Post on 09-Dec-2021
transcript
‡ 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
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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
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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