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 (> 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.

scholarly journals A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2069-2069
Author(s):  
Biagio De Angelis ◽  
Marika Guercio ◽  
Domenico Orlando ◽  
Stefano Di Cecca ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Short Term ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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 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 CD19 Redirected CAR NK Cells Are Equally Effective but Less Toxic Than CAR T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3491-3491 ◽  
Author(s):  
Concetta Quintarelli ◽  
Simona Sivori ◽  
Simona Caruso ◽  
Simona Carlomagno ◽  
Iolanda Boffa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Third Party ◽  
Advisory Committees ◽  
Car T Cells ◽  
Healthy Donors ◽  
Car T

Abstract Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells. In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16. We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL. After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets. We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs T cells), it reached the highest level when CAR-NK cells were used as effectors (7±9%, 16±30% and 22±16% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs non-transduced NK cells). Importantly, INF-g production was significantly lower upon CAR-NK activation compared to CAR-T cells (DAUDI 384±194 ng/ml vs 1860±678 ng/ml, p=0.002). Functional analysis on primary Bcp-ALL blasts, demonstrate that CAR-NK cells exert high degree of leukemia control (on average 2.1±2% vs 5.4±1.6% with non-modified NK cells as effectors; p=0.04). An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05). These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for 'off-the-shelf' immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. Figure. Figure. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, 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 Decade-Long Remissions of Leukemia Sustained By the Persistence of Activated CD4+ CAR T-Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 166-166
Author(s):  
J. Joseph Melenhorst ◽  
Gregory M Chen ◽  
Meng Wang ◽  
David L. Porter ◽  
Peng Gao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion

Abstract The adoptive transfer of chimeric antigen receptor (CAR)-reprogrammed T lymphocytes has demonstrated significant potential in various malignancies. Functional persistence of CAR T-cells in chronic lymphocytic leukemia (CLL) is a key predictor of durable remissions, yet the characteristics of long-term persisting CAR-engineered T cells have not been extensively studied. We here studied the fate of CD19-specific (CAR19) T-cells in two leukemia patients who achieved and sustained a complete remission over a decade ago. Molecular fate mapping was carried out on long-term persisting CAR T cells using lentiviral vector integration site sequencing across multiple time points up to 9.0 and 7.2 years post-infusion in patients 1 and 2, respectively. This analysis revealed little if any CAR T-cell clonal stability in the first 1.6 years in patient 1. Close to year 2 the CAR T cell repertoire stabilized in the first patient, with a strong clonal focusing until the last follow-up. The second patient had episodes of repertoire stability from the first month to approximately 12 months later, as well as from year 1 to 5. The CAR T cell repertoire in both patients were dominated by a few clones which were detected across a multiyear time span. CAR T-cells were still detectable using flow and mass cytometry 10+ years post-infusion. Deep immunophenotyping using a 40-marker mass cytometry panel identified divergence in effector but convergence in memory CAR T cell characteristics, with a prominent highly activated effector-memory CD4+ population developing late after infusion, expressing immune regulatory molecules. The CD4+ CAR T-cells were notable for a subpopulation highly expressing Ki67, suggestive of a proliferative phenotype. Ki67[hi] CD4+ CAR T-cells steadily emerged as the dominant population in both patients: this population constituted 15.9% of CAR T-cells at month 1.8 in patient 1, increasing to 97.0% by year 9.3; and constituted 0.2% of CAR T-cells in patient 2 at month 2.4, increasing to 87.2% by year 7.2. We assessed Ki67 expression in the CD4+ CAR T-cells compared to the CAR-negative T cells from these patients at matched time points, finding that this level of Ki67 expression was strongly CAR T-cell specific. CD8+ CAR T-cells also exhibited a proliferative trend overall, but Ki67 expression was generally lower and less robustly observed compared to the CD4+ CAR T-cell subset. These Ki67[hi] CD4+ T cells expressed a distinct marker profile, including activation markers CD38, HLA-DR, and CD95; transcription factors EOMES and TOX; checkpoint markers CTLA-4, LAG-3, TIGIT; and memory markers CD27 and CCR7. Together, these data suggest two major phases of CAR T-cell therapy responses in these patients: an initial response phase dominated by cytotoxic CD8+ T cells and double-negative Helios[hi] CAR T-cells, and a long-term remission phase dominated by a uniquely proliferative CD4+ CAR T-cell phenotype. To characterize these long-persisting CAR T-cells at a single-cell resolution, we performed CITE-Seq with single-cell T cell receptor-beta VDJ profiling on CAR T-cells obtained 9.3 years post-infusion. These long-persisting CAR T-cells exhibited strong evidence of ongoing activation, proliferation, and aerobic glycolysis despite evidence of chronic antigen-mediated signaling. Our data in aggregate provide important insight into and the development of a long-term memory anti-tumor response, necessary for sustained remission in leukemia following CAR T-cell therapy. Disclosures Porter: Wiley and Sons Publishing: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Unity: Patents & Royalties; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; GenenTech: Current Employment, Current equity holder in publicly-traded company; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; American Society for Transplantation and Cellular Therapy: Honoraria. Pruteanu-Malinici: Novartis: Current Employment. Frey: Sana Biotechnology: Consultancy; Novartis: Research Funding; Syndax Pharmaceuticals: Consultancy; Kite Pharma: Consultancy. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding. Davis: Tmunity Therapeutics: Consultancy, Patents & Royalties, Research Funding; Cellares Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties. Brogdon: Novartis Institutes for Biomedical Research: Current Employment. Young: Novartis: Patents & Royalties; Tmunity Therapeutics: Patents & Royalties. Levine: Immuneel: Membership on an entity's Board of Directors or advisory committees; In8bio: Membership on an entity's Board of Directors or advisory committees; Immusoft: Membership on an entity's Board of Directors or advisory committees; Akron: Membership on an entity's Board of Directors or advisory committees; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Vycellix: Membership on an entity's Board of Directors or advisory committees; Avectas: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Other: Co-Founder and equity holder. Siegel: Vetigenics, LLC: Other: Co-Founder and Equity Holder; Verismo Therapeutics, Inc: Other: Co-Founder and Equity Holder. Wherry: Merck: Consultancy; Marengo: Consultancy; Janssen: Consultancy; Related Sciences: Consultancy; Synthekine: Consultancy; Surface Oncology: Consultancy. June: AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: 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.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p&lt;0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p&lt;0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: 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.

Sign in / Sign up

Export Citation Format

Share Document