Analytical

Characterization
of Dolaflexin-
ADCs
David Lee
WCBP, Washington DC
January 30 2018
Unleashing the Targeted Power of Antibody Drug Conjugates

Dolaflexin designed to overcome the efficacy and tolerability

Highly Differentiated Platform limitations of existing ADC technologies

A HER2 Targeted Dolaflexin ADC addressing large unmet

Lead Program, XMT-1522 in Ph 1 patient needs in breast, NSCLC and gastric

A NaPi2b Targeted Dolaflexin ADC addressing large unmet

XMT-1536 in Ph1 patient needs in ovarian and NSCLC

Robust Discovery Effort 1 IND every 12-24 months; new platform innovations

Technology licensing partnerships with Takeda and Merck

Up to $2 Billion in Partnerships KGaA; Major Strategic Expansion of Takeda partnership

Successful IPO in June 2017 raised $75mm gross proceeds,

Strong Balance Sheet Current cash balance adjusted for IPO proceeds is $137mm

NEA, Wellington, Arrowmark, Cormorant, F Prime, Pfizer

World Class Investors Ventures, Rock Springs, Takeda Pharmaceuticals
2
Schematic Structure of a Dolaflexin-Based ADC

• High DAR, DAR=10-15

• Hydrophobicity of payload offset by polar polyacetal backbone enabling higher DAR
• Novel proprietary auristatin payload
3
 XMT-1522 Achieves Durable Complete Regressions
Across Models with Range of HER2 Expression Levels

HIGH MEDIUM LOW

N87 Gastric Cancer JIMT-1 Breast Cancer SNU5 Gastric Cancer
800,000 HER2/cell 80,000 HER2/cell 22,000 HER2/cell
HER2 3+ HER2 2+ HER2 0/1+

1000 1000

1600
800 Tumor Volume (mm3) 800
Tumor Volume (mm3)

Tumor Volume (mm3)

1200
600 600
Vehicle

400 400 IgG1-dolaflexin (2 mg/kg) 800

Vehicle
IgG1-dolaflexin (3 mg/kg) T-DM1 (20 mg/kg) Vehicle
200 T-DM1 (10 mg/kg) 200 400 IgG1-dolaflexin (5 mg/kg)
XMT-1522 (2 mg/kg) T-DM1 (10 mg/kg)
XMT-1522 (1 mg/kg)
XMT-1522 (0.67 mg/kg)
0 0 0
0 20 40 60 0 20 40 60 0 20 40 60
Day (Dosing on Day 1) Day (Dosing on Day 1) Day (Dosing on Day 1)

5
 Dolaflexin Intracellular Processing

Bystander Killing No Bystander Killing

Intra-tumor

AF-HPA
metabolism AF

Primary release product IC50 > 20 nM

sub-nanomolar potency; freely cell permeable Non cell-permeable; not a Pgp substrate 5
Selected Analytical Considerations for Dolaflexin-
ADCs

Selected Direct Stochastic Conjugation Dolaflexin ADC

Attributes
Linker-drug • Small simple structure • Large complex structure
• Comparability of Dolaflexin
batches important for ADC
comparability
Average DAR • Typically by HIC • HIC currently not useful for DAR

Positional • Limited number of positional • Heterogeneity and reactivity of

Isomers / isomers, can be identified and Dolaflexin complicates structural
structure quantitated, conjugation sites analysis
identified • ADC structure defined in part by
• % DAR=0, 2, 4, 6, 8 number of Dolaflexins attached,
conjugation sites occupied by
Dolaflexin
6
Dolaflexin Process

7
Sources of Heterogeneity in Dolaflexin ADCs

• Heterogeneity due to regiochemistry and loading factors:

Unit 1: 2 – 4 mole% (~ 2 units/polymer)

Unit 4: 8 – 9 mole% (3 – 4 warheads/polymer)

Unit 3: 20 mole% (10 – 12 units/polymer)

Unit 2: ≤ 70 mole% (~ 35 – 40 units/polymer)

8
MW Determined by SEC

9
NMR Spectrum of Dolaflexin

H H H H
H H H H
O O O O O O OO
H H H H HH
H H H H H H H H n4
n3 H n1 H n2
H OH O H OH OH H OH O H OH O
O O O
HN HN HN

O
O O
HN HO HN
O
O
O NH
H H H H
H H O
Backbone H H H H H
H HN O
H H O OCH3 O OCH3 O
+ others O
O
N N
N N
H N N N
H H H H O O H H O H
Maleimide H

AF-HPA

10
Selected Batch Data

Batch #
Attribute Assay 1 2 3 4 5 6 7
MW (kDa) SEC 8.3 12.5 10.8 11.2 11.4 9.3 11.3
PDI SEC 1.3 1.6 1.5 1.4 1.4 1.3 1.4
Drug Load % NMR 9.4 9.5 9.0 9.1 9.0 9.1 9.0
Free Drug % LC-MS 0.4 0.7 0.1 0.2 0.2 0.4 0.1
Linker Load % NMR 2.4 3.5 3.6 3.5 3.8 3.5 3.5

11
Controlling Dolaflexin Heterogeneity
Conclusions

• Heterogeneity in Dolaflexin can be minimized and controlled

by:

– Control of raw material properties (e.g. – Dextran MW)

– Precise control and monitoring of reaction conditions
– Chromatographic fractionations with established pooling criteria

• Dolaflexin Critical Quality Attributes have shown good batch-

to-batch reproducibility

12
Characterization of Dolaflexin
ADCs

13
Dolaflexin-ADC Process

Buffer Exchange Reduction of Conjugation

of Antibody Antibody and Quench

Formulation and Buffer Exchange Purification by

Bottling of ADC Chromatography

14
Selected Characterization Test Results
Attribute Assay Result
1 DAR RP-HPLC DAR=11-13

2 ADC covalent structure MALDI-TOF of cross-linked Verified intact MW of ADC and conjugated
ADC polypeptides
3 ADC covalent structure MSSV (AUC) Dolaflexin:mAb ~ 3:1

4 ADC covalent structure Western blot Verified cross-linking of LC and HC by Dolaflexin

5 ADC covalent structure Peptide map / MS / MS Verified correct conjugation sites
6 Secondary structure Circular dichroism Verified antibody-like structure, comparable to
spectroscopy unconjugated mAb
7 Higher order structure Disulfide bond mapping Verified antibody-like SS bond pattern

8 Higher order structure DSC Verified ADC has similar thermal stability as
mAb
9 Higher order structure Analytical ultracentrifugation Confirmed monomeric nature of ADC
10 Biological activity Biolayer interferometry Confirmed comparable binding kinetics as mAb

11 Impurities Free Dolaflexin by HPLC Dolaflexin not detected

15
DAR Determination for Dolaflexin ADCs
HIC of a Dolaflexin ADC RP-HPLC of a Dolaflexin ADC
100000
50
mAb

40 ADC 75000

AF-HPA
30
mAu

uV
50000

20
25000

10

0
0
6 10 14 18 22 26 30 4 5 6 7 8 9 10
Minutes mi n
Minutes

• Current HIC method is uninformative with respect to • AF-HPA cleaved from ADC by base hydrolysis
average DAR • Protein and polymer is precipitated
• AF-HPA concentration is quantitated by RP-HPLC
• DAR is calculated using known molar protein
concentration 16
MALDI-TOF of Chemically Cross-linked ADC
Suggests 1-3 Dolaflexins per Antibody

Intact ADC, z=+2

Intact ADC, z=+1

LC
HC

17
Multisignal Sedimentation Velocity Analysis for
Average Dolaflexin:mAb

3 Dolaflexin: 1 mAb

In collaboration with Peter Schuck, NIH

18
Dolaflexin-ADCs are Likely Intramolecularly Cross-
linked by Dolaflexin
R-CE-SDS WB Anti-Kappa Anti-FC Anti-1267
(Light chain) (Heavy chain) (Payload)
50975

XMT-1536_rQuality MW (kDa)
PDA - 220nmXMT-1535_r 250 mAb ADC Dfx mAb ADC Dfx mAb ADC Dfx
0.16
HC mAb 0.16
24008

150
LC
0.14 0.14
100
0.12 0.12
42143

59778

75734
31467

48090
11099

75
23586

0.10 0.10
ADC
AU

AU
0.08 0.08
52015

50

0.06 0.06

37
0.04 0.04
127031
107976

147135
62614

85597
74607

0.02 0.02
38989

261299
11099

25

0.00 20
0.00
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Minutes
19
In-Source MS Fragmentation of Dolaflexin

Theoretical Mass Observed Mass Mass Accuracy O O O O O O O O O O

(M+H)+ (M+H)+ (ppm) n3 n2 n5 n4
OH O OH OH n1 OH O OH O OH O
O O O
602.2668 602.2660 1.33 HN HN
O
HN
HN

803.5641 803.5627 1.74 HN

O
O O O
HN HO O
1105.6755 1105.6757 0.18 HO
O
728.4957 728.4943 3.28 O
NH

NH
O
O O O
553.3407 N HN
O OMe O OMe O Me
N
N
N Me
z=2 N N
100 602.2660 O O
H O H

728.4943

1105.6757
Relative Abundance

624.3776
z=2
696.4684 765.9219 1134.7047 1239.7346
z=1 z=2 z=1 z=1
658.4532 803.5627 1197.7226
z=1 z=1
874.6017 1281.7456
z=2 985.3913 1033.6617 z=1
z=? z=1
0
500 m/z 1300 20
 Verification of Interchain Cysteine Conjugation
Through In-Source Fragmentation of Df Peptides
Observed + 601 Da
Theoretical Mass
Base Formula Sequence Z Mass
Mass (m/z) Error
(m/z)
C155H238N36O45 THTCPPCPAPELLGGPSVFLFPPKPK 3 1130.2362 1130.2367 0.44
S2 or
THTCPPCPAPELLGGPSVFLFPPKPK
C178H274N40O56 THTCPPCPAPELLGGPSVFLFPPKPK 4 983.9885 983.9889 0.41
S2
C57H88N16O24S SFNRGEC 2 707.3004 707.3012 1.13
C41H68N10O20S SCDK 2 527.2239 527.2239 0.00
C= Maleimide-conjugated Cys C= Unconjugated Cys • Conjugated peptides
containing intrachain
100
1130.9053
z=3
1130.5717
984.4891
984.2402z=4 100
707.3004
z=2
527.2239
100 z=2
cysteines not detected
z=3
100 z=4
Relative Abundance

984.7426
1131.2388
z=3
z=4 707.8020
z=2 • ID and conjugation site
1130.2362
z=3 1131.5724
z=3
527.7253
z=2 verified by MS/MS
983.9885
z=4
984.9905
z=4
985.4914
708.3032
z=2 sequencing (data not
1131.9073
z=3 z=4
708.8033
z=2
528.2262
z=2
528.7252
shown)
z=2
0 0 0 0 22
1130 m/z 1132 984.0 m/z 985.5 707 m/z 70921 527.5 m/z 528.5
Characterization of Dolaflexin ADC
Conclusions

• Dolaflexin ADC has high DAR compared to typical DAR=4

ADCs
• ~ 3 Dolaflexins per ADC
• mAb likely intramolecularly cross-linked by Dolaflexin
• Conjugation occurs only at interchain cysteines

22
Acknowledgements

Analytical ADC Dolaflexin

• Dmitry Gumerov • Tim Lowinger • Michael Kauffman
• Venu Reddy
• Susan Clardy • Mao Yin
• Reddy Bollu
• Kenneth Avocetien • Lei Zhu • Jacques LeBlanc
• Mark Nazzaro • Dorin Toader • Tom Wagler
• Alex Johnson • Dan Custer
• Yuanyuan Li

Collaborators
• Peter Schuck, NIH

Special thanks to our patients in clinical trials and their families

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Thank you!

January 2017 24