Expanding the Ligand Binding Repertoire of Chimeric Antigen Receptors Using Lamprey Variable Lymphocyte Receptors
Authors: Moot Robert , Raikar Sunil , Fleischer Lauren C , McCarty David , Querrey Melissa , Doering Christopher B , Spencer H. Trent 
Published: October 2019

Abstract
Although chimeric antigen receptors (CARs) are yielding promising clinical results, the technology remains limited by the availability of conjugate cancer cell target antigens. As a means to increase both the identification of cancer cell target antigens and simultaneously generate unique single chain binding domain structures we utilized the immune system of jawless vertebrates. Sea lamprey possess an adaptive immune system primarily characterized by variable lymphocyte receptors (VLRs) as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins (Ig). VLRs have a fundamentally different structure and geometry than Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures with their variable region consisting of multiple assembled repeating sequences termed leucine rich repeats. These repeats can be directly inserted onto a CAR scaffold by genetic engineering. To test this platform technology, a yeast display method previously described (Tasumi et al., PNAS 2009; Xu et al., Humana press 2011) was used as a means of assaying and selecting appropriate VLRs. VLRs meeting the set criteria are sequenced and cloned into a lentiviral vector (LV) CAR transgene cassette plasmid. We constructed a CAR containing a well characterized VLR specific for the B cell receptor of a murine B cell leukemia (BCL) cell line. The CAR design incorporates the anti-BCL-VLR, Myc tag, CD28 transmembrane domain, and the intracellular CD3ζ signaling domain. SIN VLR-CAR LV was produced at high titer (~1x108) and used to transduce Jurkat cells. Transduced Jurkat cells showed successful CAR protein expression confirmed via Western Blot as well as persistent surface CAR expression for over 2 months with cell viability remaining over 85%. To determine whether the VLR was capable of signaling through the CAR, transduced Jurkat cells were incubated with the BCL cell line expressing the target B cell receptor. Using this assay, we demonstrated potent T cell activation via the VLR-CAR. CAR expressing T-cells demonstrated activation in ~80% of the cells. Furthermore, NK-92 cells expressing the VLR-CAR demonstrated an ability to recognize and kill BCL cells at target to effector ratios as low as 1:1, with 30% target cell killing. Little to no killing was observed with a control B cell line. Additionally, we created a CAR using a published VLR sequence targeting the human surface antigen CD5 (Yu et al., J. Immunol Methods, 2012). CD5 is primarily a T-cell marker, thus an anti-CD5-VLRCAR could be potentially used to target T-cell malignancies. Jurkat cells were transduced with high titer SIN VLR-CAR LV at various MOIs. Since Jurkat cells express CD5 on their surface, we tested the different transduced groups for self-activation at several time points. The corresponding transduced copy number was determined using quantitative PCR. The degree of activation directly correlated with the LV copy number, with highest activation being seen when the MOI approach 50, with >60% highly activated cells. No activation was observed in the GFP control groups. Additionally, we engineered three different CAR constructs consisting of two CD5 VLRs connected by either a helical linker with a 180° rotation, a helical linker with a 360° rotation, or a non-rigid linker allowing for flexibility among the two VLRs. Transduced Jurkat cells expressing the double CD5-VLR CARs indicated, via flow cytometry, that linking two VLRs does not provide any benefit in terms of activation compared to the single CD5-VLR-CAR. Current testing includes the use of NK-92 cells expressing the anti-CD5-VLR-CAR against T cell leukemia cell lines. Collectively, these results show that VLRs can be used to increase the repertoire of CAR binding motifs. Furthermore, these data suggest that VLRs provide both a unique and effective method for activating CAR-T cells and can expand the number and variety of antigens that may be targeted.

Disclosures
Doering: Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.