(T1530-01-01) Phosphatidylserine Targeting for Enhancing Immunochemotherapy of Cancer

Purpose: Immunotherapy has significantly improved cancer treatment. However, poorly immunogenic tumors, such as melanoma, remain resistant to current immunotherapy. The difficulty is partly due to the highly immunosuppressive tumor microenvironment (TME). For example, tumor cells use phosphatidylserine (PS) to escape immune activation against them. PS externalized on the outer leaflet of apoptotic cell membrane attracts phagocytic cells to quickly remove the dying cells via a process called efferocytosis. Simultaneously, PS turns on anti-inflammatory (tolerogenic) signals to prevent further damage and maintain homeostasis. Tumor cells hijack the latter process to induce immunosuppressive TME. Recent studies show that PS is upregulated in TME and further increases following chemotherapy. For effective immunotherapy of tumors, PS needs to be antagonized to relieve immunosuppressive TME and sensitize tumors to immune stimulants. The objective of the study is to find a strategy to relieve the immunosuppressive TME caused by the PS exposure and boost anti-tumor immune responses in tumors to enhance immunochemotherapy of tumors. Toward this goal, we have developed a formulation that (i) blocks the PS on tumor and peritumoral vessels to downregulate an immunosuppressive environment and (ii) delivers an immune stimulant such as cyclic dinucleotide (CDN) to further stimulate antitumor immune responses.

Methods: A synthetic compound dipicolylamine zinc (DPA-Zn) was identified as anti-PS reagent and its selective affinity for PS was confirmed by testing its binding to PS liposome and measuring dissociation constant by isothermal titration calorimetry (ITC). To test the anti-PS effect of DPA-Zn, bone marrow derived macrophages (BMM), pretreated with PS liposomes, were stimulated by lipopolysaccharide (LPS) with and without DPA-Zn, and TNF-α secretion from the stimulated BMM was measured. The anti-PS activity of DPA-Zn and the effect of its combination with an immune stimulant (STING agonist, CDN) on TME modulation and tumor growth inhibition were tested in the immune cold B16F10 melanoma tumor model following doxorubicin chemotherapy. To prolong the local retention of the formulation (DPA-Zn and CDN), CDN has been formulated in a nanoparticle form (CDN-Zn NPs) and loaded with DPA-Zn in an alginate-based hydrogel.

Results: DPA-Zn specifically bound to PS in the liposome with a dissociation constant K_D of 1.76 μM.  The blockade of the exposed PS by DPA-Zn restored the responsiveness of BMM to immune stimulant LPS in vitro (Fig. 1). Furthermore, the DPA-Zn-mediated PS blockade following the doxorubicin (DOX) treatment activated immune activity of the TME, making tumors more responsive to CDN, as evidenced by immunophenotyping and tumor growth in B16F10 melanoma tumor-bearing mice (Fig. 2). The combination of DOX (chemotherapy) + DPA-Zn (PS blockade) + CDN (immune stimulant) significantly increased the survival time of B16F10 melanoma tumor-bearing mice compared with DOX + DPA-Zn treated group (Fig. 3). When combined with Zn²⁺, CDN formed nanoparticles (CDN-Zn NPs) with particle size of 120 nm.

Conclusion: The PS blockade by DPA-Zn with high affinity for PS restored the ability of macrophages to respond to LPS in vitro. In B16F10 melanoma tumor bearing mice, DPA-Zn enhanced the effect of DOX. Furthermore, the combination of DOX (chemotherapy), DPA-Zn (PS blockade), and CDN (immune stimulant) increased the average survival time of B16F10 tumor bearing mice from 14 days to 24.5 days. To prolong intratumoral release of DPA-Zn and CDN and improve the anti-tumor immunity, CDN was formulated in a nanoparticle form (CDN-Zn NPs) and loaded with DPA-Zn in an alginate hydrogel.

Fig 1. LPS induced TNF-α production by BMM after 24 h incubation with PS liposomes (50% PS liposome) with or without DPA-Zn. Data are presented as mean± SD, and statistical analysis was performed using Tukery's test.



Fig 2. DPA-Zn improves anti-tumor efficacy of Doxil. (a) When B16F10 tumors grew to 50 mm3 (day 0), mice were treated with Doxil equivalent to 2.5 mg/kg doxorubicin or 5% dextrose (D5W) (n=5 per group) intravenously (i.v.) on days 0 and 3. Mice were treated intratumorally (i.t.) with 10 mg/kg DPA-Zn or D5W on days 1, 2, 4, 5. (b) B16F10 tumor growth in different treatment groups over time. Data are presented as mean± SD, and statistical analysis was performed using two tailed t-test.



Fig 3. (a) C57BL/6 mice were inoculated with 106 B16F10 tumor cells in the s.c. flank. When tumor reached 50 mm3, mice were intravenously treated with Doxil equivalent to 2.5 mg/kg DOX at day 0, 3, followed by 2 consecutive DPA-Zn (10 mg/kg) with D5W or CDN (10 μg/mouse) per day on day 1, 2, 4, 5. (b) Survive curve of B16F10 tumor bearing mice. Statistical analysis was performed using long-rank (Mantel-Cox) test.