methods & results - in vivo & in vitro · the genetic signature of the transcriptional...

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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 transcription 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 1.00E13 1.00E12 1.00E11 1.00E10 1.00E09 1.00E08 1.00E07 1.00E06 1.00E05 1.00E04 1.00E03 1.00E02 1.00E01 1.00E+00 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-2/10 6 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 (1x10 4 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 mm 3 . 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 1x10 4 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 8.18E005 DC maturation MIR424/MicroRNA 424 8.54 1.33E007 Monocyte Differentiation & Activation CSF2/GMCSF (Granulocyte Macrophage Colony Stimulating Factor) 7.33 2.09E014 Stimulates the growth and differentiation of hematopoietic precursor cells IFNG/Interferon Gamma 5.47 5.66E004 Antitumor CTL response ISG15/InterferonStimulating Gene 15 Ubiquitinlike Molecule 2.78 2.85E004 Cellcell signaling & enhance CTL function IL23A/Interleukin 23 Subunit Alpha 8.35 4.25E013 Innate & adaptive immunity (memory Tcells) CCL2/Monocyte Chemoattractant Protein1 (MCP1) 2.62 7.47E004 Immune cell recruitment LAMP3/Dendritic Cell Lysosomal Associated Membrane Protein 3 3.69 6.64E006 Processing & presentaion of antigen CCR7/CC Motif Chemokine Receptor 7 2.34 1.50E003 DC migration, homing, activation of T, B, NK cells in lymphoid tissue IL15RA/Interleukin15 Receptor Alpha 3.63 7.43E004 CD8+ T and natural killer cell activation TNFSF9/41BBLigand/ CD137L 4.60 4.18E005 Costimulatory Ligand for CTL Activation. ZNF366/Zinc Finger 366/DC SCRIPT (Dendritic Cell Speci]ic Transcript) 3.12 2.93E004 Transcriptional reglulator involved in DC differentiation RND1/Rho Family GTPase 1 6.84 9.37E006 Supresses Ras signaling & EMT BATF3/Basic Leucine Zipper ATF like Transcription Factor 3 3.98 1.20E003 Essential for presentation of tumor antigens to CD8+ T cells & ef]icacy of immunostimulatory MAb’s (PD1 & 41BB) Enriched GO Biological Processes of PIN-2 activity in human monocytes ________________________________________________________________

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Page 1: Methods & Results - in vivo & in vitro · The genetic signature of the transcriptional changes induced by PIN-2 through activation of the early innate immune response and subsequent

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

1.00E-­‐13  

1.00E-­‐12  

1.00E-­‐11  

1.00E-­‐10  

1.00E-­‐09  

1.00E-­‐08  

1.00E-­‐07  

1.00E-­‐06  

1.00E-­‐05  

1.00E-­‐04  

1.00E-­‐03  

1.00E-­‐02  

1.00E-­‐01  

1.00E+00  

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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