ProTriTAC: A Protease-Activatable T Cell Engager Platform

that Links Half-Life Extension to Functional Masking

SITC 2018

PLATFORM

INTRODUCTION

Anticipated Mode of Action of ProTriTAC

αAlbumin

αTarget

αCD3

Long-lived,target-binding

prodrug

Local activation and T cell-mediated

tumor killing

Rapid clearance in circulation

Tumor CirculationCirculation

• Tumor-associated proteolytic activation reveals active T cell engager with minimal off-tumor activity after activation

IN VITRO POC

1 2 3αAlbumin sdAb

Protease-cleavable linker

…GGGGXXXXXXXXXXGGGG…

αCD3 scFv

αEGFR sdAb

protease A protease B

1. ProTriTAC alone2. + protease B3. + protease A

SDS-PAGE gel

55 kD (Prodrug)

42 kD (Active Drug)

13 kD (αAlbumin sdAb)

Potent, Protease-Dependent, Anti-Tumor Activity in

HCT116 Colorectal Tumor Xenograft Model in NCG Mice

αAlbumin

αTarget

αCD3

Control #1

No maskNon-cleavable

Control #2

MaskedNon-cleavable

Activated

ProTriTAC

ProTriTAC

MaskedCleavable

Test Article Terminal t1/2 Cmax AUC, 0-last Clearance

(hr) (nM) (hr*nM) (mL/hr/kg)

Control #1 (No mask, non-cleavable) 118 48.2 2490 0.735

Control #2 (Masked, non-cleavable) 211 58.0 7000 0.238

ProTriTAC 101 42.7 2670 0.686

Activated ProTriTAC 0.969 66.6 41.5 58.5

Prodrug Active Drug

Clearance

t1/2 = 194 hr

(calculated)

t1/2 = 0.97 hr

(empirical)

t1/2 = 211 hr

(empirical)

Clearance

Non tumor-mediated conversion in vivo

PHARMACOKINETICS

SUMMARY• ProTriTAC is a T cell engager prodrug designed to be preferentially active in the

tumor and enables targeting of a wider selection of solid tumor antigens

• Combines the best attributes of several prodrug approaches:• Steric (albumin) + specific (non-CDR loop) masking• Half-life differential of prodrug vs. active drug = additional safety• Plug-and-play with different tumor target binders

• Platform proof-of-concept:• Potent, protease-dependent, anti-tumor activity in mice• Evidence of functional masking in vitro and in vivo• Initial CMC assessments suggest feasible large-scale production

• ProTriTAC pipeline established: first clinical candidate to be nominated in 2019

250x CD3 Binding Differential in ELISA

>1000x Human Primary T Cell Binding Differential in Flow Cytometry

550x Functional Differential in T Cell Killing Assay

ProTriTACs Are Activatable by Tumor-Associated Proteases

Biological Activity In Vitro Is Dependent

on Protease Activation

IN VIVO EFFICACY

• Activated ProTriTAC not detected in circulation• Consistent with intratumoral activation of ProTriTAC

Non-Reduced Reduced

- 200

- 116- 97

- 66

- 45

- 31

- 21

- 14

- 6

1 2 3 1 2 3

MANUFACTURABILITY

CM

Filtration

Protein A

Desalt

Ion Exchange

Desalt(Formulation)

Analytical SEC+/- SDS-PAGEFraction pooling

• Yields from stable CHO cell pools comparable to regular TriTACs• Stable after repeated freeze-thaws, and at 37oC for 1 week• Comprehensive formulation development ongoing

SDS-PAGE Analysis of Three Purified ProTriTACs

GFP control TriTAC

EGFR ProTriTAC (0.03 mg/kg)

EGFR non-cleavable ProTriTAC

(0.03 mg/kg)

• Substrate linker is sufficiently stable in circulation: 50% conversion every 194 hr• Active drug does not accumulate in circulation: below 0.5% of prodrug at all times

Functional Masking and Stability of ProTriTAC Demonstrated

in a Three-Week Single-Dose Cyno PK Study

Dual Protection Against On-Target, Off-Tumor Activity

• Limited peripheral T cell binding (as demonstrated by improved PK from masking)• Rapid clearance of active drug in circulation

• Plug-and-play: made ProTriTACs with >20 binders to 5 different targets

EC50(nM)

Masking Ratio

Active Drug 0.16 -Prodrug 11.91 74Prodrug (non-cleavable) 39.44 247

EC50(nM)

Masking Ratio

Active Drug 1.19 -Prodrug >1000 n/aProdrug (non-cleavable) >1000 n/a

EC50(nM)

Masking Ratio

Active Drug 0.004 -Prodrug 0.485 121Prodrug (non-cleavable) 2.197 549

• Proprietary library of substrate linkers with different cleavability engineered

0.01 0.1 1 10 100 10000.0

0.5

1.0

1.5

2.0

Concentration (nM)

CD

3 B

indi

ng (A

bs 4

50nm

)

ELISA

Prodrug

Active Drug

Prodrug (non-cleavable)

0.001 0.01 0.1 1 100.0

5.0×106

1.0×107

1.5×107

2.0×107

Concentration (nM)

Cel

l Via

bilit

y (R

LU)

HCT116 TDCC

Prodrug

Active Drug

Prodrug (non-cleavable)

• T cell engagers transiently tether T cells to tumor cells and mediate T cell-directed tumor killing

• T cell engagers, such as blinatumomab (Blincyto®), have demonstrated clinicalactivity in several hematological malignancies

• Harpoon has developed a proprietary half-life extended T cell engager format(TriTAC™), with lead asset HPN424 targeting PSMA/CD3 in Ph1 clinical testing

• Adoption of T cell engagers in solid tumors is limited by the scarcity of tumorantigens with sufficient differential expression between tumor and normal tissue

• T cell engagers that are preferentially active in the tumor microenvironment mayenable the safe targeting of more solid tumor antigens

• ProTriTAC™ represents a new and improved approach to engineer conditionallyactive T cell engagers

ProTriTACs Are Stable and Can Be Expressed at Scale

Analytical SEC of a ProTriTACAfter Different Stress Conditions

Acquity BEH SEC 200 1.7u 4.6 x 150mm0.25 ml/min 12 mins

Condition % HMW % Main % LMW

T0 1.7 96.0 2.3

5x FT 1.6 97.0 1.4

37C 1w 2.6 95.4 2.0

ProTriTAC #

0.1 1 10 100 10000

5000

10000

15000

20000

25000

Concentration (nM)H

uman

T c

ell b

indi

ng (M

FI)

Prodrug

Active Drug

Prodrug (non-cleavable)

0 100 200 300 400 500 6000.01

0.1

1

10

100

Time (h)P

lasm

a C

once

ntra

tion

(nM

)

PSMA ProTriTAC Cyno Single-Dose PK

ProTriTAC (empirical)

Activated ProTriTAC (calculated)

ProTriTAC (empirical)

Converted active drug (calculated)

>200x

Engineering of Inhibitory Non-CDR Loops in the Anti-Albumin

sdAb Domain that Confers Half-Life Extension

CDR loops

Binds to Albumin via existing CDR loops

Binds and masks αCD3 (or αTarget) domain

via engineered non-CDR loops

αAlbumin sdAb

αTarget sdAb

αCD3 scFv

Albumin

CD3

TumorAntigen

X

• Combines both steric masking (via binding to bulky serum albumin) and specific masking (via non-CDR loops binding to the CDRs of anti-CD3 scFv domain)

• Modifying non-CDR loops does not affect albumin binding

• Single proteolytic event required for activation = more efficient conversion in tumor

Non-CDR loops

masking improves t1/2and exposureActivated ProTriTAC

=> rapid clearance

N = 2 animals per group, each at 0.1 mg/kg

protease site

5 10 15 20 25 300

200

400

600

800

1000

1200

Day, post tumor implantation

Tum

or v

olum

e, m

m^3

Xenograft HCT116-003 in NCG MiceEGFR G8 ProTriTAC Mask 27 (C01486) Dose Response

Effect of Treatment on Tumor Volume

C00646 (GFP TriTAC) 0.3 mg/kg

C01756 (EGFR G8 NCLV Mask 27) 0.03 mg/kg

C01486 (EGFR G8 PTT Mask 27) 0.03 mg/kg

Final Dose (qdx10)

0 100 200 300 400 500 6000.1

1

10

100

Time (h)

Pla

sma

Con

cent

ratio

n (n

M)

ProTriTAC

Control #2 (Masked, non-cleavable)

Control #1 (No mask, non-cleavable)

Activated ProTriTAC

S. Jack Lin, Maria Rosalyn Dayao, Kendrick J. Kim, Sony S. Rocha, Kathryn Kwant, Timothy Yu, Thomas Evans, Stephen Yu, Michael Cremin, Wade Aaron, Maria Gamez-Guerrero, Evan Callihan,

Golzar Hemmati, Kevin J. Wright, Yinghua Xiao, Manasi Barath, Che-Leung Law, Bryan Lemon, Richard Austin, Holger Wesche. Harpoon Therapeutics Inc., South San Francisco, CA