Depletion of tumor associated macrophages enhances local and systemic platelet-mediated anti-PD-1 delivery for post-surgery tumor recurrence treatment

Abstract

Immunosuppressive cells residing in the tumor microenvironment, especially tumor associated macrophages (TAMs), hinder the infiltration and activation of T cells, limiting the anti-cancer outcomes of immune checkpoint blockade. Here, we report a biocompatible alginate-based hydrogel loaded with Pexidartinib (PLX)-encapsulated nanoparticles that gradually release PLX at the tumor site to block colony-stimulating factor 1 receptors (CSF1R) for depleting TAMs. The controlled TAM depletion creates a favorable milieu for facilitating local and systemic delivery of anti-programmed cell death protein 1 (aPD-1) antibody-conjugated platelets to inhibit post-surgery tumor recurrence. The tumor immunosuppressive microenvironment is also reprogrammed by TAM elimination, further promoting the infiltration of T cells into tumor tissues. Moreover, the inflammatory environment after surgery could trigger the activation of platelets to facilitate the release of aPD-1 accompanied with platelet-derived microparticles binding to PD-1 receptors for re-activating T cells. All these results collectively indicate that the immunotherapeutic efficacy against tumor recurrence of both local and systemic administration of aPD-1 antibody-conjugated platelets could be strengthened by local depletion of TAMs through the hydrogel reservoir.

Introduction

Surgery remains the foremost treatment option for patients with solid tumors, while tumor recurrence frequently occurs, leading to a low rate of long-term survival^1,2. Cancer immunotherapy, especially immune checkpoint blockade, has been demonstrated as one of the most potent anti-cancer recurrence strategies either as monotherapies or in combinations with other treatment modalities^3,4,5,6,7. Specifically, inhibition of binding between programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has been demonstrated to regain suppressed anti-tumor immunity, which has great potential to effectively treat cancer recurrence in the clinical settings^8,9. However, intricate physiological environment changes after surgery, especially wound healing-triggered inflammatory condition and inflammation-responsive immunosuppression, could diminish the efficacy of cancer immunotherapy, leading to the low objective response rate in the clinic^10,11,12. It is worth noting that a large number of inflammatory cells represented by macrophages participate in the process of surgical wound healing and tissue repair. Moreover, tumor-associated macrophages, especially M2 type macrophages, secrete a large amount of anti-inflammatory factors, such as IL-10, to form an immunosuppressive microenvironment, thereby helping the remaining tumor cells escape immune surveillance, and accelerate their recurrence and metastasis. Furthermore, immune-related adverse events due to over-activated T cells by immune checkpoint blockade remain a concern^13,14.

The connection between TAMs and cancer recurrence after surgery has been established in both preclinical studies and clinical settings. It has been demonstrated that surgery could enhance the proliferation of tumor cells locally and in distant metastases, which is correlated with the increased macrophage density in the tumor tissues after surgery¹⁵. Additionally, the depletion of TAMs resulted in inhibited tumor growth after surgery. For example, a single-cell protein activity analysis was applied to identify one specific macrophage population that correlated positively with clear cell renal carcinoma (ccRCC) recurrence after surgery¹⁶, which has also been validated in a large clinical validation cohort, in which this sub-population of macrophages were significantly enriched in tumors from patients who experienced tumor recurrence following surgery. Furthermore, in clinical settings, TAMs have been frequently applied as prognostic markers for multiple cancer recurrences after surgery including non-small cell lung cancer, gastric cancer, and non-functional pancreatic neuroendocrine tumor^17,18,19. Collectively, all these pre-clinical and clinical studies validate that there is a strong correlation between enriched TAMs and tumor recurrence after surgery, and TAM depletion could have a positive impact on preventing post-surgical tumor recurrence.

One more mechanism that limits the immune checkpoint blockade therapy in recurrent cancer is the hindered T cell migration towards and within the tumor site due to tumor immunosuppressive microenvironment, resulting in the low availability of tumor infiltrating lymphocytes and progressive tumor growth^20,21. Studies have shown that immunosuppressive cells, especially TAMs, are highly implicated in suppressing anti-tumor immune functions of T cells and directly facilitate tumor cell immune escape^22,23. Specifically, a recent study has identified that CD8⁺ T cell accumulation and infiltration at tumor sites have been primarily enhanced by depleting TAMs, resulting in improved immunotherapy efficacy²⁴. Pexidartinib is an FDA-approved small molecule drug that shows remarkable selectivity to block the CSF1 receptors on TAMs with negligible cytotoxicity against normal cells, favoring itself as a potential target for TAM depletion for modulating the tumor immunosuppressive microenvironment and enhancing immunotherapy by improving T cell infiltration^25,26. Thus, the combination of TAM depletion and immune checkpoint blockade could be critical for augmenting anti-tumor immunotherapy outcomes.

Here, we show a biocompatible alginate-based hydrogel encapsulating PLX-loaded nanoparticles (designated PLX-NP) and anti-PD-1-conjugated platelets (designated P-aPD-1) for post-surgery tumor recurrence treatment, to achieve local and sustained release of PLX and anti-PD-1 antibodies for depleting TAMs and re-activating infiltrated T cells, respectively (Fig. 1a). A biodegradable dextran nanoparticle is formulated to encapsulate and bioresponsively release PLX to block CSF1R to eliminate TAMs in the tumor microenvironment. Anti-PD-1 antibodies are conjugated on the surface of platelets, where the inflammatory environment secondary to the surgical exposure could activate platelets to release anti-PD-1 antibodies to block PD-1 receptors on infiltrated T cells in the format of PMP-aPD-1 (platelet-derived microparticles with anti-PD-1 antibodies)^27,28. Both PLX-NP and P-aPD-1 are harbored in the hydrogel to form a local delivery reservoir, in which PLX is gradually released to deplete TAMs to recruit T cells toward tumor parenchyma, favoring the subsequent efficacy of the anti-PD-1 immunotherapy. To further extend the application of this combination immunotherapy strategy, P-aPD-1 is systemically administered to synergize with local PLX-NP-loaded hydrogel to promote a sustained immune response against tumor recurrence, where less than 20% of patients are benefiting from a systemic injection of checkpoint inhibitors clinically²⁹. Taken together, this hydrogel-based delivery system could enhance post-surgery tumor recurrence treatment by gradually releasing PLX to deplete TAMs, facilitating T cell recruitment and infiltration, and promoting both local and systemic platelet-mediated immune checkpoint inhibitor delivery…