CAR T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T-cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Amongst 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase and occasionally hemophagocytosis. Development of carHLH was associated with pre-infusion NK-cell lymphopenia and higher bone marrow T/NK-cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK-cell lymphopenia amplified pre-infusion differences in those with carHLH without evidence for defects in NK-cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.
Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma
Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; 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; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.
103 Background: CD19-targeted CAR T cells show CR rates of 70-95% in B-ALL. Yet a subset of patients do not respond or relapse due to poor CAR T cell expansion and persistence. We hypothesized that PD-1 checkpoint pathway inhibition may improve CAR T cell expansion, function and persistence. Methods: Four children with relapsed B-ALL treated with murine (CTL019) or humanized (CTL119) anti-CD19 CAR T cells received 1-3 doses of the PD-1 inhibitor pembrolizumab (PEM) for partial/no response or prior history of poor CAR T cell persistence starting 14d-2mo post CAR T cell infusion. Results: PEM increased and/or prolonged detection of circulating CAR T cells in all 4 children, with objective responses in 2/4. It was well tolerated, with fever in 2 pts and no autoimmune toxicity. Pts 1-3 received CTL119 for CD19+ relapse after prior murine CD19 CAR T cells. Pt 1 had 1.2% CD19+ residual disease despite expansion with detectable CTL119 by D28 and received PEM at 2mo for progressive disease with decreasing circulating CTL119. CTL119 became detectable at 0.2% of CD3+ cells by flow cytometry, but disease progressed. Pt 2 had no response after initial CTL119 expansion with a rapid disappearance by D28. After CTL119 reinfusion with PEM added 14d later, circulating CAR T cells remained detectable at 4.4% by D28, but disease progressed with decreased CD19 expression. In Pt 3, prior treatment with both CTL019 and CTL119 produced CR with poor CAR T cell persistence followed by CD19+ relapse. CTL119 reinfusion combined with PEM at D14 resulted in CR with prolonged CTL119 persistence (detectable at D50 compared to loss by D36 after 1st CTL119 infusion). Pt 4 received PEM for widespread extramedullary (EM) involvement at D28 post CTL019 infusion despite marrow remission. Initial CTL019 expansion peaked at 63% at D10 and fell to 20% at D28. Resurgence of CTL019 expansion, with a 2nd peak of 70% 11d after PEM, was associated with dramatic reduction in PET-avid disease by 3mo post CTL019. Conclusions: PEM was safely combined with CAR T cells and increased or prolonged CAR T cell detection, with objective responses seen. Immune checkpoint pathways may impact response to CAR T cell treatments and warrant further investigation. Clinical trial information: NCT02374333, NCT02906371.
Abstract BACKGROUND: Chemotherapy followed by autologous T cells that are genetically modified to express a CD19-specific chimeric antigen receptor (CAR) has shown promise as a novel therapy for patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL); however, the risk of severe cytokine release syndrome (sCRS) and neurotoxicity has tempered enthusiasm for widespread application of this approach. The functional heterogeneity that is inherent in CAR-T cell products that are manufactured from undefined T cell subsets has hindered definition of dose-response relationships and identification of factors that may impact efficacy and toxicity. METHODS: We are conducting the first clinical trial that administers CD19 CAR-T cells manufactured from a defined composition of T cell subsets to adults with relapsed or refractory B-ALL. CD8+ and CD4+ T cells were enriched from each patient, transduced with a CD19 CAR lentivirus and separately expanded in vitro before formulation for infusion in a 1:1 ratio of CD8+:CD4+ CAR+ T cells at 2x105, 2x106 or 2x107 CAR-T cells/kg. Prior to CAR-T cell infusion, patients underwent lymphodepletion with a high-dose cyclophosphamide (Cy)-based regimen with or without fludarabine (Flu). RESULTS: Twenty-nine adults with B-ALL (median age 40, range 22 - 73 years; median 17% marrow blasts, range 0 - 97%), including 10 patients who had relapsed after allogeneic transplantation, received at least one CAR-T cell infusion. Twenty-four of 26 restaged patients (92%) achieved bone marrow (BM) complete remission (CR) by flow cytometry. CD4+ and CD8+ CAR-T cells expanded in vivo after infusion and their number in blood correlated with the infused CAR-T cell dose. Thirteen patients received lymphodepletion with Cy-based regimens without Flu. Ten of 12 restaged patients (83%) achieved BM CR by flow cytometry; however, 7 of these (70%) relapsed a median of 66 days after CAR-T cell infusion. Disease relapse correlated with a loss of CAR-T cell persistence in blood. We observed a CD8 cytotoxic T cell response to the murine scFv component of the CAR transgene that contributed to CAR-T cell rejection, and resulted in lack of CAR-T cell expansion after a second CAR-T cell infusion in 5 patients treated for persistent or relapsed disease. To minimize immune-mediated CAR-T cell rejection 14 patients were treated with Cy followed by Flu lymphodepletion (Cy/Flu, Cy 60 mg/kg x 1 and Flu 25 mg/m2 x 3-5) before CAR-T cell infusion. All patients (100%) who received Cy/Flu lymphodepletion achieved BM CR after CAR-T cell infusion. CAR-T cell expansion and persistence in blood was higher in Cy/Flu-lymphodepleted patients compared to their counterparts who received Cy alone (Day 28 after 2x106 CAR-T cells/kg: CD8+ CAR-T cells, mean 55.8/μL vs 0.10/μL, p<0.01; CD4+ CAR-T cells, 2.1/μL vs 0.02/μL, p<0.01), enabling reduction in CAR-T cell dose for Cy/Flu-treated patients. Patients who received Cy/Flu lymphodepletion appear to have longer disease-free survival (DFS) than those who received Cy alone (Cy/Flu, median, not reached; Cy alone, 150 days, p=0.09). CAR-T cell infusion was associated with sCRS, characterized by fever and hypotension requiring intensive care in 7 of 27 patients (26%) and neurotoxicity (≥ grade 3 CTCAE v4.03) in 13 of 27 patients (48%). Two patients died following complications of sCRS. Patients with sCRS or neurotoxicity had higher peak serum levels of IL-6, IFN-γ, ferritin and C-reactive protein compared to those without serious toxicity. Importantly IL-6, IFN-γ and TNF-α levels in serum collected on day 1 after CAR-T cell infusion from those who subsequently developed neurotoxicity were higher than those collected from their counterparts who did not develop neurotoxicity (IL-6, p<0.01; IFN-γ, p=0.05; TNF-α, p=0.04), providing potential biomarkers to test early intervention strategies to prevent neurotoxicity. The risks of sCRS and neurotoxicity correlated with higher leukemic marrow infiltration and increasing CAR-T cell dose. We have now adopted a risk-stratified approach to CAR-T cell dosing in which the CAR-T cell dose inversely correlates to the patient's bone marrow tumor burden. CONCLUSION: Risk-stratified dosing of CD19 CAR-T cells of defined subset composition is feasible and safe in a majority of patients with refractory B-ALL, and results in a CR rate of 92%. Addition of Flu to Cy-based lymphodepletion improves CAR-T cell expansion, persistence and DFS. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding.
Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy.
Abstract Background: CD19-directed CAR-T cell therapy has shown promising efficacy in relapsed/refractory (R/R) B-cell malignancies in clinical trials resulting in the approval and commercialization of two products (tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel) for R/R diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). However, relapses occur in 60-65% of patients (pts) and thus a better understanding of the early determinants of response is critical to improve long-term survival in the real-world scenario. Aims of the study: To assess whether CAR-T cell expansion after infusion represents a crucial determinant to sustain effective anti-tumor responses to both Tisa-cel and Axi-celTo evaluate differences in CAR-T cell kinetics due to the use of CD28 or 4-1BB costimulatory moleculesTo identify immune phenotypic features of infusion products accounting for CAR-T cell expansion and survival probability Methods: We analyzed samples from 43 pts [29 DLBCL, 8 high grade B-cell lymphoma (HGBCL) and 6 PMBCL] treated with Axi-cel (n=22) and Tisa-cel (n=21) at the Fondazione IRCCS Istituto Nazionale Tumori prospectively collected between November 2019 and April 2021. CAR-T cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 10, 14, 21, 28 and monthly post infusion by flow cytometry (FCM). Cells were stained with CD19 CAR Detection Reagent (Miltenyi), CD3, CD4, CD8, CD45, CD14, CD45RO, CD62L, CD197, CD279, CD223 and CD366. Residual cells obtained from washings of 32 infused commercial CAR-T bags (10 Tisa-cel and 22 Axi-cel) were also analyzed by FCM. Data were acquired on a BD FACSCanto II (BD Biosciences) and a MACSQuant® Analyzer MQ10 (Miltenyi) and analyzed using FlowJo software, version 10. Results: The median time to maximal expansion of CAR-T cells was at day 10 post infusion with no differences between Axi-cel and Tisa-cel [median concentration at day 10 (C 10) 25 for Axi-cel vs 26 CAR-T cells/µl for Tisa-cel; p, ns], nor among the different histologies (median C 10 33 for DLBCL vs 19 for HGBCL vs 18 CAR-T cells/µl for PMBCL; p, ns). On the contrary, CAR-T peak concentration (C max) was higher in responders at 3 months post infusion (RE, n=28) (defined as pts achieving complete or partial response by PET/CT) than in non responders (NR, n=13) (median C max 87 in RE vs 26 in NR CAR-T cells/µl; p<0.01; Fig 1A). Consistently, the magnitude of CAR-T cell expansion in the first 30 days was higher in RE than in NR [median area under the curve (AUC 0-30) 189 vs 50; p<0.005; Fig 1B]. Circulating CAR-T cells were enriched in subpopulations representing naïve T cells (CD8+ T N; CD45RO−/CD62L+) in RE (median 0.4% in RE vs 0.04% in NR, p<0.05) while NR had significantly higher levels of effector memory T cells (CD8+ T EM; CD45RO+/CD62L+) (median 26.5% in RE vs 66.2% in NR, p<0.05). Additionally, the extent of CAR-T cell expansion predicted the progression free survival (PFS), but not the overall survival (OS), irrespective of the product used (Fig 2, p<0.05) and the overall survival was improved by salvage treatment with bispecifc antibodies. Finally, we evaluated whether CAR-T cell expansion was influenced by the immune phenotypic attributes of the infused products. A significant enrichment of central memory populations (CD8+ T CM; CD45RO−/CCR7+/CD62L+) among CAR-T cells within the infusion products of pts with longer PFS was documented, as compared with those with shorter PFS (CD8+ T CM; median 15.2% vs 3.1%; p<0.005). Conclusion: To the best of our knowledge, this is the first study assessing the clinical utility of early CAR-T cell monitoring in lymphoma pts receiving both commercial anti-CD19 CAR-T cell therapies. We provide evidence that in pts treated with Axi-cel and Tisa-cel: i) the in vivo kinetics of the CAR-T cell products are similar, consistent with the fact that no differences in the outcome of Axi-cel and Tisa-cel treated pts were detected; ii) CAR-T cell expansion is critical for efficacy and predicts the PFS; iii) circulating CAR-T cells in responders have a naïve phenotype; iv) a memory signature in the CAR-T cell product before infusion is associated with in vivo expansion and survival. Figure 1 Figure 1. Disclosures Chiappella: Celgene Bristol Myers Squibb: Other: lecture fee, advisory board; Incyte: Other: lecture fee; Novartis: Other: lecture fee; Astrazeneca: Other: lecture fee; Servier: Other: lecture fee; Takeda: Other: advisory board; Gilead Sciences: Other: lecture fee, advisory board; Clinigen: Other: lecture fee, advisory board; Roche: Other: lecture fee, advisory board; Janssen: Other: lecture fee, advisory board. Corradini: AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; BMS: Other: Travel and accommodation; Sanofi: Consultancy, Honoraria; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; Incyte: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations.
Abstract The advent ofadoptive T-cell therapy using CD19 Chimeric Antigen Receptor (CAR) T cells has revolutionized the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). CAR T cells have shown encouraging results in clinical trials, with complete remissions in 90% of patients with refractory B-cell ALL. However, CD19 CAR T cell therapy is associated with significant side effects, including cytokine release syndrome (CRS), encompassing fevers, myalgias, hypotension, respiratory distress, coagulopathy as well as neurologic toxicity, ranging from headaches to hallucinations, aphasia, seizures and fatal cerebral edema. Our understanding of CRS and neurologic toxicity has been significantly limited by the lack of animal models that faithfully recapitulate these symptoms. We chose the non-human primate (NHP), Macaca mulatta, given that it closely recapitulates the human immune system, to create an animal model of B-cell-directed CAR T cell therapy targeting CD20. Rhesus macaques (n=3) were treated with 30-40mg/kg cyclophosphamide followed 3-6 days later by an infusion of CAR T cells at a dose of 1x107 transduced cells/kg. Recipient animals were monitored for clinical signs and symptoms of CRS and neurotoxicity, and data were collected longitudinally to determine CAR T cell expansion and persistence, B cell aplasia, as well as clinical labs of CRS and cytokine levels. Prior to testing the CD20 CAR T cells, we performed a control experiment, in which 1x107/kg control T cells, transduced to express GFP only (without a CAR construct), were infused following cyclophosphamide conditioning. This infusion resulted in short-lived persistence of the adoptive cellular therapy, with disappearance of the cells from the peripheral blood by Day +14 (Figure 1, green traces) and no clinical signs of CRS (Figure 2) or neurologic toxicities. In contrast, recipients of 1x107 cells/kg CD20 CAR-expressing T cells (n = 3) demonstrated significant expansion of the CAR T cells, and persistence for as long as 43days post-infusion, which corresponded to concurrent B cell aplasia (Figure 1). These recipients also developed clinical signs and symptoms of CRS as well as neurologic toxicity which was manifested by behavioral abnormalities and extremity tremors, beginning between days 5 to 7 following CAR T cell infusion, with the onset of clinical symptoms coinciding with maximum CAR T cell expansion and activation. The neurologic symptoms were responsive to treatment with the anti-epileptic medicationlevetiracetam. The clinical syndrome was accompanied by elevations in CRP, Ferritin, LDH and serum cytokines, including IL-6, IL-8 and ITAC (Figure 2 A and B), recapitulating data from clinical trials using CD19 CAR T cells. An expansion of CD20 CAR T cells on day 7 following infusion was also observed in the CSF in the animals, and coincided with the onset of neurotoxicity. Strikingly, we also detected CD20 CAR T cells in multiple regions of the brain via flow cytometry, including the frontal, parietal, and occipital lobes, as well as the cerebellum, and demonstrated an increased number of infiltrating T cells by immunofluorescence in the brains of animals treated with CD20 CAR T cells when compared to healthy controls. These data demonstrate the successful establishment of a large animal model of B-cell directed CAR T cell therapy that recapitulates the most significant toxicities of CAR T cell therapy, including CRS and neurotoxicity. This model will permit a detailed interrogation of the mechanisms driving these toxicities as well as the pre-clinical evaluation of therapies designed to prevent or abort them after CAR T cell infusion. Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Disclosures Kean: Juno Therapeutics, Inc: Research Funding. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.
122 Background: JCAR017 is a defined composition, CD19-directed 4-1BB CAR T cell product administered at a precise dose of CD8 and CD4 CAR T cells in a seamless design Ph1 pivotal trial of R/R B-cell NHL (TRANSCEND NHL 001; NCT02631044). Methods: Blood samples were collected for biomarker analyses at protocol-defined time points. PK (CAR T cell expansion and persistence) was measured using flow cytometry. Cytokines were measured on a Luminex platform. Additional analytes will be presented. All reported p-values are 2-sided without multiplicity adjustment. Results: Safety (n = 59) and efficacy (n = 54) outcomes were analyzed for correlations with patient (pt) characteristics and biomarkers. Dose level did not correlate with cytokine release syndrome (CRS) or neurotoxicity (NT) despite higher median Cmax and median AUC0-28 at DL2. In pts with NT or ≥Gr 2 CRS, CD4 and CD8 CAR T cell levels were 5-10 fold and 3-5 fold higher, respectively, than median DL2 levels. Pt factors that correlated with any grade CRS and NT were ECOG 2 (p = 0.03) and high disease burden (p < 0.05). Higher levels of IL-8, IL-10, and CXCL10 before CART cell infusion were associated with Gr 3-4 NT (each p< 0.05), suggesting that inherent pt factors may result in higher CAR T expansion and associated CRS and NT. Lower pre-CAR T cell ferritin, LDH, CXCL10, G-CSF, and IL-10 were associated with CR/PR, and lower pre-CAR T cell ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β, TNF-α were associated with 3-month durable response (each p< 0.05). Median Cmax and AUC0-28 of CD8 CAR T cells were higher in responding patients and with durable response at Month 3 (CD8 Cmax median = 20.8 vs 5.5; CD8 AUC median = 235 vs 55 in CR/PR vs PD at Month 3). Of pts evaluable for persistence at 3 months (n = 29), 90% and 93% had detectable CD8+ and CD4+ CAR+ T cells; of those with available PK results at time of relapse (n = 11), 82% had persistence at time of relapse. Conclusions: JCAR017 demonstrated increased CAR T cell expansion and persistence and higher durability of response at higher dose levels, with manageable toxicities. CAR T cells were also detected at time of relapse, suggesting potential opportunities for future combination clinical trials. Clinical trial information: NCT02631044.
Adoptive immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) is a promising approach to improve outcomes for cancer patients. While CAR T cell therapy is effective for hematological malignancies, there is a need to improve the efficacy of this therapeutic approach for patients with solid tumors and brain tumors. At present, several approaches are being pursued to improve the antitumor activity of CAR T cells including i) targeting multiple antigens, ii) improving T cell expansion/persistence, iii) enhancing homing to tumor sites, and iv) rendering CAR T cells resistant to the immunosuppressive tumor microenvironment (TME). Augmenting signal 3 of T cell activation by transgenic expression of cytokines or engineered cytokine receptors has emerged as a promising strategy since it not only improves CAR T cell expansion/persistence but also their ability to function in the immunosuppressive TME. In this review, we will provide an overview of cytokine biology and highlight genetic approaches that are actively being pursued to augment cytokine signaling in CAR T cells.
