scholarly journals Bispecific CAR-T Cells Targeting Both BCMA and CD24: A Potentially Treatment Approach for Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2802-2802
Author(s):  
Fumou Sun ◽  
Yan Cheng ◽  
Bailu Peng ◽  
Hongwei Xu ◽  
Frits van Rhee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Car T Cells ◽  
Co Activation ◽  
Car T

Abstract Introduction: Anti-myeloma BCMA-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising new treatment strategy, with high response rates observed in the early stages of therapy. However, the responses are not durable. One known mechanism of relapse has been traced to the loss of BCMA expression following long-term CAR-T therapy. Another potential reason is that while BCMA CAR-T cells eliminate the bulk of BCMA-positive MM cells, a small subset of BCMA-negative, very drug-resistant MM cells, such as tumor-initiating cells (TICs) survive and seed relapses. There is a strong correlation between the presence of MM TICs with minimal residual disease, acquired drug resistance and relapse. This suggests that TIC-targeted therapies could improve outcomes. We have previously demonstrated that MM cells expressing CD24 also exhibit features of TICs, e.g. self-renewal, increased expression of embryonic stem cell genes and drug resistance. We have generated bispecific CAR-T cells which recognize both BCMA and CD24 antigens and have tested their therapeutic efficacy in MM cells in vitro and in vivo models. Methods: We constructed a bispecific BCMA-CD24 CAR vector, with 2 complete CAR units: BCMA CAR and CD24 CAR. P2A was inserted between these two CARs. The BCMA CAR contains a safety switch in the hinge region, and a CD28 co-activation domain with CD3ζ. The CD24 CAR contained a 4-1BB co-activation domain with CD3ζ. To decrease the risk of severe immunological side effects, we integrated RQR8, an immunological safety switch with epitopes for CD34 and CD20 as a suicide molecule into the hinge region. Lentivirus particles were used to transduce primary human T cells. CAR-T cells were detected on day 7 by flow cytometry using antibodies to CD34. We performed co-culture killing assays, detected the T cell activation marker CD69 and measured the cytokines in the supernatant. We determined whether BCMA-CD24 CAR-T cells targeted the TIC population by flow cytometry and microscopy. The NOD. Cg-Prkdc scidIl2rg tm1Wjl/SzJ (NSG) xenograft mouse model was used for in vivo studies. 8-week-old NSG mice were administered 2 × 10 6 MM cells by intravenous injection. On day 7 after MM cells injection, 1 × 10 6 CAR-T cells were administered. Mice were weighed and monitored for signs of distress every two days. Bioluminescence images were acquired 10 min after D-luciferin injection. Myeloma progression was monitored every 7 days until the mice develop hind limb paralysis or the bioluminescence signal (ROI) is more than 2 × 10 10. Results: CAR-T cells were detected by flow cytometry using the RQR8-specific CD34 antibody. The BCMA-CD24 CAR was found to be expressed on roughly 13% of T-cells. To determine the selective lysis by the CAR-T cells, we performed co-culture killing assays in which MM cell lines over-expressing CD24 (ARP-1 CD24OE or OCI CD24OE cells) were incubated with CAR-T cells. When the CAR-T: MM ratio was 5:1, the lysis percentage of target cells was 99% (ARP-1 CD24OE) and 89% (OCI CD24OE). CAR-T cell activation was determined by increased CD69 expression and IL-2 production. As expected, exposure to CD24 + MM cells resulted in strong activation of CAR-T cells, and CAR-T cells did target and kill the TIC population. Bioluminescence imaging showed CAR-T mediated antitumor activity, yielding near-complete tumor clearance. Additionally, mice treated with CAR-T cells exhibited increased survival compared with mice in the control groups. Conclusion and Significance: This study developed a BCMA-CD24 CAR-T, a novel MM immunotherapy. We have demonstrated strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. Future studies will be aimed at determining if BCMA-CD24 CAR-T can target TIC-mediated relapses. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1690-1690
Author(s):  
Xiaomeng Hu ◽  
Mo Dao ◽  
Kathy White ◽  
Corie Gattis ◽  
Ryan Clarke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T

Abstract Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction as well as the ability to generate a final product from healthy T cells. While TCR editing can effectively prevent graft-versus-host reactions, the significant host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells (N Engl J Med. 2021;384(7):673-674). We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced by this strategy. Since CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to delete b2m, CIITA, and TCR expression and lentiviral transduction was used to overexpress CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CAR T cells. Control T cells were unmanipulated except for lentiviral transduction used to overexpress the same CD19CAR and the deletion of the TCR. When transplanted into allogeneic humanized mice, hypoimmune CD19CAR T cells evade immune recognition by T cells even in previously sensitized animals as evidenced by a lack of T cell activation measured using ELISPOT analysis. In contrast, transplantation of non-hypoimmune-edited CD19CAR T cells generated from the same human donor resulted in a significant T cell activation (see figure: mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and non-edited CD19CAR T cells, respectively; p<0.0001 unpaired T-test). In addition to evading T cells, immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmune CAR T cells susceptible to macrophage and NK cell killing in vitro and in vivo, confirming the importance of CD47 overexpression to evade innate immune clearance. The hypoimmune CD19 CAR T cells retained their antitumor activity in both the Daudi and Nalm-6 B cell leukemia models, in vitro and in vivo. This indicated that the hypoimmune technology-i.e. isolated CD47 overexpression, deletion of b2m, CIITA, and TCR- did not show any effect on the cytotoxic potential of CD19 CAR T cells (see figure). These studies demonstrate that in vivo clearance of leukemic cells in NSG mice occurs across a range of tumor cell toCD19 CAR T cell ratios in a manner comparable to control, unedited CD19 CAR T cells (see figure). This result was validated using T cells from 3 different donors These findings show that, in these models, hypoimmune CD19 CAR T cells are functionally immune evasive in allogeneic humanized mouse recipients and have cytotoxic anti-tumor capacity. They suggest that hypoimmune CAR T cells could provide universal CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that hypoimmune CD19 CAR T cells can be used in sensitized patients and for re-dosing strategies. Figure 1 Figure 1. Disclosures Hu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dao: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. White: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Gattis: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Clarke: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Landry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Basco: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tham: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tucker: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Luo: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Bandoro: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Chu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Young: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dowdle: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Rebar: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Schrepfer: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Mouse CD19-Targeted Chimeric Antigen Receptors That Include a 41BB Co-Stimulatory Domain Induce NFkB Signaling to Enhance T Cell Proliferation, Viability, and Leukemia Killing

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 843-843
Author(s):  
Gongbo Li ◽  
Nolan Beatty ◽  
Paresh Vishwasrao ◽  
Justin C. Boucher ◽  
Bin Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Car T Cells ◽  
Apoptosis Protein ◽  
Car T

Abstract CD19 targeted 2nd generation chimeric antigen receptor T (CAR T) cells have been successful against relapsed and/or refractory B cell malignancies. The pending FDA-approval of 2 separate CD19 targeted CAR T products highlight the need to understand the biology behind this novel therapy. CAR design includes a single-chain variable fragment, which encodes antigen-binding, fused to a transmembrane domain, co-stimulatory domain, and CD3ζ activation domain. The two CARs likely to be approved as standard of care include a 41BB or CD28 co-stimulatory domain. CD28 is a critical co-stimulatory receptor required for full T cell activation and persistence, while 4-1BB is a member of the tumor necrosis factor receptor family and also a critical T cell co-stimulatory factor. Early evaluation of the co-stimulatory domains role in CAR design confirmed that they are required to enhance T cell function, but lacked insight regarding their mechanism for this enhancement. Furthermore, clinical outcomes suggest that the co-stimulatory domains in CARs support different T cell functions in patients. For example, while overall outcomes are similar between 41BB (19BBz) and CD28-containing CARs (1928z), 19BBz CAR T cells can persist for years in patients, but functional 1928z CAR T cells rarely persist longer than a month. Recent studies are providing insight to these differences and have demonstrated that 4-1BB-containing CARs reduce T cell exhaustion, enhance persistence, and increase central memory differentiation and mitochondrial biogenesis, while CD28-containing CARs support robust T cell activation and exhaustion, and are associated with effector-like differentiation. However, these studies have been performed mostly in vitro or in immune deficient mice, which limits their ability to model complex immune biology. Therefore, we evaluated murine CD19-targeting CARs with a 4-1BB (m19BBz) or CD28- (m1928z) co-stimulatory domain in relevant animal models of immunity. We directly compared m19BBz and m1928z CAR T cell immune phenotype, cytotoxicity, cytokine production, gene expression, intracellular signaling, and in vivo persistence, expansion, and B cell acute lymphoblastic leukemia (B-ALL) eradication. In vitro assays revealed that m1928z CAR T cells had enhanced cytotoxicity and cytokine production compared to m19BBz CAR T cells. Also, evaluation of m1928z and m19BBz CAR T cells displayed similar immune phenotypes, but markedly different gene expression with m1928z CAR T cells upregulating genes related to effector function and exhaustion, while m19BBz CAR upregulated genes critical for NFkB regulation, T cell quiescence and memory. In vivo, both m1928z and m19BBz CAR T cells supported equivalent protection against B-ALL. Similar to patients, in our mouse models there are functional differences between the mouse CD19-targeted CAR T cells. At 1 week post-infusion m19BBz CAR T cells are present in the blood of mice at significantly greater levels than m1928z CAR T cells. Furthermore, m19BBz CAR T cells enhance proliferation and/or anti-apoptosis protein expression to enhance B cell killing, which is evidenced by our observation that irradiation significantly weakens the in vivo efficacy of m19BBz but not m1928z CAR T cells. Our results suggest that B cell killing by m1928z CAR T cells is not impacted by irradiation because of their efficacious cytotoxicity of B cells. In contrast, m19BBz CAR T cells have enhanced viability and anti-apoptosis protein expression, which allows them to compensate for reduced effector function. We investigated potential mechanisms for the enhanced viability and anti-apoptosis of m19BBz CAR T cells and determined that NFkB signaling is upregulated much greater by m19BBz than m1928z. We have observed this difference in both a reporter cell line and primary mouse T cells. We are now dissecting what cellular components mediate increased NFkB signaling by the m19BBz CAR. Our animal models recapitulate equivalent anti-leukemia efficacy of CD19-targeted CAR T cells regardless of co-stimulatory domain, but underscore that anti-leukemia killing is mediated by different methods depending on the co-stimulatory domain. Our work sheds light on how 4-1BB mechanistically regulates and impacts CAR T function and has implications for future CAR design and evaluation. Disclosures No relevant conflicts of interest to declare.

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).

scholarly journals 206 The development of ‘chimeric CD3e fusion protein’ and ‘anti-CD3-based bispecific T cell activating element’ engineered T (CAB-T) cells for the treatment of solid malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A217-A217
Author(s):  
Andy Tsun ◽  
Zhiyuan Li ◽  
Zhenqing Zhang ◽  
Weifeng Huang ◽  
Shaogang Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vivo Studies ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

BackgroundCancer immunotherapy has achieved unprecedented success in the complete remission of hematological tumors. However, serious or even fatal clinical side-effects have been associated with CAR-T therapies to solid tumors, which mainly include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome, etc. Furthermore, CAR-T therapies have not yet demonstrated significant clinical efficacy for the treatment of solid tumors. Here, we present a novel T cell therapeutic platform: a Chimeric CD3e fusion protein and anti-CD3-based bispecific T cell activating element (BiTA) engineered T (CAB-T) cells, which target tumor antigens via the secretion of BiTAs that act independently of MHC interactions. Upon BiTA secretion, CAB-T cells can simultaneously achieve anti-tumor cytotoxic effects from the CAB-T cells and simultaneously activate bystander T cells.MethodsCAB-T cells were generated by co-expressing a chimeric CD3e fusion protein and an anti-CD3-based bispecific T cell activating element. The chimeric CD3e contains the extracellular domain of CD3e, a CD8 transmembrane domain, 4-1BB costimulatory domain, CD3z T cell activation domain and a FLAG tag, while the BiTA element includes a tumor antigen targeting domain fused with an anti-CD3 scFv domain and a 6x His-tag. CAR-T cells were generated as a control. Cytokine release activity, T cell activation and exhaustion markers, T cell killing activity and T cell differentiation stages were analysed. We also tested their tumor growth inhibition activity, peripheral and tumor tissue distribution, and their safety-profiles in humanized mouse models.ResultsCAB-T cells have similar or better in vitro killing activity compared with their CAR-T counterparts, with lower levels of cytokine release (IL-2 and IFNγ). CAB-T cells also showed lower levels of exhaustion markers (PD-1, LAG-3 and TIM-3), and higher ratios of naive/Tscm and Tcm T cell populations, after co-culture with their target tumor cells (48h). In in vivo studies, CAIX CAB-T and HER2 CAB-T showed superior anti-tumor efficacy and tumor tissue infiltration activity over their corresponding CAR-T cells. For CLDN18.2 CAB-T cells, similar in vivo anti-tumor efficacy was observed compared to CAR-T after T cell infusion, but blood glucose reduction and animal mortality was observed in the mice administered with CAR-T cells.ConclusionsThe advantages of CAB-T in in vitro and in vivo studies may result from TCR signal activation of both the engineered CAB-T cells and the non-engineered bystander T cells via cross-bridging by the secreted BiTA molecules, thus offering superior anti-tumor efficacy with a potential better safety-profile compared to conventional CAR-T platforms.

scholarly journals Engineering genetic devices for in vivo control of therapeutic T cell activity triggered by the dietary molecule resveratrol

2021 ◽  
Vol 118 (34) ◽  
pp. e2106612118
Author(s):  
Linfeng Yang ◽  
Jianli Yin ◽  
Jiali Wu ◽  
Longliang Qiao ◽  
Evan M. Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Transgene Expression ◽  
Cell Activation ◽  
Cell Activity ◽  
Cell Therapies ◽  
Car T

Chimeric antigen receptor (CAR)–engineered T cell therapies have been recognized as powerful strategies in cancer immunotherapy; however, the clinical application of CAR-T is currently constrained by severe adverse effects in patients, caused by excessive cytotoxic activity and poor T cell control. Herein, we harnessed a dietary molecule resveratrol (RES)–responsive transactivator and a transrepressor to develop a repressible transgene expression (RESrep) device and an inducible transgene expression (RESind) device, respectively. After optimization, these tools enabled the control of CAR expression and CAR-mediated antitumor function in engineered human cells. We demonstrated that a resveratrol-repressible CAR expression (RESrep-CAR) device can effectively inhibit T cell activation upon resveratrol administration in primary T cells and a xenograft tumor mouse model. Additionally, we exhibit how a resveratrol-inducible CAR expression (RESind-CAR) device can achieve fine-tuned and reversible control over T cell activation via a resveratrol-titratable mechanism. Furthermore, our results revealed that the presence of RES can activate RESind-CAR T cells with strong anticancer cytotoxicity against cells in vitro and in vivo. Our study demonstrates the utility of RESrep and RESind devices as effective tools for transgene expression and illustrates the potential of RESrep-CAR and RESind-CAR devices to enhance patient safety in precision cancer immunotherapies.

94 The T cell antigen coupler (TAC) redirects T cell oncolysis while limiting tonic signaling to create a safer engineered T cell product with a higher threshold for activation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A105-A105
Author(s):  
Duane Moogk ◽  
Vivian Lau ◽  
Arya Afsahi ◽  
Anna Dvorkin-Gheva ◽  
Ksenia Bezverbnaya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Binding Domain ◽  
Antigen Binding ◽  
Car T Cells ◽  
Car T

BackgroundThe T cell Antigen Couper (TAC) is a chimeric receptor that redirects the endogenous T cell receptor (TCR) against a tumor target via an extracellular antigen-binding domain to induce activation and oncolysis. TAC-engineered T cells (TAC-T cells) showed a similar capacity to activate T cells against the tumor-associated antigen HER2 as their classical chimeric antigen receptor (CAR)-engineered counterparts in vitro. However, in a xenograft model, anti-HER2 CAR-T cells gave rise to lethal off-target toxicity while TAC-T cells were efficacious and well-tolerated, despite utilizing the same antigen-binding domain.1 2 Here, we describe differences in T cell activation by TAC (canonical via endogenous TCR) and CAR (non-canonical independent of TCR) that make CAR-T cells less discriminate towards an off-target stimulus than TAC-T cells.MethodsPaired sets of TAC- and CAR-engineered human T cells, utilizing a variety of antigen-binding domains, were compared in vitro to determine their propensity for tonic signaling and requirements for triggering T cell activation.ResultsTranscriptional profiling of CAR- and TAC- T cells in the absence of antigenic stimulus revealed an elevated basal activation status in CAR-T cells. Unstimulated CAR-T cells displayed elevated expression levels of activation and exhaustion markers, as well as basal cytokine production, versus their TAC-T cell counterparts. The degree of basal activation varied with the binding domain incorporated into the CAR, where some binding domains triggered functional exhaustion. Regardless of the binding domain, unstimulated TAC-T cells were indistinguishable from control T cells that expressed no synthetic receptor. Further, TAC-T cells displayed no evidence of functional exhaustion. TCR knock-out studies confirmed that TAC receptors signal via the endogenous TCR, whereas CAR signaling is TCR-independent. Consistent with TCR-dependent signaling, ligation of TAC receptors resulted in the formation of conventional immunological synapses, whereas ligation of CARs produced unconventional synapses. Despite these functional differences, CARs and TAC receptors demonstrated a similar capacity to activate T cells against antigen-positive tumor cell targets. However, CAR-T cells displayed reactivity to antigen-negative cells, due to interaction with a cross-reactive antigen; TAC-T cells displayed no reactivity to antigen-negative cells.ConclusionsTonic signaling in CAR-T cells reduces their activation threshold and increases their propensity to be activated by cross-reactive antigen. In contrast, TAC receptors do not deliver tonic signals, which increases the stringency of activation and reduces the likelihood of off-target responses. This feature of the TAC platform is advantageous to safeguard against the unexpected cross-reactivity that may occur when a new antigen-binding domain is deployed in vivo.Ethics ApprovalUse of human materials was approved by the Hamilton Integrated Research Ethics Board (HiREB).ReferencesHelsen C, Hammill JA, Lau VWC, Mwawasi KA, Afsahi A, Bezverbnaya K, Newhook L, Hayes DL, Aarts C, Bojovic B, Denisova GF, Kwiecien JM, Brain I, Derocher H, Milne K, Nelson BH, Bramson JL. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 2018;9:3049.Hammill JA, Kwiecien JM, Dvorkin-Gheva A, Lau VWC, Baker C, Wu Y, Bezverbnaya K, Aarts C, Helsen CW, Denisova GF, Derocher H, Milne K, Nelson BH, Bramson JL. A cross-reactive small protein binding domain provides a model to study off-tumor CAR-T cell toxicity. Mol Ther Oncolytics 2020;17:278–292.

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

107 Effects of IL-2 and IL-15 on the proliferative and antitumor capacities of allogeneic CD20 CAR-engineered γδ T cells in a 3D B cell lymphoma spheroid assay

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A119-A119
Author(s):  
Lu Bai ◽  
Kevin Nishimoto ◽  
Mustafa Turkoz ◽  
Marissa Herrman ◽  
Jason Romero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Car T

BackgroundAutologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells.MethodsMolecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine.ResultsWhen compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production.ConclusionsTogether, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

scholarly journals P07.02 Regulation of CD19 CAR T- cell activation based on engineered Nuclear factor of activated T cells artificial transcription factors

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A23-A23
Author(s):  
D Lainšček ◽  
V Mikolič ◽  
Š Malenšek ◽  
A Verbič ◽  
R Jerala
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Activity ◽  
External Control ◽  
Car T Cells ◽  
Car T Cell ◽  
Artificial Transcription Factors ◽  
Car T

BackgroundCD19 CAR T- cells (Chimeric antigen receptor T cells that recognize CD19) present a therapeutic option for various malignant diseases based on their ability to specifically recognize the selected tumour surface markers, triggering immune cell activation and cytokine production that results in killing cancerous cell expressing specific surface markers recognized by the CAR. The main therapeutic effect of CAR is a specific T cell activation of adequate cell number with sequential destruction of tumorous cells in a safe therapeutic manner. In order to increase T cell activation, different activation domains were introduced into CAR. CAR T-cells are highly efficient in tumour cell destruction, but may cause serious side effects that can also result in patient death so their activity needs to be carefully controlled.1 Several attempts were made to influence the CAR T cell proliferation and their activation by adding T cell growth factors, such as IL-2, into patients, however this approach of increasing the number of activating T cells with no external control over their number can again lead to non-optimal therapeutic effects. Different improvements were made by designing synthetic receptors or small molecule-inducible systems etc., which influence regulated expansion and survival of CAR T cells.2Material and MethodsIn order to regulate CD19 CAR-T cell activity, different NFAT2 based artificial transcription factors were prepared. The full length NFAT2, one of the main players in T cell IL2 production, a key cytokine for T cell activation and proliferation was truncated by deletion of its own activation domain. Next, we joined via Gibson assembly tNFAT21-593 coding sequence with domains of different heterodimerization systems that interact upon adding the inductor of heterodimerization. The interaction counterparts were fused to a strong tripartite transcriptional activator domain VPR and/or strong repressor domain KRAB resulting in formation of an engineered NFAT artificial transcription (NFAT-TF) factors with external control. To determine the activity of NFAT-TF HEK293, Jurkat or human T cells were used.ResultsBased on luciferase assay, carried out on NFAT-TF transfected HEK293 cells we first established that upon adding the external inductor of heterodimerization, efficient gene regulation occurs, according to VPR or KRAB domain appropriate functions. Findings were then transferred to Jurkat cells that were electroporated with appropriate DNA constructs, coding for NFAT-TF and CD19 CAR. After Raji:Jurkat co-culture ELISA measurements revealed that IL2 production and therefore CD19 CAR-T cell activity can be controlled by the action of NFAT-TF. The same regulation over the activity and subsequent proliferation status was also observed in retrovirally transduced human T-cells.ConclusionWe developed a regulatory system for therapeutic effect of CD19 CAR-T cells, a unique mechanism to control T cell activation and proliferation based on the engineered NFAT2 artificial transcription factor.ReferencesBonifant CL, et al. Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 2016;3:16011.Wu C-Y, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 2015;80:350.Disclosure InformationD. Lainšček: None. V. Mikolič: None. Š. Malenšek: None. A. Verbič: None. R. Jerala: None.

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