Poster CC17 - Lymph node-mimicking hydrogels presenting dual co-stimulation modulate T cell phenotype and anti-tumor function in vivo

Introduction: T cell activation in lymphoid organs is driven by stimulation with three signals between the T cell and the antigen presenting cell (APC): signal 1, interaction between the T cell receptor and cognate peptide-MHC (pMHC), signal 2, co-stimulation mediated by several different receptor-ligand interactions, and signal 3, cytokine-mediated stimulation. Recently, our lab has developed a lymph node-mimicking hydrogel comprised of hyaluronic acid (HA) and conjugated to functional molecules that provide these three activating signals. This HA-based hydrogel is biocompatible, allowing us to co-inject it with naïve T cells subcutaneously.

Although we have previously demonstrated robust antigen-specific T cell expansion/ activation with this lymph node-mimicking hydrogel in vivo, there is potential for further modulation of T cell phenotype. The first iteration of this platform used anti-CD28 as the signal 2, as it is the canonical co-stimulatory molecule. However, the T cell-APC interaction is highly complex, and providing co-stimulation through additional signal 2 molecules like CD27, 41BB, OX40, and CD40L may enable modulation of T cell phenotype. By including these signaling molecules in our hydrogel, we hope to better recapitulate endogenous T cell stimulation, which in turn will enable modulation of cell expansion, activation, function, and memory phenotype both in vitro and in vivo. Moreover, we have found that hydrogels presenting multiple co-stimulatory ligands elicits differential T cell phenotype/function compared to hydrogels with only one ligand in vitro. We hope to leverage these findings about biomaterial-based T cell modulation to enhance adoptive T cell cancer immunotherapy.

Materials and

Methods: An overview of the hydrogel microparticle fabrication process can be seen in Figure 1A, whereby, HA, different signal 1, 2, and 3 molecules, and a crosslinker are mixed and passed through a mesh to produce ~150 um particles. For in vitro experiments, T cells were stimulated with microparticles conjugated to aCD3 (S1) and different S2 molecules. Co-stimulatory molecules investigated here were anti-CD28 (aCD28), aCD27, a41BB, aOX40, and aCD40L. The effect of dual co-stimulation was investigated by conjugating both aCD28 and one of the other S2s mentioned above at a 1:1 ratio. Hydrogels were added to B6 CD8+ T cells and cultured for 7 days with exogenous IL2 (S3). On D7, cell count, surface markers, and cytokine release were analyzed to evaluate expansion, activation/memory phenotype, and function, respectively.

The first in vivo assay performed was an in vivo killing assay, whereby hydrogels conjugated to KbSIY pMHC (S1), different S2 combinations, and an immuno-cytokine (S3) were co-injected subcutaneously with naïve CD8+ T cells on D0. On D6 and D20, mice were injected with CFSE-labeled peptide pulsed-splenocytes. Mice spleens were harvested on D7 or D21 and and on-target killing function was assessed via flow cytometry. Finally, a tumor study was performed whereby mice were inoculated with a B16-SIY tumor s.c. on D0. On D6, hydrogels conjugated to a KbSIY pMHC, different S2 molecules, and an immuno-cytokine were co-injected s.c. with naïve CD8+ T cells. Mouse survival and tumor volumes were measured over time.

Results, Conclusions, and Discussions: First, the effect of in vitro T cell dual co-stimulation was examined. All conditions achieved robust expansion over 7 days. Interestingly, different co-stimulation combinations produced different T cell memory profiles, with CD28+CD27 and CD28+OX40 stimulation biasing cells towards effector-like phenotypes, while CD28+41BB biased cells to being more memory-like (Figure 1B). Analysis of functionality demonstrated that CD28 alone or CD28+41BB stimulation elicited higher TNFa and IFNy secretion (1C).

These in vitro results highlight dual co-stimulation as a viable method for modulating T cell phenotype and function. Given that this hydrogel is biocompatible, the effect of expanding naïve T cells with differential co-stimulation was evaluated in vivo. An in vivo killing assay was performed to assess the persistence and cytotoxic function of T cells co-injected with hydrogels expressing either CD28, CD28+CD27, or CD28+41BB. At the D7 timepoint, there were no significant differences in on-target killing between groups (1D). Interestingly, at the D21 timepoint, there was significantly higher target cell killing observed in the spleen by T cells co-injected with the CD28+CD27 and CD28+41BB hydrogels, compared to CD28 alone. The effect of adoptive transfer with multiple co-stimulatory molecules was then evaluated in a B16-SIY melanoma model. After tumor inoculation, mice were treated with naïve CD8+ T cells and CD28, CD28+CD27, or CD28+41BB hydrogels . Gels expressing dual co-stimulation demonstrated improved survival and reduction in tumor volume compared to transfer with CD28 alone (1F,1G).

Ultimately, these results highlight the development and characterization of a functional, biocompatible hydrogel for expanding antigen-specific T cells in vitro and in vivo. By tuning the co-stimulatory signals conjugated to the hydrogel, we can modulate T cell phenotype. Moreover, dual co-stimulation leads to enhanced on-target killing and reduction of solid tumor burden. Ultimately, this lymph node-mimicking hydrogel is an exciting platform for modulating immune cell phenotype, streamlining adoptive T cell therapy workflow, and improving cell potency.

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