Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals.

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

Predicting the Efficacy and Safety of TACTICs (Tumor Angiogenesis-Specific CAR-T Cells Impacting Cancers) Therapy for Soft Tissue Sarcoma Patients.

Soft tissue sarcomas (STSs) are heterogeneous and aggressive malignancies with few effective therapies available. We have developed T cells expressing a vascular endothelial growth factor receptor 2 (VEGFR2)-specific chimeric antigen receptor (CAR) to establish a tumor angiogenesis-specific CAR-T cells impacting cancers (TACTICs) therapy. In this study, we optimized the manufacturing and transportation of mRNA-transfected anti-VEGFR2 CAR-T cells and collected information that allowed the extrapolation of the efficacy and safety potential of TACTICs therapy for STS patients. Although 5-methoxyuridines versus uridines did not improve CAR-mRNA stability in T cells, the utilization of CleanCap as a 5' cap-structure extended the CAR expression level, increasing VEGFR2-specific cytotoxicity. Furthermore, 4 °C preservation conditions did not affect the viability/cytotoxicity of CAR-T cells, contrarily to a freeze-thaw approach. Importantly, immunohistochemistry showed that most of the STS patients' specimens expressed VEGFR2, suggesting a great potential of our TACTICs approach. However, VEGFR2 expression was also detected in normal tissues, stressing the importance of the application of a strict monitoring schedule to detect (and respond to) the occurrence of adverse effects in clinics. Overall, our results support the development of a "first in humans" study to evaluate the potential of our TACTICs therapy as a new treatment option for STSs.

Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold.

Chimeric antigen receptor (CAR)-T cells have demonstrated significant clinical potential; however, their strong antitumor activity may cause severe adverse effects. To ensure efficacy and safe CAR-T cell therapy, it is important to understand CAR's structure-activity relationship. To clarify the role of hinge and transmembrane domains in CAR and CAR-T cell function, we generated different chimeras and analyzed their expression levels and antigen-specific activity on CAR-T cells. First, we created a basic CAR with hinge, transmembrane, and signal transduction domains derived from CD3ζ, then we generated six CAR variants whose hinge or hinge/transmembrane domains originated from CD4, CD8α, and CD28. CAR expression level and stability on the T cell were greatly affected by transmembrane rather than hinge domain. Antigen-specific functions of most CAR-T cells depended on their CAR expression levels. However, CARs with a CD8α- or CD28-derived hinge domain showed significant differences in CAR-T cell function, despite their equal expression levels. These results suggest that CAR signaling intensity into T cells was affected not only by CAR expression level, but also by the hinge domain. Our discoveries indicate that the hinge domain regulates the CAR signaling threshold and the transmembrane domain regulates the amount of CAR signaling via control of CAR expression level.

Development and functional analysis of an anticancer T-cell medicine with immune checkpoint inhibitory ability.

Adoptive cell therapy using patients' own T-cells is expected to be an ideal cancer treatment strategy with excellent antitumor effects and low side effects. However, this therapy targeting solid tumors is unlikely to be effective because tumor tissues have an environment that suppresses T-cell function. In particular, interaction between programmed death-1 (PD-1) and its ligand (PD-L1) inhibits T-cell activation by which T-cells eliminate tumor cells. Here, we attempted to develop T-cells that can exert potent antitumor activity even in tumor tissues by genetically modifying them to express the anti-PD-L1 membrane-anchoring type single chain variable fragment (M-scFv) that can inhibit PD-L1/PD-1 interaction. Anti-PD-L1 M-scFv could be expressed on T-cells while maintaining PD-L1-binding ability. Although T-cell proliferation induced by CD3 stimulation was decreased depending on the PD-L1 stimulation intensity, M-scFv-expressing T-cells showed high proliferative activity even in the presence of PD-L1 by avoiding the PD-L1/PD-1-mediated suppression. Furthermore, M-scFv-expressing T-cells showed higher cytotoxic activity against PD-L1high tumor cells than that of mock T-cells. The effect of PD-L1/PD-1 blockade was more pronounced when the therapeutic target was low-antigenic tumor cells with low major histocompatibility complex expression, presenting only the shared antigen. These results indicated that anti-PD-L1 M-scFv expression was functional in avoiding T-cell dysfunction by PD-L1/PD-1 interaction. Our concept of anti-PD-L1 M-scFv-expressing T-cells is thus expected to improve the efficacy of T-cell therapy and contribute to simplify the treatment system and reduce treatment costs compared with the combination therapy of T-cells and antibodies.

Impact of scFv structure in chimeric antigen receptor on receptor expression efficiency and antigen recognition properties.

Gene-modifying T cells expressing chimeric antigen receptor (CAR) with an extracellular domain consisting of single chain variable fragment (scFv) and an intracellular domain with a T cell activation motif, are promising cancer immuno-medicines that can exert long term potent antitumor activity. However, CAR-T cells have a high risk of causing fatal side effects. Thus, more effective and safer CAR-T cells are urgently needed. Although antigen specificity and reactivity of CAR-T cells are defined by CAR expression level and affinity, information on optimizing the scFv structure that defines CAR avidity is lacking. Here, we investigated the impacts of scFv substitution and structural modification in CAR on receptor expression and antigen recognition properties. Four CARs with distinct scFvs targeting the same antigen were unexpectedly separated into a CAR expressed on T cells and bound to the antigen, CARs that did not show antigen-binding because of cell surface aggregation, and a rarely expressed CAR. Among the scFv structural modifications of CARs, changes in the Fv order and linker did not noticeably affect CAR expression or antigen-binding. In contrast, complementarity-determining region (CDR)-grafting to the stable framework region in Fv dramatically improved the surface expression level of non-producible CAR. These results revealed that CAR expression efficiency and stability on T cells are influenced by the Fv structure. Therefore, stabilization of the Fv structure by CDR-grafting may be an effective means for expressing scFvs, which have excellent antigen specificity and appropriate affinity but low structural stability, as a CAR on T cells.

Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research.

We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR) specific for vascular endothelial growth factor receptor 2 (VEGFR2), demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP) and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6-12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy.

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells.