Adrienne H. Long

4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor 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 show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

The efficacy of chimeric antigen receptor (CAR)–modified T cells against solid tumors is not proven. Retinoids are clinically accessible and were found to modulate tumor myeloid-derived suppressor cells, enhancing the efficacy of CAR therapies targeting solid tumors. Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869–80. ©2016 AACR.

Comparison against 186 canid whole-genome sequences reveals survival strategies of an ancient clonally transmissible canine tumor.

Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells. Little is understood about the mechanisms that converted an ancient tumor into the worlds oldest known continuously propagating somatic cell lineage. We created the largest existing catalog of canine genome-wide variation and compared it against two CTVT genome sequences, thereby separating alleles derived from the founders genome from somatic mutations that must drive clonal transmissibility. We show that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations in oncogenesis- and immune-related genes that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVTs dogged perseverance in canids around the world.

Lessons learned from a highly-active CD22-specific chimeric antigen receptor

CD22 is an attractive target for the development of immunotherapeutic approaches for the therapy of B-cell malignancies. In particular, an m971 antibody-derived, second generation chimeric antigen receptor (CAR) that targets CD22 holds significant therapeutic promise. The key aspect for the development of such a highly-active CAR was its ability to target a membrane-proximal epitope of CD22.

Neutralization of murine myeloid-derived suppressor cells enhances the efficacy of a chimeric antigen receptor T-cells directed against pediatric solid tumors

Genetically engineered T-cells that express chimeric antigen receptors (CARs) to directly target tumor-expressed antigens have shown remarkable activity in clinical trials for hematologic malignancies, but remain unproven for the treatment of solid tumors. We find that in addition to its well known high expression in neuroblastoma, the disialoganglioside GD2 is also highly expressed on pediatric sarcomas. Some human sarcoma cell lines have GD2 expression levels equivalent to that of the prototypical neuroblastoma cell line LAN5, and immunohistochemical staining of primary human tumor tissue samples taken from essentially all patients with metastatic osteosarcoma and a subset of patients with alveolar rhabdomyosarcoma demonstrate robust expression of GD2 on the cell surface. Based on these results, we have developed models to test the ability of GD2-CAR T-cells to target and lyse human sarcomas in vitro and in vivo. We find that GD2-CAR modified T-cells induce specific lysis of GD2-expressing solid tumors in vitro even at effector:target ratios as low as 1:1, but fail to induce a significant response in vivo using the same human cell lines. Interestingly, we discovered that the pediatric sarcoma xenografts induce a large expansion of murine CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) that inhibit human T-cell responses in vitro. These results lead us to adopt a combinatorial therapeutic strategy in which we first neutralized the suppressive potential of MDSC by administration of all-trans retinoic acid (ATRA) followed by GD2-CAR therapy. This combinatorial approach results in significant improvements in both overall survival and tumor growth. Given that retinoids are already available in the clinic, these results suggest that the effectiveness of CAR T-cell therapy for solid tumors could be enhanced by coadministration of retinoids to modulate the myeloid derived suppressor cells.

Abstract 2648: Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression

ALK is overexpressed on the surface of neuroblastoma (NB) and is associated with high risk disease. We developed a second generation CAR based on a monoclonal antibody against ALK. ALK CAR T-cells significantly delay the growth of human NB cell lines in murine xenograft models, but animals eventually succumb to tumors. In order to understand how target antigen density limits the effectiveness of this CAR, we created a library of NALM-6 B-cell leukemias with variable amounts of ALK expressed on the surface of each clone. In vitro, ALK CAR T-cells lyse high ALK expressing leukemias but have reduced activity against leukemias with low expression of ALK. In co-culture assays, we found that there is a minimum target antigen density required for CAR T-cells to produce appreciable amounts of Th1 cytokines. This threshold of ALK expression is above the expression on human NB lines. There is also a threshold target antigen density at which maximum cytokine production occurs. Above this density, no additional cytokines are produced. In xenograft models, ALK CAR T-Cells are significantly more effective against high ALK expressing leukemia than against low ALK expressing leukemia. To our knowledge, this is the first report of greater in vivo efficacy of CAR T-cells against tumors with higher antigen expression. The level of surface expression of the CAR on T-cells also alters the function of CAR T-cells. We have identified a phenomenon in which both ALK and CD19 CAR T-cells lose surface expression of CAR over time in culture. Additionally, both the ALK CAR and the CD19 CAR downregulate quickly after T-cells are exposed to antigen. Using a fluorescently tagged CD19 CAR, we demonstrate that CARs are rapidly internalized upon antigen encounter. We created an assay to understand the interplay of target density and CAR surface expression. We transduced T-cells with different amounts of supernatant to achieve different ALK CAR surface densities and then exposed these T-cells to varying amounts of immobilized protein-L in order to crosslink the CAR. T-cell activation (measured by an NFAT reporter) is greatest when both CAR surface expression and target antigen density are highest. As both variables are decreased, there is a significant loss of T-cell activity. Thus, diminished CAR surface density due to antigen-dependent and independent CAR downmodulation may limit CAR T-cell efficacy, especially in the context of low tumor antigen expression. In conclusion, we have created a novel CAR targeting ALK that demonstrates in vivo efficacy against NB. Efficacy is limited by low target antigen density on NB and low CAR surface expression. We have identified phenomena in which CAR T-cells lose surface expression of CAR over time and also quickly internalize their receptors in response to antigen. These factors contribute to the efficacy of other CAR T-cells and this data provides important insights into future CAR target selection. Citation Format: Robbie G. Majzner, Alec J. Walker, Meera Murgai, Ling Zhang, Adrienne H. Long, Kelsey M. Wanhainen, Rimas J. Orentas, Crystal L. Mackall. Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2648.

Chimeric Antigen Receptors for Cancer: Progress and Challenges

Chimeric antigen receptors (CARs) genetically link an antigen-binding domain with cell-signaling domains to redirect immune cell specificity toward antigens expressed on the surface of cancer cells. Progress in CAR engineering over the last two decades has elucidated fundamental principles impacting CAR potency, and today CARs can be readily generated toward essentially any cell surface target on cancer cells. Efficacy thus far has been most impressive using CD19-CAR expressing T cells to treat B cell lymphoblastic leukemia, although clear activity has also been observed using CD19-CARs in patients with chronic lymphocytic leukemic and B cell lymphoma. Much less data is available regarding CAR efficacy for solid tumors. Here, we summarize current concepts of CAR design, with a focus on the relationship between structure and function, a review of the clinical results reported thus far, and the challenges to be addressed in future studies.

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

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 3141: Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has proven effective at treating hematologic malignancies, however similar success has not been observed using CARs targeting other antigens. We have observed that several CARs, including a GD2-targeted CAR, are limited in efficacy due to T cell exhaustion induced by tonic, antigen-independent signaling. This phenomenon is triggered by oligomerization of scFvs on the surface of transduced T cells, leading to chronic CAR zeta signaling and early exhaustion characterized by a diminished proliferative potential, poor cytokine production, and overexpression of inhibitory receptors including PD1, TIM3 and LAG3. Early CAR T cell exhaustion is also associated with an altered cellular metabolism that is more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced, activated control cells. Using a Seahorse Extracellular Flux Analyzer, we found that the rate of glycolysis (ECAR) in GD2 CAR T cells was twice that of CD19 CAR T cells or untransduced controls, and no deficiencies in oxidative phosphorylation in GD2 CAR T cells were observed (normal OCR and spare respiratory capacity). To evaluate whether increased glycolytic metabolism contributes to the development of CAR T cell exhaustion, we sought to restrict glycolysis by culturing GD2 CAR T cells with the hexokinase inhibitor 2-deoxyglucose (2DG) or in media supplemented with galactose instead of glucose. Restricting CAR T cell glycolysis was associated with decreased TIM3 and LAG3 expression and enhanced cytokine production of GD2 CAR T cells but a modest decrease in cytokine production of CD19 CAR T cells. Given the previously reported role of mTOR on promoting glycolytic metabolism, we used rapamycin (sirolimus) in the cell culture during transduction and expansion. Culturing GD2 CAR T cells with rapamycin similarly resulted in slower acidification of cell culture medium and diminished expression of TIM3 and LAG3. Rapamycin treated GD2 CAR T cells retain specific lytic ability of GD2-positive tumor cells compared to non-treated GD2 CARs as measured by chromium release, and these cells persist longer than non-treated cells in a murine xenograft model of GD2-positive 143b osteosarcoma. These results suggest that modulation of T cell metabolism may provide functional benefit and enhance antitumor activity of adoptively transferred tumor antigen specific T cells by diminishing the susceptibility of highly activated T cells to exhaustion. Citation Format: Jillian P. Smith, Adrienne H. Long, Crystal L. Mackall. Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified 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 3141. doi:10.1158/1538-7445.AM2015-3141

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

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