Development of off-the-shelf hematopoietic stem cell-engineered invariant natural killer T cells for COVID-19 therapeutic intervention

Abstract

Background New COVID-19 treatments are desperately needed as case numbers continue to rise and emergent strains threaten vaccine efficacy. Cell therapy has revolutionized cancer treatment and holds much promise in combatting infectious disease, including COVID-19. Invariant natural killer T (iNKT) cells are a rare subset of T cells with potent antiviral and immunoregulatory functions and an excellent safety profile. Current iNKT cell strategies are hindered by the extremely low presence of iNKT cells, and we have developed a platform to overcome this critical limitation. Methods We produced allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells through TCR engineering of human cord blood CD34+ hematopoietic stem cells (HSCs) and differentiation of these HSCs into iNKT cells in an Ex Vivo HSC-Derived iNKT Cell Culture. We then established in vitro SARS-CoV-2 infection assays to assess AlloHSC-iNKT cell antiviral and anti-hyperinflammation functions. Lastly, using in vitro and in vivo preclinical models, we evaluated AlloHSC-iNKT cell safety and immunogenicity for off-the-shelf application. Results We reliably generated AlloHSC-iNKT cells at high-yield and of high-purity; these resulting cells closely resembled endogenous human iNKT cells in phenotypes and functionalities. In cell culture, AlloHSC-iNKT cells directly killed SARS-CoV-2 infected cells and also selectively eliminated SARS-CoV-2 infection-stimulated inflammatory monocytes. In an in vitro mixed lymphocyte reaction (MLR) assay and an NSG mouse xenograft model, AlloHSC-iNKT cells were resistant to T cell-mediated alloreaction and did not cause GvHD. Conclusions Here, we report a method to robustly produce therapeutic levels of AlloHSC-iNKT cells. Preclinical studies showed that these AlloHSC-iNKT cells closely resembled endogenous human iNKT cells, could reduce SARS-CoV-2 virus infection load and mitigate virus infection-induced hyperinflammation, and meanwhile were free of GvHD-risk and resistant to T cell-mediated allorejection. These results support the development of AlloHSC-iNKT cells as a promising off-the-shelf cell product for treating COVID-19; such a cell product has the potential to target the new emerging SARS-CoV-2 variants as well as the future new emerging viruses.

Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors

“…The cell product harvested from transduced BLT mice displayed a significant increase in the HSC-iNKT population, and their anti-tumor capacity was confirmed against an MM.1S human multiple myeloma cell line using in vitro and in vivo models. However, to produce a more massively scalable platform for HSC-iNKT generation, a modified ex vivo artificial thymic organoid (ATO) culture system was designed to grow HSCs [ 18 , 19 ]. While the ATO platform was designed for the in vitro generation of conventional T cells from HSCs using a 3D stromal cell matrix, iNKT TCR transduction combined with αGC stimulation successfully produced a high-yield and high-purity HSC-iNKT cell product [ 18 , 19 ].…”

“…However, to produce a more massively scalable platform for HSC-iNKT generation, a modified ex vivo artificial thymic organoid (ATO) culture system was designed to grow HSCs [ 18 , 19 ]. While the ATO platform was designed for the in vitro generation of conventional T cells from HSCs using a 3D stromal cell matrix, iNKT TCR transduction combined with αGC stimulation successfully produced a high-yield and high-purity HSC-iNKT cell product [ 18 , 19 ]. These cells retained proper iNKT morphology and functional profile, showing significant anti-tumor capacity against multiple human solid tumor cell lines [ 18 , 19 ].…”

“…While the ATO platform was designed for the in vitro generation of conventional T cells from HSCs using a 3D stromal cell matrix, iNKT TCR transduction combined with αGC stimulation successfully produced a high-yield and high-purity HSC-iNKT cell product [ 18 , 19 ]. These cells retained proper iNKT morphology and functional profile, showing significant anti-tumor capacity against multiple human solid tumor cell lines [ 18 , 19 ]. The capacity of HSC-iNKT to tolerate CAR engineering was also evaluated against a CD1d-engineered MM.1S cell line, wherein BCMA-targeting CAR (BCAR)-engineered HSC-iNKT (BCAR-iNKT) cells showed enhanced cytotoxicity in comparison to mock-transduced HSC-iNKT cells [ 18 , 19 ].…”

“…These studies have demonstrated the effective expansion and activation of iNKT cells when stimulated by αGC alone, αGC-pulsed DCs, or other αGC-pulsed APCs [ 14 , 16 , 17 ]. Our previous preclinical studies utilizing BCMA chimeric antigen receptor (CAR)-engineered and αGC-expanded iNKT cells indicated a feasible safe and tolerable adoptive therapy capable of eliminating multiple myeloma [ 18 , 19 ]. Enhanced tumor killing and more beneficial patient outcomes have been evidenced by αGC-boosted iNKT cell-based therapy; however, in some solid tumors, iNKT cells are still restricted in their antitumor capabilities due to difficulties in infiltrating solid tumors [ 20 ].…”

Targeting Immunosuppressive Tumor-Associated Macrophages Using Innate T Cells for Enhanced Antitumor Reactivity

“…Previous studies have identified that innate T cells, such as iNKT cells, can remediate immunosuppression through the targeting of anti-inflammatory TAMs [ 23 ]. Therefore, this study seeks to examine the efficacy of MAIT, iNKT, and γδT cells in targeting alternatively activated macrophages in vitro, and to assess their application in CAR therapies.…”