scholarly journals Enhancing the Effect of CLL-1 CAR T Cells with Interleukin-15 for Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3912-3912 ◽  
Author(s):  
Pinar Ataca Atilla ◽  
Haruko Tashiro ◽  
Mary Kathryn McKenna ◽  
Madhuwanti Srinivasan ◽  
Brian Wesley Simons ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tnf Alpha ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Introduction: C-type lectin 1 (CLL-1, CD371) is highly expressed on the malignant cells from many patients with AML, and CAR T cells directed to this antigen can selectively target both leukemic progenitor cells (LSC) as well as AML blasts whilst sparing normal tissues. We previously showed (1) that such CAR-Ts can recognize and eliminate both AML blasts and primitive AML colony-forming cells in a low tumor-burden model. We have now modified the structure of the CLL-1 CAR and added transgenic expression of IL15 to enhance performance sufficiently for activity even against more extensive disease. Material and Methods: We assessed the phenotype and cytolytic ability of T cells transduced with 5 CLL-1 CAR constructs, varying in their spacer, transmembrane and costimulatory sequences (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3), and compared these with the effects of our published construct (4-1BBz-CD8)(1). We used flow cytometry to determine the effects of each construct on T cell phenotype and differentiation, and sequential (recursive) co-culture assays with tumor-cell targets to determine the durability of the anti-tumor activity. The most active constructs (CD28z-CD8 and 4-1BBz-CD8) were then evaluated in NOD.SCID IL-2Rg-/- (NSGS) mice engrafted with 1.5x10ˆ6 FFLuc-modified HL 60 AML cells, which received 2x10ˆ6 CLL-1 CAR T cells on day 3. To determine if we could further potentiate the in vivo expansion, persistence and anti-tumor activity of the CLL-1 CAR-T cells, we used a second retroviral vector to co-express transgenic IL15, measuring the effects in vitro and in vivo. Mice engrafted with 1.5x10ˆ6 tumor cells and received 2.5x10ˆ6 CLL-1 CAR T cells on week 3 in patient derived xenograft (PDX) model. We determined antitumor activity by bioluminescence imaging and weekly bleeding and measured serum cytokines by multiplex analysis (Luminex, TX). After euthanasia, we examined formalin-fixed/paraffin embedded sections. Results: Modified CLL-1 CAR constructs were expressed by 70-80% of cells irrespective of CAR sequence, but CD28z-CD8 CAR T cell expansion was significantly higher than CAR T cells with 4-1BBz endodomains (p<0.001), in part because of a higher death rate/lower viability in 4-1BBz cells (p<0.001). Consistent with these differences, both CD4 and CD8 T cell populations had more terminally differentiated cells (CCR7-CD45RA+) in CD28z versus 41BBz CAR T cells. In sequential co-culture assays against HL 60 (E:T=1:4) and THP-1 (E:T=1:4), CD28z-CD8 CAR T cells continued to expand well producing the greatest antitumor effect. In vivo models showed reduction in tumor signal in mice receiving either CD28z-CD8 CAR T or 4-1BBz-CD8 CAR T cells, but that only CD28z-CD8 CAR T cells prolonged survival (p<0.01). Nonetheless, all mice ultimately relapsed, usually with extramedullary disease, in association with limited CAR T persistence. We therefore incorporated transgenic IL15 as a "signal 3" for CD28z-CD8 CAR T cells, and determined the effects of forced IL15 expression on T cell phenotype, expansion, and antitumor activity in vitro and in vivo. In vitro, CD28z-CD8 CAR T cells with IL15 were less terminally differentiated and had superior expansion compared to CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CD28z-CD8 CAR T co-expressing transgenic IL15 initially (week 1) expanded better than CD28z-CD8 CAR T without IL15 (p<0.001) (Fig 1a), but produced severe acute toxicity associated with high level production of human IL15, TNF alpha and IFN gamma (Fig 1b). Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by 2 strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody (1mg/kg/mouse) (BioLegend, CA USA) weekly, while excessive T cell expansion could be arrested by activation of an inducible caspase 9 safety switch by administration of dimerizing drug (2). Both strategies successfully prolonged tumor free survival (Fig 2,b). Conclusion: Addition of transgenic IL15 to CLL-1-CD28z-CD8 CAR augmented activity against AML in a range of cell line and PDX models, and toxicity associated with exuberant CART expansion could be prevented by cytokine blockade and/or an inducible safety switch. References: 1. Tashiro H, et al. Mol Ther. 2017 2.Straathof KC et al. Blood. 2005 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.

scholarly journals CAR-T Cells with High CAR Expression Intensity Have Improved In Vitro and In Vivo Anti-Lymphoma Effect without Functional Exhaustion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
ANA Carolina Carolina CABALLERO González ◽  
Laura Escribà-García ◽  
Paula Pujol-Fernández ◽  
Eva Escudero-López ◽  
Rosanna Montserrat ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Antitumor Effect ◽  
Advisory Committees ◽  
Expression Intensity ◽  
Car T Cells ◽  
Car T

Background While immunotherapy with anti-CD19 chimeric antigen receptor (CAR) T cells has shown significant efficacy in B-cell malignancies, CAR T cells directed against CD30 (CAR30) for the treatment of Hodgkin lymphoma (HL) showed modest antitumor effect, with more than 50% of patients being unresponsive. Several factors related to the infused product and persistence may be relevant to increase clinical efficacy, but further investigation is needed. In this way, CAR expression intensity may play an important role on CAR T cell function, but this has not been systematically explored. Aim We have evaluated the impact of CAR expression intensity on T cell function, cell exhaustion and antitumor efficacy against HL and B cell lymphoma. Methods T cells were generated as previously described (Alvarez-Fernández C et al. 2016) and transduced with third generation lentivirus encoding a 4-1BBz CAR (either CAR30 or CAR19). Two populations of CAR+ T cells were sorted according to mean fluorescence intensity (MFI) of CAR: CARHI (MFI&gt; 5x103) and CARLO (MFI &lt;3x103). Cytotoxicity assays were performed using Raji (CD19+) or L540 (CD30+) tumor cell lines. Multiparametric flow cytometry was used to analyze T-cell inhibition and activation markers. CARHI and CARLOin vivo antitumor effect was tested under stringent therapeutic conditions using 5x106 T cells/mice (iv) in a HL NSG model. Results CAR30+ T cells were sorted into CARLO (MFI: 1064±124.7) and CARHI (MFI: 7068±1377) (p=0.01). TSCM were highly represented in CARLO compared to CARHI (CD4+: 70.14±1.78% vs. 55.61±5.5%, CD8+: 83.78±3.8% vs 72.2±5.47%, respectively) (p&lt;0.01). However, these differences disappear after 24h co-culture with tumor cells due to an increase of TSCM in CARHI (CD4+: 72.52±7.54%, CD8+: 80.26±5.3%). CARHI showed a significantly higher in vitro antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio: 96.6±1.86% vs. 89.1±3.83%; 1.25:1 E:T ratio: 84.61±4.7% vs. 31.15±19.79%; CARHI vs. CARLO, respectively) (p&lt;0.0001). No differences were observed in expression of activation markers (i.e.: CD25, CD69, and HLA-DR) among both populations. Generalizability of this finding was studied using a CAR19. Similarly, CAR19+ T cells were arranged into CARLO (MFI: 1610±187) and CARHI (MFI: 10810±1486) subgroups (p&lt;0.01). TSCM represented the most frequent subtype in both populations (CD4+: CARHI 70,22±9,87%, CARLO 69,22±9,33%; CD8+: CARHI 65,1±10,5%, CARLO 60,9±9,5%) and no differences in T cell subset composition between CARHI and CARLO were found. Again, CARHI exhibited superior antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio:59.9±8.72% vs. 28.8±8.7%; 1.25:1 E:T ratio: 21.6±11.4% vs. 2.9±2.9%, CARHI vs. CARLO, respectively) (p&lt;0.0001). At 24h and 72h of antigen encounter, expression of inhibitory markers was determined in both CAR30+ populations. While CD4+ T cells showed significantly higher PD1 and TIM3 co-expression in CARHI compared to CARLO (p&lt;0.05), CD8+ T cells showed similar co-expression (p=0.4 and p=0.8, at 24h and 72h, respectively). A similar kinetics was observed in CAR19+ T cells, suggesting that it could be related to an inhibitory control of activation, but not cellular exhaustion. To confirm this, functional performance of CAR30HI and CAR30LO T cells was evaluated by continuous tumor exposure. CAR30HI function persisted after sequential re-exposition (n=5) to tumor cells; in contrast, the CAR30LO subpopulation showed progressive loss of cytotoxic activity (i.e., tumor death at ratio E:T 5:1 after 4 expositions: 0% vs. 91.96%, CAR30LO and CAR30HI respectively; representative of 2 independent studies with different donors). To assess if these results were consistent in vivo, the antitumor effect of CAR30HI and CAR30LO were evaluated in a xenograft model of HL. Mice treated with CAR30HI T cells showed reduced tumor growth compared to those treated with CAR30LO T cells, which translated into an improved survival. Conclusion We have shown that high expression of a CAR (either CAR30 or CAR19) confers an enhanced in vitro antitumor effect against HL and B cell lymphoma. This effect is maintained after repetitive exposures to tumor cells and is not associated with T cell exhaustion or differentiation. Notably, this enhanced antitumor effect was also found in vivo. Our data shows that CAR expression intensity should be considered as an additional important factor to improve the efficacy of CAR T cells. Disclosures Sierra: Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1974-1974 ◽  
Author(s):  
Adam D. Cohen ◽  
J. Joseph Melenhorst ◽  
Alfred L. Garfall ◽  
Simon F Lacey ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

scholarly journals Clonal Dynamics of Early Responder and Long-Term Surviving CAR-T Cells in Humans

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 52-52
Author(s):  
Christine Rivat ◽  
Natalia Izotova ◽  
Rachel Richardson ◽  
Danilo Pellin ◽  
Rachael Hough ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Post Infusion ◽  
Equity Ownership

CD19 CAR-T cells show unprecedented responses in relapsed/refractory Acute Lymphoblastic Leukaemia, but long-term persistence appears critical for their use as a stand-alone therapy. The origin of long-term persisting CAR-T cells has yet to be defined and will be critical in designing manufacturing protocols to optimise long-term persistence. Previous data are conflicting with groups showing prolonged persistence of CAR-T cells from cell products with a predominantly effector memory (TEM) phenotype, whereas others suggesting that the dominant clones originate instead from infused stem cell memory (TSCM) and central memory (TCM) T cells. To date, it has not been possible to isolate long-term (&gt; 1 year) persisting CAR-T cells in patients. In the CARPALL Phase I study, the use of an improved low-affinity CD19 CAR resulted in enhanced expansion and persistence of CAR-T cells in vivo (Ghorashian et al, Nature Medicine, in press). Combining this unique experimental setting with our well-established clonal tracking platform based on high-resolution integration sites (IS) analysis has enabled us to track the fate of the infused CAR-T cells. We analysed 2 patients with long-term persistent CAR-T cells detectable by flow cytometry in peripheral blood. CAR-T cells comprised 13 and 53% circulating CD3+ cells respectively at day 14 post-infusion, 7.1 and 7.7% circulating at 1 month, 0.7 and 1.3% at 6 months and 0.1% for both at the latest follow-ups (24-28 months). Blood samples taken at early (14d, 30d) and later (6m to 28m) time points were flow-sorted for CAR+ TCM/TEM mixed population and TSCM T cells, while the corresponding infused gene-modified products were separated into three subpopulations: TSCM, TCM and TEM. The integration profile of each sorted cell populations was established using linear amplification mediated-PCR (LAM-PCR) combined with high throughput sequencing. We identified a total of 7,105 and 4,692 IS from 2 patients overtime before infusion and up to 28 months after infusion. The infused CAR-T cell population was highly polyclonal before infusion. Although the total number of CAR-T cell clones decreased substantially upon in vivo selection, we did not observe any sign of aberrant clonal drifts and diversity was preserved long-term. Early after infusion during the response peak, TSCM underwent two waves of transient oligoclonal expansion. In both patients two distinct sets of individual TSCM clones contributed to the 73% and 97% of the whole analysed TSCM population at day 14 and 74% and 99% at day 30. Conversely, the largest memory/effector clones detected at the same timepoints spanned from 4% to 21% of the total TCM/TEM population. These TSCM clones subsequently contracted and were not observed at 6-28 months after infusion suggesting that different clones are responsible for the early response and prolonged immune surveillance. After 6 months post-infusion, when the IS profile of circulating CAR-T cells was compared with selected populations from the infused product, only 1.8%-6.1% of long term clones were derived from the infused TCM population, despite this accounting for the majority of IS in the products (72.7/75.8% of clones). Conversely, in both patients the majority of IS associated with long term persistence (90.7%/55.5%) were derived from the TSCM compartment. Our preliminary results raise two hypotheses on the clonal dynamics of infused CAR T cells: 1) There is an early expansion of a defined group of clones during the first 30 days, which is more pronounced in the precursor TSCM compartment. These early waves do not seem to be originated from clones that have substantially expanded in vitro such that their clonal mark could not be retained in the batch of the infused cell product analysed. Further, these clones rapidly disappear after the early anti-tumour response. 2) The long-term population of CAR-T cells seem to have a higher relation with TSCM clones that have expanded in vitro before infusion, supporting the notion that such cells in the infused batch would be the one primarily responsible for the preservation of circulating CAR-T cells in the treated patients. This study suggests for the first time that anti-leukemic response occurs along rapid waves of clonal succession and that TSCM are primarily responsible for the long-term survival of CAR-T cells. Disclosures Ghorashian: novartis: Honoraria; UCLB: Patents & Royalties: UCLB; Celgene: Honoraria. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Thrasher:4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Amrolia:UCLB: Patents & Royalties.

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p&lt;0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P&lt;0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p&lt;0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 742-742 ◽  
Author(s):  
Eric L Smith ◽  
Sham Mailankody ◽  
Arnab Ghosh ◽  
Reed Masakayan ◽  
Mette Staehr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (&gt;90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p&lt;0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (&gt;10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased &gt;10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had &gt;50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1934-1934 ◽  
Author(s):  
Eduardo Huarte ◽  
Roddy S O'Connor ◽  
Melissa Parker ◽  
Taisheng Huang ◽  
Michael C. Milone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

scholarly journals Phase 2 Study of the Response and Safety of P-Bcma-101 CAR-T Cells in Patients with Relapsed/Refractory (r/r) Multiple Myeloma (MM) (PRIME)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3184-3184 ◽  
Author(s):  
Caitlin L. Costello ◽  
Tara K. Gregory ◽  
Syed Abbas Ali ◽  
Jesus G. Berdeja ◽  
Krina K. Patel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 2 ◽  
Employment Equity ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

P-BCMA-101 is a novel chimeric antigen receptor (CAR)-T cell product targeting B Cell Maturation Antigen (BCMA). P-BCMA-101 is produced using the piggyBac® (PB) DNA Modification System instead of the viral vector that is used with most CAR-T cells, requiring only plasmid DNA and mRNA. This makes it less costly and produces cells with a high percentage of the favorable T stem cell memory phenotype (TSCM). The higher cargo capacity of PB permits the incorporation of multiple genes in addition to CAR(s), including a safety switch allowing for rapid CAR-T cell elimination with a small molecule drug infusion in patients if desired, and a selection gene allowing for enrichment of CAR+ cells. Rather than using a traditional antibody-based binder, P-BCMA-101 has a Centyrin™ fused to a CD3ζ/4-1BB signaling domain. Centyrins are fully human proteins with high specificity and a large range of binding affinities, but are smaller, more stable and potentially less immunogenic than traditional scFv. Cumulatively, these features are predicted to result in a greater therapeutic index. A Phase 1, 3+3 dose escalation from 0.75 to 15 x 106 P-BCMA-101 CAR-T cells/kg (RP2D 6-15 x 106 cells/kg) was conducted in patients with r/r MM (Blood 2018 132:1012) demonstrating excellent efficacy and safety of P-BCMA-101, including notably low rates and grades of CRS and neurotoxicity (maximum Grade 2 without necessitating ICU admission, safety switch activation or other aggressive measures). These results supported FDA RMAT designation and initiation of a pivotal Phase 2 study. A Phase 2 pivotal portion of this study has recently been designed and initiated (PRIME; NCT03288493) in r/r MM patients who have received at least 3 prior lines of therapy. Their therapy must have contained a proteasome inhibitor, an IMiD, and CD38 targeted therapy with at least 2 of the prior lines in the form of triplet combinations. They must also have undergone ≥2 cycles of each line unless PD was the best response, refractory to the most recent line of therapy, and undergone autologous stem cell transplant or not be a candidate. Patients are required to be >=18 years old, have measurable disease by International Myeloma Working Group criteria (IMWG; Kumar 2016), adequate vital organ function and lack significant autoimmune, CNS and infectious diseases. No pre-specified level of BCMA expression is required, as this has not been demonstrated to correlate with clinical outcomes for P-BCMA-101 and other BCMA-targeted CAR-T products. Interestingly, unlike most CAR-T products patients may receive P-BCMA-101 after prior CAR-T cells or BCMA targeted agents, and may be multiply infused with P-BCMA-101. Patients are apheresed to harvest T cells, P-BCMA-101 is then manufactured and administered to patients as a single intravenous (IV) dose (6-15 x 106 P-BCMA-101 CAR-T cells/kg) after a standard 3-day cyclophosphamide (300 mg/m2/day) / fludarabine (30 mg/m2/day) conditioning regimen. One hundred patients are planned to be treated with P-BCMA-101. Uniquely, given the safety profile demonstrated during Phase 1, no hospital admission is required and patients may be administered P-BCMA-101 in an outpatient setting. The primary endpoints are safety and response rate by IMWG criteria. With a 100-subject sample, the Phase 2 part of the trial will have 90% power to detect a 15-percentage point improvement over a 30% response rate (based on that of the recently approved anti-CD38 antibody daratumumab), using an exact test for a binomial proportion with a 1-sided 0.05 significance level. Multiple biomarkers are being assessed including BCMA and cytokine levels, CAR-T cell kinetics, immunogenicity, T cell receptor diversity, CAR-T cell and patient gene expression (e.g. Nanostring) and others. Overall, the PRIME study is the first pivotal study of the unique P-BCMA-101 CAR-T product, and utilizes a number of novel design features. Studies are being initiated in combination with approved therapeutics and earlier lines of therapy with the intent of conducting Phase 3 trials. Funding by Poseida Therapeutics and the California Institute for Regenerative Medicine (CIRM). Disclosures Costello: Takeda: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Gregory:Poseida: Research Funding; Celgene: Speakers Bureau; Takeda: Speakers Bureau; Amgen: Speakers Bureau. Ali:Celgene: Research Funding; Poseida: Research Funding. Berdeja:Amgen Inc, BioClinica, Celgene Corporation, CRISPR Therapeutics, Bristol-Myers Squibb Company, Janssen Biotech Inc, Karyopharm Therapeutics, Kite Pharma Inc, Prothena, Servier, Takeda Oncology: Consultancy; AbbVie Inc, Amgen Inc, Acetylon Pharmaceuticals Inc, Bluebird Bio, Bristol-Myers Squibb Company, Celgene Corporation, Constellation Pharma, Curis Inc, Genentech, Glenmark Pharmaceuticals, Janssen Biotech Inc, Kesios Therapeutics, Lilly, Novartis, Poseida: Research Funding; Poseida: Research Funding. Patel:Oncopeptides, Nektar, Precision Biosciences, BMS: Consultancy; Takeda, Celgene, Janssen: Consultancy, Research Funding; Poseida Therapeutics, Cellectis, Abbvie: Research Funding. Shah:University of California, San Francisco: Employment; Genentech, Seattle Genetics, Oncopeptides, Karoypharm, Surface Oncology, Precision biosciences GSK, Nektar, Amgen, Indapta Therapeutics, Sanofi: Membership on an entity's Board of Directors or advisory committees; Indapta Therapeutics: Equity Ownership; Celgene, Janssen, Bluebird Bio, Sutro Biopharma: Research Funding; Poseida: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Nkarta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teneobio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Martin:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Ghoddusi:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Spear:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Orlowski:Poseida Therapeutics, Inc.: Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding.

scholarly journals In Vitro-Selected Nanobody-Based Cellular Therapy Targeting CD72 for Treatment of Refractory B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1337-1337
Author(s):  
Matthew Nix ◽  
Yu-Hsiu T. Lin ◽  
Huimin Geng ◽  
Makeba Marcoulis ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Board Of Directors ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified &gt;900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's &lt; 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents & Royalties. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4546-4546 ◽  
Author(s):  
Raedun Clarke ◽  
Sjoukje Van Der Stegen ◽  
Chia-Wei Chang ◽  
Mushtaq Husain ◽  
Yi-Shin Lai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Abstract The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape. Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5' junction, 3' junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16). Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated. In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4). In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. Disclosures Clarke: Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

scholarly journals Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3222-3222
Author(s):  
Kathy Karasiewicz ◽  
Shuyang He ◽  
Mary Ng ◽  
Kristina Tess ◽  
Weifang Ling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership ◽  
Robust Process

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

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