BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Cancer cells exhibit increased use of nutrients including glucose and glutamine to support the bioenergetic and biosynthetic demands of proliferation. We tested the small molecule inhibitor of glutaminase CB-839 in combination with erlotinib on EGFR mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMPK pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-Glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following CB-839 + erlotinib treatment. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

Background: Despite advances in the treatment of multiple myeloma (MM) almost all patients relapse and high risk features continue to portend a short median survival. The adoptive transfer of B-Cell Maturation Antigen (BCMA) chimeric antigen receptor (CAR) T cells is demonstrating early promise in MM, but the durability of response has not been established. The infusion of genetically modified CD8+ and CD4+ T cells of a defined composition facilitates the evaluation of each subset's function and has contributed to reproducible efficacy and safety in clinical trials with CD19-specific CAR T cells. In this phase I first-in-human study employing a human scFv containing BCMA CAR T cell construct, we report rapid and deep objective responses at a low CAR T cell dose level (5 x 107) suggesting that construct specific features and differences in product formulation may substantially impact efficacy. Methods: Eligible patients had relapsed or treatment refractory MM, ≥10% CD138+ bone marrow (BM) plasma cells (PC), and ≥5% BCMA expression by flow cytometry (FC). Patients were stratified by tumor burden (CD138+ IHC) into two cohorts; 10-30% MM cells [cohort A] or >30% BM involvement [cohort B] to facilitate assessment of impact of disease burden on outcome. Eligible patient's CD8+ and CD4+ T cells were isolated via positive selection, enriched separately by immunomagnetic selection and cryopreserved. The CD8+ and CD4+ T cells were stimulated in independent cultures with anti-CD3/anti-CD28 paramagnetic beads and transduced with a 3rd generation lentiviral vector encoding a fully human BCMA scFv and 4-1BB and CD3 zeta signaling domains. After in vitro expansion, the cell product for infusion was formulated in a 1:1 ratio of CD4+:CD8+ BCMA CAR T cells. A truncated non-functional human epidermal growth factor receptor (EGFRt) was encoded in the transgene cassette to identify transduced T cells. Lymphodepleting chemotherapy preceded infusion of EGFRt+ CAR T cells at a starting dose of 5 x 107 EGFRt+ cells (n=5) for each cohort. Results: Seven patients (median age of 63 years; range 49 to 76) with a median of 8 prior regimens (range 6 to 11) have received treatment. The median %PC in BM (IHC) at enrollment was 58% (range 20% to >80%). In cohort B the dose has been escalated to 15 x107 EGFRt+ cells (n=2). All patients (7/7) had at least one high risk cytogenetic feature (17p- [n=4], t(4;14) [n=2], t(14;16) [n=1]), 71% had ≥ 2 high risk cytogenetic features, 71% had prior autologous stem cell transplant, 43% had prior allogeneic transplant, and one patient (14%) had PCL. The median involved free light chain (FLC) at enrollment was 180 mg/dL (range 40.37 to 502.96 mg/dL; n=5) and the median monoclonal protein was 3.8 g/dL (range 1.6 to 6.5 g/dL; n=5). The overall response rate at 28 days was 100%; at this time all evaluable patients (n=6) had no detectable abnormal BM PC clone by both IHC and high sensitivity FC. Within 28 days of treatment the involved FLC was normal or sub-normal in all patients and the M-protein had decreased by a median of 73% (range 56.25 to 83% reduction). BCMA CAR T cells remained detectable 90 days after infusion, representing up to 41.5 percent of CD3+ lymphocytes. All patients were surviving at a median of 16 wks (range 2 to 26 wks). One patient relapsed (day +60) and a tumor biopsy demonstrated the presence of a BCMAneg PC population, a 70% reduction in the fraction of MM cells expressing BCMA by FC and a fivefold reduction in BCMA antigen binding capacity on MM cells retaining target expression. A cytotoxic T lymphocyte response to the trans-gene product was not identified in this patient. No dose limiting toxicity has been observed during the 28 day monitoring window and treatment has been well tolerated with no cytokine release syndrome (CRS) observed in one patient and grade 2 or lower CRS (Lee Criteria) for all other patients. No neurological toxicity has been observed. Conclusion: BCMA CAR T cells harboring a fully human scFv with a defined composition of CD4+:CD8+ T cells were well tolerated and potent, demonstrating complete objective responses in heavily pretreated high-risk MM at total cell doses as low as 5 x 107. Next generation sequencing and multiparameter high sensitivity flow cytometry studies to evaluate for minimal residual disease are ongoing. Peak expansion levels and persistence of the CAR T cells are being monitored with early findings suggesting an absence of transgene product immunogenicity. Disclosures Green: Juno Therapeutics: Patents & Royalties, Research Funding. Sather:Juno Therapeutics: Employment. Cowan:Janssen: Research Funding; Abbvie: Research Funding; Juno Therapeutics: Research Funding; Sanofi: Research Funding. Turtle:Caribou Biosciences: Consultancy; Gilead: Consultancy; Bluebird Bio: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptevo: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Consultancy, Research Funding; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy. Till:Mustang Bio: Patents & Royalties, Research Funding. Becker:GlycoMimetics: Research Funding. Blake:Celgene: Employment, Equity Ownership. Works:Juno Therapeutics: Employment. Maloney:GlaxoSmithKline: Research Funding; Juno Therapeutics: Research Funding; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria; Janssen Scientific Affairs: Honoraria. Riddell:Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy.

Elevation of intracellular cyclic adenosine monophosphate (cAMP) concentrations and subsequent regulation of downstream target gene expression through phosphorylation of cAMP-responsive element binding protein (CREB) is hypothesized to underlie the mechanism(s) of long-term memory (LTM) formation. The phosphodiesterase 4 (PDE4) enzyme family is believed to play a key role in LTM by regulating cAMP levels. Thus far, four PDE4 isoforms have been identified (PDE4A, B, C and D); however, the requisite involvement of each of these isoforms in mediating LTM has yet to be elucidated. In the present study, genetic knockout mice were used to investigate the involvement of the PDE4D isoform in both in vitro and in vivo models of learning and memory. Hippocampal synaptic transmission measured electrophysiologically in CA1 slice preparations was similar between wild-type and PDE4D (-/-) mice yet, relative to wild-type controls, knockout mice displayed enhanced early long-term potentiation (LTP) following multiple induction protocols. Interestingly, the PDE4D (-/-) animals exhibited significant behavioral deficits in associative learning using a conditioned fear paradigm as compared with control littermates. The impairment in fear conditioning observed in the PDE4D (-/-) mice could not be attributed to differences in acquisition of the task, alterations in locomotor activity or effects on shock sensitivity. Overall, the in vitro and in vivo alterations in synaptic plasticity observed in the PDE4D (-/-) mice may be explained by adaptive responses occurring throughout development, and suggest that the PDE4D isoform may be an important mediator of LTM formation.

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making Janus kinase (JAK) inhibition an appealing strategy for the treatment of psoriasis. Here we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and Tyrosine Kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose-dependently (1 nM-10 μM) inhibited JAK1 and/or TYK2 dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild type mice with SAR-20347 significantly reduced IL-12 induced IFN-γ production and IL-22-dependent Serum Amyloid A (SAA) to similar extents, indicating that in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes, and proinflammatory cytokine levels compared to both TYK2 mutant mice and wild type controls. Taken together, these data indicate that targeting both JAK1 and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

Background: Although the median survival for patients with multiple myeloma has improved dramatically, almost all patients will eventually relapse and become resistant to standard therapies. Chimeric antigen receptor T cells (CAR T cells) targeting B cell maturation antigen (BCMA) have shown early promise in MM, with high initial response rates. Responses are often incomplete and durability has been a key concern, with most patients relapsing within 1 year (Raje N et al NEJM 2019). We have previously demonstrated that gamma secretase inhibitors (GSI) increase BCMA surface density, decrease soluble BCMA levels and augment anti-tumor efficacy of BCMA CAR T cells in preclinical models. In a phase I first-in-human trial (NCT03502577), we combined CAR T cells expressing a fully human BCMA scFv with an orally administered gamma secretase inhibitor (JSMD194). Methods: Eligible patients had relapsed/refractory MM, with ≥ 10% plasma cells in the bone marrow by CD138 IHC, and measurable disease by IMWG criteria. BCMA was measured on CD138+ plasma cells by flow cytometry. CD8+ and CD4+ T cells were isolated via positive selection. The T cells were stimulated in separate cultures and transduced with lentiviral vector encoding a fully human BCMA scFv in conjunction with 41BB and CD3 zeta signaling domains. Following expansion, the cell product was formulated in a 1:1 ratio of CD4+:CD8+ BCMA CAR T cells. To assess the discreet impact of the GSI on plasma cell BCMA expression, patients received a GSI (JSMD194) monotherapy "run-in" involving three oral doses (25 mg) administered 48 hours apart over 5 days. A bone marrow aspirate was obtained on day 5 and BCMA expression on tumor cells was compared to baseline. Then, after lymphodepleting chemotherapy, BCMA CAR T cells were infused at a total starting dose of 5 x 10^7 EGFRt+ cells, in combination with JSMD194 dosed at 25 mg thrice weekly for three weeks, starting on the day of CAR infusion. Results: Eight patients, with a median age of 64.5 (range, 50-70 years) and a median of 10 prior regimens (range, 4-23), were screened, and seven patients have been treated. One patient had not responded to prior treatment with BCMA CAR T cells using a different construct, and another had progressed on a clinical trial employing a BCMA bispecific antibody. Median bone marrow plasma cell involvement by IHC was 32.5% (range, 10-80%) at enrollment. High-risk features were present in 75% of patients. Median involved serum free light chain at screening was 68.7 mg/dL (range18.45 - 365.61 mg/dL) and median monoclonal protein was 2.55 g/dL (range 0.1 - 5.1 g/dL). Following 3 oral doses (run in) of JSMD194, the percent of plasma cells expressing BCMA increased from 75% to 99% (7.6 to 98% pre, 75 to 100% post), soluble BCMA decreased by 2.0 fold (range 1.6 to 2.6 fold) after 3 oral doses, and BCMA antigen binding capacity increased from a median of 718 receptors to 13355 receptors per cell, or a median of 20-fold (range, 7.55-fold to 156.68-fold). Among 6 assessable patients, the best overall response rate was 100% (5 VGPR, 1 PR), with 5/6 patients MRD negative by flow. At data cutoff of July 15, 2019, no patient has relapsed, with a median follow-up of 5 months (range 1-11 months). One patient died at day 33 post-CAR T cell in the setting of cytokine release syndrome and concurrent fungal infection. The most common non-hematologic ≥ Grade 3 AE was neutropenic fever in 70%. CRS occurred in 100% of patients, primarily grades 1-2 (Lee Criteria), and neurotoxicity in 70%. Conclusions: Although BCMA CAR T cell therapy has demonstrated potent anti-tumor efficacy in multiple myeloma, a significant proportion of patients relapse. The mechanism of myeloma recrudescence requires further study, however BCMA antigen loss has been observed after CAR T cell therapy and is a putative pathway for tumor escape. In this study we demonstrate that gamma secretase inhibition with JSMD194 routinely increases BCMA surface density on myeloma cells in treated patients and reduces soluble BCMA. The combination of a gamma secretase inhibitor with BCMA CAR T cells leads to rapid responses including in patients that have failed prior BCMA targeted therapy. These responses are achieved with low CAR T cell doses. Longer follow up is required to determine if the durability of response is improved. Disclosures Cowan: Cellectar: Consultancy; Juno: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Abbvie: Research Funding. Pont:Fred Hutchinson Cancer Research Center: Other: Inventor on a patent. Sather:Lyell Immunopharma: Employment. Turtle:T-CURX: Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Humanigen: Other: Ad hoc advisory board member; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member. Till:Mustang Bio: Patents & Royalties, Research Funding. Libby:Alnylam: Consultancy; Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy. Becker:AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; Accordant Health Services/Caremark: Consultancy; The France Foundation: Honoraria. Blake:Celgene: Employment, Equity Ownership. Works:Celgene: Employment, Equity Ownership. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria; A2 Biotherapeutics: Honoraria, Other: Stock options . Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Green:Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; GSK: Consultancy; Celgene: Consultancy; Seattle Genetics: Research Funding; Cellectar: Research Funding. OffLabel Disclosure: Gamma secretase inhibitor to increase BCMA expression in multiple myeloma

Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have shown promising clinical responses in patients with relapsed/refractory multiple myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, drives antimyeloma activity, and alters the suppressive microenvironment; these properties may effectively combine with anti-BCMA CAR T cells to enhance function. Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell function in a concentration-dependent manner. Lenalidomide increased CAR T effector cytokine production, particularly under low CAR stimulation or in the presence of inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced CAR T cytokine production, cytolytic activity, and activation profile relative to untreated CAR T cells in chronic stimulation assays. This unique potentiation of both short-term CAR T activity and long-term functionality during chronic stimulation prompted investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures from RNA sequencing and assay for transposase-accessible chromatin using sequencing indicated that pathways associated with T-helper 1 response, cytokine production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a murine, disseminated, multiple myeloma model indicated that lenalidomide increased CAR T cell counts in blood and significantly prolonged animal survival. In summary, preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in low-antigen or suppressive environments and delayed onset of functional exhaustion. These results support further investigation of lenalidomide and anti-BCMA CAR T cells in the clinic.

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