Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers

Abstract

Background: The purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers.

Methods: Immune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis.

Results: Treatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy.

Conclusions: Our data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.

Keywords: Breast cancer; Checkpoint inhibitor; Combination immunotherapy; Phosphatidylserine; Triple-negative breast cancer.

Fig. 1

Expression of PD-L1 and PD-1 in breast tumors. FACS and immunohistochemistry (IHC) analysis of a E0771 and b EMT-6 cells grown in vitro to determine whether PD-L1 is constitutively expressed and/or is inducible by IFNγ treatment. PD-1, programmed death 1, PD-L1 programmed death-1 ligand 1

Fig. 2

Anti-tumor effects of PS and PD-1 targeting alone and in combination in murine BC models. a Top panel: growth kinetics of E0771 TNBC tumors in C57/Bl6 mice treated with control (C44 ), mch1N11, anti-PD-1, or mch1N11 + anti-PD-1 antibody combinations. Treatments started at 10 days post inoculation when tumors were approximately 100 mm³. All data points are expressed as median and SD. Bottom panel: analysis of E0771 final tumor volumes from the TGI study in upper panel. Statistical analysis (Student’s t test) demonstrates that combinational treatment with PS-targeting antibody and ant-PD-1 antibody has significant inhibitory effects compared with all other treatments. b Top panel: growth kinetics of EMT-6 murine breast tumors in Balb/c mice treated with control (C44), mch1N11, anti-PD-1, or mch1N11 + anti-PD-1 antibody combinations. Treatments started at 3 days post inoculation when tumors were approximately 100 mm³. All data points are expressed as median and SD. Bottom panel: analysis of EMT-6 final tumor volumes from TGI study in upper panel. Statistical analysis demonstrates that combinational treatment with PS-targeting antibody and anti-PD-1 antibody has significant inhibitory effects compared with all other treatments. Statistically significant differences between groups were identified by Student’s t test, with 10 animals in each group. TGI tumor growth inhibition

Fig. 3

Effect of PS-targeting and anti-PD-1 antibody therapy on survival in a TNBC murine model. Mice with E0771 tumors were treated a total of six times once tumors reached approximately 100 mm³ (days 1, 4, 8, 11, 15, and 18) with single antibody or a combination of mch1N11 and anti-PD-1, and survival times were determined. Once tumors reached approximately 1500 mm³ or animals encountered a tumor-related health problem, animals were euthanized. Survival times were plotted and determined by Kaplan–Meier analysis, statistical analysis was determined by log rank (Mantel–Cox) analysis

Fig. 4

Effect of mch1N11 and anti-PD-1 therapy on establishing an immune-mediated resistance to E0771 rechallenge and presence of functional T cells. a Animals that received mch1N11 + anti-PD-1 treatment and underwent a complete tumor regression (Fig. 4) were rechallenged, along with naïve C57BL/6 mice, with E0771 cells (1.0 × 10⁶). Cells were inoculated in the opposing mammary fat pad (4/5) from the previous initial inoculation (8/9). The presence of palpable tumors and growth kinetics was determined for each group. b EliSpot analysis of IFNγ in splenocytes cultured with and without irradiated E0771 cells. Splenocytes from nontumor-bearing control animals (no treatment, C44, and mch1N11 + anti-PD-1 treated), tumor-bearing animals (C44 treated), and mice resistance to E0771 rechallenge (Fig. 4a, 4b) were utilized. All analyses were performed in triplicate

Fig. 5

Effect of PS-targeting and anti-PD-1 therapies on TILs in E0771 and EMT-6 tumors. a Mice with E0771 tumors were treated on days 10, 14, 17, 21, and 25 post implantation with single antibody treatments or a combination of mch1N11 and anti-PD-1. Five tumors from each treatment group were excised on day 26 and single-cell preparations were stained with antibodies specific to CD45⁺, CD8⁺, CD4⁺, and CD3⁺. Data are expressed as the group median and percentages of an individual animal positive for a specific surface marker by FACS analysis. CD8⁺, CD3⁺, and CD4⁺ cells were all subpopulations of CD45⁺ cells. b Mice with EMT-6 tumors were treated on days 10, 14, 17, 21, and 25 post implantation with single antibody treatments or a combination of mch1N11 and anti-PD-1. Five tumors from each treatment group were excised on day 26 and single-cell preparations were stained with antibodies specific to CD45⁺, CD8⁺, CD4⁺, and CD3⁺. Data are expressed as the group median and percentages of individual animal positive for a specific for a surface marker by FACS analysis. CD8⁺, CD3⁺, and CD4⁺ cells were all subpopulations of CD45⁺ cells

Fig. 6

NanoString immune-profiling analysis of tumor samples from control (C44), PS (mch1N11), and anti-PD-1 therapy. RNA isolated from tumors obtained at the study termination (shown in Fig. 3) was subjected to analysis utilizing the NanoString™ nCounter PanCancer Immune Profiling Panel. Expression values, expressed in log₂ are graphically represented. a Profiling of tumor-associated immune cell type markers, including the markers for PD-1 and its ligand PD-L1. b Expression profiling of immune-activating-associated cytokines in each treatment group. c Expression profiling of immune-suppression-associated cytokines in each treatment group. DC dendritic cell, PD-1 programmed death 1, PD-L1 programmed death-1 ligand 1, Th T-helper, Treg regulatory T cell