methods & results - in vivo & in vitro · the genetic signature of the transcriptional...
TRANSCRIPT
PIN-2: A novel immunopriming peptide with immunomodulatory activity linking the innate and adaptive immune systems. Joshua B. Goldberg, Sophie J. Hanscom, & Colin B. Bier
PIN Pharma, Inc., New York, NY
Objectives
Conclusions
1. Demonstrate antitumor activity of PIN-2 in the metastatic 4T1 model of murine mammary carcinoma.
2. Determine temporal relationship of PIN-2 administration to illicit the “immunopriming” effects.
3. Increase presence of CD8+ tumor infiltrating lymphocytes (TIL’s) in primary tumor.
4. Demonstrate molecular MOA through gene expression signature in human monocytes exposed to PIN-2 in vitro.
Printed by
• Potentiating innate immunity is a promising strategy to overcome cancer mediated immunosuppression.
• PIN-2 is a transactivating immunomodulator that enhances innate immunity
& subsequent activation APCs via cellular penetration & reprograming of transcription in monocytes.
• Dendritic cells are professional APC’s providing the essential link between
innate & adaptive immunity- PINS stimulate maturation of DC’s leading to presentation of the individuals unique profile of tumor associated antigens.
• Demonstrated synergy with other counter suppressive anticancer
therapeutics based on physiological mechanisms within the innate & adaptive immune responses.
• Impact on solid tumor growth & promotes CD8+ cytotoxic T lymphocyte maturation in the spleen & subsequent infiltration in primary tumors - indicative of an enhanced endogenous adaptive effector T-cell response.
• PIN-2 activity results upregulation numerous immune response genes that promote APC function & T-cell proliferation.
• The immunodynamic effects of PIN-2 activity expose molecular
mechanisms & pathways that traverse innate & adaptive immune signaling- opportunity to identify novel immune-based biomarkers & provide complementary function to other immune targeting agents.
• These data support clinical investigation of PIN-2 as a novel
immunomodulatory agent.
PIN-2 increases CD8+ cells in the spleen and primary tumor.
Background
• PINS drive the DC-APC innate immunity complex to stimulate T-cell mediated immune
responses.
• PIN-2 is a cell penetrating peptide that reprograms cellular transcriptionè and promotes maturation and function of monocyte derived dendritic cells.
• Upregulation of co-stimulatory ligands CD80 & CD86 • PIN-2 has demonstrated antitumor activity in 3 different metastatic mammary tumor models with varying degrees of immunogenicity.
• 4T1<TS/A<SM1
• PINS act upstream of checkpoint inhibitors & cell-based therapies by enhancing innate immunity.
Methods & Results - in vivo & in vitro Treatment modalities capable of enhancing a patient’s innate immune response represent a promising strategy to promote anticancer activity in the host. Currently approved immunotherapies primarily function to modulate adaptive immunity in T-cells and have limited clinical activity in patients with advanced solid tumors. Next-generation cancer immunotherapy combination strategies are being utilized to improve clinical outcomes in advanced solid tumors. PIN-2 is a novel immunomodulator derived from a transactivator protein that initiates activity by enhancing innate immune signaling de novo. Experiments with PIN-2 demonstrate its ability to trigger monocyte differentiation into activated antigen presenting cells (APC) thereby bridging adaptive immunity resulting in endogenous killer T-cell immune responses. PIN-2 demonstrated antitumor activity in a murine model of poorly immunogenic mammary carcinoma (4T1) and synergizes with additional anticancer therapeutics that relieve T-cell mediated immunosuppression. Next Generation Sequencing (NGS) of isolated CD14+ primary human monocytes following PIN-2 exposure in vitro, reveals a pattern of gene expression indicative of innate immune activation as evidenced by upregulation of mRNA involved in cytokine signaling pathways, co-stimulatory ligands, and coding and non-coding RNA’s. The genetic signature of the transcriptional changes induced by PIN-2 through activation of the early innate immune response and subsequent transition toward adaptive immunity enables discovery & development of novel immune-based biomarkers, targets for therapeutic intervention, and design of rational combination immunotherapies.
Temporal administration of PIN-2 impacts tumor progression and increases survival
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Immune Response
Taxis
Chemotaxis
Locomotory Behavior
Inflammatory Response
Response to Wounding
Defense Response
Regula?on of Leukocyte Prolifera?on
Regula?on of Mononuclear Cell Prolifera?on
Posi?ve Regula?on of Leukocyte Prolifera?on
Posi?ve Regula?on of Mononuclear Cell
Posi?ve Regula?on of Cell Prolifera?on
Regula?on of Lymphocyte Prolifera?on
Regula?on of T Cell Prolifera?on
Posi?ve Regula?on of Lymphocyte
Regula?on of Cell Ac?va?on
Regula?on of Cytokine Biosynthe?c Process
Posi?ve Regula?on of T Cell Prolifera?on
P value
GO Biological Process
Func?onal Annota?on Clustering
GO Enrichment of Biological Processes of PIN-2 stimulation in human monocytes. Representative biological processes from the top 4 enrichment clusters obtained from “functional annotation clustering” from DAVID Bioinformatics Resources 6.7. Categories from “GOTERM_BP_FAT” were used. Represents PIN-2 differentially expressed genes with ≥2X fold-change compared to unstimulated monocytes.
Primary isolated CD14+ healthy human monocytes were stimulated with 500ng of PIN-2/106 cells at 37°C for 3h. Illumnia sample preparation guide for Directional mRNA-Seq was followed. Mappable reads were aligned to genome using Tophat_v1.4.1. For pairwise differential expression analysis at the gene level, The abundance was normalized and evaluated in Fragments Per Kilobase of transcript per Million mapped reads (FPKM) using the Cuffdiff module of Cufflinks_v2.0.2. Differentially expressed genes showing significant difference with adjusted p values < 0.05 and a fold-change >2 were selected as signature genes.
Overview of NGS
Differentially Expressed Genes (771 mRNA’s)
PIN-2 vs. No Stimulation (3h)
Human CD14+ Monocytes
Depicts orthotopic 4T1 tumor growth curve (1x104 cells) of individual mice (n=10) following therapy with repeat dosing in alternating 10-day treatment cycles with PIN-2 (40ng i.v.) and Cyclophosphamide/CY (80mg/kg i.p.). Treatment begins on day 10 when primary tumor was established at 4-5 mm3.
Depicts increased survival in mice receiving alternating cycles of PIN-2 & Cyclosphamide. Treatment was terminated on Day 50. Increased survival was observed in mice receiving a PIN-2 bolus at treatment initiation compared with that received CY at the start of treatment, owing to an “immunopriming” effect of PIN-2 in this regimen.
Representative list of differentially expressed genes
Representative set of differentially expressed genes from primary monocytes following 3hr PIN-2 exposure with description of associated biologic function.
Poster # B076
Depicts IHC staining intensity of CD8+ cells in FFPE spleen & primary 4T1 tumor tissue. Mice were implanted with 1x104 4T1 cells in the mammary fatpad. Treatment was initiated 7 days after tumor inoculation. Spleen & tumor tissue were resected on Day 30 for histological comparison. PBS control (A&C) PIN-2 Rx (B&D).
Effector T-cell Response
Gene Symbol/Description
Log2-‐ Fold Change
p Value Biological Function
CD83 3.34 8.18E-‐005 DC maturation MIR424/MicroRNA 424 8.54 1.33E-‐007 Monocyte Differentiation &
Activation CSF2/GM-‐CSF (Granulocyte-‐Macrophage Colony Stimulating Factor)
7.33 2.09E-‐014 Stimulates the growth and differentiation of hematopoietic precursor cells
IFNG/Interferon Gamma 5.47 5.66E-‐004 Antitumor CTL response ISG15/Interferon-‐Stimulating Gene 15-‐ Ubiquitin-‐like Molecule
2.78 2.85E-‐004 Cell-‐cell signaling & enhance CTL function
IL23A/Interleukin 23 Subunit Alpha
8.35 4.25E-‐013 Innate & adaptive immunity (memory T-‐cells)
CCL2/Monocyte Chemoattractant Protein-‐1 (MCP1)
2.62 7.47E-‐004 Immune cell recruitment
LAMP3/Dendritic Cell Lysosomal Associated Membrane Protein 3
3.69 6.64E-‐006 Processing & presentaion of antigen
CCR7/C-‐C Motif Chemokine Receptor 7
2.34 1.50E-‐003
DC migration, homing, activation of T, B, NK cells in lymphoid tissue
IL15RA/Interleukin-‐15 Receptor Alpha
3.63 7.43E-‐004 CD8+ T and natural killer cell activation
TNFSF9/4-‐1BB-‐Ligand/ CD137L 4.60 4.18E-‐005 Costimulatory Ligand for CTL Activation.
ZNF366/Zinc Finger 366/DC-‐SCRIPT (Dendritic Cell Speci]ic Transcript)
3.12 2.93E-‐004 Transcriptional reglulator involved in DC differentiation
RND1/Rho Family GTPase 1 6.84 9.37E-‐006 Supresses Ras signaling & EMT BATF3/Basic Leucine Zipper ATF-‐like Transcription Factor 3
3.98 1.20E-‐003 Essential for presentation of tumor antigens to CD8+ T cells & ef]icacy of immunostimulatory MAb’s (PD-‐1 & 4-‐1BB)
Enriched GO Biological Processes of PIN-2 activity in human monocytes
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