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Don't get lost in translation: How smart

design and technology are enabling bench-

to-bedside in translational research

October 14, 2015

Webinar Series

Sponsored by

Bruce Levine, Ph.D.

University of Pennsylvania

Philadelphia, PA

Laurence Cooper, M.D., Ph.D.

MD Anderson Cancer Center, Houston, TX

ZIOPHARM Oncology, Boston MA

Participating experts

Chris Ramsborg, Ph.D.

Juno Therapeutics

Seattle, WA

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October 14, 2015

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Sponsored by

Bench to Bedside Translation of

Redirected Immunity to Treat

Relapsed/Refractory Cancers

Bruce Levine, Ph.D.

Barbara and Edward Netter Professor in Cancer Gene Therapy

Department of Pathology and Laboratory Medicine


• Declaration of financial interest due to intellectual

property and patents in the field of cell and gene

therapy.

• Funding support for trials: ACGT, LLS, NCI,

Lustgarten Foundation and Novartis

• Conflict of interest is managed in accordance with

University of Pennsylvania policy and oversight

Conflict of Interest Statement

Cancer Immunotherapy

Problem 1: The enemy is ourselves

Problem 2: Cancer-specific immune

cells are very rare, if present at all

Problem 3: Cancer induced immune

suppression and immune evasion

Overcoming the Scarcity of Tumor Specific Immunity and Tumor Suppression: Creation of Re-directed T cells

TCR heterodimer “CAR” – a molecular chimera - off the shelf

- MHC independent

Extracellular

Intracellular

Chimeric Protein

Tumor binding domain

Evolution of Chimeric Antigen Receptor Design

Using Synthetic Biology to Overcome Tolerance

Extracellular

Intracellular

ζ

First Generation

CD4 / CD8z CARs

VH

VLV

L

VHV

H

VLV

L

VH

ζ ζ

First Generation

scFv CARs

VH

VLV

L

VHV

H

VLV

L

VH

Second Generation

scFv BBz CARs

ζ ζ

4-1BB

Finney, 2003

Imai, 2004

Milone, 2009

Carpenito, 2009

CD28

VH

VLV

L

VHV

H

VLV

L

VH

Second Generation

scFv CD28z CARs

ζζ

Roberts, 1995

Finney, 1998

Maher, 2002

CD27

ICOS

VH

VLV

L

VHV

H

VLV

L

VH

Second Generation

scFv CD27z CARs

scFv ICOSz CARs

ζζ

Song, 2012

Guedan, 2014

Duong, 2013

Kuwana, 1987

Eshhar, 1993

Irving & Weiss, 1991

Letourneur, 1991

Romeo, 1991

J Immunol 1997; 159: 5921

Science 1997; 276: 273

Immunol. Rev. 1997; 160: 43

Anti-CD3 Anti-CD28

Artificial APC: Bead

Signal 1

Growth

CD28 CTLA4 TcR/CD4

+

Bead Based in vitro T Cell Culture System

Gene Delivery For Permanent CAR Expression

Lentiviral (HIV-1 based) vectors- advantages over other vector types

including murine leukemia viruses:

•Efficiently transduce primary cells

•Long term stable gene expression without gene silencing that

occurs at high levels in oncoretroviral vectors

•Favorable safety profile

Levine, BL, PNAS 2006;103(46):17372

McGarrity, GJ, J Gene Med 2013; 15: 78

Gene Delivery For Temporary CAR Expression

•Currently much faster and cheaper than manufacture of viral

vectors

• In some settings, repeated infusion of temporary CAR’s may

be sufficient to treat disease

•Currently primarily a tool for assessing new tumor targets,

CAR designs, trial designs

Zhao, Y, Cancer Res. 2010;70(22):9053

Beatty, GL, , Cancer Immunol Res. 2014;2(2):112

CAR Expression via RNA

Targeting CD19+ CLL with CAR-Modified T cells

• CARs combine an

antigen recognition

domain of antibody with

intracellular signaling

domains into a single

chimeric protein

• Gene transfer (lentiviral

vector) to stably express

CAR on T cells confers

novel antigen specificity

CAR, chimeric antigen receptor

TCR, T-cell receptor.

T cell

CD19

Native TCR

Tumor cell


• CARs combine an

antigen recognition




chimeric protein







Lentiviral vector

T cell

CD19

Native TCR

Tumor cell


• CARs combine an

antigen recognition




chimeric protein







T cell

CD19

Native TCR

Tumor cell

Lentiviral vector


• CARs combine an

antigen recognition




chimeric protein







T cell

CD19

Native TCR

Tumor cell


• CARs combine an

antigen recognition




chimeric protein







Anti-CD19 CAR construct


• CARs combine an

antigen recognition




chimeric protein







Anti-CD19 CAR construct

CTL019 cell

Dead tumor cell

Robust CAR T Cell Production Process

Complete & Partial Responses in

Relapsed/Refractory CLL

New Engl J Med 365:725, 2011, Science Translational Med 3:95ra73, 2011

CLL is an indolent disease – Can CAR T cells work in rapidly

growing malignancies?

April, 2012 Pediatric ALL 1st Patient

Karyotype: high risk

Dx 5/2010, R1: 10/2011,R2: 2/2012

3/2012: high dose

cytoxan/clofaribine: persistent ALL

Marrow 4/16/2012: 60% blasts

w/kidney, liver, spleen lesions

CAR T cells infused with no

additional chemotherapy

April, 2012 May, 2015

May, 2015

Center For Advanced Cell Therapy

Developing a Clinical Algorithm:

Management of CAR T Cell Toxicities

• B cell aplasia

• observed in all responding patients to date

• managed with IV immune globulin replacement

therapy

• Tumor Lysis syndrome

• Cytokine release syndrome (CRS)

• reversible, on-target toxicity

• Controlled with anti-IL-6 therapy (tocilizumab) • Grupp, SA et al. N Engl J Med. 2013 Apr 18;368(16):1509

• Lee, DW et al. Blood. 2014 Jul 10;124(2):188

• Severity related to tumor burden

The First CAR Assembly Lines

Modern CAR Assembly Lines

Penn Platform Technology:

Allows design and targeting against other tumors

Chimeric Antigen Receptor T Cell Translation:

Key Points (I)

•Engineered CAR T cells are a dividing drug and

persist for years providing functional immunity –Sci Transl Med. 2015 Sep 2

•CTL019 CARs have potent activity in refractory

ALL, CLL, DLBCL, FL, myeloma –Sci Transl Med. 2011, NEJM 2011, NEJM 2013, NEJM 2014, NEJM

2015

•CARs targeting antigens in solid tumors are

promising in early stage trials

Chimeric Antigen Receptor T Cell Translation:

Key Points (II)

•Tech transfer from academia (Penn) to industry

(Novartis) accomplished

•Novartis now manufacturing for the pediatric r/r

ALL global clinical trial and the DLBCL global

clinical trial in the US

–Will be expanded into other countries in the

second half of 2015.

CAR Clinical Trials in Philadelphia

http://www.penncancer.org/Tcelltherapy

Lentiviral vector CAR’s • Adult Acute Lymphoblastic Leukemia NCT02030847

• Chronic Lymphocytic Leukemia NCT01747486

• Adult Lymphomas: NCT02030834

• Myeloma: NCT02135406 (CART19), NCT02546167 (BCMA)

• Pediatric Leukemia and Lymphoma: NCT01626495

• Glioblastoma: NCT02209376

• Mesothelin expressing cancers: NCT02159716 (CARTmeso),

NCT02465983 (CARTmeso19)

• Multi-center CTL019 trials 8 sites enrolling NCT02228096

RNA CAR’s • Mesothelioma: NCT01355965

• Pancreatic Cancer: NCT01897415

• Breast Cancer: NCT01837602

• Hodgkin’s Lymphoma: NCT02277522

• AML: pending

• Neuroblastoma: pending


CAR Clinical Trials in Philadelphia


To Date

>200 CTL019 Patients

>40 other CAR’s

Lentiviral vector CAR’s • Adult Acute Lymphoblastic Leukemia NCT02030847

• Chronic Lymphocytic Leukemia NCT01747486

• Adult Lymphomas: NCT02030834

• Myeloma: NCT02135406 (CART19), NCT02546167 (BCMA)

• Pediatric Leukemia and Lymphoma: NCT01626495

• Glioblastoma: NCT02209376

• Mesothelin expressing cancers: NCT02159716 (CARTmeso),

NCT02465983 (CARTmeso19)

• Multi-center CTL019 trials 8 sites enrolling NCT02228096

RNA CAR’s • Mesothelioma: NCT01355965

• Pancreatic Cancer: NCT01897415

• Breast Cancer: NCT01837602

• Hodgkin’s Lymphoma: NCT02277522

• AML: pending

• Neuroblastoma: pending


Study Participants

Abramson Cancer Center

Center for Cellular Immunotherapies

Director, Carl June

Clinical Cell and Vaccine Production Facility

Process Development and Correlative Studies Laboratory

Department of Medicine

Department of Pathology and Laboratory Medicine Division of Transfusion Medicine and Therapeutic Pathology





October 14, 2015

Webinar Series

Sponsored by

Bruce Levine, Ph.D.


Philadelphia, PA



Juno Therapeutics

Seattle, WA




Cost-effective T-cell-based immunotherapies for

cancer October 14, 2015

Webinar Title: Don't get lost in translation: How smart design and technology are enabling bench-to-bedside in

translational research

Science/AAAS

Some of technology described was advanced through research conducted at the MD Anderson Cancer Center by Laurence Cooper, M.D., Ph.D. Both MD Anderson Cancer Center and Dr. Cooper have a financial interest in ZIOPHARM Oncology, Inc., and Intrexon Corporation. On May 7, 2015, Dr. Cooper was appointed as the Chief Executive Officer at ZIOPHARM. Dr. Cooper is now a Visiting Scientist at MD Anderson where he will continue to supervise the development of this technology.

T cells genetically modified with tumor-specific CAR or TCR

Clinical & Translational Immunology (2014) 3, e16;

Organization Immunology

Bioprocessing

Manufacturing Clinical conduct

Correlative studies

Regulatory

Bench Bedside

Approaches to manufacture and distribution of engineered T cells

The 3 “P”s

Procurement Process Product

The 3 “P”s


Avoid technology that relies on single source vendors

The 3 “P”s



Innovation versus

continuation

The 3 “P”s



Innovation versus

continuation Scalable

Procurement

• Tumor infiltrating lymphocytes

• Venipuncture

• Apheresis

• Select/sort • T-cell “starting” population

Process #1: Gene transfer

• Viral • Retrovirus

• Lentivirus

• Non viral • DNA

• mRNA

Process #2: Propagation

• Non-specific • Cross-link CD3

• Cross-link co-stimulatory molecules

• Antigen-specific • Tumor-associated antigen

• CAR

• Cell-free or cell-based • Beads

• K-562

K-562-derived activating and propagating cells (AaPC)

Genetically-modified K-562 cells

NK cells gd T cells

Treg cells ab T cells (TIL)

TH17 cells NKT cells

Immunol Rev. 2014 Jan;257(1):181-90

Criteria to produce a master cell bank of aAPC

Parameter Test

Endotoxin LAL <5 EU/mL

Sterility Direct inoculation method

Viruses

Replication-competent lentivirus

In vitro and in vivo culturing

Bovine 9CFR in vitro assay

PCR for human viruses and Bovine polyoma virus

Quantitative product enhanced reverse transcriptase (Q-PERT)

assay for retroviruses

Mycoplasma Test for presence of agar-cultivable and non-agar cultivable

mycoplasma

Identity

Transmission electron microscopic examination

Isoenzyme Analysis

DNA fingerprinting

Immunophenotyping (gated flow cytometry)

Viability 70%

Introduced cell surface antigen(s) 40%

CD32 75%

Product

3rd

party T cells RecipientsDonor

RecipientDonor

Autologous

Allogeneic

Dual distribution approaches for T-cell immunotherapy

Off-th

e-sh

elf (O

TS) P

atie

nt

Spe

cifi

c

Centralized Point-of-care (POC)

Genetically modified T cells

One patient Multiple Patients

Autologous T cells Allogeneic T cells

Near real time In advance of need

Disease cure Disease control

CAR

Patient

TCRαβ

Eliminating TCR on CAR+ T cells

Blood. 2013 Aug 22;122(8):1341-9 Blood. 2012 Jun 14;119(24):5697-705

CAR

Patient

TCRαβ

Normal cells

HLAs



CAR

Patient

CD19

Intended response

B-cell leukemia/lymphoma HLAs

TCRαβ

Normal cells

HLAs



CAR

Patient

CD19

Intended response


TCRαβ



CAR

Patient

CD19

Intended response


TCRαβ

Gene insertion:

Retrovirus/lentivirus

Sleeping Beauty

mRNA

Artificial nuclease



Personalized Therapy for Disease and Patient

Infuse T cells with more than one specificity Personalized for the disease

Intra-tumor Inter-tumor

Heterogeneity of tumor-associated antigen (TAA)

Infuse T cells with one or more specificity Personalized for the patient

“N=1” trial paradigm

Power-law curve The new industrialization of T cells

Number of trials

Nu

mb

er o

f p

atie

nts


Number of trials

Nu

mb

er o

f p

atie

nts

Traditional Med Centers


Number of trials

Nu

mb

er o

f p

atie

nts


Immuno-oncology at Med Centers


Cost of distribution

Number of trials

Nu

mb

er o

f p

atie

nts


Immuno-oncology at Med Centers


Cost of distribution

Number of trials

Nu

mb

er o

f p

atie

nts


Immuno-oncology at Med Centers 1

Points to consider

• No-single source failure

• Keep-it-simple

• Non viral

• Keep it simple

Put in un graphic

Acknowledgements





October 14, 2015

Webinar Series

Sponsored by

Bruce Levine, Ph.D.


Philadelphia, PA



Juno Therapeutics

Seattle, WA




September 2015 Juno Therapeutics

How product knowledge enables process scale-up/out.

Proprietary Materials

Juno Therapeutics 70 Proprietary Materials

Forward-Looking Statements

This presentation and the accompanying oral commentary contain forward-looking statements that involve risks, uncertainties, and assumptions. If

the risks or uncertainties ever materialize or the assumptions prove incorrect, our results may differ materially from those expressed or implied by

such forward-looking statements. All statements other than statements of historical fact could be deemed forward-looking, including, but not limited

to, any expectations regarding investment returns; any projections of financial information; any statements about historical results that may suggest

trends for our business; any statements of the plans, strategies, and objectives of management for future operations, including our manufacturing

and process development; any statements of expectation or belief regarding future events, potential markets or market size, technology

developments, our product pipeline, clinical data, enforceability of our intellectual property rights, competitive strengths or our position within the

industry; any statements regarding the anticipated benefits of our Celgene collaboration or other strategic transactions; and any statements of

assumptions underlying any of the items mentioned.

These statements are based on estimates and information available to us at the time of this presentation and are not guarantees of future

performance. Actual results could differ materially from our current expectations as a result of many risks and uncertainties, including but not limited

to, risks associated with: the success, cost, and timing of our product development activities and clinical trials; our ability to obtain regulatory

approval for and to commercialize our product candidates; our ability to establish a commercially-viable manufacturing process and manufacturing

infrastructure; regulatory requirements and regulatory developments; the effects of competition and technological advances; our dependence on

third-party collaborators and other contractors in our research and development activities, including for the conduct of clinical trials and the

manufacture of our product candidates; our dependence on Celgene for the development and commercialization outside of North America of product

candidates for which Celgene exercises an option; our ability to obtain, maintain, or protect intellectual property rights related to our product

candidates; among others. For a further description of the risks and uncertainties that could cause actual results to differ from those expressed in

these forward-looking statements, as well as risks relating to our business in general, see our Quarterly Report on Form 10-Q filed with the

Securities and Exchange Commission on August 14, 2015 and our other periodic reports filed from time to time with the Securities and Exchange

Commission. Except as required by law, we assume no obligation and do not intend to update these forward-looking statements or to conform these

statements to actual results or to changes in our expectations.


FOUNDING INSTITUTIONS

ACADEMIC PARTNERS

Translating Research into Clinical and Commercial Products

• Juno Therapeutics has a network of founding institutions and academic partners.

• This network provides insights into the process of translating novel research into clinical products (including clinical manufacturing)

• My expertise is in product and process development. – Research -> Early Phase – Early Phase -> Late Phase – Late Phase -> Commercial


Scaling-Up/Out Requires Product and Process Knowledge

Product Knowledge: Which product attributes are important for biological activity (safety and efficacy) of product.

Process Knowledge: Understanding the combination of process parameters and material attributes required to manufacture the desired product.

Moving any bioprocess (cell therapy or otherwise) into a multi-product manufacturing setting requires the product/process knowledge to establish a process robust enough that it can be repeatably executed (process control).

Building process knowledge and a process control strategy to enable transfer to a moderately-sized multi-product facility is resource intensive.

In most cases it is not feasible or cost effective to build this amount of process knowledge prior to demonstrating efficacy in the clinic.


Recommendations for institutions and companies in early-phase development

1. Focus on building product knowledge.

2. Invest is translational infrastructure and knowledge management.

– Centralized databases to store and compare clinical, translational and manufacturing data.

– Organizational structures to enable cross-functional collaboration between scientific and non-scientific functions.

3. Document, Document, Document

– Manufacturing Batch Records – Store analytical data (manufacturing, translational) in a thoughtful way. – Know and document source of all raw materials (e.g. cell lines, plasmids).


Engineered T-Cell Product Knowledge

Adoptive immunotherapy for cancer: harnessing the T cell response

Nicholas P. Restifo, Mark E. Dudley & Steven A. Rosenberg

Nature Reviews Immunology 12, 269-281 (April 2012)

doi:10.1038/nri3191

Product Knowledge can be developed via a product characterization effort.

• Phenotype

• In Vitro Function

• In Vivo Function

http://www.nature.com/nri/journal/v12/n4/full/nri3191.html


Invest is translational infrastructure and knowledge management

Median DFS for patients in CR:

Cy±VP16 = 150 days

Flu/Cy, not reached

CR = 17/17

MRD negative CR = 16/17

DFS = disease-free survival; DL2 = dose level 2; Flu/Cy = fludarabine & cyclophosphamide; CR = complete response; MRD =

minimal residual disease

Data for JCAR014 in Adult R/R ALL


Recommendations for institutions and companies in early-phase development

4. Invest in translational sample inventory system – Take multiple drug product, in process, raw material, translational (Clinical)

samples from all products. – Aliquot and store samples in multiple formats (Cell-based assays,

transcriptional analysis (e.g. NGS), mass spectroscopy). – Run small proof of concept studies to demonstrate sample suitability and

stability.

5. Use stored samples to help elucidate mechanisms and

attributes that discriminate response and safety in products demonstrating activity.

Product Knowledge can be developed after clinical proof-

of-concept has been established using sample inventory.


Clinical evidence and product knowledge will justify investment in process knowledge

Resolu' on)of)Advanced)Lymphoma)with)CD19)CAR)T)Cells))

Pre)CD)19)CAR?T)cell)infusion) 28)Days)Post)CD)19)CAR?T)cell)infusion)

Resolu' on)of)Advanced)Lymphoma)with)CD19)CAR)T)Cells))

Pre)CD)19)CAR?T)cell)infusion) 28)Days)Post)CD)19)CAR?T)cell)infusion)

Day 37 After CD19

CAR T Cells

ALL Patient Before CD19

CAR T cells

Day 28 After CD19

CAR T Cells

NHL Patient Before CD19

CAR T cells

Our early experience

in leukemia trials demonstrates 87%-

93% complete

remission rates

Our early experience

in lymphoma demonstrates 56%

overall response

rate (1)

(1) ORR of 62% for patients treated following cyclosphosphamide / fludarabine conditioning regimen.

.


Ease of Scale-Up/Out scales with Product Knowledge

Building Product Knowledge early enables process changes between early- and late-phase trials as well as building process knowledge.

Process changes allow the deployment of new technologies that are suitable for a multi-product large-scale manufacturing environment. – Closed Systems – Fully-Defined Raw Materials

Wait until clinical proof-of-concept has been established before

investing in process knowledge, unless a platform process has been established for product class.


To ask a question, type it into the text box and click Submit

Bruce Levine, Ph.D.


Philadelphia, PA



Juno Therapeutics

Seattle, WA




October 14, 2015

Webinar Series

Sponsored by


MDACC, Houston, TX


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