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Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKsupTROG+/sup) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROGsup+/supsiblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Simple Summary The immune system is the first line of protection against infected and tumor cells. Macrophages are specialized immune cells that recognize these abnormal cells and eliminate them by a mechanism called phagocytosis. All normal cells express a protein called CD47 or “don’t eat me signal” to prevent their elimination through phagocytosis. Cancer cells, including leukemic cells, express higher levels of CD47 as a mechanism of protection against macrophage phagocytosis. CD47 blockade leads to an increase in phagocytosis of leukemic cells and better control of the disease. In this review, we explore CD47 function in normal conditions, its role in acute myeloid leukemia progression, and possible ways to block CD47 to enhance elimination of the leukemic cells improving the therapeutic options for patients with acute myeloid leukemia. Abstract CD47 is a surface membrane protein expressed by all normal tissues. It is the so-called “don’t eat me signal” because it protects the cells against phagocytosis. The CD47 interacts with the signal regulatory protein alpha (SIRPα) on the surface of macrophages, leading to downstream inhibitory signaling that dampens phagocytic capacity. Since macrophages exert immune surveillance against cancers, cancer cells overexpress CD47 to defend themselves against phagocytosis. Acute myeloid leukemia (AML) is a cancer of hematopoietic stem/progenitor cells (HSPC), and similar to other types of cancers, leukemic blasts show enhanced levels of CD47. In patients with AML, CD47 has been associated with a higher disease burden and poor overall survival. Blockage of CD47-SIRPα signaling leads to improved phagocytosis of AML cells and better overall survival in xenograft models. However, the introduction of a pro-phagocytic signal is needed to induce greater phagocytic capacity. These pro-phagocytic signals can be either Fc receptor stimulants (such as monoclonal antibodies) or natural pro-phagocytic molecules (such as calreticulin). Based on these pre-clinical findings, various clinical trials investigating the blockade of CD47-SIRPα interaction have been designed as monotherapy and in combination with other anti-leukemic agents. In this review, we will discuss CD47 biology, highlight its implications for AML pathophysiology, and explore the potential clinical translation of disrupting CD47-SIRPα to treat patients with AML.

The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p Disclosures Jabbour: Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding. Teachey:Sobi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; La Roche: Consultancy. Rezvani:Takeda: Other: Licensing agreement; GemoAb: Membership on an entity's Board of Directors or advisory committees; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Virogen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Formula Pharma: Membership on an entity's Board of Directors or advisory committees. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Lorenzi:Precision Pathways: Consultancy. Konopleva:Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; Eli Lilly: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Agios: Research Funding; AstraZeneca: Research Funding; Ablynx: Research Funding; Forty-Seven: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Ascentage: Research Funding.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing., Graphical abstract, Basar et al. demonstrate that SARS-CoV-2 T cells can be efficiently generated from COVID19-recovered donors with specificity against multiple structural SARS-CoV-2 proteins, including the Spike protein. Moreover, these cells can be genetically modified to render them resistant to corticosteroids, making their application clinically feasible.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing. Funding: Supported in part by the generous philanthropic contributions to The University of Texas MD Anderson Cancer Center AML Moonshot Program, by the grants from National Institute of Health, National Cancer Institute 5R01CA211044-04 and Cancer Center Support (CORE) Grant (CA016672) that support the Flow Cytometry and Cellular Imaging Facility and the RNA sequencing core facility at MD Anderson Cancer Center. Conflict of Interest: The authors declare no competing interests. Ethical Approval: Buffy coat units were processed from 500mL of whole blood collected from each of the 10 COVID-19 recovered donors (CoV-RD) and 20 mL of peripheral blood from 5 healthy donors were collected under local Institutional Review Board approved protocols (Lab02-0630 and PA13-0647) and following informed consent.

Background Perturbation of the major UGT2B17-dependent androgen catabolism pathway has the potential to affect prostate cancer (PCa) progression. The objective was to evaluate UGT2B17 protein expression in primary tumours in relation to hormone levels, disease characteristics and cancer evolution. Methods We conducted an analysis of a high-density prostate tumour tissue microarray consisting of 239 localised PCa cases treated by radical prostatectomy (RP). Cox proportional hazard ratio analysis was used to evaluate biochemical recurrence (BCR), and a linear regression model evaluated variations in circulating hormone levels measured by mass spectrometry. The transcriptome of UGT2B17 in PCa was established by using RNA-sequencing data. Results UGT2B17 expression in primary tumours was associated with node-positive disease at RP and linked to circulating levels of 3α-diol-17 glucuronide, a major circulating DHT metabolite produced by the UGT2B17 pathway. UGT2B17 was an independent prognostic factor linked to BCR after RP, and its overexpression was associated with development of metastasis. Finally, we demonstrated that distinctive alternative promoters dictate UGT2B17-dependent androgen catabolism in localised and metastatic PCa. Conclusions The androgen-inactivating gene UGT2B17 is controlled by overlooked regulatory regions in PCa. UGT2B17 expression in primary tumours influences the steroidome, and is associated with relevant clinical outcomes, such as BCR and metastasis.

Introduction: A number of Clinical trials have demonstrated the feasibility, safety and efficacy of cell and gene therapy for cancer, autoimmune disorders and infectious disease. Strategies that enhance the function and survival of immune cells are critical for the success of immunotherapy. We have developed a strategy for the ex vivo expansion of off-the-shelf viral-specific T cells (VSRs) from healthy donor buffy coat which have been extremely effective in eradicating refractory cytomegalovirus (CMV), polyomavirus and adenovirus infections in immunocompromised patients. Glucocorticoids commonly used to treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are a common cause of iatrogenically-induced immunosuppression and contribute the risk of life-threatening viral-infections. To render VSTs resistant to the lymphocytotoxic effect of glucocorticoids, we have developed a novel strategy to silence the expression of the glucocorticoid receptor using RNA-guided endonucleases CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) 9 gene editing.. Methods: The technique involves the expansion from donor blood of CMV, BKV or adenoviral-specific T cells using peptide libraries from the immunodominant viral proteins followed by CRISPR knockout of exon 2 of the GR gene on chromosome 5 of the human NR3C1 gene. Cells are electroporated with the RNP (Cas9 plus guide RNA) complex (IDT pre-designed alt-R crispr Cas9 platform) using Neon electroporation and the Amaxa 4-D nucleofector system. Results: GR knockout efficiency in ex vivo expanded virus-specific T cells was consistently > 90%. In vitro experiments confirmed the resistance of VSTs to corticosteroid treatment as assessed by annexin V assay. GR KO VSTs maintained potent antiviral activity as assessed by their ability to proliferate and release effector cytokines in response to viral antigens. Conclusions: CRISPR gene-editing to knock-out the glucocorticoid receptor gene in viral-specific T cells can preserve the activity of VSTs in the presence of corticosteroid-induced immunosuppression. Engineering runs using GMP-compliant Cas9 protein and gRNA are underway in anticipation of a clinical trial. Disclosures Champlin: Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

264 Background: Latent tuberculosis infection (LBTI) affects about one quarter of the world population. Treating LTBI is the most effective way of preventing active TB. Patients with hematologic malignancy (HM) and LTBI are at heightened risk (2-40 times baseline) of progressing to active TB. Universal TB screening is recommended for this population. In 2016, a patient receiving chemotherapy for myeloma at our center developed active TB. This triggered an intensive contact tracing investigation involving local public health units, infection control, and specialists. Over 800 patients had one or more exposures to the index case. No cases of secondary TB were identified; however, the contact tracing was resource intensive and stressful for patients and staff. This project aims to prevent future incidents by developing a standardized process for completing and documenting TBSTs, ensuring LTBIs are identified and managed prior to systemic therapy for HM. We aimed to complete and document TBSTs in ≥85% of patients starting systemic therapy for HM by Feb 2018. Methods: Baseline data was determined via retrospective review of 25 patients receiving chemotherapy for HM Jul-Nov 2017. An order set, work flow process map and standard operating procedure were created over a series of plan-do-study act cycles. TBST was added to a pre-existing “ready for treatment” checklist employed by pharmacists at our site. When pharmacists could find no record of a TBST result prior to treatment, the treating physician was notified. The go-live date was Aug 15, 2018. The impact of change was evaluated via chart review of patients who started chemotherapy for HM between Aug 27, 2018-Feb 27, 2019. Results: Prior to implementation of the standardized process, 3/25 patients (12%) had documentation of prior TBST. Following implementation of the standardized process, 14/30 patients (47%) had documentation of TBST prior to starting systemic therapy for HM. All 14 patients had negative TBSTs. Conclusions: Developing a standardized process for ordering, completing, and documenting TBST for patients starting systemic therapy for HM is feasible and effective. Additional revisions of the process are required to meet our target.

Background: Hodgkin lymphoma (HL) and aggressive non-Hodgkin lymphoma (NHL) both have relatively high curative-potential with contemporary treatment strategies. Despite this, a substantial number of patients will relapse, and a principal objective of follow-up is to identify recurrent disease. The optimal method for detection of relapsed disease has been debated extensively in the literature. Previous guidelines recommended routine surveillance imaging (SI) as a means to detect relapsed disease; however, recent recommendations have de-emphasized routine SI. Nonetheless, controversy remains and approaches vary dramatically across different regions. Aim:We completed a comprehensive systematic review to evaluate whether routine surveillance with computed tomography (CT) and/or positron emission tomography (PET) for patients in complete remission following curative intent therapy for HL and/or aggressive NHL identifies more relapses than clinical vigilance strategies, and if it improves progression free (PFS) and/or overall survival (OS). Methods: We searched Medline, [1946 to Oct 18, 2015] andEMBASE [1980 to Oct 18, 2015] for English-language studies assessing one or more surveillance strategies in adult and/or pediatric patients in remission from HL and/or aggressive NHL. Reviews, conference abstracts, case reports, letters, comments, books, and editorials were excluded. Results: Our search identified 8719 potentially relevant titles for review. Two authors reviewed all titles and abstracts. A total of 120 papers were selected for full text review; of these, thirty-two papers (30 adult, 2 pediatric) were included and four were unavailable for full text review. Twenty-seven retrospective studies, four prospective cohort studies, and one randomized control trial were included. A majority of adult studies (29/30) reported on surveillance strategies in NHL/HL survivors (16/16 NHL, 11/12 HL, 2/2 evaluating both NHL and HL) as well as all pediatric studies (1/1 NHL, 1/1 HL). Almost all studies (15/16 adult NHL, 11/12 adult HL, 2/2 pediatric) reported patient-level data on the number of asymptomatic relapses identified through SI versus clinical signs or symptoms. There were a total of 797 documented relapses in the adult NHL studies and 523 relapses in the adult HL studies. Of the relapsed adult NHL patients, 78.2% (623/797) were diagnosed clinically and 19.6% (156/797) were diagnosed by SI. Among the adult HL patients, 63.1% (330/523) of relapses were diagnosed clinically and 36.5% (191/523) were diagnosed by SI. Of the two pediatric studies included, one NHL study showed that all 3 relapses were detected clinically, and one HL study showed 76.0% (19/25) of relapses were detected clinically or due to lab abnormalities, and 24.0% (6/25) were detected by SI. Fifteen adult studies reported comparative data on PFS and/or OS rates with SI versus clinical surveillance strategies (9 NHL studies, 4 HL studies, 2 NHL/HL studies). The majority of studies (12/15) documented no difference in PFS and/or OS with imaging versus clinical surveillance strategies (8/9 NHL, 3/4 HL, 1/2 NHL/HL). In the remaining three studies, the first demonstrated a significant reduction in risk of death (HR 0.47 for HL, 0.57 for DLBCL) with SI on univariate analysis, but this was not seen on multivariate analysis for DLBCL (data not analyzed for HL). A second study in NHL patients demonstrated a non-significant trend towards improved PFS (p = 0.12) and OS (p = 0.13) with SI, while the third suggested that asymptomatic individuals with HL do not benefit from SI, with the exception of those with a morphological residual mass (p < 0.00016, HR = 3.84) or advanced stage (p = 0.03644, HR 1.99). Conclusions: The preliminary analysis of this systematic review indicates that even when routine imaging surveillance is employed, a majority of NHL and HL relapses come to attention due to the development of clinical signs and/or symptoms. A small number of comparative studies have reported on PFS and/or OS, with the vast majority demonstrating no improvement in PFS or OS with routine imaging surveillance. Our study supports clinical vigilance as the predominant strategy for follow-up in asymptomatic patients following curative intent therapy for HL and aggressive NHL. Disclosures No relevant conflicts of interest to declare.