scholarly journals Tisagenlecleucel and Axicabtagene Ciloleucel Expansion Kinetics and CAR T Cell Attributes in the Infusion Products Are Early Predictors of Clinical Efficacy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3877-3877
Author(s):  
Cristiana Carniti ◽  
Chiara Monfrini ◽  
Vanessa Aragona ◽  
Martina Magni ◽  
Cristina Vella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Expansion ◽  
B Cell Lymphoma ◽  
Advisory Board ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

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.

Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 651-651 ◽  
Author(s):  
Agne Taraseviciute ◽  
Leslie Kean ◽  
Michael C Jensen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
Cell Expansion ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

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.

scholarly journals P04.01 Immunomonitoring of CD19. CAR T-cells in Large B-Cell Lymphoma- a two-center experience

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A16-A16
Author(s):  
V Blumenberg ◽  
S Völkl ◽  
G Busch ◽  
S Baumann ◽  
M Winkelmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Large B Cell Lymphoma ◽  
Car T ◽  
T Cell Expansion

BackgroundCD19. CAR T-cells for the treatment of relapsed and refractory (r/r) Diffuse Large B-Cell Lymphoma (DLBCL) demonstrated complete responses in 40%-58% of the patients. Recently, others could associate high tumor volume and low CAR T-cell expansion in vivo with poor outcome. We hypothesize, that the expansion and immunphenotype of (CAR) T cells in vivo determine treatment response and depend on patient- and disease associated factors.Materials and MethodsPatients with r/r DLBCL (n=34) were treated with either Axi-cel or Tisa-cel at the University Hospitals of Erlangen and Munich (LMU). The CAR T-cell product and peripheral blood were collected on day 0, 4, 7, 14, 30, 60 and 90 post transfusion. CAR T-cells were detected through flow cytometry utilizing a two step-staining with a biotinylated CD19 protein. Effector:Target (E:T) Ratios were estimated as absolute peak expansion of CAR T-cells (/ul) per tumorvolume (cm3). Responder (R, complete or partial remission) were compared to Non-Responder (NR, stable or progressive disease) according to response assessment with PET-CT three months after transfusion.ResultsCAR T-cell expansion peaked between day 7 and day 14 after transfusion with a greater expansion of CD8+compared to CD8- CAR T-cells on day 14 (59.27% vs 37.42%, p=0.021). The ratio of CD8+ and CD8- CAR T-cells did not differ between R and NR, however R exhibited higher E:T ratios of CD3+ CAR T-cells compared to NR (20.94 vs 12.81, p=0.015) and an increased E:T ratio of CD8+ CAR T-cells correlated with better progression-free survival (p=0.033). Interestingly, high CRP and ferritin levels at baseline were inversely associated with the E:T ratio (p=0.048 and p=0.017). CD3+ CAR T-cells of R showed earlier peak expression of PD-1 than NR (day 7 vs day 21). Further, peak expansion of CD3+ CAR T-cells correlated with higher PD-1 expression in R but not in NR (p=0.003 vs p=0.12). In addition, R revealed an increased relative frequency of effector memory differentiated CD3+ CAR T-cells (CCR7-CD45RA-, p=0.02), whereas CAR T-cells in NR showed an increased relative frequency of a naïve phenotype (CCR7+CD45RA+, p=0.001) on day 7 post infusion.ConclusionsFlow-based immunomonitoring with longitudinal characterization of CAR T-cells demonstrated a correlation of the E:T ratio with treatment response and survival. Increased inflammatory conditions at baseline correlated with diminished E:T ratios. Notably, in R CAR peak expansion was positively associated with higher PD-1 expression suggestive for superior CAR T-cell activation. In addition, greater memory differentiation was associated with efficacy during the time of peak expansion. Multiparameter analysis with other clinical covariates will show, whether CAR T-cell expansion and immunphenotypes can predict patient outcome.Disclosure InformationV. Blumenberg: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Novartis, Gilead Sciences, Janssen, BMS/Celgene. S. Völkl: None. G. Busch: None. S. Baumann: None. M. Winkelmann: None. B. Tast: None. D. Nixdorf: None. G. Hänel: None. L. Frölich: None. C. Schmidt: None. R. Jitschin: None. F. Vettermann: None. W. Kunz: None. D. Mougiakakos: None. M. von Bergwelt: None. V. Bücklein: None. A. Mackensen: None. M. Subklewe: None.

scholarly journals Understanding Outcomes of CD19-Targeted CAR T Cell Immunotherapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-14-SCI-14
Author(s):  
Cameron J. Turtle
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Advisory Board ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Counts ◽  
Car T ◽  
Peak Car ◽  
T Cell Expansion

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in patients with relapsed and/or refractory B cell malignancies, but factors that impact toxicity and efficacy have been difficult to define because of heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 ratio of CD4+:CD8 + CAR-T cells to adults with CD19+ acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL) after lymphodepletion chemotherapy. The defined composition product was remarkably potent, with bone marrow CR rates exceeding 90% in B-ALL patients, and overall response rates of 70-80% in NHL and CLL patients. In vivo CAR-T cell expansion and persistence were enhanced by the combination of cyclophosphamide (Cy) and fludarabine (Flu) lymphodepletion compared to Cy-based regimens without Flu, and were associated with better response. The enhanced CAR-T cell expansion and persistence in NHL patients receiving Cy/Flu lymphodepletion was due in part to an increase in available homeostatic cytokines, such as IL-15, and in B-ALL and NHL patients abrogation of anti-CAR transgene immune responses, which promoted CAR-T cell rejection and early relapse in a subset of patients. The depth of response appeared important in CLL, in which we found that absence of the malignant IGH clone in marrow of patients who responded by IWCLL imaging criteria was associated with longer progression-free survival (PFS) after CAR-T cell infusion compared to those in whom the malignant IGH clone was detected. CD19 CAR-T cell immunotherapy can be complicated by cytokine release syndrome (CRS), neurotoxicity, and B cell depletion. Endothelial activation was observed in patients with severe CRS and neurotoxicity, which may account for clinical manifestations, including vascular instability, capillary leak, and blood-brain barrier permeability. In B-ALL, NHL and CLL patients, we found that more severe CRS and neurotoxicity were associated with factors that increase CAR-T cell expansion, resulting in higher concentrations of distinct cytokines in serum after infusion. In B-ALL, CAR-T cell expansion and the risk of toxicity were also higher in patients with high tumor burden. Probability curves defined the likelihood of CRS, neurotoxicity or response in each disease cohort according to in vivo peak CAR-T cell concentrations in blood. The data indicate that a therapeutic window between CR and CRS or neurotoxicity can be defined in B-ALL within a distinct range of peak CAR-T cell counts. Reduction of the infused CAR-T cell dose in those with high marrow burden minimized the risk of high peak CAR-T cell counts that were associated with an increased risk of toxicity. In NHL patients, probability curves indicated that CAR-T cell dose reduction to reduce toxicity could be associated with loss of anti-tumor efficacy, suggesting that CAR-T cell dosing at the maximum tolerated dose combined with early intervention strategies in patients at high risk of CRS or neurotoxicity might be a suitable strategy to minimize toxicity while maintaining efficacy. Using a classification-tree algorithm we identified clinical and serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. These data will inform strategies that facilitate safe and effective clinical application of CD19 CAR-T cell therapy. Disclosures Turtle: Juno Therapeutics: Other: Advisory board, Patents & Royalties, Research Funding; Celgene: Other: Advisory board; Precision Biosciences: Other: Advisory board; Adaptive Biotechnologies: Other: Advisory board; Bluebird Bio: Other: Advisory board; Gilead Sciences: Other: Advisory board.

Characterization of HLH-Like Manifestations as a CRS Variant in Patients Receiving CD22 CAR T-Cells

Blood ◽  
2021 ◽  
Author(s):  
Daniel A Lichtenstein ◽  
Fiorella Schischlik ◽  
Lipei Shao ◽  
Seth M Steinberg ◽  
Bonnie Yates ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Nk Cell ◽  
Severe Neutropenia ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

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.

scholarly journals CD229 CAR T Cell Therapy for the Treatment of Relapsed B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2800-2800
Author(s):  
Michael Olson ◽  
Tim Luetkens ◽  
Fiorella Iglesias ◽  
Sabarinath Radhakrishnan ◽  
Jennie Y. Law ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract B cell lymphoma is the most common hematologic malignancy in the United States. Although treatment options have greatly improved in the past several decades, outcomes for patients with relapsed B cell lymphoma remain poor. Chimeric antigen receptor (CAR) T cells have recently entered the clinic with promise to address the gap in effective therapies for patients relapsed B cell lymphoma. However, antigen loss and poor CAR T cell persistence has been shown to drive resistance to the widely approved CD19-targeted CAR in some patients, demonstrating the need for additional therapies. Here, we demonstrate CD229-targeted CAR T cell therapy as a promising option for the treatment of relapsed B cell lymphoma, addressing an important group of patients with typically poor outcomes. CD229 is an immune-modulating receptor expressed on the surface of B cells that we recently found to be highly expressed in the plasma cell neoplasm multiple myeloma (Radhakrishnan et al. 2020). We utilized semi-quantitative PCR and flow cytometry to assess whether CD229 is also expressed on malignant B cells earlier in development as found in B cell lymphoma. Expression analysis revealed the presence of CD229 in a panel of 11 B cell lymphoma cell lines and 45 primary B cell lymphoma samples comprising several subsets of disease including aggressive B cell lymphomas such as diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and Burkitt lymphoma as well as indolent subtypes of B cell lymphoma including chronic lymphoblastic leukemia (CLL) and follicular lymphoma. Of note, CD229 was found to be overexpressed on primary B cell lymphoma cells when compared to autologous normal B cells. Given the high levels of CD229 expression throughout all B cell lymphoma subtypes analyzed, we generated CD229 CAR T cells in order to determine whether CAR T cell therapy is an effective way to target CD229 expressing B cell lymphoma cells. CD229 CAR T cells exhibited robust cytotoxicity when cocultured with B cell lymphoma cell lines and primary samples characterized by significant production of TH1 cytokines IL-2, TNF and IFNγ and rapid loss of B cell lymphoma cell viability when compared to control CAR T cells lacking an antigen binding scFv domain (∆scFv CAR T cells). In vivo analysis revealed effective tumor control in NSG mice carrying B cell lymphoma cell lines JeKo-1 (MCL) and DB (DLBCL) when treated with CD229 CAR T cells versus ∆scFv CAR T cells. Finally, we sought to determine the efficacy of CD229 CAR T cells in the context of CD19 CAR T cell therapy relapse. Here, a 71-year-old patient with CLL had an initial response when treated with CD19 CAR T cells but quickly relapsed only 2 months after treatment. Malignant cells from the CLL patient retained CD229 expression as identified by flow cytometry and an ex vivo coculture with CD229 CAR T cells revealed robust killing of CLL cells by CD229 CAR T cells. Transfer of antigen from target cell to CAR T cell by trogocytosis was recently suggested to drive relapse following CAR T cell therapy by decreasing antigen on tumor cells and promoting CAR T cell fratricide (Hamieh et al. 2019). We cocultured CD19 and CD229 CAR T cells with primary CLL cells and assessed CD19 and CD229 expression as well as CAR T cell viability by flow cytometry. In contrast with CD19 CAR T cells, CD229 CARs did not strip their target antigen from the surface of CLL cells. The transfer of CD19 from CLL cells to CD19 CAR T cells resulted in poor CAR T cell viability while CD229 CAR T cell viability remained high following coculture. In summary, we demonstrate that CD229 is a promising therapeutic target in B cell lymphoma due to its high levels of expression throughout many subtypes of disease. CD229 CAR T cells effectively kill B cell lymphoma cells in vitro and control growth of aggressive B cell lymphomas in vivo. Finally, CD229 CAR T cells are effective against primary CLL cells from patients that have relapsed from CD19 CAR T cell therapy and do no exhibit antigen loss by trogocytosis. Taken together, these data suggest that CD229 CAR T cell therapy may be a promising option to address the poor outcomes for patients with relapsed B cell lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fatal late-onset CAR T-cell–mediated encephalitis after axicabtagene-ciloleucel in a patient with large B-cell lymphoma

2021 ◽  
Vol 5 (19) ◽  
pp. 3789-3793
Author(s):  
Susanne Jung ◽  
Jochen Greiner ◽  
Stephanie von Harsdorf ◽  
Pavle Popovic ◽  
Roland Moll ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Large B Cell Lymphoma ◽  
Car T ◽  
Large B Cell

Abstract Treatment with CD19-directed (CAR) T cells has evolved as a standard of care for multiply relapsed or refractory large B-cell lymphoma (r/r LBCL). A common side effect of this treatment is the immune effector cell–associated neurotoxicity syndrome (ICANS). Severe ICANS can occur in up to 30% to 40% of patients treated with axicabtagene-ciloleucel (axi-cel), usually within the first 4 weeks after administration of the dose and usually responding well to steroids. We describe a case of progressive central neurotoxicity occurring 9 months after axi-cel infusion in a patient with r/r LBCL who had undergone a prior allogeneic hematopoietic cell transplant. Despite extensive systemic and intrathecal immunosuppression, neurological deterioration was inexorable and eventually fatal within 5 months. High CAR T-cell DNA copy numbers and elevated levels of interleukin-1 (IL-1) and IL-6 were found in the cerebral spinal fluid as clinical symptoms emerged, and CAR T-cell brain infiltration was observed on autopsy, suggesting that CAR T cells played a major pathogenetic role. This case of unexpected, devastating, late neurotoxicity warrants intensified investigation of neurological off-target effects of CD19-directed CAR T cells and highlights the need for continuous monitoring for late toxicities in this vulnerable patient population.

scholarly journals Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

2021 ◽  
Vol 12 ◽  
Author(s):  
Ulrich Blache ◽  
Ronald Weiss ◽  
Andreas Boldt ◽  
Michael Kapinsky ◽  
André-René Blaudszun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cytotoxic Agents ◽  
Clinical Staging ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

scholarly journals Case Report: Sirolimus Alleviates Persistent Cytopenia After CD19 CAR-T-Cell Therapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Limin Xing ◽  
Yihao Wang ◽  
Hui Liu ◽  
Shan Gao ◽  
Qing Shao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
B Cell Lymphoma ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor T (CAR-T) cells show good efficacy in the treatment of relapsed and refractory B-cell tumors, such as acute B-cell leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). The main toxicities of CAR-T include cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, cytopenia, and severe infection. It is still very difficult for CAR-T to kill tumor cells to the maximum extent and avoid damaging normal organs. Here, we report a case of DLBCL with persistent grade 4 thrombocytopenia and severe platelet transfusion dependence treated with CD19 CAR-T cells. We used sirolimus to inhibit the sustained activation of CAR-T cells and restore normal bone marrow hematopoiesis and peripheral blood cells. Moreover, sirolimus treatment did not affect the short-term efficacy of CAR-T cells, and DLBCL was in complete remission at the end of follow-up. In conclusion, sirolimus can represent a new strategy for the management of CAR-T cell therapy-related toxicity, including but not limited to hematotoxicity. However, further controlled clinical studies are required to confirm these findings.

The effect of pembrolizumab in combination with CD19-targeted chimeric antigen receptor (CAR) T cells in relapsed acute lymphoblastic leukemia (ALL).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 103-103 ◽  
Author(s):  
Shannon L. Maude ◽  
George E Hucks ◽  
Alix Eden Seif ◽  
Mala Kiran Talekar ◽  
David T. Teachey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Lymphoblastic Leukemia ◽  
Prior History ◽  
Cell Detection ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

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.

Long-term follow-up of anti-CD19 CAR T-cell therapy for B-cell lymphoma and chronic lymphocytic leukemia.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3012-3012 ◽  
Author(s):  
Kathryn Cappell ◽  
Richard Mark Sherry ◽  
James C. Yang ◽  
Stephanie L. Goff ◽  
Danielle Vanasse ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Long Term Adverse Effects

3012 Background: T cells expressing anti-CD19 chimeric antigen receptors (CARs) can cause complete remissions of relapsed lymphoma. We conducted the first clinical trial of anti-CD19 CAR T cells to show responses against lymphoma. This CAR was later developed as axicabtagene ciloleucel. Here, we aimed to assess the long-term durability of remissions and the long-term adverse effects after anti-CD19 CAR T-cell therapy. Methods: Between 2009 and 2015, we treated 43 patients with anti-CD19 CAR T cells preceded by conditioning chemotherapy of cyclophosphamide plus fludarabine (NCT00924326). Three patients were re-treated for a total of 46 CAR T-cell treatments. Twenty-eight patients had aggressive lymphoma (diffuse large B-cell lymphoma or primary mediastinal B cell lymphoma), eight patients had low-grade lymphoma (five with follicular lymphoma and 1 each with splenic marginal zone lymphoma, mantle cell lymphoma, and unspecified low-grade non-Hodgkin lymphoma), and seven patients had chronic lymphocytic leukemia (CLL). Patients were treated in three cohorts that differed in the CAR T-cell production process and conditioning chemotherapy dose. Results: Of the 43 treated patients, 63% had chemotherapy-refractory lymphoma. Patients had received a median of 4 previous lines of therapy. The median CAR+ T cell dose per kilogram was 2X10^6. The overall remission rate was 76% with 54% complete remissions (CR) and 22% partial remissions (PR). Patients with CR had higher median peak blood CAR levels (86 CAR+ cells/µL) than those who did not have CR (16 CAR+ cells/µL, P= 0.0041). Long-term adverse effects were rare except for B-cell depletion and hypogammaglobulinemia, which both improved over time. Conclusions: This is the longest follow-up study of patients who received anti-CD19 CAR T cells. Anti-CD19 CAR T cells cause highly durable remissions of relapsed B-cell lymphoma and CLL, and long-term adverse effects of anti-CD19 CAR T cells were rare and usually mild. Clinical trial information: NCT00924326 . [Table: see text]

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