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Finney: bluebird bio: Current Employment, Current equity holder in publicly-traded company. Yeri:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Mao:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Pandya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Alonzo:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hopkins:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hymson:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hu:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Foos:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bhadoriya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hintzen:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Gioia:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Timm:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Massaro:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hause:Bristol-Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Shah:BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding; GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy. Raje:Caribou: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Amgen: Consultancy. Berdeja:Novartis: Research Funding; Lilly: Research Funding; Legend: Consultancy; Takeda: Consultancy, Research Funding; Servier: Consultancy; Teva: Research Funding; Vivolux: Research Funding; Bluebird: Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Research Funding; BMS: Consultancy, Research Funding; Bioclinica: Consultancy; CRISPR Therapeutics: Consultancy, Research Funding; Constellation: Research Funding; Cellularity: Research Funding; Celgene: Consultancy, Research Funding; Glenmark: Research Funding; Genentech, Inc.: Research Funding; EMD Sorono: Research Funding; CURIS: Research Funding; Kite Pharma: Consultancy; Kesios: Research Funding; Karyopharm: Consultancy; Janssen: Consultancy, Research Funding; Prothena: Consultancy; Poseida: Research Funding. Grande:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bitter:Novartis AG, Predicant Biosciences, Biospect, F Hofmann-La Roche: Ended employment in the past 24 months; bluebird bio: Current Employment, Current equity holder in publicly-traded company; Novartis: Ended employment in the past 24 months, Patents & Royalties. Petrocca:bluebird, bio: Current Employment, Current equity holder in publicly-traded company. Friedman:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Sangurdekar:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Biogen: Ended employment in the past 24 months.

Introduction:

Cancer is thought to arise as a consequence of multiple insults to a cell. Mutations that lead to increased expression or activity of proto-oncogenes or decreased expression of tumor suppressors are common insults that have been identified to date. However, when considering tumor viruses, viral proteins that modify cellular gene expression, alter host immune surveillance, or affect signaling pathways are also common players. Notably, several of these tumor viruses encode proteins containing an immunoreceptor-associated tyrosine-based activation motif (ITAM), a signaling motif recently implicated in epithelial cell oncogenesis. As expression of proteins bearing this motif is normally restricted to hematopoietic cells, recent work highlighting the consequences of ITAM expression in epithelial cells suggests it may play a role in solid tumor formation.

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

The PD-1 pathway regulates dysfunctional T cells in chronic infection and cancer, but the role of this pathway during acute infection remains less clear. Here, we demonstrate that PD-1 signals are needed for optimal memory. Mice deficient in the PD-1 pathway exhibit impaired CD8+T cell memory following acute influenza infection, including reduced virus-specific CD8+T cell numbers and compromised recall responses. PD-1 blockade during priming leads to similar differences early post-infection but without the defect in memory formation, suggesting that timing and/or duration of PD-1 blockade could be tailored to modulate host responses. Our studies reveal a role for PD-1 as an integrator of CD8+T cell signals that promotes CD8+T cell memory formation and suggest PD-1 continues to fine-tune CD8+T cells after they migrate into non-lymphoid tissues. These findings have important implications for PD-1-based immunotherapy, in which PD-1 inhibition may influence memory responses in patients.

Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLBmegaTAL resulted in >90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLBdisruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLBgene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLBmegaTAL-treated CAR T cells in vivo.While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLBmegaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLBknockout enhances the potency of CAR T cells. In summary, the CBLBmegaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitroand in vivo,and thus could potentially improve the efficacy of CAR T therapy.

Hooper: bluebird bio: Employment, Equity Ownership. Havens:bluebird bio: Employment, Equity Ownership. Krostag:bluebird bio: Employment, Equity Ownership. Magee:bluebird bio: Employment, Equity Ownership. Martin:bluebird bio: Employment, Equity Ownership. Gupta:bluebird bio: Employment, Equity Ownership. Smurnyy:bluebird bio: Employment, Equity Ownership. Pechilis:bluebird bio: Employment, Equity Ownership. Rode:bluebird bio: Employment, Equity Ownership. Chavkin:bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership.

Chimeric antigen receptor T cells (CAR T) have demonstrated robust clinical efficacy in B cell and plasma cell malignancies. In contrast to success in these hematological malignancies, the application of CAR T cells as a treatment for solid tumors has yielded limited efficacy. While there are multiple mechanisms whereby CAR T cells targeting solid tumors could be hindered in their efficacy, of primary concern is the specificity of recognition. Ideally, investigators desire to select target tumor antigens that are uniquely expressed by the neoplasm to avoid normal tissue (“on-target/off-tumor”) toxicity, which can have serious clinical compilations, including death. We investigated the consequences of on-target/off-tumor CAR T cell recognition of the Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) gene, which has been proposed as a clinical target for CAR T cells. The potential for CAR T-mediated toxicity can be informed by the preclinical application of rigorous bioinformatics, and direct expression analysis using in situ hybridization (ISH), immunohistochemistry, (IHC), and flow cytometry. Using these approaches, we found that in humans and mice, ROR1 was expressed in the lungs and other normal tissues. As a specificity control, we used B-cell maturation antigen (BCMA) and confirmed that BCMA expression was limited to plasma cell-containing lymphoid tissues. Next, we generated anti-ROR1 CAR T cells (cross reactive with both human and mouse ROR1) and human-specific anti-BCMA CAR T cells. Using in vitro culture models, we demonstrated that both the ROR1 and BCMA CAR T cells robustly recognized and killed antigen expressing tumor cell lines. To follow the fate of CAR T cells in vivo, we applied ISH and IHC to study T cell activation and trafficking of anti-ROR1 and control anti-BCMA CAR T cells. ROR1-expressing multiple myeloma cells (ROR1+/BCMA+-RPMI-8226) were injected into the flanks of immune deficient NSG mice, and after tumor establishment, CAR T cells were administered by IV injections. Immediately following administration, CAR T cells traffic to the lung before release and subsequent trafficking to tumor. We observed markedly different behavior of the two CAR T cell populations. The anti-ROR1 CAR T cells accumulated in lung where they caused vasculitis and interstitial pneumonia, whereas anti-BCMA CAR T cells did not. Mice receiving anti-ROR1 CAR T cells lost weight and appeared ill. Within the lung, anti-ROR1 CAR T cells expressed the T cell activation / exhaustion marker PD-1. Lung accumulation and activation required a functional CAR molecule, as no pathology was observed with T cells expressing a CAR containing ROR1 binding elements in which the T cells signaling domains were deleted. Anti-ROR1 CAR T cell trafficking to the tumor was delayed and, compared to anti-BCMA CAR T cells, T cell infiltration of xenografts was sparse and tumor growth inhibition was not observed. By contrast, mice receiving anti-BCMA CAR T cells had robust T cell infiltration of xenografts and tumor clearance within 12 days. These findings raise concern about the effectiveness and safety of anti-ROR1 CAR T cells in humans and demonstrate that this type of preclinical safety assessment paradigm may be useful to de-risk potential CAR targets and for modeling CAR T cell toxicity and efficacy.

Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivoculture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivoculture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivoactivity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitroculture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivoefficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivoexpansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies.