Introduction: Immunotherapies such as immune checkpoint inhibitors have had limited success in treating breast cancers. We observed that women with HIV infection and even AIDS have an unusually low incidence of breast cancer. The Tat protein of HIV-1 modulates innate immunity through activation of antigen presenting cells (APCs), and thus could be the reason for this finding. A Tat protein derivative designed to eliminate elements contributing to HIV-1 mediated immunosuppression was expressed as a recombinant protein in E. coli and found to stimulate monocytes to differentiate into antigen presenting cells. This protein, designated PIN-2, was tested in the syngeneic 4T1 murine breast cancer model, a poorly immunogenic and invasive triple negative breast cancer (TNBC) cell line. Methods: In vitro activity of PIN-2 was confirmed using a human monocyte activation assay that determined cell surface expression of CD80 and CD86 by flow cytometry after 72h of incubation with PIN-2, compared with controls. BALB/c mice were implanted orthotopically in the mammary fat pad with 10000 4T1 cells (ATCC No. CRL-2593). Treatment was initiated 7 days after implantation. Mice were administered 100ng PIN-2 intravenously 3x/week for 3 weeks. Tumors were measured 2x/week starting 12 days after implantation using a standard caliper method. Mice were sacrificed 29 days after implantation and examined macroscopically for spontaneous pulmonary metastases. Results: PIN-2 significantly increased human monocyte cell surface expression of costimulatory molecules CD80 and CD86 in vitro at concentrations as low as 30ng/mL culture compared to the negative control (P<0.01). The activation was dose dependent. In vivo, PIN-2 treatment reduced tumor volume to less than 60% of the tumor volume of untreated mice 29 days after tumor implantation (686 mm3 tumor size in placebo vs. 407mm3 in treated mice, p<0.01). Moreover, PIN-2 significantly inhibited the development of spontaneous 4T1 lung metastases (p< 0.05 when compared to placebo). Preliminary examination indicates that 90% of the mice in the control group had visible nodules on the surface of the lungs. Macroscopically visible metastases were reduced to only 50% of the PIN-2 treated mice. There was no evidence of weight loss or apparent toxicity by PIN-2. Conclusion: PIN-2 stimulates cell surface expression of costimulatory molecules that serve as markers for activated antigen presenting cells and are essential for cytotoxic T-cell activation. It reduces primary and metastatic tumor burden in an orthotopic murine model of TNBC. These data suggest that PIN-2 functions as an innate immune agonist to facilitate adaptive anti-tumor immune responses in a poorly immunogenic model of breast cancer. Moreover, PIN-2 appears to be well tolerated, supporting further evaluation of PINS in human breast cancer clinical trials. Citation Format: Christoph M. Hotz-Behofsits, Sophie J. Hanscom, Joshua B. Goldberg, Colin B. Bier. A new class of immunomodulators derivatized from the Tat protein of HIV-1 in a murine breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4279. doi:10.1158/1538-7445.AM2015-4279
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.
PIN-2 is a novel immunomodulatory agent derivatized from a transactivator protein. We have demonstrated its activity to both stimulate and induce maturation of monocytes into dendritic cells and translational proof of principle using an aggressive metastatic murine mammary cancer model.PIN-2 initiates activity by enhancement of innate immune signaling de novo. Next Generation Sequencing of CD14+ primary human monocytes after 3-hour exposure to PIN-2 in vitro, reveals a unique profile of gene expression indicative of innate immune activation which is necessary to drive adaptive immunity towards killer T-cell immune responses. Genetic programs in the monocyte impacted by PIN-2 exposure include cytokine signaling pathways, co-stimulatory ligands, and coding and non-coding RNAs.Understanding the genetic signature of the changes by PIN-2 through the innate immune response supports the rational development of immune based combination therapies such as cell based immunotherapies and checkpoint blockade that function to modulate adaptive immunity in T-cells in a clinical setting. Citation Format: Joshua B. Goldberg, Sophie Hanscom, Colin B. Bier. PIN-2: A novel immunopriming peptide with immunomodulatory activity linking the innate and adaptive immune systems [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B076.
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