PIN-2 is a novel immunomodulatory agent derivatized from a transactivator protein. We have shown its activity to induce maturation of monocytes into dendritic cells and translational proof of principle using an aggressive metastatic murine mammary cancer model. The purpose of the present studies was to further elucidate the activity and mechanism of action of PIN-2 alone or in combination with other agents in the treatment of solid malignancy.
Murine mammary carcinoma was established with syngeneic 4T1 cells orthotopically implanted in the mammary fat pad of female BALB/c mice and allowed to seed 7 or 10 days before starting treatment. PIN-2 was given as an intravenous bolus of 40-100 ng/mouse on alternating weeks.
Experiment 1: Combination therapy with PIN-2 and cyclophosphamide (CTX). Treatment began on d10, when tumor diameter was ~ 4-5mm. There were 4 treatment arms (n=10): Placebo control (PBS IV every 3 days), CTX control (CTX 80 mg/kg IP once/wk), combination sequence 1 (Seq1:PIN-2 40ng IV every 3d/CTX, alternating 10d cycles), and combination sequence 2 (Seq2: CTX/PIN-2, alternating 10d cycles). Treatment was stopped on day 50. Tumor size was measured 3 times/wk. End points were tumor volume and overall survival.
Experiment 2: PIN-2 (100ng IV 3x/wk) and α-CTLA-4 (200µg IP 3x/wk) were administered to tumor bearing mice. There were 4 treatment arms (n=10): control, α-CTLA-4 (wk 2 only), PIN-2 (wks 1 and 3) and PIN-2 (wks 1 and 3) followed by α-CTLA-4 (wk 2). Animals were killed on day 29/30. End points were tumor progression and number of spontaneous lung surface nodules. Pharmacodynamic end points were primary tumor staining for PD-L1 and CD8. FFPE primary tumors from control and PIN-2 treated mice were stained using IHC for PD-L1 and CD8. Determination of PD-L1 reduction is based on in vivo tumor measurements together with PD-L1 staining intensity.
Experiment 1: Both PIN-2 and CTX impacted tumor growth and increased overall survival followed the order Seq1 (PIN-2/CTX)>Seq2 (CTX/PIN-2)> CTX. All mice treated with PIN-2 had reduced tumor burden vs. CTX alone or control.
Experiment 2: Both PIN-2 and combination therapy, but not α-CTLA-4 alone, were effective in reducing tumor burden vs. control. Additionally, the PIN-2/α-CTLA-4 combination therapy resulted in fewer lung surface nodules than α-CTLA-4 alone or control.
IHC showed that PD-L1 expression is reduced in primary tumor tissue from mice receiving PIN-2 vs. control. Tumor edge contained PD-L1+ staining was largely absent in PIN-2 treated animals vs. control. Rarely, CD8+ cells were seen in PBS control tumors whereas significant CD8+ staining was observed around tumor edges in PIN-2 treated mice.
CTX activity is improved when given in combination with PIN-2. CTX is most effective when administered after PIN-2. The MOA of PIN-2 suggests a priming effect linking innate and adaptive immunity.
PIN-2 and α-CTLA-4 were both effective in reducing the average lung surface nodule count. However, when PIN-2 is combined with α-CTLA-4, the combination is more effective than either compound alone. This suggests a priming effect of innate immunity that enhances α-CTLA-4 activity.
PD-L1 expression is reduced in PIN-2 treated primary 4T1 tumor tissue and promotes the influx of tumor infiltrating CD8+ CTL. Tumor-infiltrating CD8+ CTLs localize near the tumor edge in PIN-2 treated mice, whereas these CTLs are largely absent in PBS control. Since PD-L1 is a marker associated with disease progression, malignancy, and poor prognosis, the inverse correlation of tumor PD-L1 and CD8+ CTL can be explained by the antitumor CTL response seen with PIN-2 treatment.
In conclusion, PIN-2, a novel immunomodulatory peptide, demonstrated immune priming activity capable of linking the innate and adaptive immune systems thereby enhancing and promoting anti-tumor activity by triggering monocyte derived dendritic cells to stimulate CD8+ CTL responses.
Citation Format: Joshua B. Goldberg, Sophie J. Hanscom, Kenneth Gorelick, Colin B. Bier. PIN-2, a novel immunomodulatory peptide. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B080.
- ©2016 American Association for Cancer Research.