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Relapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor–derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen–pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167.

Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL+ polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents.

Introduction:Transgenic co-expression of a major histocompatibility complex class I restricted tumor associated antigen specific TCR and CD8αβ (TCR8) has been previously proposed as a strategy to redirect CD4+ T cells to tumors. However, it is unknown whether forced TCR8 expression induces more fundamental transcriptional consequences in both CD4+ and CD8+ T cells, and whether T cell lineage origin affects this outcome. Here we deeply interrogate the effects of transgenic TCR and TCR8 in human CD4+ and CD8+ T cells upon leukemia challenge by single cell RNA sequencing (scRNAseq) and investigate T cell function in vitro and in vivo. We identify profound changes of gene expression that have significant functional consequences. Methods:A previously characterized HLA-A*02:01 restricted survivin-specific TCR was used (Arber et al, JCI, 2015 Jan;125(1):157-68) and a new polycistronic vector with this TCR and CD8αβ was generated. CD4+ and CD8+ T cells were isolated and scRNAseq (25'474 cells in total) was performed on (1) freshly isolated cells, (2) retrovirally transduced (TCR or TCR8) expanded cells, and (3) TCR+CD8+, TCR8+CD8+ and TCR8+CD4+ T cells co-cultured with BV173 leukemia cells (HLA-A*02:01+survivin+). scRNAseq results were cross-validated in independent experiments with FACS analysis of selected markers, in vitro stress-killing assays, analysis of cytokine production, and assessment of anti-tumor function in vivo in xenograft mice. Results:CD4+ T cells only killed BV173 leukemia cells when redirected with TCR8 but not with TCR alone (p=0.0004, n=7), while killing by TCR+CD8+ and TCR8+CD8+ T was comparable (p=NS). To explore some of the possible underlying mechanisms, we used dimensionality reduction and unsupervised clustering of the scRNASeq data and identified 19 distinct cell clusters. CD4+ and CD8+ lineage origin clearly separated the samples, but separation by transgene type only became apparent upon co-culture. Analyzing differentially expressed genes, we found that co-cultured samples contained clusters with high expression of cytotoxic markers but with significant differences between CD4+ and CD8+ lineages (e.g. transcription of GZMB in CD4+ T cells and GNLY, NKG7, GZMK in CD8+ T cells). Next, we analyzed which genes were upregulated from the expanded to co-cultured states. Co-cultured TCR8+CD4+ T cells had more upregulated genes with a broader diversity compared to TCR+CD8+ or TCR8+CD8+ T cells. Among these upregulated pathways were cytotoxicity, co-stimulation, oxidative phosphorylation, NFkB regulation, cell growth and transcription factors. TCR8+CD4+ T cells also retained a less differentiated phenotype (e.g. high IL7R, SELL, CCR7, CXCR4) with preservation of replicative potential. Furthermore, co-cultured TCR8+CD4+ T cells expressed more co-stimulatory and less activation/ exhaustion markers. In addition, co-cultured TCR8+CD4+ T cells heavily relied on oxidative phosphorylation and had higher mitochondrial activity compared to co-cultured TCR+ or TCR8+ CD8+ T cells. In stress co-cultures with multiple rounds of tumor challenge, TCR8+CD4+ T cells outperformed TCR+CD8+ T cells (number of killings TCR8+CD4+ vs TCR+CD8+: 3.3±0.5 vs 1.3±1.1, p=0.01, n=7), but were comparable to TCR8+CD8+ T cells (TCR8+CD4+ vs TCR8+CD8+: 3.3±0.5 vs 2±1.4,p=NS, n=7). TCR8+CD4+ T cells expanded significantly better than TCR+CD8+ T cells (p=0.002) and TCR8+CD8+ T cells (p=0.015) and produced TH1 type cytokines. In the xenograft mouse model, we observed significant BV173 leukemia control in mice treated with TCR+CD8+ T cells compared to controls (NT), and further enhancement in mice treated with TCR8+CD8+ T cells (NT vs TCR: p=0.0002, NT vs TCR8: p Conclusion:Transgenic TCR8 expression has previously been proposed as a strategy to enhance TCR-pMHC recognition. Here we identify profound transcriptional changes involving multiple pathways that are important for sustained anti-tumor function upon adoptive T cell transfer in vivo, such as cytotoxicity, co-stimulation, cell cycle and metabolism. Our results point towards previously unrecognized mechanisms by which TCR8 transgenes mediate their beneficial effect in both CD4+ and CD8+ T cells. Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees. Arber:Cell Medica: Patents & Royalties.

High risk or relapsed pediatric acute myeloid leukemia (AML) is potentially curable with allogeneic hematopoietic stem cell transplantation (HSCT). The use of a matched related donor (MRD) is the standard of care for these patients, but in the majority of patients a MRD is not available. Significant improvements have been made with supportive care practices, but there are limited outcome data for AML in pediatric patients transplanted in the contemporary era. We report retrospective results of HSCT in eighty-seven pediatric patients with high risk or relapsed AML performed at a single institution between 2008 and 2018. The median age at the time of HSCT was 8 years old (range: 0.6-20 years). More than 2/3rd of patients were in remission at time of HSCT with 43 patients (49%) transplanted in CR1 and 18 patients (21%) in CR2. Twenty six patients (30%) had active disease (defined as any evidence of disease) at the time of HSCT. Patients were transplanted using a matched related donor (MRD, n=21), a matched unrelated donor (MUD, n=25), a mismatched unrelated donor (MMUD, n=15), a haplo-identical donor (haplo, n=12) or an umbilical cord blood (UCB, n=14) graft. The majority of patients (78/87, 90%) received myeloablative conditioning while 9 patients (10%) received reduced intensity conditioning regimens. MRD and UCB graft recipients did not receive serotherapy. MUD, MMUD and haplo graft recipients (n=52) received serotherapy, the majority of whom received an alemtuzumab-based regimen, 42/52 (81%). Haplo-identical transplants were performed using CD34+ selected grafts. Three-year overall survival (OS) and disease free survival (DFS) for the entire cohort were 52% (95% CI: 41-62%) and 49% (95% CI: 38-59%) respectively. OS differed significantly according to disease status at time of transplant (p=0.018), with 3 year OS: 60%, (95% CI: 43-74%) for patients in CR1, 56%, (95% CI: 31-75%) for patients in CR2 and 34%, (95% CI: 17-52%) for patients with active disease. OS differed by donor type (p=0.058). OS was comparable for patients with MRD (71%, 95% CI: 46-86%) and 10/10 MUD (59%, 95% CI: 37-75%) (p=0.67). Patients with alternate donor sources had worse outcomes (MMUD 33%, 95% CI: 10-59%; UCB 43%, 95% CI: 18-66%; Haplo 33%, 95% CI: 10-59%) as compared to MRD (MMUD vs MRD, p=0.042, UCB vs MRD, p=0.046, haplo vs MRD p=0.017). There was a low incidence of Grade III-IV GVHD (8%, n=9) and extensive chronic GVHD (9%, n=7) likely secondary to the use of alemtuzumab-based conditioning regimens for MUD, MMUD and haplo donors. The 3-year non-relapse mortality was low at 11%, (95% CI: 5-19%). The primary cause of death was relapse, with a 3-year cumulative incidence of relapse of 40%, (95% CI: 30-50%). Patients with active disease at transplant had significantly higher cumulative incidence of relapse (sHR: 2.42, 95% CI: 1.18-4.95, p=0.016) compared to patients in remission. There was no difference in cumulative incidence of relapse between patients in CR1 and CR2 (p=0.598). In the contemporary era, outcomes after allogeneic HSCT are comparable for MRD and 10/10 MUD for pediatric AML. Myeloablative conditioning regimens that incorporated alemtuzumab were well tolerated with low rates of NRM and very low rates of acute and chronic GVHD. Relapse remains the major cause of treatment failure and future trials evaluating use of cellular therapies and targeted agents post-HSCT will be needed to improve current rates of relapse. Disclosures No relevant conflicts of interest to declare.

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.

T-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell–derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell–derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.

Treatment of Epstein-Barr virus (EBV)–positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of auto-logous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at http://www.clinialtrials.gov as NCT00608257.

Epstein-Barr virus (EBV)–associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically “weak” but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

Abstract 2680 The nuclear factor-κB (NF-κB) pathway plays an integral role in Hodgkin's lymphoma (HL), and enhances survival of Hodgkin Reed Sternberg (H/RS) cells. Previous studies have shown that both the classical and alternative NF-κB pathways are constitutively active in adult HL. However, it is not known which proteins in the NF-κB pathway are constitutively active in pediatric HL, or if NF-κB pathway activation and regulation are similar between patients who respond rapidly to treatment (RER) and patients who are slow early responders (SER). We used a tissue microarray (TMA) to examine specimens from 102 pediatric HL patients (56 male, 78 Caucasian, median age 15y (range 1–21y), 85 nodular sclerosing subtype, 99 evaluable, 80 RER, 19 SER), and 10 lymphoid controls for NF-κB pathway subunit expression. All HL samples were intermediate-risk HL treated on the Children's Oncology Group protocol AHOD0031. RER was defined as a >60% shrinkage in tumor volume after two therapy cycles as assessed by CT; SER had Cytoplasmic NF-κB-inducing kinase (NIK) was overexpressed in SER patients compared to RER patients (p=0.005, Wilcoxon rank sum test). As expected, increased cyto-NIK was associated with increased cyto-IKK-α (r= 0.87). However, increased cyto-NIK was not associated with increases in either cyto-NF-κB2 (r = 0.12), cyto-Rel-B (r = 0.138) or nuclear Rel-B (r= 0.084). Data suggests a deregulation in the alternative NF-κB pathway, specifically IKK-mediated proteasomal cleavage of NF-κB2 in SER patients. In RER patients, there were several correlations between NF-κB pathway proteins (nuclear phospho-Rel-A with nuclear c-Rel [CC-0.51, p Disclosures: No relevant conflicts of interest to declare.

The nuclear factor-kB (NF-kB) pathway plays an integral role in Hodgkin’s lymphoma (HL), and may enhance survival of Hodgkin Reed Sternberg (H/RS) cells following chemotherapy by enhancing an anti-apoptotic phenotype. Previous studies have shown that both canonical (NF-kB/RelA or c-Rel) and non-canonical (NF-kB/RelB) NF-kB pathways are constitutively active in adults with HL. Epstein-Barr virus (EBV) infection has also been implicated in the constitutive activation of NF-kB in H/RS cells. Although LMP1 has been postulated to activate NF-kB through processing of the NF-kB subunit p100, it is unclear if this is the sole mechanism of EBV-induced activation of NF-kB and which specific NF-kB pathways (canonical versus non-canonical) are involved. There is also limited published information on EBV activation of NF-kB in pediatric HL. We have examined specimens from 105 pediatric HL patients (61 male, 85 Caucasian, age range 1–21y [median age 15y], 87 nodular sclerosing, 8 mixed cellularity, 7 lymphocyte predominant, 3 NOS) and 10 lymphoid controls for NF-kB and IkB subunit expression by immunohistochemistry (IHC) using a HL tissue microarray obtained from the BioPathology Center (Columbus, OH) created from samples archived by the Children’s Oncology Group. All patients had intermediate-risk HL and were treated on the same protocol (AHOD0031). After CD30 staining evaluation, 14 (4%) of the 322 HL and control microarray spots (including 3 HL patients) were excluded from analysis for either weak CD30 staining or insufficient H/RS cells. The remaining 102 HL patients (282 spots on the microarray) were examined for both canonical and non-canonical NF-kB pathway subunit expression (RelA, RelB, c-Rel, IkB-a and IKK-a). Slides were also evaluated for EBV status using LMP-1 (IHC) and EBER (in situ hybridization) (concordance: 93%). Staining intensity (3-point scale) and localization (nuclear vs. cytoplasmic) of NF-kB, IkB, and IKK subunits was assessed and compared to controls, and EBER+ HL samples (n=19) were compared to EBER− HL samples (n=83). As previously demonstrated using standard IHC, both canonical (RelA p

Despite improvements in the treatment of adults with acute lymphoblastic leukemia (ALL) during the last decade, most patients will relapse and ultimately die of disease. Although some factors that affect risk of disease and treatment outcome are known, our ability to predict response and tailor treatments is far from perfect. In particular, we observed that the majority of the ALL patients treated at our large public hospital in Houston, TX were Hispanic. In order to more closely examine this trend and attempt to identify characteristics that would predict risk or therapeutic response, we performed a retrospective analysis of all patients with precursor B-cell ALL, T-cell ALL, and Burkitt lymphoma treated at our hospital over a fifteen year period. One hundred thirteen patients were identified, including sixty-one with precursor B-cell ALL, thirty-two with Burkitt lymphoma, sixteen with T-cell ALL, and four patients with additional features. All patients were treated with HyperCVAD, alternating with methoxtrexate and cytarabine, although some patients had therapeutic modifications because of disease characteristics, particularly in the first course. The median overall survival for all patients was 357 days. Patients with precursor B-cell ALL had the poorest outcome in both overall survival and progression-free survival, and a less favorable outcome than expected from the literature with a 38% two-year survival (95% CI: 54–92%). In contrast, patients with Burkitt lymphoma had an 80% 2-year survival (95% CI: 54%–92%) and those with T-cell ALL had a 63% 2-year survival (95% CI: 32%–83%). The outcomes for Burkitt lymphoma and T-cell ALL are similar to those reported in the literature. Nearly 80% of the patients with precursor B-cell ALL were Hispanic, and most were older than thirty (mean age 36.4 years) with few comorbidities. In contrast, only about 40% of the other high grade leukemia/lymphoma patients were Hispanic, reflecting more closely the racial/ethnic distribution of the population served by the hospital. While our precursor B-cell ALL patients were predominantly Hispanic, there was no statistical difference in overall survival or progression-free survival for Hispanic versus non-Hispanic patients. Hispanic patients with pre-B ALL had a 38% 2-year survival (95% CI: 20%–55%) while non- Hispanic patients had a 41% 2-year survival (95% CI: 12%–69%). Patients younger than thirty with precursor B-cell ALL had a statistically significant survival benefit compared to older patients. Fourteen patients (22.6%) with precursor B-cell ALL had a white blood cell count greater than 50,000, and these patients had significantly poorer outcomes. There was no correlation between cytogenetic abnormalities and outcome; however, very few patients were found to harbor the 9:22 translocation. There was a high incidence of CD20 positivity (55.7%) in patients with precursor B-cell ALL; however, there was no statistically significant correlation with overall survival or progression-free survival. Thus in our multiethnic patient population, patients with precursor B-cell ALL had significantly poorer outcomes than those reported in the literature, while patients with T-cell ALL or Burkitt lymphoma had outcomes to those reported in the literature using the identical chemotherapy regimen. The older age of the precursor B-cell ALL patients may have contributed to this poorer outcome. As noted above, our precursor B-cell ALL patients were predominately Hispanic as compared to the other leukemia patients we studied. Although Hispanic ethnicity did not clearly contribute to outcome, risk of developing precursor B-cell ALL appears to be greater in the Hispanic population.

Objective Currently, thrombocytopenia is typically observed in patients undergoing hematopioetic stem cell transplantation (HSCT), high-dose chemotherapy or irradiation. Severe thrombocytopenia can cause bowel hemorrhage and intracranial hemorrhage. To transfuse ex vivo-expanded Mk cells into patients can reinforce the ability of platelet formation and shorten the time of platelet recovery. So it is one of the effective approaches to protect the danger. To explore the differences in megakaryocyte (MK) expansion between CD34+ stem cells derived from cord blood (CB) and Peripheral Blood (PB) and to establish the most optimal culture system. Methods Mononuclear cells were isolated by density gradient centrifugation over Ficoll. CD34+ cells were isolated by positive selection using an immunomagnetic separation system. selected CD34+ cells were seeded in Iscove’s modified Kulbecco’s medium (IMDM) supplemented with fetal calf serum (FCS) and all kinds of cytokines. After 15–17 days of culture, the cells were measured and determined the content of CD41+ cells by flow cytometry, and simultaneously measured the number of megakaryocyte colony-forming unit (CFU-MK). Results After the definite days of culture, the cytokine combination of TPO+FL+IL-6+IL-3 is most suitable both for PB and CB to obtain high numbers of MK, and better than any other three groups(P Conclusion For PB and CB, the cytokine combination of TPO+FL+IL-6+IL-3 is most suitable to obtain high numbers of MK and is the best combination for MK ex vivo expansion. It is suggested that MKs from CB have higher proliferation potential than that from PB, and the optimal culture duration of MK from PB is shorter than that of MK from CB. The study explore the differences in MK expansion between CD34+ stem cells derived from CB and PB and establish experimental foundation for further application to pre-clinic and clinic.