Engineering Novel Multi-Cytokine Backpacks for Manufacturing Functionally Improved CAR T Cell Products

Chimeric antigen receptor (CAR) T cell therapies have shown remarkable clinical efficacy in hematological cancers, including diffuse large B-cell lymphoma (DLBCL). Approximately 40% of patients with DLBCL who receive a CAR T cell product have a long-term response (Locke et al, 2021), which leaves ~60% of patients that do not benefit either due to lack of an initial response or a short duration of response to therapy. Furthermore, ~5% of eligible patients with DLBCL fail to receive an autologous CD19-directed CAR T product due to manufacturing (Locke et al, 2021). Emerging research suggests that the initial phenotype, specifically effector or stem cell-like memory (Lamure et al 2021; Locke et al 2021), of the CAR T cell product after manufacturing can greatly impact therapeutic outcomes. Therefore, developing novel reagents that aid in the consistent manufacturing of CAR T cells with well-defined phenotypes is needed to improve treatment outcomes in both adult and pediatric patients. Unlike traditional ex vivo expansion reagents that provide only TCR activation and co-stimulation, our novel platform utilizing multi-cytokine backpacks (MCB) can additionally provide cytokine support during CAR T cell manufacturing, which is critical for cell expansion, differentiation, and product phenotypes. We hypothesize that optimal formulations of these multi-cytokine backpacks will produce superior CAR T cell phenotypes with enhanced anti-tumor efficacy.

To assemble a compositionally diverse library of MCBs, magnetic iron oxide microparticles were decorated with agonist antibodies against CD3 and CD28 as well as those that display IL-2 and IL-15 in trans. MCB formulations were then screened by multi-dimensional flow cytometry to assess for transduction of CD19-directed CAR T cells and production of a less-differentiated phenotype (CD45RA+CCR7+). Top MCB candidates produced the highest populations of less-differentiated CAR T cells amongst both CD4+ and CD8+ subsets. Furthermore, the diverse composition of the MCB library particles allows for analysis to determine composition-function relationships that dictate CAR T cell phenotypes.

Five MCBs formulations were further assayed in vitro and in vivo compared to industry standard DynaBeads with IL-2 (DB). We first characterized the phenotypes of MCB-manufactured CAR T cells produced from healthy donors. MCBs were capable of transducing both CD28 and 41BB CAR constructs to equivalent levels as DB. MCB-manufactured CAR T cells positively increased CD8:CD4 ratios and yielded smaller proportions of exhausted phenotypes (PD-1+Tim3+Lag3+) than DB manufacturing. Metabolic profiling of these MCB-manufactured CAR T cells revealed differences in spare respiratory capacity. Antigen stimulation of MCB-produced CAR T cells demonstrated comparable polyfunctional cytokine production and cytotoxicity to DB. Functionally, MCB-manufactured CAR T cells exhibited greater proliferative capacity than control CAR T. Preliminary in vivo studies in a xenograft lymphoma model revealed significantly reduced tumor burden, enhanced in vivo CAR T expansion and persistence, and improved overall survival of mice treated with MCB-manufactured CAR T cells compared to DB-manufactured CAR T cells (p<0.05).

Next, to assess the capabilities of the MCB manufacturing platform in the context of disease, we generated CAR T cells derived from DLBCL patient samples. Similar to healthy donor-derived MCB-produced CAR T cells, DLBCL CAR T cells demonstrated lower proportions of exhausted phenotypes when manufactured with MCBs. Notably, we found that our MCBs significantly enhanced less-differentiated, stem cell-like memory (CD62L+) phenotypes in DLBCL CAR T cells (p<0.01). These findings support the idea that our novel MCB manufacturing platform can produce superior autologous CAR T cells in the context of disease.

Thus, we have identified unique multi-cytokine backpack formulations that consistently manufacture CAR T cells with higher proportions of less-differentiated phenotypes and improved anti-tumor efficacy in vivo. Ongoing studies aim to comprehensively phenotype MCB-manufactured CAR T cells at baseline and after tumor engagement. These studies also provide a proof-of-principle that this novel platform can be utilized to screen manufacturing microparticles for a wide range of cell therapy products.

Koff:Atara BioTherapeutics: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Oncternal Therapeutics: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Viracta Therapeutics: Research Funding.