WORLD ADC Conference 2015
Challenges, Opportunities and New Approaches for
The Synthesis of Well-Defined ADCs
Workshop A: Monday October 19, 2015
Dick Vandlen
Jack Sadowsky
ADC Lab
Genentech, Inc
1
Discussion Outline:
• Production of Homogeneous Conjugates with a
Drug to Antibody Ratio (DAR) of 1
• Metabolic and Stability Issues with THIOMAB™
Drug Conjugates (TDCs)
2
Why Make Homogeneous DAR THIOMAB™ Conjugates?
• Different DAR species (DAR2, 1 and 0) may impact biological activity
• In vivo studies need precise drug loading to determine optimal anti-tumor activity
• Different DAR species may impact therapeutic index, PK parameters
and/or overall activity
• For only a limited number of drugs or antibodies can we use hydrophobic interaction
chromatography to resolve DAR species
• Pure DAR1 species desired for assay development to verify that anti-drug
antibodies properly quantitate the amount of drug present in in vivo
and clinical samples
3
Usual DAR1 TDC Synthesis Procedure
Starting Material
Product Mixture (and Ratio)
Limited Addition of Drug
+
Mal-drug
THIOMAB™
+
~1
DAR0
:
:
Pros
Cons
•  DAR1 species has
free thiol similar
to DAR1 species
generated by
typical
conjugation
process
•  Free thiol may
result in increased
aggregation
•  Stability unknown
•  Requires additional
purification steps
+
~2
DAR1
:
:
~1
DAR2
• Option requires a purification scheme to separate species
• HIC often used but doesn’t work for all antibodies or drug
combinations
• Knob-into-Hole bispecific technology possible but time-consuming
4
Not All DAR 1 TDCs Can Be Successfully Purified by HIC
300 20140108 aMUC16(thio) Enriched 1 DAR #14 [modified by olsond2]
mAU
aMUC16(thio) TFF2 2 0.5X Drug
UV_VIS_2
WVL:280 nm
2 - DAR 3 - 7.120
250
DAR1
DAR2
200
1 - DAR 2 - 6.530
3 - DAR 4 - 7.753
150
DAR0
100
50
4 - DAR 6 - 8.373
0
-50
0.0
min
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
DAR0
DAR1
DAR2
DAR4
Average
DAR
26%
42%
28%
4%
1.15
18.0
19.0
20.0
21.0
22.0
23.0
• Incomplete separation of DAR species with this THIOMAB™-drug conjugate
• Average DAR of approximately 1 with ~ 40% DAR1 species present
•  Even with additional conjugation process development, unlikely to achieve
homogeneous DAR1
Olson, et al
5
DAR 1 Enhanced Separation Procedure with pI Shifter
+ pI shifter
++++
Average DAR 1 mixture pool
Higher pI Med pI
Normal pI
Separate on
ion exchange
Purify ion exchange
Remove charges
Homogeneous DAR 1 pool
6
Antibody pI Shifting by Poly-lysine Additions to Free Cysteines
NH 2
NH 2
O
O
N
O
H
N
C
N
H
O
O
H
N
C
C
N
H
H
N
C
C
O
O
N
H
C
O
C
OH
O
MC-GKKKKK
( MCK5)
NH 2
NH 2
Mass Addition
Peptide
NH 2
O
O
MC-GKKKKK (5K)
Mw 908.6
MC-GKKKK (4K)
N
OH
C
N
H
O
780.5
C
O
After CpB trimming – 268.1 Da
# lysines added
Theoretical pI
0
8.6
4
9.0
5
9.1
8
9.3
10
9.4
7
DAR 1 Enhanced Separation Procedure
•  Starting with pool of DAR 0, 1 and 2 species of THIOMAB™-drug check for free thiols with MMTS to ensure non-modified thiols are
present (MMTS adds S-methyl, 46 Da, to each free thiol)
•  React undrugged thiols with a pI-shifting reagent (MCK5)
•  Separate the various DAR species based on altered charges (pI) of
each on cation exchange columns
•  Remove pI-shifting charges from desired DAR species with
carboxypeptidase B
•  Re-purify DAR 1 species on cation-exchange column
•  Applicable to any antibody drug combination tested
8
LC/MS of DAR 1 Enriched Pool of THIOMAB™-Mal-Drug Conjugate
Count Free Cysteines with MMTS
x10 3
2.2
+ESI Scan (3.767-4.684 min, 56 Scans) Frag=360.0V [email protected] thiomab-drug DAR1 enriched pool PTD.d Subtract Deconvoluted (I
148795.47
2
1.8
1.6
DAR 1 enriched
pool
…
150110.49
147476.43
1.4
1.2
1
DAR 1
DAR 2
DAR 0
0.8
0.6
0.4
0.2
0
x10 2
9
+ESI Scan (3.778-4.645 min, 53 Scans) Frag=360.0V [email protected] thiomab-drug DAR1 enriched pool PTD +MMTS.d Subtract Deconv
148838.57
8
7
Pool + MMTS
to count free thiols
+ 2 SMe
6
(46 Da)
(92 Da)
5
150111.24
+ 1 SMe
147568.09
…
+ 0 Sme
4
3
2
1
0
146000
146500
147000
147500
148000
148500 149000
149500
150000
Counts vs. Deconvoluted Mass (amu)
150500
151000
151500
152000
Unconjugated cysteines are detected by reaction with MMTS (addition of 46 Da/SMe)
O
S
S
O
MMTS methyl methanethiolsulfonate
9
Reaction of DAR 1 Pool with Mc-GKKKKK (MCK5)
Starting pool
DAR 1
+1 MCK5
15 min reaction
with 10x MCK5
+2 MCK5
10
0
50
100
150
200
250
ml
2D12
2D10
2D8
2D6
2D4
2D2
2C12
2C10
2C8
2C6
2C4
2C2
2B12
2B10
2B8
2B6
2B4
2B2
2A12
2A10
2A8
2A6
2A4
2A2
1H12
1H10
1H8
1H6
1H4
1H2
2500
1G12
1G10
1G8
1G6
1G4
1G2
1F12
1F10
1F8
1F6
1F4
1F2
1E12
1E10
1E8
1E6
1E4
1E2
1D12
1D10
1D8
1D6
1D4
1D2
1C12
1C10
1C8
1C6
1C4
1C2
1B12
1B10
1B8
A280
Cation Exchange Chromatography of THIOMAB™-Drug DAR1 Pool
after Reaction with Mal-GKKKKK (MCK5)
mAU
DAR 1 + 1 MCK5
DAR 2
2000
DAR 0 + 2 MCK5
1500
conductivity
1000
500
SPHP
11
0
60
80
100
120
140
1G6
1G5
1G4
1G3
1G2
1G1
1F12
1F11
1F10
1F9
1F8
1F7
1F6
1F5
1F4
1F3
1F2
1F1
1E12
1E11
1E10
1E9
1E8
1E7
1E6
1E5
1E4
1E3
1E2
1E1
1D12
1D11
1D10
1D9
1D8
1D7
1D6
1D5
1D4
1D3
1D2
1D1
1C12
1C11
1C10
1C9
1C8
1C7
1C6
1C5
1C4
1C3
1C2
1C1
1B12
1B11
1B10
A280
Re-chromatography of Final DAR 1 + 1 MCK5 Pool
after Treatment with Carboxypeptidase B
mAU
3000
2500
2000
1500
1000
500
ml
• Homogeneous DAR1 Mab obtained after SPHP chromatography
12
Enrichment of DAR1 THIOMAB™-Drug
x10 3 +ESI Scan (3.767-4.684 min, 56 Scans) Frag=360.0V [email protected] thiomab-drug DAR1 enriched pool PTD.d
2.8
DAR 1
…
DAR 2
2.6
2.4
DAR 0
2.2
2
148795.62
150109.59
Starting pool of mixture
Using a 1:1 mole ratio of
MC-VC-MMAE to Mab
147476.57
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
x10 3
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
152736.80
145446.37
0
…
+ESI Scan (5.042-5.609 min, 35 Scans) Frag=360.0V [email protected] thiomab-drug DAR1 final pool.d
149062.19
DAR 1
Final Pool After
CPB treatment and
SPHP cleanup
147745.85
144617.47
145593.84
145000
146000
150109.42
147000
148000
149000
150000
Counts vs. Deconvoluted Mass (amu)
152597.12
151000
152000
Small amount of a non-drugged species in final pool due to incomplete reaction of
the DAR 0 species with two MCK5 (species has one cap and 1 free cysteine)
153000
13
Generalized Method for Production of DAR1 THIOMABS™
• Conjugate drug and MCK5 at the same time
• Determine ratio of MCK5 : Mal-drug for maximal DAR 1 species (1:1 to 1:10)
• Separate on a cation-exchange column with NaCl gradient
• Pool second Mab peak containing 1 LD + 1 MCK5 conjugate
• Remove lysine tails with carboxypeptidase B
• Rechromatograph on cation-ion exchange column
• Final pool contains DAR 1 TDC with the other thiol blocked with a small mal-ala to prevent any
further disulfide related aggregation or modification issues.
• Method has been used for several different THIOMABS™ and linker-drugs
• Concept applicable to enrich DAR 2 species for difficult linker-drugs
14
Purity of Another THIOMAB™ DAR1 Preparation
Deconvoluted Mass Spec – Final Pool
x10 4
+ESI Scan (3.314-3.552 min, 7 Scans) Frag=350.0V LC+ G206+mck5 DAR1 final SPH
…
150779.02
3
2.8
DAR1
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
DAR0
0.4
149854.14
0.2
DAR2
151991.15
0
149000
149500
150000
150500 151000 151500 152000
Counts vs. Deconvoluted Mass (amu)
152500
153000
• ~ 95% DAR1 species, <3% each of DAR2 and DAR0 species
• Free thiol on DAR1 blocked with MC-ala moiety (Mw 268 Da)
15
Summary – Homogeneous DAR Production
• Methods have been developed to allow for highly homogenous
THIOMAB™ conjugates using thiol-reactive pI shifting reagents
• Allows for rapid enrichment of specific DAR species without
developing new methods for each TDC
16
Metabolism Issues with ADCs and TDCs
• Serum
stabilities of antibody conjugates: protein adducts
• Cleavage of linker-drugs by serum hydrolases
17
Plasma and Serum Stability Issues with Antibody Conjugates
• Maleimide hydrolysis – differential HC and LC reactivities
(Shen, B-Q, et al; Nature Biotechnology, 2012, others)
• Deconjugation with transfer of drug to albumin
(Shen, B-Q, et al; Nature Biotechnology, 2012, others)
• Reduction of disulfide-linked drugs by reducing molecules
• Adduction of serum proteins onto thio-antibodies
• Enzymatic cleavage of linker-drugs in plasma
18
Adduction of Serum Proteins to THIOMAB™ Drug Conjugates
• In serum stabilities of several TDCs, higher molecular weight species
were observed as well as deconjugation and maleimide hydrolysis
• Various thio-antibody formats and sites (LC vs HC vs Fc)
with maleimide and disulfide linked drugs were tested
19
Two Examples of Drugs Used for TDC Serum Stability Studies
TDC
-Mal-GFQALSEGSTPYDINQMLNCVGDHQ
Mal-GIBL10 peptide
(S-Me)
TDC
N
S
O
O
S
OH
N
H
N
O
O
O
O
O
N
H
PBD (disulfide)
SG3231
N
O
20
Reaction of Mab S2C6-GIBL 10 with Human Serum
x101 +ESI Scan (3.834-3.992 min, 5 Scans) Frag=350.0V 101012 S2C6-GIBL 10 +PBS
153693.17
8
REACTION PROCEDURE
Mix 25ul conjugate (~50 ug)
with 75ul human serum
(IgG depleted)
PBS
6
4
2
0
162697.01
132784.33
222176.71
204280.03
180326.48
…
x102 +ESI Scan (3.797-3.955 min, 5 Scans) Frag=350.0V 101212 S2C6-GIBL only + human serum 24hr.d
Incubate at 37C up to 96 hrs
147872.96
3
S2C6 +Human serum
2
Protein A extraction
1
0
LC/MS
134266.48
156283.98
170473.44
185672.12
199948.82 210563.37 222965.22
x101 +ESI Scan (4.317-4.553 min, 7 Scans) Frag=350.0V 101512 S2C6-GIBL 10 +human serum 24hr
+1 GIBL + 66K
217624.79
3
2
S2C6-GIBL 10 +Human serum
1
158537.97
139775.31
+2 GIBL + 66K
193836.75
172020.40 183625.28
209283.24
0
140000
150000
160000 170000 180000 190000 200000
Counts vs. Deconvoluted Mass (amu)
GIBL 10 Mal-GFQALSEGSTPYDINQMLNCVGDHQ
• Size of mass addition suggested albumin as the adduct
(S-Me)
210000
220000
Mw 2962.3
21
Reaction of Mab S2C6-GIBL10 TDC with Purified Human Serum Albumin
REACTION PROCEDURE
TIC
Mix 25ul conjugate (~50 ug)
with 20 mg/ml deblocked HSA
TDC
Buffer 24 hrs
Incubate at 37C 24 hrs
Protein A extraction
LC/MS
TDC + HSA
TDC + HSA 24 hrs
x10 2 +ESI Scan (3.720-3.957 min, 7 Scans) Frag=350.0V 100312 S2C6-GIBL 10 without albumin 24
…
153640.19
Deconvoluted Masses
Buffer 24 hrs
4
DAR 2
2
0
x10 1 +ESI Scan (4.146-4.422 min, 8 Scans) Frag=350.0V 100312 S2C6GIBL 10 + albumin 24hr-2.d
…
220592.87
4
TDC + HSA 24 hrs2
DAR 1 + HSA
DAR 2 + HSA
241282.04
0
150000 160000 170000 180000 190000 200000 210000 220000 230000 240000
Counts vs. Deconvoluted Mass (amu)
S2C6-Mal-GFQALSEGSTPYDINQMLNCVGDHQ
• HSA ionizes much better than S2C6 so amount of adducted species is over-estimated by TIC
• By UV 280, ~ 20% of Mab is adducted with HSA
S-Albumin
22
Two Examples of Drugs Used for TDC Serum Stability Studies
TDC
-Mal-GFQALSEGSTPYDINQMLNCVGDHQ
Mal-GIBL10 peptide
(S-Me)
TDC
N
S
O
O
S
OH
N
H
N
O
O
O
O
O
N
H
PBD - disulfide linked
SG3231
N
O
23
Thio-­‐Tmab-­‐Disulfide-­‐SG3231 (HC Thiol) -­‐ Modifica@on in Human Serum x10 6 +ESI TIC Scan Frag=350.0V 111512 Mab +PBS 48hr.d
1.4
1.2
1
0.8
0.6
TIC
48 hrs incubation
with human serum
followed by purification 0.4
0.2
and MS
2
Heavy chain
A118C
Human serum PBS 4
3
0
2.8
3
3.2 3.4 3.6 3.8
4
4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8
Counts vs. Acquisition Time (min)
6
6.2 6.4 6.6 6.8
7
x10 2 +ESI Scan (4.413-4.929 min, 14 Scans) Frag=350.0V 111512 Mab +human serum
4 48hr.d
149560.67
2
215298.05
Peak 2
x10 1 +ESI Scan (5.802-6.437 min, 17 Scans) Frag=350.0V 111512 Mab +human serum
48hr.d
192417.85
…
Peak 3
2
159800.09
0
168574.49
+ 66K
199586.86
168199.19
0
7.2 7.4
205776.56
179122.76
217674.46
+ 43K
217763.83
+ 38K
x10 2 +ESI Scan (7.033-7.311 min, 8 Scans) Frag=350.0V 111512 Mab +human serum 48hr.d
187666.25
1
Peak 4
0.5
0
150380.48
162439.07
174121.31
197849.41
208098.35
x10 3 +ESI Scan (3.860-4.654 min, 21 Scans) Frag=350.0V 111512 Mab +PBS 48hr.d
i
149561.64
1
PBS 0.5
157648.97
0
150000
160000
166661.31
177976.18 185434.72
198493.80
170000
180000
190000
Counts vs. Deconvoluted Mass (amu)
200000
209840.34
210000
220000
• Adducts of 66K, 43K and 38K observed adduction on A118C (HC)
• Peak 2 adduct was confirmed as albumin by HPLC purification and sequencing
24
Thio Tmab-Disulfide-SG3231 (Fc Thiol) + Human serum
6
x10
+ESI TIC Scan Frag=350.0V 111512 Mab24 +PBS 48hr.
Human serum
PBS
3
2.5
2
TIC
2
1.5
1
1
0.5
0
3
x10 1
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
Counts vs. Acquisition Time (min)
5.4
5.6
5.8
6
6.2
6.4
6.6
6.8
7
Fc
Thiol
S400C
7.2
+ 37K
+ESI Scan (7.040-7.278 min, 7 Scans) Frag=350.0V 111512 Mab24+human serum 48hrt
187127.26
6
Peak 2
4
166491.09
2
133192.16
0
x10 2
141981.49
2
152222.92
+ESI Scan (3.864-4.301 min, 12 Scans) Frag=350.0V 111512 Mab24 +human serum 48hr
4
3
194395.63
182037.54
148427.19
Peak 1
1
131349.68
0
x10 2
167079.29
175428.34
185409.66
+ESI Scan (3.864-4.221 min, 10 Scans) Frag=350.0V 111512 Mab24 +PBS 48hrt
148505.54
4
3
157098.59
140651.15
PBS
2
1
0
127856.24
130000
140777.66
140000
• Predominant 37K adduct only
162343.08
150000
160000
170000
Counts vs. Deconvoluted Mass (amu)
179243.13
180000
191393.80
190000
25
Comparison of Serum Adducts for Heavy and Light Chain THIOMAB™
Conjugates of Disulfide-linked SG3231
x10 6 +ESI TIC Scan Frag=350.0V 052313 Mab80 HC +Human serum 72hr.d
4 2
3.5
3
TDC+albumin
Intact TDC
2.5
Heavy
chain
A118C
TDC+38K
2
1.5
1
0.5
0
x10 6 +ESI TIC Scan Frag=350.0V 052313 Mab80 LC +Human serum 72hr.d
4
2
3.5
Intact TDC
3
2.5
Light
chain
V205C
2
1.5
1
0.5
1
1.5
2
2.5
3
72 hrs incubation at 37C
3.5
4
4.5
5
5.5
6
6.5
Counts vs. Acquisition Time (min)
7
7.5
8
8.5
9
9.5
26
Reac@on of Thio-­‐TMab-­‐HC-­‐A118C-­‐Disulfide SG3231 with Human Serum and Human Albumin x10 6
+ESI TIC Scan Frag=350.0V 110212 CNJ2223 24hr.d
Human serum PBS 2
2.5
2
1.5
Hindered S-S
Heavy chain
3
1
0.5
0
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
Counts vs. Acquisition Time (min)
6
6.2
6.4
6.6
6.8
7
7.2
7.4
7.6
Albumin x10 3 +ESI Scan (3.916-5.146 min, 32 Scans) Frag=350.0V 110212 Mab23+albumin 24hr.d
149577.23
2
No reaction
with Albumin
only
1
0
x10 2
153434.28
166092.58
172241.83
Human serum +427
Peak 216
+ESI Scan (5.837-6.313 min, 13 Scans) Frag=350.0V 110212 Mab23 +human serum 24hr.d
192293.97
1
0.5
166143.95
157680.27
0
199317.07
186119.29 192142.02
+ 43K
Human serum
199963.18
177757.03
x10 2 +ESI Scan (6.988-7.306 min, 9 Scans) Frag=350.0V 110212 Mab23 +human serum 24hr.d
Human Peak 3
serum 2
1
0
151605.61 156980.13
163997.95
173580.44
+38K
187606.41
184877.78 190621.92
Human serum
197725.96
x10 3 +ESI Scan (3.956-4.353 min, 11 Scans) Frag=350.0V 110212 Mab23 +PBS 24hr.d
4
149576.64
PBS 3
2
PBS
1
0
154057.83
150000
155000
162571.76
160000
171584.91
181060.95
165000 170000 175000 180000 185000
Counts vs. Deconvoluted Mass (amu)
189251.61
190000
198481.98
195000
200000
27
Serum Adducts on an@-­‐HER2 Mabs with Disulfide-­‐linked SG3231 CONJUGATE
LC thiol
variants
HC
Fc
ADDUCTS
Thio Hu Anti-Her2 4D5-LC - K149C
NONE
Thio Hu Anti-Her2 4D5-LC - S121C
NONE
Thio Hu Anti-Her2 4D5-LC -V205C
NONE
Thio Hu Anti-Her2 4D5-Hc - A118C
38K, 43K and 66K
Thio Hu Anti-Her2 4D5-Hc - A118C
38K, 43K
Thio Hu Anti-Her2 4D5-Fc - S400C
38K
• None of the light chain thio-Mab variants are adducted by serum proteins
• The heavy and Fc chain thio-Mab variants become adducted with albumin and other
serum proteins
28
Summary of ADC and TDC Serum Stabili@es • Both human and murine albumin adducts have been observed on some TDCs
• Serum albumins have a free reactive cysteine and are present at nearly 1 mM
concentrations in plasma and blood
• The HC and Fc chain THIOMAB™ variants become adducted with albumin
and other serum proteins
• Light chain THIOMAB™ variants show little or no propensity for serum
protein adduction
• Observed time-dependent albumin adduction of several different TDCs
have been seen with mouse serum (up to 50% by 96 hrs)
• Effect of serum protein adduction on activity, PK, etc. is unknown. Each
protein adducted displaces a drug, thereby decreasing overall DAR
• Site of cysteine mutation in different THIOMABS™ is important for albumin and
other protein adducts
29
Metabolism Issues with ADCs and TDCs
• Serum stabilities of antibody conjugates: protein adducts
• Cleavage of linker-drugs by hydrolases
During plasma and serum stability studies, it was noted
that several linker drugs on ADCs and TDCs were specifically
hydrolyzed in a time-dependent manner
Efforts to identify and characterize this undesirable activity were
untaken
30
Unexpected Cleavage Product of Mal-GPImeLFF-QSY7
Conjugate by Mouse Plasma
Peptide linker
Mw 1581.7
Mouse plasma, pH 7.4, 37C
O
N
+
O
N
HO
Mw 941.5
N
S
O
O
• observed cleavage products are not a result of ‘normal’ proteases
Nelson, Darwish, Lee
31
Purification of Cleavage Activity From Mouse Serum
1.  Ammonium sulfate precipitation of activity from mouse serum.
2.  Protein A column to remove mouse IgG
3.  Activity flowed through a HiTrap S column
4.  Gradient elution from a HiTrap Q column.
5.  Repurified using Mono Q column
6.  Additional non-active protein retained on a Zn-IDA column.
7.  Gel filtration on Superose 12 column
8.  Proteins identified after LysC digestion by MS/MS
Nelson
32
Purification of QSY7-cleaving Activity from Mouse Serum
1 2 3 4 5 MWM 1.  HiTrap S F.T.
2.  HiTrap Q Active pool
3.  Mono Q Active Pool
66K
4.  Zn F.T. Active Pool
5.  Superose12 Active
Pool
33
Proteins Identified From LysC Digest
Protein
Liver Carboxylesterase
Corticosteroid-binding globulin
Uncharacterized protein
Serine protease inhibitor
Alpha-1-acid glycoprotein
Alpha-1-antitrypsin
Mass
61017
44740
62158
46850
23880
45946
Peptides
16
7
9
3
3
3
• Highly purified murine enzyme preps also cleaved commercially available
carboxylase substrates
• Recombinant murine carboxylesterase (mCES) was highly active in cleaving
the QSY7 substrate
34
Characteristics of Carboxylesterases
1.  Carboxylesterases are members of the serine hydrolase family of
enzymes, widely distributed throughout the body, and are capable of
acting on numerous distinct substrates, hydrolyzing ester, thio-ester,
and amide-ester linkages
2.  Carboxylesterases are associated with the endoplasmic reticulum.
Murine carboxylesterase also found in plasma – lacks C-terminal ERtargeting sequence
3.  Known to be involved in lipid degradation, cholesterol trafficking and
the removal of highly toxic organophosphate metabolites
4.  CES is capable of metabolizing numerous human drugs including
cocaine, heroin, meperidine and lidocaine.
5.  The enzymes also process several prodrugs (e.g. CPT-11 to irinotecan
and SYD983 to duocarmycin, Synthon) to their active forms in vivo
6. Antibody drug conjugates with a carbamate-bond linkage can release
drug by carboxylesterase activity within the cell (Yasunaga, et al.,
Cancer Science, 140, 231-237, 2013)
35
Carboxylesterase Substrates
CPT-11
hCES2
Irinotecan
• Caboxylesterases can hydrolyze a wide range of substrates
36
Cleavage of MC-­‐vc-­‐PAB-­‐MMAE by Carboxylases-­‐ LCMS Analyses x10 6
+ TIC Scan 042013 Mc-vcMMAE 56755+buffer.d
Buffer 50 mM Tris, pH 7.5 +18 hydrolyzed maleimide
6
Overnight incubation
at 37C
Tris adducts
4
MC-vc-PAB-MMAE
2
0
x10 6
+ TIC Scan 042013 Mc-vcMMAE 56755+huCES1.d
huCES1
MMAE
6
4
2
0
x10 6
+ TIC Scan 042013 Mc-vcMMAE 56755+mCES.d
6
mCES MMAE 4
2
0
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
Counts vs. Acquisition Time (min)
O
O
MC-vc-PAB-MMAE
O
O
N
O
N
H
H
N
O
O
N
H
O
N
5.2
O
N
H
N
O
5.4
O
O
N
5.6
5.8
6
O
N
H
OH
NH
H 2N
•
O
Human (hCES1) and murine CES enzymes release MMAE from free linker-drug and from TDCs
37
Time Course of Cleavage Mc-VC-PAB-MMAE on Intact
THIOMAB™ by Carboxylesterases
2 hrs incubation
23 hrs incubation
151324.7
…
3
…
151163.2
Buffer
0
…
150476.1
DAR0 149467.0
0
…
149466.1
151163.6
DAR2
C
…
150475.7
0
152490.7
7
0
151162.8
149464.9
0
151164
0
…
148000 148500 149000 149500 150000 150500 151000 151500 152000 152500 153000
Counts vs. Deconvoluted Mass (amu)
Human CES1 new
Human CES1 old
0
Mouse CesC1
0
…
…
152671.8
1
148000 148500 149000 149500 150000 150500 151000 151500 152000 152500
153000
Counts vs. Deconvoluted Mass (amu)
• Partial cleavage of MMAE after 2 hrs leaving only Mc-VC nubs on Mab; complete after 23 hrs
• Not readily cleaved by muCES1 at this site
38
Cleavage of anti-gD ADC-vc-PAB-MMAE by Human Carboxylesterase 1
ADC incubated with
hCES1 for 23 hrs at 37;
Sample reduced for LCMS
x104 +ESI Scan (3.516-3.834 min, 9 Scans) Frag=350.0V 031413 Mab18 only 23hrs.
25154.12
1
0.8
+1 DAR
LC
0.6
DAR 3.3; LC isolated
for analysis
0.4
24392.52
0.2
0
24504.62
24060.03
25293.87
24645.6824774.47
x104 +ESI Scan (3.248-3.525 min, 8 Scans) Frag=350.0V 031413 Mab18+huCES1 23hrs.d
Decreased mass for
LC of 845 Da suggests
cleavage between cit and PAB
with immolative release of
MMAE
…
6
5
4
3
24305.13
2
Mal-VC nub on LC
1
0
24096.79
25141.73
24100 24200 24300 24400 24500 24600 24700 24800 24900 25000 25100 25200 25300
Counts vs. Deconvoluted Mass (amu)
• CES enzymes cleave MMAE from ADC and TDC conjugates leaving the Mal-val-cit nub
on the antibody
• Activity seen only at neutral pH values
39
THIOMAB™ HC A118C with Carbamate-Containing Linker Drug
x10 3
+ESI Scan (4.336-4.870 min, 34 Scans) Frag=350.0V 041615 Mab912+buffer-42hr.d Subtract Deconvoluted (Isotope Width=25.3)
151949.95
3
Buffer
2
152111.69
1
0
x10 3
152273.79
151802.46
150664.09
+ESI Scan (4.359-4.974 min, 39 Scans) Frag=350.0V 041615 Mab912+human Ces1 new-42hr.d Subtract Deconvoluted (Isotope Width=25.3)
151950.10
2
Human Ces1
1.5
1
0.5
150663.28
0
x10 3
1.5
150974.14
151507.86
151799.61
152111.77
152272.89
+ESI Scan (4.250-4.898 min, 41 Scans) Frag=350.0V 041615 Mab912+human Ces1 old-42hr.d Subtract Deconvoluted (Isotope Width=25.3)
151950.76
Human Ces1
1
151794.79
0.5
150662.59
0
x10 3
150979.92
150387.14
151248.76
152111.81
152273.93
151632.45
152555.24
+ESI Scan (4.435-4.969 min, 34 Scans) Frag=350.0V 041615 Mab912+murine Ces1C-42hr.d Subtract Deconvoluted (Isotope Width=25.3)
151628.82
1
Murine CesC1
0.8
0.6
151788.25
0.4
0.2
150502.70
0
150400
150600
151444.16
150822.72 151053.75
150800
151000
151200
151947.84
152066.33
151400 151600 151800 152000
Counts vs. Deconvoluted Mass (amu)
152200
152400
152600
152800
153000
• Only mu CES1 cleaves this carbamate so mouse studies are not informative of activity in humans –
similar to response reported by Dokter, et al., Mol Cancer Ther, 2014
40
Cleavage of Carbamate-containing Linkers by muCesC1
Fab domains from
intact THIOMABS™ were
generated by limited digestion
by LysC
Site
1
2
3
4
THIOMAB™ Fab
33
0
0
54
75
0
80
~95
Values in % of starting conjugate for Thio-Fabs or THIOMABS™
•  Intact THIOMAB™ structure protects sites 2 and 3 from carbamate cleavage
•  Generation of Fab structure results in exposure of site 3 to carbamate
cleavage, whereas the site 2 remains protected
41
Structures of a Mab and Carboxylesterase Showing Relative Sizes
Fab
Tamoxifen
Mab
S400C
CES1
A118C
V205C
Fab
hu CES1 + tamoxifen 1YA4
Fc
anti HIV-1 IgG 1HZH
42
Improved
Stability
Can
Better Efficacy
Improved
Stability
Can Lead
toLead
BettertoEfficacy
Shen B-Q et al Nature Biotech 30(2) 184
43
Summary – THIOMAB™ Drug Conjugate Modifications
• Conjugation site impacts stability of TDCs in multiple ways
• Antibody drug conjugates can undergo several different modifications in blood
• Carboxylesterases may cleave certain linker-drugs decreasing overall
activity in some tumor models
• Different carboxylesterases have different specificities for substrates
• Albumin and other plasma proteins containing free thiols may react with linkerdrugs on TDCs resulting in protein adducts to the TDCs
• These reactions may affect the activity of the TDCs or alter PK properties
• The three dimensional structure of antibodies may impact accessibility to linkerdrugs thereby limiting enzymatic cleavage or protein adduction
• Different THIOMAB ™ sites allow for exquisite control over TDC properties in
tumor models both in vitro and in vivo
44
Acknowledgements
•  ADC Conjugation Lab
Martine Darwish
Chris Nelson
Rachana Ohri
Byoung-Chul Lee
Craig Blanchette
Jack Sadowsky
Pragya Adhikari
Neelie Zacharias
Breanna Vollmar
Hans Erickson
•  ADC Team at Genentech
•  Seattle Genetics and Spirogen Colleagues
45