Abstract
Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.
Highlights
chimeric antigen receptor (CAR) are synthetic chimeric receptors consisting of an antibody based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containingstimulatory signals derived from CD3ζ, CD28 and/or 4_1BB [1]
Current CAR T cells target CD19, and other B cell-related antigens and antigens associated with non-B cell malignancies [12]
To expand the usage of nanobodies in the generation of CAR T cells, here, we report an optimized protocol to speed up the process of generating CAR constructs
Summary
CARs are synthetic chimeric receptors consisting of an antibody based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containing (co)stimulatory signals derived from CD3ζ, CD28 and/or 4_1BB [1]. 39 days post RL inoculation while mice treated with CD20 nanoCAR T cells were kept for six weeks post CAR T injection, until they had to be sacrificed according to ethical guidelines, due to graft versus host disease During this period, we did not observe any outgrowth of residual tumor cells. For the CD33 antigen, we tested two different CAR backbones in vitro Both CD33-specific nanoCARs induced similar T cell activation, cytokine production and tumor cell lysis when incubated with CD33+ cells. We believe that not the spacer but rather the intracellular signaling domain influences the efficacy of our nanoCAR T cells, since in short term experiments we did not observe great differences in activity but the 4_1BB:ζ nanoCAR T cells outperformed the CD28:ζ nanoCAR T cells in our long-term in vitro stress test. We strongly believe that the use of nanobodies is advantageous over the use of scFvs, since nanobodies are monomeric structures that (i) will probably not aggregate on the T cell surface and not induce premature T cell activation and exhaustion [19]; (ii) will not lose affinity, a possible and known side effect in the design of scFvs [14,15]
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