Abstract: Chimeric antigen receptor T cell (CART) therapy has developed as a powerful and potentially curative therapy in hematological malignancies over the last few years. CD19 directed CART cells have resulted in impressive complete response rates of ~90% in acute lymphoblastic leukemia that are durable for the majority of patients. However, the overall response rates in other malignancies such as chronic lymphocytic leukemia are around 50%. This could be partially related to CART exhaustion and dysfunction induced by leukemia cells. In this study, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction and exhaustion in hematological malignancies. As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 directed CART cells (second generation CARs using 4-1BB and CD3z signaling domains and a lentiviral vector). Incubation of primary AML samples or MOLM14 cell line with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PD-L1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P 〈 0.001), and up-regulation of PD-1 and TIM-3 on T cells 3-7 days post co-culture (8% of T cells expressed PD-1 on day 0 vs 43% on day 3, P=0.03 and 13% of T cells expressed TIM-3 on day 0 vs 71% on day 3, p=0.001, Figure 1). For in vivo experiments, we used NSG (NOD-SCID-g-/-) mice and engrafted them with the MOLM14 cell line. Treatment of these AML xenografts with suboptimal doses of CD33 or CD123 CARTs resulted in initial anti-tumor responses, followed by disease relapses in 40-60% of the mice (Figure 2A). T cells were isolated from the bone marrow of these mice and analyzed for differential expression of inhibitory receptors. There was a significantly increased up-regulation of TIM-3 receptors on T cells isolated from mice with relapsed disease compared with T cells isolated from mice in remission after CART cell therapy (Figure 2B). Next, we investigated the role of adding checkpoint blockade to improve T cell function ex vivo after CART cell therapy. Marrows of mice that relapsed after CART cell therapy contained both residual CART cells and leukemia and were used to model the administration of checkpoint blockade in the setting of CART cell exhaustion. Cells were cultured with PD-1 or TIM-3 blocking antibodies or the combination of both (10 ug/ml for 72 hours). CART cell effector functions such as cytokine production and Ki-67 proliferation marker improved in the presence of checkpoint antagonists especially when both PD-1 and TIM-3 blocking antibodies were combined (figure 2C). Finally, we tested the combination of checkpoint blockade with CARTs in AML xenografts. In this approach, we treated MOLM14 xenografts with suboptimal doses of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD), with or without checkpoint blocking antibodies. NSG mice bearing MOLM14 AML xenografts were established. Engraftment was confirmed by bioluminescent imaging. The tumor bearing mice were then treated with suboptimal doses (0.25-0.5 x106 total T cells I.V) of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD). Mice also received PD-1 blockade, TIM-3 blockade or the combination of both on days 3, 6, 9 and 12 post T cell therapy, with the rationale for early checkpoint blockade being based on our in vitro observations of early upregulation of inhibitory ligands on AML after exposure to CART cells. Mice were then followed with serial imaging to assess disease burden. The addition of checkpoint antagonists to untransduced T cells, in particular anti-TIM3, did not lead to an anti-leukemic effect. However, the addition of PD-1 or TIM-3 blockade to CART cell therapy resulted in a synergistic anti-tumor activity as shown in Figure 3. The durable complete response rate was: 45% for treatment with CART123 alone, 80% for treatment with CART123+PD-1 blockade, 100% for treatment with CART123+TIM-3 blockade, and 80% for treatment with CART123+ both PD-1 and TIM-3 blockade). Our preclinical results indicate that PD-1 and TIM-3 pathways are involved in CART exhaustion and dysfunction in AML. Combination of checkpoint inhibitors with CART cells may lead to enhanced efficacy in AML and other hematological malignancies. Current studies are investigating mechanisms of synergy and the role of these combinations in other hematological malignancies. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract: Relapsing/refractory (r/r) B-cell Acute Lymphoblastic Leukemia (ALL) is associated with a poor prognosis in both pediatric and adult patients. Novel therapies targeting CD19 on leukemic blasts, such as anti-CD19 Chimeric Antigen Receptor T cells (CART19, CTL019) or bi-specific anti-CD19/CD3 antibodies (blinatumomab) induce significant responses in this population. However, CD19-negative relapses have been reported in 5-10% of patients following CART19 or blinatumomab therapies. This is likely due to selective pressure on leukemia sub-clones by these potent anti-CD19 agents. Hence, novel effective immunotherapies are needed in order to treat these patients. In order to identify potential additional B-ALL antigens, samples from 20 r/r patients (including two that relapsed with CD19-negative disease after treatment with CART19 therapy) were screened using a custom Quantigene RNA panel (Affymetrix) and expression on cell surface was confirmed by multiparametric flow cytometry. The IL-3 receptor α (CD123) was one of the most highly and homogeneously expressed antigens in the blasts of 16/20 r/r ALL patients, and 2/2 CD19-negative relapses. Therefore, we sought to investigate the role of CART targeting CD123 (CART123) against r/r B-ALL, focusing on treating patients with CD19-negative relapses after prior anti-CD19 directed therapy. CART123 was shown to be effective in eradicating acute myeloid leukemia in xenograft mouse models but its role in ALL has not been investigated (Gill et al, Blood, 2014). We used a 2nd generation CAR123 construct that comprised a 4-1BB (CD137) co-stimulatory domain. T cells were lentivirally transduced and expanded using anti-CD3/CD28 beads. Head-to-head in vitro comparisons between CART123 and CART19 revealed similar rates of proliferation, CD107a degranulation, cytokine production and cytotoxicity when CART were co-cultured with the CD19+CD123+ B-ALL cell line NALM-6 and with primary B-ALL blasts. For in vivo evaluation, we utilized the primary ALL model that was developed by our group (Barrett et al, Blood, 2011). In this model, primary blasts obtained from ALL patients were passaged in NOD-SCID-γ chain KO (NSG) mice, and transduced with GFP/luciferase. We injected NSG mice with 2 million primary ALL blasts i.v. (CD19+, CD123+) and after engraftment, mice were treated with CART19, CART123 or control untransduced T cells (1 million i.v.). Mice treated with control T cells succumbed quickly to disease, while mice treated with either CART19 or CART123 showed tumor eradication and long term survival (Figure 1). We then evaluated the role of CART123 in the treatment of leukemia obtained from an ALL patient that relapsed with CD19-negative disease after CART19 treatment. Both CART123 and CART19 were incubated with CD19-negative ALL blasts; CART123, but not CART19 resulted in significant degranulation, robust cytokine production, and potent cytotoxicity. To confirm these results in vivo, we established a unique model of CD19-negative B-ALL xenograft. We used primary CD19-negative blasts obtained from a pediatric patient that relapsed after CART19 therapy; CD19-negative blasts were passaged in vivo in NSG mice and stably transduced with GFP/luciferase. Importantly, the blasts retained their CD19-negative phenotype. After engraftment, mice were treated with CART19, CART123 or control T cells. CART19 and control T cells had no anti-tumor activity, while CART123 resulted in a complete eradication of the disease and long term survival in these mice (Figure 2). In conclusion, CART123 represents an important additional approach to treating B-ALL, in particular due to its activity against CD19-negative relapses. Since we have previously shown that treatment with CART123 can lead to myelosuppression, CART123 should be employed to eradicate disease prior to allogeneic transplantation. Future direction may include combining CART123 with CART19 preemptively in order to avoid CD19 antigen escapes. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Ruella: Novartis: Research Funding. Kenderian:Novartis: Research Funding. Shestova:Novartis: Research Funding. Scholler:Novartis: Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Nazimuddin:Novartis: Research Funding. Kalos:Novartis: CTL019 Patents & Royalties, Research Funding. Porter:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Research Funding.

Abstract: Acute myeloid leukemia (AML) is challenging to treat with antigen-specific immunotherapy due to the lack of leukemia-specific surface antigens that are absent from normal hematopoiesis. We propose a novel solution to this problem by removing CD33, an AML-associated antigen, from normal hematopoietic stem and progenitor cells (HSPCs) by gene editing, thus rendering them resistant to CD33-targeted therapy. This enables powerful CD33-directed immunotherapy such as chimeric antigen receptor T cells (CAR T cells) to be employed against AML without disrupting normal myeloid function. We developed an optimized protocol to generate CD33 KO HSPCs with CRISPR/Cas9 and obtained up to 90% CD33 KO in primary human CD34+ cells as confirmed by flow cytometry and DNA sequencing. To test whether CD33 KO HSPCs retained multilineage potential, we engrafted NSG mice with CD34+ cells treated with Cas9 and either a CD33-targeting gRNA or a control gRNA. CD33 KO HSPCs were able to engraft and differentiate into both lymphoid and myeloid lineages in the mice to the same degree as controls. Human myeloid cells in the peripheral blood (PB) of the CD33 KO-engrafted mice were deficient for CD33, consistent with the KO efficiency measured in vitro, while expression of other myeloid markers such as CD11b and CD14 were similar to controls (Figure 1A, B). To ensure that long-term repopulating stem cells were present in the CD33 KO HSPCs, we performed secondary transplants of bone marrow from mice after four months of primary engraftment. Persistent engraftment for up to three additional months was observed in both CD33 KO HSPCs and controls, proving that gene editing with CRISPR/Cas9 and the resulting CD33 KO does not affect the self-renewal capability of human hematopoietic stem cells. To determine whether CD33 loss would lead to any functional defects in the myeloid progeny, we investigated the properties of myeloid cells derived from CD33 KO HSPCs differentiated with SCF, GM-CSF, G-CSF, IL-3, IL-6 and EPO. Cell morphology and immunophenotype of the myeloid cells after in vitro differentiation were consistent with normal neutrophils and macrophages. Phagocytosis ability, as measured by uptake of E. coli particles, was identical in CD33 KO myeloid cells as compared to controls. CD33 KO myeloid cells were also able to secrete inflammatory cytokines (IL-1b, IL-8, IL-8, IL-10, IL-12, TNF) in response to LPS stimulation to the same degree as controls. RNA-seq showed a highly concordant gene expression profile of CD33 KO HSPCs and controls (R2=0.99), thus excluding any major impact of CD33 loss on downstream gene expression. Prior studies have shown that CAR T cells targeting CD33 (CART33) can eliminate AML in mouse models but also cause toxicity to normal HSPCs (Kenderian et al, Leukemia 2015). To test our hypothesis that CD33 KO HSPCs can evade CART33-mediated toxicity, we treated mice engrafted with either control or CD33 KO HSPCs with CART33. CD33-expressing cells were eliminated in both control and CD33 KO-engrafted mice, which led to the expected myeloablation in control mice; in contrast CD33 KO-engrafted mice continued to sustain human myeloid cells (Figure 2A). Bone marrow (BM) analysis showed significantly more human stem cells (CD34+38-) and myeloid progenitors (CD34+38+) in CD33 KO-engrafted mice compared to controls after CART33 treatment (Figure 2B). We then proceeded to engraft Molm14, an AML cell line that was engineered to express luciferase, into mice bearing control or CD33 KO hematopoiesis, and studied the effect of CART33 treatment in this model. As expected, AML was eliminated in both groups as measured by bioluminescent imaging (BLI) (Figure 2C), as were peripheral blood monocytes in the control group, while monocytes were still present in the CD33 KO-engrafted mice (Figure 2D). In conclusion, we show that CD33 is dispensable in hematopoietic differentiation, and that absence of CD33 from myeloid progeny does not cause any discernible functional changes. Gene editing to remove CD33 from normal HSPCs allows persistent myelopoiesis during CART33-mediated eradication of AML, thus paving the way to a novel system that maximizes efficacy while minimizing toxicity. We envision future clinical translation of this approach by administering CD33 KO HSPCs as an allogeneic stem cell transplant in combination with CART33 in patients with AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract: Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.

Abstract: Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients & lt;50kg and 1-5x108 for pediatric patients ≥50kg and adult patients,. infused after lymphodepleting chemotherapy. Patient characteristics are described in Table 1. For the adult trial, 5 patients were screened, 4 enrolled (1 patient withdrew consent) and 3 infused (1 manufacturing failure). For the pediatric trial, 9 patients were screened, 8 enrolled (1 screen failure) and 6 infused (two patients were not infused for disease progression). For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

Abstract: We recently conducted a clinical trial of CD22-directed chimeric antigen receptor (CAR) T cells in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). While we did observe some transient responses, overall outcomes were inferior to another recent trial of CD22 CAR T cells in ALL performed at the NCI (Fry, T.J. et al. Nat Med, 2018). Intriguingly, these trials used a CAR that employed the same antigen-binding and intracellular signaling domains, and differed only in the length of linker connecting the variable regions of the single chain variable fragment (scFv). Based on these clinical observations, we sought to identify how the scFv linker impacts CAR biology and regulates CAR-driven T cell activity. The University of Pennsylvania's CD22 CAR contained a long 20 amino acid scFv linker ("CAR22-L") while the NCI's CAR had a 5 amino acid linker ("CAR22-S"). We began by investigating the impact of linker length on CAR biochemistry. Both CAR22-L and CAR22-S had similar antigen-binding affinities (KD of 1.67nM and 6.05nM, respectively). Chromatography revealed that CAR22-L remained monomeric in solution while CAR22-S formed homodimers. To explore how dimerization influenced surface-membrane biology, we developed GFP-tagged versions of each CAR and performed confocal microscopy on CAR+ T cells. CAR22-L exhibited homogenous surface membrane expression, while CAR22-S appeared to self-aggregate and cluster (Fig. 1a). We investigated the impact of this clustering on receptor signaling and found that CAR22-S demonstrated high levels of signaling molecule activation (i.e. Akt, p70-S6 and STAT3) in the absence of antigen engagement. This is consistent with previous reports establishing that CAR clustering can lead to tonic signaling (Long, A.H. et al. Nat Med, 2015). Importantly, this tonic signaling did not lead to autonomous T cell proliferation. We proceeded to evaluate how clustering and tonic signaling impacted CAR function upon antigen engagement. Microscopic evaluation of CAR T cells combined with CD22+ Nalm6 cells revealed greater actin and microtubule organizing complex polarization (P = 0.02 and 0.01, respectively) in CAR22-S cells, consistent with superior immune synapse formation. This was accompanied by increased phosphorylation of PI3K, MAPK and calcium signaling proteins (Fig. 1b) after CAR engagement. RNA sequencing revealed significantly greater activation of immune response gene programs in CAR22-S cells as compared to CAR22-L after overnight exposure to Nalm6. We next investigated the impact that this enhanced receptor-driven activity had on CAR T cell anti-tumor function. CAR T cells were combined with Nalm6 in vitro and residual Nalm6 was serially quantified, revealing that CAR22-S mediated greater tumor control than CAR22-L, particularly at later time periods (P & lt; 0.001). This was associated with greater secretion of IFNg, IL-2 and TNFa (all P & lt; 0.001). Finally, we compared anti-tumor efficacy in xenograft models of systemic Nalm6. NOD/SCID/cg-/- mice were engrafted with Nalm6 and received 1x106 CAR T cells 7 days later. CAR22-S demonstrated greater in vivo expansion (P & lt; 0.0001) and enhanced control of systemic disease (Fig. 1c,P = 0.017), resulting in prolongation of animal survival (Fig. 1d,P = 0.013). Based on these observations, we have designed a novel, affinity-enhanced CD22 CAR and confirmed that shorter linker length improves anti-tumor activity of this CAR. T cells expressing this CAR are currently undergoing evaluation in a phase I clinical trial (ClinicalTrials.org Identifiers NCT03620058 and NCT02650414). Thus far, 4 children and 2 adults have been infused with manageable toxicity. Early outcomes are promising, with 67% achieving complete remission at day 28, compared to 50% in our original CART22 trials. In summary, by investigating the potential mechanisms for an apparent discrepancy in outcomes between two different clinical trials, we demonstrate that reducing the length of the scFv linker results in significant changes to CAR biochemistry that directly lead to antigen-independent receptor activity. In contrast to previously published data demonstrating that tonic signaling of CD28-costimulated CARs is detrimental to T cell function (Long, A.H. et al. Nat Med, 2015), we found that tonic signaling of 4-1BB-costimulated CARs may be beneficial, possibly by priming T cells for rapid response to antigen. Disclosures Singh: University of Pennsylvania: Patents & Royalties. Frey:Novartis: Research Funding. Engels:Novartis: Employment. Zhao:Novartis: Employment. Peng:Novartis: Employment. Granda:Novartis: Employment. Ramones:Novartis: Employment. Lacey:Novartis: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding. Young:novartis: Research Funding. Brogdon:Novartis: Employment. Grupp:Roche: Consultancy; GSK: Consultancy; Novartis: Consultancy, Research Funding; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Maude:Novartis: Consultancy; Kite: Consultancy. Gill:Novartis: Research Funding; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Research Funding; Amphivena: Consultancy; Aro: Consultancy; Intellia: Consultancy; Sensei Bio: Consultancy; Carisma Therapeutics: Equity Ownership. Ruella:AbClon: Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer.

Abstract: Early T-lineage progenitors (ETPs) arise after colonization of the thymus by multipotent bone marrow progenitors. ETPs likely serve as physiologic progenitors of T-cell development in adult mice, although alternative T-cell differentiation pathways may exist. While we were investigating mechanisms of T-cell reconstitution after bone marrow transplantation (BMT), we found that efficient donor-derived thymopoiesis occurred before the pool of ETPs had been replenished. Simultaneously, T lineage–restricted progenitors were generated at extrathymic sites, both in the spleen and in peripheral lymph nodes, but not in the bone marrow or liver. The generation of these T lineage–committed cells occurred through a Notch-dependent differentiation process. Multipotent bone marrow progenitors efficiently gave rise to extrathymic T lineage–committed cells, whereas common lymphoid progenitors did not. Our data show plasticity of T-lineage commitment sites in the post-BMT environment and indicate that Notch-driven extrathymic Tlineage commitment from multipotent progenitors may contribute to early T-lineage reconstitution after BMT.

Abstract: Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field. In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC. To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2). In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract: Hodgkin lymphoma (HL) generally carries a good prognosis. However, 10-15% of patients relapse or are refractory to first-line therapy. These patients have a poor prognosis and would benefit from innovative approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor redirected T cells (CART19, CTL019) for refractory B cell malignancies. Despite the B-cell origin of the malignant Hodgkin Reed-Sternberg (HRS) cells, B-cell antigens, in particular CD19, are typically not expressed in HL. We sought to define a HL-associated cell membrane antigen that could be targeted by CAR T cells. Given the relative paucity of the malignant cells and the importance of the immunosuppressive tumor microenvironment in HL, the ideal target would be expressed on neoplastic cells as well as on infiltrating immune cells in order to provide robust stimulation of the CAR T cells. Immunohistochemistry for novel HL targets on 10 patient samples revealed that 5/10 patients expressed CD123 on the HRS cells. CD123 was also seen on immune cells of the microenvironment in most samples. CD123 is the α chain of the receptor for interleukin-3 (IL-3), an important cytokine in hematopoietic growth and differentiation that has been previously shown to promote HL cell line growth (Aldinucci et al, Leuk & Lymph, 2005). As primary HL is non-engraftable in mice we turned to immortalized HL cell lines and confirmed that CD123 is expressed by flow cytometry and Q-PCR in four different HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). To determine the role of IL-3 signaling in HL we engrafted NOD-SCID-γ-chain KO mice that overexpress human cytokines including IL-3 (NSG-S mice) with the luciferase-expressing HDLM-2 cell line. After i.v. injection, the neoplastic cells progressively formed disseminated soft tissue masses. Serial injections of a neutralizing anti-IL3 antibody slowed the growth of tumor, suggesting that CD123 may be a particularly relevant target in HL. We therefore sought to investigate the utility of anti-CD123 CAR T cells (CART123) for the treatment of HL. We have recently described the activity of CART123 in human acute myeloid leukemia (Gill et al, Blood, 2014). Our construct is a 2nd generation CAR, comprising 4-1BB co-stimulatory and CD3-ζ chain signaling domains with an anti-CD123 scFv. In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells (Table 1). Moreover, long-term co-culture (20 days) of CART123 with HDLM-2 cells at a 1:1 ratio led to T cell proliferation and complete elimination of HL cells by day 4. To confirm these in vitro data, we developed a rigorous in vivo model injecting 1 million luciferase+ HDLM-2 cells i.v. on day 0. Serial bioluminescent imaging (BLI) demonstrated low level of tumor on day 7, which was followed by gradual increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day 43 when the tumor burden was 20-fold higher than baseline, mice were treated with 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 6 months (Figure 1 and 2). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In summary, we show for the first time that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. We have previously demonstrated that CART123 lead to myelosuppression, suggesting that our findings could be translated to treat patients with refractory HL with a combined CART123 and rescue autologous bone marrow transplantation. Table 1 In vitro activity of CART123 compared to untransduced control T cells (UTD) against a HL cell line (HDLM-2). IN VITRO EXPERIMENT CART123* UTD CD107a Degranulation (4 hrs, E:T = 1:5) 59.3% 2.69% Specific Killing (24 hrs) E:T = 2:1 57% 5% E:T = 0.25:1 27% 1% Proliferating cells (CFSE based) (5 days, E:T = 1:1) 96.4% 20% Cytokine production (24 hrs, E:T = 1:1) (Luminex, MFI) INF-γ 38,265 42 IL-2 85,604 0 TNF-α 10,684 55 MIP-1β 40,038 111 IL-6 16,425 110 GM-CSF 99,915 285 *All P values are 〈 0.05 when compared to UTD Figure 2 Figure 2. Disclosures Ruella: Novartis: Research Funding. Kenderian:Novartis: Research Funding. Shestova:Novartis: Research Funding. Chen:Novartis: Research Funding. Scholler:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Gill:Novartis: Research Funding.

Abstract: Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies. Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%). Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.