Multi-Ganglioside Targeting to Overcome Antigen Heterogeneity in DMG

Rationale: Diffuse midline glioma (DMG) is a lethal brain tumor with no effective treatments. My research group discovered previously that H3K27M-altered DMG express high levels of ganglioside GD2 and demonstrated the potential of GD2 CAR T cells in treating these patients. However, while at least one durable response was observed, most patients progressed, and little is understood about mechanisms of resistance. In preliminary data, I have found that although GD2 is present in all DMG tumors, it is expressed at heterogenous levels, which may limit the efficacy of GD2 CARs. By studying the expression and regulation of gangliosides in DMG patients, we discovered heterogeneity in B4GALNT1, the enzyme catalyzing biosynthesis of GD2, and we hypothesized that this bottleneck would lead to higher levels of precursor GD3. In preliminary data, I have found high expression of precursor GD3 on GD2-low tumors. Importantly, GD3 has already been safely targeted in multiple clinical trials. 

Hypothesis: Based on preliminary data, I hypothesize that GD2 heterogeneity can limit GD2 CAR T cell therapy and co-targeting of GD2 and GD3 can potentially enhance therapeutic efficacy. 

Specific Aims: Aim 1: Define ganglioside heterogeneity in DMG to establish GD2/GD3 as a compelling target combination and define the basic biologic mechanisms driving GD2 heterogeneity. Our preliminary data indicate that GD2 is heterogeneously expressed in H3K27M-altered DMG and we previously reported on lower GD2 levels in H3wt tumors. I have found that GD3 is highly expressed in GD2-low tumors and hypothesize that co-targeting of GD2 and GD3 can efficiently target all tumor cells. We will further study GD2 and GD3 co-expression on a larger panel of primary samples, utilizing specially developed IHC and flow cytometry I developed. We will also perform spatial transcriptomics to reveal underlying biologic factors. 

Aim 2: Develop bispecific CAR T cells that efficiently co-target GD2 and GD3 in DMG. I have already developed a proof-of-concept CAR construct that effectively eliminates both GD2 and GD3-expressing cells in vitro. I will optimize the GD2-GD3 bispecific CAR construct and demonstrate efficacy and safety in xenograft models of DMG. 

Significance: This study draws on the combined expertise of the Majzner and Filbin Laboratories in both cancer immunotherapy and DMG tumor biology, respectively, which uniquely positions us to carry out this proposal. Our laboratory is currently embarking on a new GD2 CAR T cell trial in DMG. This trial utilizes a novel signaling chassis meant to overcome limitations of T cell exhaustion that contribute to poor persistence, which patients with heterogeneous GD2 are unlikely to benefit from. The results of my study will instead credential co-targeting of GD2 and GD3 with CAR T cells to extend the benefit of GD2 CAR T cells to patients whose tumors do not express high GD2, which we will similarly aim to translate into clinical trials.