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. 2024 Dec 29;20(2):155–166. doi: 10.1080/17435889.2024.2446008

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

Salma Althobaiti a,*, Prahlad Parajuli a,b,*, Duy Luong a, Samaresh Sau a, Lisa A Polin b,c, Seongho Kim c, Yubin Ge b,c, Arun K Iyer a,d,✉, Navnath S Gavande a,b,✉
PMCID: PMC11731328  PMID: 39764733

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

GRAPHICAL ABSTRACT

graphic file with name INNM_A_2446008_UF0001_OC.jpg

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1. Introduction

Pancreatic cancer is one of the most lethal solid tumors. It accounts for ~ 3% of all cancers in the US and 7% of all cancer deaths, with a 5-year overall survival of 3% for distant (metastatic) stage and 12% for all stages combined [1–4]. Pancreatic ductal adenocarcinoma (PDAC), arising from the exocrine pancreas, is the most common and aggressive subtype due to the lack of early diagnosis and limited response to treatments. Currently, gemcitabine (GEM), in combination with nab-paclitaxel (PTX) and/or erlotinib, comprises the frontline chemotherapy (CT) regimen for locally invasive and metastatic PDAC, albeit with only a modest benefit [1–3]. Many solid tumors respond well to immunotherapy (IT) targeting specific checkpoints in the T cell activation pathway [5]. However, PDAC is considered immunologically ‘cold’ due to low tumor mutation burden resulting in low neoantigen production, and lack of immune infiltration, especially by effector T cells [6–8]. Therefore, IT targeting the PD-1/PD-L1 checkpoint have resulted in an unimpressive survival benefit, ranging merely from 3 weeks (0.7 months) to 3.8 months, in PDAC [7,8].

PDAC features a desmoplastic stroma comprised of a heterogenous population of highly proliferative cancer-associated fibroblasts (CAFs), pancreatic stellate cells, and deposition of thick extracellular matrix (ECM) proteins. Desmoplasia contributes to chemoresistance by hindering tumor penetration by CT drugs [7,9]. Desmoplasia also hinders tumor infiltration by immune cells, thus making PDAC unresponsive to IT [6,10]. In pre-clinical and clinical studies, treatment of PDAC with CD40 ligand (CD40L) or agonistic CD40 Ab (referred to as CD40a) have been shown to break the desmoplastic ECM to a great extent [11,12].

In this context, first-generation strategies to stimulate CD40 via CD40-L (recombinant or membrane-bound) were not able to sufficiently stimulate immune activity due to inadequate receptor crosslinking [13]. Next-generation tetra- or hexa-valent CD40L or CD40a activate various effector functions in CD40+ macrophages, B cells, and dendritic cells (DC), and promote antitumor CD8+ T-cell immunity [12–15]. Clinical studies show modest but encouraging improvement in survival following treatment with CD40a, especially in combination with standard CT (GEM/PTX) and IT (checkpoint inhibitors) [16–19]. However, therapy with CD40a also results in undesirable systemic toxicity, including cytokine release syndrome (CRS) mediated by IL-6, elevated transaminases, and hepatitis, due to overstimulation of the immune system [13,15–19]. Moreover, abundant CD40-expressing peripheral immune cells create an antigen sink that reduces the pharmacokinetics (PK) of CD40a in the tumor, thus reducing its therapeutic efficacy [20]. Therefore, developing novel strategies for selective delivery of CD40a into the tumor microenvironment (TME) is a crucial need for improving the efficacy and safety of CD40a therapy.

Liposome-based nanolipids (NLs) have demonstrated tremendous clinical success with the ability for retention and sustained delivery of both hydrophobic and hydrophilic therapeutics due to their core-shell nanostructure [21]. NL has significantly enhanced efficacy and reduced side effects of CT, because of its prolonged stability in the plasma and passive targeted delivery of CT into tumors by taking advantage of the enhanced permeability and retention (EPR) effect [21,22]. Several clinical studies have utilized NL formulations for passive or active delivery of anti-cancer drugs in various cancers, including pancreatic cancer, with various degrees of success [23–25]. Our group has successfully adopted NL for tumor-specific drug delivery in pre-clinical studies for enhanced efficacy with minimal systemic toxicity [26–28]. The purpose of this study was to develop and evaluate low extracellular pH (pHe)-triggered tumor/stromal cell membrane-adhesive nanolipid encapsulated CD40a (pHTANL-CD40a) for targeted delivery into the TME to enhance efficacy while minimizing systemic toxicity.

2. Materials and methods

2.1. Lipids, therapeutic antibodies, and other reagents

Lipids were obtained from Avanti Polar Lipids [L-α-phosphatidylserine (DSPS), CAS number: 383907-32-2; DSPE-PEG2000-DMA, Lot Number: 791849-01-013] or from Fisher Scientific [Cholesterol (Chol), CAS number: 57-88-5]. Anti-mouse CD40 agonistic antibody (FGK4.5/FGK45) and Anti-mouse PD-L1 inhibitor antibody B7-H1(10F.9G2) were obtained from BioXCell (NH, USA). All other reagents, unless specifically mentioned, were obtained from Fisher Scientific.

2.2. Preparation of pHTANL and loading with CD40a cargo

Preparation of pHTANL-CD40a was performed by thin film hydration followed by membrane extrusion, following our published protocols [27,29], with some modifications. Quality by Design (QbD) software was used to theoretically predict the best possible optimized formulation with good stability and high drug loading [30]. In brief, all lipid components, DSPS, Chol, DSPE-PEG2000-DMA, at indicated ratios, were dissolved in chloroform, then the organic solvent was evaporated by using a rotary evaporator in a warm water bath at 45°C and high pressure to form the phospholipid film, followed by 30 minutes of incubation in a vacuum desiccator. The desired volume of CD40 antibody was prepared in phosphate-buffered saline (PBS) and swirled at 60°C in a water bath for 30 minutes. The hydrated film was incubated at 4°C overnight and then the liposomes were extruded 21 times through polycarbonate membranes with pore sizes of 400, 200, and 100 nm sequentially, followed by the separation of antibody-encapsulated liposomes from free antibodies by using a 200-kDa molecular weight cutoff (MWCO) centrifuge tube. For some experiments, pHTANL was loaded with bovine serum albumin (BSA) or fluorescein isothiocyanate (FITC), instead of CD40a antibody.

2.3. Evaluation of physical characteristics, and stability of pHTANL-CD40a

Dynamic light scattering (DLS) (Beckman Coulter Delsa Nano-C DLS Particle Analyzer, Fullerton, CA) was used to measure the average particle size, polydispersity index (PDI), and zeta potential of the liposomes at room temperature, following our published protocol [31]. Briefly, samples were diluted in PBS (10 mm phosphate buffer, 150 mm NaCl, 300 mOsm) pH 7.4 and potassium phosphate buffer 1 M pH 6.5 to measure particle size and zeta potential. To evaluate the shape of nanoparticles, samples were further examined by a JEOL Transmission Electron Microscope (TEM) equipped with a LaB6 fiber gun (JEM 2010, Tokyo, Japan) at a voltage of 200 kV. Samples were prepared by layering a small drop of appropriately diluted nanoparticles on copper grid with a 200-mesh carbon coating, stained with 5% uranyl acetate, and any extra staining solution was absorbed with filter paper. BSA release by the nanolipid formulations were measured using a BCA kit (ThermoFisher, Catalog # A53225) following the manufacturer’s instructions.

2.4. Cell lines and culture

PDAC cell lines, Panc02 and AsPC-1, were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S). Mouse macrophage cells (J774-A) were maintained in RPMI-1640 containing 10% FBS and P/S.

2.5. In vitro cell membrane adhesion study

A published protocol [32] was followed, with some modifications. Briefly, J774-A macrophage cells were cultured overnight in a glass-bottomed cell culture dish (CELLview, Greiner) and then incubated with 1 mg/ml pHTANL- FITC in fresh media for 2 h at 37°C. Cells were then fixed in 4% formaldehyde for 15 minutes, and stained with Hoechst 33,542 (Invitrogen, USA), 1 mg/ml for 5 min. After washing with PBS, the cells were observed and imaged under a confocal microscope (LSM 780, Zeiss, Germany).

2.6. Syngeneic PDAC transplant model in C57BL/6J (BL6) mice

All animal procedures were conducted in accordance with the U.S. Public Health Service Policy on Use of Laboratory Animals and with approval by Wayne State University Institutional Animal Care and Use Committee. BL6 mice (7 to 8 weeks old) were purchased from Jackson Laboratory and were allowed to acclimatize for three days before any experiment. Frozen Panc02 tumor stocks were first resurrected using SCID mice [33] and then serially transplanted into host BL6 mice. Tumor fragments (30 to 50 mg) were subcutaneously (s.c.) implanted via trocar and non-selectively distributed to control and treatment groups, as previously described [34,35]. When the tumors were palpable (day 3), mice (n = 8/group) were intraperitoneally (i.p.) administered pHTANL- CD40a (8 mg/kg in PBS) or controls (empty-pHTANL, free-CD40a, or PBS), every three days for a total of 5 doses. Tumor growth was measured with a vernier caliper two to three times per week. Tumor volume was calculated with the formula XY2/2, where X is the long axis and Y is the short axis. Blood, tumor, spleen, liver, and kidney were harvested 48 h after the final treatment, to analyze systemic cytokine/liver enzyme levels, immune phenotype/function, and organ toxicity. For the survival experiment, the mice were euthanized when the tumor volume reached ~1500 mm3.

2.7. Immune profiling

Flow cytometry was performed as described elsewhere [36]. Briefly, harvested tumor tissues were enzymatically digested, and single cell suspensions were incubated with antibodies against various immune cell phenotypic/functional markers, as indicated. Flow cytometry data was acquired using a spectral flow cytometer (Cytek, Northern Lights) and analyzed using FCS Express 7.1 software (DeNovo). The antibodies used are listed in Supplementary Table S1.

2.8. Evaluation of systemic toxicity by histopathology, serum enzyme, and cytokine analyses

Liver and kidney sections were stained with hematoxylin & eosin and imaged following our published protocol [37]. To quantitatively assess the liver toxicity and systemic inflammation, serum samples were analyzed, using ELISA kits, for alanine transaminase (ALT) (MyBioSource, MBS264717), IL-6, (BioLegend 431,304) and IL-10 (BioLegend,431411), following the manufacturers’ instructions.

2.9. Statistical analysis

Data analysis was performed using GraphPad Prism version 10.1.2 (GraphPad Software). All in vitro experiments and ex vivo assays were performed at least twice, each time with three replicates, where applicable. The distribution of continuous outcomes was checked, and if needed, a data transformation (e.g., square root transformation) was applied to meet normality assumptions. Murine survival was graphically summarized using Kaplan-Meier curves and compared using the log-rank test. For tumor volume and in vitro data, unpaired student’s t-test was used to compare groups. The area under the tumor growth curve (AUC) was calculated using the trapezoidal method, and tumor growth rates were compared using a linear mixed-effects model. Multiple comparison corrections and post-hoc analyses were performed using Holm’s procedure. A p-vale of < 0.05 was considered significant.

3. Results

3.1. Preparation and characterization of CD40a-pHTANL

A small library of nanoliposomes (NL) with various lipid compositions were synthesized as listed in Supplemental Table S2. Based on the average hydrodynamic particle size and the ability for Zeta potential conversion in pH 7.4 and pH 6.5, NL number 3 (pHTANL) with a lipid composition of DSPS:Chol:DSPE-PEG2000-DMA (molar of 1:0.1:0.08) was selected for further studies. DLS measurement of pHTANL showed the average hydrodynamic size of 155 ± 70.7 nm. A polydispersity index value of 0.18 indicated uniformity of the particle size distribution (Figure 1(a)). TEM imaging showed the morphology of the pHTANL with a uniform size (Figure 1(b)). The zeta potential determination estimated the surface charges of pHTANL to be −14.7 mV and 37.17 mV at pH 7.4 and pH 6.5, respectively (Figure 1(c)), suggesting that the pHTANL was physically stable at physiological pH and undergoes DMA cationic conversion at low pH. In principle, protonation, and cationic conversion of DMA and DSPS in the acidic TME will enable pHTANL to adhere to cell membranes and initiate subsequent sustained release of CD40a in the tumor interstitial space, respectively.

Figure 1.

Figure 1.

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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.

3.2. At low extracellular pH (pHe), pHTANL adheres to the cell membrane and releases the ‘cargo’ extracellularly

pHTANL was loaded with FITC and incubated with macrophages in media at pH 6.5. After 2 h, membrane adherence or internalization of pHTANL-FITC was detected by fluorescent and confocal microscopy. In cultures incubated with pHTANL-FITC, the FITC was detected on the membrane of cells (Figure 2(a) and (c)). On the other hand, FITC loaded in the pH-insensitive control NL that lacked adhesive lipid DSPE-PEG2000-DMA (ContNL), was detected inside the cells (Figure 2(b) and 2(d)). These results show that cationic conversion of DMA at acidic pHe condition enables membrane adherence of pHTANL for subsequent release of cargo in the interstitial space.

Figure 2.

Figure 2.

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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.

To demonstrate sustained drug retention and release as a proof-of-principle, the release of BSA from pHTANL-BSA formulation was analyzed over several hours. We observed complete retention of BSA for over 6 h, followed by sustained release (>80%) over a 48 h period at physiological pH (Figure 2(e)), which agrees with the hydrodynamic size results from DLS, suggesting stability of pHTANL with minimal release of cargo in the circulation. On the contrary, a considerable amount of released BSA (23 ± 0.8%) was detected as early as 6 h following exposure to pHe condition (Figure 2(e)). The results indicated that cationic conversion of DSPS at pH 6.5 initiates release of cargo significantly earlier than at pH 7.4, and the release is sustained over a period of 48 hours.

3.3. Encapsulation in pHTANL significantly enhances anti-tumor efficacy of CD40a while reducing systemic toxicity in a mouse PDAC transplant model

Treatment of Panc02-transplanted mice with free CD40a (8 mg/kg, i.p.) significantly reduced the growth of tumors (Figure 3(a) and (b); Supplementary Table S3), but the tumors grew rapidly after the treatment was stopped and there was no survival benefit compared to the control groups (Figure 3(c)). On the other hand, pHTANL-CD40a showed significantly higher inhibition of overall tumor growth rate and median tumor volume on day 17 with a treatment efficacy of 61.5% (compared to 27.3% in free CD40a group (Figure 3(a) and (b)). The area under the curve (AUC) analysis also showed significant reduction of tumor growth by pHTANL-CD40a compared to control (Mean±SD: 5699.84 ± 865.21 vs. 2641.97 ± 942.36, p = 0.0001, Supplementary Table S3) and a considerable reduction compared to free-CD40a (Mean±SD: 3454.57 ± 1239.87 vs. 2641.97 ± 942.36, p = 0.3274, Supplementary Table S3). Importantly, the anti-tumor effect continued beyond the final treatment, with a significantly prolonged survival of 29 days in the pHTANL-CD40a group, compared to 20 and 22 days in the control and free CD40a groups, respectively (Figure 3(c)).

Figure 3.

Figure 3.

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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.

Liver and kidney tissues from pHTANL-CD40a treatment group showed noticeably fewer signs of deformity (toxicity) than the tissues from the free-CD40a group (Figure 4(a)). In a more objective assessment of systemic toxicity, the serum level of liver enzyme ALT was measured, and was found to be significantly enhanced following the treatment with free CD40a; whereas in the pHTANL-CD40a group, it was comparable to that of the control group (Figure 4(b)). Similarly, there was a significant increase in serum level of inflammatory cytokine IL-6, the main mediator of cytokine release syndrome (CRS), in the free-CD40a group, while it was significantly reduced in the pHTANL-CD40a group, compared to the control as well as the free CD40a group (Figure 4(c)). On the other hand, the serum level of IL-10 remained unaltered (Figure 4(c)).

Figure 4.

Figure 4.

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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.

Finally, an immediate reduction in body weight was observed in animals after the first round of free CD40a administration (>15%), and the weight did not recover throughout the study period (Figure 4(d)). Whereas there was no significant change in body weight in mice treated with pHTANL-CD40a relative to controls (Figure 4(d)). These results demonstrated that pHTANL-CD40a causes minimal systemic CRS or organ toxicity and significantly improves the safety profile of CD40a.

3.4. pHTANL-CD40a significantly enhances the frequency and activation profile of antigen presenting cells (APCs) and CD8+ T cells in PDAC transplants

Flow cytometry analysis revealed a significant enhancement in the relative frequency of CD11b+ MHC class II (IA/IE) expressing APCs in the tumor from pHTANL-CD40a treatment group compared to the controls (Figure 5(a) and (b)). Notably, treatment with CD40a considerably enhanced the expression of MHC class II in the APCs (35.6 ± 11%), relative to controls (22.7 ± 7%), and the expression levels were even higher (41.41 ± 10%) in the pHTANL-CD40a treatment groups (Figure 5(a) and (b)). These data suggest that pHTANL encapsulation of CD40a further enhances the antigen presenting capacity of myeloid cells in the TME. Similarly, while the frequency of tumor-infiltrating CD3+ T cells were modestly enhanced by both free CD40a and pHTANL-CD40a, compared to the control; the relative frequency of activated (CD107a+) CD8+ T cells in the pHTANL-CD40a treated tumors were considerably higher than in free-CD40a treated tumors (p = 0.09) and significantly higher than in the control tumors (p = 0.05) (Figure 5(c) and (d)). The expression of death receptor PD-1 in the CD8+ T cells was also considerably lower in the pHTANL-CD40a group (p = 0.6) and the free CD40a group (p = 0.48) than in the control. Moreover, the CD8/CD4 T cell ratio in the pHTANL-CD40a group was considerably higher than in the free-CD40a group (p = 0.27) and the control group (p = 0.09).

Figure 5.

Figure 5.

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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.

4. Discussion

CD40-targeted IT has shown tremendous promise against solid tumors, including PDAC, in several pre-clinical and clinical studies due to its ability to: (1) induce cell mediated (T cell) and humoral (B cell) immune responses [13,14,38,39], (2) directly affect the proliferation of CD40-expressing tumor cells [40]; and (3) indirectly alter the tumor stroma [11,13,14,41]. However, attempts to reprogram the TME by CD40L or CD40a have been met with challenges related to lower-than-expected efficacy and undesirable systemic toxicity. Therefore, Intratumoral administration of CD40a, peritumoral injection of CD40a-hydrogel or use of bispecific Abs for selective targeting of DCs has been attempted to enhance efficacy of CD40a therapy and reduce associated systemic toxicity in pre-clinical models of PDAC and melanoma [42–45]. In this study, we encapsulated CD40a in tumor extracellular pH (pHe)-triggered membrane-adhesive nanoliposomes (pHTANL) to enhance anti-tumor efficacy of CD40a while reducing systemic toxicity.

Studies with about 15 NL-based anti-cancer therapeutics either approved by the FDA or in clinical trials have shown improved response rate due to enhanced tumor accumulation of the drug (>10-fold) [46], despite very low accumulation of administered non-targeted NL in the tumor via EPR effect. Due to tumor heterogeneity among PDAC, various degrees of EPR has been reported based on the degree of vascularity and desmoplasia [47]; and the EPR effect can be augmented by vesico-vacuolar organelle (VVO) or iRGD mediated transcytosis [48]. Recently, two FDA-approved NL-based drugs Abraxane and Onyvide have shown modest survival benefit in patients with pancreatic cancer compared to conventional therapy [24,25]. Moreover, in recent pre-clinical studies, antigen-targeted or pH-sensitive NL have been shown to preferentially accumulate (~60%) in the tumor, versus ~ 20% accumulation in the liver and spleen [49–51]. These observations suggest that a smart NL-based, preferential intratumoral delivery and sustained release will significantly enhance the tumor availability and hence the efficacy of CD40a. Antibody (Ab) ‘conjugated’ NL (‘immunoliposomes,’ where the Ab sits on the outer surface of NL) or nanoparticle-based hydrogels, which provides a depot effect for sustained release, have been reported [44,52,53]. These immunoliposomes or NP/hydrogel-CD40a preparation were not designed to sequester the cargo (CD40a) in the circulation. Therefore, one study tested administration of NP/hydrogel-CD40a in peritumoral space [44], rather than systemic administration. To the best of our knowledge, we are the first to describe a pH-triggered, cell-adhesive NL design which encapsulates high molecular weight antibody, such as CD40a, as a cargo.

Our pHTANL-CD40a formulation was stable at physiological pH and protected the entire payload for more than six hours, which is sufficient time for the accumulation of pHTANL-CD40a in the tumor by escaping through leaky intratumoral vesicles [22,54]. The payload protection in the first 6 h along with the preferential accumulation of pHTANL-CD40a and subsequent sustained release of CD40a in the acidic TME makes the pHTANL-CD40a safer than free CD40a treatment by reducing the on-target, off-tumor effect of CD40a in healthy tissues. In a parallel study using a kidney cancer transplant model, we have observed preferential accumulation of red fluorescent pHTANL in the tumor than in the liver (data not shown). Further PK/PD studies need to be performed to confirm the same in mice models of pancreatic cancer. Our pHTANL formulation was designed to enable pHe-triggered cationic conversion and adherence to cell membranes in the acidic TME. Our results demonstrated adherence of pHTANL on the macrophage cell membrane in vitro at acidic pH. Upon membrane adherence, the release of cargo in the extracellular space would enable binding of CD40a to its receptors on the surface of stromal cells in the TME. This occurrence was also reflected in the PDAC transplant model, as indicated by a higher number of activated (MHC class II high) APCs in the tumor following treatment with pHTANL-CD40a. This led to infiltration of T cells with a high CD8/CD4 ratio and activated (CD107a+) CD8 T cells, and ultimately resulted in reduction of tumor volume and prolongation of survival in the pHTANL-CD40a treatment group. More importantly, there was a significant reduction of systemic inflammatory cytokine release, as indicated by lower serum levels of IL-6 and unchanged levels of IL-10, which was accompanied by lower levels of liver enzyme ALT, low organ deformity in the liver and kidney, and no significant loss of body weight in the pHTANL-CD40a treated group compared to the control or free CD40a treated group. These results indicate that pHTANL-CD40a not only avoids systemic toxicity by reducing IL-6 induction, but also is more efficacious than free CD40a in inducing infiltration and activation of APCs (monocytes/macrophages) and effector T cells in the TME.

There are a few limitations of this study, such as 1) the survival analysis was performed with a relatively small group (n = 5) of animals for a short duration (29 days). 2) The cytokine/ALT toxicity analysis in the pHTANL control group was not included, as there was no significant difference between the control and empty-pHTANL groups in tumor growth and survival. Therefore, we excluded the sera from the Empty-pHTANL group in the cytokine analysis. More extensive studies will be conducted in the future to evaluate the toxicity profile and efficacy, especially long-term survival benefits, of pHTANL-CD40a in combination with standard CT/IT regimen in spontaneous PDAC or PDX mice models.

Treatment of spontaneous PDAC-bearing KP (KRAS and p53 mutant) mice with CD40L or CD40a has been shown to alter desmoplasia via macrophage-mediated degradation of collagen 1 and reduction in fibrosis, via elaboration of collagenase, IFN-g and CCL2 [11]. Moreover, clinical studies with small patient cohorts have also shown reduced fibrosis and desmoplastic stroma, along with higher activation/infiltration of monocytes/macrophages and T cells in the surgical specimen from patients with PDAC following treatment with CD40a and chemotherapy [11,12]. Another clinical study evaluating the combination of a CD40L-expressing oncolytic virus with standard CT (GEM/PTX) and IT (PD-L1 inhibitor) has been underway [55]. A recent study has revealed expression of CD40 in cancer-associated fibroblasts (CAFs), although in low levels [56]. Since CAFs also play a critical role in inducing desmoplasia, TME-targeted delivery of CD40a may also contribute to reduction of desmoplasia by directly or indirectly altering the phenotype/functions of CAFs, along with the activation/polarization of tumor associated monocytes/macrophages (TAM). These observations support the notion that tumor-targeted delivery of pHTANL-CD40a reprograms the TME to benefit IT/CT strategy and enhance survival in PDAC.

5. Conclusion

In this study, we have optimized the preparation of pHTANL-CD40a. We have demonstrated that pHTANL adheres to the cell membrane at TME-like pHe conditions, followed by sustained cargo release in interstitial space. This ensures binding of CD40a to the surface membrane, rather than being internalized, and alters the phenotype/functions of CD40 expressing cells. In a PDAC transplanted mouse model, pHTANL-CD40a exhibited significantly higher anti-tumor efficacy than free CD40a as indicated by late but significant reduction in tumor growth, a reduced median tumor volume, lower area under the curve over time, and a significantly prolonged survival, which was accompanied by a significantly higher activation/infiltration of APCs and CD8+ T cells in the tumor. Importantly, pHTANL-CD40a is stable at physiological pH, thereby minimizing the release of CD40a in the periphery, and avoiding inflammatory CRS, as indicated by significantly reduced plasma IL-6 level, and systemic toxicity. Finally, our results suggested that pHTANL-CD40a is more effective than free CD40a in altering the TME immune landscape for a potential therapeutic benefit against PDAC in combination with immune checkpoint (PD-1/PD-L1) inhibitors.

Supplementary Material

Supplemental Material
INNM_A_2446008_SM2811.docx (23.3KB, docx)

Acknowledgments

The research work in Gavande laboratory is supported by the National Institutes of Health (R01CA247370 and R01AI161570), the Department of Defense (W81XWH-22-1-0369), the VA, KCI’s Michigan SPORE, Richard Barber Interdisciplinary Research Program, DMC Foundation, WSU Applebaum Faculty Research Award (FRAP) and the Wayne State University. AKI acknowledges US Department of Defense CDMRP KCRP Idea Development Award # W81XWH1810471 for funding Iyer lab. The Animal Model & Therapeutics Evaluation Core (AMTEC) and Microscopy, Imaging and Cytometry Resources Core are supported, in part, by NIH Center grant P30 CA22453 to the Karmanos Cancer Institute and R50 CA251068 to Dr. Moin (MICR), Wayne State University. We are thankful to Jeremy Kelm and Amirreza Samarbakhsh for thorough editing of the manuscript for its language and scientific content.

Funding Statement

The authors gratefully acknowledge the funding from the WSU Applebaum Faculty Research Award Program (FRAP) to generate preliminary data and the National Institutes of Health NCI (R01CA247370) and the Department of Defense (W81XWH1810471) to partially support this project. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Article highlights

  • Pancreatic ductal adenocarcinoma (PDAC) is resistant to immunotherapy due to highly desmoplastic stroma and lack of immune infiltration.

  • Agonistic CD40 mAb (CD40a) has capacity to break the desmoplastic stroma and induce immune activation and tumor infiltration, but therapeutic studies have shown only modest efficacy and high systemic toxicity due to lack of tumor-targeted delivery.

  • Tumor-targeted delivery of CD40a via encapsulation in low extracellular pH (pHe)-triggered membrane adhesive nanoliposome (pHTANL) could enhance anti-tumor immune responses while reducing systemic toxicity

  • pHTANL-CD40a was prepared and characterized via dynamic light scattering (DLS), Transmission Electron Microscopy (TEM), confocal microscopy

  • Cargo retention and release, at pH7.4 and pH6.5, respectively, was assessed via BSA-release assay and adhesion to macrophage membrane was analyzed via flow cytometry and confocal microscopy.

  • In a PDAC transplanted mouse model, pHTANL-CD40a exhibited significantly higher anti-tumor efficacy than free CD40a as indicated by a reduction in tumor growth, a significantly reduced median tumor volume, lower area under the curve over time, and a significantly prolonged survival.

  • Importantly, pHTANL-CD40a induced significantly higher activation/infiltration of APCs and CD8+ T cells in the tumor while avoiding inflammatory CRS, as indicated by significantly reduced plasma IL-6 level, and systemic toxicity.

  • Ther results suggest that pHTANL-CD40a is more effective than free CD40a in altering the tumor immune landscape for a potential therapeutic benefit against PDAC in combination with immune checkpoint inhibitors.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Author contributions

NSG, PP and AKI conceptualized and designed the study and wrote the manuscript; SA and PP performed the experiments, analyzed data, prepared the figures, and wrote the manuscript; DL and SS prepared the nanolipids, characterized them, prepared the figures, and helped write the manuscript. LAP and YG helped SA in animal studies and data analysis. All authors contributed essential portions of the manuscript, reviewed, and approved the final manuscript.

Ethical declaration

All animal procedures were conducted in accordance with the U.S. Public Health Service Policy on Use of Laboratory Animals and with approval by Wayne State University Institutional Animal Care and Use Committee (IACUC-23-01-5454).

Data availability statement

Data presented in this study is available from the corresponding author upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2024.2446008.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
INNM_A_2446008_SM2811.docx (23.3KB, docx)

Data Availability Statement

Data presented in this study is available from the corresponding author upon reasonable request.

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