In Situ Therapeutic Engineering of the Immune Cell Surface

Introduction:: Engineering of immune cells as a therapeutic modality has been primarily focused on redirecting T cells to target cancer by genetically introducing specific antigen receptors. Although establishing itself as part of a new pillar of cancer care, the current adoptive T cell therapies require complex procedures which raises costs considerably. The characteristic expression of glycan sialyl Lewis X (sLeX) on leukocytes offers a unique opportunity to engineer the surface of all of the subpopulations of leukocytes for therapeutic purposes. We created E-selectin (ES)-tagged therapeutic entities that directly adhere to sLeX on the surface of circulating immune cells in the blood. We report the applications of this novel engineering approach in models of cancer and inflammation.


Materials and Methods:: To address the problem of metastasis, ES was chemically conjugated with TNF-related apoptosis-inducing ligand (TRAIL), a recombinant protein that kills cancer cells while sparing healthy ones. An in vitro cone-and-plate shear assay was used to study the adherence of the ES/TRAIL conjugates to leukocyte surfaces, and their potential to kill circulating tumor cells (CTCs) in whole blood for the prevention of cancer metastasis. This assay was developed by us to mimic the shearing conditions in regions of the vasculature. We also synthesized ES-conjugated nanoscale liposomes loaded with the broad-spectrum antibiotic Cefoperazone sodium (Cefo-Na) as a model drug. A migration assay using Transwell (Corning) that consists of a lower and an upper chamber separated by a porous membrane was used to test the efficiency of leukocyte mediated transendothelial drug delivery in vitro. An acute lung injury model in mouse induced by Lipopolysaccharide (LPS) was used to study the migration and accumulation of leukocytes carrying the liposomes in the lung after passing through the blood-air barrier.


Results, Conclusions, and Discussions:: We found CTCs are associated with leukocytes in peripheral blood of PCa patients (Figure 1A). The leukocyte engineering approach with ES/TRAIL conjugates and ES liposomes is illustrated in Figure 1B. The conjugates were synthesized by crosslinking six TRAIL monomers and two E-selectin (ES) monomers (Figure 1C). The conjugates were then purified and characterized by size exclusion spectroscopy (SEC) (Figure 1D). The ES/TRAIL conjugates adhered to multiple leukocyte subpopulations after exposure to shear flow of whole blood. Flow cytometry further revealed that the percentage of TRAIL-coated leukocytes among total leukocytes was dependent on the concentration of ES/TRAIL conjugate (Figure 1E). In another experiment, fluorescently labeled COLO 205 cells and ES/TRAIL conjugates was co-incubated with whole blood at the same shearing rate, with blood incubated only with COLO 205 cells as control. Flow cytometry analysis revealed that ~85% more COLO 205 cells were killed by the conjugates than the non-conjugate treated control (Figure 1F). In the case of ES liposomes, the content of Cefo-Na reached ~25% by weight. In the in vitro transmigration studies, we found that >80% of the leukocytes migrated to the bottom chamber while carrying the liposomes, and each leukocyte was able to transport ~10⁴ liposomes in average in the process. In mouse studies, we found nearly half Cefo-Na of the injected dose in the groups of ES liposomes were accumulated in the lung, much higher than that in the groups of naked liposomes. Our lab first invented the leukocyte surface engineering approach using liposomes conjugated with both ES and TRAIL.¹ We previously demonstrated that the liposomes can prevent bloodborne metastasis in orthotopic PCa and breast cancer models, and kill CTCs in blood samples of patients with late stage PCa.² The current project created new ES-tagged biomaterials and demonstrate their therapeutic applications in two distinctly different diseases. The new ES-tagged materials provided a general in situ molecular engineering method that takes advantage of the targeting power of leukocytes for various therapeutic purposes without interfering with their native immune functions. New therapies developed using this approach would be much more straightforward to practice in clinic than current adoptive cell therapies.


Acknowledgements (Optional): :

References (Optional): : 1. M.J. Mitchell, E. Wayne, K. Rana, C.B. Schaffer, M.R. King, TRAIL-coated leukocytes that kill cancer cells in the circulation, Proceedings of the National Academy of Sciences, 111 (3) (2014), pp. 930-935.
2. a) N. Jyotsana, Z. Zhang, L.E. Himmel, F. Yu, M.R. King, Minimal dosing of leukocyte targeting TRAIL decreases triple-negative breast cancer metastasis following tumor resection, Science Advances, 5 (7) (2019), Article eaaw4197; b) E. C. Wayne, S. Chandrasekaran, M. J. Mitchell, M. F. Chan, R. E. Lee, C. B. Schaffer, M. R. King, TRAIL-coated leukocytes that prevent the bloodborne metastasis of prostate cancer, J Control Release. 2016 Feb 10; 223: 215–223. c) N. Ortiz-Otero, J. R. Marshall, A. Glenn, J. Matloubieh, J. Joseph, D. M. Sahasrabudhe, E. M. Messing, M. R. King, TRAIL-coated leukocytes to kill circulating tumor cells in the flowing blood from prostate cancer patients, BMC Cancer 21, 898 (2021).