Ryan S. Cross

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

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.

Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer.

MYB Is Essential for Mammary Tumorigenesis

MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.

CAR-T Cells Inflict Sequential Killing of Multiple Tumor Target Cells

Davenport, Jenkins, and colleagues used time-lapse microscopy and CD8+ T cells coexpressing TCRs and CARs for different antigens to show that CAR T cells can kill multiple tumor cells; engagement via CAR or TCR did not affect killing kinetics; T cells detached faster when CAR was engaged; and CARs are downregulated over time. 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

Therapeutic DNA vaccination against colorectal cancer by targeting the MYB oncoprotein

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.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity

Significance Davenport et al. discovered that the chimeric antigen receptor (CAR) immune synapse structure is different from the T cell receptor (TCR) synapse. The CAR immune synapse formed a disorganized pattern of Lck and more rapidly recruited lytic granules compared with the TCR. The differing immune synapse correlated with faster killing of tumor target cells and detachment from dying tumor cells by CAR-T cells. These findings provide a mechanism whereby CAR-T cells can effectively reduce large tumor burden in patients. This study will form a basis upon which to compare future receptor design to modulate signaling and programming of cytotoxic CAR-T cells to improve treatment of solid cancers. Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Myb via TGFβ is required for collagen type 1 production and skin integrity

Abstract Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb+/− mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers–Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfβ−1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.

Hsp70 Architecture: The Formation of Novel Polymeric Structures of Hsp70.1 and Hsc70 after Proteotoxic Stress

Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

Immunomodulation by MYB is associated with tumor relapse in patients with early stage colorectal cancer

ABSTRACT The presence of tumor immune infiltrating cells (TILs), particularly CD8+ T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8+ T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8+ and CD45RO+ -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8+ TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8+ TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.

Abstract B10: Individual CAR-T cells, when activated via their CAR or TCR, kill tumor cells with similar kinetics and can kill multiple sequential tumor targets

Introduction: Chimeric antigen receptor (CAR) T cells have had spectacular success in the clinic, particularly in B cell malignancies. This clinical success was built on years of pre-clinical development; despite this there remains aspects of CAR-T cell responses to antigen activation, which have not been fully explored. In this study we compared the kinetics of CAR- versus TCR-mediated activation, immune synapse formation and tumor cell killing. This study has particular clinical relevance to CAR-T cell therapy, including conventional CAR-T cells and the more recently described dual antigen receptor T cells. Experimental procedures/strategy: To address these questions, we developed an in vitro model system where T cells could be activated via a CAR or TCR in response to cognate antigen on tumor cells. We used CD8+ T cells from a novel transgenic mouse model where both the CAR and OT-I TCR were constitutively expressed (CAR.OT-I cells), the CAR comprised an scFv directed to human HER-2 (hHER-2) and intracellular signaling domains (CD28 and CD3ζ). Effector CAR.OT-I cells were activated via their TCR using MC57 cells pulsed with SIINFEKL (MC57 OVA257), or via their CAR using MC57 cells, which expressed hHER-2. Interactions between individual effector CAR.OT-I cells and the tumor cells were monitored via time lapse live video (TLLM) microscopy and the kinetics of effector cell activation, delivery of lethal hit, tumor cell killing and effector cell detachment recorded. In addition, short term (four and eight hour Cr-release assay) and long term (50 hour xCELLigence assay) killing assays were performed to compare killing capacity and kinetics. Key data: When effector CAR.OTI cells recognized targets via their TCR or CAR, they displayed the same kinetics for activation, delivery of lethal hit and tumor cell killing to OTI CTL. In contrast, time for CAR.OT-I cell detachment from tumor cells was significantly shorter when activated via the CAR rather than the TCR. Short term killing assays (4-8 hours) showed no difference in bulk CAR.OT-I cell killing of tumor cells. In contrast, longer term killing assays (50 hours) showed there was a significant difference in tumor cell killing rate between CAR- versus TCR-activated. The difference in killing rate was only apparent from 20-50 hours, when CAR-activated CAR.OT-I cells had a significantly reduced tumor cell killing rate compared to TCR-activated CAR.OT-I cells. Finally, we showed for the first time, that individual CAR. OT-I cells were capable of killing multiple tumor target cells (serial killing). This occurred whether the CAR.OT-I cells were activated via the CAR or TCR. Conclusion: In conclusion, this study provides novel information regarding CAR-T cell killing of tumor cells and has practical implications for the use of CAR-T cells in the clinic, whether the therapy is using single or dual antigen receptor T cells. Citation Format: Alex J. Davenport, Misty R. Jenkins, Ryan S. Cross, S Yong Carmen, H Miles Prince, David S. Ritchie, Joseph A. Trapani, Michael H. Kershaw, Phillip K. Darcy, Paul J. Neeson. Individual CAR-T cells, when activated via their CAR or TCR, kill tumor cells with similar kinetics and can kill multiple sequential tumor targets. [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 B10.