Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating advanced B-cell malignancies by targeting CD19, but antigen-negative relapses and immune responses triggered by murine-derived antibodies remain significant challenges, necessitating the development of novel humanized multitarget CAR-T therapies. Here, we engineered a second-generation 4-1BB-CD3ζ-based CAR construct incorporating humanized CD19 single-chain variable fragments (scFvs) and BAFFR single-variable domains on heavy chains (VHHs), also known as nanobodies. The resultant CAR-T cells, with different constructs, were functionally compared both in vitro and in vivo. We found that the optimal tandem and bicistronic (BI) structures retained respective antigen-binding abilities, and both demonstrated specific activation when stimulated with target cells. At the same time, BI CAR-T cells (BI CARs) exhibited stronger tumour-killing ability and better secretion of interleukin-2 and tumour necrosis factor-alpha than single-target CAR-T cells. Additionally, BI CARs showed less exhaustion phenotype upon repeated antigen stimulation and demonstrated more potent and persistent antitumor effects in mouse xenograft models. Overall, we developed a novel humanized CD19/BAFFR bicistronic CAR (BI CAR) based on a combination of scFv and VHH, which showed potent and sustained antitumor ability both in vitro and in vivo, including against tumours with CD19 or BAFFR deficiencies.

Wu S ，Luo Q ，Li F ，Zhang S ，Zhang C ，Liu J ，Shao B ，Hong Y ，Tan T ，Dong X ，Chen B ... -  《-》

A Humanized Lym-1 CAR with Novel DAP10/DAP12 Signaling Domains Demonstrates Reduced Tonic Signaling and Increased Antitumor Activity in B-Cell Lymphoma Models.

The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent in vivo antitumor efficacy.

Zheng L ，Ren L ，Kouhi A ，Khawli LA ，Hu P ，Kaslow HR ，Epstein AL ... -  《-》

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line.

Kang CH ，Kim Y ，Lee HK ，Lee SM ，Jeong HG ，Choi SU ，Park CH ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Development and functional characterization of novel fully human anti-CD19 chimeric antigen receptors for T-cell therapy.

Impressive outcomes have been achieved by chimeric antigen receptor (CAR)-T cell therapy using murine-derived single-chain variable fragment (scFv) FMC63 specific for CD19 in patients with B cell malignancies. However, evidence suggests that human anti-mouse immune responses might be responsible for poor persistence and dysfunction of CAR-T cells, leading to poor outcomes or early tumor recurrence. Substituting a fully human scFv for murine-derived scFv may address this clinically relevant concern. In this study, we discovered two human anti-CD19 scFv candidates through an optimized protein/cell alternative panning strategy and evaluated their function in CAR-T cells and CD19/CD3 bispecific antibody formats. The two clones exhibited excellent cytotoxicity in CAR-T cells and bispecific antibodies in vitro compared with the benchmarks FMC63 CAR-T cells and blinatumomab. Furthermore, Clone 78-BBz CAR-T cells exhibited similar in vivo antitumor activity to FMC63-BBz CAR-T cells. Our results indicate that Clone 78-BBz CAR has excellent efficacy and safety profile and is a good candidate for clinical development.

Dai Z ，Hu X ，Jia X ，Liu J ，Yang Y ，Niu P ，Hu G ，Tan T ，Zhou J ... -  《-》

Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected by Hinge and Transmembrane Domains.

Anti-CD19 chimeric antigen receptor (CAR) T cells have caused remissions of B cell malignancies, but problems including cytokine-mediated toxicity and short persistence of CAR T cells in vivo might limit the effectiveness of anti-CD19 CAR T cells. Anti-CD19 CARs that have been tested clinically had single-chain variable fragments (scFvs) derived from murine antibodies. We have designed and constructed novel anti-CD19 CARs containing a scFv with fully human variable regions. T cells expressing these CARs specifically recognized CD19+ target cells and carried out functions including degranulation, cytokine release, and proliferation. We compared CARs with CD28 costimulatory moieties along with hinge and transmembrane domains from either the human CD28 molecule or the human CD8α molecule. Compared with T cells expressing CARs with CD28 hinge and transmembrane domains, T cells expressing CARs with CD8α hinge and transmembrane domains produced lower levels of cytokines and exhibited lower levels of activation-induced cell death (AICD). Importantly, CARs with hinge and transmembrane regions from either CD8α or CD28 had similar abilities to eliminate established tumors in mice. In anti-CD19 CARs with CD28 costimulatory moieties, lower levels of inflammatory cytokine production and AICD are potential clinical advantages of CD8α hinge and transmembrane domains over CD28 hinge and transmembrane domains.

Alabanza L ，Pegues M ，Geldres C ，Shi V ，Wiltzius JJW ，Sievers SA ，Yang S ，Kochenderfer JN ... -  《-》