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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.

Supplementary Figure 2

Figure S4 shows the effect of CXCR3 blockade on the number and phenotype of tumor-infiltrating immune cells post combination therapy

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

Cytokines present in serum of ACTIV-treated mice.

CARaMEL T cells persist in long term surviving mice.

Purpose:Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies.Experimental Design:The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses.Results:The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB.Conclusions:These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Supplementary Figure Legends

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

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.

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.

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478–90. ©2016 AACR.

ACTIV therapy induces cytokine secretion and apoptosis of tumor cells.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic., Activation of the adenosine receptor A2AR is associated with suppression of T cell function in the tumor microenvironment. To overcome immunosuppression, here the authors show that CRISPR/Cas9 mediated deletion of A2AR enhances CAR T cell effector functions without altering memory or persistence properties, improving CAR-T mediated tumor control in pre-clinical models.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8(+)CD62L(+)TCF7(+)IRF4(–) population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7(+) phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Purpose: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. Experimental Design: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. Results: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. Conclusions: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

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.

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging. RESULTS Cytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D). CONCLUSIONS Here we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. Figure 1 Figure 1. Disclosures Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model. Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors. Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient. Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478–90. ©2016 AACR.

The development of new cancer immunotherapies including checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment. CAR T cells have shown tremendous success in certain B cell malignancies, resulting in U.S. Food and Drug Administration (FDA) approval of this approach for certain types of leukemia and lymphoma. However, response rates against solid cancer have been less successful to date. Approaches to modulate the immunosuppressive tumor microenvironment including targeting checkpoint pathways, modulating metabolic pathways, and generating cytokine-producing T cells have led to considerable enhancement of adoptive T cell immunotherapy, first in preclinical models and now in patients. This review provides a discussion of the most recent strategies to enhance the efficacy of CAR T cell antitumor responses in solid cancers.

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.

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19, CD22, or B cell maturation antigen (BCMA). Antigen density is an important factor modulating CAR T cell responses, and therefore antigen expression below a certain threshold may fail to induce optimal CAR-T cell response. This presents a significant problem as antigen expression varies highly in many cancers including lymphoma, leukemia, multiple myeloma, and solid tumors, both between and within patients. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two antigens will result in enhanced CAR T cell signaling, allowing them to attack low antigen density (LAD) tumors that would otherwise escape CAR T cells. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 (FMC63), CD22 (CD22-12), or both, herein referred to as CAR19, CAR22, or CAR19/22, respectively. Tumor cells expressing variegated antigen densities were generated using lentivirus transduction or mRNA electroporation. For in vitro functional characterization, co-culture assay of T cells and tumor cells was performed, and CAR T cell anti-tumor potency was determined by assessing T cell functional parameters including T cell cytotoxicity in real-time using the Incucyte® systems for live-cell imaging and analysis. In addition, cytokine production and CFSE-based proliferation were also assessed using flow cytometry. RESULTS Using CAR19/22, we found LAD tumors are indeed more susceptible to bi-specific CAR T cells compared to either CAR19 or CAR22 T cells, as demonstrated by significantly higher amounts of cytokines produced by bi-specific CAR T cells such as IL-2 (p CONCLUSIONS Here we showed that antigen density on tumors modulates T cell transcriptional profiles. Our results demonstrated that bi-specific CAR19/22 T cells are superior than mono-specific CAR19 or CAR22 T cells against LAD tumors, as demonstrated by their enhanced cytokine-producing function, cytotoxic capacity and proliferation. Results from this study will provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing the risk of antigen-negative escape. Disclosures Ruella: Abclon, BMS, NanoString: Consultancy; Abclon: Consultancy, Research Funding; UPenn/Novartis: Patents & Royalties. Gill:Tmunity Therapeutics: Research Funding; Sensei: Consultancy; Aileron: Consultancy; Fate: Consultancy; Carisma Therapeutics: Patents & Royalties, Research Funding; Novartis: Research Funding.

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

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

Adoptive immunotherapy utilising chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematological cancers, with up to 90% complete remission being reported in patients with acute lymphoblastic leukemia (ALL). However, this success is not currently transferrable to solid malignancies due in part to the immunosuppressive tumor microenvironment limiting CAR T cell responses. Given that activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong co-stimulatory signals for augmenting and diversifying T cell responses, we hypothesized that the combination of α-4-1BB and CAR T cell therapy would result in improved anti-tumor responses. Using a human-Her2 self-antigen mouse model, we found that α-4-1BB significantly enhanced CAR T cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Investigation into the mechanism revealed increased expression of IFNγ and the proliferation marker Ki67 by tumor-infiltrating CAR T cells when combined with α-4-1BB. The importance of IFNγ was demonstrated in vivo where synergistic effects of the combination therapy were abrogated when IFNγ was depleted. Strikingly, we also found that α-4-1BB significantly reduced host immunosuppressive cells at the tumor site including regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), correlating with an increased therapeutic response. Investigation in immunocompromised RAG -/- mice showed reduced synergistic effects following the combination therapy, suggesting some contribution by host T cells. Furthermore, we found increased frequency of endogenous T cells and dendritic cells in the tumor-draining lymph nodes, supporting potential engagement of host immune cells. We therefore conclude α-4-1BB has a multifunctional role for enhancing CAR T cell responses including engagement of endogenous immune cells, and this combination approach has high translational potential given that CAR T cell therapy has recently been FDA approved for pediatric ALL, and that α-4-1BB is currently being tested in clinical trials against various types of cancer. Citation Format: Sherly Mardiana, Liza B. John, Michael H. Kershaw, Paul A. Beavis, Phillip K. Darcy. A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1530.