scholarly journals Effects of Prior Exposure to Tec Kinase(BTK/ITK) Inhibitors on Kte-X19 Products

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3849-3849
Author(s):  
Irene Scarfò ◽  
Kathleen M.E. Gallagher ◽  
Mark B. Leick ◽  
Michael C. Kann ◽  
Justin Budka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stock Options ◽  
Effector Function ◽  
Prior Exposure ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Post Infusion ◽  
Privately Held

Abstract Introduction: Frequent and durable responses were recently reported in relapsed or refractory (R/R) mantle cell lymphoma (MCL) patients treated with KTE-X19, an autologous CD19-targeted chimeric antigen receptor-engineered T-cell (CAR-T) product (Wang et al. N Engl J Med. 2020). Most patients enrolled had received at least one line of Tec kinase inhibitor prior to KTE-X19 manufacturing, either in the form of ibrutinib, a Bruton's tyrosine kinase (BTK) and Inducible T cell kinase (ITK) inhibitor, or acalabrutinib, a more selective BTK inhibitor. Pharmacokinetic expansion of KTE-X19 was higher in ibrutinib-treated patients relative to acalabrutinib-treated patients. We previously showed that prolonged exposure to ibrutinib enhanced T cell effector function and proliferation in patients with CLL (Fraietta et al, Blood, 2016). To assess the impact of Tec kinase inhibitor on KTE-X19 products and downstream clinical outcomes, we examined the phenotype, transcriptional profile and cytokine production of KTE-X19 infusion products and post-infusion lymphocytes from patients with R/R MCL treated on the Zuma-2 study. Study Design and Methods: We evaluated biospecimens from MCL patients who enrolled on the Zuma-2 clinical trial (NCT02601313) and who were previously treated with ibrutinib (n=14) or acalabrutinib (n=6). Samples analyzed consisted of KTE-X19 CAR T products and peripheral blood mononuclear cells (PBMC) collected 7 days after infusion. Lymphocytes were assessed for CAR expression, T cell phenotype, transcriptional profile and cytokine production. In addition, CAR T cell phenotypes and cytokines were profiled following co-culture of KTE-X19 with CD19 + Toledo cells (DLBCL). Results: Flow cytometric analysis of KTE-X19 demonstrated similar distributions of CD4+ and CD8+ T cells and comparable frequencies of central and effector memory populations in the CAR+ T cells derived from patients with prior exposure to ibrutinib vs. acalabrutinib. T helper subset analysis trended towards enrichment of Th1/Th17 populations within the CAR+ CD4+ cells of the ibrutinib cohort. This finding was further supported by transcriptional profiling of sorted CAR+ T cells from infusion products, where Th1/Th17, Jak/STAT and activation-related genes were enriched in the cohort with prior ibrutinib exposure. In addition, the Th1 phenotype was more frequent in PBMC of ibrutinib-exposed patients (8/14) compared to acalabrutinib-exposed patients (1/4). Interestingly, a shift from a central memory-dominant product towards an effector memory phenotype was observed in peripheral CD4+ and CD8+ CAR T cells in the ibrutinib cohort, whereas acalabrutinib post-infusion CAR T cells maintained a central memory phenotype. In vitro stimulation of KTE-X19 CAR-T infusion products with tumor cells resulted in a significant enrichment of the Th1 population in patients who had received ibrutinib compared to those that received acalabrutinib (p=0.0058). Following stimulation, CAR-T cells from the acalabrutinib cohort produced higher levels of Th2 cytokines, including IL-4, IL-5, and IL-13 as well as GM-CSF compared to the ibrutinib cohort. Conclusions: Analysis of KTE-X19 infusion products and day 7 post-infusion PBMC demonstrated that CAR T cells from patients with prior ibrutinib exposure have a Th1 predominant phenotype, suggesting that ibrutinib but not acalabrutinib promotes Th1 differentiation and effector function, potentially through the inhibition of ITK. Furthermore, our data suggest that inhibition of non-BTK targets such as ITK may play a role in driving a Th17 phenotype. Prior exposure to ibrutinib may increase CAR T cell effector function to a greater extent than exposure to acalabrutinib to enhance clinical outcome in patients with MCL. Disclosures Budka: Kite Pharma: Current Employment. Sowrirajan: Kite Pharma: Current Employment. Nguyen: Kite Pharma: Current Employment. Shen: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties; Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

scholarly journals Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer Immunotherapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2771-2771
Author(s):  
Mark A Wallet ◽  
Toshinobu Nishimura ◽  
Christina Del Casale ◽  
Andriana Lebid ◽  
Brenda Salantes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stock Options ◽  
Γδ T Cells ◽  
Differentiation Process ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T ◽  
Privately Held

Abstract Introduction Allogenic CAR-T cell therapies for cancer provide a new option to reduce barriers faced by autologous cell therapies, but several challenges remain. One challenge is the risk of graft versus host disease (GvHD) caused by the infused T cells. A potential solution is the use of a subset of gamma delta (γδ) CAR-T cells whose T cell receptors (TCRs) recognize invariant antigens rather than hypervariable MHC molecules. Here we describe an off-the-shelf, induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ CAR-iT) for treatment of cancer and a process for deriving such cells. Methods T cell-derived iPSCs (TiPSC) are generated by reprogramming γδ T cells to yield pluripotent stem cells. For proof-of-concept studies, TiPSC were engineered using CRISPR gene editing to deliver a CD19 CAR transgene. TiPSC are then subjected to a two-stage differentiation process. First, TiPSC are differentiated into CD34-expressing hematopoietic progenitor cells (HPCs). HPCs are then exposed to a feeder-free differentiation process that results in uniform γδ CAR-iT cells. The purity and identity of γδ CAR-iT cells were assessed by flow cytometry and the ability of γδ CAR-iT cells to respond to homeostatic growth factors was determined by intracellular staining of phosphorylated signaling proteins and mRNA transcriptome analysis. Cytokine production by CAR-iT cells was measured by immunoassays following stimulation of the CAR. Tumor cell killing by γδ CAR-iT cells was performed using IncuCyte cytotoxicity assays. In vivo control of tumors by γδ CAR-iT in immunodeficient mice was determined using a NALM-6 B cell lymphoblastic xenograft model. Results A research-grade γδ TiPSC line was used to develop an iT differentiation process. This γδ TiPSC line was engineered to express a CD19 CAR molecule and then subjected to the differentiation process after which >95% of cells were CD3 + γδ TCR + CAR + iT cells. These γδ CAR-iT cells responded to IL-2 and IL-15. STAT5 phosphorylation levels were similar but STAT3 phosphorylation levels were greater in response to IL-15 compared to IL-2 at equimolar concentrations of cytokine. IL-2 and IL-15 elicited qualitatively similar transcriptional responses, but the magnitude of cytokine-induced gene expression was generally greater in IL-15-treated cells. Upon activation, γδ CAR-iT cells released markedly less IFN-γ and other inflammatory cytokines than conventional blood-derived ab CAR-T cells. In an IncuCyte serial killing assay, γδ CAR-iT cells exhibited sustained killing of NALM-6 tumor cells for at least one week in the presence of IL-15. In vivo, γδ CAR-iT cells caused a significant reduction in NALM-6 tumor burden with a single dose of γδ CAR-iT resulting in >95% tumor growth inhibition. To establish an efficient method for derivation of clinical grade γδ TiPSC lines, we investigated methods to isolate, expand, and reprogram human γδ T cells. When γδ T cells were expanded by exposure to the chemical zoledronic acid (zoledronate) and IL-2, we found a large disparity between donors; some donors exhibit robust expansion while others are seemingly resistant to zoledronate. In order to enhance γδ T cell expansion we screened dozens of activation conditions and eventually established a universal activation protocol that can elicit robust expansion of γδ T cells from all donors tested. When expanded γδ T cells were subjected to reprogramming conditions, dozens to hundreds of individual TiPSC colonies were obtained from each donor. The identity of the rearranged γδ TCR locus was confirmed using molecular assays. New γδ TiPSC lines were engineered with a CD19 CAR molecule and killing activity was confirmed in an in vitro serial killing assay. Conclusions γδ CAR-iT cells provide a new opportunity to treat cancers with an off-the-shelf universal T cell platform without the risk for GvHD. γδ CAR-iT cells are readily manufacturable, and we have derived an end-to-end process that enables new TiPSC line reprogramming, genetic modification of TiPSC lines, and feeder-free differentiation. γδ CAR-iT cells exhibit potent antigen-specific tumor killing and they release less inflammatory cytokine than conventional CAR-T cells, potentially reducing the risk for cytokine-mediated toxicities. We believe that this off-the-shelf platform will enable safer and more accessible allogenic cell therapies for hematologic and solid cancers. Disclosures Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Nishimura: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Del Casale: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lebid: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Salantes: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Santostefano: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bucher: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Thompson: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company.

scholarly journals A Phase II Trial of Anakinra for the Prevention of CAR-T Cell Mediated Neurotoxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2814-2814
Author(s):  
Matthew J. Frigault ◽  
Kathleen M.E. Gallagher ◽  
Marc Wehrli ◽  
Betsy Valles ◽  
Keagan Casey ◽  
...  
Keyword(s):  
T Cell ◽  
Stock Options ◽  
Research Funding ◽  
Leadership Role ◽  
Publicly Traded ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion ◽  
Grade 3 ◽  
Privately Held

Abstract Introduction: Chimeric antigen receptor (CAR)-T cell therapy is limited in most cases to inpatient use due to risk of severe treatment-related toxicities. The two primary toxicities observed with CAR-T therapy, cytokine release syndrome (CRS) and neurotoxicity, are associated with increased circulating inflammatory cytokines such as IL-6 and IL-1. Targeting IL-6 with tocilizumab is effective for treating CRS but not neurotoxicity. Anakinra is an FDA-approved recombinant IL-1 receptor antagonist that competitively inhibits IL-1 receptor signaling and therefore blocks downstream production of inflammatory cytokines including IL-6. Leveraging support from Kite Pharma, we opened an investigator-initiated clinical trial (NCT04150913) with the hypothesis that anakinra could be administered prophylactically to prevent severe CRS and neurologic events (NE) in patients receiving axicabtagene ciloleucel (axi-cel). Here we report preliminary outcomes of this study. Study Design and Methods: This is a phase II single center, open-label study for patients ≥18 years old with relapsed or refractory large cell lymphoma. Patients must have progressed after ≥2 lines of systemic therapy but could not have CNS disease or have been previously treated with CAR-T therapy. Following leukapheresis and manufacturing, patients received 3 days of lymphodepleting chemotherapy (LDC, cyclophosphamide 500mg/m 2 and fludarabine 30 mg/m 2) and 200 mg of subcutaneously administered anakinra starting 4 hours prior to axi-cel infusion and daily thereafter for a total of 7 days. CRS and NE were graded based on the Lee 2013 criteria and the CTCAE 4.03 criteria, respectively, to enable direct comparison to the pivotal Zuma-1 cohorts. The primary endpoint is the rate and severity of NE within the first 30 days of infusion; secondary endpoints include the incidence and severity of CRS and disease response. CAR-T cell expansion, serum cytokines, and circulating biomarkers of toxicity were measured at baseline, day 3, 7, 14, 21, and 28 post CAR-T cell infusion. Results: Interim analysis of the first 6 patients demonstrated a median age of 68 (range 59-72). Patients included a diverse group of histologies including double-hit lymphoma (n=2), transformed indolent NHL (n=3), and DLBCL NOS (n=1). Two patients were considered primary refractory at time of enrollment. Pre-LDC baseline characteristics included a median SPD of 2819 mm 2 (range 1063-5802), median LDH of 415 (range 147-497) which were comparable to the pivotal ZUMA-1 cohorts. Baseline ferritin, CRP, SAA and IL-15 were similar to the pivotal ZUMA-1 cohorts. While low-grade CRS was observed in 5/6 patients, no patients experienced severe CRS and median onset occurred on day +8 (range 1-8). Four patients did not experience any NE, while two patients experienced grade 3 NE on days +6 till +9 (somnolence) and +12 (global aphasia only, for one day) respectively. With a median follow-up of 4 months, the day +28 overall response rate was 100% (4 CRs, 2 PRs), with 4/6 patients having an ongoing complete response at last disease assessment. One patient was re-infused at progression and remains in a CR 3 months from re-infusion. Responses were seen despite varying CAR-T peak level with most patients demonstrating expansion in the lower quartile of the historic ZUMA-1 cohort. Median post-infusion peak of CRP, ferritin, IL-2, GM-CSF, IFNγ, IL-10, IL-6 and SAA were lower than that observed in the pivotal ZUMA-1 cohorts. All patients remain alive at time of data analysis. Conclusions: With a limited number of patients analyzed thus far, anakinra appears to provide benefit to the toxicity profile of axi-cel, presenting reduced and/or delayed CRS and NE and a decrease in post-infusion inflammatory analytes, when compared to ZUMA-1 pivotal cohorts. No severe CRS was observed in this initial analysis and 2/6 patients experienced grade 3 NE (somnolence and global aphasia) after day 6. Despite CAR-T expansion in the lower quartile of that of ZUMA-1, we observed a 100% ORR with 4 patients remaining in CR at a median follow-up of 4 months. Additional subjects will be assessed to investigate the role of prophylactic anakinra in the management of CRS and NE, which has potential for making axi-cel treatment an outpatient therapy. Disclosures Frigault: BMS: Consultancy; Editas: Consultancy; Iovance: Consultancy; Arcellx: Consultancy; Takeda: Consultancy; Kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Wehrli: CSL Behring: Patents & Royalties; Nestle: Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Chou: Kite Pharma: Current Employment. Shen: Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties; Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties. Filosto: Kite, a Gilead Company: Current Employment; Gilead Sciences: Other: stock or other ownership ; Tusk Therapeutics: Patents & Royalties: or other intellecular property. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

scholarly journals Phase 1 Study of CD37-Directed CAR T Cells in Patients with Relapsed or Refractory CD37+ Hematologic Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 653-653
Author(s):  
Matthew J. Frigault ◽  
Yi-Bin Chen ◽  
Kathleen M.E. Gallagher ◽  
Nora K. Horick ◽  
Areej El-Jawahri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stock Options ◽  
Research Funding ◽  
Ad Hoc ◽  
Cell Lymphoma ◽  
Publicly Traded ◽  
Car T Cells ◽  
Advisory Boards ◽  
Car T

Abstract Background: CD37 is a tetraspanin molecule expressed in B-cell and some T-cell lymphomas. We designed a Chimeric Antigen Receptor (CAR) targeting CD37 and with 4-1BB and CD3z intracellular signaling domains (CART37). Preclinical studies indicated comparable anti-tumor activity to CD19-directed CAR-T cells against B-cell lymphomas, promising activity against CD37 + T-cell lymphomas, and no evidence of off-tumor activity (PMID: 30089630). The lentiviral vector used in the clinical study includes a truncated EGFR reporter gene. NCT04136275 is a Phase 1, single-site, open-label, dose-escalation trial enrolling subjects with lymphoma who have received ≥ 2 prior regimens, and whose tumor cells express CD37. Methods: We developed a clinical assay to assess CD37 expression on patient tumor samples using flow cytometry and IHC. Peripheral blood mononuclear cells are collected via leukapheresis and manufactured using the CliniMACS Prodigy®. Following release testing, fresh or cryopreserved cell product is infused. Subjects undergo lymphodepletion with fludarabine and cyclophosphamide, then receive CART37 as a single infusion. Planned starting dose was 100 x 10 6 CAR + T cells, with options to dose-escalate to 300 x 10 6 CAR + T cells or dose de-escalate to 30 x 10 6 CAR + T cells in the event of dose-limiting toxicities (DLT) using a 3+3 design. The primary outcome measure is incidence of adverse events (AEs), including DLTs. Additional outcome measures are clinical response, progression-free and overall survival; correlative studies focus on quantification and persistence of CAR + cells in blood, residual tumor, and cytokine modulation. Results: As of July 13, 2021, 4 subjects (ages 35-70 years) have received CART37. Subjects had a median of 5.5 prior lines of systemic therapy. Two subjects had primary refractory double-hit high-grade B cell lymphoma (HGBCL) that had relapsed after commercial CD19 CAR-T; one subject had cutaneous T cell lymphoma relapsed after extracorporeal photopheresis, alemtuzumab, total skin electron beam radiation, allogeneic hematopoietic stem cell transplant (HSCT), brentuximab, and donor lymphocyte infusion, and one subject had Hodgkin's lymphoma refractory to six prior regimens, including chemotherapy, brentuximab vedotin, nivolumab and everolimus. All subjects were infused in the DL1 cohort, but one subject (with cutaneous T cell lymphoma) received only 19 x 10 6 CAR + due to limited ex vivo expansion. Three subjects developed low-grade CRS and ICANS, and one subject developed refractory Grade 3 CRS and Grade 3 ICANS which resolved with medical management. One patient with HGBCL developed CD19 neg and CD37 neg progressive disease prior to the day 28 evaluation. The three other subjects demonstrated deep responses (2 CR, 1 PR that converted to CR) as best response. Two subjects are alive 208 and 272 days from CAR37 infusion. All subjects had detectable expansion of CART37 by flow cytometry and molecular assays. Two subjects (who received ≥ 100 x 10 6 CART37 had robust expansion and developed prolonged pancytopenia with marrow aplasia; cetuximab infusion decreased detection of truncated EGFR on circulating T cells but had no impact on vector copy number. Both subjects underwent allogeneic HSCT after conditioning with flu/cy/TBI(400) and post-transplant cyclophosphamide (PTCy) based GVHD prevention and successfully engrafted, and had no detectable CART37 after HSCT. Conclusions: In this initial cohort, CART37 infusion resulted in CRS or ICANs as is common for CAR T cell products. Bone marrow aplasia was unexpected and was observed in two subjects who received at least 100 x 10 6 CART37; this was successfully rescued with allogeneic HSCT. Three of four subjects had deep clinical responses in heavily pretreated, refractory disease of diverse lymphoma subtypes. The protocol is open to enrollment with dose de-escalation to 30 x10 6 CART37 and has been amended to require identification of a potential donor prior to treatment in the case that rescue allogeneic HSCT is needed. CART37 has a potential role in enabling allogeneic transplantation in patients with relapsed or refractory hematologic malignancies. Disclosures Frigault: Takeda: Consultancy; Editas: Consultancy; BMS: Consultancy; Novartis: Consultancy, Research Funding; Iovance: Consultancy; Arcellx: Consultancy; Kite: Consultancy, Research Funding. Chen: Gamida: Consultancy; Incyte: Consultancy. Wehrli: CSL Behring: Patents & Royalties; Nestle: Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Spitzer: Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy; Qihan Bio: Consultancy; Syneos Health: Consultancy. Preffer: Cytek Biosciences: Other: Unspecified Relationship. Shaw: Orchard Therapeutics, Ltd: Current equity holder in publicly-traded company. Nikiforow: Kite/Gilead: Other: Ad hoc advisory boards; Novartis: Other: Ad hoc advisory boards; Iovance: Other: Ad hoc advisory boards; GlaxoSmithKline (GSK): Other: Ad hoc advisory boards. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Maus: Novartis: Consultancy; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Kite Pharma: Consultancy, Research Funding; GSK: Consultancy; Intellia: Consultancy; In8bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; Cabaletta Bio (SAB): Consultancy; BMS: Consultancy; Bayer: Consultancy; Atara: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; WindMIL: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

scholarly journals Characteristics of Post-Infusion Chimeric Antigen Receptor (CAR) T Cells and Endogenous T Cells Associated with Early and Long-Term Response in Lisocabtagene Maraleucel (liso-cel)-Treated Relapsed or Refractory (R/R) Large B-Cell Lymphoma (LBCL)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3834-3834
Author(s):  
Jerill Thorpe ◽  
Yue Jiang ◽  
Julie A Rytlewski ◽  
Ana Kostic ◽  
Yeonhee Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rna Seq ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion ◽  
Term Response

Abstract Background: Liso-cel is an autologous, CD19-directed, defined composition, 4-1BB CAR T cell product administered at equal target doses of CD8 + and CD4 + CAR + T cells. In the pivotal TRANSCEND NHL 001 study (NCT02631044) in patients (pts) with R/R LBCL after ≥ 2 prior lines of therapy, liso-cel demonstrated significant clinical activity with an objective response rate of 73%, complete response (CR) rate of 53%, and median duration of response (DOR) not reached at 12 months' median DOR follow-up, and a low incidence of severe cytokine release syndrome (2%) and neurological events (10%; Abramson et al. Lancet 2020;396:839-852). While CAR T cells have shown remarkable efficacy in treating LBCL, with a well-understood mechanism of direct CAR T cell tumor killing, the contribution of the endogenous immune system in this response is currently unclear. Previously, histologic and gene expression analysis of pre- and posttreatment tumor biopsies from pts who received liso-cel in TRANSCEND NHL 001 showed a relationship between tumor-infiltrating CAR-negative (endogenous) T cells and early and durable response [Reiss et al. Blood 2019;134(suppl 1):202]. Here we examine, by gene expression and flow cytometry, post-infusion characteristics of liso-cel and peripheral endogenous T cells in pts from TRANSCEND NHL 001 with early (CR at 1 or 3 months) and long-term (CR at 9 or 12 months) response versus pts with progressive disease (PD) before or at the same time points. Methods: CAR and endogenous T cells were assayed using a novel cell-sorting and low-input RNA-seq method. CAR and endogenous T cells were isolated from peripheral blood mononuclear cell samples from 39 pts at time to maximum concentration of liso-cel (T max; Day 8-22), 1 month, and 2 months after liso-cel infusion. In addition, samples from 44 pts were assayed only at T max by RNA-seq. A subset of these samples was assessed by flow cytometry for expression of the T cell differentiation markers CD4, CD8, CD45RA, CCR7, CD27, and CD28. All RNA-seq analyses were performed in R version 4.0.2. Differential expression analysis was performed with the DESeq2 package with gene set enrichment analysis using clusterProfiler and GSVA packages with gene sets obtained from MSigDB. Comparisons of 2 groups were made with a Wilcoxon rank sum test. Association with the time-to-event outcome of progression-free survival (PFS) was assessed using Cox proportional hazards model or log rank test. Results: Characteristics consistent with less differentiated CAR T cells and endogenous T cells were significantly associated with early and long-term response. Elevated CAR T cell expression of CD27 at T max was associated with CR at 1 month and longer PFS. Expression of CCR7 by CAR T cells at 1 and 2 months was associated with CR at 9 months. Additionally, at T max, gene sets associated with T cell effector differentiation and metabolism were enriched in CAR T cells from pts with early relapse (pts with CR vs PD at 3 months). In endogenous T cells, elevated expression of CCR7 and CD28 at T max was associated with early response (CR at 1 and 3 months). Additionally, expression of CCR7 and CD28 in T cells isolated at 1 and 2 months was associated with long-term response (CR at 9 and 12 months). Furthermore, expression of the immune checkpoint receptor LAG3 by endogenous T cells was associated with PD from month 3 to month 12. Conclusions: In summary, the data from our study show the relationship between CAR T cell differentiation status and achieving response over time, and provide new evidence for the involvement of endogenous T cells in the therapeutic efficacy of CAR T cell therapy. Disclosures Thorpe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jiang: Bristol Myers Squibb: Ended employment in the past 24 months. Rytlewski: Adaptive Biotechnologies: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Kostic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kim: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Peiser: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Recombinant CD19 Glycomutant Accurately and Reproducibly Detects CD19-Directed CAR-T Cells By Flow Cytometry

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1724-1724
Author(s):  
Delaney R. Kirchmeier ◽  
Alyssa Sheih ◽  
Salvatore Fiorenza ◽  
Alexandre V. Hirayama ◽  
Cassie Chou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stock Options ◽  
Advisory Board ◽  
Car T Cells ◽  
Scientific Advisory Board ◽  
Car T Cell ◽  
Car T ◽  
Scientific Advisory ◽  
Privately Held

Abstract Background CD19-directed chimeric antigen receptor (CAR)-modified T cell therapy expansion and persistence in peripheral blood has been shown to correlate with responses against B cell malignances (Hay, Blood, 2018; Ayuk, Blood Adv, 2021). CAR-T cell enumeration is typically achieved through quantitative PCR (qPCR) or flow cytometry (FC). qPCR, however, requires techniques that may not be locally validated and, unlike FC, cannot simultaneously address T cell immunophenotype. Existing FC reagents to detect CD19 CAR-T cells are mostly specific for a given CAR sequence or bind non-specifically to the introduced protein (e.g. Protein A). Utilizing glycosylation data from the structure of CD19(Teplykov, Proteins, 2018), we created a stable, biotinylated CD19 ectodomain glycomutant reagent (gmCD19) that binds to the CD19-directed scFv in CAR constructs and can be detected by FC. Methods The gmCD19 ectodomain (amino acids 21-227, N138Q) was expressed in 293 Freestyle™ cells with a C-terminal 6x Histidine-AviTag™ to facilitate expression, purification, FC detection (with fluorophore-bound streptavidin) and enable tetramer formation. We used FC to determine limit of detection (LoD), defined as the percent of detectable CAR-T cells when spiked into peripheral blood mononuclear cells (PBMC) at known concentrations. We used a CD19-directed CAR-T cell that co-expresses truncated EGFR (EGFRt) to benchmark gmCD19 and compare our reagent with commercially available detection reagents. This was quantitated by Pearson correlation coefficient and Bland-Altman measure of bias - defined as the mean of differences between tests on the same sample. We also tested reagent stability by assessing for decrease in mean fluorescence intensity (MFI) and the percent of CAR-T cells detected after multiple freeze-thaw cycles of the gmCD19 reagent. We compared CAR-T cell detection by gmCD19 with qPCR from actual patient samples treated with axicabtagene ciloleucel (axi-cel) and collected into three different anticoagulants. Results gmCD19 detected as few as 0.25% CAR-T cells by FC and was highly correlated with EGFRt expression (r=0.9993, p<0.0001). When benchmarking gmCD19 with EGFRt expression and comparing gmCD19 quantitation with other commercially available CD19 scFv detection reagents (Acro and BioSwan), gmCD19 showed the least bias (0.04 vs -1.225 and 61.66, respectively), and was the only method that demonstrated statistically significant agreement with EGFRt. Following each freeze-thaw cycle of gmCD19, there was no statistically significant decrease in percent of CAR-T cells detected with only a slight decrease in MFI (~2% decrease per cycle). Detection of axi-cel from patient samples was highly correlated with CAR copy number determined by qPCR, regardless of the anticoagulant in which the patient sample was collected (r=0.9387, 0.9849 and 0.9373 for sodium heparin, sodium citrate and EDTA, respectively) with equivalent coefficients of variation (11.0% vs 11.4% for gmCD19 and qPCR, respectively). Conclusion The availability of multiple CD19-directed CAR-T cell products and the importance of monitoring CAR-T cell expansion and persistence in patients undergoing CAR-T cell therapy creates the necessity for an easily applied, stable, and reliable quantitation FC method. We show that our gmCD19 accurately measures CD19-directed CAR-T cells across a variety of CAR-T cell constructs, including commercially available products. Disclosures Sheih: Umoja Biopharma: Current Employment. Fiorenza: Bristol Myers Squibb: Research Funding; Link Immunotherapeutics: Consultancy. Hirayama: Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Honoraria. Chou: Genentech: Current Employment. Gauthier: Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy. Correnti: Link Immunotherapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Turtle: PACT Pharma: Consultancy; Amgen: Consultancy; Asher Bio: Consultancy; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Nektar Therapeutics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; TCR2 Therapeutics: Research Funding; Allogene: Consultancy.

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 Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2776-2776
Author(s):  
Salvatore Fiorenza ◽  
George S. Laszlo ◽  
Tinh-Doan Phi ◽  
Margaret C. Lunn ◽  
Delaney R. Kirchmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Set Domain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

scholarly journals Decade-long remissions of leukemia sustained by the persistence of activated CD4+ CAR T-cells

2021 ◽  
Author(s):  
Jan Joseph Melenhorst ◽  
Gregory M Chen ◽  
Meng Wang ◽  
David . L Porter ◽  
Peng Gao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphocytic Leukemia ◽  
Late Time ◽  
Sustained Remission ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion

The adoptive transfer of T lymphocytes reprogrammed to target tumor cells has demonstrated significant potential in various malignancies. However, little is known about the long-term potential and the clonal stability of the infused cells. Here, we studied the longest persisting CD19 redirected chimeric antigen receptor (CAR) T cells to date in two chronic lymphocytic leukemia (CLL) patients who achieved a complete remission in 2010. CAR T-cells were still detectable up to 10+ years post-infusion, with sustained remission in both patients. Surprisingly, a prominent, highly activated CD4+ population developed in both patients during the years post-infusion, dominating the CAR T-cell population at the late time points. This transition was reflected in the stabilization of the clonal make-up of CAR T-cells with a repertoire dominated by few clones. Single cell multi-omics profiling via Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) with TCR sequencing of CAR T-cells obtained 9.3 years post-infusion demonstrated that these long-persisting CD4+ CAR T-cells exhibited cytotoxic characteristics along with strong evidence of ongoing functional activation and proliferation. Our data provide novel insight into the CAR T-cell characteristics associated with long-term remission in leukemia.

scholarly journals RG6076 (CD19-4-1BBL): CD19-Targeted 4-1BB Ligand Combination with Glofitamab As an Off-the-Shelf, Enhanced T-Cell Redirection Therapy for B-Cell Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-40
Author(s):  
Sylvia Herter ◽  
Johannes Sam ◽  
Claudia Ferrara Koller ◽  
Sarah Diggelmann ◽  
Esther Bommer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Clinical Development ◽  
Bispecific Antibodies ◽  
Publicly Traded ◽  
Humanized Mouse Model ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T

Synthetic T cell redirecting therapies, using chimeric antigen receptor (CAR)-T cells or CD3-bispecific antibodies targeting B-cell surface antigens such as CD19 and CD20, currently in clinical development, are emerging as promising, potential therapeutic approaches for the treatment of non-Hodgkin lymphomas (NHL). CD3-bispecific antibodies and first generation CAR-T cells only provide T cell receptor stimulation, so-called "signal 1", to the redirected T cells, but lack costimulatory, so-called "signal 2", support of those T cells. Agonism of costimulatory receptors on T cells, such as CD28 and/or 4-1BB, can increase the strength and durability of a T cell-mediated response via multiple mechanisms. Co-stimulation can enhance T cell specific cytotoxicity, proliferation, secretion of Th1-polarizing cytokines, recruitment of additional T cells via increased chemokine secretion, T cell metabolic fitness, and resistance to T-cell exhaustion and to activation-induced T-cell death. Indeed, 2nd generation CAR-T cells that incorporate CD28 or 4-1BB co-stimulation have replaced 1st generation ones in clinical development. However, complex manufacturing logistics and the need of specialized clinical centers for the administration of CAR-T cells significantly limit their broad application. In order to provide an off-the-shelf, synthetic T cell redirection approach delivering both signals 1 and 2 to T cells, CD3-bispecific antibodies would need combination with systemically administered T-cell costimulatory agonists. Yet, clinical development of 1st generation costimulatory agonists has not been successful to date due to on-target, off-tumor immune-mediated toxicity, such as hepatotoxicity. To overcome this limitation, we have generated a novel 4-1BB costimulatory agonist, CD19-targeted 4-1BBL (CD19-4-1BBL, RG6076, RO7227166), and are developing it in combination with a potent CD20xCD3 T cell bispecific antibody, CD20-TCB (RG6026 or glofitamab). CD19-4-1BBL consists of a trimeric, human 4-1BBL fused to a monovalent CD19-targeting IgG1 antibody with an engineered Fc region devoid of FcgR binding. As effective agonism of 4-1BB receptor requires crosslinking of more than three receptor units on a T cell, CD19-4-1BBL is systemically inactive unless it binds to CD19 and clusters on the surface of targeted B-cells to hyper-crosslink multiple 4-1BB receptors on redirected T cells. In our off-the-shelf, combination approach, glofitamab binds to CD20 on B-cells and engages CD3 on redirected T cells, providing signal 1 and inducing the expression of 4-1BB on those T cells. CD19-4-1BBL can then target those activated T cells and provide them with signal 2. In preclinical experiments, we show that CD19-4-1BBL can boost glofitamab-mediated cytokine release by activated T cells in healthy donor as well as DLBCL patient-derived PBMCs. Using a human diffuse large B cell lymphoma (DLBCL) tumor-bearing (WSU-DLCL2) fully humanized mouse model, we observed a CD19-4-1BBL dose-dependent, synergistic combination effect with glofitamab, leading to strongly increased T cell accumulation in tumors, tumor growth inhibition and regression. Importantly, CD19-4-1BBL was also able to prevent tumor escape to glofitamab monotherapy at late treatment time points in a fully humanized mouse model bearing large OCI-Ly18 human DLBCL tumors. Glofitamab monotherapy has recently demonstrated encouraging activity in relapsed/refractory NHL patients with reported complete response rates in DLBCL in the same range as those of 2nd generation CAR-T cells that already incorporate both T cell signals 1 and 2. The preclinical data we report here provide a strong rationale for adding CD19-4-1BBL-mediated T cell signal 2 to glofitamab in the clinic to further boost treatment efficacy and deliver an off-the-shelf, enhanced T cell redirection approach alternative to CAR-T cell therapy. CD19-4-1BBL is currently in clinical trials (NCT04077723). Disclosures Herter: Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Sam:Roche Glycart AG:Current Employment.Ferrara Koller:Roche Glycart AG:Current Employment.Diggelmann:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company.Bommer:Roche Glycart AG:Current Employment.Schönle:Roche Glycart AG:Current Employment.Claus:Roche Glycart AG:Current Employment.Bacac:Roche Glycart AG:Current Employment, Patents & Royalties.Klein:Roche:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Umana:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

scholarly journals CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2942-2942
Author(s):  
Christie Ciarlo ◽  
Zach Frye ◽  
Andre DeGroot ◽  
Walter Flores ◽  
Kutlu Elpek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Transduction Efficiency ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Integrating Vector

Abstract Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells. Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg). Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers >1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo. Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer. Disclosures Ciarlo: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. DeGroot: Sana Biotechnology: Current Employment. Flores: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Johnson: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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