scholarly journals CD4/CD8 T-Cell Selection Enhances CD22 CAR-T Cell Transduction and in-Vivo CAR-T Expansion: Updated Results on Phase I Anti-CD22 CAR Dose Expansion Cohort

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 809-809
Author(s):  
Nirali N Shah ◽  
Steven L Highfill ◽  
Haneen Shalabi ◽  
Bonnie Yates ◽  
Eli Kane ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Complete Remission ◽  
Cell Selection ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Selection

Abstract Background: We previously demonstrated that non-T cells in apheresis products collected from patients with cancer, particularly in those with circulating leukemic cells or low CD3 counts, can substantially affect T cell expansion and transduction and, potentially, in vivo efficacy. Flask adhesion and elutriation methods to remove monocytes and granulocytes, and separation using CD3/CD28 activation beads are inefficient at removing tumor and other inhibitory cells. In the context of an ongoing CD22 CAR clinical trial, we introduced magnetic CD4/CD8 selection prior to activation and lentiviral transduction which resulted in enhanced in vivo CAR T-cell potency compared to products generated prior to the introduction of CD4/CD8 T cell selection. Design: Children and young adults with relapsed/refractory CD22+ hematologic malignancies eligible for our phase I dose escalation anti-CD22 CAR protocol were enrolled on study (NCT02315612). All had bone marrow evaluations at baseline, prior to lympho-depleting chemotherapy (Fludarabine 25 mg/m2 x 3 days and Cyclophosphamide 900 mg/m2 x 1 day) and again at day 28 (+/- 4 days) post-CAR infusion. Three dose levels were explored during dose-escalation (3 x 10e5; 1 x 10e6 and 3 x 10e6 transduced CAR T-cells/kg) prior to treating in the expansion phase at the 1 x 10e6 dose level, which is the focus of this report. As a result of a manufacturing failure in an enrolled patient, as of January 2017, the CAR-T manufacturing process was modified to include CD4/CD8 bead selection (TCS) for all subsequent patients. CAR T cell activation, transduction and expansion processes were otherwise unchanged. Results: From December 2014 to July 2017, 30 patients with ALL were treated, of which 22 (73%) were treated at 1 x 10e6 transduced CAR T cells/kg. All patients had active bone marrow involvement at baseline and 18 (82%) had an M2 marrow (>5% blasts) or higher disease burden. Eighteen had a prior transplant and 14 were previously treated with CAR. The first 15 patients were treated using an unselected apheresis product and 7 patients were treated using CD4/CD8 TCS. The first patient treating using TCS initially had a product failure when CAR-T cells were manufactured without TCS, due in part to a low CD3 at the time of collection resulting in substantial numbers of non-T cells in the apheresis product. Following CD4/CD8 TCS, the CD3 T-cell content in the starting product increased from 47% to 90%, T cell proliferation increased from 1.97-fold to 24.2-fold, transduction increased from 2.57% to 33.4% and an MRD negative complete remission was achieved. Following TCS, median transduction efficiencies were significantly higher (33.4% (pre-TCS) vs 40.7%, p=0.02). Cytokine release syndrome (CRS) occurred in 19 of 22 patients and was grade 1 CRS in 12 (4 in the TCS arm); grade 2 in 6 (2 in the TCS arm) and grade 4 in one patient. Amongst those developing CRS, tocilizumab was utilized in 2 of 13 (15%) patients pre-TCS and in 3 of 6 (50%) patients in the TCS arm to prevent higher grade CRS. Steroids were administered to 1 of 13 patients pre-TCS versus 3 of 6 (50%) patients in the TCS arm. Clinical parameters suggestive of a more robust inflammatory response in TCS patients included statistically significantly higher peak IL-6, IL-8, IL-10 and IL-15 levels as well as higher ferritin (Figure), and higher peak CAR-T% (75% vs 82%, p=0.03) with a trend towards higher absolute CAR values. Furthermore, 3 of the 5 responders in the TCS arm had evidence for a secondary expansion with a late rise in ferritin, WBC and CAR-T% with 2 patients having confirmed hemophagocytosis on the bone marrow at day 28, which was not seen in patients enrolled pre-TCS. For the entire cohort, 16 of 21 (76%) patients evaluable for response attained a complete remission (11 of 15 enrolled pre-TCS and 5 of 7 post-TCS). Amongst the TCS group, 5 of 5 who were CD22 CAR naïve attained MRD negative remission; two patients pre-treated with CD22 CAR T at an outside institution experienced poor in vivo CAR expansion possibly due to immune rejection. Conclusion: Ongoing experience on our expansion cohort confirms activity of CD22 CAR T cells resulting in high remission induction rates in relapsed refractory ALL, including responses in patients previously treated with CD19 CAR T cells. T cell selection using CD4/CD8 positive selection led to enhanced in vivo CAR-T expansion resulting in a 100% MRD negative complete remission rate (5 of 5 patients) in CD22 CAR naïve patients. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD4/CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial

2020 ◽  
Vol 38 (17) ◽  
pp. 1938-1950 ◽  
Author(s):  
Nirali N. Shah ◽  
Steven L. Highfill ◽  
Haneen Shalabi ◽  
Bonnie Yates ◽  
Jianjian Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase I ◽  
Chimeric Antigen Receptor ◽  
Cd8 T Cell ◽  
Cell Selection ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Selection

PURPOSE Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells. PATIENTS AND METHODS We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22+ malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling. RESULTS Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis–like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation. CONCLUSION In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.

scholarly journals Single-cell imaging of CAR T cell activity in vivo reveals extensive functional and anatomical heterogeneity

2019 ◽  
Vol 216 (5) ◽  
pp. 1038-1049 ◽  
Author(s):  
Marine Cazaux ◽  
Capucine L. Grandjean ◽  
Fabrice Lemaître ◽  
Zacarias Garcia ◽  
Richard J. Beck ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cell Interactions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

CAR T cells represent a potentially curative strategy for B cell malignancies. However, the outcome and dynamics of CAR T cell interactions in distinct anatomical sites are poorly understood. Using intravital imaging, we tracked interactions established by anti-CD19 CAR T cells in B cell lymphoma–bearing mice. Circulating targets trapped CAR T cells in the lungs, reducing their access to lymphoid organs. In the bone marrow, tumor apoptosis was largely due to CAR T cells that engaged, killed, and detached from their targets within 25 min. Notably, not all CAR T cell contacts elicited calcium signaling or killing while interacting with tumors, uncovering extensive functional heterogeneity. Mathematical modeling revealed that direct killing was sufficient for tumor regression. Finally, antigen-loss variants emerged in the bone marrow, but not in lymph nodes, where CAR T cell cytotoxic activity was reduced. Our results identify a previously unappreciated level of diversity in the outcomes of CAR T cell interactions in vivo, with important clinical implications.

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).

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

scholarly journals AAV-Mediated In Vivo CAR Gene Therapy for Targeting Human T Cell Leukemia

2021 ◽  
Author(s):  
Waqas Nawaz ◽  
Bilian Huang ◽  
Shijie Xu ◽  
Yanlei Li ◽  
Linjing Zhu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Human T Cell ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV)vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NCG tumor mouse model of human T cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ex vivo processes of traditional CAR T cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.Significance StatementAAV can generate enough CAR cells within the host. That act as a living drug, distributed throughout the body, and persist for weeks, with the ability to recognize and destroy tumor cells.

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

scholarly journals A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells in Vivo

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 567 ◽  
Author(s):  
Susana Pascoal ◽  
Benjamin Salzer ◽  
Eva Scheuringer ◽  
Andrea Wenninger-Weinzierl ◽  
Caterina Sturtzel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Therapy ◽  
Xenograft Model ◽  
Model Systems ◽  
Preclinical Model ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

scholarly journals Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaojuan Shi ◽  
Daiqun Zhang ◽  
Feng Li ◽  
Zhen Zhang ◽  
Shumin Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inhibitory Effects ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractAsparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.

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.

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