scholarly journals Clonal Kinetics and Single Cell Transcriptional Profiling of Adoptively Transferred CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 702-702
Author(s):  
Alyssa Sheih ◽  
Laila-Aicha Hanafi ◽  
Valentin Voillet ◽  
Hannah A. DeBerg ◽  
Reed M. Hawkins ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Adoptive Transfer ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Clones ◽  
Car T

Abstract INTRODUCTION: When introduced into polyclonal T cells, chimeric antigen receptors (CAR) redirect specificity of the engineered T cells to an antigen recognized by the CAR. We conducted a phase I/II clinical trial of treatment of relapsed and refractory CD19-positive B cell malignancies using a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells (NCT01865617). Little is known about the transcriptional heterogeneity of CAR-T cells in the infused product and their clonal kinetics after adoptive transfer. METHODS: To understand the factors that impact clonal CAR-T cell behavior in vivo, we performed TCRBV sequencing and single cell transcriptional profiling (10X Genomics) on CD8+ CAR-T cells isolated from infused products and the blood of treated patients. TCRBV sequencing was performed on 0.8 to 1.5 million cells from the infused product and 700-65,000 CAR-T cells from blood after CAR-T infusion. For single-cell RNA sequencing (scRNAseq), we obtained paired 5' gene expression and V(D)J data from individual CAR-T cells isolated from infused products and from the blood at the peak of in vivo expansion, after contraction, and at a late time point. RESULTS: High-throughput sequencing of the TCRBV genes revealed that CAR-T cells were polyclonal in the infused products, and during in vivo expansion and contraction, and at late times (≥ 3 months) after adoptive transfer. We evaluated the diversity of the TCRBV repertoire using the Shannon entropy index, and found that clonal diversity was highest in the infused product and declined at later time points after adoptive transfer. Loss of diversity after adoptive transfer was due to both expansion of higher frequency CAR-T cell clones and loss of low-frequency clones. We identified distinct CAR-T cell clones in the infused product and in blood at multiple time points after infusion that exhibited different kinetics of expansion and contraction. To examine the transcriptional programs that regulate the fate of CAR-T cells after infusion, we performed scRNAseq on CD8+ CAR-T cells, and found transcriptional heterogeneity in the infused products, which declined in CD8+ CAR-T cells isolated from patient blood after adoptive transfer. Gene set enrichment analysis showed that the infused products expressed higher levels of genes associated with hypoxia, glycolysis, and proliferation, and lower levels of genes associated with cytotoxicity compared to CAR-T cells isolated after adoptive transfer. In the infused product, genes associated with cytotoxicity were expressed at higher levels in CAR-T cells harboring clonotypes that were subsequently represented at relatively higher levels in vivo after adoptive transfer. CONCLUSIONS: There is transcriptional heterogeneity in the infused product and distinct CAR-T cell clones exhibit different kinetics of expansion and contraction after infusion. A better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy. Disclosures Turtle: Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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<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<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<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 Long-Term Remission of CLL Sustained By Pauciclonal Anti-CD19 Chimeric Antigen Receptor T (CTL019) Cell Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 699-699 ◽  
Author(s):  
J. Joseph Melenhorst ◽  
David L. Porter ◽  
Lifeng Tian ◽  
Simon F Lacey ◽  
Christopher L Nobles ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Integration Site ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Clones ◽  
Car T

Abstract We recently demonstrated that sustained remission in 41 CLL patients treated with the CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR19) T-cells correlated strongly with the expansion and persistence of the engineered T cells and that important pathways such as T cell exhaustion, glycolysis and T cell differentiation segregated responders from non-responders (Fraietta et al., 2018, Nature Medicine). We here report two advanced, chemotherapy-resistant CLL patients with the longest (7 years) follow-up on any trial of CART19 cells. Both patients had received five therapies before being treated at the University of Pennsylvania with autologous, murine CTL019 (tisagenlecleucel) cells for their CLL in 2010, receiving 1.1e9 and 1.4e7 CAR19+ T cells, respectively. Both patients have persistence of CAR-engineered T cells and both patients are still in remission as determined by flow cytometry and deep sequencing of IgH rearrangements for 5.5-7 years. Thus, the infused CAR-T cells have maintained these patients in deep molecular remission of their disease for the longest period of time that has been reported to date. To understand the fate of the infused CAR-T cells we determined the phenotype, function, and clonal nature of the persisting CTL019 cells. Flow cytometric CART19 cell analyses demonstrated that early during the anti-leukemia response, activated, HLA-DR-expressing CD8+ CAR-T cells rapidly expanded, followed by similarly activated CD4+ CAR-T cells. With tumor clearance the CAR-T cell population contracted, but an activated CD4+ CAR-T cell population was maintained and was still detectable at the last follow-up of 7 years. The CD8+ CAR-T cell pool remained present at low frequencies. Both populations had acquired and maintained an effector memory phenotype, a phenotype most consistent with active disease control. Furthermore, the analysis of the classical immune checkpoint inhibitory markers PD1, TIM3, LAG3, and CTLA4 showed that only PD1 was expressed from the earliest to the latest time point on >80% of all CAR-T cells, whereas LAG3 and TIM3 were expressed only early on but lost after tumor clearance. These data suggest that the initial tumor clearance was mediated by CD8+ CAR-T cells, but sustained by a CD4+ CAR-T cell population that still actively engages with target cells. To understand the clonal nature of these long-term persisting CAR-T cells we used two complementary methods: a) CAR T cells were sorted from post-infusion aliquots during the first two years for T cell receptor-beta deep-sequencing (TCR-seq); b) the CAR integration sites in the genome were sequenced in the infusion product and in circulating CAR-T cells. TCR-seq analysis of early post-infusion time points demonstrated that the circulating CAR-T cell populations consisted of hundreds to thousands of distinct clones which in patient 1 and 2 displayed clonal focusing by 21 and 1 month post-infusion, respectively, with some clones making up as much as 12% (patient 1) and 48% (patient 2) of the CAR-T cell repertoire. The analysis of clonotype sharing at the various time points via Morisita's overlap index analysis similarly showed repertoire stabilization late (21 months; patient 1) and early (1 month; patient 2) after infusion. Lastly, fate mapping of the infused CART19 cells via CAR integration site analysis in the infusion product until the latest time point indicated that the infusion products for both patients had a very diverse, non-clonal make-up, containing over 8,000 and 3,700 integration sites in patients 1 and 2, respectively. The higher degree of clonality in patient 2 but not 1 CAR-T cells as seen by TCR-seq was confirmed by integration site analysis, as was the sharing of CAR-T cell clones over time. Importantly, whereas the CAR integration site repertoire in patient 1 was diverse in the first two years, it stabilized and trended towards oligoclonality 21 months after infusion. Lastly, CAR integration site analysis revealed a high degree of clonal persistence, suggesting that tumor control and B cell aplasia were maintained by few, highly functional CD4+ CAR-T cell clones. In summary, we demonstrate that in both patients with the longest persistence of CAR-T cells reported thus far, early and late phases of the anti-CLL response are dominated by highly activated CD8+ and CD4+ CAR-T cells, respectively, largely comprised of a small number of persisting CD4+ CAR-T cell clones. Disclosures Melenhorst: Parker Institute for Cancer Immunotherapy: Research Funding; Incyte: Research Funding; Casi Pharmaceuticals: Consultancy; novartis: Patents & Royalties, Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Lacey:Novartis Pharmaceuticals Corporation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Young:Novartis: Patents & Royalties, Research Funding. Siegel:Novartis: Research Funding. 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; 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; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 776-776
Author(s):  
Claire Roddie ◽  
Maeve A O'Reilly ◽  
Maria A V Marzolini ◽  
Leigh Wood ◽  
Juliana Dias Alves Pinto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Peak Car ◽  
Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 196-196
Author(s):  
Bishwas Shrestha ◽  
Kelly Walton ◽  
Jordan Reff ◽  
Elizabeth M. Sagatys ◽  
Nhan Tu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Graft Versus Host ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

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 (>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<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 (>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 >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 >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 Third-Generation CAR T Cells Targeting CD19 Are Associated with an Excellent Safety Profile and Might Improve Persistence of CAR T Cells in Treated Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 51-51 ◽  
Author(s):  
Maria-Luisa Schubert ◽  
Anita Schmitt ◽  
Brigitte Neuber ◽  
Angela Hückelhoven-Krauss ◽  
Alexander Kunz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
University Hospital ◽  
Third Generation ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Manufacturing ◽  
Car T

Introduction T cells transduced with a chimeric antigen receptor (CAR) have demonstrated significant clinical efficacy in patients with lymphoid malignancies including relapsed or refractory (r/r) B-lineage acute lymphoblastic leukemia (ALL) or r/r B-cell non-Hodgkin's lymphoma (NHL). Second-generation CAR T cells comprising 4-1BB or CD28 as costimulatory domains have become commercially available for the treatment of patients with CD19+ lymphoid malignancies. However, achievement of durable clinical responses remains a challenge in CAR T cell therapy. Consequently, third-generation CARs incorporating both elements might display short-term efficacy with potent and rapid tumor elimination (CD28) as well as long-term persistence (4-1BB). So far, only two clinical trials employing third-generation CAR T cells have been reported. Both enrolled 31 patients in summary and demonstrated favorable results for third-generation CAR T cells. Here, we report on first results of our investigator-initiated trial (IIT) on third-generation CD19-directed CAR T cells: The Heidelberg CAR trial 1 (HD-CAR-1; NCT03676504; EudraCT 2016-004808-60) is a phase I/II trial initiated in September 2018 with in-house leukapheresis and CAR T cell manufacturing in full compliance with European Good Manufacturing Practice (GMP) guidelines at the University Hospital Heidelberg. Methods Adult and pediatric patients with r/r ALL and patients with r/r chronic lymphocytic leukemia (CLL) or NHL including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL) are treated with autologous T lymphocytes transduced with a CD19 targeting third-generation CAR retroviral vector (RV-SFG.CD19.CD28.4-1BBzeta). The main purpose of HD-CAR-1 is to evaluate safety and feasibility of escalating third-generation CAR T cell doses (1-20×106 transduced cells/m2) after lymphodepletion with fludarabine (30 mg/m2/d on days -4 to -2) cyclophosphamide (500 mg/m2/d on days -4 to -2). Patients are monitored for cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and/or other toxicities. In vivo function, survival and anti-tumor efficacy of CAR T cells are assessed. Results To date, 10 patients (3 adult ALL, 2 CLL, 2 MCL, 2 DLBCL, 1 transformed FL) have been enrolled and subjected to leukapheresis. Transduction efficiency of T lymphocytes ranged between 33%-66% and high numbers of transduced CAR T cells were harvested (70-123x106 CAR T cells). No production failure occurred. CAR T cell products were sterile and free from mycoplasma and endotoxins. The copy number per CAR T cell did not exceed 7. Eight patients (2 adult ALL, 2 CLL, 1 MCL, 2 DLBCL, 1 transformed FL) have received the CAR T cell product (6 patients: 106 transduced cells/m2; 2 patients 5×106 transduced cells/m2). No signs of CRS or ICANS > grade 2 have been observed. Only one patient required tocilizumab. No neurological side-effects occurred, even not in patients with involvement of the central nervous system (CNS). In quantitative real-time PCR, CAR T cells were detectable in the peripheral blood (PB) in 3 of 4 analyzed patients or the cerebrospinal fluid (CSF) of an ALL patient with CNS involvement. The CAR T cell frequency reached up to 200,000 copies/µg DNA, in some patients beyond end-of-study at day 90 after CAR T cell administration. Clinical responses to treatment were observed in 6/8 (75%) treated patients so far (2/8 patients have received CAR T cells recently and are not yet evaluable for response). Conclusion Leukapheresis and CAR T cell manufacturing were effective for all patients enrolled in the HD-CAR trial to date. Patients responded clinically to treatment despite low numbers of administered CAR T cells. CAR T cells displayed an excellent safety profile and were detectable for more than 3 months following administration. Furthermore, CAR T cells migrated into different compartments including the CSF in case of CNS involvement. For HD-CAR-1 leukapheresis, CAR T cell manufacturing, CAR T cell administration, patient monitoring and follow-up are performed in-house, providing autarky from transport or production sites outside the University Hospital Heidelberg. Altogether, HD-CAR-1 accounts to clinical evaluation of third-generation CAR T cells that might contribute to long-term CAR T cell persistence, hence improving efficient and durable responses in treated patients. Disclosures Schmitt: Therakos Mallinckrodt: Other: Financial Support . Sellner:Takeda: Employment. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:Therakos Mallinckrodt: Other: Financial Support; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia.

scholarly journals Efficacy and Toxicity of JCAR014 in Combination with Durvalumab for the Treatment of Patients with Relapsed/Refractory Aggressive B-Cell Non-Hodgkin Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1680-1680 ◽  
Author(s):  
Alexandre V. Hirayama ◽  
Jordan Gauthier ◽  
Kevin A. Hay ◽  
Alyssa Sheih ◽  
Sindhu Cherian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Group 2

Abstract Introduction Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have shown high overall response rates (ORR) in otherwise treatment-refractory CD19+ B-cell non-Hodgkin lymphoma (NHL); however, not all patients (pts) achieve complete remission (CR). PD-L1 expression on tumor cells and/or other tissues could impair the function of PD-1+ CAR-T cells and the efficacy of CD19 CAR-T cell immunotherapy. PD-1 pathway blockade may enhance the function and antitumor activity of CD19 CAR-T cells. Here we report preliminary data from a phase 1 dose-finding study (NCT02706405) of the safety and feasibility of combination therapy with JCAR014 CD19-specific 4-1BB-costimulated CAR-T cells and escalating doses of durvalumab, an anti-PD-L1 monoclonal antibody, in adults with relapsed/refractory aggressive B-cell NHL. Methods Pts are treated in one of two groups. All pts receive lymphodepletion chemotherapy with cyclophosphamide and fludarabine followed by infusion of JCAR014. Pts in group 1 receive the first infusion of durvalumab (225 mg, 750 mg, or 1500 mg) 21-28 days after treatment with JCAR014. Pts in group 2 receive the first dose of durvalumab (7.5 mg, 22.5 mg, 75 mg, 225 mg, 750 mg, or 1500 mg) 1 day prior to JCAR014 infusion. Up to 10 doses of durvalumab are administered after JCAR014 at the highest identified safe dose at 4-week intervals until toxicity or disease progression. We evaluated the safety, tolerability, and efficacy of the combination therapy and the pharmacokinetic profile of JCAR014 after infusion. Adverse events were graded using the Common Terminology Criteria for Adverse Events (CTCAE) v4.03, with the exception of cytokine release syndrome (CRS), which was graded according to consensus criteria (Lee, Blood 2014). Positron emission tomography/computed tomography was performed approximately 1, 2, 4, 6, 9, and 12 months after JCAR014 infusion and the best anti-tumor response was reported according to the Lugano criteria (Cheson, JCO 2014). Results Patient characteristics are shown in Table 1. Fifteen pts have been treated, including 6 in group 1 who received post-JCAR014 durvalumab doses of 225 mg (n = 3) and 750 mg (n = 3), and 9 in group 2 who received pre-JCAR014 durvalumab doses of 7.5 mg (n = 1), 22.5 mg (n = 1), 75 mg (n = 3), or 225 mg (n = 4). Durvalumab dose escalation is ongoing. JCAR014 manufacturing was successful for all pts. All pts received 2 x 106 JCAR014 CAR-T cells/kg, except the first 2 pts treated on the study who received 7 x 105 CAR-T cells/kg. Of the 13 pts who received JCAR014 at 2 x 106 CAR-T cells/kg, 5 pts (38%) developed CRS (2 grade 1, 2 grade 2, and 1 grade 4) and one (8%) developed grade 1 neurotoxicity. CRS and/or neurotoxicity occurred within 4 weeks of JCAR014 infusion, and were not observed when durvalumab was administered after JCAR014. With the exception of B cell aplasia, no autoimmune adverse events were observed. Twelve of 13 pts who received 2 x 106 CAR-T cells/kg were evaluable for response. One patient, who had grade 4 CRS and biopsy evidence of extensive CAR-T cell infiltration into persistent sites of disease, elected to receive hospice care and died on day 32 after JCAR014 infusion without full response evaluation. The overall response rate was 50% (5 CR, 42%; 1 PR, 8%). Of the 5 pts who achieved CR, 3 were in CR at the first restaging after JCAR014 and 2 subsequently converted to CR after the first post-JCAR014 durvalumab infusion. Only one patient who achieved CR has relapsed (median follow-up 10.6 months, range 3.7-11.8). Continued stable disease or evidence of regression was seen in 4 of 6 (67%) initially non-responding pts who continued durvalumab therapy (median 5 doses, range 1-6). CAR-T cell counts expanded in the peripheral blood within 14 days of JCAR014 infusion in all pts. Higher peak and day 28 CAR-T cell copy numbers in blood by qPCR were observed in responding pts. CAR-T cells were detected for a median of 5.1 months (range, 1.7 to 9.1 months) in responding pts. In vivo re-accumulation of CAR-T cells after the first post-JCAR014 durvalumab dose was observed in the blood of two patients in group 2. Conclusion The combination of JCAR014 with durvalumab for the treatment of adult pts with aggressive B-cell NHL appears safe; however, dose escalation is ongoing. Complete responses were observed both at initial restaging after JCAR014 infusion, and also subsequently in pts continuing durvalumab therapy after initially failing to achieve CR. Disclosures Hirayama: DAVA Oncology: Honoraria. Hay:DAVA Oncology: Honoraria. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy. Shadman:Verastem: Consultancy; Beigene: Research Funding; Mustang Biopharma: Research Funding; Gilead Sciences: Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy; Genentech: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy; Genentech: Consultancy; AstraZeneca: Consultancy; Acerta Pharma: Research Funding. Cassaday:Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Pfizer: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Kite Pharma: Research Funding; Incyte: Research Funding. Acharya:Juno Therapeutics: Research Funding; Teva: Honoraria. Riddell:Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy. Maloney:Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; GlaxoSmithKline: Research Funding; Seattle Genetics: Honoraria. Turtle:Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Bluebird Bio: Consultancy; Gilead: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Caribou Biosciences: Consultancy; Aptevo: Consultancy.

scholarly journals CAR Density Influences Antitumoral Efficacy of BCMA CAR-T Cells and Correlates with Clinical Outcome

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 735-735
Author(s):  
Paula Rodriguez-Marquez ◽  
Maria Erendira Calleja-Cervantes ◽  
Guillermo Serrano ◽  
Maria Luisa Palacios-Berraquero ◽  
Diego Alignani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Outcome ◽  
Single Cell ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Cd8 Cell

Abstract Background: Chimeric Antigen Receptor-modified T cell (CAR-T) therapies have revolutionized cancer immunotherapy, especially in hematological malignancies. Although great results have been achieved during the last years, long-term efficacy is still compromised in some cases and factors behind CAR-T cell disfunction are not fully understood. Recent studies have shown that the control of CAR expression influences CAR-T fitness and antitumoral efficacy 1. Therefore, we hypothesized that CAR density on the membrane of CAR-T cells could directly affect CAR-T cell function. In this study we perform a functional and genomic analysis of FACS-isolated subpopulations of CAR-T cells with different CAR densities (CAR High and CAR Low). Methodology: Second generation CAR-T cells with 4-1BB costimulatory domain targeting BCMA were generated by lentiviral transduction of αCD3/αCD28 activated T cells that were expanded for 12-14 days in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry before and after coculture with MM cells. Cytotoxic activity and cytokine production were measured by standard procedures. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. Transcriptomic (RNA-seq) and epigenetic (ATAC-seq) analysis were performed following stablished protocols 2. Single cell analysis was performed using the Chromium Single Cell Immune Profiling solution from 10x Genomic that allows simultaneous analysis of gene expression and paired T-cell receptors from a single cell. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method that infers regulatory dissimilarities 3. Results: RNA-seq and ATAC-seq analysis revealed completely different profiles between CAR High- and CAR Low-T cells in both CD4 +and CD8 + cell subsets, with >3500 differentially expressed genes (2086 for CD4 + and 1553 for CD8 +) that were related with increased tonic signaling, T cell activation and proliferation in CAR High-T cells. Functional studies at resting state (before antigen encounter) corroborated that CAR High-T cells presented increased tonic signaling, that lead to a higher basal activation and a more differentiated phenotype with skewed presence of CCR7 +/CD45RA +/CXCR3 + T SCM cells. After antigen-driven activation, increased cytotoxicity and cytokine production was observed in CAR High-T cells, that also presented higher percentage of terminally differentiated effector cells (CCR7 -/CD45RA +), along with increased exhaustion (PD1 +/LAG3 +/TIGIT +). This effect was also observed in the infusion products of CARTBCMA-HCB-01 clinical trial for patients with R/R MM (NCT04309981), where products enriched in CAR High-T cells presented increased cytotoxic activity. Although no significant differences were observed in the antitumoral efficacy in vivo, CAR Low-T cells presented increased persistence, suggesting that higher CAR levels could reduce long-term efficacy. Further characterization of CAR-T cells at single cell level (scRNA-seq) showed enrichment of CAR High-T cells in activated CD4 + and exhausted CD8 + cell clusters. The analysis of regulatory dissimilarities driven by different CAR densities with SimiC revealed an increased activity of the regulon associated to NR4A1 transcription factor (a well-known TF driving T cell exhaustion 4) in CAR High-T cells, providing mechanistic insights of the regulatory networks behind differential functionality of CAR High-T cells. Finally, to evaluate the impact of CAR density in the clinical outcome of CAR-T therapies, we developed a gene signature associated to increased CAR density, that was applied to transcriptomic data available from public studies 5. We score the infusion products of several clinical trials testing CTL019 (NCT01029366, NCT01747486 and NCT02640209) and we observed an enrichment on CAR High signature in the products from non-responder patients. Conclusions: Our data demonstrate that CAR density on the membrane of engineered T cells plays important roles in CAR-T activity with a significant impact on clinical outcome. Moreover, the comprehension of regulatory mechanisms driven by CAR densities at the single cell level offer an important tool for the identification of key regulatory factors that could be modulated for the development of improved therapies. Figure 1 Figure 1. Disclosures Rodríguez-Otero: Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel and other expenses. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, Takeda: Consultancy, Other: Advisory board. Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding.

scholarly journals AUTO1, a Novel Fast Off CD19CAR Delivers Durable Remissions and Prolonged CAR T Cell Persistence with Low CRS or Neurotoxicity in Adult ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 226-226 ◽  
Author(s):  
Claire Roddie ◽  
Maeve A O'Reilly ◽  
Maria A V Marzolini ◽  
Leigh Wood ◽  
Juliana Dias Alves Pinto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Split Dose ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Grade 3

Introduction: In adults, prognosis for B-ALL is poor, patients are more vulnerable to CD19 CAR immunotoxicity and there is currently no CD19 CAR therapeutic with acceptable toxicity and durable efficacy. We have developed a novel second generation CD19CAR (CAT-41BBz CAR), with a faster off-rate but equivalent on rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 9s vs 4.2 hours) designed to result in more physiological T-cell activation, reduce toxicity and improve engraftment. Preliminary paediatric clinical data of this novel CD19 CAR (AUTO1) supports this assertion. We here describe preliminary data from ALLCAR19 (NCT02935257), a multi-centre, Phase I clinical study of AUTO1 as therapy for r/r adult B-ALL. Methods: Manufacturing: AUTO1 utilises non-mobilised autologous leucapheresate. The first 6 trial products were generated using a standard dynal bead/WAVE Bioreactor process and subsequent products using a semi-automated closed process. Study design: ALLCAR19 is a phase I/II study recruiting subjects 16-65y with r/r B ALL. Lymphodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) is followed by split dose CAR T cell infusion (Day 0: if ≥20% BM blasts, infuse 10 x 106 CAR T cells ; if <20% BM blasts, infuse 100 x 106 CAR T cells. Day +9: if no Grade 3-5 CRS/CRES, infuse Dose 2, to a total dose of 410 x 106 CAR T cells). Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months Results: As of 24 July 2019, 16 patients have been leukaphresed, 14 products manufactured (one failed leukaphresis and one currently in manufacture) and 13 patients have received at least 1 dose of AUTO1. Of the 16 patients, median age was 35.5 (range 18-63), 10/16 (63%) had prior blinatumomab or inotuzumab ozogamicin and 12/16 (75%) had prior HSCT. At the time of pre-conditioning, 9/13 (69%) patients were in morphological relapse with >5% leukemic blasts of which 6/13 (46%) had ≥50% blast. 9/13 patients (69%) received the total target split dose of 410 x 106 CAR T cells while 1/13 patients (8%) received a reduced split total dose of 51.3 x 106 CAR T cells due to manufacturing constraints. 3/13 patients (23%) received only a first dose of 10 x 106 CAR T cells. The dose was administered safely to date: No patients experienced ≥Grade 3 CRS (using Lee criteria) and only 1/13 (8%) experienced Grade 3 neurotoxicity (dysphasia) that resolved swiftly with steroids. All patients had robust CAR expansion (median peak expansion 172 CAR/uL blood). Of the 13 patients dosed (1/13 pending 28 day follow up), 10/12 (83%) achieved MRD negative CR at 1 month and all patients had ongoing CAR T cell persistence at last follow up. Two patients experienced CD19 negative relapse (one at M3, one at M6), 1 patient died on D17 before first response evaluation, 1 died in molecular CR from sepsis, and 1 died from persistent disease. Currently, 7/12 remain on study and continue in flow/molecular MRD negative remission with a median follow up of 9.0 months (range 1.2-14.8). Conclusions: AUTO1 delivers excellent early remission rates with initial data showing 83% MRD negative CR and robust CAR expansion and persistence. Despite high tumour burden, the safety profile compares favourably to other CD19 CARs, with no cases of severe CRS and only one case of Gr3 neurotoxicity. This is consistent with experience in the paediatric cohort. Updated results will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: 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> 5x103) and CARLO (MFI <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<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<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<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<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<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.

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