PD-L1 Immune Checkpoint Targeted Photoactivable Liposomes (iTPALs) Prime the Stroma of Pancreatic Tumors and Promote Self-Delivery

Abstract

Desmoplasia in pancreatic ductal adenocarcinoma (PDAC) limits the penetration and efficacy of therapies. It has been previously shown that photodynamic priming (PDP) using EGFR targeted photoactivable multi-inhibitor liposomes remediates desmoplasia in PDAC and doubles overall survival. Here, bifunctional PD-L1 immune checkpoint targeted photoactivable liposomes (iTPALs) that mediate both PDP and PD-L1 blockade are presented. iTPALs also improve phototoxicity in PDAC cells and induce immunogenic cell death. PDP using iTPALs reduces collagen density, thereby promoting self-delivery by 5.4-fold in collagen hydrogels, and by 2.4-fold in syngeneic CT1BA5 murine PDAC tumors. PDP also reduces tumor fibroblast content by 39.4%. Importantly, iTPALs also block the PD-1/PD-L1 immune checkpoint more efficiently than free α-PD-L1 antibodies. Only a single sub-curative priming dose using iTPALs provides 54.1% tumor growth inhibition and prolongs overall survival in mice by 42.9%. Overall survival directly correlates with the extent of tumor iTPAL self-delivery following PDP (Pearson's r = 0.670, p = 0.034), while no relationship is found for sham non-specific IgG constructs activated with light. When applied over multiple cycles, as is typical for immune checkpoint therapy, PDP using iTPALs promises to offer durable tumor growth delay and significant survival benefit in PDAC patients, especially when used to promote self-delivery of integrated chemo-immunotherapy regimens.

Keywords: PD‐L1 targeting; cancer; drug delivery; immunogenic cell death; photoactivation.

Figure 1:

Schematic representation of the utility of PD-L1 immune checkpoint targeted photoactivable liposomes (iTPALs (17α)) presented in this study. iTPALs (17α) contain a lipidated BPD-PC PS variant in the hydrophobic bilayer and α-PD-L1 antibodies on the surface. These iTPALs (17α) have following salient features: (a) facilitate tumor cell targeted delivery through PD-L1 receptor-mediated endocytosis, (b) induce immunogenic cell death upon light activation, which is characterized by the release of Damage Associated Molecular Patterns (DAMPs e.g. calreticulin), (c) block the PD-1/PD-L1 immune checkpoint between cytotoxic T cells and cancer cells, (d) prime the tumor stroma by photomodulating tumor collagen and fibroblasts to promote the self-delivery through PDAC tumors, and (e) inhibit tumor growth and improve survival even with a single sub-curative priming dose. Created with BioRender.com.

Figure 2:. α-PD-L1 antibody-to-liposome ratios impact cellular binding and uptake.

(A) Schematic representation of the targeted photoactivable liposomes (iTPALs) entrapping the BPD-PC PS on the hydrophobic bilayer and α-PD-L1 antibodies on the surface (Created with BioRender.com and ChemDraw 22.0.0). (B) Conjugation of varying α-PD-L1 antibody-to-liposome ratios on iTPALs (17α). (C) Median BPD emission from iTPALs (17α) obtained from the flow cytometry analysis on the cellular uptake of different α-PD-L1 antibody-to-liposome ratios (0, 3, 9, 17, 27, 35 antibodies per liposome) at 1 h, 6 h, 9 h, 24 h and 48 h of incubation in CT1BA5 cells. (D) Bar graph depicting the cellular uptake of iTPALs (17α) which shows a significant improvement on the cellular uptake of iTPALs (17α) at 9 h (E) % Improvement in cellular uptake of iTPALs with respect to untargeted PALs at different time points mentioned. (F) iTPALs (17α) exhibited the higher percentage improvement in uptake at 9 h in comparison to other iTPALs. (Data are mean ± S.D.; statistical significance was calculated on GraphPad Prism v9.2.0, ***: P<0.001, ****: P< 0.0001)

Figure 3:. iTPALs are physically stable during storage and incubation.

The hydrodynamic diameter (A) and polydispersity indices (B) of iTPALs (17α) was unchanged during storage in DPBS at 4 °C and. incubation in serum containing media at 37 °C for 8 days. (Data are mean ± S.D.)

Figure 4:. PD-L1 antibody conjugation does not impair RMS photoproduction by iTPALs.

(A, B) Singlet oxygen generation by iTPALs (17α), PALs, and conventional liposomal BPD (Lipo-BPD), as measured by the colorimetric singlet oxygen probe ADPA. (C and D) Singlet oxygen generation by the three formulations, as measured by fluorogenic singlet oxygen probe SOSG. (E and F) Hydroxyl radical and peroxynitrite anion generation by the three formulations, as measured using the fluorogenic probe HPF. (Data are mean ± S.D.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v9.2.0, *: P<0.1, **: P< 0.01)

Figure 5:. iTPALs are more photostable than other formulations.

(A) Decreasing fluorescence emission of Lipo-BPD, PALs and iTPALs (17α) in DPBS with increasing fluence of 690 nm irradiation. (B) Photobleaching rate kinetics of Lipo-BPD, PALs and iTPALs (17α) demonstrating lower rates of photobleaching in iTPALs (17α) than in other formulations. (Calculated by GraphPad Prism v9.2.0. (Data are mean ± S.D.; statistical significance was calculated on GraphPad Prism v9.2.0, ****: P< 0.0001)

Figure 6:. iTPALs (17α) bind specifically to PD-L1 expressing murine cancer cells.

(A) mIFN-γ increased the PD-L1 expression on murine cell lines: LLC (Lewis lung carcinoma), CT1BA5 (PDAC), MC38 (colorectal cancer), AT84 (head and neck cancer), BMFA3 (PDAC) and ID8 (ovarian cancer). (B) Binding specificity of iTPALs (17α) to murine cancer cell lines, with respect to untargeted PALs. (C) PD-L1 expression in all cell lines tested correlates positively with the binding specificity of iTPALs (17α). (Data are mean ± S.D; Correlation was analyzed using Pearson r correlations on GraphPad Prism v9.2.0, ****: P<0.0001)

Figure 7:. iTPALs are internalized in PDAC cancer cells through endocytosis.

Confocal microscopy images of CT1BA5 cells using a 100× objective after 24 h incubation with iTPALs (17α) (red, left). Lysosomes are labeled with LysoTracker^™ Green DND-26 (green, middle). The first two images (left and middle) were merged on ImageJ to identify regions of colocalization between iTPALs (17α) and lysosomes (yellow, right).

Figure 8:. iTPALs block the PD-1/PD-L1 immune checkpoint.

(A) Representative confocal microscopy images showing cytotoxic T cell-mimicking microspheres tagged with recombinant mouse PD-1 binding to CT1BA5 cells (left). The cells are also pre-incubated with PALs (middle) and iTPALs (17α) (right) to assess the blocking of PD-1 microsphere binding to PD-L1 expressing CT1BA5 cells. (B) Relative PD-1 microsphere binding to CT1BA5 cancer cells demonstrating that iTPALs (17α) are more effective at inhibiting PD-1/PD-L1 contact than free α-PD-L1 at the same antibody equivalent (7.5 nM). Increasing the ratio of α-PD-L1 per iTPALs from 17 to 35 does not further inhibit PD-1/PD-L1 contact in CT1BA5 cells (C), but does further inhibit PD-1/PD-L1 contact in AT84 cells (D). (Data are mean ± S.D.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v 9.2.0, *: P< 0.05, **: P< 0.01, ****: P< 0.0001)

Figure 9:. iTPALs induce immunogenic cell death in PDAC cells.

(A) Schematic representation of PDP-induced translocation of calreticulin (CRT) from the endoplasmic reticulum to the cell surface as a marker of immunogenic cell death (Created with BioRender.com). (B) Increase in the surface exposure of calreticulin in CT1BA5 cells is light dose dependent following incubation with iTPALs (17α). The median fluorescence intensity was obtained using flow cytometry after background subtraction of baseline fluorescence signals. (Data are mean ± S.D.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v9.2.0, **: P<0.01, **P<0.001)

Figure 10:. PDP using iTPALs photomodulates collagen.

(A) Schematic representation of the photomodulation of collagen hydrogels followed by imaging. (B) Representative SHG images of collagen hydrogels on the untreated control (left), with iTPALs (17α) incubation and no irradiation (middle), and with iTPALs (17α) incubation and 690 nm irradiation (right). Graphs depicting the reduction in collagen fiber area fraction(C), collagen fiber density (D), and comparison of reduction in collagen fiber area fraction with iTPALs (17α) + 690 nm irradiation and iTPALs (35α) + 690 nm irradiation (E) demonstrating the physical effects of iTPALs (35α) + 690 nm on collagen. Images were quantified using the image segmentation tool CT-FIRE and analyzed on the topmost slice of the z-stack. (Data are mean ± S.D.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v9.2.0, *: P<0.05, **: P<0.01, ***: P< 0.001)

Figure 11:. iTPALs promote self-delivery through collagen following photoactivation.

(A) Representation of the self-delivery of iTPALs (17α) through collagen that is potentiated by 690 nm irradiation. Fluorescence of iTPALs (17α) obtained by two-photon excitation imaging (B) Quantification of the penetration of iTPALs (35α) through the collagen hydrogel from the top where they were added to the hydrogel. (C) Graph demonstrating the improvement in self-delivery of iTPALs (35α) into the collagen hydrogel following 690 nm irradiation. Image quantification was performed by averaging the signal intensity on the plane of interest using a custom MATLAB script. (Data are mean ± S.D.; statistical significance was calculated using unpaired t-test on GraphPad Prism v9.2.0, n=3, ****: P<0.001)

Figure 12:. iTPALs promote self-delivery through PDAC tumors following photoactivation.

(A) Schematic representation of the timeline of the animal study conducted in C57BL/6 mice bearing CT1BA5 tumors. (B) Representative images of the mice bearing CT1BA5 tumors, administered with iTPALs (17α) without (top) and with (bottom) PDP using 690 nm irradiation at 0.5 h following intravenous administration. (C) Quantification of longitudinal tumor fluorescence imaging depicting the delivery and retention of iTPALs (17α) with 0 h, 9 h PDP and without PDP. (D) Area Under Curve (AUC) analyses showing the total accumulation of iTPALs (17α) into the tumor as calculated using GraphPad Prism v9.2.0. (Data are mean ± S.E.M.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v9.2.0, *: P<0.05, **: P< 0.001).

Figure 13:. A single PDP dose using iTPALs (17α) controls CT1BA5 PDAC tumor growth and prolongs the survival.

(A) Spline plot of the tumor volumes of mice until day 40 when all mice died, (B) area under curve of tumor volumes until day 16 (when a first mouse from the groups died) depicting a delay in tumor growth when treated with a single dose PDP (0 h) using iTPALs (17α) in comparison to sham IgG-PALs (0 h PDP) and the untreated control. (C) Spline plot of the tumor volumes of mice until day 40 when all mice died, (D) area under curve of tumor volumes until day 23 (when the first mouse from the respective treatment groups died) depicting a delay in tumor growth when treated with a single dose of PDP (0 h) using iTPALs (17α) in comparison to iTPALs (17α) with 9 h PDP, iTPALs (17α) without PDP, and the untreated control. (E) Kaplan-Meier plots representing the probability of progression free survival (tumor volume <500m³) in mice where iTPALs (17α) with 0 h PDP significantly improves the progression free survival in mice. (F) Kaplan-Meier plots representing the probability of overall survival in mice where iTPALs (17α) with 0 h PDP significantly improves the overall survival in mice. (Data are mean ± S.E.M.; statistical significance was calculated using one-way ANOVA with a Tukey post-test on GraphPad Prism v9.2.0; statistical significance for survival data was calculated using Log-rank (Mantel-cox test) on GraphPad Prism v9.2.0; *: P<0.05, **: P< 0.01, ***: P< 0.001).

Figure 14:. iTPAL self-delivery directly correlates with improved overall survival.

Scatter plots of the cumulative tumor uptake of iTPALs (A) and sham IgG-PALs (B) following PDP reveal a statistically significant correlation between iTPALs self-delivery and overall survival (P = 0.034). No relationship between tumor delivery of IgG-PALs and overall survival is found. (P values (two-tailed test) correspond to the statistical significance of the Pearson’s r correlation calculated using GraphPad Prism v9.2.0.)

Figure 15:. A single PDP dose using iTPALs (17α) reduces fibroblast content and collagen density in CT1BA5 PDAC tumors.

(A) Representative immunofluorescence images depicting decreased fibroblast contents on the cyrosectioned CT1BA5 tumor tissues when treated with iTPALs (17α) + PDP. (B) Quantitative analysis of fluorescence specific to fibroblasts from 10 ROIs spanning the entire tumor cross-section from 6 tumor samples for each condition. Mean fluorescence is calculated using Image J. (C) Representative second harmonic generation (SHG) images of cryosectioned CT1BA5 tumor tissues showing reduced collagen area fraction when treated with iTPALs (17α) + PDP. (D) Quantitative collagen area fraction on the control tumor tissues and iTPALs (17α) + PDP treated tumor tissues depicting significant reduction in collagen fiber area fraction with the treatment. (Data are mean ± S.E.M.; statistical significance was calculated using a two-tailed t test on GraphPad Prism v9.2.0; *: P<0.05, ****: P< 0.0001)