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

Supplementary Figure 2

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

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.

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

CARaMEL T cells persist in long term surviving mice.

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.

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

Cytokines present in serum of ACTIV-treated mice.

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.

Background Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8

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.

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.

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.

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

Mucosal-associated invariant T (MAIT) cells are T cells that recognise vitamin-B derivative Ag presented by the MHC-related-protein 1 (MR1) antigen-presenting molecule. While MAIT cells are highly abundant in humans, their role in tumour immunity remains unknown. Here we have analysed the frequency and function of MAIT cells in multiple myeloma (MM) patients. We show that MAIT cell frequency in blood is reduced compared to healthy adult donors, but comparable to elderly healthy control donors. Furthermore, there was no evidence that MAIT cells accumulated at the disease site (bone marrow) of these patients. Newly diagnosed MM patient MAIT cells had reduced IFNγ production and CD27 expression, suggesting an exhausted phenotype, although IFNγ-producing capacity is restored in relapsed/refractory patient samples. Moreover, immunomodulatory drugs Lenalidomide and Pomalidomide, indirectly inhibited MAIT cell activation. We further show that cell lines can be pulsed with vitamin-B derivative Ags and that these can be presented via MR1 to MAIT cells in vitro, to induce cytotoxic activity comparable to that of natural killer (NK) cells. Thus, MAIT cells are reduced in MM patients, which may contribute to disease in these individuals, and moreover, MAIT cells may represent new immunotherapeutic targets for treatment of MM and other malignancies.

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.

Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours.

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

Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.

Aim: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). Material & methods: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. Results: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. Conclusion: This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.