Exploring the effects of linker composition on site-specifically modified antibody-drug conjugates

Article abstract of DOI:10.1016/j.ejmech.2014.08.062

In the context of antibody-drug conjugates (ADCs), noncleavable linkers provide a means to deliver cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release of the free drug. Additionally, noncleavable linker

Full text of DOI:10.1016/j.ejmech.2014.08.062

European Journal of Medicinal Chemistry xxx (2014) 1e7
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the effects of linker composition on site-specifically
modified antibodyedrug conjugates
Aaron E. Albers, Albert W. Garofalo, Penelope M. Drake, Romas Kudirka,
Gregory W. de Hart, Robyn M. Barfield, Jeanne Baker, Stefanie Banas, David Rabuka
*
Redwood Bioscience, 5703 Hollis Street, Emeryville, CA 94608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2014
Received in revised form
In the context of antibodyedrug conjugates (ADCs), noncleavable linkers provide a means to deliver
cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release
of the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemical
properties of the small molecule to improve potency or diminish affinity for multidrug transporters,
thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino-iso-Pictet-
Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in the
noncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligated
through one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADC
panel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functional
properties. We observed that slight differences in linker design affected these parameters in disparate
ways, and noted that efficacy could be improved by selecting for particular attributes. These studies serve
as a starting point for the exploration of more potent noncleavable linker systems.
21 August 2014
Accepted 22 August 2014
Available online xxx
Keywords:
Antibodyedrug conjugate
Noncleavable linker
Aldehyde tag
©
2014 Elsevier Masson SAS. All rights reserved.
1
. Introduction
There are essentially two broad classes of ADC linkers; those
that are chemically labile or enzymatically-cleavable, and those
that are chemically stable or noncleavable [12]. Labile/cleavable
linkers are designed to keep the ADC intact when in circulation but
release the drug payload upon internalization by the target cell.
Some cytotoxic payloadsdfor example, MMAEdrequire a cleavable
linker, as they do not tolerate substitutions [13,14]. By contrast,
other cytotoxic payloadsdfor example, maytansinedcan accom-
modate substitutions while maintaining potency [15]. Such drugs
are good substrates for the development of noncleavable linkers. By
design, noncleavable linkers do not contain chemical functional-
ities that are readily susceptible to intracellular degradation.
Therefore, after an internalized ADC is trafficked to the lysosome,
the antibody moiety is proteolytically degraded into amino acids
while the cytotoxic drug remains attached via the linker to an
amino acid residue [16]. The retention of the linker as part of the
active metabolite allows for the modulation of the overall proper-
ties of the metabolite (e.g., by altering hydrophobicity, length, and
charge) in order to improve potency.
Antibodyedrug conjugates (ADCs) promise to alter the land-
scape of anti-cancer therapeutics by targeting highly cytotoxic drug
molecules directly to cancer cells. The success of currently
approved ADCs has inspired a spate of research and development
efforts in the area; dozens of new ADCs are in pre-clinical or clinical
trials [1]. ADCs comprise a monoclonal antibody, a cytotoxic
payload, and a linker that joins them together [2]. The monoclonal
antibody targets the payload to cells expressing the antigen on their
surface, and the cytotoxic payload kills the cells upon internaliza-
tion of the ADC. The linker is literally the central component of an
ADC; it contains the reactive group that governs the conjugation
chemistry, and serves as a chemical spacer that physically connects
the drug payload to the antibody. As such, the linker is also the most
versatile aspect of the ADC. It can be modified in any number of
ways to influence various drug/linker characteristics (e.g., solubil-
ity) [3,4] and ADC properties (e.g., potency, pharmacokinetics,
therapeutic index, and efficacy in multidrug resistant cells) [5e11].
We previously reported a novel site-specific ligation chemistry
that takes advantage of an aldehyde-tagged protein [17]. The
aldehyde tag is a straightforward means of site-specifically func-
tionalizing proteins for chemical modification. The genetically-
encoded tag consists of a pentapeptide sequence (CXPXR) that is
*
Corresponding author.
E-mail address: drabuka@redwoodbioscience.com (D. Rabuka).
http://dx.doi.org/10.1016/j.ejmech.2014.08.062
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Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062

2
A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
specifically recognized by formylglycine-generating enzyme (FGE)
18-20]. During protein expression in cells, the cysteine residue in
2.4. Bioconjugation, purification, and HPLC analytics
[
the sequence is recognized by FGE and oxidized co-translationally
to formylglycine. The resulting aldehyde affords a bioorthogonal
chemical handle for ligation (Fig. 1). Linkers terminating in a 2-
Humanized anti-HER2 IgG antibodies (15 mg/mL) bearing the
aldehyde tag (LCTPSR) at the C-terminus of the heavy chain were
conjugated to maytansine-containing drug linkers (8 mol equiva-
ꢀ
(
(1,2-dimethylhydrazinyl)methyl)-1H-indole react with the alde-
lents drug:antibody) for 72 h at 37 C in 50 mM sodium citrate,
hyde by way of a hydrazino-iso-Pictet-Spengler (HIPS) reaction to
form an azacarboline, resulting in a stable CeC bond joining the
antibody and payload.
50 mM NaCl pH 5.5 containing 0.85% DMA and 0.085% Triton X-100.
Free drug was removed using tangential flow filtration. Unconju-
gated antibody was removed using preparative-scale hydrophobic
interaction chromatography (HIC; GE Healthcare 17-5195-01) with
mobile phase A: 1.0 M ammonium sulfate, 25 mM sodium phos-
phate pH 7.0, and mobile phase B: 25% isopropanol, 18.75 mM so-
dium phosphate pH 7.0. An isocratic gradient of 33% B was used to
elute unconjugated material, followed by a linear gradient of
41e95% B to elute mono- and diconjugated species. To determine
the DAR of the final product, ADCs were examined by analytical HIC
(Tosoh #14947) with mobile phase A: 1.5 M ammonium sulfate,
25 mM sodium phosphate pH 7.0, and mobile phase B: 25% iso-
propanol, 18.75 mM sodium phosphate pH 7.0. To determine ag-
gregation, samples were analyzed using analytical size exclusion
chromatography (SEC; Tosoh #08541) with a mobile phase of
The aldehyde tag platform allows for site-specific conjugation
that yields a highly homogenous product. Accordingly, this tech-
nology is well-suited for performing structure activity relationship
studies in the context of an intact ADC. Here, we isolated linker
composition as a single variable for optimization while the other
ADC componentsdantibody backbone, cytotoxic payload, conju-
gation site, drug-to-antibody ratio, and conjugation chemis-
trydwere held constant. By characterizing a panel of five drug/
linkers and their corresponding conjugates, we explored the impact
of small changes in linker design on ADC potency and stability.
2
. Materials and methods
300 mM NaCl, 25 mM sodium phosphate pH 6.8.
2.1. Linker synthesis
2.5. In vitro cytotoxicity
Synthetic routes and analytical data are provided in the
Supplemental materials.
The HER2-positive breast carcinoma cell line, NCI-N87, was
obtained from ATCC and maintained in RPMI-1640 medium (Cell-
gro) supplemented with 10% fetal bovine serum (Invitrogen) and
Glutamax (Invitrogen). 24 h prior to plating, cells were passaged to
ensure log-phase growth. On the day of plating, 5000 cells/well
2
.2. Microtubule polymerization assay
We used the Tubulin Polymerization Assay Kit (Cytoskeleton)
were seeded onto 96-well plates in 90
supplemented with 10 IU penicillin and 10
Cellgro). Cells were treated at various concentrations with 10
m
L normal growth medium
g/mL streptomycin
L of
diluted analytes, and the plates were incubated at 37 C in an at-
mosphere of 5% CO . After 6 d, 100 L/well of CellTiter-Glo reagent
Promega) was added, and luminescence was measured using a
according to the manufacturer's instructions for the fluorescence-
based test. All test articles were used at 3 M.
m
m
(
m
ꢀ
2
m
2.3. Direct ELISA antigen binding
(
ꢀ
Molecular Devices SpectraMax M5 plate reader. GraphPad Prism
software was used for data analysis, including IC50 calculations.
Maxisorp 96-well plates (Nunc) were coated overnight at 4 C
with 1 g/mL of human HER2-His (Sino Biological) in PBS. The plate
was blocked with ELISA blocker blocking buffer (ThermoFisher),
and then the HER2 wild-type antibody and ADCs were plated in an
-step series of 2-fold dilutions starting at 100 ng/mL. The plate was
m
a
2.6. In vitro stability
8
incubated, shaking, at room temperature for 2 h. After washing in
PBS 0.1% Tween-20, bound analyte was detected with a donkey
ADCs were spiked into rat plasma at ~1 pmol (payload)/mL. The
ꢀ
samples were aliquoted and stored at ꢁ80 C until use. Aliquots
ꢀ
anti-human Fc-
gated secondary antibody. Signals were visualized with Ultra TMB
Pierce) and quenched with 2 N H SO . Absorbance at 450 nm was
g-specific horseradish peroxidase (HRP)-conju-
were placed at 37 C under 5% CO
2
for the indicated times and then
were analyzed by ELISA to assess the anti-maytansine and anti-Fab
signals. A freshly thawed aliquot was used as a reference starting
value for conjugation. All analytes were measured together on one
plate to enable comparisons across time points. First, analytes were
(
2
4
determined using a Molecular Devices SpectraMax M5 plate reader
and the data were analyzed using GraphPad Prism.
Fig. 1. The aldehyde tag coupled with HIPS ligation yields site-specifically modified antibodies. Using standard molecular biology techniques, a formylglycine-generating
enzyme (FGE) recognition sequence (CXPXR) is site-specifically inserted into the backbone of the antibody. FGE co-translationally oxidizes the cysteine residue to formylglycine.
The aldehyde of formylglycine can then be reacted with nucleophiles to form a stable CeC bond.
Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062

A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
3
diluted in blocking buffer to 20 ng/mL (within the linear range of
the assay). Then, analytes were captured on plates coated with an
anti-human Fab-specific antibody. Next, the payload was detected
with an anti-maytansine antibody followed by an HRP-conjugated
secondary; the total antibody was detected with a directly conju-
gated anti-human Fc-specific antibody. Bound secondary antibody
was visualized with TMB substrate. The colorimetric reaction was
2 4
stopped with H SO , and the absorbance at 450 nm was deter-
mined using a Molecular Devices SpectraMax M5 plate reader. Data
analysis was performed in Excel. Each sample was analyzed in
quadruplicate, and the average values were used. The ratio of anti-
maytansine signal to anti-Fab signal was used as a measure of
antibody conjugation.
2.7. Xenograft studies
The animal studies were approved by Charles River Laboratories
Institutional Animal Care and Use Committee (IACUC). Female C.B-
7
1
7 SCID mice were inoculated subcutaneously with 1 ꢂ10 NCI-N87
Fig. 2. Inclusion of amino acid residues resulted in highly soluble maytansine-
linker constructs with varied chemical composition. Five different maytansine-
conjugated linkers were synthesized (as shown in Scheme 1) and characterized,
tumor cells in 50% Matrigel. When the tumors reached an average
of 112 mm , the animals were given a single 5 mg/kg dose of ADC,
trastuzumab antibody (untagged), or vehicle alone. The animals
were monitored twice weekly for body weight and tumor size.
Tumor volume was calculated using the formula:
3
both as free drugs and after conjugation to an a-HER2 antibody.
active metabolite. We also incorporated a spacer element into the
linkersdeither PEG or n-propyldto improve conjugation effi-
2
ꢀ
ꢁ
2
w ꢂ l
3
ciency and mitigate ADC aggregation. Finally, taking advantage of
the hydrazino-iso-Pictet-Spengler (HIPS) chemistry, the linkers
terminated in either a reactive 2-((1,2-dimethylhydrazinyl)methyl)
indole (1, 2, 3, and 5) or 2-((1,2-dimethylhydrazinyl)methyl)pyrrolo
Tumor volume mm
¼
2
where w ¼ tumor width and l ¼ tumor length.
Tumor doubling times were obtained by averaging the tumor
growth rate curves from four groups of mice. Then, log10 cell kill
was estimated using the formula:
[2,3-b]pyridine (4). The latter varied from the former by a single
nitrogen atom (Fig. 2), making it slightly more hydrophilic. Both
reactive groups enabled HIPS ligation of the linker-maytansine to
aldehyde-tagged antibodies for ADC production.
A representative synthesis of the linkers is shown in Scheme 1.
In the example, a pegylated, protected amino acid, 6, is coupled to
pentafluorophenyl ester, 7. The product, 8, is then coupled to N-
deacetylmaytansine, 9, using HATU followed by hydrolysis of the
tert-butyl ester and removal of the Fmoc-protecting group with
piperidine to give the final desired product, 1.
treated group TTE ꢁ control group TTE
log10 cell kill ¼
3:32 ꢂ tumor doubling time
Treatment over control (T/C) ratios were determined by dividing
the tumor volume of the treatment group by the tumor volume of
the control group at a designated time point.
3
. Results and discussion
3.2. Linker composition did not alter the payload's ability to inhibit
3.1. Linker design and synthesis
microtubule polymerization
To examine the effect of linker composition, we tested a variety
of maytansine-linkers that contained functional groups anticipated
to aid in solubility, which improves bioconjugation yields [4].
As a first step, once the drug/linkers were in hand, we per-
formed an in vitro microtubule polymerization assay to confirm
that the incorporated structural variations and elaborations to
maytansine did not impair the drug's ability to inhibit microtubule
polymerization (Fig. 3). As anticipated, due to the known tolerance
of maytansine to substitutions at the N-acyl position [21], the panel
of drug/linkers resulted in microtubule polymerization inhibition
similar to unmodified maytansine. A small spread of values was
noted, but all were within 32% of maytansine itself. As shown in the
next section, these small differences did not appear to impact the
IC50 of the drug/linkers when formulated as an ADC.
Initially, we used PEG
n
spacers (with n ¼ 2, 4, or 6), but found that
the PEG group alone was not sufficiently hydrophilic to overcome
the very hydrophobic contributions from the maytansine and HIPS
components. The conjugation efficiencies observed with linkers
containing PEG
n
spacers alone were poor, e.g., 40% yield with a
PEG -maytansine linker conjugated to a C-terminally-tagged anti-
6
body. We found that a simple way to incorporate hydrophilicity was
by using amino acid residues as linker components (Fig. 2). In turn,
this change resulted in a significant improvement in conjugation
efficiency, e.g., 90% yield with a glutamic acid PEG
2
-maytansine
3.3. Bioconjugation and in vitro assessment of the ADC panel
linker conjugated to a C-terminally-tagged antibody. Here, we
tested the effect of using different amino acids as solubilizing
agents by evaluating glutamic acid (Linkers 1, 4, and 5), asparagine
Conjugation of the drug/linkers to a C-terminally aldehyde
tagged
a-HER2 antibody was carried out by treating the antibody at
ꢀ
(
Linker 2), and phosphotyrosine (Linker 3). The latter was meant to
37 C with 8e10 equivalents of linker-maytansine in 50 mM so-
dium citrate, 50 mM NaCl pH 5.5 containing 0.85% DMA and 0.085%
Triton X-100, and the progress of the reaction was tracked by
analytical hydrophobic interaction chromatography (HIC). Upon
completion, the excess payload was removed by tangential flow
function as a pro-drug, where the phosphorylated form would be
soluble, but not membrane permeable. Once inside a cell, the linker
was intended to be a substrate for phosphorylases, the action of
which would yield a more hydrophobic and membrane-permeable
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4
A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
Scheme 1. Representative linker synthesis.
4
3
3
2
2
1
1
000
500
000
500
000
500
000
varying concentrations of the analytes for 6 days, and then cell
viability was measured by using a CellTiter-Glo assay, which
quantifies ATP levels. All ADCs exhibited picomolar activity, with
IC50 values similar to or better than that observed after treatment
with free maytansine. By contrast to the ADCs, the free drug/linkers
were overall less potent, generally showing IC50 values that were
1000- to 2000-fold higher than the corresponding ADCs. Linkers 1
Buffer
Maytansine
Paclitaxel
Vinblastine
Linker 1
Linker 2
Linker 3
Linker 4
Linker 5
and 4, which shared a glutamic acid-PEG
2
scaffold, both had free
drug/linker IC50 values above 1 M, ~10,000-fold higher than the
m
ADC versions of those compounds. In addition to the IC50 values, we
noted that the Linker 3 ADC, in spite of its measured picomolar
activity, failed to kill more than 70% of the cells, even at the highest
doses (Fig. 5A). The free version of Linker 3 did not suffer from this
same cytotoxic plateau, reducing cell viability by >93% at the
highest dose (Fig. 5B). We observed the same trends with the un-
conjugated and conjugated versions of Linker 3 on a different
antibody and against a different cell line, suggesting that the
plateau effect of this linker is translatable across platforms.
Although the cytotoxic plateau is commonly observed in these
types of assays, the underlying mechanisms involved and the bio-
logical significance of the effect is not clear.
500
0
0
5
10
15
20
25
30
35
40
45
50
55
60
Minutes
Fig. 3. Linker selection does not hinder maytansine inhibition of microtubule
polymerization. The drug/linkers were tested in a microtubule polymerization assay.
Free maytansine was included as a positive control. Paclitaxel and vinblastine were
included as a promoter and inhibitor, respectively, of microtubule polymerization. The
buffer control indicates the polymerization rate of untreated microtubules. Relative to
free maytansine, the drug/linkers inhibited polymerization by the following amounts:
Linker 1, 68%; Linker 2, 92%; Linker 3, 79%; Linker 4, 105%; Linker 5, 73%.
As a final in vitro characterization of the ADC panel, we exam-
ined the stability of the HIPS-conjugates in plasma for 14 days at
ꢀ
3
7 C. The assay consisted of an ELISA-based method that compared
the ratio of anti-payload to anti-Fc signals. As a group, the conju-
gates exhibited a high degree of stability, with ꢃ85% payload
remaining after 7 days and ꢃ74% payload remaining after 14 days
filtration, and the unconjugated antibody was removed by pre-
parative HIC. These reactions were high yielding, with >90%
conjugation efficiency (Table S1). After purification, the ADCs
contained an average drug-to-antibody ratio (DAR) of 1.6 as
determined by hydrophobic interaction chromatography
(
2
Table 1). The glutamic acid-PEG -containing scaffolds were the
most stable over 14 days, both demonstrating more than 80%
retention of payload. The most labile linker, Linker 2, only differed
from the most stable linker by about 10% over 14 days.
(Figure S2). The drug distribution (ratio of DAR 1e2) was very
similar among the ADCs made with different linkers (Table S2). The
preparations were ꢃ95% monomeric as assessed by size-exclusion
chromatography (data not shown).
3.4. In vivo efficacy of the ADC panel
With the ADCs in hand, we first asked whether conjugation with
the drug/linkers had altered the antibody affinity for the HER2
antigen. To test this, we performed a direct-binding ELISA assay
using plates coated with human HER2-His, and compared the EC50
To test the in vivo efficacy of the ADC panel, we assessed the
conjugates using an NCI-N87 xenograft model in SCID mice. Com-
pounds were administered as a single 5 mg/kg dose at the onset of
the study. All ADCs were well-tolerated with no animal showing
>10% weight loss up to 40 days post-treatment (Figure S1). Tumor
growth was arrested, and some tumors were reduced in size after
of the wild-type (untagged and unconjugated)
a-HER2 to the values
obtained for the panel of -HER2 ADCs (Fig. 4). Only minimal dif-
a
ferences in affinity were noted, with most of the ADCs appearing to
bind with slightly higher affinity than the wild-type antibody.
Next, we tested the in vitro cytotoxicity of the ADCs against the
HER2-overexpressing gastric cell line, NCI-N87 (Fig. 5A). As a
comparator, we also tested the cytotoxic activity of the corre-
sponding free drug/linkers (Fig. 5B). Cell cultures were exposed to
treatment with the a-HER2 ADCs (Fig. 6A), but not after treatment
with the isotype control ADC (conjugated using Linker 1). Eventu-
ally, tumors began to regrow in all animals, sooner in some groups
than others, depending on the ADC used for treatment. By 60e70
days post-dose, there were clear differences in mean tumor vol-
umes among groups treated with an a-HER2 ADC; specifically, the
Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062

A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
5
Table 1
3
2
1
0
Wild-type αHER2
Linker 1
Linker 2
Linker 3
Linker 4
ꢀ
ADCs made with different linkers show similar stability in plasma at 37 C.
ADC % Conjugate remaining after 7 % Conjugate remaining after 14
days
93
days
81
a
a
a
a
a
HER2-
Linker 1
HER2-
Linker 2
HER2-
Linker 3
HER2-
Linker 5
85
93
97
95
74
77
83
77
Linker 4
HER2-
Linker 5
of the study, reducing tumor volume more than any other treat-
ment (Fig. 6A). Linker 3 had the poorest in vivo efficacy (p < 0.007,
by the log-rank, ManteleCox, test). It is interesting to consider
whether the incomplete in vitro killing of NCI-N87 target cells by
ADCs conjugated to this linker is related todor perhaps predictive
ofdits reduced in vivo efficacy as compared to the other ADCs.
Next, we selected two linkers from the initial panel to take into a
multidose efficacy study. We chose Linker 1 on the merits of its
overall potency, as measured by tumor growth, log10 cell kill, and
survival. We chose Linker 2 because it showed the fastest initial
tumor reduction, and we reasoned that perhaps this quick response
would translate into increased efficacy in a multidose setting. The
multidose study employed NCI-N87 tumors in SCID mice. Animals
were dosed (10 mg/kg) once a week for four weeks. The experiment
employed two armsdwith dosing beginning when tumors reached
average volumes of either 180 or 400 mm .
with both Linkers 1 and 2 were highly active against the smaller
tumors (Fig. 7A), and resulted in very similar levels of tumor con-
trol. By contrast, against the larger tumors, the a-HER2 ADC made
with Linker 2 showed superior efficacy, resulting in a greater level
of tumor inhibition as compared to the ADC made with Linker 1
(Fig. 7B). Specifically, the treated/control tumor volumes at day 42
were 0.39 and 0.26 for Linkers 1 and 2, respectively.
In conclusion, we developed a panel of C-terminally-conjugated
a-HER2 ADCs bearing highly similar linkers, and observed that
0.1
1
10
100
[Ab ng/ml]
Fig. 4.
affinity for HER2 antigen. A direct-binding ELISA assay, using immobilized human
HER2-His protein as the capture reagent and an anti-human Fc- -specific HRP-
conjugated antibody as the detection reagent, was used to monitor the affinity of
a-HER2 ADCs conjugated with the panel of drug/linkers maintain their
g
a
-
HER2 conjugates relative to the wild-type antibody. Standard curves of each analyte
were generated and the EC50 value of each curve was calculated using GraphPad Prism.
The calculated EC50 values (nM) were: Wild-type antibody, 8.72; Linker 1, 6.20; Linker
2
, 10.80; Linker 3, 7.10; Linker 4, 5.95; Linker 5, 7.23.
mean tumor volumes ranged from 249 to 487 mm3 at day 60
Fig. 6A). In order to investigate this effect, we looked at the
log10 cell kill for tumors dosed with the various treatments
Table 2). The results indicated that treatment with ADCs conju-
gated to Linkers 1 and 4 killed more tumor cells as compared to
treatment with the other ADCs. Notably, these two linkers repre-
sented the absolute minimum amount of chemical diversity, both
(
3
a
-HER2 ADCs made
(
2
contained the glutamic acid-PEG scaffold and differed from each
other by only a single nitrogen group in the azacarboline that forms
during ligation. This increased potency translated into a survival
advantage for animals treated with ADCs conjugated to Linkers 1 or
4
(Fig. 6B).
The efficacy of Linker 5 was reduced as compared to Linkers 1
and 4, with which it shared the glutamic acid moiety. The results of
this series of linkers suggest that, in this context, inclusion of the n-
relatively minor structural changes led to dramatic differences in
potency both in vitro and in vivo against the NCI-N87 tumor model.
Other biophysical parameters were less impacted. Specifically, we
observed only minor effects of linker architecture on inhibition of
microtubule polymerization (at the free drug/linker level), and
antibody affinity (at the ADC level). With respect to in vitro cyto-
toxicity, as a group, the ADCs were highly efficacious and yielded
very similar IC50 values, with less than a 2-fold difference
propyl spacer reduced efficacy as compared to the PEG
other two linkers, which incorporated different amino acids on the
PEG scaffold, had varying efficacy. Linker 2 showed an interme-
2
spacer. The
2
diate log10 cell kill value (reflecting total cells killed throughout the
course of the study), but was the best performer in the first 10 days
Fig. 5. Small changes in linker composition do not influence the in vitro cytotoxicity of aHER2 ADCs. NCI-N87 cells, which overexpress HER2, were used as targets for in vitro
cytotoxicity in a 6 day assay. Free maytansine (black line) was included as a positive control, and an isotype control ADC (gray line) conjugated to Linker 1 was used as a negative
control to indicate specificity. (A) ADC IC50 values (reflecting the antibody concentrations except in the case of the free drug) were measured as follows: free maytansine, 250 pM;
Linker 1, 170 pM; Linker 2, 160 pM; Linker 3, 110 pM; Linker 4, 96 pM; Linker 5, 120 pM; isotype control ADC, could not be determined. (B) Free drug/linker IC50 values were
measured as follows: free maytansine, 405 pM; Linker 1, 1.58 mM; Linker 2, 342.5 nM; Linker 3, 125.8 nM; Linker 4, ~1 mM; Linker 5, 274.9 nM.
Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062

6
A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
A.
1200
B.
Isotype ADC, Linker 1
Linker 1
1
00
1000
Linker 2
800
600
400
200
0
Linker 3
Linker 4
Linker 5
5
0
0
1
8
15 22 29 36 47 53 60 67
50
100
Days post-dose
Days post-dose
Fig. 6. Linker composition affects the in vivo efficacy of aldehyde-tagged
with NCI-N87 cells. When the tumors reached ~113 mm , the animals were given a single 5 mg/kg dose of an
a
-HER2 ADCs in an NCI-N87 tumor model. CB.17 SCID mice (8/group) were implanted subcutaneously
3
a
-HER2 conjugated to Linkers 1-5 or of an isotype control antibody
conjugated to Linker 1. (A) Tumor growth was monitored twice weekly. (B) The differences in efficacy among the ADCs tested were reflected in survival curves. Animals were
euthanized when tumors reached 800 mm3 or on day 112 of the study, whichever occurred first.
Table 2
“completeness” of the cell cytotoxicity was not always the same
between the corresponding ADC and free drug/linker analytes. For
example, treatment with free Linker 3 abrogated all but 7% of the
viable cells. With respect to in vivo cytotoxicity, ADCs made with all
of the linkers inhibited growth of the HER2-overexpressing NCI-
N87 xenograft to some extent. However, the log10 cell kill values
achieved by the ADCs varied by up to 2-fold, and the median sur-
vival time among the groups differed by 17 days, indicating that
linker structure affected efficacy. Furthermore, we observed a dif-
ference in the kinetics of tumor response to ADCs made with
distinct linkers, e.g., Linker 2 vs. Linker 1, whereby Linker 2 was
more efficacious in the short term (1 wk), but the response was
short lived. We were able to capitalize on this difference in a follow
up multidose xenograft study, in which the ADC with faster cyto-
toxic kinetics showed superior efficacy against larger tumors.
Therefore, we demonstrated that the sensitivity of our system to
linker design affords an opportunity to engineer next-generation
ADCs with optimized characteristics for improved efficacy.
In vivo log10 cell kill of NCI-N87 tumor cells achieved by a single 5 mg/kg ADC
dose.
a
-HER2 ADC linker composition
Log10 cell kill
Linker 1
Linker 2
Linker 3
Linker 4
Linker 5
1.24
0.82
0.65
1.22
0.92
encompassing the entire panel. However, one ADCdconjugated
with Linker 3dexhibited a striking viability plateau in vitro, with
2% viable cells remaining at the highest doses. By contrast to the
ADCs, the potency of the free drug/linkers varied more widely, with
a 12-fold difference encompassing the range of IC50 values. Inter-
estingly, the rank order potency of the ADC did not directly corre-
late with that of the free drug/linker. Furthermore, the
3
1
1
200
000
1200
Vehicle
Linker 1
Linker 2
B.
Vehicle
A.
Linker 1
1000
800
600
400
200
0
Linker 2
8
6
4
2
00
00
00
00
0
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
Days
Days
Fig. 7. Multidose xenograft studies reveal differences in efficacy against larger tumors between ADCs made with Linkers 1 and 2. CB.17 SCID mice (8/group) were implanted
3
subcutaneously with NCI-N87 cells. Tumors were allowed to grow to either ~180 or 400 mm (Panels A and B, respectively) and then treatment was initiated. Animals were dosed
once a week for four weeks with 10 mg/kg of an
euthanized when tumors reached 800 mm .
a
-HER2 ADC conjugated to Linkers 1 or 2. Arrows indicate dosing days. Tumor growth was monitored twice weekly. Animals were
3
Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062

A.E. Albers et al. / European Journal of Medicinal Chemistry xxx (2014) 1e7
7
Appendix A. Supplementary data
[11] P. Strop, S.-H. Liu, M. Dorywalska, K. Delaria, R.G. Dushin, T.-T. Tran, W.-H. Ho,
S. Farias, M.G. Casas, Y. Abdiche, D. Zhou, R. Chanrasekaran, C. Samain, C. Loo,
A. Rossi, M. Rickert, S. Krimm, T. Wong, S.M. Chin, J. Yu, J. Dilley, J. Chaparro-
Riggers, G.F. Filzen, C.J. O'Donnell, F. Wang, J.S. Myers, J. Pons, D.L. Shelton,
A. Rajpal, Location matters: site of conjugation modulates stability and
pharmacokinetics of antibody drug conjugates, Chem. Biol. 20 (2013)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.062.
1
61e167.
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Please cite this article in press as: A.E. Albers, et al., Exploring the effects of linker composition on site-specifically modified antibodyedrug
conjugates, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.08.062