Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

Abstract

Chimeric antigen receptors (CARs) are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs). VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig)-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

“…21 The complementary DNA sequence was codon optimized for human cell expression and then cloned into our previously used vector encoding a secondgeneration CAR with CD28 as the costimulatory domain. 22 The vector allowed for dual expression of enhanced green fluorescent protein and the CD5-CAR using a P2A sequence. High-titer, recombinant, self-inactivating HIV lentiviral vector was produced using a 4-plasmid system as we have previously described.…”

“…High-titer, recombinant, self-inactivating HIV lentiviral vector was produced using a 4-plasmid system as we have previously described. 22 Titering of the concentrated recombinant lentiviral vector was performed on HEK-293T cell genomic DNA using quantitative polymerase chain reaction with resulting titers of ;1 3 10 7 TU/ mL. Primary T cells were transduced on day 10 of expansion by incubating cells with the CAR-expressing lentiviral vector in the assigned culture media supplemented with 4 mg/mL polybrene (EMD Millipore, Billerica, MA).…”

Programming CAR-T cells to kill cancer

“…Conversely, a disadvantage of using scFv-binding domains is their tendency for oligomerization, which can lead to tonic signalling and T-cell exhaustion 17 . Alternative binding domains have been used in preclinical studies, including receptors 18 , ligands 19 , cytokines 20,21 , DARPins (designed ankyrin repeat proteins) 22 , adnectins 23 , Fc receptor fragments 24 , nanobodies 25 , peptides 26 and variable lymphocyte receptors 27 . The relative efficacy, safety and immunogenicity of these alternative binders have not yet been fully investigated and are the focus of substantial ongoing work.…”

Engineering Chimeric Antigen Receptors

“…A related approach is to use CARs whose antigen-recognition domain consists of designed ankryrin repeat proteins (DARPins) [29, 30], nanoantibodies (VHH) [31-34], or variable lymphocyte receptors (VLRs) [35]. DARPins are compact and stable protein modules selected for high-affinity binding to one or several targets.…”