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Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis-induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.

We previously developed human CAR macrophages (CAR-M) and demonstrated redirection of macrophage anti-tumor function leading to tumor control in immunodeficient xenograft models. Here, we develop clinically relevant fully immunocompetent syngeneic models to evaluate the potential for CAR-M to remodel the tumor microenvironment (TME), induce T cell anti-tumor immunity, and sensitize solid tumors to PD1/PDL1 checkpoint inhibition. In vivo, anti-HER2 CAR-M significantly reduce tumor burden, prolong survival, remodel the TME, increase intratumoral T cell and natural killer (NK) cell infiltration, and induce antigen spreading. CAR-M therapy protects against antigen-negative relapses in a T cell dependent fashion, confirming long-term anti-tumor immunity. In HER2+ solid tumors with limited sensitivity to anti-PD1 (aPD1) monotherapy, the combination of CAR-M and aPD1 significantly improves tumor growth control, survival, and remodeling of the TME in pre-clinical models. These results demonstrate synergy between CAR-M and T cell checkpoint blockade and provide a strategy to potentially enhance response to aPD1 therapy for patients with non-responsive tumors. Anti-PD1 monotherapy shows limited efficacy against HER2+ tumors. Here, the authors show that murine CAR macrophages (CAR-M) induce tumor microenvironment remodeling, T-cell mediated immunity and synergy with PD1 blockade, improving survival in immunocompetent female-mouse models of HER2+ solid tumors.

Gabrilovich and colleagues show that monocytic myeloid-derived suppressor cells (MDSCs) differentiate into polymorphonuclear MDSCs in individuals with tumors, demonstrating a demonstrating a distinct regulation of myeloid cell development in cancer. Two major populations of myeloid-derived suppressor cells (MDSCs), monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) regulate immune responses in cancer and other pathologic conditions. Under physiologic conditions, Ly6C hi Ly6G − inflammatory monocytes, which are the normal counterpart of M-MDSCs, differentiate into macrophages and dendritic cells. PMN-MDSCs are the predominant group of MDSCs that accumulates in cancer. Here we show that a large proportion of M-MDSCs in tumor-bearing mice acquired phenotypic, morphological and functional features of PMN-MDSCs. Acquisition of this phenotype, but not the functional attributes of PMN-MDSCs, was mediated by transcriptional silencing of the retinoblastoma gene through epigenetic modifications mediated by histone deacetylase 2 (HDAC-2). These data demonstrate a new regulatory mechanism of myeloid cells in cancer.

BackgroundOX40 is a costimulatory receptor upregulated on antigen-activated T cells and constitutively expressed on regulatory T cells (Tregs). INCAGN01949, a fully human immunoglobulin G1κ anti-OX40 agonist monoclonal antibody, was designed to promote tumor-specific immunity by effector T-cell activation and Fcγ receptor-mediated Treg depletion. This first-in-human study was conducted to determine the safety, tolerability, and preliminary efficacy of INCAGN01949.MethodsPhase I/II, open-label, non-randomized, dose-escalation and dose-expansion study conducted in patients with advanced or metastatic solid tumors. Patients received INCAGN01949 monotherapy (7–1400 mg) in 14-day cycles while deriving benefit. Safety measures, clinical activity, pharmacokinetics, and pharmacodynamic effects were assessed and summarized with descriptive statistics.ResultsEighty-seven patients were enrolled; most common tumor types were colorectal (17.2%), ovarian (8.0%), and non-small cell lung (6.9%) cancers. Patients received a median three (range 1–9) prior therapies, including immunotherapy in 24 patients (27.6%). Maximum tolerated dose was not reached; one patient (1.1%) receiving 350 mg dose reported dose-limiting toxicity of grade 3 colitis. Treatment-related adverse events were reported in 45 patients (51.7%), with fatigue (16 (18.4%)), rash (6 (6.9%)), and diarrhea (6 (6.9%)) being most frequent. One patient (1.1%) with metastatic gallbladder cancer achieved a partial response (duration of 6.3 months), and 23 patients (26.4%) achieved stable disease (lasting >6 months in one patient). OX40 receptor occupancy was maintained over 90% among all patients receiving doses of ≥200 mg, while no treatment-emergent antidrug antibodies were detected across all dose levels. Pharmacodynamic results demonstrated that treatment with INCAGN01949 did not enhance proliferation or activation of T cells in peripheral blood or reduce circulating Tregs, and analyses of tumor biopsies did not demonstrate any consistent increase in effector T-cell infiltration or function, or decrease in infiltrating Tregs.ConclusionNo safety concerns were observed with INCAGN01949 monotherapy in patients with metastatic or advanced solid tumors. However, tumor responses and pharmacodynamic effects on T cells in peripheral blood and post-therapy tumor biopsies were limited. Studies evaluating INCAGN01949 in combination with other therapies are needed to further evaluate the potential of OX40 agonism as a therapeutic approach in patients with advanced solid tumors.Trial registration numberNCT02923349.

BackgroundImmune homeostasis is regulated by a balance of pro- and anti-inflammatory cytokine signals. Dysregulated cytokine expression can cause deleterious immunosuppression or inflammation, which drives disease pathology. In solid tumors, cytokines such as IL10 and TGFβ induce an immunosuppressive tumor microenvironment (TME) that blunts endogenous and therapeutic anti-tumor immunity. Therapeutic strategies to block immunosuppressive cytokines have primarily focused on monoclonal antibodies targeting cytokines/cytokine receptors. While this approach can reduce immunosuppressive signaling, it fails to provide an inflammatory signal that could initiate anti-tumor immunity. Here, we engineered macrophages with synthetic cytokine switch receptors (SR) to develop a cell therapy platform for modulation of pro-/anti-inflammatory signals. Macrophages are homeostatic regulators capable of both initiating inflammation and infiltrating solid tumors, and we leveraged this natural proficiency using SRs that convert tumor-related immunosuppressive (M2) signals into pro-inflammatory (M1) responses for tumor microenvironment (TME) modulation. We termed this engineered myeloid cell platform ’Engineered Microenvironment Converters’ (EM-C) and evaluated its modular ability to target several tumor-associated cytokines.MethodsEM-Cs targeting IL10 or TGFβ were generated by expressing SR in primary human macrophages and monocytes. M2-to-M1 SR were designed to convert IL10 or TGF-β into pro-inflammatory signals based on interferon or toll-like receptor (TLR) signaling pathways. The response of EM-Cs to target cytokines was monitored using phenotypic characterization of surface molecules, measurement of cytokine release, mRNA profiling, and biochemical analysis of downstream signaling. Co-culture assays with bystander immunosuppressive cells were used to assess the ability of EM-Cs to alter their microenvironment. Additionally, combinatorial EM-C were designed to target both IL10 and TGFβ for multiplexed TME conversion.ResultsPro-inflammatory EM-Cs efficiently sequestered IL10 and TGFβ, two prevalent immunosuppressive cytokines in the TME, and converted them into pro-inflammatory signals by upregulating M1 markers, cytokines, and pathways in a dose-dependent manner. EM-Cs furthermore repolarized bystander M2 macrophages towards a pro-inflammatory phenotype following co-culture.ConclusionsWe present a novel immunotherapy platform that harnesses macrophages as ‘living converters’ to locally regulate inflammation in solid tumors. We establish EM-C that convert IL10 or TGFβ into pro-inflammatory signals, showcasing a modular ability to control the inflammatory status of microenvironments without systemic cytokine antagonism. EM-Cs enable the development of target antigen agnostic myeloid cell immunotherapies for solid tumors.

BackgroundMacrophages expressing chimeric antigen receptors (CAR-M) have been shown to reduce tumor burden, remodel the tumor microenvironment (TME), and coordinate a systemic immune response in pre-clinical solid tumor models. Solid tumors overexpress immunosuppressive molecules, such as CD47, which reduces macrophage tumor phagocytosis. We have previously demonstrated that CD47 is a checkpoint that reduces CAR-M function, and have shown that CRISPR-mediated SIRPα knockout (KO) CAR-M are refractory to the anti-phagocytic checkpoint protein CD47.1 Here, we have enhanced our ability to control gene expression in CAR-M by creating a single vector system that incorporates synthetic shRNA into the CAR intron, enabling simultaneous CAR expression with knockdown of SIRPα. The intronic shRNA platform allows gene-silenced CAR-M and CAR-Monocytes to be manufactured using a single vector in a streamlined single-day process.MethodsTo generate gene-silenced CAR-M, we transduced primary human macrophages or monocytes with a novel adenoviral vector comprising a CAR transgene with custom intronic shRNA expressed under a shared promoter. We characterized shRNA-modified CAR-M using a model anti- human epidermal growth factor receptor 2 (HER2) CAR paired with shRNA targeting SIRPα. CAR-M phenotype was characterized using flow cytometry. The anti-tumor efficacy of CAR-M in vitro was monitored by quantifying killing, phagocytosis, and cytokine production in co-culture assays with HER2+ tumor cell lines. The in vivo efficacy of CAR-M was characterized using metastatic solid tumor xenograft models.ResultsIntronic shRNA enabled concomitant CAR expression with target SIRPα knockdown. Reduction in SIRPα expression by shRNA was comparable to that achieved by CRISPR/Cas9 ribonucleoprotein. Additionally, the inclusion of an intron significantly augmented expression of the neighboring CAR transgene. Compared to unmodified CAR-M, SIRPα-knockdown CAR-M exhibited enhanced killing, phagocytosis, and cytokine production against HER2+ tumor cells in vitro. Furthermore, SIRPα-knockdown CAR-M significantly delayed tumor growth and prolonged survival in vivo.ConclusionsWe show the feasibility of generating gene-silenced primary CAR-M in a single transduction step by integrating CAR delivery with custom intronic shRNA, and we demonstrate that targeted gene knockdown of SIRPα can enhance the anti-tumor activity of CAR-M in vivo. The intronic shRNA design is a generalizable platform that will be valuable for future CAR designs, target tumor antigens, and gene knockout targets.ReferenceSloas C, Gabbasov R, Anderson N, Abramson S, Klichinsky M, Ohtani Y. 144 SIRPα deficient CAR-Macrophages exhibit enhanced anti-tumor function and bypass the CD47 immune checkpoint. J Immunother Cancer. 2021;9:A152-A152.

BackgroundMacrophages are abundant in the solid tumor microenvironment (sTME) and can promote tumor growth (M2) or enhance anti-tumor immunity (M1). CAR expression can redirect macrophage function to selectively target and phagocytose antigen overexpressing cancer cells. CAR-M can reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and anti-tumor immunity. CT-0508 is comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies demonstrated that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden, prolonged survival, and was safe. Notably, anti-HER2 CAR-M treatment led to activation of the sTME, with infiltration of CD8+ and CD4+ T cells, NK cells, dendritic cells, and increased activated CD8+ tumor infiltrating lymphocytes. In a pre-clinical anti-PD1 resistant solid tumor model, mice that received anti-HER2 CAR-M and anti-PD1 demonstrated improved tumor control, overall survival, and TME activation compared to single treatment alone, indicating synergy and capacity for CAR-M to sensitize solid tumors to checkpoint blockade.1 MethodsThis Phase 1, First in Human study evaluates the safety, tolerability, cell manufacturing feasibility, trafficking, TME activation, and preliminary evidence of efficacy of investigational product CT-0508 in 18 participants (pts) with locally advanced (unresectable)/metastatic solid tumors overexpressing HER2. Pts previously treated with anti-HER2 therapies are eligible. Filgrastim mobilized autologous CD14+ monocytes are collected by apheresis, without the need for lymphodepleting chemotherapy, followed by manufacturing and cryopreservation. Group 1 pts (n = 9; enrollment complete) received fractionated doses of CT-0508 over Days 1, 3, and 5. Group 2 pts (n = 9) receive CT-0508 as a single infusion on D1. Additional cohorts include: CT-0508 co-administered with pembrolizumab and CT-0508 monotherapy administered intraperitoneally in pts with peritoneal predominant disease. Correlative assessments include pre- and post-treatment biopsies and blood samples for safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others.ReferenceKlichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. 2020;38:947–953.

BackgroundCT-0508 is an autologous monocyte-derived pro-inflammatory macrophage cell product engineered with Ad5f35 to express an anti-HER2 CAR. In pre-clinical studies CT-0508 was safe and effective. This abstract contains preliminary results from the first-in-human experience with CAR macrophages (CAR-M).MethodsThis First-In-Human Phase 1, multi-center, open-label study is evaluating the safety, tolerability, manufacturing feasibility, pharmacokinetics and mechanism of action of CT-0508 in 18 subjects with advanced solid tumors overexpressing HER2 who have progressed on prior therapies, including HER2 targeted therapies if indicated.Patients receive four doses of filgrastim for monocyte mobilization prior to apheresis. CT-0508 CAR-M is manufactured from autologous apheresis products and delivered as a cryopreserved cell product. Group 1 subjects enter an intra-patient fractionated dose escalation regimen, receiving CT-0508 on D1, D3 and D5, followed by Group 2 subjects who receive CT-0508 on D1. There is no preparative chemotherapy prior to CT-0508 infusion.Pre and post treatment biopsies and blood samples are collected to investigate correlates of safety, serum cytokines and chemokines, pharmacokinetics, TME modulation, and induction of an adaptive anti-tumor immune response.ResultsTo date, two subjects have been treated with CT-0508 (esophageal adenocarcinoma and extrahepatic cholangiocarcinoma). Patient product was successfully manufactured, CT-0508 treatment was well tolerated, with no dose limiting and no major organ toxicities observed.One subject experienced Grade 2 CRS on Day 3 which resolved on the same day.Grade 3 AEs included anemia (present at baseline for both subjects) and lymphopenia (present at baseline in one subject). One subject experienced one SAE of Grade 4 tumor bleeding which was unrelated to CT-0508, 88 days after the last infusion.CAR-M were transiently detected in the peripheral blood following each infusion, demonstrating rapid egress from the periphery into tissues within hours. Transient cytokine/chemokine elevations were observed (peak: 2 hours, back to baseline at 48 hours). Single cell RNAseq analysis of dissociated tumor tissue samples (pre-treatment, day 8 and week 4) demonstrated dynamic TME reprogramming, with recruitment of inflammatory innate immune cells and naïve T cells at day 8, and significant CD8+ T cell infiltration, activation, and proliferation at week 4.ConclusionsCT-0508 has been administered to two subjects thus far, exhibiting safety, good tolerability, T cell repertoire modulation, and reprogramming of the TME consistent with the induction of anti-tumor immunity. The study continues to recruit patients and updated data will be presented.Trial RegistrationNCT04660929ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953.Ethics ApprovalEthics approvals have been obtained from the clinical sites enrolling patients: the University of Pennsylvania (844106/IORG0000029), the University of North Carolina and City of Hope Comprehensive Cancer Center (20201732/IORG0000432).

BackgroundMacrophages, monocytes, and dendritic cells are sentinel cells of the innate immune system that play a central role in phagocytosis, inflammation, immune cell recruitment, and antigen presentation. Genetically redirecting myeloid cells against tumor-associated antigens represents a novel strategy for cancer immunotherapy. Ex vivo chimeric antigen receptor (CAR) macrophage and monocyte cell therapies have demonstrated robust anti-tumor immunity via targeted phagocytosis, cytokine/chemokine release, activation of the tumor microenvironment (TME), T cell recruitment, and epitope spreading in pre-clinical models. Here, we describe a novel strategy to deliver modified messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs) to generate CAR-M (macrophages and monocytes) directly in vivo.MethodsHuman and murine primary monocytes and macrophages were utilized to characterize mRNA/LNP driven CAR expression kinetics and anti-tumor functionality in vitro using flow cytometry, live cell imaging, single cell RNA sequencing (scRNAseq), and cytokine release assays. The efficacy, safety, and myeloid cell tropism of CAR-encoding mRNA/LNP were evaluated in vivo in syngeneic and humanized mouse models of solid tumors. Tissues were collected and CAR expression along with dynamic changes in immune cell frequency and phenotype were assessed using flow cytometry and scRNAseq. All animal studies were conducted in accordance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Wistar Institute.ResultsHuman macrophages and monocytes engineered with CAR-encoding mRNA/LNP in vitro demonstrated high CAR expression and viability. CAR expression conferred antigen specificity leading to target-specific proinflammatory cytokine secretion and tumor cell killing, with serial killing demonstrated upon tumor rechallenge. The myeloid tropism of the LNP was demonstrated both in vitro and in vivo, with significant CAR expression observed in macrophages, monocytes, and dendritic cells compared to immune cells of non-myeloid origin in mice. In vivo, regional and systemic administration of CAR-encoding mRNA/LNP led to significant tumor regression in subcutaneous and systemically disseminated metastatic solid tumor models, respectively. Treated animals tolerated repeat mRNA/LNP administration with no signs of toxicity.ConclusionsThese data demonstrate that CAR-M can be directly produced in vivo and directed against tumor associated antigens using mRNA/LNP technology. This novel cancer immunotherapy platform offers an off-the-shelf solution that has the potential to increase access to CAR-based therapies and can be applied to numerous target antigens and indications.Ethics ApprovalAll animal studies were conducted in accordance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Wistar Institute.