LIVE – Afternoon Session added – Gene Therapy Development & Cell Therapy Bioprocessing – Overcoming Scientific and Development Challenges @Biotech Week Boston, October 5, 2016 Boston Convention and Exhibition Center | Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Lymphoeid Leukemia – Persisting CTL019 Cells remains – Pediatrics ALL – Relapsed and Refractory B-celll
Bedside back to Bench lessons: Days post infusion
28 days vs 51 cells
quality of cells: related to Clinical Response
93% CR rate for r/r ALL after CTL019
Novartis and UPenn Cell Center Building

LIMPHOMA

Diffuse Large B Cell Lymphoma
Engineered T Cells and CHeckpoint ANtibody Therapies: Potential Synergies
Days post-tumor injection: Non-responding patients: infusion of PD1 Inhibitor
After treatment: Durable responses necrosis no tumor left
Single Arm, Open-Label

MYELOMA

CART19
CART BCMA – cells for Multiple Myeloma – Bone Marrow: Pre-treatment 70% myeloma cells
Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in Leukemia
Synthetic Biology – Penn Platform Technology: Academic CAR Clinical Trials

Academic-Industry COllaboration with Novartis: Human Cell Therapy: 1996-2011
FromGene therapy to >>> Immunotherapy, to >>>>>> Gene Editing
TMUNITY – new mission with Leukmia and Lymphoma Foundation

9 am
30 mins

Novartis Pharma: Stabilizing Lentiviral (LV) vector formulation for CART Application

LV development of Vector Production – for Ex-vivo Therapy
mamamalian cells + plasmid
high titer, sequences liability &FTO
PROCEESS

aggregation
Trunsduction
purification –
Phys-chem profile,
stable – – Stability in buffers for recovery of infectious particles – Purification
low cost

LV: Optimize transient production:

EARLY PROCESS-CHECK,
FORMULATION SCEEEN – Preformulation of LVs: Drug DIscovery
ANALYTICS DEVELOPMENT
TECHNICAL-GRADE VECTOR SUPPLY

High throughput screen
stability promoting buffers
stability in process intermediates
Attributes: Preformulation – success criteria – stable in transduction media/conditions’minimal loss of infectious particles
LV with Transgene 1Preformulation: Effect of buffer and salts – 96 different conditions (buffer +salts)
LV Aggregation & Loss of Infectivity – Accelerated Stree Studies
Size Interpretations – Dynamic Light scattering (DLS): Buffer A, E, H
Robustness: Preformulation buffers: Small, Medium, Large Rh:

pH 6.0 – 8.5 – X axis and
Salt 0 – 200nM – V Axis

9. Freeze-Thaw Studies – Accelerated Stress Studies: w/o Carbohydrate

number of Freeze-thaw cycles X axis vs
% Control to standard – V Axis

10, Infectious Titer: Primary T Cells

Case study: LV with Transgene #2 –

Platform formulation for LV is possible
Transduction in primary T cells showed high titer and minimal toxicity
stability studies of in-process intermediates and LV-DS provided additional evidence
4 promising conditions identified for preformulation of LV vectors from HTS screens

9:30 am
30 mins

Mesenchymal Stem Cells (MSCs) on Steroids

Oren Levy, HMS, Brigham and Women’s Hospital

I. Mesenchymal Stem Cells – readily accessible potent immunomodulatory secretome already used in >600 clinical trials

Uncontrolled cargo’Inefficient targeting to disease sites – A microparticle-based cellular
Particle-in-a cell approach in Prostate Cancer: PSA – Thapsigargin – PLGA – Poly Lactic co Glycol Acid – encapsulated agent’The cellular carriers MSCs
Glioblastoma, Prostate
Drug-loaded MSCs kill prostate cancer cells
Drug release from G114 MP-loaded cells
MDA-MB; LNCaP – Coculture with pretreated MSCs
Drug-loaded MSCs inhibit tumor growth: days post-inoculation vs Probabilirt of Tumor-free SUrvival vs. Tumor Volume
Particle Payloading: Boosting Potency of Administrated Cell Populations – for Drug delivery
Controlled inhibition of the MSC proinflammatory
Donor variability leads to Functional Variability – variability between 7 donors
Budesonide microparticles boosted MSC
Improved imaging of transplanted MSCs
Retention of PLGA

MSC: Therapy
Betacells – Islet transplantation Macrophages – immunotherpy

II. Targeting cells to disease site – Surface of MSC – lacks standard “homing”

tetharing – rolling – firm adhesion – transmigration- endothelial cells, basement membrane: Healthy vs Inflammed
lucocytes
Controlling MSC fate via small molecule pretreatment
RNA transfection
Bone marrow: PSGL-1/SLex MSCs exhibit enhanced homing to healthy and irradiated bone marrow

A multi-step screening platform for identification of small molecules that improve MSC homing: Surface expression, FIrm adhesion, MSC homing
A medium throughput screen identified Ro-31-8425
Upregulated of CD11a, in response to pretreatment increases MSC firm adhesion
Pretreated MSCs exhibit superior therapeutic impact post systemic adm – suppression of local skin inflammation
Ro-31-8425 Treated MScs va Vehicle treated MScs
boosted clinical impacet in a MS Model
Bradly applicable cell-based therapeutic platform
mRNA transfection
drug microparticles

Gene Therapy Development & Production – Emerging Research: Delivery Strategies and Genome Engineering Technologies

10:30 am
30 mins

Genetic Engineering Red Blood Cells for Therapeutic Function

Robert J. Deans, Ph.D.,Chief Scientific Officer, Rubius Therapeutics

Rubius Therapeutics – Whitehead Institute/DARPA legacy
Flagship Venture founded
RCTs – development of ALternative Therapeutics
Platfoms: Transfusion /donor selection, risk of oncogenicity – uncontrolled division and secretion
Allogenic RCTs grown in Culture from Hemopoeitic Stem Cells (HSCs) In vitro and engineered to Purpose along the way
Expansion: CD34 + HSCs: Mobilized, cord blood
Cytoplasm: Native cytoplasmic, Tethered cytoplasmic , tethered surface native surface
Visualizing Red Cell Therapeutics – RCT during differentiation
Reliablly use cell surface markers to track the cells as hey differentiate
Flow Cytometry
Automated platform foe creating Master cell BioBank

Apheresis Transduction
clinical scale
Rapid prototyping capability for candidate Selection, Vector generation, Human CD34+
Prototyping for Pipeline
Exogenous protein is retained: Cultured RBCs circulating in vivo like Human RBCs
Experimental Setup: RCT circulation in similar to HuRBCs
RCTs can possess antibody drugs on their surface for clearance and therapeutic action
Target: scFv antiVirusAg – capture circulating antigen in vitro and in vivo – Virus Antigen – label antibody
Phenylketonuria (PKU) – CNS morbidity from Serum PHE – it is easily measured, gold standard diagnostic and clinical biomarker

Novel protein, cell & gene therapy treatment
Existing PAL-RBCs have clinical grade potency at currently achievable manufacturing scale: Serum PHE vs Units PAL-RBS IRON
dose dependent PHE expression

11 am
30 mins

Novel Approaches to Gene Editing and Gene Delivery

In Vivo gene therapy

Andrew Scharenberg, M.D.,Professor of Pediatrics, Adjunct Immunology / Co-director, Program in Cell and Gene Therapy, University of Washington / Seattle Children’s Research Institute

Viral vector tranfer
cell mediated immunity
Humoral immunity
Evasion/tolerance – neonatal adm of vector
Challenges: Unmet need Goal – Primary cell: DNA delivery to nucleus

Cells have mupliplr mechanisms to prevent foreign DNA from entering the nucleus
Viruses evade these mechanisms

Non-enveloped Virions – classic gene therapy vector AAV

Viral capsid serves multiple roles:
AAV has no mechanism to neutralize nuclear silencing mechanism

Enveloped Virions: Retro and Lenti-Virus

Re-dosable in vivo gene transfer

LNP’s mimic the endosomal uptake and escape functions of AAV Capsid

mRNA delivery to build a capsid “life boat” – reverse transcribe the gemone and send it in
RNA packing, minus strand systhesis, plus strand synthesis, budding, ER vesicular transport ENTRY repair

HBV-based Non-Viral RNA Gene Transfer System: Electric Poration (EP) or LNP

Pregenomic (pgRNA) Vector RNA architecture

Viral RNA – Pol transfers to 3′ DR1 for _ strand sysnthesis
Vector RNA – payload cap… pA
pgRNA with GFP cassette flanked by SB Tn sites – Inhibition and Translation areas
BFP vs GFP
Validation of pgRNA reverse transcription: HepG2 cells transfected with indicated RNAs plus POL/CORE/X mRNAs, then cultured for 2 weeks
Plasma DNA vs Tn-pgRNA, Tn-pgRNA + 5B vs COntrol
pgRNA/SB achieves stable luciferase expression
No-Viral RNA Gene Transfer System: Status and Future
Encouraging evidence of functional synthetic recycling RT vector: SERT – Synthetic encoded reverse transcription)
Transposon IR/DR – flaked pgRNA payload

11:30 am
30 mins

Development of Stem Cell Derived Extracellular Vesicles into a Non-Living Regenerative Therapeutic Drug Candidate

Keil Ph.D., Capricor, Inc.

Gene Therapy Development & Production – Manufacturing Strategies and Solutions for Gene Therapies

Cardioshere-Derived Cells – DOnors of cells – Primary cardiac tissue’Key functions – paracrine:
CDC Manufacturing
DOnor Heart from organ procurement —
Cell Therapy: Autologous vs Allogenics
Cell engraftment
Autologous vs Allogenics Clinical Trials:
Caduceus vs Dynamic
Scar tissue

What are exosomes? as a therapeutics

rich in RNA
CDC EVs: By the numbers
Exosomal Markers vs CDC Marker – CD 105

Acute MI Model

Viable mass increased, Myocyte size increased, EF increased,
Identified 146a as a major player in cardio-protection
CAP2003 manufacturing Process
CDC-EV Manufsacturing Overview vs CDC Product
Conditioned Medium collection
day 1 vs 15 days
Drug Product Formulation
Choosing a Formulation Buffer: pH
30 days stability
Process Robustness: Particle Size and pH: 4 diffrent donors (variability is related to culture and to patients) – 10 formulation runs: EV size vs diafiltrate pH (average =7)
Process Robustness: Concentration and Cargo
Human mir panel v3.0
nCounter miRNA analysis
Particle foes not increase linearly with protein
Process Robustness: miRNA 146 & 210
Process Impurities & Residual: Residual Fibronectin in CM vs BSA wash-out
CDC-EVs have anti-apoptotic, anti fibrotic, immunomodulatory and pro-regenerative activities similar to CDCs
New indications
Pathway to iND: Pre-Clinical Efficacy
Average Total clinical Score

conjunctival injection
corneal opacification
corneal area affected
corneal neovascularization
aqueous flare
Ongoing Pre-Clinical: biodistribution,
Upscaling Exosomes: Bioreactor CDCs
Current Process vs Target Process

Next Gen Storage: Lyophilization (Astraunatu EV

State of Capricor’s Art

Cell Therapy Bioprocessing – Innovations in Cell Processing – Part 2

2:10 pm
5 mins

Chairperson’s Remarks

2:15 pm
30 mins

CAR T production in G-Rex: Less is More

Juan Vera, M.D., Associate professor Center for Cell and Gene Therapy, Baylor College of Medicine., Baylor College of Medicine

Adoptive T-cells Transfer
Donor Lymphocytes blood draw — antigen specificities == cell expansion == infusion back to aptient – antigen specific T-cells to iincrease immunogenecity
Low seeding density results in greater fold expansion
Low cell density + Greater cell expansion: Optimal time for cell harvest vs maximum cell density
Volume of Media vs Cells density
Upfront media addition resulted in shortest culture period
Conventional cultureware – risk for contamination
G-Rex – Plate vs G-Rex – G-REX 5; G-Rex 100; G-REX -500 (flacks)
superior cellnumber not due to increase cell number – improved cell output

phase 1 – what is optimal seeding density – cells expansion in 12 days
phase 2 – optimal volume in media – I=1L
phase 3 –

are observations reproducible?
Cell harvest with The GatheRex – in 5 minutes you collect one billion cells (density
CAR-T cells: Preparation of genetically-modified T cells
PSCA – solid Tumor
NT T cells – suppress tumor cells by immunoresponse to antigen specific to CAR-T
CAR-T cell expansion in G-REX – day in culture vs Glucose concentration – inverse correlation
Cell performance of production of CAR=t in G-REX
Phynotype of CAR-T cells: 24 wells vs G-REX
CCR7 vs CD62L: Conventional vs G-REX – superior Phynotype of CAR-T Cells expanded inside G-REX
stimulation beads = not used i=with G-REX
media vs cell ratio – serology
Persistance of CAR-T cells 6 month post transfer – rechallenge the cells ramping up withstand multiple re-challenge
Post transfection – transduction 3 days after