Poster H2 - Conditionally-Activatable Fusogenic Liposomes for Targeted Cytosolic Delivery of Granzyme B

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ananth P. Shyamal (he/him/his)

Undergraduate student
Massachusetts Institute of Technology
Cambridge, Massachusetts, United States

Co-Author(s)

QZ

Qian Zhong

Research Scientist
Massachusetts Institute of Technology, United States
HF

Heather Fleming

Research mentor
Massachusetts Institute of Technology, United States

Primary Investigator(s)

SB

Sangeeta Bhatia

Professor
Massachusetts Institute of Technology, United States

Introduction: Immunotherapies often show limited efficacy against immunologically inert tumors characterized by a lack of T cell infiltration and activation signals such as chemokines. Approaches that inflame immunosuppressive tumor microenvironments are promising therapeutic options to sensitize these tumors to various immunotherapies. Pyroptosis is an inflammatory form of programmed cell death in which released immunogenic cell death biomarkers promote the recruitment of antigen-presenting cells and T cells. Pyroptosis can be induced in gasdermin E (GSDME)-expressing tumor cells by granzyme B (GzmB), a serine protease released by cytotoxic T cells into the cytoplasm of target cells (Figure 1). Here, we engineer a class of conditional fusogenic liposomes (C-FLIPs) that overcome cell membrane barriers and enable efficient delivery of GzmB into the cytoplasm upon catalytic reaction with dysregulated proteases in tumors. C-FLIPs fuse with the plasma membrane of tumor cells after tumor-associated proteases remove fusion inhibitors. This fusion reaction subsequently releases membrane-impermeable GzmB directly into the cytoplasm (Figure 2). In contrast, C-FLIP fusion capability is largely masked in normal tissues and circulation due to limited expression of target proteases.

Materials and

Methods: C-FLIPs comprise fusion inhibitors, tumor-specific protease substrates, and fusogenic liposomes. We synthesized and optimized matrix metalloproteinase-cleavable C-FLIPs loaded with recombinant GzmB (C-FLIP/GzmB). We assessed the cytotoxicity of C-FLIP/GzmB to a cultured lung cancer cell line from a Kras(G12D);Trp53(fl/fl) (KP) mouse, both in the presence and absence of candidate proteases. We analyzed the expression of various immunogenic cell death (ICD) markers on the surface of the cancer cells and culture media post-treatment. Additionally, we analyzed the cleavage states of caspase 3 and GSDME in the treated cells by performing Western blots. In vivo, we evaluated the antitumor efficacy of C-FLIP/GzmB in the KP mouse lung cancer model by administering it intravenously as a monotherapy and in combination with an immune checkpoint inhibitor (ICI) therapy (αPD-1). Furthermore, we mechanistically examined how the pyroptosis elicited by C-FLIP/GzmB modulates the tumor immune microenvironment.

Results, Conclusions, and Discussions: C-FLIPs that can be conditionally activated by disease-specific proteases demonstrate an in vitro functional capacity to fuse with mouse tumor cells and deliver GzmB into their cytosols, requiring no concomitant pore-forming motif (e.g., perforin). As a result, conditionally-activated C-FLIP/GzmB exhibits high cytotoxicity when incubated with cultured cancer cells. Additionally, treated cancer cells and conditional media upregulate the expression of various ICD markers. In particular, we observe a bimodal distribution of expression in the treated cells, indicating a mixture of apoptotic and pyroptotic cells. Western blotting of treated cells shows the presence of cleaved caspase 3 and N-terminal proteolytic fragments of GSDME, indicating the occurrence of non-canonical pyroptosis via a GzmB-caspase 3-GSDME axis.

C-FLIP/GzmB administered intravenously substantially suppresses tumor growth in genetically-modified lung tumors by triggering GSDME-mediated pyroptosis. Strikingly, combining C-FLIP/GzmB with an ICI therapy attains tumor regression in the treated mice. Immune cell profiling reveals that the pyroptosis triggered by the C-FLIP/GzmB alone or in combination with ICI therapy substantially increases the ratio of the number of effector CD8+ T cells to immunosuppressive regulatory T cells and reduces CD8+ T cell exhaustion. This suggests that C-FLIP/GzmB can reprogram the tumor immune microenvironment towards a more inflamed state, boosting the efficacy of ICI therapy.

The delivery of GzmB using non-viral, cell-free C-FLIPs represents a promising cancer treatment strategy, especially for treating immunosuppressive tumors, since they can induce pyroptosis and synergize with ICI therapy to suppress tumor growth. Broadly, C-FLIPs represent a promising platform for delivering membrane-impermeable biomacromolecules to the cytosol, particularly for gene editing and therapeutic delivery applications.

Acknowledgements (Optional): Zhang, Zhibin, et al. “Gasdermin E Suppresses Tumour Growth by Activating Anti-Tumour Immunity.” Nature News, Nature Publishing Group, 11 Mar. 2020.