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Abstract Background The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. Results We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. Conclusions The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

Background: The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. Results: We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. Conclusions: The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment. [ABSTRACT FROM AUTHOR]

Fusion of therapeutic proteins with the antibody Fc domain is a strategy widely applied to increase protein half-life in plasma. In our previous study, we generated a recombinant human lactoferrin (hLF)-immunoglobulin G1 Fc fusion protein (hLF-hinge-CH2-CH3) with improved stability, biological activity, and pharmacokinetics ( Shiga , Y. et al. Eur J Pharm Sci. , 2015 , 67 , 136 - -143 ). However, the Fc domain in fusion proteins can potentially induce antibody-dependent and complement-dependent cytotoxicity and serious side effects. To overcome these drawbacks, we engineered an hLF-Fc fusion protein (hLF-CH2-CH3) without the Fc hinge region which is essential for engaging Fc receptors on immune cells and inducing complement-mediated cell lysis. The hLF-CH2-CH3 protein was stably expressed in Chinese hamster ovary (CHO) DG44 cells and compared for in vitro activities, thermal stability, pharmacokinetics, and attenuation of Fc-mediated immune effector functions with the conventional hinge-containing Fc fusion protein. Both hLF-hinge-CH2-CH3 and hLF-CH2-CH3 exhibited iron-binding activity, superior uptake by Caco-2 cells, similar thermal stability, and longer plasma half-life compared to recombinant hLF. However, in contrast to conventional hLF-hinge-CH2-CH3, hinge-deficient hLF-CH2-CH3 did not elicit Fc-mediated effector response potentially damaging for the target cells. Our findings demonstrate that conjugation of hinge-deficient Fc to therapeutic proteins is a promising strategy for improving their pharmacokinetic properties without enhancing effector functions. Cell-expressed hinge-deficient hLF-CH2-CH3 is a potential drug candidate with improved plasma half-life for parenteral administration.