scholarly journals A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 327-327
Author(s):  
Ranjan Maity ◽  
Sacha Benaoudia ◽  
Franz Zemp ◽  
Holly Lee ◽  
Elie Barakat ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Antigenic Stimulation ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Chronic Antigenic Stimulation ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D). Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. Figure 1 Figure 1. Disclosures Neri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

scholarly journals Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lintao Liu ◽  
Enguang Bi ◽  
Xingzhe Ma ◽  
Wei Xiong ◽  
Jianfei Qian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Effector Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

scholarly journals Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4301
Author(s):  
Marianna Csaplár ◽  
János Szöllősi ◽  
Stephen Gottschalk ◽  
György Vereb ◽  
Árpád Szöőr
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Cytolytic Activity ◽  
Effector Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Differentiation Status

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

scholarly journals 145 Comparison of CAR-T cell manufacturing platforms reveals distinct phenotypic and transcriptional profiles

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A153-A153
Author(s):  
Hannah Song ◽  
Lipei Shao ◽  
Michaela Prochazkova ◽  
Adam Cheuk ◽  
Ping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Effector Memory ◽  
Cell Phenotype ◽  
Gene Clustering ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Manufacturing ◽  
Car T ◽  
And Function

BackgroundWith the clinical success of chimeric antigen receptor (CAR)-T cells against hematological malignancies, investigators are looking to expand CAR-T therapies to new tumor targets and patient populations. To support translation to the clinic, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity while using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses that can number in the billions. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, it is currently unknown whether the manufacturing platform itself significantly influences the output T cell phenotype and function.MethodsStatic bag culture was compared with 3 widely-used commercial CAR-T manufacturing platforms (Miltenyi CliniMACS Prodigy, Cytiva Xuri W25 rocking platform, and Wilson-Wolf G-Rex gas-permeable bioreactor) to generate CAR-T cells against FGFR4, a promising target for pediatric sarcoma. Selected CD4+CD8+ cells were stimulated with Miltenyi TransAct, transduced with lentiviral vector, and cultured out to 14 days in TexMACS media with serum and IL2.ResultsAs expected, there were significant differences in overall expansion, with bag cultures yielding the greatest fold-expansion while the Prodigy had the lowest (481-fold vs. 84-fold, respectively; G-Rex=175-fold; Xuri=127-fold; average of N=4 donors). Interestingly, we also observed considerable differences in CAR-T phenotype. The Prodigy had the highest percentage of CD45RA+CCR7+ stem/central memory (Tscm)-like cells at 46%, while the bag and G-Rex cultures had the lowest at 16% and 13%, respectively (average N=4 donors). In contrast, the bag, G-Rex, and Xuri cultures were enriched for CD45RO+CCR7- effector memory cells and also had higher expression of exhaustion markers PD1 and LAG3. Gene clustering analysis using a CAR-T panel of 780 genes revealed clusters of genes enriched in Prodigy/de-enriched in bag, and vice versa. We are currently in the process of evaluating T cell function.ConclusionsThis is the first study to our knowledge to benchmark these widely-used bioreactor systems in terms of cellular output, demonstrating that variables inherent to each platform (such as such as nutrient availability, gas exchange, and shear force) significantly influence the final CAR-T cell product. Whether enrichment of Tscm-like cells in the final infusion product correlates with response rate, as has been demonstrated in the setting of CD19 CAR-Ts, remains to be seen and may differ for FGFR4 CAR-Ts and other solid tumors. Overall, our study outlines methods to identify the optimal manufacturing process for future CAR-T cell therapies.

scholarly journals Early Clinical Experience of CD19 x CD22 Dual Specific CAR T Cells for Enhanced Anti-Leukemic Targeting of Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 278-278 ◽  
Author(s):  
Rebecca Gardner ◽  
Colleen Annesley ◽  
Olivia Finney ◽  
Corinne Summers ◽  
Adam J. Lamble ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dose Level ◽  
T Cell Subsets ◽  
Preclinical Testing ◽  
Car T Cells ◽  
Car T Cell ◽  
Dual Specificity ◽  
Car T

Abstract Introduction: Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile. Methods: Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS. Results: Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone. Conclusions: Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. Figure. Figure. Disclosures Park: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

scholarly journals A Novel Second Generation CD19 CAR for Therapy of High Risk/Relapsed Paediatric CD19+ Acute Lymphoblastic Leukaemia and Other Haematological Malignancies: Preliminary Results from the Carpall Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4026-4026
Author(s):  
Sara Ghorashian ◽  
Anne Marijn Kramer ◽  
Sarah Jayne Albon ◽  
Catherine Irving ◽  
Lucas Chan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Transduction Efficiency ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion ◽  
Equity Ownership

Abstract Introduction: Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) unprecedented anti-leukemic responses. We have developed a novel CD19CAR with a new scFv in the 41BBz format (CAT-41BBz CAR) which confers enhanced cytotoxicity and cytokine secretion in response to stimulation with CD19+ targets in vitro as well as equivalent in vivo anti-tumour efficacy to the FMC63 41BBZ CAR in use in clinical studies. We have designed, optimized and validated GMP-grade CAR T cell production using this novel CAR. Based on these data, we have recently initiated a Phase I clinical study (CARPALL) of this novel CAR in pediatric patients with relapsed ALL and other CD19+ hematological malignancies to determine the safety profile and durability of responses to CD19CART therapy. This will be critical in determining whether CD19CAR T cells are best used as a stand-alone therapy or as a bridge to stem cell transplant (SCT). Methods: We initially optimized our GMP production methodology in terms of activation method, cytokine milieu and expansion conditions on healthy donor peripheral blood mononuclear cells (PBMCs) to give optimal transduction efficiency and preserve early memory subsets within the CAR T cell product. We have subsequently validated this methodology using unstimulated leucaphereses from 5 lymphopenic patients with ALL. PBMCs were activated with anti-CD3/CD28 microbeads (Dynabeads CTS) and then lentivirally transduced with the CAT CAR vector. T cells were then expanded in the WAVE bioreactor before bead removal on a magnetic system and cryopreservation. Patients on study receive lymphodepletion with fludarabine and cyclophosphamide followed by a single dose of 106 CAR+ T cells/kg and are then monitored as an in-patient for 14 days post infusion for toxicities such as cytokine release syndrome or neurotoxicity. The primary end-points of the study are toxicity and the proportion of patients achieving molecular CR at 1 month post CD19CAR T cell infusion. Following this, patients undergo intensive monitoring of disease status for a total of 2 years post infusion. To determine the durability of responses, patients achieving a molecular CR will be monitored closely for the re-emergence of molecular level disease without additional consolidative therapy or SCT Results: We were able to generate the target dose of 1x106 CAR+ T cells/kg in 6 of 7 production runs (involving 2 healthy donors and 5 patients) to date, all of which met sterility release criteria. Transduction efficiency was on average 37% (range 7-84%, see table 1). Mean viral copy was 4.2 (range 1.2-5.8). Memory T cells of stem cell-like phenotype (CAR+ CCR7+ CD45RA+ CD95+ CD127+) formed a mean of 9% (range 0-31%), central memory T cells (CAR+ CCR7+ CD45RA-) formed a mean of 43% (range 16-70%) and effector memory T cells formed a mean of 31% (range 0-77%) of the final CAR T cell product. The percentage of CAR T cells expressing dual exhaustion markers (TIM3+ PD-1+) was on average 5% (range 2-8%). So far 2 patients have been treated. Conclusions We have optimized and successfully validated a robust GMP production method for CD19CAR T cells lentivirally transduced with a novel CD19CAR. Preliminary results of therapy with CAT-41BBz CAR T cells in initial patients on the clinical study will be presented. Disclosures Qasim: Autolus: Consultancy, Equity Ownership, Research Funding; Cellectis: Research Funding; Calimmune: Research Funding; Catapult: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

scholarly journals Lenalidomide Enhances the Function of CS1 Chimeric Antigen Receptor Redirected-T Cells Against Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 812-812 ◽  
Author(s):  
Xiuli Wang ◽  
Ryan Urak ◽  
Miriam Walter ◽  
Lihong Weng ◽  
Laura Lim ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Multiple myeloma (MM) is an incurable malignancy of plasma cells even with great advances in treatment. Chimeric Antigen Receptor (CAR) directed T cell therapy, which can specifically recognize tumor associated antigens and kill tumor cells in an MHC independent manner, is a promising approach for hematological malignancy. There are several candidate antigens for CAR T cell targeting of multiple myeloma, including BCMA and CS1. Our goal is to develop novel CARs for the treatment of MM and explore the potential benefits of combinatorial therapy of CAR T cells and immunomodulatory drugs (IMiDs) such as lenalidomide. In the present study, we redirected central memory T cells to express second-generation CARs specific for either CS1 or BCMA that incorporate CD28 signaling moieties. Central memory T cells were activated by CD3/CD28 bead stimulation, transduced with lentivirus encoding the CAR construct, and expanded ex vivo. The engineered and expanded CS1 and BCMA CAR T cells exhibited similar phenotypes and comparable in vitro effector function. However, once adoptively transferred into MM tumor-bearing NOD/Scid IL2RγCnull (NSG) mice by intravenous injection of 1x10^6 CAR T cells, CS1 CAR T cells exhibited superior antitumor activity over BCMA CART cells and significantly prolonged mouse survival (P<0.01). To further improve the anti-MM activity of CAR T cell therapy, we investigated the effects of lenalidomide on CS1 CAR T cell function against MM. Central memory T cells were activated and transduced with lentivirus encoding CS1 CAR and then expanded in vitro in the presence of 0, 1 or 10mM lenalidomide for 3-4 weeks and then effector function was evaluated. We found that CD8+ CAR T cells were preferentially expanded over CD4+ CAR T cells in a dose-dependent manner. Lenalidomide-treated CAR T cells secreted higher levels of Th1 cytokines such as IFN-gamma, TNF-alpha, and IL-2, but reduced Th2 cytokines such as IL-4 and IL-10 upon antigen stimulation as compared with untreated CAR T cells. Meanwhile we observed that lenalidomide greatly improved the maintenance of T cell memory markers (CD62L, CD28, and CD27) in the culture and enhanced the formation of immune synapses between CAR T cells and MM cells. RNA-seq analysis revealed that more than 600 genes were differentially expressed among the lenalidomide treated and un-treated CD8+CAR+ T cells. Among those, expression of immune synapse related genes such as cell junction and biological assembly is significantly increased with lenalidomide treatment. Moreover, lenalidomide results in elevated gene transcrips characteristic of memory, homing and cytolytic function of CAR T cells. To test the synergistic effects, MM bearing mice were treated with a single infusion of 1x10^6 CS1 CAR T cells (i.v) on day 0 and/or 5-7.5mgkg-1 of lenalidomide daily (i.p.) initiating on day 0 for 30 days. CS1 CAR T cells and lenalidomide exhibited synergistic anti-MM activity in vivo when MM mice received combinatorial treatment. The combination therapy significantly inhibited tumor growth in vivo, prolonged mouse survival (P<0.01) and improved CAR T cell persistence in mice as compared to single-agent treatment. Taken together, these findings indicate that lenalidomide plays a co-stimulatory role in immune modulation of CAR T cells and strengthens the anti-tumor activity of CS1 CAR T cells in vivo. Rational combination of these immunotherapeutic regimens is an effective strategy and the planned clinical trial will use a combination of lenalidomide and CS1 CAR T cells for increasing treatment efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity

2021 ◽  
Vol 6 (57) ◽  
pp. eabd4344
Author(s):  
Morgane Boulch ◽  
Marine Cazaux ◽  
Yann Loe-Mie ◽  
Ronan Thibaut ◽  
Béatrice Corre ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cross Talk ◽  
T Cell Subsets ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy relies on the activity of a large pool of tumor-targeting cytotoxic effectors. Whether CAR T cells act autonomously or require interactions with the tumor microenvironment (TME) remains incompletely understood. Here, we report an essential cross-talk between CAR T cell subsets and the TME for tumor control in an immunocompetent mouse B cell lymphoma model of anti-CD19 CAR T cell therapy. Using single-cell RNA sequencing, we revealed substantial modification of the TME during CAR T cell therapy. Interferon-γ (IFN-γ) produced by CAR T cells not only enhanced endogenous T and natural killer cell activity but was also essential for sustaining CAR T cell cytotoxicity, as revealed by intravital imaging. CAR T cell–derived IFN-γ facilitated host interleukin-12 production that supported host immune and CAR T cell responses. Compared with CD8+ CAR T cells, CD4+ CAR T cells were more efficient at host immune activation but less capable of direct tumor killing. In summary, CAR T cells do not act independently in vivo but rely instead on cytokine-mediated cross-talk with the TME for optimal activity. Invigorating CAR T cell interplay with the host represents an attractive strategy to prevent relapses after therapy.

scholarly journals CD19-CAR Therapy Using Naive/Memory or Central Memory T Cells Integrated into the Autologous Stem Cell Transplant Regimen for Patients with B-NHL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 610-610 ◽  
Author(s):  
Leslie Popplewell ◽  
Xiuli Wang ◽  
Suzette Blanchard ◽  
Jamie Wagner ◽  
Araceli Naranjo ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Central Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidative therapy for patients with recurrent non-Hodgkin lymphoma (NHL), but is limited by the high incidence of NHL relapse. We report a Phase I clinical trial of ASCT followed by CD19-specific CD28-costimulatory chimeric antigen receptor (CD19:28z-CAR) T cells, with the goal of reducing NHL relapse rates. Safety and feasibility were the primary objectives, with CAR T cell persistence and expansion in the myeloablative ASCT setting as secondary objectives. This study examines safety and feasibility for two manufacturing platforms that differed in the T cell subset composition used for CAR engineering. Initially, the T cell population for CAR transduction was central memory (Tcm)-enriched: participants' peripheral blood mononuclear cells (PBMC) were depleted for CD14+ monocytes, CD25+ Tregs, and CD45RA+ naïve and stem-memory T cells, after which they were selected for CD62L+ Tcm (Wang et al. Blood;127:2980). Based on comparative preclinical data, a second arm was added to the trial to evaluate a Tn/mem-derived manufacturing platform that enriched central memory, naïve, and stem memory T cells (no CD45RA+ depletion). Either Tcm- or Tn/mem-enriched T cells were activated with CD3/CD28 beads, transduced with lentiviral vector encoding the CD19:28z-CAR, and expanded ex vivo. This phase I trial used the toxicity equivalence range design (Blanchard and Longmate. Contemp Clin Trials; 32;114) with an equivalence range for DLTs of 0.20-0.35 and a target toxicity rate of 0.25. The first 3 participants on each arm were followed one at a time, with later accrual in cohorts of 3. Twenty-three participants underwent ASCT and received CD19:28z-CAR T cells 2 days post stem cell infusion at the assigned dose level (DL): 17 on the Tcm arm (DL 50 million [M] CAR+ T cells [n=3], 200 M [n=5], 600 M [n=9]); 6 on the Tn/mem arm (DL 200 M). Participants were followed for dose limiting toxicity (DLT) for 28 days. Table 1 shows results by arm and DL. Both arms demonstrated safety and feasibility. There was no delayed hematopoietic reconstitution on either arm. On the Tcm arm, the only DLT was at DL 600 M (1 of 9 at 600 M). The Tn/mem arm was opened at 200 M and 6 participants were treated with no DLTs. The dose was not escalated as the protocol management team had seen activity at the 200M level in 2 other trials using the Tn/mem product. Tcm Arm: Fourteen of 17 participants (82%) had a diagnosis of diffuse large B-cell lymphoma (DLBCL) and 3 had mantle cell lymphoma. The mean age of the participants on the Tcm arm was 57 (35-75). The median number of prior chemotherapy regimens was 2 (1-5). The median progression-free survival (PFS) was 34.6 months 95% CI [21.8, undefined]. Seven of 17 participants (41%) have progressed, 1 died in remission of unrelated intracranial hemorrhage (6%), 7 (41%) remain in CR and are still in follow-up, and 2 are lost to follow-up (12%). All 17 participants achieved a CR or a continuing CR after ASCT and T cells. Tn/mem arm: Five of 6 participants (83%) had a DLBCL diagnosis, and 1 was NHL not otherwise specified. The mean age of the participants was 50 (40-72). The median number of prior chemotherapy regimens was 2.5 (1-3). The median follow-up time for the Tn/mem arm was 12 months, with median PFS not yet reached. One of 6 (17%) has progressed, 4 (66%) remain in CR and are still in follow-up, and 1 is lost to follow-up (17%). Five of 6 (83%) participants achieved a best response of CR or continuing CR after therapy. Several differences were observed between the manufacturing platforms. Since the Tn/mem production platform has fewer depletion steps, it resulted in a higher product yield, which shortened the ex vivo expansion period by 4.1 days (95% CI [1.5%, 6.6%]) from 18.9 days (15-24) for Tcm to 14.8 days (12-18) for Tn/mem (P<0.005). Notably in the ASCT minimal disease burden setting, the Tn/mem-derived CD19:28z-CAR T cell products exhibited significantly higher in vivo CAR T cell expansion compared to Tcm products at the 200M DL (Figure 1). We conclude that although both Tcm- and Tn/mem-enriched CD19CAR T cell therapies are safe, the Tn/mem product is more promising due to its 1) shorter production time, 2) higher cell yield, and 3) better in vivo expansion, despite the low antigen drive in these patients post-salvage and ASCT therapy. Longer follow-up for the 2-year PFS secondary objective will indicate if improved Tn/mem expansion impacts tumor control. Disclosures Wang: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Budde:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Brown:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

scholarly journals Exhausted CLL T Cells Mediated By PD1 Expression an Important Mechanism for CD19 CAR Efficacy in CLL in the Adoptive Transfer TCL1 Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4537-4537
Author(s):  
Robin Sanderson ◽  
Arantxa Romero-Toledo ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Research Funding ◽  
Complete Response ◽  
T Cell Subsets ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background: The first two autologous CD19 chimeric antigen receptor T (CAR T) cells targeting CD19 have now been approved for the treatment of ALL and refractory lymphomas. Despite impressive responses in these diseases, results remain inconsistent in chronic lymphocytic leukaemia (CLL). It is unknown if this reflects CAR design or an effect of the underlying function of CLL T cells. These 2nd generation CAR T cells require CD28 or 41BB co-stimulatory signalling domains, but these have not been compared directly in humans. Pre-clinical models afford the opportunity to do this, however, modelling of CAR T cells has mostly been performed in vitro or using immunodeficient mice, limiting the ability to study more complex immune biology. CLL is associated with a tumour supportive microenvironment and T cells exhibit multiple functional defects and features of exhaustion. These T cell defects in CLL are closely recapitulated in Eμ-TCL1 (TCL1) mice, and induced in healthy mice by adoptive transfer (AT) of murine CLL cells. We aimed to demonstrate the effect of CLL T cell dysfunction on CAR T cell efficacy and compare CD28 and 41BB directly. Methods: Immunocompetent C57BL/6 mice (WT) received AT of pooled 20 x106 TCL1 cells from fully leukemic TCL1 mice from the same background. Syngeneic donor CAR T cells were either pooled spleens from WT mice or WT mice given AT CLL with CLL load >80%. Both groups were aged matched (approx. 3 months). Splenoctyes were enriched for CD3+ with magnetic beads then activated with anti CD3/CD28 Dynabeads (Thermofisher) and rIL-2 (Roche). They were transduced with retroviral supernatant from either SFG-m19BBmZ-GFP (CD19-41BB) or MSGV-1D3-28Z-1.3mut (CD19-CD28) and cultured for 4 days when they were injected into 48 mice in total. Mice were given 100mg/kg intraperitoneal cyclophosphamide on D-1 followed by 6-8 x106 CAR T cells (or untransduced T cells). Mice were bled weekly to assess CLL load and T cell subsets and were culled when they appeared sick or peripheral blood (PB) CLL>70%. Results: CAR T cells derived from WT and AT T cells exhibit different phenotypes. WT CAR T cells proliferate more readily in culture and exhibit significantly higher transduction efficiencies in the CD8 subset although CD4 transduction is preserved. Following activation and transduction WT CAR T cells have a CD4: CD8 ratio of 1:1 whilst those from AT are heavily skewed to CD8. In both groups >90% T cells are CD44+. PD1+ expression in both CD4 and CD8 subsets is significantly higher in AT compared to WT CAR T cells. Mice treated with the CD19 -41BB CAR derived from WT and AT T cells or untransduced T cells did not respond, whereas 100% of mice treated with CD19-CD28 CAR derived from WT T cells had a complete response with loss of normal B cells 1 week post CAR T cells injection compared to 50% of mice treated with CD19-CD28 from AT T cells. All non-responding mice were culled at week 8 due to progressive leukaemia as were control mice treated with untransduced T cells. All mice with an established response had a continued complete response for 5 weeks following CAR T cell injection. Half of these mice were culled for phenotypic comparison and the other half observed for survival analysis. Those mice that responded and culled at week 8 had equal spleen size (0.1g) to age matched WT mice controls whilst non-responding mice had significantly larger spleens (0.5-3.3g). CAR T cells were only detectable in the PB +1 week post injection. In the PB there was restoration of CD4: CD8 ratios in responding mice compared to leukemic mice. PD1 expression in the spleen and bone marrow in CD3+CD8+ and CD4+ T cells normalised in responding mice compared to non-responding mice. Conclusion: AT of TCL1 CLL into immunocompetent mice is a viable model to study in vivo CAR T cell function and the host immune response. CAR T cells derived from WT T cells lead to a complete response in all of the mice but this response is significantly reduced if T cells exposed to CLL are used. Time to relapse for these responding mice has not been reached. We postulate that failure of the CD19 -41BB CAR in vivo relates to rejection of the GFP construct. There are significant differences in PD1 expression between WT and AT derived CAR T cells, which suggest strategies to repair exhausted T cells may improve the clinical response to CAR T cells in CLL. This provides the rationale for our on going studies of PD1/PDL1 blocking drugs in combination with CAR T cells in this immunocompetent pre-clinical model. Disclosures Gribben: Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria; Pharmacyclics: Honoraria; NIH: Research Funding; Kite: Honoraria; TG Therapeutics: Honoraria; Wellcome Trust: Research Funding; Cancer Research UK: Research Funding; Unum: Equity Ownership; Roche: Honoraria; Abbvie: Honoraria.

Anti-CD19 Chimeric Antigen Receptor-Modified T Cell Therapy for B Cell Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia: Fludarabine and Cyclophosphamide Lymphodepletion Improves In Vivo Expansion and Persistence of CAR-T Cells and Clinical Outcomes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 184-184 ◽  
Author(s):  
Cameron J Turtle ◽  
Carolina Berger ◽  
Daniel Sommermeyer ◽  
Laila-Aicha Hanafi ◽  
Barbara Pender ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
T Cell Subsets ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Clinical Responses

Abstract BACKGROUND: Autologous T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) have demonstrated activity in patients with relapsed or refractory B cell NHL and CLL. 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, such as the lymphodepletion regimen and infused cell dose. We manufactured anti-CD19 CAR-T cells from a defined composition of CD4+ and CD8+ T cell subsets to treat adults with relapsed or refractory B cell NHL or CLL. T cell subsets 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 one of three dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg). CAR-T cells were administered 48-96 hours after lymphodepletion with either cyclophosphamide (Cy, 60 mg/kg)+/- etoposide or Cy (60 mg/kg) and fludarabine (25 mg/m2 daily for 3-5 days (Cy/Flu). RESULTS: Adult patients with relapsed/refractory CD19 expressing B cell NHL (n=28, median age 59 years, range 36-70) or CLL (n=6, median age 60 years, range 54-64) were treated with at least one CAR-T cell infusion. NHL histologies include diffuse large B cell or transformed NHL (DLBCL, n=18), follicular NHL (FL, n= 6) or mantle cell lymphoma (MCL, n=4). 15 patients had failed prior autologous (n=13) or allogeneic (n=3) transplants. Twelve of the 28 NHL patients received lymphodepletion with Cy-based regimens without fludarabine. Expansion of CAR-T cells and clinical responses were observed in 50% (CR=1 (DLBCL), PR=5 (2 FL, 2 DLBCL, 1 MCL), no response=6). Patients were treated at all three dose levels without dose limiting toxicity or severe cytokine release syndrome (sCRS). With this regimen, we observed short CAR-T cell persistence in most patients and demonstrated a CD8-mediated immune response to the murine scFv component of the CAR transgene that correlated with loss of CAR-T cells. Retreatment with CAR-T cells with or without chemotherapy in 5 patients led to no significant T cell expansion or clinical responses. To minimize transgene rejection fludarabine was added to the lymphodepletion regimen administered to the subsequent 16 NHL patients. Clinical responses were evaluated in 12 of 16 patients (2 not yet evaluable, 2 early deaths). Addition of Flu to the lymphodepletion regimen increased the CR rate to 42%, compared to 8% with Cy alone. Clinical responses were identified in 6 of 8 patients with DLBCL (3 CR, 3 PR) and 2 of 3 patients with FL (2 CR). The overall response rate was 67%. We noted higher peak CAR-T cell levels in blood in the Cy/Flu group (n=13) compared with the Cy only group (n=11) (CD8+ CAR-T cells, median 31.9 cells/ml vs 0.55 cells/ml, p = 0.009; CD4+ CAR-T cells, median 16.5 cells/ml vs 0.31 cells/ml, p= 0.007), and CAR-T cell persistence was longer in Flu-treated patients (see Figure 1 for patients treated at 2 x 107/kg). Surprisingly, 2 of 7 patients who received 2 x 107 CAR-T cells/kg experienced dose-limiting toxicity necessitating dose de-escalation. Markedly elevated IL-6 levels were observed within the first day after CAR-T cell infusion in patients who subsequently developed severe toxicity, which may provide an opportunity to test early interventional approaches to minimize toxicity. Six patients with relapsed and refractory CLL received CAR-T cells. Five of 6 restaged patients had complete clearance of blood and/or marrow disease by high-resolution flow cytometry 4 weeks following treatment. Overall clinical responses included 3 CR, 1 PR and 2 no response. One patient with a PR died from refractory pulmonary aspergillus infection. Patients with CR remain in remission at 1-10 months after therapy. CONCLUSION: Immunotherapy with CD19 CAR-T cells of defined subset composition is feasible in patients with NHL and CLL and has potent anti-tumor activity. Toxicity is related to cell dose. The addition of Flu to a Cy-based lymphodepletion regimen results in greater CAR-T cell expansion and persistence, and improves the CR rate after CD19 CAR-T cell therapy. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Maloney:Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria.

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