ProTriTAC: A Protease-Activatable T Cell Engager Platform ... · •Single proteolytic event...
1
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 Circulation Circulation • Tumor-associated proteolytic activation reveals active T cell engager with minimal off-tumor activity after activation IN VITRO POC 1 2 3 αAlbumin sdAb Protease-cleavable linker …GGGGXXXXXXXXXXGGGG… αCD3 scFv αEGFR sdAb protease A protease B 1. ProTriTAC alone 2. + protease B 3. + 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 mask Non-cleavable Control #2 Masked Non-cleavable Activated ProTriTAC ProTriTAC Masked Cleavable Test Article Terminal t 1/2 C max 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 t 1/2 = 194 hr (calculated) t 1/2 = 0.97 hr (empirical) t 1/2 = 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 -6 123 123 MANUFACTURABILITY CM Filtration Protein A Desalt Ion Exchange Desalt (Formulation) Analytical SEC +/- SDS-PAGE Fraction pooling • Yields from stable CHO cell pools comparable to regular TriTACs • Stable after repeated freeze-thaws, and at 37 o C 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 EC 50 (nM) Masking Ratio Active Drug 0.16 - Prodrug 11.91 74 Prodrug (non-cleavable) 39.44 247 EC 50 ( nM ) Masking Ratio Active Drug 1.19 - Prodrug >1000 n/a Prodrug (non-cleavable) >1000 n/a EC 50 (nM) Masking Ratio Active Drug 0.004 - Prodrug 0.485 121 Prodrug (non-cleavable) 2.197 549 • Proprietary library of substrate linkers with different cleavability engineered 0.01 0.1 1 10 100 1000 0.0 0.5 1.0 1.5 2.0 Concentration (nM) CD3 Binding (Abs 450nm) Prodrug Active Drug Prodrug (non-cleavable) 0.001 0.01 0.1 1 10 0.0 5.0×10 6 1.0×10 7 1.5×10 7 2.0×10 7 Concentration (nM) Cell Viability (RLU) 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 clinical activity 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 tumor antigens with sufficient differential expression between tumor and normal tissue • T cell engagers that are preferentially active in the tumor microenvironment may enable the safe targeting of more solid tumor antigens • ProTriTAC™ represents a new and improved approach to engineer conditionally active T cell engagers ProTriTACs Are Stable and Can Be Expressed at Scale Analytical SEC of a ProTriTAC After Different Stress Conditions Acquity BEH SEC 200 1.7u 4.6 x 150mm 0.25 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 1000 0 5000 10000 15000 20000 25000 Concentration (nM) Human T cell binding (MFI) Prodrug Active Drug Prodrug (non-cleavable) 0 100 200 300 400 500 600 0.01 0.1 1 10 100 Time (h) Plasma Concentration (nM) 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 Tumor Antigen 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 t 1/2 and exposure Activated ProTriTAC => rapid clearance N = 2 animals per group, each at 0.1 mg/kg protease site 5 10 15 20 25 30 0 200 400 600 800 1000 1200 Day, post tumor implantation Tumor volume, mm^3 Final Dose (qdx10) 0 100 200 300 400 500 600 0.1 1 10 100 Time (h) Plasma Concentration (nM) 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
Transcript of ProTriTAC: A Protease-Activatable T Cell Engager Platform ... · •Single proteolytic event...
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
![CDR FoodLab Line [FOODLAB - Brownstone Asia-Tech 2 of 9 [OxiTester] (Code CDR - 225006Z01) Analysis system for test of Acidity (FFA), Peroxides (PV) and Poliphenols/stability index](https://static.fdocument.org/doc/165x107/5ea63c60ff517674815cb116/cdr-foodlab-line-foodlab-brownstone-asia-2-of-9-oxitester-code-cdr-225006z01.jpg)
![arXiv:1606.08415v4 [cs.LG] 8 Jul 2020Figure 1: The GELU ( = 0;˙= 1), ReLU, and ELU ( = 1). Masking inputs in this fashion re-tains non-determinism but maintains dependency upon the](https://static.fdocument.org/doc/165x107/5f62fa87c0efba7c406f83f0/arxiv160608415v4-cslg-8-jul-2020-figure-1-the-gelu-0-1-relu-and.jpg)
![Index []Beer foam carbonation, 314 fatty acids, 71 hops, 46 proteolytic enzymes, 264 total soluble nitrogen, 29 Beer industry advertising, 15 beer sales, 7 beer segments, 12 classification](https://static.fdocument.org/doc/165x107/5f1733991d40922a93492bd1/index-beer-foam-carbonation-314-fatty-acids-71-hops-46-proteolytic-enzymes.jpg)
