‡ Jian Chen, ‡

Brian Melo, ‡ Matthew Myers, ‡

Martha Vallejo, ‡ Xinqun Wu, Priya Sriraman, Yongjun Xue and Sekhar Surapaneni

Drug Metabolism and Pharmacokinetics (DMPK) , Non Clinical Development (NCD) , Celgene Corporations, Summit, NJ

Characterization of Pharmacokinetic Properties for a Site-Specific Antibody Drug Conjugate

(ADC) Using Multiple-Platform Bioanalysis Assays

Conclusions

Methods

Introduction

Results

Methods (cont’d) Discussion

Comparing Total-conjugated Drug and Total Antibody in Circulation

Acknowledgments

ADC: Mechanism of Action and PK Questions

Modified from Thudium et al., Mabs 2012.

Payload Release

Enzymatic or Chemical

degradation of ADC to release

payload

CDR Mediated Binding

to Target expressing

cell

Can the released payload leave

the cell and get back into

circulation ?

?

Does the ADC fall apart in

circulation?

Multiple Platform Bioanalytical Assays to Assess Different PK

Properties of ADC

Total Antibody Assay by ELISA

Standard conjugation techniques yield a heterogeneous mix of ADCs

Joseph Piccotti and Nate Collins : toxicology study design and conduct

Eric Schwartz, Xiao-Ping Dai for reagents. Jason Kahana for anti-toxin antibody.

Jason Kahana and the ADC team

Steve Maxwell, and Gondi Kumar for discussions and resources

‡ : Author contributed independently for each of the five assays

Concentration – time profiles for total conjugated drug correlated well with those from total antibody

assays, suggesting that this site-specific ADC candidate is stable in circulation.

DAR measurement confirmed in vivo linker stability and maintenance of drug antibody ratio (DAR)

of 2 - Both ELISA- and MS-based assays performed reliably during sample analysis, with the

consistent results among multiple assays (from multiple analytical platforms).

The ADC drug compound was stable in circulation for at least up to the 10 days tested.

Concentrations of free catabolite in circulation were minimal, less than 5 ng/mL at low or mid dose

and less than 25 ng/mL at high dose. No detectable free drug/toxin, or other drug containing

moieties in circulation.

All animals that survived past 2 weeks were identified as positive for the presence of anti-drug

antibodies

The total conjugated antibody assay utilizes robust tryptic digestion and provides a highly

reproducible DAR-bias-free assay. It’s internal standard undergoes all steps including

immunocapture and tryptic digestion which increases reproducibility and robustness of the assay

Total antibody, total conjugated drug, DAR, catabolite and immunogenicity assays collectively

provided comprehensive PK data to support the multi-phased exploratory monkey tox study.

Site-specific conjugation Advantages:

Specific location of payloads

Precise drug/antibody ratio (DAR)

Adapted from Kaur et al. (2014)

Range: 100-3000 ng/mL; in Monkey Serum

0

0.5

1

1.5

2

2.5

3

0 500 1000 1500 2000 2500 3000 3500

DAR 2

D2 QC

D1 QC

D0 QC

OD

Units (

ELIS

A a

ssay R

eadout)

ng/mL Antibody

Site Specific Conjugation

Catabolite (released by cells via Ab digestion) concentrations are in the low ng/mL range in circulation. These

concentrations are in the range of highly stable ADCs, and expected to be relatively ‘safe’

No free toxin was detected in circulation (data not shown). Free toxin is expected only if the toxin ‘falls-off’ during circulation,

due to linker instability, or if there was free toxin contamination in the dosing material

We have demonstrated that the ADC is stable in circulation, and the drug : antibody ratio (DAR) of 2 is maintained

throughout. There is no detectable de-conjugated ADC in circulation.

1h post dose

24h post dose

72h post dose

96h post dose

Dar0

Dar1

Dar2

All human antibodies

circulating in monkey

serum were DAR 2 at all

time-points tested.

Samples collected from

high dose animals were

tested up to 96 hours

post dose

Each DAR species is

indicated by the colored

arrows

Molar ratio of

Total-conjugated-drug

Total-antibody

at all time-points on the PK curve and at all

doses in the monkey study, indicating a DAR

2 is maintained.

≈ 2

Conjugated drug is defined as drug that is still linked to the antibody. Concentrations of conjugated drug were much higher

(over 1000 fold) than concentrations of released catabolite. Conjugated drug is typically a driver of efficacy, while free

catabolite or toxin in circulation is a driver of non-target mediated toxicities.

Assay should detect all antibody species equally

αhF(ab)-HRP Detection

ADC

Immuno- Capture

•Quantitation of peptide containing the tryptic peptide-linker-toxin complex

•Standard curve range 100 to 25000 ng/mL of ADC (DAR-2) in monkey serum

•Internal Standard , an analog ADC with a slightly different linker, was spiked at the

beginning of Immunocapture

Total Conjugated Drug Assay by Hybrid IP-LC/MS/MS

Trypsin

Digestion

Captured ADCs

and its Internal

Standard

Immuno-capture of human antibody from monkey serum

samples using biotinylated anti-Human Fc antibody; then

pulled down by Streptavidin coated magnetic beads

Antidrug Antibody (ADA) Assay by ELISA

Immuno-capture of human antibody from monkey serum

samples using biotinylated anti-Human Fc antibody; then

pulled down by Streptavidin coated magnetic beads

Captured

ADCs

DAR 2

DAR 1

DAR 0

Drug-Antibody Ratio (DAR) measurement was conducted

using high-resolution mass spectrometry (LC/HR-MS) on a

Shimadzu HPLC coupled with Sciex 5600 Q-TOF.

m/z spectrum of each sample was de-convoluted by

software (BioPharmaView) to produced mass distribution

graphs of intact ADC showing DAR distribution

High Resolution MS for Drug-Antibody Ratio (DAR) Analysis

No digestion, Intact

protein MS

Exploratory Toxicity Study in Monkey

Sample Name: "1 017 CC-DISC-ET-2140_catabolite STD1 1 1" Sample ID: "17" File: "Run 1.wiff"Peak Name: "TCRS-358" Mass(es): "780.669/749.500 Da"Comment: "none" Annotation: ""

Sample Index: 17

Sample Type: Standard

Concentration: 1.00 ng/mL

Calculated Conc: 1.09 ng/mL

Acq. Date: 7/1/2015

Acq. Time: 7:57:56 PM

Modified: No

Proc. Algorithm: Analyst Classic

Bunching Factor: 2

Noise Threshold: 30.55 cps

Area Threshold: 152.77 cps

,Num. Smooths: 1

Sep. Width: 0.20

Sep. Height: 0.01

Exp. Peak Ratio: 5.00

Exp. Adj. Ratio: 4.00

Exp. Val. Ratio: 3.00

RT Window: 30.0 sec

Expected RT: 2.61 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 2.64 min

Area: 1232.403 counts

Height: 4.95e+002 cps

Start Time: 2.57 min

End Time: 2.71 min

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300

310

320

330

340

350

360

370

380

390

400

410

420

430

440

450

460

470

480

490

500

510

Intensi

ty, cps

2.64

1.01

2.36

2.232.75

1.741.15 1.80

1.451.192.03

1.52 2.841.931.03

Sample Name: "1 017 CC-DISC-ET-2140_catabolite STD1 1 1" Sample ID: "17" File: "Run 1.wiff"Peak Name: "TCRS-450_IS(IS)" Mass(es): "785.630/754.400 Da"Comment: "none" Annotation: ""

Sample Index: 17

Sample Type: Standard

Concentration: 1.00 ng/mL

Calculated Conc: N/A

Acq. Date: 7/1/2015

Acq. Time: 7:57:56 PM

Modified: No

Proc. Algorithm: Analyst Classic

Bunching Factor: 1

Noise Threshold: 28.93 cps

Area Threshold: 144.67 cps

,Num. Smooths: 1

Sep. Width: 0.20

Sep. Height: 0.01

Exp. Peak Ratio: 5.00

Exp. Adj. Ratio: 4.00

Exp. Val. Ratio: 3.00

RT Window: 30.0 sec

Expected RT: 2.62 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 2.64 min

Area: 49785.55 counts

Height: 1.98e+004 cps

Start Time: 2.58 min

End Time: 2.72 min

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.6e4

1.7e4

1.7e4

1.8e4

1.8e4

1.9e4

1.9e4

2.0e4

Intensi

ty, cps

2.64

2.902.19

Catabolite Assay by LC-MS/MS

•Catabolite is defined as the active species that is

released from the ADC, upon internalization

•The Catabolite generated from our ADC (non-

Cleavable linker) is : NN-aa + Linker + Toxin

• Requires very low LLOQ due to low catabolite

levels, but highly toxic

•LLOQ=1 ng/mL

• Capture reagent: ADC

• Testing sample: predose and last

sample(2% serum)

• Detection reagent: mouse anti-

monkey IgG HRP (1:12000 dilution)

• Substrate: TMB (3,3', 5,5"-

tetramethylbenzidine)

Drug-Antibody Ratio Analysis

Antidrug Antibody (ADA)

Pre-dose samples were ADA negative; No ADA was detected in samples collected prior to Day 10

Animals dosed with ADC were ADA positive on Day 29. The Presence of ADA may have an impact PK parameters

Hamilton STAR on Day 1

•STD/QC preparation

•STD/QC/ sample aliquot

•IS addition

•Addition of immuno -

capture reagent

•Incubate

overnight at

4°C

Hamilton STAR on Day 2

•Add beads

•Incubate 1hr at RT on deck

•Wash beads multiple times ( 3X)

•Shake plate between washes

• Elute from beads

• Reduction and Alkylation

• Add Trypsin

Incubate

overnight at

37°C

LC-MS/MS

Automated Sample Preparation for Total-Conjugated Drug Assay

• Study includes single dose and multi-dose arms

• Approximately 230 serum samples need to be analyze for

Total Antibody (Elisa)

Total Conjugate drug

DAR

Catabolite

ADA (Elisa)

•Quick turn around time to support program decision

DAR-adjusted to show

overlapping of total

conjugate and total

antibody in multiday arm

Surrogate analyte Surrogate IS

Catabolite