scholarly journals Monocytes Are Required for Both Optimal Anti-Leukemic Efficacy and the Cytokine Release Syndrome By CAR-T Cells: Lessons from an Innovative Xenotolerant Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 997-997
Author(s):  
Margherita Norelli ◽  
Monica Casucci ◽  
Barbara Camisa ◽  
Laura Falcone ◽  
Catia Traversari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Secondary Transfer

Abstract Background: Chimeric antigen-receptor (CAR)-engineered T cells promise to cure chronic and acute leukemias refractory to standard treatments. Before this promise is fulfilled, however, two crucial issues need to be solved: i) how to circumvent the emergence of secondary resistance (e.g. due totarget-antigen loss; leukemic lineage switch); ii) how to manage associated toxicities (e.g. the cytokine release syndrome, CRS; lineage aplasias). Unfortunately, all these issues cannot be addressed pre-clinically in currently available NSG mouse models, because they lack human hematopoiesis and, furthermore, ultimately develop xenograft-versus-host disease (X-GVHD), preventing the evaluation of long-term effects. Methods: We have developed an innovative xenotolerant model by transplanting human hematopoietic stem cells (HSCs) intraliver in newborn NSG mice triple transgenic for human SCF, GM-SCF and IL-3 (SGM3). Differently from "classical" NSG, SGM3 mice reconstituted high levels of human T cells (>1000 cells per microL at week 8), which, once transferred in secondary recipients, persisted up to 200d without causing X-GVHD, even after irradiation. Robust and specific xenotolerance was confirmed by in vitrohyporesponsiveness to NSG, bot not to C57/Bl6 antigens (irradiated splenocytes) or human HLAs (PBMCs). Secondary transfer experiments in leukemic and/or HSC-humanized SGM-3 mice have been then designed for studying the determinants of CAR-T cell efficacy and associated toxicities in the absence of confounding xenoreactivity. Results: SGM3-derived T cells were transduced ex vivo with either a CD19 or a CD44v6 CAR (both having a CD28 2G design) after activation with CD3/CD28-beads and IL-7/IL-15, resulting in a preferential and functional CD45RA+/CD62L+/CD95+ stem memory T cell (TSCM) phenotype. Once transferred in secondary recipients previously engrafted with a CD19+/CD44v6 leukemic cell line, CD19 or CD44v6 CAR-T cells equally mediated rapid tumor clearance both in low and high tumor-burden settings, in the absence of malaise or elevated human IL-6 levels in vivo. At later time points (after 100d), however, approximately 50% of responding mice relapsed despite significant CAR-T cell persistence in vivo (>50 cells per microL). A significant fraction of leukemia relapses were characterized by post-transcriptional down-regulation of CD44v6 expression or CD19 loss, respectively. Conversely, secondary transfer of SGM3-derived CAR-T cells in leukemic SGM3 mice that had been previously humanized with HSCs resulted in the development of a clinical syndrome similar to the CRS observed in clinical trials (high fevers, elevated IL-6, TNF-alpha and serum amyloid A levels - mouse analog of C-reactive protein in humans), resulting in 30% lethality. This CRS was anticipated and shortened for CD44v6 compared with CD19 CAR-T cells and worse in the case of 4-1BB compared with the original CD28 2G CAR designs. Strikingly, mice recovering from the CRS benefited from durable leukemic remissions, yet experienced long-lasting CD19+ B-cell or CD44v6+ monocyte aplasias. Deepness of remission was confirmed in "tertiary" recipients, which did not develop leukemia after the infusion of bone-marrow cells from mice in remission 150d since CAR-T cell infusion. Interestingly, in this model, tocilizumab administration at the time of either CD19 or CD44v6 CAR-T cell infusion efficiently prevented the CRS, but did not interfere with their comparable and long-term anti-leukemic effects. Conversely, depleting monocytes/macrophages before therapeutic CAR-T cell infusion by either lyposomal clodronate or by the prophylactic CD44v6 CAR-T cells inhibited CRS development, but also resulted in significantly worse leukemia-free survival (at 250d, 0% vs 80%, P<0.0001). Conclusions: A number of lessons can be learned from this innovative xenotolerant mouse model of CAR-T cell immunotherapy: monocytes are required for both i) optimal anti-leukemic efficacy, and ii) the occurrence of CRS; iii) tocilizumab prevents the CRS without interfering with efficacy; iv) monocyte aplasia induced by CD44v6 CAR-T cells does not impact on their efficacy, at least in the theraeputic setting, and may ameliorate CRS toxicity. As for CD44v6 CAR-T cells, this model could be used for effectively predicting the efficacy and associated toxicities of new CAR-T cell therapies, speeding up their clinical development. Disclosures Traversari: MolMed SpA: Employment. Bordignon:MolMed SpA: Employment. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Bondanza:Formula Pharmaceuticals: Honoraria; TxCell: Research Funding; MolMed SpA: Research Funding.

scholarly journals Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1934-1934 ◽  
Author(s):  
Eduardo Huarte ◽  
Roddy S O'Connor ◽  
Melissa Parker ◽  
Taisheng Huang ◽  
Michael C. Milone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

scholarly journals Development of molecular and pharmacological switches for chimeric antigen receptor T cells

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Bill X. Wu ◽  
No-Joon Song ◽  
Brian P. Riesenberg ◽  
Zihai Li
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Intervention Strategy ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract The use of chimeric antigen receptor (CAR) T cell technology as a therapeutic strategy for the treatment blood-born human cancers has delivered outstanding clinical efficacy. However, this treatment modality can also be associated with serious adverse events in the form of cytokine release syndrome. While several avenues are being pursued to limit the off-target effects, it is critically important that any intervention strategy has minimal consequences on long term efficacy. A recent study published in Science Translational Medicine by Dr. Hudecek’s group proved that dasatinib, a tyrosine kinase inhibitor, can serve as an on/off switch for CD19-CAR-T cells in preclinical models by limiting toxicities while maintaining therapeutic efficacy. In this editorial, we discuss the recent strategies for generating safer CAR-T cells, and also important questions surrounding the use of dasatinib for emergency intervention of CAR-T cell mediated cytokine release syndrome.

Pre-Emptive Donor Lymphocyte Infusion with CD19-Directed, CAR-Modified T Cells Infused after Allogeneic Hematopoietic Cell Transplantation for Patients with Advanced CD19+ Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 862-862 ◽  
Author(s):  
Partow Kebriaei ◽  
Stefan O. Ciurea ◽  
Mary Helen Huls ◽  
Harjeet Singh ◽  
Simon Olivares ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cell Transplantation ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Lymphoid Malignancies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: Allogeneic hematopoietic cell transplantation (HCT) can be curative in a subset of patients with advanced lymphoid malignancies but relapse remains a major reason for treatment failure. Donor-derived, non-specific lymphocyte infusions (DLI) can confer an immune anti-malignancy effect but can be complicated by graft-versus-host-disease (GVHD). Chimeric antigen receptor (CAR)-modified T cells directed toward CD19 have demonstrated dramatic efficacy in patients with refractory ALL and NHL. However, responses are often associated with life-threatening cytokine release syndrome. Aim: We hypothesized that infusing CAR-modified, CD19-specific T-cells after HCT as a directed DLI would be associated with a low rate of GVHD, better disease control, and a less severe cytokine release syndrome since administered in a minimal disease state. Methods: We employed a non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-specific CAR (designated CD19RCD28 that activates via CD3z & CD28) in donor-derived T cells for patients with advanced CD19+ lymphoid malignancies. T-cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T-cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The AaPC were derived from K562 cells and genetically modified to co-express CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound version of IL-15. Results: To date, we have successfully treated 21 patients with median age 36 years (range 21-62) with advanced CD19+ ALL (n=18) or NHL (n=3); 10 patients had active disease at time of HCT. Donor-derived CAR+ T cells (HLA-matched sibling n=10; 1 Ag mismatched sibling n=1; haplo family n=8; cord blood n=2) were infused at a median 64 days (range 42-91 days) following HCT to prevent disease progression. Transplant preparative regimens were myeloablative, busulfan-based (n=10) or reduced intensity, fludarabine-based (n=11). All patients were maintained on GVHD prophylaxis at time of CAR T-cell infusion with tacrolimus, plus mycophenolate mofeteil for cord, plus post-HCT cyclophosphamide for haplo donors. The starting CAR+ T-cell dose was 106 (n=7), escalated to 107 (n=6), 5x107 (n=5), and currently at 108 (n=3) modified T cells/m2 (based on recipient body surface area). Patients have not demonstrated any acute or late toxicity to CAR+ T cell infusions. Three patients developed acute grades 2-4 GVHD (liver n=1, upper GI n=1, skin=1) which was within the expected range after allogeneic HCT alone. Of note, the rate of CMV reactivation after CAR T cell infusion was 24% vs. 41 % previously reported for our patients without CAR T cell infusion (Wilhelm et al. J Oncol Parm Practice, 2014, 20:257). Nineteen patients have had at least 30 days follow-up post CAR T-cell infusion and are evaluable for disease progression. Forty-eight percent of patients (n=10) remain alive and in complete remission (CR) at median 5.2 months (range 0-21.3 months) following CAR T cell infusion. Importantly, among 8 patients who received haplo-HCT and CAR, 7 remain in remission at median 4.2 months. Conclusion: We demonstrate that infusing donor-derived CD19-specific CAR+ T cells, using the SB and AaPC platform, in the adjuvant HCT setting as pre-emptive DLI may provide an effective and safe approach for maintaining remission in patients at high risk for relapse. Graft-vs-host disease did not appear increased by administration of the donor derived CAR-T cells. Furthermore, the add-back of allogeneic T cells appears to have contributed to immune reconstitution and control of opportunistic viral infection. Disclosures Huls: Intrexon and Ziopharm: Employment, Equity Ownership. Singh:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Olivares:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Su:Ziopharm and Intrexon: Employment. Figliola:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Kumar:Ziopharm and Intrexon: Equity Ownership. Jena:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted); Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted). Ang:Intrexon and Ziopharm: Equity Ownership. Lee:Intrexon: Equity Ownership; Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership.

Chimeric Antigen Receptor T-Cell Therapies for Aggressive B-Cell Lymphomas: Current and Future State of the Art

2019 ◽  
pp. 446-453 ◽  
Author(s):  
Jeremy S. Abramson ◽  
Matthew Lunning ◽  
M. Lia Palomba
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Refractory Disease ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Aggressive B-cell lymphomas that are primary refractory to, or relapse after, frontline chemoimmunotherapy have a low cure rate with conventional therapies. Although high-dose chemotherapy remains the standard of care at first relapse for sufficiently young and fit patients, fewer than one-quarter of patients with relapsed/refractory disease are cured with this approach. Anti-CD19 chimeric antigen receptor (CAR) T cells have emerged as an effective therapy in patients with multiple relapsed/refractory disease, capable of inducing durable remissions in patients with chemotherapy-refractory disease. Three anti-CD19 CAR T cells for aggressive B-cell lymphoma (axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene ciloleucel) are either U.S. Food and Drug Administration approved or in late-stage development. All three CAR T cells produce durable remissions in 33%–40% of treated patients. Differences among these products include the specific CAR constructs, costimulatory domains, manufacturing process, dose, and eligibility criteria for their pivotal trials. Notable toxicities include cytokine release syndrome and neurologic toxicities, which are usually treatable and reversible, as well as cytopenias and hypogammaglobulinemia. Incidences of cytokine release syndrome and neurotoxicity differ across CAR T-cell products, related in part to the type of costimulatory domain. Potential mechanisms of resistance include CAR T-cell exhaustion and immune evasion, CD19 antigen loss, and a lack of persistence. Rational combination strategies with CAR T cells are under evaluation, including immune checkpoint inhibitors, immunomodulators, and tyrosine kinase inhibitors. Novel cell products are also being developed and include CAR T cells that target multiple tumor antigens, cytokine-secreting CAR T cells, and gene-edited CAR T cells, among others.

CD4 CAR T Cells Mediate CD8-like Cytotoxic Anti-Leukemic Effects Resulting in Leukemic Clearance and Are Less Susceptible to Attenuation By Endogenous TCR Activation Than CD8 CAR T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 100-100 ◽  
Author(s):  
Yinmeng Yang ◽  
Tasha Lin ◽  
Elad Jacoby ◽  
Haiying Qin ◽  
Elizabeth Grier Gardner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Tcr Activation ◽  
The Impact

Abstract Adoptive immunotherapy using T cells armed with chimeric antigen receptors (CAR) has proven extremely effective against CD19+ B-lineage acute lymphoblastic leukemia (ALL) with remission rates as high as 70-90% in recent clinical trials in relapsed/refractory patients. CD8 T cells are typically thought of as the primary antitumor effector cells in an adoptively transferred product due to their potent cytolytic capabilities, whereas CD4 T cells are thought to primarily provide "help" to enhance CD8 T cell activity via cytokine production. However, CARs are synthetic constructs that likely alter the functionality of T cells. Further, CAR T cells are endowed with 2 specificities, one through the CAR and one through the endogenous T cell receptor (TCR). To evaluate the biology of CAR T cells, we sought to evaluate the impact of CAR expression on the functionality of T cells and to study the impact of TCR on CAR T cell activity using both human CAR T cells in a xenograft model and murine CAR T cells in a syngeneic murine model. A human or murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR was transduced into human or mouse CD8 (CAR8) and CD4 (CAR4) T cells, respectively, and tested against pre-B ALL cell lines in human murine xenografts or syngeneic models. Surprisingly, human CAR4 cells alone had equivalent ability to eradicate the Nalm6 ALL in vivo as CAR8 cells. Although CAR8 cells more rapidly cleared leukemia (2 vs 4 days), relapse eventually occurred. In contrast, CAR4 cells eradicated leukemia more slowly, but persisted longer and prevented relapse. In mice receiving CAR4+CAR8 cell products, only CAR4 cells were detectable at day 55. Next we utilized our syngeneic murine system to study CAR4 and CAR8 activity in an immunocompetent system using TCR transgenic T cells with known TCR specificity against the male histocompatibility antigen, HY. As expected, when stimulated through the TCR, CAR8 cells produced cytokines and degranulated, as manifested by CD107a expression, whereas CAR4 cells only demonstrated a cytokine production response. However, murine CAR4 cells activated through the CAR receptor develop "CD8-like" cytolytic features characterized by degranulation and were able to completely eradicate leukemia in vivo when administered without CAR8 cells, similar to the human CAR4 cells. We then evaluated the effect of TCR antigen in vivo on CAR T cell function by comparing activity in male (HY+) vs female (HY-) recipients. CAR4 cells were curative in both male and female recipients. However, CAR8 treatment has no anti-tumor activity in male recipients indicating that CAR8 cells are very susceptible to the negative effects of TCR signaling. TCR antigen availability in vivo greatly increased the number of infused CAR4 cells (P=0.003, male vs female recipients), while they significantly decreased the number of CAR8 cells (P=0.0023, male vs female recipients) after 1 week. Interestingly, we found that there was significant down-regulation of CAR expression on both CAR4 and CAR8 cells in male recipients (HY+). PD-1 and Tim3 were expressed at low levels on CAR4 cells and CAR8 cells in female recipients. However, there was significantly higher PD-1 (P=0.0079) and TIM3 (p=0.014) expression on CAR8 cells in male vs female recipients, whereas there was no difference in PD-1 or Tim3 on CAR4 cells recovered from male vs female recipients. These data indicate that CAR4 cells are more functional and become less exhausted than CAR8 cells in the presence of TCR antigen. Finally, we evaluated the long-term persistence of CAR4 and CAR8 cells in our syngeneic model in which there is no xenogeneic reactivity allowing for long-term monitoring. At day 60 and day 80, CAR4 cells were detectable in bone marrow and spleen, whereas CAR8 cells had completely disappeared. Despite the long-term persistence of CAR4 cells in recipients with or without TCR antigen, CAR4 cells are not completely immune to effects of TCR activation. In male recipients after 60 days, CAR4 cells also start to express high PD-1 levels and have decreased CAR T cell counts. These findings have important implications for CAR therapy, particularly in the allogeneic setting where the presence of TCR antigen may be more likely to be present in the recipient. In addition, these results suggest that CAR4 cells alone are cytolytic, equally potent to CAR8 cells at eradicating leukemia in vivo, and may be superior due to better persistence and reduced susceptibility to exhaustion. Disclosures No relevant conflicts of interest to declare.

CAR-T Therapy for Lymphoma with Prophylactic Tocilizumab: Decreased Rates of Severe Cytokine Release Syndrome without Excessive Neurologic Toxicity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31 ◽  
Author(s):  
Paolo F Caimi ◽  
Ashish Sharma ◽  
Patricio Rojas ◽  
Seema Patel ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Advisory Board ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Significant Difference ◽  
Car T

INTRODUCTION: Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES/ICANS) are well-known complications of CAR-T cell therapy. Tocilizumab, a humanized monoclonal antibody targeting the interleukin 6 (IL-6) receptor, is approved for treatment of CRS. Our institutional standard was modified to administer prophylactic tocilizumab before infusion CAR-T cell products. We present the outcomes of subjects treated with locally manufactured antiCD19 CAR-T cells (TNFRSF19 transmembrane domain, CD3Zeta/4-1BB costimulatory signaling) with and without prophylactic tocilizumab. METHODS: Relapsed / refractory (r/r) lymphoma patients (pts) treated with anti-CD19 CAR-T cells at our institution were included. Baseline demographic and clinical characteristics, as well as laboratory results were obtained from our Hematologic Malignancies and Stem Cell Therapy Database. Prior to institution of prophylactic tocilizumab, pts received this agent only if they presented evidence of CRS grade 2 or higher. In May 2019, our institutional practice changed to provide tocilizumab 8mg/kg, 1 hour prior to infusion of CAR-T cell product. CRS was measured according to the ASTCT Consensus Grading, whereas CRES was measured using the CARTOX-10 criteria. Comparisons between groups were done with the Mann-Whitney U test for continuous variables and Fisher's exact test for categorical variables. RESULTS: Twenty-three relapsed / refractory lymphoma pts were treated with antiCD19 CAR-T cells; 15 pts received prophylactic tocilizumab. Median follow up was 312 days (range 64 - 679) days. Baseline characteristics are listed in table 1. Both groups were similar: There were no statistically differences in the rate of bulky, refractory disease, prior ASCT or number or prior lines of therapy. Baseline lymphocyte counts, C - reactive protein (CRP) and were also comparable between groups (Table 2). We did not observe immune adverse reactions to tocilizumab infusion. There were no differences in the incidence of cytopenias or infectious complications between groups. CRS of any grade was observed in 6/8 (75%) of pts without prophylactic tocilizumab vs. 6/15 (40%) in pts treated with prophylactic tocilizumab (p = 0.23), whereas CRS grade &gt;1 was observed in 5 pts (62.5%) without prophylactic tocilizumab and in 3 pts (20%) treated with prophylactic tocilizumab (p = 0.02). There was no significant difference in the incidence of all grade CRES (no prophylaxis, 3/8 [38%] pts; prophylaxis 5/15 [30%] pts, p = 0.2969). There was a statistically significant difference in the peak CRP and peak ferritin without difference in the peak lymphocyte count after CAR-T infusion (Table 2, Figure 1). Patients given prophylactic tocilizumab had higher IL-6 plasma concentrations on day 2 after infusion (Figure 2). Complete response was observed in 4/8 (50%) pts without prophylactic tocilizumab vs. 12/15 (80%) pts with prophylactic tocilizumab (p = 0.18). All pts had detectable Anti-CD19 CAR-T cells on day 30, both groups had peak CAR-T expansion on day 14, with no statistically significant differences in expansion rates between groups. All evaluable subjects have had CAR-T persistence on days 60, 90, 180, and 365. CONCLUSIONS: Use of prophylactic tocilizumab prior to infusion of antiCD19 CAR-T cells is associated with reduced incidence of severe CRS and decreased levels of clinical laboratory markers of inflammation, despite increases in plasma concentration of IL-6. This decreased rate of grade ≥2 CRS is not associated with impaired disease control and did not result in increased rates of neurologic toxicity. Prophylactic tocilizumab does not appear to affect CAR-T cell expansion or persistence. Figure 1 Disclosures Caimi: ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau; Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board. Worden:Lentigen, a Miltenyi biotec company: Current Employment. Kadan:Lentigen, a Miltenyi biotec company: Current Employment. Orentas:Lentigen Technology, a Miltenyi Biotec Company: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. de Lima:Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding; Kadmon: Other: Personal Fees, Advisory board; Incyte: Other: Personal Fees, advisory board; BMS: Other: Personal Fees, advisory board. OffLabel Disclosure: Use of tocilizumab as prophylaxis for CRS is not approved, whereas use for treatment is approved and on label.

scholarly journals Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-24
Author(s):  
Ameet K. Mishra ◽  
Iris Kemler ◽  
David Dingli
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Nsg Mice ◽  
Human T Cells ◽  
Car T

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p&lt;0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p&lt;0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p&lt;0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity. Disclosures Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.

Remissions of Multiple Myeloma during a First-in-Humans Clinical Trial of T Cells Expressing an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. LBA-1-LBA-1 ◽  
Author(s):  
Syed Abbas Ali ◽  
Victoria Shi ◽  
Michael Wang ◽  
David Stroncek ◽  
Irina Maric ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Plasma Cells ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Bone Marrow Plasma

Abstract B-cell maturation antigen (BCMA) is a protein expressed by normal and malignant plasma cells. We are conducting a phase I clinical trial of an anti-BCMA chimeric antigen receptor (CAR-BCMA) that incorporates an anti-BCMA single-chain variable fragment, a CD28 domain, and a CD3-zeta T-cell activation domain (Carpenter et al. Clinical Cancer Research 2013). Autologous T cells are genetically modified to express the CAR with a gamma-retroviral vector. Patients receive a single infusion of CAR-BCMA T cells. Before the CAR T-cell infusions, patients receive a chemotherapy regimen of 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine with each chemotherapy agent given daily for 3 days. The purpose of the chemotherapy is to enhance activity of the CAR T cells by depleting endogenous leukocytes. Twelve patients have been enrolled, and 11 patients have been treated on one of 4 dose levels, 0.3x106, 1x106, 3x106, and 9x106CAR+ T cells/kg of bodyweight. Patients had advanced multiple myeloma (MM) with a median of 7 prior lines of therapy. Of the 6 patients treated on the lowest 2 dose levels, one patient had a transient partial remission (PR) of 2 weeks duration; the other 5 patients had responses of stable disease (SD). On the 3rddose level, 2 patients obtained responses of stable disease, and one patient obtained a response of very good PR (VGPR) with complete elimination of MM bone disease on positron emission tomography (PET) scan, normalization of serum free light chains, and clearance of bone marrow plasma cells. Toxicity among patients on the first 3 dose levels was mild and included cytopenias attributable to chemotherapy, fever in 3 patients, and signs of cytokine release syndrome including tachycardia and hypotension in Patient 8 who had a VGPR. Two patients have been treated on the highest dose level of 9x106CAR+ T cells/kg. The first patient on this dose level, Patient 10, had MM making up 90% of total bone marrow cells before treatment. Starting 4 hours after infusion of CAR T cells, Patient 10 exhibited signs of cytokine release syndrome including fever, tachycardia, dyspnea, acute kidney injury, coagulopathy, hypotension requiring vasopressor support, and muscle damage manifesting as an elevated creatine kinase level and weakness. His neutrophil count was less than 500/µL before the CAR-BCMA T-cell infusion and remained below 500/µL for 40 days after the CAR T-cell infusion before recovering. He also experienced prolonged thrombocytopenia. Patient 10’s myeloma was rapidly eliminated after CAR-BCMA T-cell infusion. By immunohistochemistry staining for CD138, bone marrow plasma cells decreased from 90% before treatment to 0% one month after the CAR T-cell infusion. The serum M-protein decreased from 1.6 g/dL before treatment to undetectable 2 months after treatment. The serum and urine immunofixation electrophoresis tests were negative 2 months after the CAR T-cell infusion. Patient 10’s current myeloma response is stringent complete remission. The second patient treated on the 9x106CAR+ T cells/kg dose level, Patient 11, had IgG lambda MM with 80% bone marrow plasma cells before treatment. Patient 11 experienced signs of cytokine release syndrome with toxicities including fever, tachycardia, hypotension, delirium, hypoxia, and coagulopathy. Patient 11’s M-protein decreased from 3.6 g/dL before treatment to 0.8 g/dL 4 weeks after treatment. His serum lambda free light chain decreased from 95.9 mg/dL before treatment to 0.15 mg/dL 4 weeks after treatment. Four weeks after CAR T-cell infusion, bone marrow plasma cells were undetectable. T cells containing the CAR-BCMA gene were detected in the blood of all 10 patients evaluated with peak levels of 0.04 to 18.2% of blood mononuclear cells. Patient 10 had the highest peak absolute number of blood CAR T cells with 51 CAR+ T cells/µL. Blood levels of IL-6 and other inflammatory cytokines were highest in patients with clinical signs of cytokine release syndrome, and the 3 patients with the highest serum IL-6 levels also had the most impressive anti-myeloma responses. Before treatment, the mean serum BCMA level of treated patients was 243 ng/mL. In responding patients, serum BCMA levels decreased after treatment. Toxicities in patients receiving CAR-BCMA T cells were similar to toxicities in leukemia patients treated with anti-CD19 CAR T cells. Our findings demonstrate strong anti-myeloma activity in the first clinical trial of a CAR targeting BCMA. Disclosures: Wang: Celgene: Research Funding. Kochenderfer:bluebird bio Inc.: Research Funding. Off Label Use: Use of cyclophosphamide and fludarabine as a conditioning regimen for adoptively-transferred T cells will be part of the presentation.

scholarly journals Brain capillary obstruction as a novel mechanism of anti-CD19 CAR T cell neurotoxicity

2021 ◽  
Author(s):  
Lila D Faulhaber ◽  
Kendra Jae Hartsuyker ◽  
Anthea Q Phuong ◽  
Yeheun Cho ◽  
Katie K Mand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Therapeutic Interventions ◽  
High Dose ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T Cell ◽  
Syngeneic Mouse Model ◽  
Car T

Immunotherapy for hematologic malignancies with CD19-directed CAR T cells is associated with neurotoxicity in about 40% of patients. Systemic cytokine release syndrome, endothelial activation, and disruption of endothelial integrity have all been associated with neurotoxicity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We developed a syngeneic mouse model which manifests systemic cytokine release and behavioral abnormalities within 3-5 days after infusion of high-dose murine CD19-CAR T cells. Histologic examination revealed widespread brain hemorrhages, diffuse extravascular IgG deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. In vivo two-photon imaging of blood flow revealed plugging of >10% of capillaries by leukocytes, associated with regions of localized hypoxia. These data reveal capillary obstruction and associated brain hypoxia and microvascular decline as a potential basis for neurotoxicity during CD19-CAR T cell treatment in humans, which may be amenable to therapeutic interventions.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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