloney:Roche: Consultancy; Genentech: Consultancy. Off Label Use: Lentiviral vector encoding a CD20-specific chimeric antigen receptor, used to re-direct autologous T cells to recognize B cell lymphoma cells.