•

Sleeping Beauty: Current applications and future strategies

CAR-TCR Summit 2017

Partow Kebriaei, MD

Outline

• Chimeric antigen receptor (CAR) technology

– Viral versus nonviral vectors

• Results of current clinical trials utilizing the Sleeping Beauty (SB) platform

• Future strategies utilizing the SB platform to target hematologic malignancies with CARs and solid tumors with T- cell receptor (TCR)s

• Support provided by ZIOPHARM Oncology

Current CAR, TCR T-cell technology • Uses a genetically-engineered CAR or

TCR that is:

– Transduced into T cells using viral and non-vectors and

– Expressed in T cells which are expanded ex vivo and then administered to patients to target tumor cells in the body

• The introduced CAR and TCR redirect T-cell specificity to target cancer cells

Recipient

Endogenous immune

response fails to halt cancer

T cell genetically modified ex vivo to redirect specificity

ab

TCR

CAR

Intracellulartargets

Cell surfacetargets

T cell

T cell

Allogeneic, autologous CAR T-cells

Allogeneic

Autologous

Donor RecipientsEngineered

T cells

Off-the-shelf (OTS)

Barriers:· Requirement for

lymphodepletion· Rejection of infused T

cells curtails anti-tumor effect

· Only applicable to CARs (not solid tumors)

Donor RecipientEngineered

T cells

Autologous

Barriers:· Requirement for

lymphodepletion· Cost· Time to manufacture

(during which patients condition deteriorates)

One allogeneic donor into multiple recipientsMade in advance of need and infused on demand

One autologous donor/recipientMade as needed

Viral gene transfer

Viral gene transfer

Curr

ent

prac

tice

• Readily available product.

• Potential for rejection of modified T cells.

• Very little clinical data.

• Most commonly used.

• Potential for manufacture failure.

• Long time to make.

Non-viral versus viral transduction

Non-viral gene transfer: Sleeping Beauty Viral gene transfer: Retrovirus & Lentivirus

Cost effective High cost

Slower transduction rate Faster transduction rate

More nimble production: Customizable Labor intensive, challenging to customize

Packaging cells Lentivirus

Release

Release

DNA plasmid

Sleeping

Beauty

DNA plasmidEx vivo gene transfer

Limitations of current approach to CAR+ (& TCR+) T Cells • Heterogeneity in end-product after

ex vivo culture

• Failure to manufacture.

• Risk of insertional mutagenesis

• Time for manufacture

• Lack of control of T cells after infusion

• Need for lymphodepletion

• Specialized treatment centers for administration

SLEEPING BEAUTY: CLINICAL TRIALS AT MD ANDERSON CANCER CENTER

SB transposon/transposase

Nucleus

Transposase

Transposon

CAR

Cytoplasm

IR/DR

IR/DR

hEF1a

CAR

CMVIE

SB11

Transposase

Co-delivery into cells by nucleofection

Transposon DNA plasmid Transposase DNA plasmid

(or in vitro transcribed mRNA)

Release

DNA plasmid

Sleeping

Beauty

a b

T-cell receptor(TCR)

scFv

Extracellular scaffold

Intracellular domainsShowing signaling motifs

Chimeric antigen receptor(CAR)

Membrane bound IL-15

(mbIL15)

Non-viral gene transfer using Sleeping Beauty

system to express CAR, TCR and membrane-

bound IL-15 (mbIL15)

First-in-human application of SB system CAR+ T cells infused after hematopoietic stem-cell transplantation

Methods available at: J Vis Exp. 2013 Feb 1;(72):e50070. Irradiated AaPC (feeder cells) derived from K-562 cells and modified to co-express CD19, CD86, CD137L, membrane-bound IL-15 (mbIL15, and CD64)

9

Trial schema • Study populations

– CD19+ lymphoid malignancies beyond first remission, induction failure, or relapse at time of HSCT

– 1-65 yrs-old for allo-HSCT; up to 75 yrs-old for auto-HSCT

• Preparative treatment

– Auto-HSCT • BEAM prep

• PBSC day 0, CAR T cells day +2

– Allo-HSCT • HSCT prep per MD choice

• Donor-derived T cells 6-12 weeks

post HSCT

• GVHD prophylaxis maintained

Dose Level Single T-cell dose*

A >5 x 107/m2 but < 5 x 108/m2

B >5 x 108/m2 but < 5 x 109/m2

Dose Level Single T-cell dose*

A Not to exceed 106/m2

B >106/m2 but < 107/m2

C >107/m2 but < 5x 107/m2

D >5x107/m2 but < 108/m2

*Per body surface area

J Clin Invest. 2016 Sep 1;126(9):3363-76.

Trial Summary I

• Successful manufacture of T-cell products – 200 mL of peripheral blood (avoiding costs & inconvenience of apheresis)

• Safely infuse patients – No immediate or late toxicity

– Decreased GVHD rate at 11% – Administered up to 108/m2 genetically modified haplo-identical T cells

– Decreased CMV reaction, 24% vs. 41%1

– Outpatient infusions

• Cytokines – Low levels of cytokine at time of T-cell infusion

– Mild elevation, peak at ~2 weeks

– No cytokine storm

1. J Oncol Parm Practice, 2014, 20:257

1st generation complete

First-in-human use of SB system: Summary II • Persistence of CAR+ T cells

• Survival of recipients after CAR+ T cells

Average, days Max, days

Autologous 201 360

Allogeneic 51 180

PFS OS

Autologous 83%, 3-yr 100% 3-yr

Allogeneic (all) 53%, 1-yr 63%, 1-yr

Allo, haploidentical 75%, 1-yr 100%, 1-yr

• CAR+ T cells exhibit longer persistence in the auto-HSCT group.

• No apparent positive correlation with T-cell dose or disease burden.

J Clin Invest. 2016 Sep 1;126(9):3363-76

1st generation trial: Evaluate SB system (first-in-human studies)

1st generation SB platform in two trials infusing CAR+ T cells after HSCT showed favorable PFS and OS in both autologous and allogeneic cohorts1 compared with historical controls. 2,3

1. J Clin Invest. 2016 Sep 1;126(9):3363-76

2. Blood 125, 2579-2581 (2015) 3. Curr Hematol Malig Rep 7, 144-152 (2012)

Shortening manufacturing time for CD19-specific CAR+ T cells

1st generation complete 2nd generation ongoing

Shortening manufacturing process time from 4 weeks to ~ 2 weeks as T cells co-cultured on feeder cells J Clin Invest. 2016 Sep 1;126(9):3363

2nd generation trial with SB-modified T cells for active CD19+ malignancies • clinicaltrials.gov: NCT02529813

• CD19-specific CAR+ T cells; SB-based gene transfer

– T cells modified to express CD19-specific CARs with a CD8 stalk region that signals through CD28 and CD3z

– Shortened manufacturing time

– CAR+ T cells infused following lymphodepletion

in active disease

– Phase 1, dose escalation

• Actively recruiting

Dose level CAR+ T-cell dose/kg

-1 105

+1 > 105, but 106

+2 > 106, but 107

+3 > 107, but 108

+4 > 108, but 109

SLEEPING BEAUTY: COMBINING CAR WITH CYTOKINE SIGNALING

High serum levels of IL-15 associated with tumor regression

J Clin Oncol. 2017 Jun 1;35(16):1803-1813

Improving CAR+ T cells by co-signaling through IL-15 receptor

Co-expression of CAR and mbIL-15

T cell

Signal 3

Signals 1 & 2

Combination

IL-15

Type I cytokine

CAR

Membrane bound IL-15

(mbIL15)

scFv

Extracellular scaffold

Intracellular domainsShowing signaling motifs

JAK STAT

Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7788-E7797

Membrane bound IL-15 augments antitumor activity of CAR+ T cells

3rd generation SB technology based on point-of-care (POC)

• Rapid manufacture: Manufactured as needed in <2 days at POC without need to ex vivo expand cells

• Target tumors with CARs or TCRs

• Sleeping Beauty non-viral system

• mbIL15 to keep T-cells in naive, powerful state

• Distributed manufacture and infusion similar to blood-banking practices

• Safety switch will allow T-cells to be tuned to patient’s need

3rd generation progressing

Pre-clinical data supporting POC

21

Standard

Manufacture (4-

Stim)

P-O-C: < 2 days

Mononuclear Cells 2-Stim

Days: 0 1 7 14 21 28

Nucleofection

(DNA transposon & DNA transposase) Stimulation with K562 AaPC

TN TSCM TCM TEM TEff

Nucleus

Transposase

Cytoplasm

CAR

Transposon

IR/DR

IR/DR

hEF1a

CAR

CMVIE

SB11

Transposase

Co-delivery into cells by electroporation

Transposon DNA plasmid Transposase DNA plasmid

(or in vitro transcribed mRNA)

1 2

POC-generated T cells co-expressing CAR and mb IL-15

Tu

mo

r fl

ux

(p

/s/c

m2/s

r)

5 10 15 20 25 30 35 40 45 50

105

106

107

108

109

1010

1011 Tumor only

Days after tumor injection

POC-CARneg

T cells

Tu

mo

r fl

ux

(p

/s/c

m2/s

r)

5 10 15 20 25 30 35 40 45 50

105

106

107

108

109

1010

1011

Days after tumor injection

POC-mbIL15-CAR T cells

POC-CAR T cells

Days after tumor injection

Tum

or

flux (

p/s

/cm

2/s

r)

POC-CARneg T cells

Tumor only POC-mbIL15-CAR T cells

POC-CAR T cells

5 10 15 20 25 30 35 40 45 50 5 10 15 20 25 30 35 40 45 50

1011

1010

109

108

107

106

105

Harnessing mbIL15 so that infused T cells propagate inside, removing the need to pre-expand before administration

0 10 20 30 40 500

25

50

75

100

Days after tumor injection

Dis

ease

-fre

e s

urv

ival (%

) Tumor only

POC-CAR T cells

POC-mbIL15-CAR T cells

POC-CARneg

T cells

**

(3/5)

*

(3/5)

ASH: December 4 Publication Number: 2807

• Electroporated (Day 0) • <2 days infusion • 106 CAR+ T cells/mouse (Day 1

expression*) *Integrated & episomal expression

POC

CAR T cells

POC

mbIL15-CAR T cells

POC

CARneg

T cells

Competitive advantage achieved by POC

23

• Reduce concerns regarding heterogeneity of product

• Major reduction in costs

• Improve scalability

• Very rapidly deliver T-cell therapy

• T cells targeting solid tumors, as CAR is replaced with TCR

Viral-based POC Solutions

SLEEPING BEAUTY: TARGETING SOLID TUMORS WITH TCR-MODIFIED T CELLS

Personalized TCR-modified T cells

• Tumor antigen is not known before the patient arrives

• Tumor and normal cells are interrogated to determine the neoantigen

• TCRs against known tumor antigen are prepared in real time

• TCRs rapidly expressed in autologous T cells using SB platform via POC

Donation RecipientGenerated using P-O-C

IntroducedTCR

Genetically modified

a b

a b

EndogenousTCR

T cell

a b

TumorTargeting

neoantigen(s)

mbIL15

Control persistence

TCR+ T cells specific for neoantigens using SB system

Mol Ther. 2016 Jun;24(6):1078-89

Competitive advantage of POC

• Reduce concerns regarding heterogeneity of product

• Major reduction in costs

• Improve scalability

• Rapidly deliver T-cell therapy

• T cells targeting solid tumors, as CAR is replaced with TCR

27

Harnessing genetically modified T cells using SB system

Potential of point-of-care (P-O-C)

1st era • Viral-based gene

transfer

2nd era • Sleeping Beauty non-viral gene

transfer

3rd era • Shorten manufacture

with IL-15 signaling

Final era • Point-of-

care

CAR+

Hematologic cancers

TCR+

Solid tumors

28

Summary

• “First generation” trial demonstrated safety and feasibility of the SB platform

• “Second generation” trial is testing a modified CAR with shorter manufacturing time using SB platform

• “Third generation” trial to rapidly infuse CAR+mbIL15+ T cells in < 2 days using SB platform

THANK YOU

pkebriae@mdanderson.org