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Patients who receive chimeric antigen receptor (CAR)–T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8 + memory T cell progenitors that can become either functional stem-like T (T STEM ) cells or dysfunctional T progenitor exhausted (T PEX ) cells. To that end, we demonstrated that T STEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate T STEM -like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, T STEM -like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4 + T cells during T STEM -like CAR-T cell production. Adoptive transfer of T STEM -like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of T STEM -like CAR-T cells and an increased memory T cell pool. Last, T STEM -like CAR-T cells and anti–programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8 + CAR + T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated T STEM -like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

Movie 2. CAR.OT-I cells co-cultured with MC57-OVA257 cells.

This figure shows the proliferation and phneotype of tumor lines in response to NECA.

Supplementary Figure 2

This supplementary Figure shows the phenotype and function of CD8+ T cells and tumor cells following A2A blockade single agent therapy.

This supplementray Figure shows the expression of IFNy and Granzyme B on tumor-infiltrating lymphocytes 7 days post anti-PD-1/ anti-PD-1/A2A blockade.

This Supplementary Figure shows the expression of CD73 and adenosine receptors by tumor lines.

Supplementary Figure 2. OVA257 peptide dose does not affect effector cell maturation status.

Movie 4. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the TCR

This document contains the Figure Legends for the Supplementary data Figures.

This Supplememntary Figure shows the effect of combined PD-1 and A2A blockade on in vitro effector functions of OT-I cells.

This supplementary Figure shows the expression of A2A and IFNy on tumor infiltrating lymphocytes following PD-1 blockade.

Movie 3. CAR.OT-I cells co-cultured with MC57-HER2 cells.

Supplementary Figure 1. TCR activated OT-I and CAR. OT-I cells display similar expression levels of cytotoxic granules.

This supplementary figure shows the phenotype of tumor-infiltrating lymphocytes following PD-1 or PD-1/A2A blockade.

Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2Kb–presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells (“serial killing”), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T–tumor cell interactions, with implications for single- or dual receptor–focused T-cell therapy. Cancer Immunol Res; 3(5); 483–94. ©2015 AACR.See related commentary by June, p. 470

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3− cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3− T cell–mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3− cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069–81. ©2018 AACR.

Movie 5. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the CAR

Supplementary Methods. Time lapse live video (TLLV) microscopy

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8+ T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8+ T cells and, accordingly, increased growth inhibition of CD73+ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.

This supplementary figure shows the expression of PD-1 on tumor infiltrating immune subsets.

Supplementary Figure 1 - PDF file 73K, Tumor cells express high levels of PD-L1

CARaMEL T cells persist in long term surviving mice.

Supplementary Figure 3. Pano induced pancreatic tumor cell apoptosis.

Supplementary Data from Toll-Like Receptor Triggering and T-Cell Costimulation Induce Potent Antitumor Immunity in Mice

Supplementary Figure 2 - PDF file 20K, Expression of PD-1 is upregulated on CAR T cells following antigen stimulation with e0771-Her2+ tumor cells

Supplementary Figure 2. Pancreatic cells are resistant to VV-gp100 oncolysis but sensitive to CAR T cell-mediated cytotoxicity.

upplementary Figure 3 - PDF file 17K, Gr1+ cells express PD-1 and PD-L1

Supplementary Figure 4. Pano did not enhance the cytotoxicity of CARaMEL cells nor oncolysis of VV-gp100.

Purpose: To determine the antitumor activity of a novel combination of two immunomodulatory agents that simultaneously direct multiple components of immunity against cancer.Experimental Design: We combined the Toll-like receptor agonist CpG 1826 with a T-cell costimulatory antibody specific for CD137 in an optimal treatment route and dosing schedule against established tumors in two mouse models. Mechanistic insight was gained using gene-deficient mice and cell-depleting antibodies.Results: The combination was shown to eradicate tumors in a large proportion of mice. Crucial roles for CD8+ T cells, natural killer cells, and IFNs were shown. CpG and anti-CD137 injection led to activation of dendritic cells and optimal expansion of activated T cells in the blood. Macrophages were not necessary for therapeutic effect, and indeed depletion of macrophages in vivo enhanced therapy leading to tumor rejection in 100% of mice, which has not been previously reported in the immunotherapeutic setting. Long-term surviving mice were resistant to tumor rechallenge, demonstrating immunologic memory. In addition, we show, for the first time, that mice lacking B cells have a total loss of a recall response against tumor, suggesting a role for B cells in the induction of antitumor immunologic memory.Conclusion: This study provides support for the use of a novel combination of immunomodulatory agents stimulating multiple facets of immunity for the effective immunotherapy of cancer. (Clin Cancer Res 2009;15(24):7624–33)

Supplementary Figure 1. Panc02-Her2, KPC-Her2 and 24JK-Her2 cells express comparable levels of Her2.

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression.Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach.Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen.Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR.

Supplementary Figure 5. Pano did not alter Her2 or H2-Db expression on Panc02-Her2 tumor cells in vivo and CARaMEL cells were detectable over 100 days in ACTIV+ Pano treated mice.

CAR T cells lacking the pMEL TCR are less effective than CARaMEL T cells at inhibiting tumor growth.

Supplementary Figure Legends - PDF file 66K, Supplementary Figure legends

Dual-specific T cells respond against both gp100 and Her2.

Supplementary Figure S1. Expression of activation and memory markers on transduced mouse T cells. Supplementary Figure S2. CAR T cell cytotoxic responses are not modulated by anti-4-1BB mAb therapy. Supplementary Figure S3. Her2 expression level on the mouse breast cancer cell line e0771-Her2 is comparable to the Her2-overexpressing human breast cancer cell line SKBR3. Supplementary Figure S4. Tumor weight is significantly reduced following CAR T cell and alpha-4-1BB therapy. Supplementary Figure S5. Anti-4-1BB therapy does not modulate the frequency of transferred or endogenous CD8+ tumor-infiltrating T cells. Supplementary Figure S6. Anti-4-1BB therapy modulates the frequency of mature dendritic cells in tumors and draining lymph nodes. Supplementary Figure S7. Anti-4-1BB does not modulate the frequency of MDSCs and DCs in RAG -/- mice. Supplementary Figure S8. Anti-4-1BB therapy drives maturation of NK cells.

High dose IL-2 enables durable tumor regression and proliferation of T cells.

Cytokines present in serum of ACTIV-treated mice.

Supplementary Figure 8. Proposed mechanisms and clinical application for the ACTIV+Pano treatment.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296–309. ©2017 AACR.