Impact of Glycine-Serine Linker on Target Antigen Binding and Subsequent CD37CAR-T Performance
Authors: Khopanlert Wannakorn , Prompat Napat , Saetang Jirakrit , Maneechai Kajornkiat , Okuno Shingo , Terakura Seitaro , Julamanee Jakrawadee 
Published: December 2023

Introduction/Objectives:
Gene-modified therapy expressing CD19-chimeric antigen receptor (CD19CAR) has shown promising outcomes in B-cell malignancies. However, some patients experience relapses due to poor CAR-T persistence and antigen escape. Various approaches have been studied to address these issues, including a novel signaling domain; CD28/CD40 to potentiate CAR-T effectiveness, linker modification to improve antigen-binding affinity, and new target identification such as CD37 in mature T-/B-cell malignancies. Consequently, we developed the third-generation CD37CAR incorporating the dual T- and B-cell costimulatory molecules; CD28/CD40 with two different single-chain variable fragment (scFv) linkers to improve CAR-T efficacy against CD37 + malignancies.
Methods:
The two different scFv linkers, GSTSGSGKPGSGEGSTKG (18aaL) and GGGGSGGGGSGGGGSGGGGS (GS4L), were fused into the CD37CAR; V L-linker-V H-H-CD28TM-CD28/CD40-CD3z-tEGFR backbone. CAR genes were subcloned into retroviral packaging cells to produce viral particles and transduced into primary CD3 + cells. The CAR +-Tcells were enriched using biotinylated anti-EGFR mAbs and further expanded with anti-CD3/CD28 microbeads. The cells were then used for subsequent experiments compared with untransduced T-cells. The in-silico studyinvestigated the structural behavior and binding characteristics of scFv at the atomistic level.
Results:
All CD37CAR genes were successfully transduced into primary CD3 + cells with significantly higher transduction efficiency in the glysine-serine linker (CD37.GS4L) CAR than CD37.18aaL. We initially evaluated the influence of linkers on CAR-T proliferation by stimulating once with gamma-irradiated Raji cells and found the robust CD37.GS4L CAR expansion regardless of IL-2. Short- and long-term co-culture assays against various CD37 + malignancies were then performed to examine tumoricidal activity. The overnight specific cytolysis demonstrated greater tumor eradication in CD37.GS4L CAR-T cells against Ramos (burkitt's lymphoma), Jeko-1 (mantle cell lymphoma; MCL), MM1S, KMS12BM (multiple myeloma), Hut78 (cutaneous T-lymphocyte), SNU449 (hepatocellular carcinoma), NCI-H522 (lung adenocarcinoma), primary-MCL and primary-chronic lymphocytic leukemia at various E:T ratios. In addition, we observed significant tumor suppression against Raji cells (burkitt's lymphoma) in CD37.GS4L CAR throughout the 12-day long-term cytotoxicity assay. Concerning the possibility of fratricide, CTV-labeled CD3 + were co-cultured with CARs and found that CD37.18aaL CAR revealed significant autologous T-cell killing compared to others. In terms of cytokine secretion, similar levels of IL-2, IFN-g, perforin, and granzyme B positive cells were noted after being stimulated with target cells. To replicate the effects of prolonged antigen exposure in humans, three consecutive weeklytarget cell stimulations revealed substantial expansion with significant preservation of central memory T-cells and less expressed exhaustion phenotypes in CD37.GS4L CAR compared to CD37.18aaL. Furthermore, the potential anti-tumor efficiency in vivo was studied using Raji-bearing NSG mice model. CD37.GS4L CAR-T effectively suppressed tumor cell growth and prolonged overall survival with persistent T CM positive cells in mice's spleen, liver, and bone marrow on day 60 compared to tEGFR-T and CD37.18aaL CAR-T (Figure 1). Additionally, the preliminary results of MM1S- and HUT78-inoculated mice models illustrated greater in vivo cytotoxicity in CD37.GS4L CAR-T than tEGFR-T. Regarding the in-silico study, molecular dynamic simulation and docking were evaluated. The scFv-specific CD37.GS4L CAR displayed more flexibility with higher binding affinity compared to CD37.18aaL CAR with ∆G = -11.1 kcal/mol, K d = 1.4 x 10 -8 molar, and ∆G = -9.7 kcal/mol, K d of 1.5 x 10 -7 molar, respectively.
Conclusions: The considerable flexibility and affinity of CD37.GS4L CAR augment the CAR-T function in terms of proliferation, persistence, and tumoricidal activity. The glysine-serine linker could facilitate scFv activity and maximize CAR-T effectiveness.