Targeting CXCR4 potentiates anti-PD-1 efficacy modifying the tumor microenvironment and inhibiting neoplastic PD-1

BACKGROUND: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. METHODS: Mice were subcutaneously injected with MC38 (1 × 10(6)) or B16-hCXCR4 (5 × 10(5)). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1–14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. RESULTS: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC(+) cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. CONCLUSION: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.