Development of a novel LRP1 antibody that identifies upregulation of membrane LRP1 on activated and chimeric antigen receptor T cells

Abstract

Abstract Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of the LDL receptor family which plays a variety of different roles in many cell types, including lipid metabolism, cell signaling, and scavenger uptake (Sizova et al. 2023). While LRP1 may have a role in motility and modulating proliferation of T cells, the regulation, timing, and role of LRP1 on T cells is largely unknown (Sizova et al. 2023; Yang et al. 2018). Because commercial anti-LRP1 antibodies are generally low affinity and may be cross-reactive, we developed novel monoclonal antibodies (mAb) with higher affinity and greater specificity to characterize LRP1 on T cells, including major T cell subsets and chimeric antigen receptor (CAR) T cells. Using recombinant protein consisting of the LRP1 N-terminal extracellular domain as the immunogen, we generated a panel of six monoclonal antibodies that specifically bound recombinant LRP1 via enzyme-linked immunosorbance assaying (ELISA) with four showing improved affinity compared to commercially available anti-LRP1 mAb, 8G1. Antibody binding affinity was further determined via Octet analysis revealing that five antibodies showed high affinity to LRP1 with antibody dissociation (KD) ranging from 1.19x10-9 to 3.67x10-10M. Furthermore, in a competition binding assay with commercially available anti-LRP1 mAb 8G1, three of the four high affinity antibodies prevented binding to highly expressed LRP1 on CD14+ cells. From the candidate antibody pool, we selected 2A7 based on affinity (KD = 3.67x10-10) and highly specific binding to recombinant and surface membrance LRP1 to characterize the role of LRP1 on T cells. Others have shown that LRP1 is weakly expressed on the surface of unstimulated T cells due to continuous cleavage of the membrane protein in the absence of antigen stimulation. However, LRP1 surface expression increases upon T cell activation due to inhibition of LRP1 shedding (Panezai et al. 2017). We sought to fully characterize the expression of LRP1 on healthy adult T cells prior to and after activation with CD3/CD28 soluble antibodies. We observed that LRP1 expression was indeed low on unstimulated human T cells; however, high expression of LRP1 was observed 24 hours after T cell receptor stimulation and increased over a 5-day time course after activation measured by flow cytometry with 2A7. Specifically, LRP1 expression on CD4+ T cells increased 1.8-fold 1-day post-activation, 69-fold 3 days post-activation, and 288-fold 5-days post-activation. Similarly, LRP1 expression on CD8+ T cells increased 1.69-fold 1-day post-activation, 26-fold 3 days post-activation, and 299-fold 5 days post-activation. These results show that LRP1 expression continuously increases over several days after activation via CD3/CD28. Furthermore, we found higher LRP1 expression on effector memory and central memory T cells compared to naïve T cells, supporting LRP1 upregulation on antigen-experienced T cells, particularly with more robust expression on CD4+ T cells which suggests LRP1 is a marker for T cell activation. As LRP1 expression showed a marked and consistent increase after activation on primary human T cells, we sought to profile the expression of LRP1 on CAR-T cells, as many cells maintain an elevated activation status even in the absence of antigen. Using a CAR-T cell developed within our group named Hu8F4-CAR, which uses the scFv of the Hu8F4 mAb targeting the leukemia-associated PR1/HLA-A2 complex on AML, we profiled the expression of LRP1 on these CAR-T cells (Ma et al. 2016). Interestingly, LRP1 expression was indeed high on Hu8F4-CAR-T cells from multiple donors compared to non-transduced activated T cells (NT) from the same donors, with MFI (median fluorescence intensity) = 283 ± 225.3 on CAR-T cells as opposed to MFI = 13 ± 9.5 on NT T cells. Moreover, LRP1 expression was five-fold higher on PD-1+ CAR-T cells (MFI = 459 ± 365.7) compared to PD-1- CAR-T cells (MFI = 88 ± 54.7). The data suggests that LRP1 expression is a marker for a subset of CAR-T cells that maintain a state of activation. Furthermore, because LRP1 is coexpressed with PD-1 on activated CAR-T cells, their surface expression my be coordinately regulated. Overall, we report the development of a novel LRP1 monoclonal antibody, 2A7, which readily identifies upregulation of LRP1 preferentially on the surface of effector memory, central memory, activated CD4 and CD8 T cells, and a subset of CAR-T cells that maintain an activated state.