(357v) Carbon Nanotube-Protein Conjugate for Photothermal Therapy Combined with Checkpoint Inhibition for the Immunomodulatory Treatment of Metastatic Breast Cancer

Research Interests: Pharmaceutical development, protein engineering, protein-drug conjugation, bionanotechnology, photothermal therapy for cancer treatment, combination of therapeutic strategies to enhance immunotherapy.

Introduction:

The lack of targeting treatments for triple negative breast cancer (TNBC) leads to poor prognosis, especially when metastasis has occurred, which spurs on the development of novel treatment strategies.

Although the development of checkpoint inhibition, such as anti-PD-1, has established a breakthrough in cancer treatment, the lack of responsivity to immunotherapy has been correlated with the absence of tumor associated CD8+ T cells, which characterize non-immunogenic tumors. There is evidence that the ablation of solid tumors generates danger signals and tumor antigens that convert it into an immunogenic environment. Thus, the combination of an ablation method to immunotherapy is promising for the generation of a synergistic effect that is capable of not only treating the primary tumor, but also generating a systemic immune response that suppresses metastasis.

We have developed a targeted photosensitizer for photothermal therapy (PTT) for the efficient ablation of solid tumors based on the functionalization of single-walled carbon nanotubes (SWCNTs) to annexin A5 (ANXA5): SWCNT-ANXA5 conjugate. The peak absorbance of SWCNT in the near-infrared (NIR) range efficiently convert light into thermal energy during PTT, aiming to ablate the primary tumor. ANXA5 is a protein that has specificity for cancer due to the strong binding (K_d ~ 1 nM) to externalized phosphatidylserine (PS) on the surface of tumor cells and tumor vasculature. PS is a biomarker of various types of cancers, including TNBC, but is absent on the surface of healthy cells.

Here, we study the in vivo parameters that better generate an immune activation through SWCNT-ANXA5 mediated PTT: injection mode and tumor surface temperature. Additionally, we combined the optimized therapy with checkpoint inhibition of anti-PD-1, for a systemic immune response against a metastatic tumor model.

Methods:

ANXA5 was produced in E. coli and purified by affinity liquid chromatography in an immobilized-nickel column. The high purity protein (>95%, confirmed by SDS-PAGE electrophoresis) is then linked to (6,5) CoMoCAT SWCNTs with a linker (DSPE-PEG-MAL), which interacts hydrophobically with SWCNTs and bonds to ANXA5 through the cysteine reactive maleimide group.

EMT6 cells, a TNBC tumor model, were orthotopically injected in BALB c/J female mice. When the tumors were an average of 5 mm in diameter, the mice were treated with a 980 nm NIR laser at 1 W/cm². For the first experiment, SWCNT-ANXA5 was injected either intravenously (IV) or intratumorally (IT), and levels of cytokine TNF-α in the serum 1 day after PTT were measured by ELISA. Secondly, using IT injection, the tumors were subjected to PTT, and treatment ceased when the tumor surface reached different peak temperatures (45, 50, 55 and 60ËšC), which was measured by a thermal camera. Blood serum was collected 1 day and 7 days after irradiation for analysis of proinflammatory cytokines (TNF-α and IL-12). A long-term survival test was performed using IT injection and irradiation until the tumor surface temperature reached 45ËšC in combination with checkpoint inhibition of anti-PD-1 (three intraperitonial injections of 200 g/mouse, 1 and 4 days before irradiation, and 4 days after irradiation). Lastly, to study primary tumor control, EMT6 tumor were irradiated with 980 nm NIR laser at 1 W/cm² by holding the tumor surface temperature at 45 1 for longer period of times (60, 120 and 300 s compared to 0 s) by cycles of on/off with the laser. Tumor volumes were recorded for 15 days after irradiation.

Additionally, a biodistribution study of IT injection and another long-term survival study are being performed. Firstly, EMT6 tumor bearing mice were injected intratumorally with the SWCNT-ANXA5 conjugate treated with the PTT until 45. Those mice will be euthanized at 1, 2, 3 and 4 weeks after irradiation and major organs harvested for the measurement of SWCNT. Secondly, either EMT6 or 4T1 tumor bearing mice will be treated similarly to the forementioned long-term survival study, but the PTT will be performed holding the tumor surface temperature at 45 1 for 300 s (5 min) by cycling the laser irradiation on and off.

Results:

IT injection was the only method that induced a statistically significant increase in the level of TNF-α (n = 4-5, p<0.001). When comparing the irradiation temperatures, 45˚C was the group that was more effective in increasing the level of cytokines TNF-α (at 1 day and 7 days after PTT) and IL-12 (1 day after PTT), respectively p<0.05, p<0.01 and p<0.01 for n=4-5. Furthermore, the optimized combination therapy resulted in complete tumor recession and survival at 100 days for 50% of mice in the anti-PD-1 + PTT group. Additionally, the group where the primary tumor was kept at 45˚C for 300 s had statistically significant smaller tumors at 15 days compared to the other groups with the tumor held at this temperature for shorter times (p<0.05, n=5).

The ongoing biodistribution experiment aim to explore whether the SWCNT remain in the tumor region after intratumoral injection. The long-term survival study intend to assess wheter the irradiation at 45ËšC for 5 min in combination with anti-PD-1 is efficient at increasing the survival rate of EMT6 tumor model, additionally comparing with the 4T1 tumor model, which is considered less immunogenic.

Implications:

There is evidence that IT injection and PTT at 45°C are more effective at generating immune responses compared to the higher temperatures tested. The lower temperature is important for the safety of the PTT and reduces the chances of damage to surrounding tissue. Lowering PTT temperature could also improve chances of translating this therapy strategy into clinical trials. Increasing the irradiation time holding the tumor surface at 45°C improves the ablation of deeper layers of the solid tumor, improving tumor control. Because EMT6 tumor models are known to establish metastatic tumors very early in the tumor development, the long-term survival is one evidence that the combination therapy of PTT and anti-PD-1 elicits a systemic antitumoral response that efficiently suppresses metastatic tumors.