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Plenary Keynotes TUESDAY | AUGUST 30 4:00PM – 5:30PM @CHI’s IMMUNO-ONCOLOGY SUMMIT, Marriott Long Wharf Hotel in Boston

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

Leaders in Pharmaceutical Business intelligence (LPBI) Group

covers in Real Time the IMMUNO-ONCOLOGY SUMMIT using Social Media

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

Plenary Keynotes TUESDAY | AUGUST 30

4:00 Personalized, NeoantigenBased Immunotherapy

Edward Fritsch, Ph.D., Chief Technology Officer, Neon Therapeutics, Inc.

Multiple lines of evidence have demonstrated the critical role that Neoantigens have in the immune response to cancer and the availability of next-generation sequencing to identify personal, neoantigen-creating mutations has opened the door to directly enhance the power and breadth of host immunity to overcome this deadly disease.

  • Yervoy approved for melanoma ipilimumab
  • ipilimumab and Nivolumab combination
  • Cancer Vaccine for infections disease – PREVENTIVE NOT TREATMENT HPV
  • CLASSES OF TUMOR ANTIGENS CT many tumors: Methylation pattern
  • Selectively expressed: Melanomas
  • Over expressed antigens – some tumor

Personalized

Scientific Advance I:

  • Tumor DNA sequences: Kras, PIK3CA, FBXW7
  • Somatic mutations potential to generate neoantigens
  • Neoantigents: Native antigens (mage) vs NEOANTIGENS 0 tumor specific Antigens

Scientific Advance II:

  • Ipilimumab
  • anti PL-1
  • single neoantigen reactive CD4+ T cell clone mediates tumor rejection in adoptive therapy

A Neoantigen Vaccine – The Opportunity

  • T -cell capable of tumor infiltration

DFCI/Broad Institute: Tumor procurement, Target selection Personal caccine manufacturing Vaccine administration

  • Identify targets: accuracy, epitope features multipla epitopes
  • Personalised GMP manufacturing – 20 long peptide regulatory acceptance, time and cost

FDA approval:

  • High risk Melanoma (IIIB/C; IVM1a)
  • cancer with documented immune responsiveness
  • 12 patients enrolled – Vaccine prepared for 8, 6 dosed
  • Immunological Responses: DeNovo – detectable directly ex-vivo: ICS Primarily CD4+ Poly-functional
  • CD8+ Responses – 1 Pre-stim: Pre vs 16 weeks after
  • Immunizing peptide (IMP)

NEON Therapeutics, Inc.:

  • Personalized Neoantigens vs Shared Neoantigens
  • Vaccines vs T-Cells
  • Moving into Advanced Metastatic disease — COmbination Checkpoint blockade
  1. immediate response
  2. synergies by combination therapy
  3. Neoantigets

 

4:30 Merck Sasso 

Emerging Innate Immune Targets for enhancing 

  • Additional component of the Immune system
  1. Combination of Checkpoint inhibitors – Targeting functionality of T-Cells
  2. Standard of care moving to Goal State by Check point blocade
  3. Monoclonal antibodies blocking PD-1 and CTLA4
  4. Immunogenic cell death
  5. Effectors that promote cross presentation of antigens
  6. recruitment of T cells
  7. reversing the pathways driving a repressing tumor environment

MERCK – Keytruda

Study with Dinociclib – Immunogenic Cell Death – induced by Radiation, combined with CTLA4  – median response overall survival – 20 month

MORE OPTIONS:

  • OV therapies – enhance tumor Combination:
  1. TVEC +Keytruda – disease control 68%
  2. CAVATAK +Keytruda
  3. CAVATAK + pembrolizumab
  • TLR agonism
  1. Two Phase I in melanoma
  2. TC-1 Anti-IL-10 MK-1966 induced by SD-101
  3. Innate Immune Triggers against Pathogens and Damaged selt

 

  • STING agonism
  1. DNA Sensing cGAS/STING – another approach to viral mechanism for Cancer
  2. Non-nuclear dsDNA is a ‘danger
  3. DMXAA – can’t stimulate HUman only moth STING pathway
  • RNA – RIG like receptors –
  1. leveraging anti-viral Mechanisms to eliminate Tumors 
  2. Activation of RIG-like receptor

SHARED MECHANISMS: INFgamma, TNF alpha

  • TLR9
  • RIG-I
  • STING
  • Oncolytic viruses

Summary

  • Merck – Keytruda – will be combined with different strategies to leverage innate immunity in combination with traditional T cell approaches
  • expend beyond T cell
  • limitation of checkpoint blockade therapies can be due to aberrant T cell localization and the suppressive microenvironment of the tumor

 

5:00 Reading Tea Leaves: The Dilemma of Prediction and Prognosis in Immunotherapy

Morganna Freeman, D.O., Associate Director, Melanoma & Cutaneous Oncology Program, The Angeles Clinic and Research Institute

With the rapid expansion of immunotherapeutics in oncology, scientifically significant advances have been made with both the depth and duration of antitumor responses. However, not all patients benefit, or quickly relapse, thus much scientific inquiry has been devoted to appropriate patient selection and how such obstacles might be overcome. While more is known about potential biomarkers, accurate prognostication persists as a knowledge gap, and efforts to bridge it will be discussed here.

  • CD8+ T cell – Tumor — Imune Priming — CHeckpoint Inhibition  CD8+
  • execution is complex
  • predict who will benefit from what treatment
  • Patient and Tumor Profiling
  1. Tcell Prining: TILs, PDL1, IDO, Tcell anergy Treg- Ovarian cancer: CD8+ TILs
  2. microenvironment – immuno-scoring
  3. Immune competence -flow, biomarkers CyTOF – Mass Flow Cytometry
  4. mutation burden – chemo + Vaccine – longer time to progression
  5. mictoenvironment – Tumor Profiling
  6. tumor adaptation – serum ULBP2 NKligand – independent predictor of prognosis in Stage I-III
  7. Predictive Genomic Analysis – Immune SIgnature of Response to CHeckPoint Blockage – liquid biopsy
  8. Neoantigens: Allows analysis of T cell
  9. Multispectral imaging – Immune cell phenotypes visualized and quantified simultaneously – improve TME immune suppression, TIL harvest potential, location of the T celle impact prognosis
  10. Immune monitoring: Pre intervation vs Post Intervention
  11. Tumor heterogeneity: Cancer progression and metastasis, clinical resistance
  12. Intervention Assessments: Tissue marker of Blood which one is the best to use
  13. Cloonal Tracking: Quantifying Tumor
  14. ImmunoPET – anti CD8 immune-PET sensitive of tumor infiltrating
  15. Transcriptomic Signature: IPRES (innate PD-1 resistence) can be induced by MAPKi, furthe account for poor response – due to immune depletion
  16. Tests and immuno-toxicity, Translation to POC
  17. Data assimilation
  18. Ideal BioMarkers related to Mechanism of Action – multivariant scoring systems
  19. Gender differences, BMI differences, Age difference  — IN RESPONDING TO IMMUNOTHERAPY IN IMMUNO ONCOLOGY

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AUGUST 30 8:30 am TRANSLATIONAL AND CLINICAL UPDATES

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

Leaders in Pharmaceutical Business intelligence (LPBI) Group

covers in Real Time the IMMUNO-ONCOLOGY SUMMIT using Social Media

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

 

TUESDAY, AUGUST 30 8:00 am Morning Coffee

TRANSLATIONAL AND CLINICAL UPDATES

8:25 Chairperson’s Opening Remarks

Joe Conner, PhD, CSO, Virttu Biologics

8:30 Phase I of Intravenous Vcn-01 in Patients with Advanced Cancer: Update on Clinical & Biologic Data

Manel Cascallo, Ph.D., Co-Founder, President and CEO, VCN Biosciences

A first-in-human Phase I dose escalation study of intravenous administration of VCN-01 (an oncolytic adenovirus with RB tumour-targeting properties and expressing hyaluronidase) with or without gemcitabine and Abraxane is ongoing for patients with advanced solid tumours including pancreatic cancer. Dose dependent tolerability data and VCN-01 levels in different biological samples (including blood and tumour biopsies) are available.

  • Phase I Intravenous
  1. VCN-01 – Pancreatic Cancer (IV)
  2. VCN-01 – Pancreatic Cancer (IT)
  3. Retinoblastoma (leV) –
  • ABD Technology 0 VCN-02
  • Immunoshift Technology VCN-03

Pancreatic tumor Treatment: Abraxane/Gemcitabine – 1st line

IHQ alpha-adenovirus

Tumor matrix composition can pose limitations to intratumor adenovirus spreading

Genetically modified adinovirus – sensitivity of replication in tumor cells –>> biodistribution slectivity (tumor targeting) –. tumor potency (fdiffusion)

modification in the fiber RGDK of the virus

  • Evidence of enhanced intratumoral spreading of oncolytic adenovirus after Hyal expression by adenovirus genome
  • Evidence of Hyal activity at long time points after systemic administration of VCN-01
  • Evidence of sensitization of chemotherapy (GE) after administration of VCN-01
  1. systemic administration in mice
  2. intratumoral administration in humster

Clinical Protocol – IV administration of VCN-01 — P-VCNA-001 ->> NCT02045602 – adenovirus in human

  • Patients with advanced tumor – Pancreatic tumor Treatment: Abraxane/Gemcitabine – 1st line
  • combination with standard
  • Part 1:
  • Part 2: recruiting ongoing

Toxicity Profile

  • Level of virus in blood – VCN-01 low dose level by day 8 no level in blood
  • increase of dose to 3,3E12 vp/patient – dose dependent – level in blood remains to day 28 then clearance
  • Immune response analysis: NAb’s generation
  • Level 1-1, 1-2, 1-3, 1-4
  • Part 2: Pancreatic tumor Treatment: Abraxane/Gemcitabine – 1st line – 28 cycle: DAy 1 highest – tumor biopsy – PET imaging day 28 post administration – 50%  (5/10) metastasis improvement — RECIST vs 1.1 – antitumor evaluation
  • Level of Toxicity: febrile neutropenia when combine adenovirus with Abraxane/Gemcitabine = virus +drug ->> febrile neuropenia observed
  • Part 2: Level in Blood Part 1 vs Part 2
  • Active replication of virus in the body cause infectionSamples positive at both dose level, Positivity observed in pancreatic lesions primary and in liver mestastatis

Immuno Markers in intratumor Biopsies

  • Pathways: PD-1/PDL1 AXIS Day 0 (+) vs CD8/T reg AXIS
  • explanatory Endpoints evaluationvs. Day 28 (-) CD8+ in tumor core

Summary

  • evidence of clinical activity has been obtained 3PR observed until now, 4/6 patients beyond stablished
  • viral replication
  • level of virus in blood not correlated with Positive effect on tumor core

 

9:00 Reolsyin: A Clinical Update of a Directed Cytotoxic Agent and Immune Modulator

Brad Thompson, Ph.D., CEO, Oncolytics Biotech – Publicaly traded

REOLYSIN was initially investigated for its potential as a selective cytotoxin. However, recent research shows that it also functions as an immune modulator. This dual mechanism of action for a single viral agent suggests that the potential of viral therapies may be broader than previously anticipated.

  • proprietary isolate of wide-type REOVIRUS SEROTYPE 3 DEARING
  • SAFETY PROFILE
  • 1,100 PATIENTS TREATED +1,000IV
  • NO MAXIMUM TOLERATED DOSE (MTD) REACHED
  • FIVE STUDIES CONFIRMED AND RANDOMIZED

REOLYSIN TWO MOA as an Oncolytic Therapy

  • REOLYSIN – DIRECTED CYTOTOXIN: RAS PATHWAY: BRAF, KRAS, NRAS, HRAS, EDFR, P53,
  • REOLYSIN – IMMUNE THERAPY – brings Immune System to baseline

Free Survival and Overall Survival: Effect of Ras Pathway Activation and /or p53 mutations on Progression Time in Month — Progression free survival 15 month vs control 5 month

By gender: Colonal rectal, Chron more prevalent in Females

REOLYSIN as an Immune Therapy

  • transcription
  • translation

TWO MOA as an Immune Therapy

  • Vaccine
  • Check point inhibitor up-reregulation

Pre-Clinical Immune Model – Steele 1995

REO 013: CHanges in Blood CHemokines/Cytokines

  • TRAIL
  • INFeron
  • activation of blood Immune Calls Post-REOLYSIN
  • REOLYSIN Increases PDL-1 Expression
  • Glioblastomas treated with REOLYSIN: productive reoviral infection showed increases PD-L1 expression
  • Multiple Myeloma: PD-L1 – Checkpoint protein

Combination Therapy 

  • REOLYSIN with Carfilzomib in Multiple Myeloma
  • Variable MARKERS: CD8, PD-L1, caspase-3, NK cells, CD68
  • IDO-1, CTLA-4
  • Intracraneal Murine Brain Cancer Model:
  • % Survival: 50 days GM-CSF/REO, GM-CSF/REO, anti PD-1 +anti-CTLA4 – now in Pediatrics
  • infiltration, proliferation, activates T-cell population

CANCER AND METASTESIS

  1. 1.2 DEATH OF LIVER METASTESIS
  2. REOLYSIN – ON LIVER METASTASIS = cross BBB, genetics: Ras Pathway defects
  3. REO 013: Liver metastasiss in REOLYSIN Monotherapy treated Pt
  4. RNA Transcription yes with REOLYSIN
  5. Post cycle 6 – vs. Post cycle 2 (radiation) vs. Pre-Treatment:
  6. Randomized Tumor-Specific Data: REOLYSIN/carboplatin/Paclitaxel/Combinations
  7. IND 210: Colon rectal : randomized Specific Data
  • 51% increased reduction in median total liver tumor volume
  • New colon rectal: Oncolytics: FOLFOX

Manufacturing – Commercial scale

Patent Portfolio: +400 patents issued Worldwide

Highlights

  • REOLYSIN treated patients +1,100
  1. Does not work on Melanoma – it is IV and does not get to skin,
  2. REOLYSIN is effective for Pancreatic and Liver, colon rectal, head and neck

 

9:30 Retroviral Replicating Vectors for Cancer-Selective Immuno/Gene Therapy: Translational and Clinical Update

Noriyuki Kasahara, M.D., Ph.D., Professor, Departments of Cell Biology and Pathology, CoLeader, Viral Oncology Program, University of Miami

Pro-drug activator gene therapy with retroviral replicating vectors is tumor-selective, and can lead to development of anti-tumor immunity. Ascending dose Phase I trials by Tocagen Inc. in recurrent high-grade glioma demonstrated favorable safety and tolerability, intratumoral virus spread, radiographic responses, and survival surpassing historical benchmarks. Based on these results, a randomized controlled Phase II/III trial is now underway.

  • Viruses as gene delivery vehicles:
  1. Adenovirus
  2. retrovirus – infect cancer cells and persists – unique RRV – Retroviral Replicating Vector – NOT lytic
  3. armed with 5-FC – CNS fungus infection – used for glioblastoma
  4. tumor produce his own drug after fungal infection
  5. RRV-CD reservoir  +5FC – become part of the CNS – continue infected cycle multiple times
  6. RRV – mediated Prodrug Survival 300 days without
  7. Immune activation, virus not immunogenic, T-cells vs Tumor Burden T-cells and B- cells infiltration causing decrease in tumor burden
  8. MDSC vs CD4 helper cells  vs CD8 cytotoxic cell
  9. Naive control vs Previosly cured at 30 days
  10. Anti tumor immunity – immunized T cells
  11. immunized unfractionated spleen cells
  12. RRV Administered — RRV spread through tumor
  13. High Grade Glioma (HGG) :
  14. Toca 511: RECURRENT gliomaindication Orphan Drug – gene present only in tumor sample

First in human injection of RRV Toca 511 in recurrent HGG

Favorable Safety Profile – for oncology drug

IV Study

Tocagen subject – Near complete response in Patients with Glioblastoma

Survival 70 weeks   – Toca511: 13.6 month

  • Higher dose cohort: 14.6 month
  • 1st and 2nd Recurrence

49 Centers; 50% in US

Toca5: Ongoing Trial – 128 patients:

  • GBM or AA with tumor <5cm and 1st and 2nd Recurrence 
  1. No vector
  2. IV
  3. Surgery intracranial local administration
  4. dose ascalation on 303 patients
  5. alive after >1 year: Comparison

Efficacy in multiple Cancer types:

  • Higher doses of RRV increased efficacy
  • Systemic 5-FU – Toxicity
  • Improve survival no trop in White blood cells
  • Toca 6 Trial: University of Miami: New indication – IV studies
  • Prodrug activator is the start killer gene added RRV MOA applied

 

10:00 Seprehvir, an Icp34.5 Deleted OHSV with Both Direct and Covert Modes of Action Joe Conner, Ph.D., CSO, Virttu Biologics

Seprehvir, an oncolytic HSV, is a complex biologic with multi-mechanistic modes of action. Lytic cytotoxicity, induction of Th1 cytokines/chemokine responses, recruitment of innate and adaptive immune cells and changes in the tumor microenvironment can enhance therapeutic efficacy in combination with other anti-cancer agents. How these modes of action intersect with PD-1 checkpoint inhibitors, CAR T cells and small molecule targeted therapies will be discussed. 10:30 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing

  • Oncolutic immunotherapy induces anti-tumor immune response in patients
  • Combination for apoptosis – direct lysis
  • Seprehvir, an oncolytic HSV – Oncolytic Immune therapy
  • Delivery Intratumoral – 83% systemic administration
  • Mesothelioma (malignant Pleural (MPM)
  • Seprehvir persistent in Pleural Fluid: HSV DNA, HMGB1, Cytokine signatures: Th1, Granzyme B, Immune Cells – three dose regime
  • Isolate imune cells – Gene expression profiling of Immune cells recruited post Seprehvir: Binding density
  • Seprehvir induces CD8+ Y cell infiltration and activity: CD3, CD8, FLT3 ligand – T-cell stimulatoe, NK cells, Fox3P,Granzyme B, Granulysin,
  • Seprehvir induces: anti-tumor IgG immune response: Proteins associated: Ferritin, D52 like 1&3 and tumor antigens (Mage A8/9) – anti tumor response and anti viral response
  • Noval IgG targets increases post Seprehvir

Combinations

  • Seprehvir and PD-1: Increase CD4+ and CD8+
  • CAR-T against GD2+ human Ewing Sarcome xenograft model – treated with PFU Seprehvir or PBS intratumorally on day 3,5,7
  • Seprehvir combines synergistically with mTOR/VEGFR signaling axis, AKT, P13K, cMET/VEGFR
  • Targeted therapies inhibit Seprehvir replication
  • mTOR/TK inhibitors and Seprehvir – induces intrinsic apoptosis: Caspases
  • Indication of synergies and apoptosis
  • Maurine 3T6 cells export a death signal – infected with Seprehvir – causes cell death
  • exported death signal MEK inhibitor and mTOR
  • did not worked with MEK (GSK)
  • Invitro in Cell lines: Seprehvir _ aurora Kinase A inhibitor Alisertib
  • Combination Seprehvir with Alisertib in vitro

 

11:15 Virus Manufacturing Comes of Age: Turning Bugs into Features Anthony Davies, Ph.D., COO, 4D Molecular Therapeutics

Viruses destroy the host in which you’re trying to produce them and then must be separated from all components of those cells. Many solutions to these challenges have been invented since the earliest production of viral vaccines in primary cells obtained directly form animals. But few have proven amenable to cost-effective, compliant and scaleable operation.

  • Glybera – AAV1 – Lipoprotein
  • Imlygic – T-VEC atenuated HSV
  • CMC = COGS (cost of Goods)
  1. PROVENCE
  2. Xtandi – Medivation bought by Pfizer
  • Head room vs cost
  • OV are diverse and have specific requirements
  1. Master Cell Banks & Master Virus Banks

Personalized Gene Therapy

  • Centralized manufacturing – cold storage
  • Distributed manufacturing – consistency across sites

Adenovirus

  • non-enveloped icosahedral nucleocaspid
  • affinity, anion

HSV

  • enveloped icosahedral caspid
  • 120-300nm
  • 152 kb dsDNA

Measles Family Virus

  • enveloped icosahedral nucleocaspid

4DMT Manufacturing Methodology – JMP Statistical Discovery Software

  • Control chart
  • long term continuous process improvement
  • Technology Transfer
  • Campaign monitoring
  • Reference standard
  • Precision makes Perfect
  • qPCR titration of AAV viral genomes (Precision for one variant 33%, closely related 22%

4DMT Analytical Processes

Design of Experiment – Design Space – range of parameters

  • JMP SW- Optimal design
  • DOE SW

 

11:45 Manufacturing Large Enveloped Oncolytic Viruses for Human Clinical Trials

Mark J. Federspiel, Ph.D., Professor and Director, Viral Vector Production Laboratory, Mayo Clinic

The large-scale production and purification of larger enveloped oncolytic viruses are particularly challenging. We have developed enveloped virus GMP production processes using suspension cells in combination with gentle but effective purification using hollow fiber tangential flow filtration that result in greater than 99.5% removal of contaminants and greater than 100-fold increases in final infectious virus titers.

  • Measles Viruses – a promising oncolytics – easy to sheer, toxic
  • Local vs systemic (more) – concentration and titer different
  • Reporter Gene: genomic contamination – Vaccine – neutralizer efficient release – Aseptic throughput
  • – size 100-300 nM – envelop Virus
  1. MV – CEA – secreted from
  2. MV-NIS
  • FDA Concerns:
  • Genomic DNA contanimation
  • risk vs benefit to patient
  • Initial Large-Scale Production method for MV-NIS – Optimized – composition of product after purification – Purification Steps – for standard titer
  • 3 micron initial filter bioprocess bags
  • FLOW FILTRATION USING HOLLOW-FIBER CARTIDGES
  • buffer control
  • anti measle vaccine getting titer dose
  • HeLA S3 – suspension – serum free – Aseptically Vialed Clinical Product
  • Protein concentration
  • DNA concentration – residual cellular DNA in MV-NIS Preps
  • 10 to power of 10 and 10 to the power of 11 dose possible 
  • Residual cellular DNA in MV-NIS Preps
  • None of HPV genomes are intact
  • analysis of MV-NIS residual DNA by qPCR: HeLA S3 dilution vs MV-NIS dilution
  • Tumorgenicity of large amount of noval agents – studies published of no risk of

WHY Patient 11.2 responded so well?

 

 

 

12:15 pm Close of Oncolytic Virus Immunotherapy

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4:30 8/29 PANEL DISCUSSION: ONCOLYTIC IMMUNOTHERAPY IN THE ERA OF CHECKPOINT BLOCKADE @IMMUNO-ONCOLOGY SUMMIT – AUGUST 29-30, 2016 | Marriott Long Wharf Hotel – Boston, MA

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

 

Leaders in Pharmaceutical Business intelligence (LPBI) Group covers in Real Time the IMMUNO-ONCOLOGY SUMMIT using Social Media

 

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

 

4:30 8/29 PANEL DISCUSSION: ONCOLYTIC IMMUNOTHERAPY IN THE ERA OF CHECKPOINT BLOCKADE

Robert Coffin, Ph.D., CEO,

Replimmune Ltd Oncolytic immunotherapy treats cancer by virus-mediated tumor cell lysis and generation of a patient specific cancer vaccine, including to neo-antigens, in situ directly in the patient. Both are likely important for the clinical efficacy seen with single agent use, and also for the clinical synergy observed with immune checkpoint blockade. Background and data supporting single agent and combination use will be discussed, and future directions described.

  • synergistic with other therapies
  • 15 years of basic research now is available

David Kirn, MD, CEO, 4D Molecular Therapeutics

  • comnibation VT with IT
  • deliver into the tumor MDs are not agents og change, oral drug IV, IT injections intratumorally by Interventional Radiology is a fight worth fighting
  • Ovarian cancer – injection was a strugle
  • injection in to the eye is now the practice
  • IV
  • Angiogenic paradigm was throughn out
  • synergies with antivascular

Mattew Mulvey, PhD, CEO, BeneVir

  • consensus was IV ot iT
  • IT dosing targeting the tumor for IV dosing –  need ne IT in present time
  • Interested in the microboome, manipulate to achieve goals,
  • consolidation in the industry is unavaidable

John Bell, PhD, University of Ottawa

  • approval of single agent will change
  • viral particles to be used

Stephen Russell, MD, PhD, Mayo Clinic and VYRID

  • OV is the main event, other IT will be used in conjunction with OT which will LEAD the Therapy 
  • vascular enthotelium
  • cell therapyas carriers will act like vessels
  • Systemic delivery from AMGEN, Phase III is a proof
  • cycle of drug approval is 15 years, good is the enemy of Better, minor improvals are nmany

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LIVE 1:30 – 4:30 8/29 BIOMARKERS AND IMPROVING VIRUS ACTIVITY @IMMUNO-ONCOLOGY SUMMIT – AUGUST 29-30, 2016 | Marriott Long Wharf Hotel – Boston, MA

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

 

Leaders in Pharmaceutical Business intelligence (LPBI) Group covers in Real Time the IMMUNO-ONCOLOGY SUMMIT using Social Media

 

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

 

1:30 – 4:30 BIOMARKERS AND IMPROVING VIRUS ACTIVITY

1:25 Chairperson’s Remarks

David Kirn, M.D., CEO & Co-Founder, 4D Molecular Therapeutics & Adjunct Professor of Bioengineering, UC Berkeley

1:30 New Biomarkers that Predict Response to Oncolytic Virus Immunotherapy

Howard L. Kaufman, M.D., FACS, Associate Director, Clinical Sciences, Rutgers Cancer Institute of New Jersey; Professor and Chief, Division of Surgical Oncology, Rutgers Robert Wood Johnson Medical School

T-VEC is the first oncolytic virus approved for the treatment of melanoma, and will soon enter clinical trials for treatment of other cancers. Further studies using T-VEC in combination with T cell checkpoint inhibitors are underway and showing promising early results. The identification of predictive biomarkers of response would be helpful for improving patient selection and optimizing therapeutic outcomes. We have recently focused on HSV-1 entry receptors and oncogenic signaling pathways within cancer cells as potential biomarkers of T-VEC response.

  • Why we wish to have biomarkers?
  1. identify patients
  2. efficacy
  3. cost/avoid over/under tretment
  4. molecular therapeutics understandin
  5. PD1 – expression is the ONLY biomarker we currently have for eligibility to participate
  6. Biomarker discovery – Biopsy or blood, many indicators to measure

Biological challenges: Tumor and lesion heterogeneity

  1. detection frequency for DNA vs. protein serum vs imaging modality
  2. prior treatment
  3. Circadian rhythm

Technical/Logistical

OV Immunotherapy

  1. Tumor cell intrisic factors
  2. Host Factore
  3. Immune Ssytem factors

Tumor cell intrisic factors

  • Viral cell surface – viral infection
  • signalling receptors – Nectin1, Nectin2 molecules
  • HVEM
  • NCB160

mRNA expression of HSV-1 receptors on NCI 60 cell line panel

Oncogenic pathways – apoptosis of cell — abberation of this process prevent apoptosis and

  • Treatment with T-VEC – response to BRAF and NRAS
  • MEK inhibition sentisizes mealnoma cell line to lysis by T-VEC
  • mutation status,
  • Oncolytic viruses trigger immunity through release of PAMPs
  • Patients with low serum HMGB1
  • Balancing the inflamed tumor microenvironment
  • Expression of PD-L1 and IDO associated with CD8+ T cell infiltrate, Fox
  • ICOS plus  plus T cells after T-VEC CD4+
  • Immune model: B16 Nectin-1 on one side
  • in 4 days Post Injection and viral infection –>> Increase in microphage – CD8+
  • Anti HSv – glycoprotein B lead to anti Herpes: Left vs right Flank
  • HSV-1 antigens – recognized by Human CD4+
  • PD-L1 Expression in Melanoma – PFS
  1. NIVO _IPI, FoxP3, CD163, DAPI, PD-L1, CD 3, CD8
  • LA-07 CALM Study: Best % of Change in tumor size T1 and 8 days later T8
  • Interferon – NanoString analysis: Immune
  • Mutation Load andneoantigen
  • Mutation Burden correlates with PF
  • Avelumab antiPD-L1
  • MCPyV and PPD-L1 therapy: MCpyV positive va negative MCC: at the extremes of mutational frequency

Conclusions

  1. predictive biomarkers
  2. Intrinsic tumor cell factors

Viralytics

Questions:

  • Intra Tumor injection – pushback by MDs – Yes, virus need be in different room than chemotherapy

2:00 Therapeutic Viral Vector Evolution: A Robust Platform for the Discovery of Optimized Vectors – Lessons in 20 yers

David Kirn, M.D., CEO & Co-Founder, 4D Molecular Therapeutics; Adjunct Professor, Bioengineering, University of California, Berkeley

Therapeutic virus vectors hold great promise for cancer gene and immunotherapy. However, novel vectors with improved efficacy are needed. Therapeutic Vector Evolution is a discovery platform from which optimized and proprietary viral vectors can be identified with beneficial characteristics of interest.

  • Translational
  • Design of 2nd generation VT
  • Adenovirus 2/5 deletion mutant Onxy-015 (d/1520) – cancer targeting selelctivity: p53 inactivation in cancer cells
  • 1996-2001: ONXY 015 experience in humans
  • first engineered OV in human: Intratumoral, Peritumor, Intraperiteneal, Intra-atrial, IV
  • Well tolerated acure, flu- like symptoms
  • Tumor specific replication of ONXY-015 in head & neck complete response vd peripheral escape and viral clearance
  • Tumor response limited IT only
  • No systemic anti-tumor efficacy
  • no IV delivery
  • No anti-tumor immune response demonstrated
  • Resistance
  • OV can sensitize tumors for chemotherapy
  • Hepatic artery
  • Pharma executives: nich unless given IV – in 2016 the position changed
  • VACCINIA Biology: The virus pharmacophore Library
  • Vaccinia Vs other visuses:
  • JX-594 (Pexa-Vec): Novel 3-pronged MOA
  • Studies:
  • Neutrophil induction correlates with GM-CSF in serum
  • Tumor liver injected – complete response
  • Systemic efficacy: carcinoma in the abdomen
  • Lytic effect: Vaccinia, other virus
  • Resist disease control: Randomized Phase 2A HCC trial design: High vs Low dose: Baseline Wek8 month 21
  • Uninjected week8 week 14 chronic inflammation
  • JX-594 (Pexa-Vec) experience in Humans (2007-2014): IV infusion vs IT injection

Biology mover to Vaccine – induce local injection in Gene Therapy with applications to CANCER

  • Therapeutic Vector Evolution DIscovery Platform: Overcoming hurdles 1st generation

4D Molecular Therapeutics:

Partnerships:

  1. Roche on Retina – opthalmology
  2. Pfizer – Cardiovascualr and
  3. Therapeutic Vector Evolution Discovery Platform
  4. D. Schaffer – Developed at UC, Berkeley – 4DMT Vector Discovery Programs: 
  5. Human Organotypic Lung POC for Vector Evolution” 4DMT variant transduction superior to 1st gen AAV
  6. Untransduceed CF vs transduced two dominant motifs – hepatocytes
  7. Summary
  8. predictive biomarkers
  9. overcome ECM
  10. overcome antiviral immunity’pharmaceutical partnerships
  11. Optimize ICI combination
  12. more tumors injected prior to activation of immune system better results

 

2:30 Enhancing Oncolytic Virus Activity by Engineering of Artificial MicroRNAs

John Bell, Ph.D., Senior Scientist, Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Professor, Departments of Medicine and Biochemistry, Microbiology & Immunology, University of Ottawa We have devised a novel strategy to enhance the ability of oncolytic viruses to infect malignant cells by expressing artificial microRNAs (amiRNAs) from the oncolytic virus genome. We have screened a variety of amiRNAs and identified a number that enhance virus replication within tumour but not normal cells. The characterization of these miRNAs and their targets will be discussed.

  • Immune adjuvant Gene therapy
  • Iv/IT agents
  • Virus
  • OV – are exquisitely specific: Cancer cells – behavior
  • multiple pahtologically activated pathways mitigate cellular antiviral response
  • Pikor, Bell, Diallo (2015) Trends in CANcer Vol 1 266-277
  1. WNT pathway
  2. MEK
  3. Vascular attack by Rhabdovirus and Vaccinia OV
  4. VEGF inhibits interferone production leading to OV replication and spread
  5. Tumor/wounded OV infected endothelium –
  6. OV: A sustemic therapy for Metastatic Cancer

Tumor Specific Anti-VIral defects: – Patient/tumor Heterogeneity will impact Tx activity of OV

  • Oncolysis
  • antitumor Immune Activity
  1. Maraba virus: Oncolytic and vaccine candidate: Rhabdovirus Structure
  2. How to increase therapeutic potency?
  • manipulate the infected Host cell with amiRNAs: Host determinants of Virus Repliccation
  • Library screen on Pancreatic CancerP) of SV-LIB (16,000 unique clones) serial Passaging of viral go to deep sequencing = Large scale genome sreening to identity virus
  • Enriched amiRNAs confer enhanced OV cytotoxicity
  • ARID1A: member of SWI/SNF gene Family
  • Helicase/ATpase – Synthetic Lethality – double mutant lethality
  • GSK126: EZH2 inhibitor (an epiegnetic) HPAF II cells – virus– GSK126 — readout
  • miRNA transfer via exosomes may mediate bystander effect
  • OV Infection of Tumor cells Stimulates exosome secretion
  • Is amiR6 present in exasomes shed by infected cells?
  • miRNA transfer via exosomes
  • Epstein-Barr virus-infected cells secrete exosomes that containn EBV-encoded miRNA (Pegtel et al, PNAS, 2010)

3:00 Refreshment Break

3:30 Immuno-Oncolytic Viruses as Cancer Therapies

Stephen Thorne, Ph.D., Professor and Scientific Advisor, Inventor, Western Oncolytics

Oncolytic viruses primarily act as immunotherapies, yet most vectors still rely on the virus’ inherent immune activation, often coupled to single cytokine transgene expression. However, for optimal activity they will need to overcome the tumor¹s immunosuppressive microenvironment, to raise anti-tumor CTL and allow repeated systemic delivery. Approaches to achieve all of these activities in a single vector are being developed.

  • insitu vaccination
  • capacity to modify immuno therapeutic activity
  • Optimizing immune activation
  • – Th1 vs Th2 response
  • overcoming local immunosuppression wihtin the Tumor – get the OV overcome the immunosuppression
  • Vaccinia infection of TLT2-/- mice resulted in reduced production of neutrlizing
  • Re-direction TLP Activation – Toll-Like Receptor Signaling
  • Deglycosylating viral practices reduced TLR2 activation
  • Deglycosylating Vacinia – does os
  • In vivo ablating TLR2 activation through Deglycosylation leads systemic delivery of the virus
  • TRIF expression alters immune response and enhances therapeutic activity in vivo after single IV delivery of virus
  • Deglycosylation evades anti-viral neutralizing antibody: Tumor Volume vs % neutralized
  • Maximize immune activation, overcoming immunosupression
  • MDSC – blocks the resistence – as tumor grows resistence increases – and cycolytic capabilities of the Viral therapy
  • G-MDSC in tumor: % of cells vs PBS< anti-PGE2, celecoxib
  • T-cell transfer
  • Immune checkpoint blockade
  • Optimized Imune activation
  • Overcoming the localized immune suppression
  • Over Effects, novel vector WO-12

 

4:00 Arming the Oncolytic Virus Enadenotucirev to Develop Tumor-Localized Combination Immunotherapeutics

Brian Champion, Ph.D., Senior Vice President, R&D, PsiOxus Therapeutics Ltd.

We have developed a systemically deliverable, oncolytic adenoviral platform for directing efficient and selective local production of a combination of biotherapeutic agents selectively within the tumor. This has the potential for enhanced efficacy while reducing side effects by limiting systemic exposure. Up to three separate biomolecules can be encoded in the same virus without affecting oncolytic properties of the virus.

  • Endenotucirev (EnAd): Oncolytic, reduces tumor burden – Carcinoma Ovarian cancer
  • “Armed” (ENAd): – Tumor-Specific Immuno
  • Research Virus Platform: antibody production: Virus Replication
  • Selectivity: Replication vs Anti-VEGF Ab vs Infectious Virus
  • HT-29 vs Hepatocytes
  • Human dendritic cells: 48h post co-culture with GFP-expressing (ENAd)
  • Payload to the Virus: Stay on or in the infected cell,
  • Next gen virus: TUmor cell activation signals: anti-tumor immune response by T-cells
  • Cytokines: INFalpha,
  • EnAd vs EnAd-CMV-EpBiTE
  • 3 unique TRANSGENES can be secreted ot inseted into the plasma membrane
  • NG-348 class immune-gene therapy
  • CAR-T/TCR Immunotherapyactivation via antigen selection
  • Activating ligand
  • IFNgamma secretion

Summary

NG-348, lead candidate –> selective expression of ligands

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LIVE 9:55 – 12:00 8/29 UNDERSTANDING MECHANISMS OF ACTION @IMMUNO-ONCOLOGY SUMMIT – AUGUST 29-30, 2016 | Marriott Long Wharf Hotel – Boston, MA

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

 

Leaders in Pharmaceutical Business intelligence (LPBI) Group covers in Real Time the 

IMMUNO-ONCOLOGY SUMMIT using Social Media

 

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

 

9:55 – 12:00 UNDERSTANDING MECHANISMS OF ACTION

9:55 Chairperson’s Remarks

Fares Nigim, M.D., Massachusetts General Hospital and Harvard Medical School

First phase:

  • OV infection/replication

Second phase

  • Immune response

Clinical considerations: viral delivery, Patient’s selection, Biomarkers, combination with Immunomodulators

 

10:00 Designing Clinical Trials to Elucidate Oncolytic Virus Mechanisms-of-Action

Caroline Breitbach, Ph.D., Vice President, Translational Development, TurnstoneBiologics

Oncolytic viruses have been shown to target tumors by multiple complementary mechanisms-of-action, including direct oncolysis, tumor vascular targeting and induction of anti-tumor immunity. Phase I/II clinical trials can be designed to validate these mechanisms. Development experience of an oncolytic vaccinia virus and a novel rhabdovirus oncolytic vaccine will be summarized.

  • Mechanism of Action:
  • Clinical trial design and choosing population
  • Pex-Vec: Oncolytic Vaccinia – Infection and SPread within tumors follwoing IV AdministrationCCRC vd Ovarian cancer
  • dose threshold for IV delivery Defined
  • Tumor-specific Trnsgene
  • dose-dependent induction of antibodies to beta-galactosidase
  • Delayed Virema and evidence of GM-CSF Expression – day 4 nad Day 6
  • Oncolytic Viral Immunotherapy: Oncolytic Virus and T-cell Vaccine
  • Maraba MG1 Oncolytic Virus – Rhabdovirus Structure – from insects not a human pathogen.
  • MG1 boosts immunity- engage memory T cells,
  • Unique Biology of T Cell Boosting:
  1. virus infects follicular B cells: Lung metastesis DCT Prima, DCT Boost
  2. COmparison MG1 to other Vaccine Platforms
  3. Immunecheck Inhibitors – combined to augment Immune activity in preclinical models
  4. T- cells recruited to tumors post Ad-GM!
  5. MAGE-A3: in Human in CLinical studies – fresh peripheral blood underwent in vitro stimulation with MAGE-A3 peptide pools for 6 hours follwoed by staining and flow
  6. First Human CLinical Trial – Status: Enrolling – 70 patients
  • Arm A, B, C and Pahse II: Prescreen MA3, Screening CT/biopsy ADvirus Biopsy CT
  • MOA – Amplification in tumor
  • transgene expresion systemic delivery induction of anti-viral antibodies/immunity
  • Efficacy ENdpoints:
  • Radiographic endpoints
  • Cancers with tumor markers
  • must ensure suggogate endpoints are approvable
  • Acute reduction of Perfusion after Pexa-Vec Treatment
  • unmasking of existing lesions
  • response in non-injected tumors: baseline, week 8,20
  • Lymphocytic inflitrate
  • Selection of Patient Population: Injectable Unresectable Stage IIIB -IV

Liver Cancer: HCC – First-lineLow and High dose

Phase 2b – Second-line: Single agent NOT approriate for advanced disease

Pharmacokinetics – unique Replication-dependent PK

  • GM-CSF – cytokine autoimmunity

10:30 T- Stealth Technology Mitigates ANtagonism between Oncolytic Viruses and the Immune System through Viral Evasion of ANti-Viral T-Cells

Matthew Mulvey, PhD, CEO, BeneVir

Virus evading immune response – resist interferon

T-Vec vs T-Stealth = viral spread continues enhanced efficacy

T-Stealth – unique ability to evade clearance by T-cells in order to permit stimultaneous co-administration of OV and immune checkpoint inhibitors

  • evades of T-cells
  • induction and systemic anti tumor T-cells
  • synergy with checkpoint
  • Efficacy of repeat dose
  • Inhibitor of innate immunity, T-cell
  • Bladder cancer MBT-2 injection bi-lateral : T-Stealath + AntiPD1 + CTLA4
  • improved T-Cell receptor diversity in untreated tumors suggestin that t-Stealth induces immune response system to target a wider range of tumor neo-antigens

DRUG PROFILE

  1. Replicate,spread
  2. mitigate antagonism with checkpoint inhibitorscan be armed with 3 additional transgenes to promote anti-tumor
  3. One stop shopping
  4. systemic OV dosing:
  5. more efficacious because virus spreads

 

11:00 Improving Oncolysis and Therapy with Pharmacologic Modulation – Glioblastoma

Antonio Chiocca, Professor & Chairman, Department of Neurosurgery , Brigham & Women’s Hospital/ Harvard Medical School

Glioblastoma (GBM) – survival 15 month – heterogenous, target therapies – FAILED, subclones – mutation burden – OV – injection into tumor

  • if inject into the Brain – bad effect – tolerated, efficacy NOT established
  • Current therapies aimed at ICP6 – Herpes+ Nestin
  • HSV strain attenuated ICP4 mutation
  • UL39
  • Mice models: Animal survival is 80% when OV is injected 7 days
  • Animal survival is 50% when OV is injected 14 days after tumor implant
  • GBM Clinical Trial
  • VPA – Valporic Acid – FDA approved – antitumor efficacy of Herpes-based OV – Histone deacetylase
  • HDAC6 – major deacetylase in cytoplasm – improves shuttling of post-entry oHSV into nuclei vs lysosomes
  • 2015 – Histone deacetylase  6 Inhinbition enhances OV replication in glioma
  • The nestin promoter in rQNestin34.5
  • VPA demethylate oncolytic HSV promoter
  • barriers to OV therapy and maneuvers to circovent
  • Transcript profile analyses of Glioma
  • NK cells are recruited to brain GBM following oncolytic HSV
  • PD-L1 expression in Glioma Stem cells after oHSV
  • immunocompetent mouse Glioma cells that replicate oHSV to high levels – testing
  • Conclusions

 

 

11:30 Moving Toward MultiFunctionality in PoxvirusBased Oncolytic Virotherapy

Eric Quemeneur, Ph.D., Pharm.D., Executive VP and CSO, Transgene

Poxviruses are powerful immunotherapeutics and tumor-targeting platforms. We recently expanded Transgene’s portfolio of armed oncolytic Vaccinia Viruses (oVV) by engineering a vector that targets anti-PD1 IgG expression into the tumor. Local concentration of virus-encoded antibody was ~10-50 times higher than the reference mAb, leading to significant improvement of survival in a sarcoma preclinical model. Such results announce the next-generation OVs, combining immunogenic oncolysis with the capacity to deliver complex therapeutic modalities in the tumor micro-environment.

  • The merit of poxyviruses for UV – envelop Virus – cytosol replocation
  • suitable for molecular engineering: large genome
  • Enzyme – Fcu1 VV(TK-RR-)-Fcu1
  • FCU1 is a chimeric bifunctional enzyme
  • Activity of TG6002 in human tumors: Growth control: SKOV (ovarian) and U87-MG (control) Ovarian
  • TG6002 also active on Cancer stem cells, compatible with Chemotherapy: Human Pancreatic Cancer

Activity in immuno-competent models –

  • TG6002 (WR) in mice synergy study and
  • Lymphocyte infiltration into the tumor
  • Complementarity with PD1 blocker
  • induce abscopal response – effect on survival
  • Combination OV/ICI in the clinic
  • Genetic recombination of mAb expression cassettes
  1. IgG
  2. Fab
  3. scFv

T cell depletion experiment – Anti- CD4 and CD8

Pre-Clinical results – in vivo expression and biodistribution of WR-mAb1

  • Tumor growth inhibition & survival (MCA205 sarcoma model
  • Overcoming the tumor access barrier
  • cavitation-enhanced ultrasonic virus delivery

Summary

  • Poxviruses – safe, potent and versatile
  • IV route
  • synergy with other immunotherapies
  • platform is customizable

 

 

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LIVE 8:25 – 9:30 8/29 REALIZING THE POTENTIAL OF ONCOLYTIC VIRUS IMMUNOTHERAPY @IMMUNO-ONCOLOGY SUMMIT – AUGUST 29-30, 2016 | Marriott Long Wharf Hotel – Boston, MA

http://www.immuno-oncologysummit.com/uploadedFiles/Immuno_Oncology_Summit/Agenda/16/2016-The-Immuno-Oncology-Summit-Brochure.pdf

 

Leaders in Pharmaceutical Business intelligence (LPBI) Group

covers in Real Time the IMMUNO-ONCOLOGY SUMMIT using Social Media

 

Aviva Lev-Ari, PhD, RN,

Founder, LPBI Group & Editor-in-Chief

http://pharmaceuticalintelligence.com

Streaming LIVE @ Marriott Long Wharf Hotel in Boston

Curation of Scientific Content @Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

 

8:25 – 9:30 REALIZING THE POTENTIAL OF ONCOLYTIC VIRUS IMMUNOTHERAPY

 

LIVE 8:25 Chairperson’s Opening Remarks

Brian Champion, Ph.D., Senior Vice President, R&D, PsiOxus Therapeutics Ltd

  • Different viruses
  • Engineering
  • Manufacturing: CMC
  • Systemic vs IT Delivery
  • Tumor Markers environment: Tumor cell lysis and immune response
  • Biomarkers Clinical Development
  • Role of Pre-clinical Models
  • regulatory affairs

8:30 T-Vec: From Market Approval to Future Plans

Jennifer Gansert, Ph.D., Executive Director, Global Development Lead, IMLYGIC, Amgen, Inc.

Talimogene laherparepvec (T-VEC) is a modified herpes simplex virus type -1 designed to selectively replicate in tumors and to promote an anti-tumor immune response. T-VEC is approved for metastatic melanoma based on a randomized phase III trial; T VEC significantly improved durable response rate vs GM-CSF. Data from the pivotal trial and combination studies with checkpoint inhibitors will be presented.

T-VEC – HSV-1

  • Viral Protein:
  1. ICP47 – Deleltion ,
  2. ICP34.5 – Deletion ,
  3. US11 – Temporal expression,
  4. GM-CSF – Insertion
  • Engineering Change:
  1. Injected Tumor
  2. Contralateral tumors
  • Dual MOA
  • Administration: Largest lesion first, 4 cycles of injections
  • OpTim – Phase III: N = 436 Stage III-IV Melanoma
  • T-VEC (N = 295)
  • GM-CSF (N=141)
  • Key ENTRY Criteria
  • END POINT: Primary and Secondary – Survival benefit
  • 2/3 – prior infection with HIV – melanoma not resectable with spread to lymph nodes
  • Response rate with T-VEC: 30% response 2/3 – control of the disease
  • Lesion-Level, Lesion-Type Response Analysis
  • Overal Survival:Over 20% reduction of burdon
  • Retrospective analysis: If not spread yet to lymph nodes: Best response to treatment
  • Early disease stage and early therapy are correlated
  • T-VEC double survival vs GM-CSF
  • Adverse effects: Cellulitis

Phase I

Phase II

Phase III

  • Regulatort Interactions for US BLA – Full approval in 10/2015
  • Rationale for Combination wiht CHeckPoint Inhibitors
  1. Immunologic response:
  2. Control
  3. OncoVEX mGM-CSF
  4. CTLA4
  5. Ipilimumab – 3
  6. Pembrolizumab -4
  7. Neoadjuvant – 2
  8. unserectable safety  – 1
  • Changes in Tumor Burden by DIsease CHange
  • Progression-Free Survival – 72%
  • Adverse events: as expected
  • Phase II design: Pembrolizumab 200mg
  • Monotherapy vs Combination
  • Address multiple Tumor type: Menaloma, RCC, mCRC, BrCA, Gastric, NSCLC, HCC
  • Other: Head & Neck (completed), Pacreatic (completed), Hepatic injection (ongoing), Pediatric study (planned)

9:00 Oncolytic Virotherapies as a Single Shot Cure?

Stephen J. Russell, M.D, Ph.D., Professor, Mayo Clinic

VYRIAD, CEO

Oncolytic virotherapy is increasingly used as a cancer immunotherapy. However, certain oncolytic viruses can also mediate wholesale tumor destruction independently of an antitumor immune response. This is the oncolytic paradigm, where a cytolytic virus with preferential tumor tropism spreads extensively at sites of tumor growth and directly kills the majority of the tumor cells in the body leaving only a few uninfected tumor cells to be controlled by the concomitant antitumor immune response.

  • Virus – does the heavy lifting – small virus inoculum, local spread, systemic virus spread – via blood stream -VIREMIA – killing of infected cells Immune response help Virus elimination
  • Engineer virus: Tropism, dose, route
  • Immune response: Killing Uninfected cells killing tumors cells
  • Second exposure – preformed antibodies: Viremia – neutrilized + Memory cytotoxic T-Cells CTL
  • Oncolytic ViroTherapyFirst dose more effecitve then subsequence
  • VSV- Vesiculat Stomatisis Virus: IFNbeta and NIS
  • SIngle dose: Intratumorally: complete regression – controlling tumor
  • Reaching mestastesis: IV delivery
  • After systemic delivery: Mode of Virus spread in Tumors: tumor distruction: density of tumors: Delivery and SPread
  • Second and subsequent – Ovarian Cancer: single dose vs six doses: no significant (three doses – NO additional therapeutic benefit
  • Pet-dog with lynphoma: Multi center – single shot
  • HUMAN: Clinical Trial in Mayo, Arizona, Redractory/Intolerant HCC: In Patient 12:necrosis of the tumors, markers: HCC – metastasis to ColonRectal Cancer – developed Day 13 Hepatorenal outcome – virus infected non-injected tumors
  • NGS – error rat 1 in 1000 of virus genome sequence – 164 mutations 103 coding and 61 are noncoding or silent mutations
  • What determined rapid virus spear in Patient12: 4 gees of thr 84 genes:
  1. antiviral state
  2. antiviral sensing and signaling
  3. IFN signaling
  4. Antigen processing and presenation

NOW Companion Diagnostics is been developed

CASE: Measles Seronegative: – Complete response to IV MV-NIS  – patient with melanoma

  • no systemic response – Oncolytic debulking and lasting immune control

Summary

  1. Single shot cure for cancer – and likely transform Cancer care
  2. Oncolytic and Immune two MOA – killing infected and uninfected tumor cells

Success: monitor viral spread

  1. exploit first dose
  2. develop tests to match with tumor
  3. combine with immuno modulatory drugs
  4. continue create better viruses

VYRIAD: Companion Diagnostic: Lung, Head & Neck, Bladder

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Oncolytic Virotherapy for Pancreatic Cancer: Overcoming Obstacles in Oncolytic Virus Delivery

Reporter: Aviva Lev-Ari, PhD, RN

 

We covered MGH’s Innovation on Tumor targeted therapy in Pancreatic Cancer in

Pancreatic Cancer Targeted Treatment?

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2016/05/18/pancreatic-cancer-targeted-treatment/

 

Below, we report on the State of the Science for Overcoming Obstacles in Oncolytic Virus Delivery and provide the source for all the references used

 

ONCOLYTIC VIROTHERAPY FOR PANCREATIC CANCER

Adenovirus

ONYX-015 was the first TOV used in a clinical trial for pancreatic cancer. ONYX-015 was administered intratumourally under endoscopic ultrasound-guidance into patients with locally advanced adenocarcinoma of the pancreas or metastatic disease in phase I/II trials[132]. The treatment was well-tolerated in most patients, however no objective responses were seen with ONYX-015 as a single agent and only 2/21 patients experienced mild responses when combined with gemcitabine[132]. A second adenovirus vector carries a deletion in the E1A gene[133]. E1A normally binds to the retinoblastoma protein, forcing cells to prematurely enter the S phase of the cell cycle. Since most pancreatic cancers harbor a mutation in CDKN2A[134], the E1A protein is unnecessary for entry of the TOV into cancer cells. Furthermore a double-deleted (E1A and E1B19) adenovirus demonstrated increase potency and selectivity in pancreatic cancer models[135,136]. This demonstrates that TOVs can be genetically engineered to increase selectivity and efficacy while maintaining their potency. Adenovirus selectivity has also been improved by engineering tumour-specific promoters such as a human CEA promoter[137] or by substituting the adenovirus serotype 5 fiber knob with the fiber knob from serotype 3[138]. The potency of TOVs can also be improved further by engineering them with therapeutic genes that stimulate the immune system and/or improve direct oncolysis. Adenovirus ZD55-IL-24 expressing IL-24 locally in pancreatic tumours in immune competent mice inhibited tumour growth and induced a stronger T cell response compared to its backbone virus, as measured by IL-6 and IFN-γ levels[139].

HSV

Two oncolytic HSV-1 vectors are currently in clinical trials for the treatment of pancreatic cancer. HF10 is a non-engineered, naturally occurring oncolytic HSV that demonstrated regression in 1/6 of the patients treated[140,141]. OncoVex GM-CSF is a ∆34.5 and ICP47-deleted mutant expressing GM-CSF, whereby the deletions allow for tumour-selective replication and inhibition of protein-kinase R activation, respectively[142]. Phase I/II trials in various solid tumours demonstrated OncoVex GM-CSF to be well-tolerated at high and repeated doses[143,144]. A phase I clinical trial with OncoVex GM-CSF in patients with unresectable pancreatic cancer is underway.

Poxviruses

The most widely studied poxvirus is VV, which is highly immunogenic and produces a strong cytotoxic T cell response[145] and circulating neutralizing antibodies which can be detected decades later[146]. For its crucial role in the eradication of smallpox, much has been learned about its potential role in immunotherapy today. The Lister strain of vaccinia remarkably showed no replication degradation even under the hypoxic conditions of PDAC[147]. A second Lister strain, thymidine kinase-deleted replicating VV armed with IL-10 demonstrated superior and long-lasting antitumour immunity in both a subcutaneous pancreatic cancer model and a Kras-p53 mutant-transgenic pancreatic cancer model after systemic delivery compared to its unarmed backbone virus[148]. Myxoma virus, a rabbit-specific poxvirus combined with gemcitabine resulted in 100% long-term survival in Pan02-engrafted immunocompetent intraperitoneal dissemination models of pancreatic cancer[149]. The only poxvirus to be tested in clinical trials is a non-replicative VV that expresses the pancreatic TAAs CEA and MUC-2[150]. The vaccine also includes a triad of costimulatory molecules, B7.1 (CD80), ICAM-1 (intra-cellular adhesion molecule-1) and LFA-3 (leukocyte function-associated antigen-3) (TRICOM) (PANVAC-VF)[150]. GM-CSF was also used as an adjuvant following each vaccination of PANVAC-VF. Phase I trials demonstrated antigen-specific antitumour responses in 62.5% of patients enrolled and antibody responses against VV was observed in all ten patients, which was associated with an increase in survival (15.1 mo vs 3.9 mo)[48]. A phase III clinical trial for the treatment of metastatic pancreatic cancer after failing treatment with gemcitabine, however, was terminated after failing to reach its primary efficacy endpoint[151].

Other pre-clinical TOVs for pancreatic cancer therapy

Parvovirus, measles virus and reovirus have also demonstrated pre-clinical activity in pancreatic cancer models. Parvoviruses particularly demonstrated enhanced IL-2-activated NK responses against PDAC cells[152,153]. An armed measles virus (MV), MV-purine nucleoside phosphorylase (PNP)-anti-prostate stem cell antigen, that expresses the prodrug convertase PNP, which then activates the prodrug fludarabine, was shown to enhance the oncolytic efficacy of the virus in gemcitabine-resistant PDAC cells[154]. Reovirus is another promising TOV for pancreatic cancer therapy, particularly because its selectivity depends on the cellular activity of Ras, which is constitutively active in pancreatic cancer[155]. Reolysin® (Oncolytics Biotech Inc., Calgary, AB, Canada) a reovirus administered intraportally resulted in decreased metastatic tumour volumes in the liver of immunocompetent animal models[156,157]. A phase II study of Reolysin® in combination with gemcitabine in patients with advanced PDAC has been completed (clinicaltrials.gov: NCT00998322). A two-armed randomized phase II study of carboplatin and paclitaxel plus Reolysin® vs carboplatin and paclitaxel alone in recurrent or metastatic pancreatic cancer is currently being conducted by the United States National Cancer Institute (NCI-8601/OSU-10045).

RATIONALIZING VIRO-IMMUNE-CHECKPOINT COMBINATION THERAPY

A understanding how antitumour immunity is regulated allows us to recognize barriers against effective immunotherapy delivery and furthermore, allow for the development of rational combination therapies aiming targeting these mechanisms[108,158,159]. This approach allows therapies to work synergistically and also has the potential to benefit a broader patient population[108]. Tumours have evolved to avoid immune recognition and/or destruction at every stage in the antitumour response, therefore targeting more than one immune resistance mechanism will enhance antitumour immunity.

An important immunological barrier in cancer immunotherapy is the tolerance towards self-antigens. Tumours downregulate their antigenicity through various mechanisms in response to selective pressure by the immune system, a process called “immunoediting”[37]. Therefore, in order to raise an effective antitumour response, the immunological tolerance must be broken to allow tumour antigen-specific cytotoxic T cell responses[158]. This can be achieved by increasing the tumour load and/or enhance antigen presentation[108]. TOVs can initiate selective infection and replication in the tumour bed, exposing TAA, disrupting the immunotolerance employed by the tumour while re-engaging adaptive immune effector responses[39]. Combining an agent that can cause disruption to the tumour bed i.e., an oncolytic virus, with a novel antitumour immunomodulating agent such as anti-PD-1/PD-L1 antibodies can maximize immune-stimulating and immune-recruiting inflammatory responses[39]. Specifically, TOV lysis induces the release of tumour antigens into the microenvironment, which are then cross-presented to T cells in the draining lymph nodes by APCs[159] (Figure (Figure1).1). This allows T cell infiltration to the tumour bed. Next, T cell dysfunction must be reversed[108,158]. Immune checkpoint inhibitors alleviate immunosuppression, allowing the elimination of the tumour by the adaptive immune system[70]. TOVs in combination with immune checkpoint inhibitors can therefore potentiate and activate the immune system synergistically, ultimately creating a pro-inflammatory environment. Pre-existing TILs are strong prognostic predictors in cancer[106]. This is extremely relevant for tumours with poor immune-cell infiltration, such as pancreatic cancer, which would depend on TOV-infection mediated lymphocyte infiltration for an enhanced response to immune checkpoint blockade. Zamarin et al[160] demonstrated constrained replication of an intratumoural-injected Newcastle disease virus in a B16 melanoma model. Lymphocytic infiltrates, however, were detected in both TOV-injected and non-TOV-injected tumours, and rendered the tumours sensitive to CTLA-4 blockade. The antitumour activity was dependent on CD8+ T cells, NK cells and type I and II IFNs[160]. Ipilimumab with or without talimogene laherparapvec, is in early clinical testing in patients with unresected melanoma (clinicaltrials.org: NCT01740297). Interestingly, an MV engineered to express CTLA-4 or PD-L1 antibodies delayed tumour progression and prolonged median OS in B16 melanoma models[161]. Finally, TOVs have demonstrated a tolerable toxicity profile, whereby flu-like symptoms are the most common adverse events, and in fact, most of the side effects seen so far in the combination regiment are related to the immune checkpoint blockade inhibitor[162]. Dias et al[163] suggested an oncolytic adenovirus expressing CTLA-4 locally might reduce systemic side effects normally induced with anti-CTLA-4 antibodies alone.

OVERCOMING OBSTACLES IN ONCOLYTIC VIRUS DELIVERY

The main issue with virotherapy is systemic delivery for targeting metastatic cancer cells. Intravenous administration is more practical, especially for treatment of a tumour in a hard-to-reach location such as the pancreas, and with the majority of patients presenting with advanced or metastatic disease. However, nonimmune human serum and existing anti-TOV antibodies may neutralize the TOV in the bloodstream. Furthermore, non-specific hepatic and splenic sequestration of the TOV and ineffective extravasation into the tumours are important issues[164]. Currently, studies in pre-clinical models aim to overcome these obstacles. These include chemical modification of viral coat proteins by conjugation of biocompatible polymers e.g. polyethylene glycosylation[165,166], using mesenchymal stem cell carrier systems to deliver the TOV to the tumour bed[167169], and increasing vessel permeabilization[170,171].

In PDAC, however, the biggest hurdle may not be the host immune system, but the TME. The TME has played a significant role in not only acting as a physical barrier to deliver treatments, but it also in the development of resistance to conventional drugs. The TME remains a problem for successful TOV treatment. The TOV must be able to spread in the hypoxic and densely stromal-rich TME in order to attract enough attention to induce antitumour immunity[172]. Breaching the stromal barrier in PDAC is needed for TOVs to access the cancer cells[173]. Paradoxically, a recent study by Ilkow et al[174] demonstrated that the cross-talk between CAFs and cancer cells actually lead to increased permissibility of TOV-based therapeutics. Tumour cells producing TGF-α reprogrammed CAFs, dampening levels of anti-viral transcripts. This allowed the cells to be more sensitive to VV, vesicular stomatitis virus and maraba MG1 TOVs. The reprogrammed CAFs produced fibroblast growth factor (FGF)-2 which suppressed levels of retinoic acid-inducible gene I and increased the susceptibility of the tumour cells to virus[175]. This study also demonstrated that an FGF2-expressing TOV has improved therapeutic efficacy by sensitizing the tumour cells to virotherapy and is particularly relevant to pancreatic cancers, where CAFs are a major component of the tumour stroma[175]. It is important to note that not only the patient’s existing immune system may impede successful TOV therapy, but that the enhanced antitumour response by combinatory approaches (e.g., the inclusion of immune-checkpoint inhibitors) may also impede successful TOV infection, spread and engagement of the immune system. This stresses the importance of determining strategic combinations, dosing and timing schedules in future studies.

CONCLUSION

The poor prognosis of pancreatic cancer due in part to the limited efficacy of conventional and targeted therapies, appeals for a novel strategy to treat this disease. It has become very clear that the immune system has the greatest potential to selectively destroy tumours, and when it is strategically induced, a durable benefit can be achieved. Past and present studies have defined means for tumour escape from immune surveillance and have developed immunotherapies to counteract these mechanisms. However, with the various escape strategies leading to low immunogenicity and highly immunosuppressive tumour beds, a successful control of tumour growth by immunotherapy does not come without various obstacles and challenges. Future steps include the development of immune-monitoring strategies for the identification of biomarkers, to establishment guidelines to assess clinical end points of immunotherapy and finally to evaluate combination therapeutic strategies to maximize clinical benefit[176]. The ability of TOVs to stimulate inflammation, deliver genes and immunomodulatory agents as well as reduce tumour burden by direct cell lysis, allows them to be important therapeutic vectors for a highly immunosuppressed tumour such as PDAC. Immune checkpoint blockade agents can then reverse T cell anergy and further boost OV-induced responses. As this combinatory approach may exist as a double-edged sword, it is crucial to determine appropriate timing, dosing and sequence schedules of each agent.

SOURCE & REFERENCES

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Vectorisation Of Immune Checkpoint Inhibitor Antibodies

Reporter: David Orchard-Webb, PhD

 

The FDA approved ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) combination in October 2015 for the treatment of advanced melanoma. The antibodies have recently been approved in the UK for the same indication. Over half of patients respond to the combination [1]. These drugs belong to the class of monoclonal antibodies known as immune checkpoint inhibitors. The binding of anti-CTLA-4 antibodies to activated T cells prevents the surface CTLA-4 receptor from binding CD80 and/or CD86 on antigen presenting cells (APCs). Normally CTLA-4 binding to APCs deactivates the T-cell. Antibodies against programmed cell death protein 1 (PD-1) work by a similar mechanism to CTLA-4. These drugs are delivered by repeated intravenous injections (iv) [2].

 

Oncolytic viruses are an emerging class of immunotherapeutics that actively stimulate the immune system by releasing tumour antigens via lysis and by virtue of anti-viral immunity. The first FDA approved oncolytic virus (Imlygic), developed by Amgen/ BioVex, was given the green light in October 2015 for advanced melanoma patients delivered via direct tumour injection. The mechanism of action of oncolytic viruses is highly complementary with checkpoint inhibitor antibodies and multiple trials combining these two classes of agent are under way.

 

At the recent American Association for Cancer Research (AACR) annual meeting in New Orleans, Louisiana, the oldest biotechnology company in France – Transgene, presented preclinical data concerning oncolytic vaccinia viruses that express whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv) against mouse PD-1 [3]. These combinations proved superior over virus alone in mouse xenografts of melanoma and fibrosarcoma cell lines. Transgene claim that “these results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs” (Figure 1) [3].

 

 698848905_d8bf7f415f_z
Figure 1: The convergence of therapeutics based on oncolytic viruses and monoclonal antibodies against immune checkpoint inhibotor proteins. Image Source: Eric Molina. No changes were made. Creative Commons Attribution 2.0 Generic (CC BY 2.0).

 

The combination of immune checkpoint inhibitors and oncolytic virus as a single molecular entity clearly has advantages in terms of manufacturing cost effectiveness. In addition viral vectors have the capacity for perfect specificity to tumours which has potential safety advantages.

 

REFERENCES

 

  1. http://www.bbc.com/news/health-365496740
  2. http://www.cancer.org/cancer/skincancer-melanoma/detailedguide/melanoma-skin-cancer-treating-immunotherapy
  3. http://www.transgene.fr/wp-content/uploads/2016/04/1604-Poster-AACR-format-122-244-v2.pdf

 

Other Related Articles Published In This Open Access Online Journal Include The Following:

 

https://pharmaceuticalintelligence.com/2016/04/12/oncolytic-virus-immunotherapy/

https://pharmaceuticalintelligence.com/2015/09/23/oncolytic-viruses-a-new-class-of-immunotherapy-drugs-against-cancer/

https://pharmaceuticalintelligence.com/2016/06/16/first-drug-in-checkpoint-inhibitor-class-of-cancer-immunotherapies-has-demonstrated-superiority-over-standard-of-care-in-the-treatment-of-first-line-lung-cancer-patients-mercks-keytryda/

https://pharmaceuticalintelligence.com/2016/05/07/durable-responses-with-checkpoint-inhibitor/

https://pharmaceuticalintelligence.com/2016/05/02/cancer-research-institute-nyc-623-6242016-will-combination-of-adoptive-t-cell-therapy-and-anti-checkpoint-inhibitor-therapies-be-the-next-wave/

https://pharmaceuticalintelligence.com/2016/02/14/checkpoint-inhibitors-for-gastrointestinal-cancers/

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Pancreatic Cancer Modeling using Retrograde Viral Vector Delivery and IN-Vivo CRISPR/Cas9-mediated Somatic Genome Editing, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Pancreatic Cancer Modeling using Retrograde Viral Vector Delivery and IN-Vivo CRISPR/Cas9-mediated Somatic Genome Editing

Curators: Larry H. Benstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Genes Dev. 2015 Jul 15; 29(14): 1576–1585.
PMCID: PMC4526740

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

1Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA;
2Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA;
3Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA;
4Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, USA;
5Department of Surgery, Rutgers Robert Wood Johnson University Medical School, New Brunswick, New Jersey 08903, USA;
6Department of Pharmacology, Rutgers Robert Wood Johnson University Medical School, New Brunswick, New Jersey 08903, USA;
7Cancer Biology Program, Stanford University School of Medicine, Stanford, California 94305, USA;
8Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California 94305, USA;
9Transgenic, Knockout, and Tumor Model Center, Stanford University School of Medicine, Stanford, California 94305, USA;
10Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA;
11Department of Pathology, University of California at San Francisco, San Francisco, California 94143, USA;
12Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA
13These authors contributed equally to this work.
Corresponding author: ude.drofnats@wolsniwm

Little is known about how these alterations contribute to the development of metastatic and therapy-refractory PDAC. Given the inability to test gene function in human cancers in vivo, genetically engineered mouse models represent tractable and biologically relevant systems with which to interrogate the molecular determinants of each stage of pancreatic cancer development.

Identification of the mutations that drive the development of human pancreatic cancer combined with the ability to alter gene function in mice has enabled the development of genetically engineered murine PDAC models. Transgenic expression of Cre-recombinase in pancreatic cells of loxP-Stop-loxP (LSL) KrasG12Dknock-in mice (KrasLSL-G12D/+) results in deletion of the transcriptional/translational Stop element, expression of oncogenic KrasG12D, and development of lesions that closely resemble early stage human pancreatic intraepithelial neoplasms (PanINs) (Hingorani et al. 2003). Concomitant expression of a point mutant p53 allele, deletion of p53, deletion of Cdkn2a, and/or deletion of Smad4 allow(s) for the development of invasive and metastatic PDAC

(Aguirre et al. 2003Hingorani et al. 2005Bardeesy et al. 2006a,bGidekel Friedlander et al. 2009Whittle et al. 2015).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526740/

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Keywords: CRISPR, genome editing, mouse model, pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal tumor type that is projected to become the second leading cancer killer in the United States by 2030 (Rahib et al. 2014). PDAC patients have a 5-year survival rate of ∼5%, underscoring the need for novel approaches to accelerate the molecular characterization of this disease. Although high-prevalence mutations have been identified in pancreatic cancer, these tumors also incur low-frequency mutations and genomic alterations, interact with their extensive and complex stromal environment, and undergo poorly characterized changes in their gene expression programs (Biankin et al. 2012; Waddell et al. 2015). Despite the potential importance of these molecular and cellular changes, very little is known about how these alterations contribute to the development of metastatic and therapy-refractory PDAC. Given the inability to test gene function in human cancers in vivo, genetically engineered mouse models represent tractable and biologically relevant systems with which to interrogate the molecular determinants of each stage of pancreatic cancer development.

Identification of the mutations that drive the development of human pancreatic cancer combined with the ability to alter gene function in mice has enabled the development of genetically engineered murine PDAC models. Transgenic expression of Cre-recombinase in pancreatic cells of loxP-Stop-loxP (LSL)KrasG12D knock-in mice (KrasLSL-G12D/+) results in deletion of the transcriptional/translational Stop element, expression of oncogenic KrasG12D, and development of lesions that closely resemble early stage human pancreatic intraepithelial neoplasms (PanINs) (Hingorani et al. 2003). Concomitant expression of a point mutant p53 allele, deletion of p53, deletion of Cdkn2a, and/or deletion of Smad4 allow(s) for the development of invasive and metastatic PDAC (Aguirre et al. 2003; Hingorani et al. 2005; Bardeesy et al. 2006a,b;Gidekel Friedlander et al. 2009; Whittle et al. 2015).

These in vivo models have been instrumental in our understanding of the genetic determinants of cancer progression as well as the functional interactions of neoplastic cells with the immune system and stromal environment. However, using conventional genetically engineered autochthonous mouse models of PDAC to interrogate gene function is complicated by inherent practical and biological limitations of these systems. Existing mouse models typically fail to model the adult onset of pancreatic cancer and induce genomic alteration in nearly every cell in the pancreas. In the decade since the first genetically engineered PDAC models were developed, few technical advances have been made, and generating the mice required to investigate a gene of interest in the established PDAC models remains a time-consuming and costly endeavor (Aguirre et al. 2003; Hingorani et al. 2003,2005; Saborowski et al. 2014).

Systems that enable in vivo functional interrogation of genes in pancreatic cancer without the financial and temporal cost of generating new mouse alleles and incorporating them into increasingly complex mouse models could have an extremely broad impact on pancreatic cancer research. To functionally investigate the molecular changes that drive each step of pancreatic cancer development, it would be desirable to have a system in which the timing of tumor initiation and the number of lesions that form in the adult pancreas can be controlled, the number of germline-encoded alleles is minimized, and genes of interest can be eliminated without having to generate a conditional allele and breed it into a complex genetically engineered mouse model.

Here we describe methods for the direct delivery of viral vectors to the pancreas and transgenic mouse lines to allow CRISPR/Cas9-mediated genomic alterations in pancreatic cells in vivo. These systems allow titratable initiation of pancreatic tumors in adult mice and functional interrogation of candidate genes in pancreatic cancer in vivo.

Pancreatic retrograde ductal injection of Adeno-Cre or Lenti-Cre induces widespread recombination and initiates the development of PanINs and ductal adenocarcinoma. (A) Diagram of the pancreatic retrograde ductal injection procedure. (B) Images of retrograde
Chronic pancreatitis is associated with an increased incidence of PDAC in humans, and experimental evidence from mouse models suggests that adult cells may be refractory to transformation in the absence of pancreatic inflammation (Guerra et al. 2007; Raimondi et al. 2010). Therefore, we assessed whether retrograde ductal viral infection induces pancreatitis. Both Ad-Cre and Lenti-Cre infection induced acute pancreatitis with morphological evidence of ADM and focal replacement of acinar cells by infiltrating mononuclear cells (Supplemental Figs. 1C,D, 4C). The induction of pancreatitis is consistent with the efficient tumor initiation observed in adult animals following retrograde ductal injection of viral-Cre.
Diverse expansion potential and metastatic ability of viral-Cre-initiated PDAC. (A) DTCs can be detected in the peritoneal cavity of Ad-Cre-infected KPTmice. Viable (DAPInegative), lineage-negative (CD31, CD45, Ter119, F4/80)negative cells are shown.
CRISPR/Cas9 enables in vivo genetic alteration in pancreatic cancer. (A) Schematic of the Cre-regulatedCas9 allele. (CAGGS) Cytomegalovirus immediate–early enhancer/chicken β-actin promoter; (hSpCas9) human codon-optimized Streptococcus

Somatic genome engineering enables rapid generation of genetically defined pancreatic cancer mouse models

To assess the impact of Lkb1 deletion on pancreatic cancer, we performed retrograde ductal injections of KT;H11LSL-Cas9/+, control KT, and KT;Lkb1flox/flox mice with Lenti-sgLkb1/Cre as well as KT;H11LSL-Cas9/+ mice with lentiviral vectors containing negative control sgRNAs. Control KT mice infected with Lenti-sgLkb1/Cre as well as KT;H11LSL-Cas9/+ mice infected with a Lenti-sgRNA/Cre vector containing either a nontargeting sgRNA (sgNT) or an sgRNA targeting an inert region of the genome (sgNeo) formed only rare Tomatopositive lesions (Fig. 3F–H). Both Lenti-sgLkb1/Cre-infected KT;H11LSL-Cas9/+ and KT;Lkb1flox/flox mice had extensive tumor growth as early as 2 mo after tumor initiation (Fig. 3I,J, respectively).

All three groups of negative control mice developed only rare PanIN lesions within almost completely normal pancreata (Fig. 4A–C; Supplemental Fig. 9A; data not shown). The tumors that formed in Lenti-sgLkb1/Cre-infectedKT;H11LSL-Cas9/+ mice were cystic lesions comprised of Tomatopositive, CK19positive tall cuboidal to columnar epithelial cells with otherwise bland cytological features (Fig. 4D; Supplemental Fig. 9B). Some smaller lesions also contained high levels of mucin (Supplemental Fig. 9B). Importantly, these features were histologically indistinguishable from those found in KT;Lkb1flox/flox mice infected with Lenti-sgLkb1/Cre (Fig. 4E; Supplemental Fig. 9C). Lenti-sgLkb1/Cre-infected KT;H11LSL-Cas9/+ mice also had a substantially higher tumor burden when compared with all three groups of negative control mice (Fig. 4F).

Cas9-mediated targeting of Lkb1 in the pancreas promotes tumor growth. (A) Control Lenti-sgLkb1/Cre-infected KT mice (n= 5) have small clusters of Tomatopositive cells and very rare ADMs and PanINs. (B,C) KT;H11LSL-Cas9/+ mice infected with Lenti-sgNT/Cre
Diverse indels at the targetedLkb1 locus were specifically detected in the neoplastic cells from these tumors (Fig. 5A,B; Supplemental Fig. 9F). The indels were all frameshift mutations or large exon deletions that included the splice acceptor or donor regions of Lkb1exon 6, further supporting the strong selective advantage of Lkb1 inactivation (Fig. 5B,C). Consistent with the presence of CRISPR/Cas9-induced frameshift mutations and large deletions, Lkb1 protein was absent from most neoplastic cells in the Lenti-sgLkb1/Cre-induced tumors in KT;H11LSL-Cas9/+ mice (Fig. 5D,E; Supplemental Fig. 9G).
Cas9-mediated targeting leads to the formation of pancreatic tumors harboring deleterious Lkb1mutations and lacking Lkb1 protein. (A) Genomic cleavage detection assay detected indels in the targeted Lkb1 locus in Tomatopositive cells isolated from Lenti-sgLkb1/Cre-infected

The development of systems that accelerate our ability to investigate pancreatic carcinogenesis at the molecular level will be a critical step toward overcoming the dismal rate of successful treatment and low survival rate of patients with this recalcitrant cancer. The ability to induce pancreatic cancer using viral vectors will be instrumental in understanding the mechanisms that sustain tumor growth, lead to metastatic spread, and drive drug resistance. Our CRISPR/Cas9-based model should allow any gene of interest to be inactivated in pancreatic cancer in vivo without the need to generate any new mouse alleles. The method developed in this study could have a profound impact on both basic and translational pancreatic cancer research. Collectively, these methods will enable a more rapid and complete understanding of the molecular regulators of all aspects of pancreatic tumorigenesis and complement the strength of existing genetically engineered models, human patient-derived xenograft models, and studies on human and murine cell lines.

Retrograde ductal injection of Adeno-Cre and Lenti-Cre vectors allows titratable pancreatic tumor initiation in the adult pancreas. This removes the requirement for the transgenic Cre(ER) alleles used in conventional genetically engineered pancreatic cancer models and enables sparse rather than widespread expression of oncogenic Kras and deletion of tumor suppressor genes, more closely recapitulating the initiating events in human PDAC. The inclusion of a fluorescent Cre reporter in transgenic Cre(ER)-induced PDAC models leads to the fluorescent labeling of not only the neoplastic cells but also most of the nontransformed pancreatic epithelial cells, making unequivocal distinction of neoplastic from normal cells difficult.

The ability to induce PDAC in Cre-lox models without having to include a transgenic Cre line will make the molecular investigation of pancreatic cancer more rapid and less expensive (Supplemental Table 2). Additionally, with the ease of generating pancreatic tumors now approaching that of lung tumors (via intranasal or intratracheal injection), comparing the impact of the same genetic alterations on each cancer type should become standard practice and will uncover the commonalities and differences between these lethal adenocarcinomas.

PDAC models often generate tumor masses of unknown clonal origin. By incorporating a multicolor fluorescent reporter, we were able to mark individual clonal lesions and identify the relationship between primary tumors and metastases. Unexpectedly, some late time point mice had only one or two large cancers, underscoring the dramatic heterogeneity in expansion potential of pancreatic lesions initiated with identical engineered genetic events.

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