Unicar: A Novel Modular Retargeting Platform Technology for CAR T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5549-5549 ◽  
Author(s):  
Marc Cartellieri ◽  
Simon Loff ◽  
Malte von Bonin ◽  
Elham P. Bejestani ◽  
Armin Ehninger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cell ◽  
Binding Domain ◽  
Single Chain ◽  
Chain Fragment ◽  
Cell Recruitment ◽  
Single Chain Fragment Variable ◽  
Car T ◽  
Single Chain Fragment

Abstract The adoptive transfer of T cells engineered with chimeric antigen receptors (CARs) is currently considered as a highly promising therapeutic option for treatment of otherwise incurable malignant diseases. CARs combine the cellular and humoral arm of the immune response by assembling a single-chain fragment variable (scFv) as binding moiety which provides the antigen-specificity and an activating immune receptor. It has been demonstrated both in vitro and in vivo, that CAR engrafted effector T cells mediate long-lasting anti-tumor responses. Despite encouraging clinical efficacy targeting CD19 in recent clinical trials, the appearance of potentially life-threatening adverse reactions and the lack of control mechanisms once initiated, prevent more widespread application of the CAR technology. To overcome limitations of conventional CAR T cells, a unique chimeric antigen receptor (UniCAR) technology was developed (Fig. 1) which allows precise control of CAR T cell reactivity, thus lowering the risk of side effects while preserving efficacy. Moreover, the UniCAR technology enables the retargeting of engrafted T cells against more than one antigen simultaneously or subsequently, thus reducing the risk for development of antigen-loss tumor variants under treatment. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CAR into two individual components. T cells are engineered to express a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein. Thus, T cells engineered to express UniCAR remain inactivated after re-infusion, since the UniCAR target is not available for binding under physiological conditions. The ultimate antigen-specificity of the system is provided separately by targeting modules (TMs) comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR binding domain. Here we provide first in vitro and in vivo prove of concept for this new approach. Antigen-specific redirection of T cells armed with the universal CAR in the presence of different targeting modules against various antigens (CD33, CD123, CD19, CD20, PSCA, PSMA,) was effective at femtomolar concentrations of the targeting module both. Taken together, the modular nature of UniCAR technology will allow retargeting of autologous, patient-derived T cells to several antigens under controlled pharmacological conditions and has the potential to become a highly effective treatment option for late stage cancer patients with reduced risks for side effects. Figure 1. Schematic representation of T cell recruitment with the modular UniCAR system. The UniCAR T cell recruitment system consists of two separated units. The first unit is the UniCAR expressed on T cells with a single-chain fragment variable (scFv) specific for a short 10 aa long peptide motif. The intracellular signalling domain of the UniCAR contains a costimulatory domain derived from CD28 and the T cell receptor z chain. The second unit is a targeting molecule (TM) which consists of a scFv fused to the peptide epitope. The cross-linkage of T cell and target cell is mediated by interaction between the UniCAR binding domain on T cells and target cell binding TM. Figure 1. Schematic representation of T cell recruitment with the modular UniCAR system. / The UniCAR T cell recruitment system consists of two separated units. The first unit is the UniCAR expressed on T cells with a single-chain fragment variable (scFv) specific for a short 10 aa long peptide motif. The intracellular signalling domain of the UniCAR contains a costimulatory domain derived from CD28 and the T cell receptor z chain. The second unit is a targeting molecule (TM) which consists of a scFv fused to the peptide epitope. The cross-linkage of T cell and target cell is mediated by interaction between the UniCAR binding domain on T cells and target cell binding TM. Disclosures Cartellieri: Cellex Patient Treatment GmbH: Employment. Loff:GEMoaB Monoclonals GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership; Cellex Patient Treatment GmbH: Equity Ownership. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.

Excessively High-Affinity Single-Chain Fragment Variable Region in a Chimeric Antigen Receptor Can Counteract T-Cell Proliferation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4799-4799 ◽  
Author(s):  
Keisuke Watanabe ◽  
Seitaro Terakura ◽  
Susumu Uchiyama ◽  
Anton C. Martens ◽  
Tom van Meerten ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Variable Region ◽  
Single Chain ◽  
Chain Fragment ◽  
Car T Cells ◽  
Single Chain Fragment Variable ◽  
Car T ◽  
Anti Cd20

Abstract Background: Single-chain fragment variable region (scFv) in a chimeric antigen receptor (CAR) is a key component that directly binds the target antigen and transmits an activating signal into the CAR-T cells, subsequently triggering its effector function against the target cell. Thus, the affinity of scFv is considered to be critically important for CAR-T-cell function. However, optimal scFv affinity to induce maximal CAR function remains unclear. Methods: In this study, we constructed anti-CD20 scFv based on the reported sequence of humanized anti-CD20 monoclonal antibody with five different affinities: 20-vv (Kd value=7.07 nM), -fv (7.21), -fa (10.09), -sv (12.56), and -sa (14.66). Each scFv was then fused to an IgG4 hinge, a CD3-zeta chain, a CD28 costimulatory domain, and a truncated version of the epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD8-positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CARs on day 3, and enriched by selection with anti-EGFR mAb. CAR-T cells were expanded using anti-CD3/28 beads and used in the subsequent analysis. Results: To determine the lytic activity according to the scFv affinity, we tested cytotoxicity against CD20-transduced K562 cells (K562-CD20) by 51Cr releasing assay. CAR-T cells with the three highest-affinity scFv (20CAR-vv, -fv, -fa) effectively and equally lysed K562-CD20, while CAR-T cells with the two lowest-affinity scFv (20CAR-sv, -sa) failed to exhibit significant cytotoxicity (20CAR-vv, -fv, -fa, -sv, and -sa: 60.2±2.85%, 57.3±2.03%, 57.0±3.82%, 2.8±0.1%, and 2.0±0.64% at an E:T ratio = 10:1, respectively). To examine CAR-T-cell activation, cytokine secretion in the supernatant after 16 hours of culture with K562-CD20 was analyzed. Similarly to the cytotoxicity, 20CAR-vv, -fv, and -fa produced cytokine, but 20CAR-sv and -sa did not. The highest-affinity CAR-T (20CAR-vv) produced significantly higher IFN-g and IL-2 than the other CAR-T cells [IFN-g (pg/ml): 7644±326, 3380±179, 4718±315, 4.9±0.4, and 5.4±1.2; IL-2 (pg/ml): 4179±177, 1071±30, 1379±118, 0, and 0, respectively]. We next analyzed cell proliferation upon stimulation with K562-CD20 over 72 hours. 20CAR-vv resulted in rather poor absolute cell proliferation, while CAR-T cells with the second and third highest-affinity scFv (20CAR-fv, -fa) resulted in preferable proliferation (1.22±0.20-, 2.52±0.12-, 2.01±0.42-, 0.65±0.11-, and 1.15±0.13-fold expansion, respectively). To explore the possible reason for the difference of CAR-T proliferation, we evaluated activation-induced cell death (AICD) at 24 hours after the K562-CD20 stimulation (Annexin-V/PI staining). 20CAR-vv underwent significantly higher AICD than the other CAR-T cells (68.4±1.1, 39.6±0.7, 37.9±5.3, 25.9±11.2, and 16.1±1.2% cell death). Finally, we tested cell proliferation and AICD of the three highest-affinity scFv CAR-T cells (-vv, -fv, and -fa) upon CD20 high and low stimulation. In this experiment, we used CD20-transduced CEM cells, which are controlled to express high and low CD20: CEM-CD20 high (CD20 = 143,000 molecules/cell, the same level as other B-cell lines) and CEM-CD20 low (5,320 molecules/cell, the same level as the rituximab refractory cell line). The replacement of the stimulator from CD20-CEM high to low resulted in partial reduction of AICD to a level comparable to those of other 20CARs (-vv, 62.7%→33.0%, -fv, 56.0%→37.4%, –fa, 55.3%→27.7%), but the cell proliferation upon stimulation did not improve (-vv, 1.2-, -fv, 2.6-, -fa, 2.7-fold expansion after CD20-CEM low stim). These results suggest that the higher-affinity scFv was not necessarily associated with better CAR-T proliferation after ligation and may inhibit cell proliferation, possibly by multiple factors besides AICD. Conclusions: We observed that scFv with affinity above a certain level is needed to induce CAR-T function. Higher-affinity scFv induced more robust cytokine production; however, excessively high-affinity scFv in CAR can counteract T-cell proliferation, which may be partially associated with increased AICD. Optimization of scFv affinity is desirable for effective CAR-T production. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tonic Signaling Leads to Off-Target Activation of T Cells Engineered with Chimeric Antigen Receptors That Is Not Seen in T Cells Engineered with T Cell Antigen Coupler (TAC) Receptors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Duane Moogk ◽  
Arya Afsahi ◽  
Vivian Lau ◽  
Anna Dvorkin-Gheva ◽  
Jonathan Bramson
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transcriptional Profiling ◽  
Binding Domain ◽  
Antigen Binding ◽  
Knock Out ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptors (CARs) are powerful tools that enable MHC-independent activation of T cells. Recent reports have indicated that constitutive, low-level (tonic) signaling by CARs can impair the utility of the engineered T cells. The single-chain antibody (scFv) binding domain was one of the features determined to promote tonic signaling. We have recently developed a novel chimeric receptor, known as the T cell antigen coupler (TAC), that is less prone to tonic signaling than second-generation CARs. The TAC consists of a scFv-based antigen binding domain, a CD3-binding domain that couples the TAC to endogenous T cell receptor (TCR), and a transmembrane and cytoplasmic coreceptor (CD4) domain. In contrast to CARs, this design enables TAC-T cells to signal through the endogenous TCR, which we propose provides a fidelity to natural T cell signal regulation. Interestingly, we have recently reported that CAR-T cells have a greater propensity for off-target activation than TAC-T cells, suggesting a safety advantage to TAC-T cells (Helsen et al., Nat. Comm., 2019). Further characterization of the differences between CAR- and TAC-T cell signal initiation and activation is required to understand how their design affects sensitivity, specificity and regulation of T cell activation. Examination of the activation requirements for BCMA-specific CAR-T cells and TAC-T cells confirmed that TAC-T cells are reliant upon the endogenous TCR for T cell activation whereas CAR-T cells are TCR-independent. TRAC knock-out CAR-T cells retained potent effector function at levels similar to CAR-T cells with intact TCR expression, whereas TRAC knock-out TAC T-cells showed significant impairment in effector function. Consistent with TCR-dependence, the immunological synapse produced by TAC-T cells displays all the hallmarks of a conventional immunological synapse, whereas CAR-T cells form unconventional synapses. Unlike TAC-T cells, immunological synapses formed by CAR-T cells display non-uniform central supramolecular activation clusters, disperse Lck distribution, a lack of an LFA-1 associated adhesion ring (Figure), as well as more disperse delivery of perforin to the cell interface. CAR-T cells also formed synapses faster than TAC-T cells. This suggests that while TAC T-cells are beholden to the requirement of organized, mature synapse formation, CAR T-cells can rapidly form less structurally organized synapses. Transcriptional profiling of CAR-T cells in the absence of antigen stimulation revealed a basal activation status associated with upregulation of Nur77, a transcription factor that is downstream of TCR activation. Transcriptional profiling of TAC-T cells failed to reveal evidence of TCR signaling in the absence of stimulation. Further evaluation of CAR- and TAC- T cells in the absence of stimulation revealed elevated levels of CD69, PD-1 and LAG-3 in CAR-T cells compared with TAC-T cells, as well as higher expression of IL-2, IFNγ, and TNF in CAR-T cells. Interestingly, the level of tonic signaling was dependent on the antigen-binding scFV, as otherwise identical BCMA-specific CAR- and TAC-T cells displayed different levels of CD69, PD-1 and LAG-3 depending on the identity of the BCMA-specific scFv. Despite different levels of basal activation, both CAR- and TAC-T cells displayed comparable activation kinetics as measured by upregulation of CD69 and Ki-67, as well as proliferation. However, the elevated level of basal activation rendered the CAR-T cells more easily activated by a cross-reactive off-target antigen that failed to stimulate TAC-T cells carrying the same binding domain. These data suggest that the TAC receptor offers a valuable alternate platform to CAR-T cells. The antigen-binding scFv domain has a direct impact on tonic signaling and basal activation in CAR-T cells. Conversely, TAC-T cells are less susceptible to basal activation and this works suggests that the TAC receptor can deploy scFv binding domains that are not suitable for CARs. This work was supported by Triumvira Immunologics and Genome Canada. Figure 1 Disclosures Bramson: McMaster University: Current Employment, Patents & Royalties; Triumvira Immunologics: Current Employment, Current equity holder in private company, Research Funding.

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