Response to Anti-Bcma CAR T Cell Therapy Correlates with T Cell Exhaustion and Activation Status in T Cells at Baseline in Myeloma

Despite intense efforts, multiple myeloma remains incurable in most patients with the standard of care therapies. The plasma cell surface receptor B cell maturation antigen (BMCA) is highly expressed by myeloma cells and we recently demonstrated that 12 out of 25 heavily pretreated myeloma patients achieved a partial response or better after anti-BCMA CAR T cell treatment (VGPR, n=5; CR, n=1; sCR, n=1; Cohen et al., 2019, JCI 129(6):2210). To better understand the biological basis of this therapy, we identified key correlates of response using the pre-manufacturing apheresed T cells, the infusion product, and post-infusion T cells from the 25 patients in this cohort. As reported before, the disease characteristics, tumor burden, and CAR transduction efficiency did not correlate with therapy response. CAR T cell expansion, measured by the area under the curve of CAR qPCR in the first 21 days (AUC[0-21]), was highest in responding, lowest in non-responding patients (Jonckheere-Terpstra test, JT = 38, p=1.8x10^-6)(Fig.1A,B). Soluble BCMA, a biomarker of disease burden, shows a similar trend with response (Jonckheere-Terpstra test, JT = 54, p=1.2x10^-4). Furthermore, AUC[0-21] for CAR T cell expansion and soluble BCMA decline also strongly correlated (Spearman's rank correlation test, rho=0.82; p=2.41x10^-6), underscoring the quantitative relationship between CAR T cell expansion and tumor reduction. We have previously shown that response to CAR T cell therapy in CLL is largely determined by T cell memory function. To find if this extends to myeloma, we immunophenotyped apheresed T cells (or CAR-T precursor cells) and infusion product from the 25 patients. Phenotypically distinct T cell subpopulations were identified using shared-nearest-neighbor clustering method (PMID: 31178118) and their correlation with response to CAR T cell treatment was evaluated. This analysis revealed that among CD4+ and CD8+ CAR-T precursor cells, subpopulations representing naive and central memory T cells were enriched in T cells from responding patients, while non-responders displayed a distinctly activated effector phenotype at baseline. Additional analyses showed that apheresed CD8+ and CD4+ T cells from responder patients were non-cycling, granzyme B-negative, CTLA4[low] but otherwise largely immune checkpoint inhibitor-negative. CD8+ CAR-T precursor cells isolated from non-responders exhibited high expression levels of TIM3 or LAG3, and/or granzyme B, but not PD1, CTLA4, CD45RO or CD27. These data confirm the high activation, potential exhaustion and end-stage differentiation state of CAR-T precursor cells in this group. Similar analyses of infusion product CAR T cells did not reveal subpopulations associated with response. Clustering analysis of CD8+ CAR T cells within 20 days after infusion revealed a BCMA CAR-expressing cluster enriched in responding patients: a non-cycling, negatively regulated, Eomes-expressing central memory subset (cluster 0; Fig. 1E). Non-responding patients CAR-T cells displayed high levels of granzyme B and PD1 expression but were otherwise devoid of signs of activation (cluster 8; Fig. 1F). Furthermore, the abundance of CD8+ CAR-T cells with cluster 0 and 8 phenotype correlated significantly with in vivo expansion (AUC[0-21]; Fig. 1C). Four patients with a sufficiently high proportion of CAR expressing cells were phenotyped up to 125 days post-infusion. This analysis showed that the highly activated CAR T cell clusters 2 and 5 dominated at early phases post infusion but was rapidly replaced by non-cycling CAR T cells with downregulated CTLA4 and LAG3 but maintained expression of PD1 and TIM3 (cluster 0; Fig. 1D). Patient 27 with VGPR had a prominent effector population four months after infusion. BCMA-redirected CD4+ CAR T cells showed an enrichment of central memory phenotype CAR T cells in responding patients early after infusion, with high expression of Eomes, TIM3, and other immune checkpoint inhibitor molecules. This cluster also dominated the CD4 T cell repertoire in the first four months after infusion in the four responding patients. In conclusion, our data suggest that strategies to promote expression of Eomes and central memory function and reduce exhaustion in BCMA CAR T cells will enhance clinical activity. Further, these results underscore the "self-sustaining" feature of successful CAR T cell therapies in myeloma. Disclosures Pruteanu: Novartis: Employment. Cohen:Poseida Therapeutics, Inc.: Research Funding. Garfall:Tmunity: Honoraria, Research Funding; Amgen: Research Funding; Novartis: Patents & Royalties: inventor on patents related to tisagenlecleucel (CTL019) and CART-BCMA, Research Funding; Janssen: Research Funding; Surface Oncology: Consultancy. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Fraietta:Tmunity: Research Funding; Cabaletta: Research Funding; LEK Consulting: Consultancy. Brogdon:Novartis: Employment. Davis:Tmunity: Research Funding; Cabaletta: Research Funding. Levine:Tmunity Therapeutics: Equity Ownership; Avectas: Membership on an entity's Board of Directors or advisory committees; Vycellix: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Cure Genetics: Consultancy; Incysus: Membership on an entity's Board of Directors or advisory committees; Brammer Bio: Membership on an entity's Board of Directors or advisory committees; CRC Oncology: Consultancy. Milone:Novartis: Research Funding; Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA. Stadtmauer:Janssen: Consultancy; Tmunity: Research Funding; Amgen: Consultancy; Abbvie: Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Stand Up to Cancer: Research Funding; Incyte: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Genentech: Speakers Bureau.

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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 ◽

10.1182/blood-2019-129640 ◽

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.

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

Blood ◽

10.1182/blood-2019-124031 ◽

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.

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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 ◽

10.1182/blood.v130.suppl_1.742.742 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 742-742 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood-2018-99-117390 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 699-699 ◽

Cited By ~ 2

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.

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

Blood ◽

10.1182/blood-2018-99-116745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1680-1680 ◽

Cited By ~ 8

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.

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Cell Adhesion of ALL to Stromal Cells May Mediate CAR T-Cell Resistance: A Novel Escape Mechanism for Immunotherapy

Blood ◽

10.1182/blood-2019-132131 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2623-2623

Author(s):

Hye Na Kim ◽

Enzi Jiang ◽

Ruan Yongsheng ◽

Heather Ogana ◽

Nour Abdel-Azim ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Integrin Α4

Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p<0.005). Further combination treatments with chemotherapy are in progress. Conclusion: In summary, our data indicate that similarly to CAM-DR, CAM-CART-R can occur resulting in relapse of ALL. Targeting adhesion molecules may be a new approach to treat or prevent relapse following CD19 CAR T cell therapy for . Disclosures Ahmed: CellMedica: Other: Royalties; Celgene: Other: Royalties; Adaptimmune: Membership on an entity's Board of Directors or advisory committees. Babak:Simurx. Inc: Other: Founder . Pulsipher:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria. Wayne:AbbVie: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding. Abdel-Azim:Adaptive: Research Funding.

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CD70-Specific CAR T Cells Have Potent Activity Against Acute Myeloid Leukemia (AML) without HSC Toxicity

Blood ◽

10.1182/blood-2019-125534 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1932-1932 ◽

Cited By ~ 2

Author(s):

Tim Sauer ◽

Kathan Parikh ◽

Sandhya Sharma ◽

Bilal Omer ◽

Stephen Gottschalk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Xenograft Model ◽

Proliferative Potential ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

The outcome for the majority of patients with AML remains dismal. Adoptive transfer of chimeric antigen receptor (CAR) modified T-cells (CARTs) holds the promise to improve outcomes since they do not rely on the cytotoxic mechanism of conventional therapies. CD70 is a promising CAR T-cell therapy target for AML since it is expressed on AML bulk cells and leukemic stem cells (LSC) whereas expression is absent on normal hematopoietic stem cells (HSCs) in contrast to other antigens, like CD33 and CD123, which are currently being explored as immunotherapy targets for AML. The goal of this project was to construct a panel of CD70-CARs and determine their effector function in vitro and in preclinical AML xenograft models. We generated a panel of CD70-CARs using a CD70-specific single chain variable fragment (scFv) as the antigen recognition domain, different spacers (intermediate [IMS] or long flexible spacer [LFS]) and signaling domains that contained CD3z combined with one of three co-stimulatory domains (CD27, CD28, or 41BB). An additional CAR comprising the human ligand of CD70, CD27 (including its costimulatory endodomain) fused to the CD3z chain (CD27z) was also included in the comparison. CD70-CARs were efficiently expressed from retroviral vectors as detected by FACS analysis. T-cells expressing IMS-CARs had lower viability and a more differentiated phenotype than T-cells expressing the LFS- or the CD27z-CARs. In sequential co-culture assays with live tumor cells, T-cells expressing the LFS-CAR with a 41BB co-stimulatory domain (LFS-41BBz) or CD27z-CARs had greater proliferative potential and an increased ability to repeatedly eliminate tumor cells than NT- and other CD70-CAR T-cell populations. In addition, CD27z-CAR T-cells secreted the highest levels of type I cytokines (TNF-a and Interferon-g) after tumor cell stimulation and expressed lower levels of markers (LAG3, TIM-3 and PD-1) that are associated with T-cell exhaustion. In vivo, only LFS-28z- or CD27z-CAR T-cells had significant anti-tumor efficacy in the murine Molm-13 xenograft model in comparison to NTs and all other CD70 CARTs resulting in a significant (p<0.001) overall survival advantage. The potent anti-tumor activity of LFS-28z- and CD27z-CARTs was confirmed in a second murine AML (THP-1) xenograft model. While LFS-28z- and CD27z-CARTs had similar anti-AML activity, adoptive transfer of fire-fly luciferase-expressing CAR T-cells into AML-bearing mice revealed that CD27z-CARTs had the greatest proliferative potential in vivo. Crucially, LFS-28z- or CD27z-CARTs did not recognize normal CD34+ HSCs in colony forming unit (CFU) assays, confirming that HSCs do not express CD70 and suggesting that treatment with CD70 CAR T cells is safe. In conclusion, CD70 is a promising target antigen for CAR T-cell therapy of AML. We demonstrate here that hinge and costimulatory domain greatly influences CD70-CAR T-cell function. LFS-28z- and CD27z-CAR T-cells had potent anti-AML activity in xenograft models with a favorable safety profile, warranting future early phase clinical testing of CD70-CAR T-cells in patients with CD70-positive AML. Disclosures Gottschalk: TESSA Therapeutics: Other: Research Collaboration; NHLBI: Research Funding; ASSISI fundation of Memphis: Research Funding; ViraCyte: Consultancy; California Institute for Regenerative Medicine: Research Funding; Sanofi: Honoraria; Merck: Consultancy; EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; Tidal: Membership on an entity's Board of Directors or advisory committees; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; America Lebanese Syrian Associated Charities: Research Funding. Rooney:Marker Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founding member; Viracyte: Other: Founding member; Tessa Therapeutics: Research Funding; Cell Genic: Membership on an entity's Board of Directors or advisory committees.

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Intestinal Microbiota Composition Prior to CAR T Cell Infusion Correlates with Efficacy and Toxicity

Blood ◽

10.1182/blood-2018-99-118628 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3492-3492 ◽

Cited By ~ 3

Author(s):

Melody Smith ◽

Eric R. Littmann ◽

John B. Slingerland ◽

Annelie Clurman ◽

Ann E. Slingerland ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Intestinal Microbiota ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Firmicutes Phylum

Abstract Introduction Cellular therapy with chimeric antigen receptor (CAR) T cells has fundamentally changed the treatment of many cancers. Unfortunately, not all patients who receive this therapy have a favorable response. Additionally, some patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. Recent studies have found a relationship between the intestinal microbiome and the response to immunotherapy with checkpoint blockade. We propose the intestinal microbiota as a factor that influences the efficacy and toxicity of CAR T cells. We hypothesize that the differences in outcomes of patients who receive CAR T cells are related to the composition of their intestinal microbiota at baseline. We report a single-center analysis of pre-CAR T cell infusion microbiota composition. Methods We collected stool samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC). A baseline sample was collected prior to CAR T cell infusion. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the operational taxonomic units (OTUs) were classified using the NCBI Reference Sequence Database. Clinical response to assess efficacy was classified as either complete response (CR) or no complete response. Given the sample size, toxicity was pooled to encompass Grade 1 to 4 CRS and Grade 1 to 4 neurotoxicity. Linear discriminant analysis effect size (LEfSe) was used to identify microbial biomarkers for efficacy and toxicity between groups using relative abundances with a linear discriminant analysis score threshold >2.5. Results We analyzed 24 baseline samples from 24 patients treated at MSKCC. The patients were all adult recipients of cellular therapy with CAR T cells. The patients varied in conditioning regimen, CAR construct and underlying diagnosis, which included solid tumors and hematologic malignancies. First, we assessed the 16S relative abundance of the intestinal microbiota of the patients at baseline. We found that the composition of the microbiota prior to CAR T cell infusion was diverse, as defined by an Inverse Simpson >4 in all of the patients, although the level of diversity amongst the patient samples varied (Fig A). An assessment of the efficacy of CAR T cells with LEfSe analysis found increased abundance in several families of the Clostridiales order (Firmicutes phylum), including Oscillospiraceae, Ruminococcacaeae, and Lachnospiraceae, in those patients who achieved a CR. For the patients who did not achieve a CR, we found an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Patients who experienced toxicity, either CRS or neurotoxicity, had an increased abundance of families within the Clostridiales or Lactobacillales order (Firmicutes phylum), which included Lachnospiraceae and Lactobacillaceae. Finally, patients who did not experience toxicity also had an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Conclusion We demonstrate that our subset of patients had diverse microbial composition prior to receiving CAR T cell therapy despite the fact that many of them were heavily pre-treated. Additionally, we observe the abundance of the family Lachnospiraceae in the patients who achieved a CR and those who experienced toxicity. Many Lachnospiraceae are butyrate producers, whose presence has been found to be protective against Clostridium difficile infection in recipients of allogeneic hematopoietic cell transplant but whose abundance is lower in colon cancer. Conversely, we observe an abundance of the family Peptostreptococcaceae in patients who did not achieve a CR or who did experience toxicity. Peptostreptococcaceae has been found to be more abundant in the intestines of patients with colon cancer. Of note, the intestinal micriobiota that we identify are not congruent with the specific bacteria that have been found to promote anti-tumor immunity to checkpoint blockade. Our data suggests a role for the intestinal microbiota in mediating the response to CAR T cells and proposes that the baseline microbial composition may correlate with efficacy and toxicity. Further studies will investigate biochemical mechanisms to understand the interplay of the intestinal microbiota and the immune system to improve patient outcomes following CAR T cell therapy. Disclosures Park: Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy. O'Cearbhaill:Juno: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Safety and Anti-Tumor Activity of CD5 CAR T-Cells in Patients with Relapsed/Refractory T-Cell Malignancies

Blood ◽

10.1182/blood-2019-129559 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 199-199 ◽

Cited By ~ 8

Author(s):

LaQuisa C. Hill ◽

Rayne H. Rouce ◽

Tyler S. Smith ◽

Lina Yang ◽

Madhuwanti Srinivasan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

T Cell Malignancies

Introduction: We describe a Phase I dose escalation study (NCT03081910) of autologous CD5-directed chimeric antigen receptor T cell (CD5 CAR T) therapy for relapsed or refractory (r/r) T-cell leukemia and lymphoma. Establishing a CAR T cell platform to target neoplasms of T-cell origin has been hindered by the shared expression of most targetable antigens on both malignant and normal T lymphocytes, which can promote CAR T cell fratricide. CD5 is one such pan-T cell surface marker present in ~85% of T-cell malignancies. We developed a second-generation CD5-specific CAR with CD28 costimulatory endodomain that produces minimal and transient fratricide when expressed in T cells. We designed this study to evaluate the safety and feasibility of treating patients with r/r T-cell malignancies with these CD5 CAR T cells as a bridge to allogeneic hematopoietic stem cell transplant (HSCT). Secondary objectives of our study included evaluating the antitumor response, in vivo expansion, persistence of CD5 CAR T cells, as well as their impact on normal T-cell numbers and function. Patients and methods: CD5 CAR T cells were generated from autologous PBMCs using gammaretroviral transduction and cryopreserved. We detected no residual malignant cells in the CD5 CAR T cell products by flow cytometry. To date, we have treated a total of 9 patients (8 adults and 1 adolescent; age 16-71 years [median 62 yrs]) with CD5+ r/r T-acute lymphoblastic leukemia (T-ALL; n=4) or T-non-Hodgkin's lymphoma (T-NHL; n=5) on dose levels 1 and 2. All patients were transplant-eligible with an identified allogeneic HSCT donor, yet unable to proceed due to residual disease. All patients had been heavily pretreated, with a median of 5 (range 2 -18) prior lines of therapy. Two patients had previously failed allogeneic HSCT. Patients received cytoreductive chemotherapy with cyclophosphamide and fludarabine followed by a single dose of CD5 CAR T cells. We evaluated adverse events, clinical responses, and in vivo expansion and persistence pre and post-infusion. Results: Three patients received CD5 CAR T cells on dose level 1 (1x107 CAR T cells/m2) and 6 on dose level 2 (5x107 CAR T cells/m2). In all patients treated, CAR T cells reached peak expansion in peripheral blood (PB) 1-4 weeks following infusion, followed by a gradual contraction in most patients (Figure 1). CD5 CAR T cells were present in lymph node and marrow biopsies in patients with T-NHL and T-ALL, respectively, and were also detected in a CSF sample in 1 T-ALL patient. After cytoreduction and CAR T cell infusion, we observed decreased PB CD3+ cell numbers but this ablation was never complete. Cytokine release syndrome (CRS) occurred in 3/9 patients (all at dose level 2). Grade 1 CRS was observed in 2 patients. One patient experienced Grade 2 CRS and Grade 2 neurotoxicity, which resolved after administration of tocilizumab and supportive care, respectively. Two patients had prolonged cytopenias at 6 weeks, 1 of whom had viral reactivation (CMV and BK virus) requiring antiviral therapy. On disease re-evaluation 4-8 weeks post-CD5 CAR T cell infusion, 4 of 9 evaluable patients obtained an objective response (1 of 3 on DL1 and 3 of 6 on DL2). Complete responses (CR) were achieved in 3 patients, one with angioimmunoblastic T cell lymphoma (AITL), one with peripheral T cell lymphoma (PTCL), and one with T-ALL. Two of these patients did not wish or were unable to proceed to planned HSCT and relapsed with their underlying CD5+ malignancy at 6 weeks and 7 months post-infusion. The remaining patient is currently undergoing work-up for HSCT (Figure 2). An additional patient with extensive AITL was classified as a mixed response (Figure 3) due to the appearance of a new PET-avid lesion. This patient received a second infusion of CD5-CAR T cells, proceeded to HSCT, and remains in CR at 125 days post-transplant. Conclusions: These results demonstrate that CD5 CAR T cells are safe and can induce clinical responses in heavily treated patients with r/r CD5+ T-ALL and T-NHL without inducing complete T-cell aplasia. Importantly, elimination of malignant T cells by CD5 CAR T cells may allow previously ineligible patients to proceed to HSCT. Disclosures Rouce: Novartis: Consultancy, Honoraria; Tessa Therapeutics: Research Funding; Kite, a Gilead Company: Consultancy, Honoraria. Grilley:Allovir: Consultancy, Equity Ownership; Marker Therapeutics: Consultancy; Tessa: Consultancy. Heslop:Marker Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allovir: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cell Medica: Research Funding. Brenner:Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; T Scan: 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.

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Allogeneic Anti-CD19 CAR T Cells Manufactured from Healthy Donors Provide a Unique Cellular Product with Distinct Phenotypic Characteristics Compared to CAR T Cells Generated from Patients with Mature B Cell Malignancies

Blood ◽

10.1182/blood-2019-123018 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3228-3228 ◽

Cited By ~ 3

Author(s):

Charlotte Graham ◽

Agnieszka Jozwik ◽

Ruby Quartey-Papafio ◽

Nikolaos Ioannou ◽

Ana M Metelo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

B Cell Malignancies ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

Despite the success of autologous anti-CD19 CAR T cell therapy in B-Acute lymphoblastic leukaemia (B-ALL) and Diffuse Large B Cell Lymphoma (DLBCL), treatment failures occur. One contributing factor may be the intrinsic T cell fitness of the CAR T cell product that is influenced by the underlying malignancy and prior treatments. With the advent of gene editing, 'off the shelf' non-HLA matched healthy donor (HD) CAR T cells are under investigation for the treatment of patients (pts) in clinical trials. UCART19 (S68587) is a first-in-class allogeneic CAR T cell product expressing a second generation anti-CD19 CAR with TALEN®-mediated gene knockouts of T cell receptor alpha chain (TRAC) and CD52 to prevent graft versus host disease and to render them resistant to anti-CD52 antibody used for lymphodepletion. Preliminary clinical trial data on the use of UCART19 in B-ALL was previously reported at ASH (Benjamin et al, 2018). The phenotypic and functional characteristics of CAR T cell products manufactured from B-ALL, Chronic Lymphocytic Leukaemia (CLL) and DLBCL pts were compared to young adult healthy donor (HD) CAR T cell products. In addition, potential effects related to knocking out TRAC in HD TCR-CAR T cells were examined. Thawed PBMCs from B-ALL, CLL, DLBCL pts and HDs underwent T cell enrichment, activation with anti-CD3/CD28 beads and IL-2, followed by transduction with anti-CD19 4-1BB CD3ζ lentiviral CAR construct and expansion. HD TCR- CAR T cells were manufactured by electroporation of HD CAR T cells with mRNA coding for TRAC TALEN® and residual TCRαβ+cells were removed by magnetic bead selection. CAR expression levels, T cell subsets, and exhaustion markers were examined by flow cytometry. Expression of activation markers CD25 and CD69 was measured in response to co-culture with the CD19+cell line NALM-6. Cytotoxicity against NALM-6 and Raji was assessed and antigen-mediated proliferation measured over 14 days. HD CAR T cells (n=11) expanded significantly more during manufacture than CAR T cells derived from B-ALL (n=9), CLL (n=8) or DLBCL (n=8) pts. As expected, the electroporation step resulted in a transient decrease in viability which recovered over time in culture (n=10). Median CAR expression level was higher on CLL CAR T cell products compared to those from B-ALL pts and HDs, thought to be due to a higher CD4:CD8 ratio in some CLL products. As a consequence of TCR knockout, CD3 expression was lost on HD TCR- CAR T cells (n=10), apart from a small population of γδ CAR T cells. CLL and DLBCL CD8+CAR+cells expressed higher levels of PD1 than HD CD8+CAR+cells. DLBCL CD4+CAR+cells also expressed significantly higher levels of PD1 than HD or HD TCR-CD4+CAR+T cells. CAR+CD8+CD27+PD1- T cells have been previously described as a functionally important population that correlated with clinical outcome in pts who received CLL CAR T cells (Fraietta et al, 2018). We found HD (n=13) and HD TCR- (n=10) CAR T cells had significantly more CD8+CD27+PD1- CAR T cells compared to those derived from CLL (n=8) and DLBCL (n=6) pts, but similar levels to B-ALL pts (n=10). In the absence of CD19 antigen, DLBCL CAR+CD8+ T cells (n=6) had greater expression of CD25 and CD69. However, in response to stimulation with CD19+ NALM-6 cells, HD (n=12), HD TCR- (n=10) and B-ALL (n=10) CAR T cells had higher fold increase in CD69+ cells compared to DLBCL (n=6) CAR T cells. On paired analysis (n=6), no difference was seen in activation in response to CD19 antigen on HD compared to HD TCR- CAR T cells. All CAR T cell products demonstrated comparable cytotoxicity against NALM-6 and Raji cell lines in short term in vitro assays. However, long-term cytotoxicity will be evaluated in a murine model. We performed a detailed comparison of the phenotypic and functional characteristics of CAR T cells derived from pts with B-cell malignancies and HDs. DLBCL CAR T cells showed lower antigen specific activation but higher baseline activation which could lead to more differentiated exhausted T cells. CAR T cells derived from HDs show a higher proportion of the therapeutically relevant CAR+CD8+CD27+PD1- cells compared to patients with mature B cell malignancies (CLL and DLBCL), which is maintained after TRAC knockout. This suggests allogeneic CAR T cells, such as UCART19, may provide a more effective product for pts with T cell dysfunction. Disclosures Graham: Gillead: Other: Funding to attend educational meeting; Servier: Research Funding. Jozwik:Servier: Research Funding. Metelo:Pfizer: Research Funding; Allogene: Research Funding. Almena-Carrasco:Servier: Employment. Peranzoni:Servier: Employment. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Dupouy:Servier: Employment. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Patten:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Benjamin:Amgen: Honoraria; Allogene: Research Funding; Gilead: Honoraria; Servier: Research Funding; Eusapharm: Consultancy; Pfizer: Research Funding; Takeda: Honoraria; Novartis: Honoraria.

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