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Background Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2–vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). Methods Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2–vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2–vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. Results Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2–vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2–vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. Conclusions Highly functional SARS-CoV-2–vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. Clinical Trials Registration NCT04896606.

Supplementary Fig.S1: Anti-CD20 CAR enhances NK92 cells in-vitro cytolytic activity against CD20+ B-NHL cells by retroviral transduction. (A) Schematic representation of the anti-CD20 chimeric receptor in an RD114-pseudotyped retroviral vector. LTR indicates long terminal repeat; AMP, ampicillin resistance. (B)-(C) Expression of anti-CD20 CAR was confirmed in infected Jurkat cells by flow cytometry after staining with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin followed by streptavidin PE (y-axes); expression of GFP is also shown (x-axes) (B) and it was also confirmed in infected PBMC by western blot analysis with an anti-human CD3� antibody (C). (D) NK92 cells expressing anti-CD20 chimeric receptors or expression GFP only were incubated with BATDA labeled targets: CD20+ NK sensitive Ramos, CD20+ NK resistant Daudi and the CD20- pre-B-ALL cell line RS4;11 at the indicated E/ T ratios for 4 hours. Europium release assays were performed according to the manufacturer's instructions. Each data point represents the mean (SEM; n=3). Average values are reported as the mean {plus minus} SEM. p values using unpaired student t test were noted in (D).

Supplementary Methods, Figure Legends

The prognosis is very dismal for patients with relapsed CD20+ B-cell non-Hodgkin lymphoma (B-NHL). Facilitating the development of alternative novel therapeutic strategies is required to improve outcomes in patients with recurrent/refractory CD20+ B-NHL. In this study, we investigated functional activities of anti-CD20 CAR-modified, expanded peripheral blood NK cells (exPBNK) following mRNA nucleofection against CD20+ B-NHL in vitro and in vivo. CAR+ exPBNK had significantly enhanced in vitro cytotoxicity, compared with CAR− exPBNK against CD20+ Ramos (P < 0.05), Daudi, Raji, and two rituximab-resistant cell lines, Raji-2R and Raji-4RH (P < 0.001). As expected, there was no significant difference against CD20− RS4;11 and Jurkat cells. CD107a degranulation and intracellular IFNγ production were also enhanced in CAR+ exPBNK in response to CD20+ B-NHL–specific stimulation. In Raji-Luc and Raji-2R-Luc xenografted NOD/SCID/γ-chain−/− (NSG) mice, the luciferase signals measured in the CAR+ exPBNK-treated group were significantly reduced, compared with the signals measured in the untreated mice and in mice treated with the CAR− exPBNK. Furthermore, the CAR exPBNK-treated mice had significantly extended survival time (P < 0.001) and reduced tumor size, compared with those of the untreated and the CAR− exPBNK-treated mice (P < 0.05). These preclinical data suggest that ex vivo–exPBNK modified with anti-CD20 CAR may have therapeutic potential for treating patients with poor-risk CD20+ hematologic malignancies. Cancer Immunol Res; 3(4); 333–44. ©2014 AACR.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System (CCS).Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of IFN-γ+ CD4 and CD8 T-cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS® CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis.93% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T-cells that were 79% ± 21% IFN-γ+ T-cells. SARS-CoV-2-vCTLs displayed a highly diverse TCR repertoire with enhancement of both memory CD8 and CD4 T-cells, especially in CD8 TEM, CD4 TCM and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways.Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors.NCT04896606, NCT03266627, NCT03266640, NCT03266653, NCT04197596.

Ewing sarcoma (ES) is a malignant pediatric bone and soft tissue tumor. Patients with metastatic ES have a dismal outcome which has not been improved in decades. The major challenge in the treatment of metastatic ES is the lack of specific targets and rational combinatorial therapy. We recently found that protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is specifically highly expressed in ES and promotes tumor growth and metastasis in ES. In the current investigation, we show that PPP1R1A regulates ES cell cycle progression in G1/S phase by down-regulating cell cycle inhibitors p21Cip1 and p27Kip1, which leads to retinoblastoma (Rb) protein hyperphosphorylation. In addition, we show that PPP1R1A promotes normal transcription of histone genes during cell cycle progression. Importantly, we demonstrate a synergistic/additive effect of the combinatorial therapy of PPP1R1A and insulin-like growth factor 1 receptor (IGF-1R) inhibition on decreasing ES cell proliferation and migration in vitro and limiting xenograft tumor growth and metastasis in vivo. Taken together, our findings suggest a role of PPP1R1A as an ES specific cell cycle modulator and that simultaneous targeting of PPP1R1A and IGF-1R pathways is a promising specific and effective strategy to treat both primary and metastatic ES.

Background: Viral infection remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Bollard/Heslop Blood 2016). Anti-viral agents for treatment of viral infection in immunocompromised patients are limited in efficacy and are associated with significant toxicities (Gerdemann BBMT 2004; Sili Cytother 2012). The use of virus-specific cytotoxic T-lymphocytes (VST) for immunocompromised patients with viral infections has been associated with therapeutic benefit and improved OS (Bollard/Heslop Blood 2016; Sutrave Cytother 2017). Methods of VST production include ex-vivo expansion and direct selection (Gottlieb Cytother 2017). Ex-vivo expansion requires prolonged manufacturing time, is associated with T-cell exhaustion, and results in a limited donor pool. Direct selection is rapid (12-24 hours), can be done locally, allows for expanded HLA matching, permits a low degree of HLA match to the recipient, and can be adapted for many viruses. A multicenter consortium, the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC) was created to investigate the safety and efficacy of VST manufactured by direct selection using the IFN-g Cytokine Capture System process automated on the CliniMACS® Prodigy device (Miltenyi Biotec) for immunocompromised patients with viral infection (Figure 1). Objective: Determine the safety and efficacy of VST for the treatment of immunocompromised child, adolescent and young adult (CAYA) patients with refractory, systemic viral infection and/or viral infection and intolerance to appropriate anti-viral medical therapy. Design/Methods: CAYA patients after allo-HSCT, solid organ transplantation (SOT), or with primary immunodeficiency (PID) with refractory adenovirus (ADV), cytomegalovirus (CMV), Epstein Barr virus (EBV) or BK virus (BKV) infections as evidenced by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance to anti-viral agents, and/or intolerance to anti-viral agents were eligible. Related donors with ≥1 HLA A, B, or DR match to recipient and with an adequate T-cell response to virus specific MACS® PepTivators were eligible. Donors were screened with viral specific antigen (PepTivator®) to predict successful VST manufacturing. Peripheral blood mononuclear cells (PBMC) were collected from eligible related donors using non-mobilized apheresis. VST were isolated using the CliniMACS® Prodigy following stimulation of PBMC with specific viral MACS PepTivator® pools, generously provided by Miltenyi Biotec. Production of CD4+ and CD8+ VST was performed as previously described (Feuchtinger Blood 2010). The target cell dose was 0.5x104 CD3+/kg for HLA mismatched haploidentical related donors and 2.5x104 CD3+/kg for matched related donors. Based on response and safety, VST were given every 2 weeks for a maximum of 5 infusions. Results: Eleven patients have been enrolled to date. Seven patients were treated for ADV, 2 for BKV, 1 for CMV, and 1 for EBV. There were 8 males and 3 females enrolled, aged 1-38 years. There were 10 patients post allo-HSCT and 1 patient post SOT. There were 8 haploidentical, related, original allo-HSCT donors and 3 haploidentical, related, third party donors. There have been no matched related donors enrolled to date. The mean±SEM %CD4+ IFN-g+ of total CD4+, %CD8+ IFN-g+ of total CD8+, and %CD3 cells recovered in the final product were 21.5±4.8, 25.0±7.0, and 50.4.2±7.2, respectively. The median number of VST infusions was 2 (1-5). The mean±SEM CD3+ cell dose was 0.49±0.001x104. Ten patients achieved complete response (PCR negative) and 1 patient achieved partial response (PCR≥1 log decrease). The overall response and complete response rates were 100% and 90.9%, respectively. The median time to maximal response was 34 days (7-141) (Table 1). No patient developed aGVHD, cGVHD, infusion reaction or CRS associated with VST. Conclusion: Preliminary results of this pilot study demonstrate that VST are safe, well tolerated and efficacious in CAYA with refractory viral infections after allo-HSCT, SOT or with PID. Manufacturing utilizing the CliniMACS® Prodigy device is rapid, reproducible and effective. Accrual is ongoing. This research is supported by FDA RO10063-01A1. Disclosures Flower: Lentigen Technology Inc/Miltenyi Biotec: Research Funding. O'Donnell:Kiadis Pharma: Other: Licensing of intellectual property. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Johnson:Cell Vault: Research Funding; Miltenyi Biotec: Research Funding. Cairo:Technology Inc/Miltenyi Biotec: Research Funding; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Miltenyi: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Unrelated cord blood (CB) is an excellent alternative as an allogeneic donor source for stem cell transplantation. CB transplantation is associated with lower incidence of severe acute graft versus host disease (GVHD) and chronic GVHD but similar rates of malignant relapse compared with other unrelated donor cell transplants. NK cells are critical innate immune components and the comparison of CB vs. peripheral blood (PB) NK cells is relatively unknown. NK cell receptor expression, cell function, and maturation may play a role in the risk of relapse after CB transplant. We investigated CB vs. PB NK cell subset cytotoxicity, function, receptor expression, and genomic and proteomic signatures. The CB CD56dim compared with PB CD56dim demonstrated significantly increased expression of NKG2A and NKG2D, respectively. CB vs. PB CD56dim NK cells had significantly decreased in vitro cytotoxicity against a variety of non-Hodgkin lymphoma targets. Various proteins were significantly under- and over-expressed in CB vs. PB CD56dim NK cells. Microarray analyses and qRT-PCR in CB vs. PB CD56dim demonstrated significantly increased expression of genes in cell regulation and development of apoptosis, respectively. In summary, CB vs. PB CD56dim NK cells appear to be earlier in development, have decreased functional activity, and increased capacity for programmed cell death, suggesting that CB NK cells require functional and maturational stimulation to achieve similar functional levels as PB CD56dim NK cells.

Children with sickle cell disease (SCD) are at high-risk of progressive, chronic pulmonary and cardiac dysfunction. In this prospective multicenter Phase II trial of myeloimmunoablative conditioning followed by haploidentical stem cell transplantation in children with high-risk SCD, 19 patients, 2.0-21.0 years of age, were enrolled with one or more of the following: history of (1) overt stroke; (2) silent stroke; (3) elevated transcranial Doppler velocity; (4) multiple vaso-occlusive crises; and/or (5) two or more acute chest syndromes and received haploidentical transplants from 18 parental donors. Cardiac and pulmonary centralized cores were established. Pulmonary function results were expressed as percent of the median of healthy reference cohorts, matched for age, sex, height and race. At 2 years, pulmonary functions including forced expiratory volume (FEV), FEV1/ forced vital capacity (FVC), total lung capacity (TLC), diffusing capacity of lung for carbon monoxide (DLCO) were stable to improved compared to baseline values. Importantly, specific airway conductance was significantly improved at 2 years (p

Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of obinutuzumab and rituximab for 24 hours (1-100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic that has taken millions of lives around the globe. Treatment of patients with moderate and severe COVID-19 disease has included dexamethasone, tocilizumab, Remdesivir, convalescent plasma, and targeted antibodies, however, currently, there are no FDA approved targeted cellular therapies in the treatment of mild or moderate SARS-CoV-2 disease. Virus-specific cytotoxic T cell lymphocytes (vCTLs) have shown therapeutic efficacy in immunocompromised patients with viral infections. We developed a multicenter and multidisciplinary Viral Cytotoxic T-Cell Consortium (VIRCTLC) to investigate the use of vCTLs manufactured by direct enrichment using the Cytokine Capture System (CCS) on the CliniMACS® Prodigy device. SARS-CoV-2 specific PepTivator Peptides consist of overlapping peptides that span the entire sequence of the protein (Protein N and M), or the length of its immunodominant domain (Protein S). The peptides can bind to either MHC class I or MHC class II molecules and are therefore able to target both CD4 and CD8 T cells. Objective: To screen, manufacture, and characterize SARS-CoV-2 vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System on the CliniMACS® Prodigy device. Methods: Donor screening was done utilizing PBMNCs from 15 convalescent COVID-19 donors after informed consent. PBMNCs were stimulated with a mix of PepTivator peptides (Miltenyi Biotech®) contained in the S, M and N proteins. IFN-γ levels were examined in CD3, CD4, and CD8 T cells by flow cytometry analysis. After informed consent, PBMNCs from three convalescent COVID-19 donors who screened positively to the PepTivator® peptide pools of SARS-CoV-2 Proteins M, N and S were collected by apheresis using the SPECTRA Optia® apheresis instrument. PBMNCs were incubated with the PepTivator® peptide pools for 4 hours. After incubation, the SARS-CoV-2 vCTLs were enriched using the CliniMACS Cytokine Capture System as we have previously described (Flower/Cairo, et al, ASTCT, 2020). Samples were taken from the enriched vCTLs and tested in gram stains, sterility cultures, cell counts, viability and IFN-γ cytokine staining (CD3/CD4/CD8/IFN-γ marker panel) by flow cytometry. Amplification and sequencing of TCRβ CDR3 regions of pre-stimulated PBMNC, stimulated PBMNCs samples taken from the QC bag (QC samples) and the enriched SARS-CoV-2 vCTLs were performed on the ImmunoSEQ platform using ImmunoSEQ® TCRB Assay kit (Adaptive Biotechnologies, Seattle, WA, USA). Characterization of immune subsets was done by mass cytometry analysis with 41 Immunophenotypic markers. Transcriptome of the immune landscape of QC samples, and enriched vCTLs was compared with the pre samples using the human nCounter PanCancer Immune Profiling Panel on the nCounter system. Results: We demonstrate that 93.3% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that consisted of 79% + 21% (mean + SEM) IFNγ + T cells (Fig.1). TCRβ sequencing showed that convalescent COVID-19 donors have a highly diverse TCR repertoire and we identified TCRβ CDR3 clones that are known to be associated with SARS-CoV-2 T cell responses. Immunophenotyping analysis demonstrated more CD4 T cells than CD8 T cells in the SARS CoV-2 vCTLs, an increase in memory CD8 and CD4 cells, especially CD8 T EM, CD4 T cm and CD4 T EMRA cells (Fig.2) and an increase DC cells in the SARS CoV-2 vCTL products as compared to pre-stimulated PBMNCs. Expression of the exhaustion markers was not enhanced in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Transcriptome analysis showed increased gene expression in T-cell function, interleukin, pathogen defense, and TNF superfamily pathway genes in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Conclusion: Our study demonstrates that highly functional SARS-CoV-2 vCTLs can be rapidly generated by direct cytokine enrichment from convalescent donor peripheral blood mononuclear cells. These data serve as pre-clinical validation for an ongoing clinical trial utilizing related HLA-matched and haplo-identical SARS CoV-2 vCTLs for the treatment of patients with mild and moderate SARS-CoV-2 disease (IND #27260, NCT# 04896606). Figure 1 Figure 1. Disclosures Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company. Johnson: Miltenyi Biotec: Research Funding. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees; Nektar: Membership on an entity's Board of Directors or advisory committees.

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Background: Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal. Objective: To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α. Design/Methods: Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P Results: The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p Conclusions: Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. Figure 1 Figure 1. Disclosures Cairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure: Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

Burkitt lymphoma (BL) is the most common histological subtype of non-Hodgkin lymphoma (NHL) in children and adolescents. Through the introduction of short intensive multi-agent chemoimmunotherapy, survival has improved significantly over the past 30 years. However, this successful approach is limited by significant chemotherapy-induced acute toxicity and risk of developing resistant disease, demonstrating the need to identify less toxic and targeted therapies. We analysed the comparative genomic signature and targetable signalling pathways in paediatric BL (PEBL) samples from the Children’s Oncology Group study (ANHL01P1) by genomic profiling and selected genes were confirmed by quantitative real time polymerase chain reaction. These results were compared to PEBL samples from public databases and utilised the Gene Expression Omnibus (GEO) Series (GSE) 10172 and 4475 (n=16), and 4732 (n=15). Three hundred and seventy-six genes (approximately 25%) were similarly expressed among three PEBL sample groups. Several target genes in Toll-like receptor signalling, JAK-STAT signalling and MAPK signalling were significantly overexpressed in PEBL. In addition, several tyrosine kinases, including Bruton tyrosine kinase (BTK), protein tyrosine phosphatase (PTP) and histone deacetylase inhibitor (HDACi) were highly expressed in PEBL. These pre-clinical results suggest that specific signal transduction pathways are overly expressed in PEBL and several pathways could serve as potential future therapeutic targets.

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p 0.05); LAMP-1 (p 0.05), granzyme B, and perforin expression (p 0.001); and in vitro cytotoxicity against B-NHL (p 0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p = 0.0086) and B-NHL cell numbers (p 0.01) at 5 weeks and significantly increased survival (p 0.001) at 10 weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting in vitro and in vivo.

Background: Graft versus host disease (GVHD) is a life-threatening complication following allogeneic stem cell transplantation (AlloSCT), which results from allo-reactive donor T-cells reacting against human leukocyte antigen (HLA) disparate host antigens. Despite the use of prophylactic immunosuppresants, 40 to 70 percent of patients undergoing HLA matched related and unrelated transplants will develop grade II-IV acute GVHD (Goker et al, Ex Hem, 2001; Nademanee, Blood, 1995; Rutuu et al, BMT, 1997).CD34+ enrichment of GCSF mobilized peripheral blood stem cells (PBSCs) obtained by apheresis is a method for T-cell depletion to reduce the risk of Grade II-IV aGVHD but unfortunately maybe concomitantly associated with delayed immune reconstitution, increased opportunistic infection and/or malignant relapse. To circumvent these latter complications, we previously demonstrated the results of a CD34+ enrichment with mononuclear cell (MNC) addback fixed at 2x105 CD3/kg of recipient weight in pediatric MUD recipients demonstrating rapid engraftment, robust immune reconstitution, and a low incidence of Grade II-IV aGVHD (Geyer/Cairo, BJH, 2012).More recently, we demonstrated a similar approach in children, adolescents, and young adults with high-risk SCD following familial haploidentical stem cell transplantation resulting in a rapid engraftment, probability of 6.7% of Grade II-IV aGVHD and 90% 1-year OS (Cairo et al, JAMA Peds, 2020). This approach of PBMNC addback with a fixed dose of 2x105 CD3/kg facilitated the infusion of additional NK, NKT, B, DC, DC2 cells at that same time (Chu/Cairo et al, ASH, 2020). Objective: To determine the safety, hematopoietic engraftment, probability of Grade II-IV GVHD following HLA related and unrelated PBSC transplantation following CD34+ enrichment with MNC cell addback (2x105 T-cell/kg fixed dose) in patients with malignant and non-malignant diseases. Design/Methods: Eligible patients were enrolled on study. Patients received individualized conditioning regimens determined by the PI stratified by disease, disease status, and donor source and received CD34+ enriched products processed by using the CliniMACSâ CD34+ Reagent System (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD34+ cell product was either infused or cryopreserved and stored until time of transplantation. A target of 5x106 CD34+/kg recipient weight with a PBMNC fixed dose of 2x105 T cell CD3 dose/kg was infused as we previously demonstrated (Cairo et al, JAMA Peds, 2020). Patients were followed for safety, engraftment, donor chimerism, probability of Grade II-IV aGVHD and chronic GVHD. Results: Thirty-eight patients underwent HSCT with median age of 35.2 years (21 months to 71 years). Patients' disease status was as follows: complete remission (CR) 3 in 1 AML patient, CR2 in 8 AML patients, CR1 in 14 AML patients, CR2 in 4 ALL patients, CR1 in 2 ALL patients, PR in 1 MDS patient, Lymphohistiocytosis (n=1), Macrophage Activating Syndrome (n=1), Diamond Blackfan (n=1), Aplastic Anemia (n=2), Sickle Cell Disease (n=1), 1 CNL patient, CR2 in 1 Non-Hodgkins Lymphoma patient. Sixteen patients received allogeneic 10/10 HLA-matched unrelated donors, 5 from 9/10 HLA-matched unrelated donors, 12 from 6/6 HLA-matched sibling donors, 5 from related haplo donors. PB-HPC products contained 2x105 CD3/kg (±0.25 x 105), and 9.72x106 CD34/kg (±0.97 x106). After CD34 enrichment, the PB-HPC product processed was 76.09% CD34+ (±2.7%) (Fig. 1A) enriched with mean ± SEM log T cell depletion of 4.01 (±0.17) (Fig. 1B). The target HSCT dose per patient was 5x106 CD34/kg. Thirty-seven patients had myeloid engraftment and 32 patients had platelet engraftment with a median of 11 and 17 days, respectively. Six patients died prior to platelet engraftment - three due to multi-organ system failure following septic shock, two due to refractory disease, and one due to adenoviremia. Early and late peripheral blood chimerism was ³ 95% at 14- and 100-days following transplantation. The probability of grade II-IV aGVHD was 28.1% (CI95: 9.3-50.7) (Fig. 2). The probability of cGVHD was 4% (CI95: 0-63.5). Conclusion: This study demonstrates safety, rapid hematopoietic engraftment, sustained donor chimerism of CD34+ enriched PBSC products with MNC cell addback with a fixed 2x105 CD3/kg dose in alloSCT recipients with a low probability of Grade II-IV aGVHD.and cGVHD. Disclosures Seiter: Novartis: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Forma: Research Funding; Sun Pharma: Research Funding; Amphivena: Research Funding; Roche: Research Funding; AbbVie: Speakers Bureau; Alexion: Speakers Bureau; Onconova: Research Funding. Flower:Lentigen Technology Inc/Miltenyi Biotec: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background & Aim Background Viral infection/reactivation after allogeneic hematopoietic stem cell (HSCT) or solid organ transplant (SOT) is a significant cause of morbidity and mortality in drug-refractory patients. Adoptive transfer of allogeneic virus-specific cytotoxic T lymphocytes (vCTLs) can rapidly restore antiviral T cell immunity. The IFN-gamma Cytokine Capture System (CCS, Miltenyi) provides a fast and automated method of isolating vCTLs by stimulating them with virus-specific peptides. Aim To evaluate the safety and efficacy of vCTLs manufactured using IFN-gamma CCS on CliniMACS Prodigy® in children and young adults (CAYA) and highlight technical aspects of vCTL manufacturing by this method. Methods, Results & Conclusion Methods CAYA HSCT or SOT recipients with refractory viral infections (CMV, ADV, EBV, BK) received adoptive transfer of vCTLs from haploidentical relatives or matched donors who screened positive for the relevant vCTL. Following non-mobilized leukocytapheresis, virus-specific CD4+ and CD8+ T cells were isolated using IFN-gamma CCS on CliniMACS Prodigy®. Results Fourteen patients have been treated to date (Table 1). Of patients treated for CMV, EBV, ADV, or their combination, 100% (9) achieved complete remission (CR). 2 out of 4 patients treated for BK achieved CR, 2 had partial remission (PR), and one patient treated for CMV/BK had resolution of CMV and no response (NR) to BK. There were no episodes of acute Grade II-IV GVHD, chronic GVHD, CRS, or infusion reactions. We noted occasional discordance between donor viral serology and their response to the stimulation by the viral Peptivator; for example, we successfully manufactured anti-ADV vCTLs from a donor who was serologically negative to adenovirus. Flow cytometry analysis of rare events in a limited sample required sequential gating strategy to maximally reduce background noise. Products thus manufactured have unique properties, such as low cell dose and high debris content, which may face additional regulatory scrutiny. Conclusion Despite its unique challenges, vCTL manufacturing is rapid, reproducible and effective, and vCTLs are safe and well tolerated in CAYA with HSCT and SOT.

There is an increase in utilizing matched unrelated donors (MUD) & haploidentical donors over the past 20 years (Cairo et al, Biol Blood and Marrow Transplant, 2008). However both of these allogeneic donor sources are associated with severe acute graft -versus -host diseases. We have previously reported the success of CD34 enrichment utilizing the Isolex® device (Geyer/Cairo et al, British Journal of Hematology, 2012). Positive selection of CD34+ cells with the CliniMACS® system has been shown as a highly effective technique for obtaining a T-lymphocyte-depleted product. We report our results with 55 procedures performed in the last two years at our Cell and Tissue Engineering Laboratory. To evaluate the impact of overnight storage on cell recovery, viability and sterility before processing, as well as to evaluate the CD34 +cells recovery in the final product. From October 2017 to August 2019, after consent, 37 donors were mobilized with G-CSF. Fifty-five leukapheresis harvests were performed using the Spectra Optia® Apheresis System. Twenty-six were stored overnight at 4°C before processing using the CliniMACS® device. WBC counts were performed using the Sysmex XE hematology analyzer. Phenotypic analysis was performed with a FACSCalibur BD flow cytometer on samples from leukapheresis products at the day of collection (Day 0), before processing (Day 1) and after the selection. The WMC Microbiology Laboratory performed the sterility testing using BACTEC FX System. Twenty-six products were stored overnight before processing with a mean ±sem nucleated cells (NC) of 19 × 108/kg (± 2.66) and CD34+ cells of 13.8 x106/kg (± 2.93) at Day 0 and NC of 16.2 × 108/kg (± 2.62) and CD34+ cells of 13.9 x106/kg (± 3.03) at Day 1. NC and CD34+ cells content were not significantly different at Day 0 and Day 1 (respectively p=0.95 and p=0.98). The median CD34 viability at Day 1 was 99% (range 93-99%). The sterility testing were all negative. Fifty-five products showed a median starting cellularity of 8.45 × 106 CD34+ cells (range 1.4-60.3 × 106). The median post selection CD34+ cells was 6.62 × 106 (range 1.2-46.2 × 106). The mean ±sem CD34 recovery was 9.64 × 106/kg (± 1.2) (Figure 1A) with a purity of 98.5%. The final T cell reduction was a mean ±sem of 0.38 × 105/kg (± 0.08) (Figure 1B). A mean ±sem of 9.75 × 106/kg (± 0.84) of CD34+ cells was infused. To enhance engraftment, 2 × 105/kg CD3+cells were added back to the selected product. All patients engrafted at a median time of 11 days. No patient developed an infusion reaction. Overnight storage of peripheral blood stem cells does not affect CD34+ cells enrichment, viability nor safety of the leukapheresis product. Our median of 80% of CD34 recovery is higher than the recovery of previous studies (Cairo et al, JAMA Pediatrics, 2019). These data demonstrate that CD34 enrichment on the following day using CliniMACS® device is reproducible and safe in pediatric and adult allogeneic recipients.

Background Sickle cell disease (SCD) is a hemoglobinopathy and vasculopathy resulting in vaso-occlusive crises, chronic end-organ damage, and shortened life span. The only curative therapy is allogeneic stem cell transplant (AlloSCT) (Talano/Cairo, EJH, 2015). Unfortunately, only approximately 16% of the sickle cell population has an HLA matched unaffected sibling (Bhatia/Cairo, BMT, 2014). We have demonstrated the safety and efficacy of familial haploidentical transplants (FHI) in this population with 100% engraftment (Cairo et al, JAMA Peds, 2019). However, the incidence of sinusoidal obstructive syndrome (SOS) in SCD AlloSCT recipients is approximately 32% (McPherson, BMT, 2011). Defibrotide primarily targets endothelium and can reduce endothelial cell injury (Falanga et al, Leukemia, 2003 and Scallia et al, Clin Pharm, 1996). Corbaciolgu et al (Lancet 2012) demonstrated the safety and efficacy of prophylactic defibrotide in high-risk pediatric AlloSCT recipients to significantly reduce the incidence of SOS. Objective To determine the safety and efficacy of defibrotide prophylaxis for SOS in children, adolescents and young adults (CAYA) with high-risk SCD following MAC and FHI AlloSCT, utilizing CD34+ selection with CD3 addback. Design/Methods Patients with SCD aged 2 to 35 years with high-risk SCD (stroke, ≥2 acute chest syndrome in past 2 years, ≥3 pain crises in last 2 years, abnormal TCD study, ≥1 silent infarct lesion on MRI) were enrolled after being consented (NCT02675959). Patients were in two cohorts (2 to 17.99 years and 18 to 34.99 years of age) for age-based analysis and treatment (Table 1 and 2). All patients received a MAC regimen prior to receipt of an FHI with CD34+ selection and CD3 addback peripheral blood stem cell transplantation (PBSCT), as we have already demonstrated (Cairo et al, ASH, 2018). Defibrotide was given day -10 during conditioning through day +21 following transplant. Baseline studies of organ function and biomarkers were obtained prior to transplantation. Results We have enrolled eight patients, five in cohort 1 (median age 9.9 years) and three in cohort 2 (median age of 24 years) with a gender ratio (M/F) of 4/4. Patients had hemoglobin SS (n=7) or hemoglobin SC (n=1). Patients had either a maternal (n=7) or sibling (n=1) donor. Patients had early neutrophil engraftment (median day +11 (day +7 to day +14)). All tolerated 109 ± 11doses of defibrotide. There were no severe adverse or bleeding events probably or directly related to defibrotide. No patient developed SOS. Conclusion The preliminary data suggests defibrotide is safe and well tolerated in CAYA patients with high-risk SCD following MAC and FHI AlloSCT, utilizing CD34+ selection with CD3 addback. A larger cohort and/or follow-up will be required to confirm these preliminary findings. Supported by FDA R01FD004090 and Jazz Pharmaceuticals.

Viral infection remains a major cause of morbidity and mortality after allo-HSCT (Bollard/Heslop Blood 2016). Antibiotics for treatment of viral infection in immunocompromised patients are limited in efficacy and associated with toxicity (Gerdemann BBMT 2004; Sili Cytother 2012). Increasing evidence suggests that use of VST for immunocompromised patients with viral infections can provide therapeutic benefit and improve OS (Bollard/Heslop Blood 2016; Sutrave Cytother 2017). Methods of VST production include ex-vivo expansion and direct selection (Gottlieb Cytother 2017). Ex-vivo expansion requires prolonged manufacturing time, is associated with exhaustion, and provides a limited donor pool. Direct selection results in rapid production and allows for expanded HLA matching. A multicenter consortium (VIRCTLC) was created to investigate the safety and efficacy of VST (Figure 1) manufactured by the IFN- CCS process automated on the CliniMACS® Prodigy device (Miltenyi Biotec) for immunocompromised patients with viral infection. To determine the safety and efficacy of VST therapy for immunocompromised CAYA patients with viral infection. CAYA patients after allo-HSCT, SOT or with PID with refractory ADV, CMV, EBV or BKV infections despite 2 weeks of appropriate anti-viral therapy, and/or resistance to antibiotics and/or intolerance to antibiotics were eligible. Donors were screened with viral specific antigen (PepTivator®) to predict successful VST manufacturing. Eligible donor PBMC were collected with non-mobilized apheresis. VST were isolated using the CliniMACS® Prodigy following specific stimulation of PBMC with specific viral MACS PepTivator® pools, generously provided by Miltenyi Biotec. Production of CD4+ and CD8+ VST was performed as previously described (Feucthinger Blood 2010). Target cell dose was 0.5 × 104 CD3+/kg for HLA mismatched related donors and 2.5 × 104 CD3+/kg for matched related donors. Based on response and safety, additional doses of VST were given every 2 weeks, for a maximum of 5 infusions. Thirteen patients were treated under IND 17449 and other single INDs. Median number of VST infusions was 2 (1-5). Mean±SEM CD3+ cell dose infused was 0.48±0.03 × 104. There were 10 CR (PCR negative), 2 PR (PCR≥1 log decrease), and 1 too early to assess (ORR 100%, CRR 83%). Median time to maximal response was 24 days (range 3-111). No patient developed aGVHD, extensive cGVHD, infusion reaction or CRS associated with VST infusions. One patient experienced recurrence of aGVHD, possibly related to VST. (Table 1) Preliminary results of this pilot study demonstrate that VST are safe and efficacious in CAYA with refractory viral infections after alloHSCT, SOT or with PID. VST manufacturing utilizing the CliniMACS® Prodigy device is rapid (24 hours), reproducible and effective. Accrual is ongoing.

Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton's tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p&nbsp

Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo. Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.

Background Allogeneic stem cell transplantation (AlloSCT) from an HLA-matched sibling donor is the only known curative therapy in high-risk SCD patients (Talano/Cairo, EJH, 2015). Unfortunately only about 15% of high-risk SCD patients have an HLA-matched unaffected sibling donor. T cell depletion has been employed to reduce AGVHD. We reported a very low incidence of GVHD in pediatric recipients receiving CD34 enriched HPC products with PBMNC addback from MUD donors (Geyer/Cairo et al, BJH, 2012). Rapid NK cell reconstitution after AlloSCT is associated with a significant improvement in 1yr OS (Pical-Izard, BBMT, 2015). Recently, we reported promising results for high-risk SCD patients at 1 yr follow-up after FHI CD34 enriched/PBMNC with addback with the probability of 1-yr OS 88.2% (Cairo, et al, JAMA Peds, 2019). Objective To investigate donor chimerism, immune reconstitution and the incidence of GVHD in high-risk SCD patients following AlloSCT using FHI CD34 enrichment/PBMNC (2 × 105 CD3/kg) addback. Methods CD34 cells were enriched using the CliniMACS® system, kindly provided by Miltenyi Biotec, with a target dose of 10 × 106 CD34+ cells/kg. PBMNC were added back to the final CD34 product at a dose of 2 × 10*5 CD3/kg. Whole blood and CD71-enriched erythroid lineage chimerism were determined by STR. Immune cell, subset reconstitution, NK function, CD107a and granzyme B were assessed by flow cytometry as previously described (Geyer/Cairo et al. BJH, 2012, Chu/Cairo et al, Can Imm Res, 2015). Results The cumulative incidence of grade II-IV and late AGVHD was 6.2 % and moderate and/or severe CGVHD incidence was 6.7%, respectively (Fig. 1A). There was 100% engraftment of neutrophils and platelets. The median day post-HISCT to neutrophil and platelet engraftment was +9 and +19, respectively. Whole blood donor chimerism (mean±SEM) at 1-yr, 2-ys, and 3-ys post-HISCT was 97±1%, 97±1%, 97±1%, respectively (Fig.1B). Donor chimerism for CD71-enriched erythroid lineage cells (mean±SEM) at 1-yr, 2-yrs, 3-yrs post-HISCT was 97±2%, 98±1%, 98±1%, respectively (Fig.1B). The time to recovery of minimal normal levels for CD3 (800 cells/ul), CD4 (400 cells/ul), CD8 (200 cells/ul), & CD19 cells (200 cells/ul), was approximately 365, 365, 270, and 60 days post-HISCT, respectively (Fig.1C). NK reconstitution was rapid and peaked at d+30 (36±9%) with higher level of activating receptors NKp46, NKG2D & KIR2DS and inhibitory receptors NKG2A, CD94 and KIR2DL2/3. NK cytotoxicity against K562 (E:T = 10:1) peaked at d+30 and d+180 vs pre-transplant (p Conclusion The PBMNC addback facilitated rapid donor chimerism & immune reconstitution with a low probability of Grade II-IV AGVHD. The rapid NK reconstitution may have in part contributed to the excellent 1yr OS in this study (FDA R01FD004090 (MSC)). This approach is comparable to the method of CD3 TCRa/b-CD19 depletion.

Background MAC or RTC followed by UCB donor allo-HSCT in children with malignant and non-malignant diseases is safe and effective (Geyer/Cairo, BJH 2011). However, concentration of CD34+ HPCs in UCB is low, leading to delayed hematopoietic reconstitution and high incidence of engraftment failure (Satwani/Cairo, BBMT 2013). HPDSCs are rich in HPCs, low in HLA Class I/II expression and T-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (Liao/Cairo, Stem Cells Trans Med 2018). Objective To determine the safety and efficacy of HPDSCs plus UCB for allo-HSCT in children. Design/Method UCB with TNC ≥ 5 × 107/kg (4/6 HLA match) or ≥ 3.5 × 107/kg (5-6/6 HLA match) were included. Patients received MAC or RTC followed by UCB plus HPDSC infusion. GVHD prophylaxis consisted of tacrolimus and MMF as previously described (Bhatia/Cairo, BBMT 2009). Results Twenty-eight patients ≤18 years were enrolled, 15 males. Mean age± SD was 6.8±5.5 years, (4 months-12 years). There were 12 patients with non-malignant diseases including ALD (2), CAT (1), CGD (1), SCID (2), DC (1), CN (1), SAA (2), LCH (1), and SDS (1) and 16 patients with malignant diseases including B-ALL- CR1 (4), B-ALL - CR2 (4), T-ALL – CR1 (1), AML-CR1 (4), JMML-CR1 (1), TLL - CR1 (1), and BL - CR2 (1). There were 13, 9, and 5 patients with 6/6, 5/6, and 4/6 HLA match, respectively. Mean UCB TNC±SD and CD34+ cell dose±SD infused were 0.77±0.5 × 108/kg and 0.48±0.59 × 106/kg, respectively. Mean HPDSC TNC±SD and CD34+ cell±SD dose were 0.96±3.51 × 108/kg and 0.05±0.05 × 106/kg, respectively. There were no SAEs associated with HPDSC infusions. Four patients were withdrawn for administration of alternative therapy after engraftment failure (3) or relapse (1). Probability of neutrophil engraftment by day 42 was 78.6%, median day 22 (13-53). Probability of platelet engraftment by day 100 in neutrophil engrafted patients was 86.4%, median day 42 (35-98) (Figure 1). Mean UCB chimerism was 92, 99, 96 and 99% at day 30, 60, 100 and 180, respectively. Average HPDSC chimerism was Conclusions These results suggest that UCB plus HPDSCs for Allo-HSCT is safe and well tolerated. Median day to platelet engraftment was lower than previously reported using UCB alone (Geyer/Cairo.BJH 2011). Probability of Grade 2-4 aGVHD was lower than previously reported using UCB alone, possibly due to the immunomodulatory effect of HPDSCs (Geyer/Cairo.BJH 2011). A larger cohort and longer follow-up is required to determine clinical significance.

Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton’s tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström’s macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p

Background Children and young adults who develop treatment-refractory infection with EBV, CMV, and/or adenovirus (AdV) after allogeneic hematopoietic stem cell transplant (allo-HSCT) have a dismal prognosis (≥90% mortality). Adoptive transfer of ex-vivo expanded virus-specific cytotoxic T lymphocytes (CTLs) is a treatment option for these patients, but the cell manufacturing process is labor-intensive and typically takes several weeks of culturing to complete. More recently, a culture-free method has been developed to directly isolate virus-specific T cells from peripheral blood for adoptive transfer. The process is fully automated using the CliniMACS Prodigy device (Miltenyi Biotec), a cGMP-compliant, temperature-controlled, closed system. Importantly, virus-specific T cells ready for infusion can be isolated with Methods Peripheral blood mononuclear cells from volunteer donors were stimulated with PepTivators (CMV, EBV, and/or AdV), followed by isolation of the virus-specific T cells using the interferon-γ (IFNγ) cytokine capture system (CCS) on the CliniMACS Prodigy device (n=11). Expression of lineage and T-cell subset markers, IFNγ, and cell viability were determined by flow cytometry. Results The viability of recovered T cells was remarkably and consistently high (92.9% ± 7.4%, Table 1). The number of recovered viable T cells was 5.03 × 106 ± 5.09 × 106. The purity of IFNγ+ cells was 69.0% ± 23.8% for CD4 T cells and 82.9% ± 15.3% for CD8 T cells. At a dose of 2.5 × 104 T cells/kg, the mean T cell collection for a 70 kg recipient would suffice for 3 separate infusions. Conclusion The results presented here show that manufacture of virus-specific CTLs by the CCS method reproducibly produced sufficient quantities of viable T cells for patient treatment. These results thus support the feasibility of the multicenter VIRCTLC trial. The trial is currently enrolling patients.