dr geoff nichol - bhiva · geoff nichol mb chb fracp executive vice-president, r&d sangamo...

BHIVA AUTUMN CONFERENCE 2014Including CHIA Parallel Sessions

Dr Geoff NicholSangamo BioSciences, USA

9-10 October 2014, Queen Elizabeth II Conference Centre, London

BHIVA AUTUMN CONFERENCE 2014Including CHIA Parallel Sessions

Dr Geoffrey NicholSangamo BioSciences, USA

9-10 October 2014, Queen Elizabeth II Conference Centre, London

COMPETING INTEREST OF FINANCIAL VALUE > £1,000:

Speaker Name Statement

Dr Geoffrey Nichol

Is an employee and US Section 16 Officer at Sangamo BioSciences;

receives salary and holds shares and share options in Sangamo

BioSciences

Date October 2014

CCR5 knockout gene therapy trials

Geoff Nichol MB ChB FRACP

Executive Vice-President, R&D

Sangamo BioSciences

BHIVA Autumn Conference 2014

HIV – an infection and an immune system disease

4

Primary infection of CD4 T cells

Loss of CD4 T cells by direct cytopathic and bystander mechanisms

“Helpless” activation of CD8 T cells fails to clear infection

Reservoir established in memory CD4 cells Damage to mucosal barriers

Chronic inflammatory state

ART

Blocking the narrow doorA lesson from Nature – the CCR532 mutation

5

Individuals homozygous for the CCR532 allele are highly resistant to HIV-1

infection

CCR5

CCR532

“Berlin patient”

6

CCR532 donor

“Berlin patient”

SB-728-T – the product

7

ZFNs cause targeted gene disruption

CCR5 target

gene disruption

NHEJerror-prone repair

8

ZFP

ZFP

• Delivered with a non-integrating, replication-deficient, chimeric adenoviral 5/35 vector or mRNA electroporation

DNA

CCR5

ZFN modificationSite 165

D32 mutation

Zinc finger nucleases (ZFNs) “Designer restriction enzyme”

9

SB-728-T: Zinc finger nuclease driven CCR5 modified autologous CD4+ T-cells

10

Enrich CD4+

Infusion

SB-728-T

SB-728

CCR5 ZFNs

CCR5 gene

disruption

Apheresis

Expand, formulate and test

Median CCR5 modification ~25%

Single Infusion of:10-30 billion SB-728-T

ZFN

ZFN

DNA

The infused product (SB-728-T) contains T-cells with a stem cell-like phenotype

11

0

5

10

15

20

25

CD45RAlow RO low

% o

f C

CR

7+ C

D27

+

pre-CCR5 modification SB-728-T

Naïve

CM

TM

EM

CCR7+CD27-

CD45RAlowROlow

CD4+

CD45RA

CD

45

RO

CD4+

Memory Stem Cell-Like

• Most frequently is a 5-bp insertion or “Pentamer Duplication” (CTGAT)• Approximately 16 to 39% (mean = 23%) of CCR5 allele disruptions

• In clonal studies bi-allelic disruption occurs in about 1/3 of disrupted cells – total CCR5 knockout• 2/3 if one allele already has the Δ32 mutation

• ZFN mediated gene disruption generate a diverse array of short insertions and deletions to the targeted CCR5 locus.

Pentamer (5bp) Duplication

How we assay for CCR5 deletions

SB-728 – key exploratory clinical studies

13

Study Study Goal

SB-728-T-902 Cohorts 1-3 (n=9)• Immune non-responders (INR) on ART; longitudinal

follow-up 3 years

Reservoir Depletion / Elimination & Immune

Reconstitution

Phase I study at U Penn (n=6)• ART-treated subjects, treatment interruption (TI)

SB-728-T-902 Cohort 5 (n=10)• CCR5 Δ32 Heterozygotes, ART, TI

Demonstrate Immunological Control of Viral Growth without ART

SB-728-T-1101 Cohorts 1-5 (n=18)• Cytoxan preconditioning, ART, TI

SB-728mR-T 1401 (commencing)• mRNA electroporation, ART, TI

SB-728-HSC (planned)• mRNA electroporation in HSPCs

SB-728-T – pharmacokinetics and pharmacodynamics

14


15

Study Study Goal


follow-up 3 years


Reconstitution

Phase I study at U Penn (n=6)• ART-treated subjects,treatment interruption (TI)






Long-term CD4 T-cell reconstitution post SB-728-T

16

0 7 14 21 28 2 3 6 7 8 9 10 11 12

0

500

1000

1500

102

103

104

201

203

302

303

305

304

Ch

an

ge o

f C

D4 T

cell

s

fro

m B

aseli

ne (

cell

s/

l)

Bas

elin

e

Day

14

Mon

th 3

Mon

th 6

Mon

th 1

2

Mon

th 2

4

0

500

1000

1500

2000

***

***

**

CD

4 c

ou

nts

(cell

/uL

)Infusion of CCR5-disrupted cells led to a sustained significant

increase in CD4 T cell counts (mean of 103 cells/µL at 12 Months)

days months

* p≤0.05 ** p≤0.01

10/9/2014 17

Long-term engraftment of CCR5 modified cells

102 103 104

201 203 302

303304305

1X106 Cells 2X10

6 Cells 3X10

6 Cells

Visits

Bas

elin

eD

ay 1

Day

7D

ay 1

4D

ay 2

1D

ay 2

8M

onth

2M

onth

3M

onth

4M

onth

5M

onth

6M

onth

7M

onth

8M

onth

9M

onth

10

Mon

th 1

1M

onth

12

LTFU

M3

LTFU

M6

LTFU

M9

LTFU

M12

LTFU

M18

LTFU

M24

Pen

tam

er D

uplic

atio

n

per

L

0

10

20

30

40

50

60

Median

Mean

18

CCR5 modified T-memory stem cells expand and persist up to 12 months

BL M 9-10 M11-12

0

20

40

60

80

100

120

102

201

203

103

302

Esti

mate

d n

um

ber o

f S

B-7

28-T

rela

tive t

o i

np

ut

TSCM (CD45ROlowRAlowCCR7+CD27+CD95+)

•Median fold expansion of CCR5-modified cells relative to amount infused was 20.7 at Month 12

•In contrast, fold expansion in modified CM and EM were ~ 3 fold and < 1 fold

Esti

mat

ed F

old

Exp

ansi

on

of

CC

R5

-mo

dif

ied

ce

lls

CCR5 gene modification level is maintained for three years in the TSCM fractions

19

SB-728-T traffics to the rectal mucosa

Time

Baseline

2-3 W

eeks

6-12 W

eeks

16-24 W

eeks

36-48 W

eeks

Per

cent

CC

R5

Dis

rupt

ion

in M

ucos

al C

D4

0

10

20

30

40

50

60

70

N=19 N=11 N=12 N=9 N=3

Outliers

Median

Mean

75 %

25 %

Std. Error

20

High levels of monocyte activation (DRhiCD86hiCD40hi) in HIV+ subjects at baseline

21

Inflammatory Monocyte CD14+ CD16+

HIV

Neg

ativ

e

ART+

HIV

+

0

20

40

60

80

100 p < 0.0001

% a

cti

vate

d C

D14+

CD

16+

Classical Monocyte CD14++ CD16-

HIV

Neg

ativ

e

ART+

HIV

+

0

20

40

60

80

100 p = 0.0066

% a

cti

vate

d C

D14+

+ C

D16-

Baseline levels of monocyte activation inversely correlate with levels of CCR5-modified cell engraftment

22

0 2 4 6 80

5

10

15

r = -0.7505 p = 0.0198

Estimated CCR5-modified PBMC countsat day 21 relative to input

% H

LA

-DR

hiC

D86h

i

in C

D14+

mo

no

cyte

s a

t B

L

Baseline levels of monocyte activation inversely correlate with levels of CD4 T-cell reconstitution

23

-200 0 200 400 6000

5

10

15

20

r = -0.8633 p = 0.0027

delta CD4 counts M32-44 (cells/uL)%

HL

A-D

Rh

iCD

86h

i

in C

D14+

mo

no

cyte

s a

t B

L

CD4 Persistence after 3 Years

0 200 400 600 800 10000

5

10

15

rho r = -0.8034 p = 0.0091

delta CD4 counts M12 (cells/uL)

% H

LA

-DR

hiC

D86h

i

in C

D14+

mo

no

cyte

s a

t B

L

CD4 Persistence after 1 Year


24

Study Study Goal


follow-up 3 years


Reconstitution




SB-728-T-1101 Cohorts 1-5 (n=18)• Cytoxan preconditioning dose-ranging, ART, TI



Higher peak CD4 T-cell reconstitution and engraftment of SB-728-T is observed at a dose of 1 gm/m2 CTX

25

CD4 change from baseline Pentamer duplication

CD

4 C

han

ge f

rom

Baseli

ne (

per

L)

0

1000

2000

3000

4000

5000

CTX(0.1

g/m2 )

CTX(0.5

g/m2 )

CTX(1.0

g/m2 )

CTX(2.0

g/m2 )

CTX(1.5

g/m2 )

902INR

Pe

nta

me

r D

up

lic

ati

on

(p

er

L)

0

20

40

60

80

100

120

140

160

180

902INR

CTX(0.1

g/m2 )

CTX(0.5

g/m2 )

CTX(1.0

g/m2 )

CTX(2.0

g/m2 )

CTX(1.5

g/m2 )

SB-728-T – effects on viral load during ART interruption

26


27

Study Study Goal


follow-up 3 years


Reconstitution







First-in-human study of CCR5 KO published in NEJM6 March 2014

• First genome edited therapy tested in man (ZFN modified CD4+ T cells)

• Infusions generally safe and well tolerated

• Marked increases in total CD4+ T cell levels

• Traffic to GALT (key battle ground of HIV infection)

• Modified cells show a selective survival advantage during ARD interruption

• One subject controlled viral load to below levels of detection prior to reinstating ARD

28

HIV viral load during treatment interruptions.

29Tebas et al, 2014

Estimated mean Biallelic Modification per L During treatment interruption

0 50 100 150 200 250

Lo

g o

f V

iral

Lo

ad D

rop

fro

m

pea

k to

en

d o

f T

reat

men

t In

terr

up

tio

n

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

rho = - 0.55, P= 0.017

01-005

01-009

01-037

04-019

07-001

01-509

01-513

04-502

10-503

12-503

201

203

205

253

Delta 32 Cytoxan UPENN

09-002

10-004

10-005

12-007 Threshold

Changes in VL correlate with levels of biallelic modification6-week bi-allelic engraftment following Cytoxan - approaching threshold?

30

Median

Mean

200 mg 500 mg/m2 1 g/m2

*

*Delta 32 Subject

*


31

Study Study Goal


follow-up 3 years


Reconstitution


SB-728-T-902 Cohort 5 (n=10) • CCR5 Δ32 Heterozygotes, ART, TI





Sustained functional control of viral load for more than one year

• Subject 04-502 (SB-728-902 Cohort 5)– Viral load controlled for more than 59 weeks (<500 VL copies/mL)

– Subject remains off ART

– Durable functional control achieved

32

Subject 04-502

Days from Baseline

0 60 120 180 240 300 360 420 480 540 600

Cell

s /

L

0

1000

2000

3000

4000

5000

6000

7000

Pen

tam

er

Du

pli

ca

tio

n /

L

0

50

100

150

200

250

300

Vir

al

loa

d C

op

ies

/ m

L

101

102

103

104

105

106

107

Absolute CD4 Count

Pentamer

VIral Load

Set Point

Treatment Interruption


33

Study Study Goal


follow-up 3 years


Reconstitution







SB-728-1101: Viral load decreases from peak during TIFour subjects with extended TI with VL <10,000 copies and CD4>500

34

TI= Treatment interruption

Red box: 32 Heterozygote

# Viral Load: Copies/mL

SubjectsL

og

Vir

al

loa

d d

rop

fro

m p

ea

k t

o e

nd

of

TI

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Cohort 1 (0.1 g/m2)

Cohort 2 (0.5 g/m2)

Cohort 3 (1.0 g/m2)

Cohort 4 (2.0 g/m2)

Cohort 5 (1.5 g/m2)

** **

01-005

01-009

09-002

10-005

07-001

01-037

12-007

10-004

04-019

01-051

04-038

01-053

04-009

01-060

01-059

254

8130

4880

2060

Cohort (CTX)

* Subject Continuing TI

Meaningful reductions in VL seen during TI in Cytoxan-treated subjects

Subject 04-019 (SB-728-1101)– CTX dose – 1.0 gm/m2

– >2 log reduction in Viral load (VL)

– Sustained control for 39 weeks


Subject 01-060 (SB-728-1101)– CTX dose – 1.5 gm/m2

– >2 log reduction in VL


35

Subject 04-019

Days from Baseline

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420

Ce

lls

/

L

0

1000

2000

3000

4000

5000

6000

7000

Pe

nta

me

r D

up

lic

ati

on

/L

0

50

100

150

200

250

300

Vir

al

loa

d C

op

ies

/ m

L

101

102

103

104

105

106

107

Treatment Interruption

Absolute CD4 Count

Pentamer

VIral Load

Viral Setpoint

Absolute C8 Count

Subject 01-060

0 30 60 90 120 150 180 210 240

Cell

s /

L

0

1000

2000

3000

4000

5000

6000

7000

Pen

tam

er

Du

pli

cati

on

/L

0

50

100

150

200

250

300

Vir

al

load

Co

pie

s/

mL

101

102

103

104

105

Absolute CD4 Count

Absolute CD8 Count

Pentamer

VIral LoadTreatment Interruption

SB-728-T – effects on the HIV reservoir

36

HIV reservoir

37

• Laid down at time of initial infection

• HIV DNA integrated within CD4 memory cells

• Reservoir size driven by time from infection to start of ART

• Highly stable on chronic ART• Maintenance is a dynamic

process• Activation cycling of CD4

reservoir cells creates a target for immunotherapies

Barouch and Deeks, Science 345, 169 (2014)


38

Study Study Goal


follow-up 3 years


Reconstitution







Step 1. “Digitize” sample into 20,000 droplets. Effectively reducing level of background gDNA

Step 2. Run PCR to endpoint. Quantification is no longer dependent upon PCR kinetics

Step 3. Fluorescent analysis drop by drop (yes or no). Copy number is calculated by Poisson distribution

Digital PCR- A new sensitive method to assess HIV proviral DNA

39

Reduction of PBMC HIV DNA (ddPCR) observed in SB-728-T treated subjects (Median 0.9 log decrease at Month 36)

40

1-01 1-02 1-03

2-01 2-02 2-03

3-01 3-02 3-03

Days Post Infusion

Log

[HIV

-DN

A/

1E6

PB

MC

]

Raltegravir/maraviroc +/- IL-7 - increased CD4 counts BUTincreased HIV pro-viral DNA

41Katlama et al CROI 2013

42

0 2 4 6 80.00

0.01

0.02

0.03

0.04

% C

D8

T-c

ells

at

M9

(p

ea

k)

0 2 4 6 80.00

0.01

0.02

0.03

0.04

0.05

% C

D8

T-c

ells

at

M9

(p

ea

k)

TNF-aSpearman r = 0.8857 p = 0.033

IFN-gSpearman r = 0.8286 p = 0.058

CD8 T-cells responsive to HIV GAG post-infusion correlate with the decay of CD4 T-cells harboring integrated HIV DNA

Reservoir ReductionRatio [Integrated HIV DNA (copies/10e6 cells)] BL/M36

Gene therapy for HIV

43

SB-728-T - Next steps

• IND for mRNA electroporation of CD4 cells is open – SB-728mR-T

– Allows potential for retreatment

• Key proof-of-concept Phase II study commencing:

– Optimal subject population

Short time from initial infection to ART

Favorable macrophage inflammatory profile

– Optimal Cytoxan dose (1 g/m2)

– 9 subjects in 2 cohorts will receive multiple doses of SB-728-mR-T

Cohort 1: SB-728-mR-T infusions of 2 equal doses 14 days apart

Cohort 2: SB-728-mR-T infusions of 3 equal doses 14 days apart

– Objective: define proportion of subjects with functional control outcome

• Reservoir assay work continues

44

Using ZFNs to protect CD34+ HSCs

T-cells

Macrophages

Hematopoietic stem cells

(HSC)X HIV

Virus

45

SB-728 CD34+ HSCs in HIV ZFN-treated HSC mice control R5-tropic HIV-1

blood

HIV-1BaL

Ctrl.

ZFN

8 9 10 12 8 9 10 12

small intestine large intestine

gut mucosa

Weeks post-infection

46

IND in 2014 in collaboration with City of Hope and California Institute of Regenerative Medicine

Summary and conclusions

• Ex vivo CCR5 knockout using ZFNs - a very appealing strategy for treatment of HIV

• T cell program has shown

– Sustained increase in total CD4 count and CCR-modified CD4 cell engraftment with tissue trafficking

Influenced by host factors related to inflammation

Optimized by conditioning with Cytoxan 1 g/m2

– Control of VL to undetectable or <1000 copies in a CCR5 Δ32 heterozygote for more than 1 year

– Two subjects with a 2-log decrease in viral load with Cytoxan conditioning, sustained in one case for >39 weeks

– Downward trends in viral reservoir in PBMCs over three years

Related to CD8 activation/numbers

• Optimized Phase II program commenced for SB-728-mR-T

• IND open for HSC program in 2014

47

Acknowledgements

48

VGTI-Florida Rafick-Pierre Sékaly, PhDJoumana Zeidan, PhDFrancesco Procopio, PhDNicolas Chomont, PhDRemi Frementin, PhDUniversity of California, San FranciscoSteven Deeks, MDSangamo BioSciencesDale Ando, MDGary Lee, PhDWinson Tang, MDShelley Wang, MDMarty Giedlin, PhDShirley CliftBaolu Chen, PhDMichael Holmes, PhDPhilip Gregory, DPhil

Quest Clinical ResearchJay Lalezari, MDUniversity of California, Los AngelesRonald Mitsuyasu, MDCircle CARE Center, LLCGary Blick, MDOrlando Immunology CenterEdwin DeJesus, MDRicky K Hsu, MD, PC Ricky Hsu, MDSouthwest CARE CenterTrevor Hawkins, MDCentral West Clinical Research, Inc.David Parks, MDClinical Research Puerto RicoJavier O. Morales-Ramírez, M.DPenn Carl June, MDPablo Tebas, MDBruce Levine, PhD

dr geoff nichol - bhiva · geoff nichol mb chb fracp executive vice-president, r&d sangamo...

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