ALBERT

Description: Background: Chimeric antigen receptor T cell (CART) therapy is revolutionizing modern cancer therapy, with two anti-CD19 CARTs FDA-approved for relapsed/refractory B cell lymphoma/leukemia and many other CARTs for solid and liquid tumors currently undergoing clinical trials. Our group recently demonstrated multiplexed CRISPR/Cas9 gene-editing of anti-CD7 CARTs to produce CD7 and T cell receptor alpha constant (TRAC)-deleted "off-the-shelf" universal (U)CART7s that effectively kill CD7+ T cell lymphoma in vivo without causing GVHD or fratricide (Cooper et al, Leukemia, 2018). However, in current clinical practice, suboptimal CART persistence and tumor killing permit tumor cell escape and, ultimately, disease relapse. Reasoning that a pro-lymphoid growth factor could promote CART efficacy, we supplemented UCART infusion with subcutaneous injections of the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19+ lymphoma xenograft model. Methods: To create anti-CD19 universal CARTs (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC-targeted gRNA, and virally transduced an anti-CD19 scFv 3rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NOD-scid IL2Rgammanull (NSG) mice with 5x105 RamosCBR-GFP cells four days prior to UCART19 (2x106 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion. Results:RamosCBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving RamosCBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While RamosCBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of RamosCBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p

Description: Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

Description: Background: Sézary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. We demonstrated effective T cell lymphoma therapy with allogeneic gene-edited anti-CD7 CARTs (Cooper et al, Leukemia, 2018). However, SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo. Methods: To generate SS PDX models, we injected NOD scid IL2Rgammanull (NSG) mice expressing SCF, GM-CSF, and IL-3 (NSG-SGM3) with ~2x106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers (Staser et al, Cytometry A, 2018). To generate UCART2s, we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9, a TRAC-targeted gRNA, and a CD2-targeted gRNA followed by viral transduction with an anti-CD2 scFv 3rd generation CAR. For initial UCART2 testing, we injected NSG mice with 5x105 cells from a human Sézary cell line transduced with click beetle red luciferase (HHCBR-GFP) four days prior to UCART2 treatment. Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART2 infusion. Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p

Description: T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD. To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing "off-target" nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses. UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice. Fratricide-resistant and allo-tolerant 'off-the-shelf' UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Despite remarkable clinical efficacy, CAR-T therapy has been limited by life-threatening toxicities in over 30% of patients (Maude, NEJM 2014 and Davila, SciTransMed 2014). Toxicities primarily manifest as Cytokine Release Syndrome (CRS) characterized by an early phase with fever, hypotension, and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6. Using a protein kinase inhibitor library containing 644 independent compounds, we aimed to identify compounds that could block CRS-related cytokine production without inhibiting CAR-T function. We identified, duvelisib (kindly provided by Verastem Oncology, Needham, MA), a novel and selective dual PI3K-δ,γ inhibitor as a potent inhibitor of CRS in vitro and in vivo without attenuating CAR-T function. Duvelisib (Copiktra) is approved for the treatment of relapsed/refractory CLL after 2 prior therapies and follicular lymphoma after 2 prior systemic therapies; the latter gained accelerated approval status based on overall response rate and continued approval may be contingent on confirmatory trials. To assess the ability of duvelisib to inhibit CAR-T mediated CRS, we performed an in vitro CRS assay (Singh, Cytotherapy, 2017). CART19 (19-28BBζ, 25,000 cells), Ramos (CD19+, 50,000 cells), and immature dendritic cells (iDC, 2,500 cells) were co-cultured in a 96 well plate for 48hrs in the presence of varying concentrations of duvelisib (0.3nM-1000nM). Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA (R&D Systems). Duvelisib reduced IL-6 levels in a dose-dependent manner with 30nM duvelisib reducing IL-6 secretion more than 10-fold (Fig 1a). To confirm that duvelisib did not inhibit CAR-T function, we performed in vitro killing assays, in which CAR-T efficacy was determined using BLI imaging of luciferase labeled CD19+ Ramos targets. At clinically relevant therapeutic doses (C max of 200 to 500nM) of duvelisib, there was no effect on CAR-T function in vitro. Treatment with 10nM duvelisib resulted in a statistically insignificant ~20% reduction of CART19 efficacy (p〉0.05) (Fig 1b). Of interest, although selective inhibitors of either or PI3Kδ (GSK2292767) or PI3Kγ (IPI549) had modest effects on blocking CAR-T induced IL-6 production in this in vitro model, the effect of combining both inhibitors had a more dramatic effect similar to the dual PI3K-δ,γ inhibitor, duvelisib. Next we assessed the ability of duvelisib to block IL-6 secretion in vivo using a fully immunocompetent murine model of CRS. Six-week old BALB/c mice were injected with the mitogenic anti-CD3ε antibody, 145-2C11 (10 µg/mouse). Duvelisib (450µg/mouse) was administered daily intraperitoneally (I.P), with the first dose of duvelisib injected 24 hours prior to injection of 145-2C11. Plasma IL-6 levels were determined using a mouse IL-6 ELISA (R&D Systems). Injection of 145-2C11 acutely elevated plasma IL-6 〉32 fold relative to non-treated controls (4hrs; Control 34.4 pg/mL versus vs. 145-2C11 1088±99.6 pg/mL). Duvelisib significantly reduced mean plasma IL-6 〉54% at 4hrs (Vehicle 1088±99.6 pg/ml vs duvelisib 492±99.6 pg/ml, p≤ 0.01) and 〉78% at 24hrs (Vehicle 220±101 pg/ml vs. duvelisib 49.3±16.1, p≤ 0.01) (Fig 1c) consistent with the effect of duvelisib in our in vitro CAR-T-induced CRS model described above. These studies demonstrate that duvelisib can inhibit CAR-T induced IL-6 production from iDC while having no inhibitory effect on CAR-T. Experiments are currently in progress to further characterize PI3K-δ,γ inhibition in humanized mouse models of CRS and CAR-T efficacy. Our preclinical data suggest that dual PI3K-δ,γ inhibition with duvelisib may represent an attractive alternative to IL-6 receptor antagonists, such as tocilizumab, for the treatment of CAR-T-associated cytokine release syndrome in the clinic which warrants further clinical evaluation. Figure 1. Dose dependent effect of duvelisib on IL-6 secretion in co-culture of CART19, Ramosluc, and iDC after 48 hours (a) Dose dependent inhibition of CART19 mediated cytotoxicity using duvelisib (b) Duvelisib effectively lowers IL-6 plasma level in an immunocompetent BALB/cJ mouse model of CRS (c). (** p≤ 0.01 and ns p〉0.05). Figure 1 Disclosures Cooper: Wugen: Consultancy, Current equity holder in private company, Patents & Royalties. Pachter:Verastem Therapeutics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.