American Association for Cancer Research Investor Meeting ...s21.q4cdn.com/250105458/files/doc_presentations/... · 4/18/2016 · 22. 0 7 14 21 0 250 500 750 1000 1250 1500 Days

American Association for Cancer ResearchInvestor Meeting

Targeting the Tumor Microenvironment

Copyright © 2016 Halozyme, Inc.

Forward-Looking Statements

1

All of the statements in this presentation that are not statements of historicalfacts constitute forward-looking statements within the meaning of the PrivateSecurities Litigation Reform Act of 1995. Examples of such statements includefuture product development and regulatory events and goals, anticipatedclinical trial results and strategies, product collaborations, our businessintentions and financial estimates and results. These statements are basedupon management’s current plans and expectations and are subject to anumber of risks and uncertainties which could cause actual results to differmaterially from such statements. A discussion of the risks and uncertaintiesthat can affect these statements is set forth in the Company’s annual andquarterly reports filed from time to time with the Securities and ExchangeCommission under the heading “Risk Factors.” The Company disclaims anyintention or obligation to revise or update any forward-looking statements,whether as a result of new information, future events, or otherwise.


Opening Remarks

Dr. Helen TorleyPresident and CEO

April 18, 2016Copyright © 2016 Halozyme, Inc.

Agenda

Time Topic Presenter

5 minutes Opening Remarks Dr. Helen TorleyPresident and CEO

10 minutes PEGPH20 Immuno-Oncology

Dr. Sanna RosengrenDirector, Immunology andCell Biology

10 minutes PEG-ADA2: PEGylated Adenosine Deaminase 2

Dr. Christopher ThanosSenior Director, Biotherapeutics Discovery

10 minutes HTI-1511: Anti-EGFR Antibody-Drug Conjugate


15 minutes Questions and Answers All

10 minutes Focus on the Tumor Microenvironment Dr. Michael LaBarre

VP, Chief Scientific Officer

3

Agenda










4



Focus on the Tumor MicroenvironmentDr. Michael LaBarreVice President, Chief Scientific Officer

April 18, 2016


Our Focus: Developing Therapeutics That Target the Tumor Microenvironment (TME)

6

• PEGylated human recombinant PH20 (PEGPH20)– to deplete hyaluronan (HA) in the TME and increase tumor access

• PEGylated adenosine deaminase 2 (PEG-ADA2)– to deplete adenosine (an immune checkpoint) in the TME

• Anti-EGFR-ADC (HTI-1511)– to bind preferentially to EGFR at low pH in TME and deliver cytotoxic

drug conjugate to tumor cells

Modify the TME Structure

Leverage the TME Biochemistry and Physiology

6

The Tumor Microenvironment

7

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA


8

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HAHA and Collagen


9

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

Immune Cells


10

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

Adenosine andMolecular Receptors


11

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

Regions of- Low O2- Low pH


12

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HAHA and Collagen

Hyaluronan (HA) Can Be a Barrier to Therapeutic and Immune Cell Access to Tumor Cells

13

• HA is a Structural Carbohydrate– Hydrophilic, viscous polysaccharide

• Stabilizes the Tumor Microenvironment– Provides structure for extracellular and

cell surface components

1 Brekken et al. Anticancer Res. 20:3503 (2000) 2 Provenzano and Hingorani, Br J Cancer 108:1 (2013) 3 Thompson et al. Mol Cancer Ther. 9:3052 (2010) 4 Stylianopoulos et al. PNAS. 110:18632 (2013)5 Singha et al. . Mol Cancer Ther. 14:523 (2015)

HA (Red) surrounding a single breast cancer cell overexpressing HAS3 (Bright Green)

Kultti, et al. JBC. 2006,281:15821

HA Surrounding a Cancer CellRed Blood Cells

Hyaluronan (HA) Can Be a Barrier to Therapeutic and Immune Cell Access to Tumor Cells

14

• HA is a Structural Carbohydrate– Hydrophilic, viscous polysaccharide

• Stabilizes the Tumor Microenvironment– Provides structure for extracellular and

cell surface components

• Compromises Access to the Tumor – Increased tumor interstitial pressure1,2

– Vasculature compression3,4

– Can decrease therapeutic and immune cell access5

1 Brekken et al. Anticancer Res. 20:3503 (2000) 2 Provenzano and Hingorani, Br J Cancer 108:1 (2013) 3 Thompson et al. Mol Cancer Ther. 9:3052 (2010) 4 Stylianopoulos et al. PNAS. 110:18632 (2013)5 Singha et al. . Mol Cancer Ther. 14:523 (2015)

HA (Red) surrounding a single breast cancer cell overexpressing HAS3 (Bright Green)

Kultti, et al. JBC. 2006,281:15821

HA Surrounding a Cancer CellRed Blood Cells

HA Accumulation Associated With Decreased Survival in Some Tumors in Clinical Trials

Negative correlation with survival also reported in gastric2, colorectal3, ovarian4, non-small cell lung5, metastatic breast6 and prostate cancers7

HA-highMedian Survival: 9.3 months

HA-low Median Survival: 24.3 months

1 Whatcott, et al. Clin Cancer Res. 21:15 (2015)2 Setala, et al. Br J Cancer 79:1133 (1999)3 Ropponen, et al. Cancer Res. 58:342 (1998)4 Antilla, et al. Cancer Res. 60:150 (2000)5 Pirinen, et al. Int J Cancer 95:12 (2001)6 Auvinen, et al. Am J Pathol. 156:529 (2000)7 Lipponen, et al. Eur J Cancer 37:849 (2001)

Pancreatic Ductal Adenocarcinoma1

15

PEGPH20 Targets Hyaluronan in the TME

PEGPH20

Removal of HA by PEGPH20 demonstrated in HA-high tumor animal models to:

Decreaseintratumoral

pressure

Decompressvasculature

Increaseperfusion

Increase access for

therapeutics

Increase access for

immune cells

16

PEGPH20 Decreased HA in an HA-high Wilms’ Tumor Animal Model

PEGPH20 37.5 µg/kg (HED)Vehicle

HA (brown) staining with HTI-601(HA-specific binding probe developed at Halozyme)

Cowell et al. (2016). AACR Annual Meeting, Poster #2463

WT-CLS1/HAS3 HA-high peritibial Wilms’ tumor model

HA StainingNuclear Staining

Tumors harvested 6h after 2 doses of Vehicle or PEGPH20 on days 0 and 3

17

PEGPH20 Reduced Tumor Interstitial Fluid Pressure in HA-high Prostate Cancer Animal Model

Thompson et al. Mol Cancer Ther. 9:3052 (2010)

- 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 00 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

1 . 2

0 . 0 1 5

0 . 1 5

1 . 5

4 . 5

1 5

V e h ic le

T i m e a f t e r T r e a t m e n t ( m i n )

No

rma

liz

ed

Tu

mo

r IF

P

~40 mmHg

IV dose

PEGPH20 Dose (mg/kg)

PC3 peritibial prostate tumor model

18

PEGPH20 Increased Tumor Perfusion inHA-high Prostate Cancer Animal Model

19

PEGPH20 (24h)Vehicle (24h)

Hyperechoic microbubbles imaged to visualize vasculature “space” or vascular area of peritibial PC3 tumors ± PEGPH20 (15 mg/kg, IV). Blue tracing is tumor area.



Vascular Decompression Increased Drug Delivery in HA-high Prostate Cancer Animal Model

20

0

5

1 0

1 5

2 0

2 5

3 0

3 5

P E G P H 2 03 hL i p o s o m a l

D o x o r u b i c in

A lo n e

*

L i p o s o m a l

D o x o r u b i c in

Tum

or

Lip

oso

ma

l Do

xo

rub

icin

(ng

/ µg

DN

A)

Increased drug accumulation not observed in normal tissues tested

Increased tumor DOXIL(measured as doxorubicin)

324%↑[doxorubicin]tum

* p < 0.05



21

0 7 1 4 2 1

0

2 5 0

5 0 0

7 5 0

1 0 0 0

1 2 5 0

1 5 0 0

D a y s o n S t u d y

Tum

or

Vo

lum

e,

mm

3 (

SE

M)

V e h i c le

P E G P H 2 0

V in + D A C T

P E G P H 2 0 + V in + D A C T

PEGPH20 37.5 µg/kg (HED), vincristine 0.415 mg/kg, dactinomycin 0.125 mg/kg 2x weekly Cowell et al. (2016). AACR Annual Meeting, Poster #2463

WT-CLS1/HAS3 HA-high peritibial tumor model

PEGPH20 Increased Activity of Vincristine and Dactinomycin in HA-high Wilms’ Tumor Animal Model

22

0 7 1 4 2 1

0

2 5 0

5 0 0

7 5 0

1 0 0 0

1 2 5 0

1 5 0 0

D a y s o n S t u d y

Tum

or

Vo

lum

e,

mm

3 (

SE

M)

V e h i c le

P E G P H 2 0

V in + D A C T

P E G P H 2 0 + V in + D A C T

PEGPH20 37.5 µg/kg (HED), vincristine 0.415 mg/kg, dactinomycin 0.125 mg/kg 2x weekly Cowell et al. (2016). AACR Annual Meeting, Poster #2463

WT-CLS1/HAS3 HA-high peritibial tumor model

With PEGPH2010/10 Regressions

Without PEGPH205/10 Regressions

PEGPH20 Increased Activity of Vincristine and Dactinomycin in HA-high Wilms’ Tumor Animal Model

PEGPH20 Increased Tumor Growth Inhibition of shIDO-ST in HA-high Pancreatic Cancer Animal Model

23

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

s h I D O - S T

P E G P H 2 0

s h I D O - S T + P E G P H 2 0

V e h i c le

p < 0 . 0 1 , A N O V A

P o s t - t u m o r i m p l a n t a t i o n ( d )

log

ph

oto

ns

/s

(x

10

6)

Manuel et al. Cancer Immunol Res. 3:1096 (2015)

KPC-derived pancreatic cancer model

PEGPH20 2.25 mg/kg on day 13 shIDO-ST 5 x 106/dose on day 14, 15 and 16(n=3/group)

shIDO-ST: Salmonella typhimurium (ST) short hairpin RNA against IDO (shIDO)

PEGPH20 With shIDO-ST Increased Immune Cell Influx in HA-high Pancreatic Cancer Animal Model

24

Manuel et al. Cancer Immunol Res. 3:1096, Figure S8 (2015)

KPC-derived pancreatic cancer model

PEGPH20 2.25 mg/kg on day 13 shIDO-ST 5 x 106/dose on day 14, 15 and 16Tumors extracted 96 hours post-ST treatment

PEGPH20 + Control-ST PEGPH20 + shIDO-STEpi-fluorescence

Counts

Neutrophil accumulation

PEGPH20 Increased Access of Various Drugs and Immune Cells in HA-high Tumor Animal Models

25

1 Singha et al. Mol Cancer Ther. 14:523 (2015), 2 Singha et al. AACR Natl Mtg(2015), 3 Manuel et al. Cancer Immunol Res. 3:1096 (2015),4 Measured as paclitaxel in Osgood et al. AACR PDA Mtg.(2014), 5 Thompson et al. Mol Cancer Ther. 9:3052 (2010), 6 Kang et al. Halozyme new data to be submitted for publication, 7Jacobetz et al. GUT 62:112 (2013)

Leukocytes (e.g., NK cells1, T cells2, neutrophils3)

shIDO-ST cellular immunotherapy3

Liposomes/nanoparticles (e.g., ABRAXANE® 4, DOXIL® 5)

Monoclonal antibodies (e.g., cetuximab6, trastuzumab1)

Small molecules (e.g., gemcitabine7)smaller

larger

Relative Size



Agenda








26



PEGPH20 Immuno-Oncology

Dr. Sanna RosengrenDirector, Immunology and Cell Biology

April 18, 2016



28

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA


29

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HAImmune Cell Involvement

Anti-Tumor T Cells Face Immunosuppressive Barriers in the TME

30

Tumorcell

Anti-tumorT cell

Antigen-presentingdendritic cell


31

Blocking interaction

Legend

Anti-tumorT cell

CTLA4

PD-1

PD-1


PD-L1PD-L1

Immune checkpoints

Tumorcell


32

Tumorcell

Anti-tumorT cell

CTLA4

PD-1

PD-1


PD-L1PD-L1

Immune checkpoints


Legend

M2macrophage

Myeloidsuppressor cell

RegulatoryT cell

Immuno-suppressive

cells


33

Immuno-suppressive

cytokines and growth factors

GRADIENT

High Levels

Tumorcell

M2macrophage

Myeloidsuppressor cell

RegulatoryT cell

Anti-tumorT cell

CTLA4

PD-1

PD-1


PD-L1PD-L1

Immuno-suppressive

cells

Immune checkpoints


Legend

Some Biomarkers of Immunosuppression Increased in HA-high Colon Cancer Animal Model

34

C T 2 6

C T 2 6 /HA S 3

0

1

2

3

4

p = 0.002

C T 2 6

C T 2 6 /HA S 3

0

1

2

3

4

p = 0.008

C T 2 6

C T 2 6 /HA S 3

0

1

2

3

4

p = 0.009

Gen

e ex

pres

sion

(Act

b-no

rmal

ized

)

Other immunosuppressive genes, including PD-L1 and IDO, were not significantly different in this model

IL10(immunosuppressive

cytokine)

FoxP3(regulatory T cell marker)

CTLA4(immune checkpoint)

CT26/HAS3 HA-high syngeneic colon tumor model

Rosengren et al. (2016). AACR Annual Meeting, Poster #4886

Does PEGPH20 Increase Activity of Anti-CTLA4 Antibodies in HA-high Tumors?

35

Cancer cell

Anti-tumorT cell

CTLA4

PD-1

PD-1


PD-L1PD-L1

CTLA4 engagement blocks co-stimulation of activated T cells

Anti-CTLA4 antibodies can lower tumor immunosuppression

PEGPH20 might enhance their efficacy in HA-high tumors

PEGPH20 Effect on Anti-CTLA4 Activity in HA-high and HA-low Colon Cancer Animal Models

36

0 5 1 0 1 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Iso ty p e c o n tro l

P E G P H 2 0

A n ti-C TL A 4

P E G P H 2 0 + a n ti-C TL A 4

D a y o n S tu d y

Tum

or

Vo

lum

e (

mm

3)±

SEM

CT26/HAS3 HA-high model

PEGPH20 37.5 µg/kg (HED) biweekly, 24h prior to anti-CTLA4 or IgG2b isotype control (4 mg/kg)

p ≤ 0.002to anti-CTLA4and PEGPH20**


PEGPH20 Effect on Anti-CTLA4 Activity in HA-high and HA-low Colon Cancer Animal Models

37

0 5 1 0 1 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0


P E G P H 2 0

A n ti-C TL A 4


D a y o n S tu d y

Tum

or

Vo

lum

e (

mm

3)±

SEM

CT26/HAS3 HA-high model

PEGPH20 37.5 µg/kg (HED) biweekly, 24h prior to anti-CTLA4 or IgG2b isotype control (4 mg/kg)

0 5 1 0 1 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0


P E G P H 2 0

A n ti-C TL A 4


D a y o n S tu d y

Tum

or

Vo

lum

e (

mm

3)±

SEM

CT26 HA-low model

Not significant

p ≤ 0.002to anti-CTLA4and PEGPH20**


Does PEGPH20 Increase the Activity of Anti-PD-1/PD-L1 Antibodies in HA-high Tumors?

38

Cancer cell

Anti-tumorT cell

CTLA4

PD-1

PD-1


PD-L1PD-L1

PD-1/PD-L1 interaction sends signal of exhaustion to T cell

Anti-PD-1/PD-L1 antibodies can lower tumor immunosuppression

PEGPH20 might enhance their efficacy in HA-high tumors

PEGPH20 Increased Activity of Anti-PD-1 in HA-high Pancreatic Cancer Animal Model

39

KPC-derived HA-high pancreatic tumor model

PEGPH20 37.5 µg/kg (HED) 2x weekly, 24h prior to anti-PD-1 (0.5 mg/kg) Rosengren et al. (2016). AACR Annual Meeting, Poster #4886

0 5 1 0 1 5 2 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

V e h ic leP E G P H 2 0

D a y o n S tu d y

Tum

or

Vo

lum

e (

mm

3)

± S

EM

A n ti- P D - 1P E G P H 2 0 / a n ti- P D - 1

* p < 0.02 to anti-PD-1and PEGPH20

PEGPH20 Increased Activity of Anti-PD-L1 in HA-high Pancreatic Cancer Animal Model

40

PEGPH20 37.5 µg/kg (HED) 2x weekly, 24h prior to anti-PD-L1 or IgG2b isotype control (2 mg/kg)



0 5 1 0 1 5 2 0 2 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Is o ty p e c o n tr o l

A n ti-P D -L 1

D a y o n S tu d y

Tu

mo

r V

olu

me

(m

m3)

± S

EM

P E G P H 2 0 / is o ty p e c o n tro l

P E G P H 2 0 / a n ti-P D -L 1

*** p < 0.0001 to anti-PD-L1and PEGPH20

- 2 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

% C

ha

ng

e T

um

or

Vo

lum

e f

rom

ba

selin

e

I so ty p ec o n tro l

P E G P H 2 0 /Iso ty p e c o n tro l

A n ti-P D -L1 P E G P H 2 0 /A n ti-P D -L1

PEGPH20 Combined With Anti-PD-L1 Induced Regressions in HA-high Pancreatic Cancer Animal Model

41


With PEGPH203/8 Regressions

PEGPH20 37.5 µg/kg (HED) 2x weekly, 24h prior to anti-PD-L1 or IgG2b isotype control (2 mg/kg) Rosengren et al. (2016). AACR Annual Meeting, Poster #4886

Human Equivalent Dose of PEGPH20 Reduced HA in HA-high Pancreatic Cancer Animal Model

42

Pre-treatment24 hours after PEGPH20

(37.5 µg/kg)HA StainingNuclear Staining


HA (brown) staining with HTI-601(HA-specific binding probe developed at Halozyme)

PEGPH20 37.5 µg/kg (HED) 2x weekly, 24h prior to anti-PD-L1 or IgG2b isotype control (2 mg/kg) Rosengren et al. (2016). AACR Annual Meeting, Poster #4886

PEGPH20 Enhanced Anti-PD-L1 Accumulation in HA-high Ovarian Cancer Animal Model

43

S al i n

e

P E GP H

2 0 37 . 5

µ pk

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

Me

an

de

ns

ity

(S

EM

)

(Ba

se

lin

e s

ub

tra

cte

d)

p = 0.006

SKOV3/HAS2 ovarian tumor model with anti-human-PD-L1-AlexaFluor 488

Anti-PD-L1PEGPH20 + Anti-PD-L1

Indi

vidu

al Tu

mor

s

Anti-PD-L1-AlexaFluor 488 Fluorescence Rosengren et al. (2016). AACR Annual Meeting, Poster #4886

PEGPH20 Immuno-Oncology Program Highlights

44

HA-high tumor status may lead to a more immunosuppressive TME

In HA-high syngeneic mouse tumors, PEGPH20 enhanced the effect of immune checkpoint inhibitors

In HA-low mouse tumors, a combinatorial effect was not observed

PEGPH20 treatment increased anti-PDL1 antibody accumulation in ovarian tumor xenograft model with elevated HA





Agenda






45



PEG-ADA2: PEGylatedAdenosine Deaminase 2Dr. Christopher ThanosSenior Director, Biotherapeutics Discovery

April 18, 2016



47

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

The Tumor Microenvironment - Adenosine

48

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

Adenosine Generation

The Adenosine Pathway: A Recognized Immune Checkpoint Interaction

Pardoll DM, Nature Reviews Cancer 12, 252-264 (April 2012)

49

Adenosine: A Key Suppressor of Immune Cells in Tumor Microenvironment

50

>100x IncreaseTumor Microenvironment Adenosine

~0.1 µM → ~50 µM

ATP

Tumor

Adapted from Stagg & Smyth, Oncogene, 2010


51


~0.1 µM → ~50 µM

ATP

ATP

Tumor



52


~0.1 µM → ~50 µM

ATP

ATP

Tumor

CD39



53


~0.1 µM → ~50 µM

ATP

ATPAMP

Tumor

CD39



54


~0.1 µM → ~50 µM

ATP

ATPAMP

Tumor

CD73CD39



55


~0.1 µM → ~50 µM

ATP

ATPAdenosineAMP

Tumor

CD73CD39



56


~0.1 µM → ~50 µMSaturation of Adenosine Receptor Checkpoints on Immune Cells

neutrophilsblocks adhesion / extravasation(-) phagocytosis(-) superoxide & nitric oxide

macrophages(-) phagocytosis(-) superoxide & nitric oxide(-) TNF-α, IL-12, MIP-1α↓ MHC class II↑ IL-10, IL-6

endothelial cells(-) ICAM-1 & E-selectin

↑ VEGF and angiogenesis↑ βFGF, IGF-1

dendritic cells↑ IL-6, IL-8, TGFβ, VEGF, IDO

(-) IL-12

NK cells(-) IFN-γ(-) cytotoxicity

T cells(-) TCR-induced NF-κβ, IL-2, IL-4, IFN-γ(-) cytotoxicity(+) anergy(+) CD4+ T cell diff. to Foxp3+ or Lag-3+ Tregs

mast cells↑ IL-4, IL-8, IL-13

↑ VEGF

NKT cells(-) IFN-γ

(+) IL-4, IL-10, TGF-β

(-) BCR-induced NF-κB activation

B cells

Tumor


ATP

ATPAdenosineAMP

CD73CD39

ADA2: An Approach to Deplete Adenosine in the TME

57

ADA2

H20 NH4

1Zavialov AV, Biochem J. 391, p51-7 (2005)2Zavialov AV, J Biol Chem. 285(16):12367-77 (2010)3Wang et al. (2016). AACR Annual Meeting, Poster #1217

Human, extracellular glycoprotein, ~120,000 MW1,2

Catalyzes the deamination of adenosine to inosine1,2

Highly resistant to inactivation in plasma3

Produced in standard CHO cells3

0

2

20

200

0 20 40 60 80

Act

ivity

(U/m

l)

Time (Hour)

ADA2-WT

PEG-ADA2-K374D

PEGylated ADA2: Improved Pharmacokinetics Profile in Animal Model

58

PEG-ADA2K374D extended PK profile in mice*• t1/2 = 37.6 hours• Less frequent dosing• Systemic dosing

Wang et al. (2016). AACR Annual Meeting, Poster #1217*Dosed at 3 mg/kg, n=9 mice/group

Tumor Growth Inhibition With PEG-ADA2 inColon Cancer Animal Model

59

0 2 4 60

2 0 0

4 0 0

6 0 0

D a y o n S tu d y

Tum

or

Vo

lum

e, m

m3 (

SEM

)V e h ic leP E G A D A 2 - K 3 7 4 D

PEGADA2-K374D, 0.3 mg/kg 2X weekly, IV(N=8)

p < 0.0001

Murine CT26 colon tumor model

Wang et al. (2016). AACR Annual Meeting, Poster #1217

PEG-ADA2 Increased T-cell Infiltration in CT26 Colon Cancer Animal Models

60

p re - d o se 6 - h o u rs p o s t - d o se0 .0

0 .1

0 .2

0 .3

0 .4

CD

3 p

osi

tive

ce

ll d

en

sity

5-fold increase

p < 0.001

Histological Assessment of T-cell infiltration

PEGADA2-K374D, 0.3 mg/kg 2X weekly, IV(N=8) Wang et al. (2016). AACR Annual Meeting, Poster #1217

Murine CT26 colon tumor model

CD73 May Be a Useful Biomarker for Identifying Tumor Types That Could Respond to PEG-ADA2

61

CD39 CD73ATP AMP Adenosine

Wang et al. (2016). AACR Annual Meeting, Poster #1217

CD73 May Be a Useful Biomarker for Identifying Tumor Types That Could Respond to PEG-ADA2

62

CD39 CD73ATP AMP Adenosine

B1 6 F 1 0 /S

C

B1 6 F 1 0 /IM

CT 2 6 /IM

L L C/S

C

L L C/IM

P a n 0 2 /SC

P a n 0 2 /IM

E MT -6

/SC

KLN

2 0 5 /SC

KLN

2 0 5 /IM

Mad is

o n -10 9 /S

C

4 T 1 /OT

MH

1 9 4 + P S C4 /IM

0 .0 1

0 .1

1

1 0

1 0 0

1 0 0 0

Fo

ld G

en

e e

xp

res

sio

nv

s C

T2

6/H

AS

3 I

M

CD39 Expression in Various Tumors*

B1 6 F 1 0 /S

C

B1 6 F 1 0 /IM

CT 2 6 /IM

L L C/S

C

L L C/IM

P a n 0 2 /SC

P a n 0 2 /IM

E MT -6

/SC

KLN

2 0 5 /SC

KLN

2 0 5 /IM

Mad is

o n -10 9 /S

C

4 T 1 /OT

MH

1 9 4 + P S C4 /IM

0 .0 1

0 .1

1

1 0

1 0 0

1 0 0 0

( , )CD73 Expression in Various Tumors*

*syngeneic mouse tumors Wang et al. (2016). AACR Annual Meeting, Poster #1217

Various solid tumor types tested

PEG-ADA2 Mediated Tumor Growth Inhibition in CD73-Positive Animal Models

63

0 5 1 0 1 5 2 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

D a y o n S tu d y

Tum

or

Vo

lum

e, m

m3 (

SEM

)

V e h ic leP E G A D A 2 - K 3 7 4 D

0 5 1 0 1 50

5 0 0

1 0 0 0

1 5 0 0

D a y o n S tu d y

Tum

or

Vo

lum

e, m

m3 (

SEM

)

V e h ic le

P E G A D A 2 - K 3 7 4 D

* p < 0.0001* p < 0.0001

*

*

KPC-derived pancreatic modelKLN-205 lung model

PEGADA2-K374D, 0.3 mg/kg 2X weekly, IV(N=8) Wang et al. (2016). AACR Annual Meeting, Poster #1217

Engineering ADA2 Yielded a Variant With a 16-fold Improvement in Enzymatic Activity

64

S265

R222

ADOAnalog PDB#

3LGG1

1Zavialov AV, J Biol Chem. 285(16):12367-77 (2010)2Wang et al. (2016). AACR Annual Meeting, Poster #1217

Engineering ADA2 Yielded a Variant With a 16-fold Improvement in Enzymatic Activity

65

kcat/KM (1/Ms)

Wild-Type 10,312

R222Q/S265N2 165,411

S265

R222

ADOAnalog PDB#

3LGG1

1Zavialov AV, J Biol Chem. 285(16):12367-77 (2010)2Wang et al. (2016). AACR Annual Meeting, Poster #1217

16x improved

10

1 0

2 0

3 0

4 0

66

Vehicle PEG-ADA2 R222Q/S265N

Study day 18, histological assessmentn=6 mice (vehicle), n=8 mice (treatment)2x weekly dosing, 0.3 mg/kg

Lung metastasesper mouse

1Wang et al. (2016). AACR Annual Meeting, Poster #1217

Mouse 4T1 breast cancer metastasis model

PEG-ADA2R222Q/S265N Inhibited Lung Metastasis in Breast Cancer Animal Model

p = 0.0183

Treatment With PEG-ADA2 Decreased TME Adenosine Levels in Pancreatic Cancer Animal Model

67

0

1 0

2 0

3 0

U n tre a te dT u m o rs

P E G -A D A 2R 2 2 2 Q /S 2 6 5 N

KPC-derived pancreatic tumor model

1Wang et al. (2016). AACR Annual Meeting, Poster #12172Protocol from: Cancer Research 57, 2602-2605(1997)

TMEAdenosine

(µM)

KPC-derived mouse tumors 10 mm probe (55 kDa MWCO) Microdialysis perfusates analyzed by LC-MS

p = 0.0127

Measuring adenosine levels in a mouse TME (solid tumor)

PEG-ADA2 Program Highlights

68

Adenosine: Attractive immune checkpoint target

• Abnormally high levels accumulate in the TME • Binds to receptor checkpoints on immune cells• Contributes to an immunosuppressive TME

PEG-ADA2: An engineered human enzyme that targets adenosine

• Improved pharmacokinetics and enzyme activity• Anti-tumor responses observed in several animal

models− Increase in T-cell infiltration− Decrease in high TME adenosine levels







Agenda




69



HTI-1511: Anti-EGFR Antibody-Drug Conjugate



April 18, 2016


71

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

The Tumor Microenvironment – Molecular Receptors

72

Collagen

Fibroblast

Vasculature

Macrophage

Treg MDSC

T cell

Adenosine

Cancer cell

MolecularReceptors

HA

Molecular Receptors

Two Limitations With Anti-EGFR Therapeutics

73

1Cunningham, NEJM 2004, 2Van Cutsem, JCO 2012, 3Amado, JCO 20084Misale, Cancer Discovery 2014, 5Barras, Biomarkers in Cancer, 20156Deerden, Annals in Oncology, 7Lee, JNCI 2013

Treatment can lead to skin rash

• May limit dosing • ~90% cutaneous side effects1-31

Downstream, activating mutations

• KRAS mutations present in over 50% of mCRC4

• BRAF mutations in ~10% of mCRC5

• EGFR mutations in ~3-19% of NSCLC in west6-72

Physicochemical Properties Offer Opportunities for TME-Specific Therapeutics

74

Adapted from Lancet Oncol 2010; 11: 661–69

Solid tumor example

Hypoxia(pimonidazole)

Proliferation(iododeoxyuridine)

Blood vessel(immunofluorescent)

Acidic pH

Tumor microenvironment ↓ pH vs. Healthy Tissue

↑ Lactate↑ Albumin

1mm

0

10

20

30

40

50

pH 6.0 pH 6.5 pH 7.4

Halozyme mAb has Attenuated In Vitro Binding to EGFR at Skin pH

75

EGFR

Bin

ding

(EC

50 n

g/m

L)

Acidic pH, TME conditions Skin pH

HALO MabCetuximab

Low Affinity

High Affinity

Huang,L. et al. (2016). AACR Annual Meeting, Poster #1472

Halozyme mAb Attenuated Human Skin Bindingvs. Human Tumor Binding in Xenograft Models

76

CetuximabControl

Comparable, strong binding between tumor

and skin

Day 1 Day 2 Day 3


Halozyme mAb Attenuated Human Skin Binding vs. Human Tumor Binding in Xenograft Models

77

CetuximabControl

HALO Anti-EGFR

mAb

Comparable, strong binding between tumor

and skin

Strong tumor, attenuated skin binding

Day 1 Day 2 Day 3


ADCs May Treat EGFR+ Mutation-Resistant Tumors

78

EGFR

KRAS

BRAF

MEK

ERK/MAPK

MigrationSurvivalAngiogenesis

Proliferation

EGFR mediated signaling promotes cell

growth

EGF

Adapted from Pao, Nature Reviews Cancer 10, 760-774 (2010)


79

EGF

NakedmAb

EGFR

KRAS

BRAF

MEK

ERK/MAPK


Proliferation


growth

Naked anti-EGFR mAbinhibits signaling

pathway

KRAS

BRAF

MEK

ERK/MAPK

No signaling

EGF



80

EGF

NakedmAb

EGFR

KRAS

BRAF

MEK

ERK/MAPK


Proliferation


growth


pathway

Mutation promotes cell growth and is resistant to

mAb therapy

KRAS

BRAF

MEK

ERK/MAPK

KRASMut

BRAF

MEK

ERK/MAPK


Proliferation

No signaling

BRAF

EGF



81

EGF

NakedmAb

EGFR

KRAS

BRAF

MEK

ERK/MAPK


ProliferationReleased Cytotoxins

Internalized ADC

ADC


growth


pathway

Mutation promotes cell growth and is resistant to

mAb therapy

ADC overcomesmutation resistance and selectively kills tumor cell

KRAS

BRAF

MEK

ERK/MAPK

KRASMut

BRAF

MEK

ERK/MAPK

KRASMut

BRAF

MEK

ERK/MAPK


Proliferation

Tumor Cell DeathNo signaling

BRAF

EGF

ADC = antibody-drug conjugate


Tumor Regressions in KRAS- and BRAF-Mutated Tumor Animal Models

82

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

H A L O V a ra n t 1 - M M A E (3 0 m g /k g )

H A L O V a r ia n t 1 - M M A E (1 0 m g /k g )

H A L O V a r ia n t 1 - M M A E (3 m g /k g )

V e h ic le

C e tu x im a b (3 0 m g /k g )


↓

T im e (D a y s )

Tum

or V

olum

e (m

m3 )±

SE

M

MDA-MB-231M (KRASG13D)

6/6 Regressions, no evidence of tumors

*Dosing stopped at Day 38

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0




H A L O V a r ia n t 1 - M M A E (1 m g /k g )C e tu x im a b (3 0 m g /k g )V e h ic le

↓

T im e (D a y s )

Tu

mo

r V

olu

me

(mm

3 )±

SE

M

HT29 (BRAFV600E)

*Dosing stopped at Day 39

Human TNBC Tumor Xenografts Human CRC Tumor Xenografts

Huang,L. et al. (2016). AACR Annual Meeting, Poster #1472n=6 mice group2X weekly dosing

Next Generation ADC Technology

83

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9

# drugs / mAb # drugs / mAb

Random Lysine Conjugation Cysteine Conjugation

0 1 2 3 4 5 6 7 8 9

# drugs / mAb

Heterogeneous More homogeneous

1st Generation Chemistries ThioBridge™ Cysteine Conjugation

Thanos, 2016 in press

HTI-1511: Anti-EGFR mAb Conjugated With Thiobridge-MMAE Highly Homogeneous

84

Chromatography Peak = HTI-1511• HALO anti-EGFR mAb• Thiobridge MMAE Chemistry• Drug: Antibody Ratio = 4

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

-2.0

5.0

10.0

15.0

20.0

25.0

mAU

Retention time (minutes)

12.4 min (1.8 %)

4 Drug Conjugates per mAb (98.2%)Analytical Hydrophobic

Interaction ChromatographyHALO

anti-EGFR mAb

4 ThiobridgeMMAE’s


Improved ADC Stability Observed in Primate Model

85

HTI-1511

Exposure Ratio (ER) = Group Mean AUC of ADC / Group Mean AUC of Total x 100

91% Intact ADC

Control HALO mAb1st Gen Conjugation

40% Intact ADC

0 10 20 300

100000

200000

300000

400000

200 400 600Time (hours)

Con

cent

rat io

n(n

g /m

L ,M

ean ±

SEM

)

0 10 20 300

100000

200000

300000

400000

200 400 600Time (hours)

Con

cent

rat io

n(n

g /m

L ,M

ean±

SEM

)

ADC Detection mAb Detection

Toxin on ADC Detection


Pilot Toxicology: Safety Profile Tested in Primate Model

86

Week 1 2 3 4

Cycle 1Endpoint

0Day 8 15 22 291

Dose

Parameters• Clinical observations and food consumption• Body weight • Dermal scoring• Clinical pathology • ECG and blood pressure• Veterinary physical examinations and ophthalmology • Histology• Pharmacokinetics

No unexpected findings observed at either dose (2.5 mg/kg and 8 mg/kg)

Safety profile met criteria for candidate nomination and further investment

Limited dermal scoring findings comparable with vehicle control group


1 Cycle, 2 Dose Study Design, N=3 animals per group

Complete Tumor Regressions Observed in Patient Derived (PDx) Tumor Models in Mice

87

NSCLC (EGFR+, KRASpG12C) PDx

0 2 0 4 0 6 0 8 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)±

SE

M

V e h ic le

H T I-1 5 1 1

last dose

Cholangiocarcinoma (EGFR+, KRASpG12A) PDx

0 2 0 4 0 6 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

S tu d y D a yT

um

or

Vo

lum

e (

mm

3)±

SE

M

V e h ic le

H T I-1 5 1 1

last dose

N=8 mice / group 2.5 mg/kg (weekly dosing) Huang,L. et al. (2016). AACR Annual Meeting, Poster #1472

HTI-1511 Anti-EGFR ADC Program Highlights

88

Engineered mAb with attenuated binding to human skin grafts

ADC mechanism targets KRAS- or BRAF-mutated tumors in mice

Utilization of next generation, Thiobridge chemistry• More homogeneous, stable

Safety profile met criteria for candidate nomination

Complete tumor responses observed in PDx tumor models

IND enabling studies underway







Agenda





89


Q&A


90

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