scholarly journals "IF-Better" Gating: Combinatorial Targeting and Synergistic Signaling for Enhanced CAR T Cell Efficacy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2774-2774
Author(s):  
Sascha Haubner ◽  
Jorge Mansilla-Soto ◽  
Sarah Nataraj ◽  
Xingyue He ◽  
Jae H Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Antigen ◽  
Normal Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Provision Of Services ◽  
Xenograft Models

Abstract CAR T cell therapy provides a potent therapeutic option in various B cell-related hematologic malignancies. One of the major efficacy challenges is escape of tumor cells with low antigen density, which has been clinically observed in several malignancies treated with CAR therapy. Novel concepts of CAR design are needed to address phenotypic heterogeneity including clonal variability of target antigen expression. In the study presented here, we focused on AML and selected ADGRE2 as CAR target due to its high rate of positivity on AML bulk and leukemic stem cells (LSC) in a molecularly heterogeneous AML patient population. We chose an ADGRE2-CAR with optimized scFv affinity and fine-tuned CD3zeta signaling to achieve an ideal killing threshold that would allow for sparing of ADGRE2-low normal cells. We hypothesized that co-targeting of a second AML-related antigen may mitigate potential CAR target antigen-low AML escape and we identified CLEC12A as preferential co-target due to its non-overlapping expression profiles in normal hematopoiesis and other vital tissues. We developed ADCLEC.syn1, a novel combinatorial CAR construct consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR). ADCLEC.syn1 operates based on what we describe as "IF-BETTER" gate: High CAR target expression alone triggers killing, whereas low CAR target expression does not, unless a CCR target is present. Additional CCR interaction lowers the threshold for CAR-mediated killing through increased avidity and costimulation, allowing for higher CAR sensitivity that is purposefully limited to target cells expressing both antigens. In the context of ADCLEC.syn1, ADGRE2-high/CLEC12A-negative AML cells can trigger cell lysis while ADGRE2-low/CLEC12A-negative normal cells are spared. Importantly, ADGRE2-low/CLEC12A-high AML cells are also potently eliminated, preventing ADGRE2-low AML escape. Using NSG in-vivo xenograft models of engineered MOLM13 AML cell line variants with low levels of ADGRE2 to model antigen escape, we found that ADCLEC.syn1 outperforms a single-ADGRE2-CAR lacking assistance via CLEC12A-CCR. Importantly, ADCLEC.syn1 also outperformed an otherwise identical alternative dual-CAR version (OR-gated ADGRE2-CAR+CLEC12A-CAR) in the setting of both ADGRE2-high and ADGRE2-low MOLM13, further underlining the importance of fine-tuned overall signaling. We confirmed high in-vivo potency against diverse AML cell lines with a wide range of ADGRE2 and CLEC12A levels reflecting population-wide AML heterogeneity. At clinically relevant CAR T cell doses, ADCLEC.syn1 induced complete and durable remissions in xenograft models of MOLM13 (ADGRE2-high/CLEC12A-low) and U937 (ADGRE2-low/CLEC12A-high). ADCLEC.syn1 CAR T cells were found to be functionally persistent for >70 days, with a single CAR T cell dose potently averting relapse modeled via AML re-challenges. In summary, we provide pre-clinical evidence that an "IF-BETTER"-gated CAR+CCR T cell (ADCLEC.syn1) can outperform a single-CAR T cell (ADGRE2-CAR) and a dual-CAR T cell (ADGRE2-CAR+CLEC12A-CAR). ADCLEC.syn1 enhances antileukemic efficacy and prevents antigen-low AML escape via detection of a rationally selected combinatorial target antigen signature that is commonly found in AML but limited in vital normal cells. Using phenotypically representative AML xenograft models and clinically relevant T cell doses, we demonstrate high therapeutic potential of ADCLEC.syn1 CAR T cells, further supporting clinical translation of an "IF-BETTER"-gated CAR concept into a phase 1 trial. Disclosures Haubner: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties: Co-inventor of IP that MSK licensed to Takeda, Research Funding. Mansilla-Soto: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Fate Therapeutics: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties. He: Takeda Pharmaceuticals Company Ltd.: Ended employment in the past 24 months, Patents & Royalties. Park: Curocel: Consultancy; BMS: Consultancy; Innate Pharma: Consultancy; Autolus: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy; Amgen: Consultancy; Kura Oncology: Consultancy; Artiva: Consultancy; Novartis: Consultancy. Rivière: Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); St. Jude Children's Research Hospital: Other: Provision of Services; Minerva Biotechnologies: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Ceramedix: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; Atara Biotherapeutics: Patents & Royalties.

PL03.3.A Development and characterization of CD317-specific CAR T cells as an innovative immunotherapeutic strategy against glioblastoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii2-ii2
Author(s):  
L Hänsch ◽  
M Peipp ◽  
R Myburgh ◽  
M Silginer ◽  
T Weiss ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Glioma Cells ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND Due to the limited success of existing therapies for gliomas, innovative therapeutic options are urgently needed. Chimeric antigen receptor (CAR) T cell therapy has been successful in patients with hematological malignancies. However, using this treatment against solid tumors such as glioblastomas is more challenging. Here, we generated CAR T cells targeting the transmembrane protein CD317 (BST-2, HM1.24) which is overexpressed in glioma cells and may therefore serve as a novel target antigen for CAR T cell-based immunotherapy. MATERIAL AND METHODS CAR T cells targeting CD317 were generated by lentiviral transduction of human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells was determined in lysis assays using different glioma target cell lines with varying CD317 expression levels. The efficiency of CD317-CAR T cells to control tumor growth in vivo was evaluated in clinically relevant orthotopic xenograft glioma mouse models. RESULTS We created a second-generation CAR construct targeting CD317 and observed strong anti-glioma activity of CD317-CAR T cells in vitro. Glioma cells with a CRISPR/Cas9-mediated CD317 knockout were resistant to CD317-specific CAR T cells, demonstrating their target antigen-specificity. Since CD317 is also expressed by T cells, transduction with a CD317-directed CAR resulted in fratricide of the transduced T cells. Silencing of CD317 in CAR T cells by integrating a specific shRNA into the CAR vector significantly increased the viability, proliferation and cytotoxic function of the CAR T cells. Importantly, intratumoral treatment with CD317-CAR T cells prolonged the survival and cured a significant fraction of glioma-bearing nude mice. CONCLUSION We demonstrate strong CD317-specific anti-tumor activity of CD317-CAR T cells against various glioma cell lines in vitro and in xenograft glioma models in vivo. These data lay a scientific basis for the subsequent evaluation of this therapeutic strategy in clinical neuro-oncology.

scholarly journals Understanding the CD8+ CAR T Cell Response to Antigen-Low Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 736-736
Author(s):  
Kole Degolier ◽  
Jennifer Cimons ◽  
Michael Yarnell ◽  
Mark Eric Kohler ◽  
Terry J. Fry
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Response ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Tumor Clearance

Abstract Chimeric antigen receptor (CAR) T cell therapy has emerged as a highly efficacious treatment for B-lineage acute lymphoblastic leukemias (B-ALL). However, downregulation of the CAR-targeted antigen on leukemia cells, predicted to reduce cellular avidity, is associated with post-CAR T cell leukemic relapse following CD22 CAR treatment (Fry et al., Nat. Med., 2017). We have observed reduced function of human CAR T cells against low target antigen site density (Ag Lo) human leukemia in immunodeficient mouse models, relative to CARs responding to high-antigen expressing leukemia. Thus, a better understanding of CAR responses to Ag Lo leukemia could help to increase the durability of remissions. We set out to develop a model system in which we could further interrogate the consequences of low-avidity interactions on CAR immunobiology, generating variants of a murine B-ALL driven by the E2A-PBX fusion protein (E2A) with different levels of target antigen to use in an immunocompetent syngeneic mouse model. We observed impaired expansion (p<0.0001) and tumor clearance (p<0.001) of CAR T cells responding to low-antigen variants of E2A (E2A-Ag Lo) as compared to wildtype E2A expressing high levels of antigen (E2A-WT). While CD8+ CAR T cell (CAR8) transcription factor (TF) expression in response to E2A-Ag Lo versus E2A-WT was largely similar early after CAR infusion, by day 9 post-CAR, CAR8s responding to E2A-Ag Lo exhibited decreased expression of multiple TFs, with Eomes (p<0.01), Irf4 (p<0.001) and Blimp1 (p<0.01) showing the largest magnitude change relative to CAR8s responding to E2A-WT. Additionally, CAR8s from mice bearing E2A-Ag Lo became enriched for cells of a "terminally exhausted" phenotype (Eomes+/PD1 Hi/TOX Hi) by day 11 post-CAR, and negatively-enriched for the "progenitor exhausted" (Tcf1+/PD1 Int) phenotype which can be functionally rescued by anti-PD1 therapy (p<0.0001, p<0.01). These data suggest that continual stimulation by low density antigen leads to a gradual reduction in the ability of CAR8s to mount an effector response, and eventually to T cell states with sub-optimal anti-tumor efficacy. Following in vitro stimulation of human CD22 CARs across a range of leukemic antigen densities, we saw that the percentage of CAR+ cells capable of producing IFNγ and IL2 corresponded to target antigen density (p<0.01, p<0.001). As human CARs are commonly manufactured from heterogenous bulk donor T cells, we hypothesized that antigen sensitivity is impacted by the prior antigen-experience of a given T cell. We predicted that T cells which had encountered cognate antigen through their TCR prior to CAR manufacturing (CAR8 AgEx) would have enhanced capacity to respond to low-avidity stimulation compared to CARs manufactured from naïve CD8+ T cells (CAR8 Naïve). We used a well-characterized ovalbumin vaccination model with OT-I TCR-transgenic T cells, allowing defined control of T cell antigen experience, to generate CAR8 AgEx. We found that CAR8 AgEx were highly antigen-sensitive relative to CAR8 Naïve, showing almost no reduction in numbers of cells capable of producing IFNγ and TNFα in vitro against E2A-Ag Lo as compared to E2A-WT. In vivo, CAR8 AgEx showed near complete depletion of E2A-Ag Lo in bone marrow by day 11 post-CAR, while mice treated with CAR8 Naïve maintained a substantial tumor burden (p<0.01). To test our hypothesis in human cells, we manufactured CD22 CAR T cells from naïve (CD45RO-) versus non-naïve (CD45RO+) starting T cell populations, and again found that CAR AgEx outperformed CAR Naïve against Ag Lo leukemia in production of IFNγ and IL2 in vitro (p<0.001, p<0.01) and in early leukemic clearance in vivo (p<0.0001, day 13). In conclusion, we have established a model to study the immunobiology of the CAR T cell response to Ag Lo B-ALL in an intact host. Preliminary findings indicate impaired expansion and tumor clearance of Ag Lo leukemia, associated with altered CAR T cell transcriptional profiles and features of T cell exhaustion. Furthermore, T cell history prior to CAR manufacturing has a drastic impact on the capacity to respond to Ag Lo leukemia. Future studies with this model will expand our characterization of CAR T cells responding to Ag Lo leukemia, with the goal of optimizing antigen sensitivity. We expect that advancing our understanding on the interplay of antigen density and CAR differentiation status will prove useful in developing more effective iterations of this therapy. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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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