Enhanced safety and efficacy profile of CD40 antibody upon encapsulation in pHe-triggered membrane-adhesive nanoliposomes

Abstract

Aim: To develop pH (pHe)-triggered membrane adhesive nanoliposome (pHTANL) of CD40a to enhance anti-tumor activity in pancreatic cancer while reducing systemic toxicity.

Materials and methods: A small library of nanoliposomes (NL) with various lipid compositions were synthesized to prepare pH (pHe)-triggered membrane adhesive nanoliposome (pHTANL). Physical and functional characterization of pHTANL-CD40a was performed via dynamic light scattering (DLS), Transmission Electron Microscopy (TEM), confocal microscopy, and flow cytometry. In vivo studies were performed using PDAC (Panc02) transplanted mice. Tumor tissue was analyzed by flow cytometry, and plasma cytokines and liver enzymes were analyzed by ELISA.

Results: pHTANL-CD40a reduced tumor growth, enhanced tumor immune infiltration/activation, and enhanced survival compared to vehicle and free-CD40a. Importantly, pHTANL-CD40a treatment resulted in significantly lower systemic toxicity as indicated by unchanged body weight, minimal organ deformity, and reduced serum levels of liver enzyme alanine transaminase (ALT) and inflammatory cytokine IL-6.

Conclusion: pHTANL-CD40a is more effective than free CD40a in anti-tumor activity, especially in altering the TME immune landscape for a potential therapeutic benefit in combination with immunotherapy.

Keywords: CD40 agonist antibody (CD40a); Immunotherapy; Pancreatic ductal adenocarcinoma (PDAC); nanoliposomes; pHe-triggered membrane adhesive.

Graphical abstract

Figure 1.

Physical characterization of the nanoliposomes. (a) Dynamic light scattering detected a homogenous particle size distribution with a narrow polydispersity index of pHTANL. (b) The transmission electron image showed a uniform morphology of the pHTANL particles. (c) The zeta potential estimated surface charges of pHTANL to be −14.7 mV and 37.17 mV at pH 7.4 and pH 6.5, respectively, suggesting physical stability at physiologic pH and cationic conversion at pH 6.5 of TEM enabling membrane adhesion.

Figure 2.

Functional characterization of the nanoliposomes. (a-d) pHTANL adheres to cell membranes and releases the ‘cargo’ extracellularly. pHTANL (a & c) and pH-insensitive ContNL (without DMA, B & D) were loaded with FITC and then added into cultures of macrophage cell line J774-A1. After 2 h of incubation, cultures were imaged by fluorescent (a & b) and confocal (c & d) microscopy. (e) Time kinetics of BSA retention/release by pHTANL-bsa formulation at physiological pH (7.4) and pHe condition of the TME (pH 6.5) was evaluated using a BCA kit at indicated multiple timepoints. *p < 0.05 versus corresponding value for pH 7.4.

Figure 3.

Encapsulation in pHTANL significantly enhances the anti-tumor efficacy of CD40a against PDAC. (a) BL6 mice transplanted with syngeneic Panc02 tumor were treated with CD40a (8 mg/kg), either free or formulated in pHTANL (pHTANL-CD40a), from day 3 through 15 (5 doses, indicated by arrowheads). Tumor volume was monitored every other day. Data are presented as mean ± SD of values from 8 mice in each group. Tumor growth rates were compared between groups using a linear mixed-effect model followed by Holm’s post-hoc procedure. (b) Median tumor volume on day 17 (8 mice per group). Comparisons between groups were conducted using an unpaired t-test followed by Holm’s post-hoc procedure. (c) Survival of mice bearing Panc02 (5 mice per group) following treatment with pHTANL-CD40a and controls. *p < 0.01 versus control and empty-pHTANL groups; #p < 0.05 versus free-CD40a group.

Figure 4.

Encapsulation of CD40a in pHTANL reduces systemic toxicity in PDAC transplanted mice. BL6 mice transplanted with syngeneic Panc02 tumor were treated with CD40a (8 mg/kg), either free or formulated in pHTANL (pHTANL-CD40a), every 3 days, from day 0 through 12 (5 doses). (A) Liver and kidney were harvested 48 h after the last treatment and analyzed by IHC. The tissues were ranked from ‘+’ (normal/minimal necrosis) to ‘+++’ (severe necrosis) based on the estimated necrotic (pink) areas; blood was collected 48 h after the last treatment and (B) serum level of ALT and (C) cytokines were measured by ELISA. Data for ALT are presented as mean ± SD of values from 4 mice in each group. Cytokine data are expressed as % change relative to the control group. (D) Body weights were monitored, as described in the methods. Comparisons between groups were performed using an unpaired t-test followed by Holm’s post-hoc procedure.

Figure 5.

pHTANL-CD40a significantly enhances the frequency and activation profile of tumor infiltrating APCs and T cells in PDAC. Tumor tissues, harvested 2 days after final treatment, as described above, were enzymatically digested, and analyzed by flow cytometry. Electronic gates were applied as: cells → singlets → viability → CD45 → CD11b/CD3/NK1.1, as indicated. Unstained cells were used as negative controls. (A and B) CD45-gated CD11b+ myeloid cells were further analyzed for the expression of CD206 and IA/IE (MHC class II) to determine antigen presentation capacity. (C and D) similarly, CD3+ T cells were further analyzed for the expression of CD4 and CD8 markers, and then CD8+ cells were analyzed for the surface expression of PD1 and CD107a. The dot plots (a and c) are representative data from one mouse in each group and the violin plots (b and d) show data from four mice in each group. Comparisons between groups were performed using an unpaired t-test followed by Holm’s post-hoc procedure.