Engineering T cells with lipid nanoparticles for cancer immunotherapy

Cancer immunotherapy has become the fourth pillar of cancer treatment apart from surgery, radiation, and chemotherapy, most notably with checkpoint blockade antibodies and chimeric antigen receptor T cell therapy. Adoptive T cell therapy of either expanded tumor-infiltrating lymphocytes or genetically modified T cells has demonstrated a big breakthrough in the treatment of some established malignancies, especially leukemia and lymphoma. Despite the success in hematological malignancies, the response is severely limited in treating solid tumors due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of target. Thus, co-administration of adjuvant drugs or genetic modification of T cells are often required. However, the methods for T cell engineering have so far met with challenges of unprecise modification, being complex and expensive. In addition, limited efficacy and significant toxicity have been major issues for the broader success of T cell therapy. To address these issues and enhance the anticancer efficacy of T cell therapy, in this Ph.D. thesis we developed a novel approach to engineer T cells by equipping the cells with lipid nanoparticles containing therapeutic agents. Using this strategy, we have loaded supportive small-molecule immunomodulators and mRNA in T cells via two different conjugation techniques.
In the first part, we developed and characterized a remote-loading method for loading small molecule immunomodulatory drugs into liposome carriers. A TLR-7 agonist Gardiquimod and SHP2 inhibitor SHP099 were tested as the candidate drugs, respectively. Both drugs were efficiently loaded into liposomes of various lipid compositions using the remote-loading method via an ammonium sulfate gradient. Complete drug loading was achieved at a drug-to-lipid ratio of 0.25/1 (mol/mol), and the drugs were entrapped inside the liposomes and sustainedly released out for more than 1 week in the cell culture medium. We further assessed the biological effects of the drugs in vitro. The liposomes exhibited prolonged effect and alleviated toxicity, and the capacity for co-loading drugs as combination therapy. The approach to efficiently load high concentrations of Gardiquimod and SHP099 allowed the use of lipid-based drug carriers for loading T cells with immunomodulators.
The second part of the Ph.D. thesis describes the strategy to load T cells with liposomal SHP099 nanocrystals, with the aim to improve the efficacy of adoptive T cell therapy. A novel formulation containing tri-arginine motifs (SR3) was designed to enable T cells attachment and loading through electrostatic interaction and membrane penetration. Using the remote-loading method developed previously, SHP099 was loaded into lipid vesicles and formed nanocrystals in the core. By incubating T cells with SR3-SHP099, a high loading efficiency into T cells was achieved without affecting the T cell viability, and the nanocrystals formed a depot to sustainedly release SHP099 over more than 5 days. We found that SHP099 enabled prolonged inhibition of the PD-1/PD-L1 signaling compared to the anti-PD1 antibody. The SHP2 inhibition resulted in superior cytotoxicity of loaded OT.1 T cells against the target tumor cells in a co-culture cells lysis assay. In addition, loading the T cells did not affect the biodistribution of infused T cells in vivo, and the tumor-homing T cells were able to carry the lipid vesicles into the tumor tissue, enhancing the tumor accumulation of the cargos. On an established solid tumor model, adoptively transferred T cells loaded with SR3-SHP099 induced complete tumor eradication and a durable immune memory against re-challenging tumor formation on all treated mice. The efficacy was equivalent to that resulting from the repeated administered anti-PD-L1 antibody. These findings demonstrate that the combination of adoptive T cell therapy with SHP2 inhibition is a promising therapeutic strategy. Moreover, our T cells loading technique provides a precise and efficient drug delivery platform for targeting T cells.
In the third part, we explored another T cell loading technique by conjugating lipid nanoparticles (LNPs) to the surface of bioorthogonal glycoengineered T cells through SPAAC click chemistry. Conjugating drug-loaded nanoparticles to living cells represent a promising strategy for targeted drug delivery, because of the tissue homing properties in the specific cell types to overcome in vivo barriers. And covalent conjugation has advantages in cellular loading regarding stability and specificity. In this study, T cells were decorated with azide groups on their surface through metabolic glycoengineering, followed by reacting with dibenzylcyclooctyne (DBCO) modified LNPs. We first demonstrated highly specific and robust conjugation of liposomes to T cells, and the conjugation efficiency can be well-tuned by changing the azide and DBCO liposome presence. Based on the optimized procedure, we further developed DBCO functionalized mRNA-LNPs that can be conjugated to T cells. Preliminary results showed the conjugated LNPs delivered mRNA into T cells and subsequently transfected the T cells with the encoded gene.
Taken together, the work in this Ph.D. thesis integrated the topics of nanomedicine and T cell-based cancer immunotherapy, presenting a novel and versatile strategy for engineering therapeutic T cells by loading the cells with lipid-based nanoparticles encapsulating immunomodulatory drugs.