Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα)

Article abstract of DOI:10.1016/j.bmcl.2019.126907

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Full text of DOI:10.1016/j.bmcl.2019.126907

Journal Pre-proofs
Antibody-Mediated Delivery of Chimeric Protein Degraders Which Target Es-
trogen Receptor Alpha (ERα)
Peter S. Dragovich, Pragya Adhikari, Robert A. Blake, Nicole Blaquiere, Jinhua
Chen, Yun-Xing Cheng, Willem den Besten, Jinping Han, Steven J. Hartman,
Jintang He, Mingtao He, Ellen Rei Ingalla, Amrita V. Kamath, Tracy
Kleinheinz, Tommy Lai, Douglas D. Leipold, Chun Sing Li, Qi Liu, Jiawei Lu,
Ying Lu, Fanwei Meng, Lingyao Meng, Carl Ng, Kaishan Peng, Gail Lewis
Phillips, Thomas H. Pillow, Rebecca K. Rowntree, Jack D. Sadowsky, Deepak
Sampath, Leanna Staben, Steven T. Staben, John Wai, Kunpeng Wan, Xinxin
Wang, BinQing Wei, Ingrid E. Wertz, Jianfeng Xin, Keyang Xu, Hui Yao,
Richard Zang, Donglu Zhang, Hao Zhou, Yongxin Zhao
PII:
S0960-894X(19)30885-6
DOI:
https://doi.org/10.1016/j.bmcl.2019.126907
BMCL 126907
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 October 2019
9 December 2019
10 December 2019
Please cite this article as: Dragovich, P.S., Adhikari, P., Blake, R.A., Blaquiere, N., Chen, J., Cheng, Y-X., den
Besten, W., Han, J., Hartman, S.J., He, J., He, M., Rei Ingalla, E., Kamath, A.V., Kleinheinz, T., Lai, T., Leipold,
D.D., Sing Li, C., Liu, Q., Lu, J., Lu, Y., Meng, F., Meng, L., Ng, C., Peng, K., Lewis Phillips, G., Pillow, T.H.,
Rowntree, R.K., Sadowsky, J.D., Sampath, D., Staben, L., Staben, S.T., Wai, J., Wan, K., Wang, X., Wei, B., Wertz,
I.E., Xin, J., Xu, K., Yao, H., Zang, R., Zhang, D., Zhou, H., Zhao, Y., Antibody-Mediated Delivery of Chimeric
Protein Degraders Which Target Estrogen Receptor Alpha (ERα), Bioorganic & Medicinal Chemistry Letters
(2019), doi: https://doi.org/10.1016/j.bmcl.2019.126907
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Antibody-Mediated Delivery of Chimeric Protein Degraders
Which Target Estrogen Receptor Alpha (ER)
Peter S. Dragovicha,*, Pragya Adhikaria, Robert A. Blakea, Nicole Blaquierea, Jinhua Chenc,
Yun-Xing Chengd, Willem den Bestena, Jinping Hand, Steven J. Hartmana, Jintang Hea,
Mingtao Hed, Ellen Rei Ingallaa, Amrita V. Kamatha, Tracy Kleinheinza, Tommy Laic,
Douglas D. Leipolda, Chun Sing Lic, Qi Liud, Jiawei Lub, Ying Luc, Fanwei Mengd, Lingyao
Menga, Carl Nga, Kaishan Pengb, Gail Lewis Phillipsa, Thomas H. Pillowa, Rebecca K.
Rowntreea, Jack D. Sadowskya, Deepak Sampatha, Leanna Stabena, Steven T. Stabena, John
Waic, Kunpeng Wanc, Xinxin Wangc, BinQing Weia, Ingrid E. Wertza, Jianfeng Xind, Keyang
Xua, Hui Yaoc, Richard Zanga, Donglu Zhanga, Hao Zhouc, Yongxin Zhaoc
aGenentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
bWuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
China
cWuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
China
d
Pharmaron Beijing, Co. Ltd., 6 Tai He Road, BDA Beijing, 100176, P. R. China
1

Abstract
Chimeric molecules which effect intracellular degradation of target proteins via E3
ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal
chemistry. However, these entities are relatively large compounds that often possess
molecular characteristics which may compromise oral bioavailability, solubility, and/or in
vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric
degraders to monoclonal antibodies using technologies originally developed for cytotoxic
payloads might provide alternate delivery options for these novel agents. In this report we
describe the construction of several degrader-antibody conjugates comprised of two distinct
ER-targeting degrader entities and three independent ADC linker modalities. We
subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the
degrader payloads that are incorporated into these conjugates. We also provide evidence for
efficient intracellular degrader release from one of the employed linkers. In addition,
preliminary data are described which suggest that reasonably favorable in vivo stability
properties are associated with the linkers utilized to construct the degrader conjugates.
2

The use of synthetic molecules to induce the intracellular degradation of targeted
proteins is a rapidly expanding area of medicinal chemistry research.1 These entities often
function by forming a ternary complex between a protein of interest and an E3 ubiquitin
ligase which results in ubiquitination of the target and its subsequent destruction via
trafficking to the proteasome.2 Degraders which operate in this manner are typically
comprised of (1) a moiety which binds to the target of interest, (2) an E3 ligase recognition
element, and (3) a spacer group which connects the first two components (Figure 1).2 These
entities have been previously named PROTACs (proteolysis targeting chimeras),3 SNIPERS
(specific and nongenetic IAP-dependent protein erasers),4 and degronimers in the chemical
literature. However, for consistency and simplicity purposes, the term “chimeric degrader”
will be used to describe such molecules throughout the course of this work.
Figure 1. General description of chimeric degrader construction and biological interactions.
Biologically active chimeric degraders that utilize a variety of distinct E3 ligases have
been reported to effectively degrade many different target proteins.1,2,5 These results suggest
that the degradation approach to protein modulation may have broad application in the
biology and medicinal chemistry fields. Given their mechanisms of action, chimeric
degraders offer several advantages over conventional small-molecule inhibitors including the
3

potential to operate catalytically,6 the ablation of target protein scaffolding properties,6 and
the ability to function by binding to virtually any site on a target protein.1,2 However, such
degraders are also relatively large entities that often possess molecular characteristics which
may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties.
The majority of in vivo experiments conducted with chimeric degraders to date employ
subcutaneous or intraperitoneal dosing regimens,7 although reports of intravenously-
delivered8 and orally bioavailable9 entities are now beginning to emerge.10 Accordingly, we
wished to explore whether conjugation of chimeric degraderss to monoclonal antibodies
might provide alternate delivery options for the degrader molecules.11 In this report, we
broaden our initial studies involving BET-targeting chimeric degrader antibody-conjugates12
by successfully expanding the conjugation concept to entities which degrade a different
intracellular protein target.13
Modulation of the estrogen receptor (ER) is known to have beneficial effects in
patients with ER-positive breast cancers, and the selective estrogen receptor modulator
(SERM) tamoxifen (1) is widely prescribed to such individuals as an adjuvant therapy
following surgery (Figure 2).14 More recently, the selective ER down-regulator (SERD)
fulvestrant (2), which strongly induces degradation of the ER protein, was introduced as an
improved treatment for ER-positive breast cancers in patients with disease progression
following traditional anti-estrogen therapy.15 The clinical success of fulvestrant has spurred
the identification of additional ER degraders, and multiple examples of similarly functioning
small molecules have recently appeared in the literature.15 Several chimeric degraders which
target the alpha-isoform of the ER protein (ER) have also been described, and
representative examples of these entities are shown in Figure 2 (3 and 4).4a,16 Importantly,
these molecules illustrate the diversity with respect to ER binding elements and ligase
ligands (3 = VHL, 4 = XIAP) that can be tolerated in these types of chimeric ER degraders.
4

Figure 2. Molecules which modulate estrogen receptor activity and/or protein levels.
OH
O
N
H
O
F
F
H
H
S
HO
CF3
1
2
OH
S
H
O
H
N
N
O
H
H
H
N
HO
N
O
O
OH
3
N
O
O
N
O
O
O
S
HN
N
O
NH
3
O
4
OH
As part of our own exploration of ER-modulating compounds, we profiled the ability
of chimeric degraders 5 and 6 to degrade the ER protein in cells (Figure 3). Like compound
4 above, these molecules both contain the known tamoxifen metabolite endoxifen (9) that
was prepared as a roughly 1:1 mixture of E and Z olefin isomers.17 In addition, compound 5
incorporates a ligase binding element that recognizes the baculoviral IAP repeat 2 (BIR2)
domain of the XIAP protein.18 In contrast, compound 6 contains a peptidomimetic moiety
5

known to potently associate with an alternate E3 ligase: the von Hippel-Lindau tumor
suppressor protein (VHL).19,20 As shown in Table 1 and Figure S1, compounds 5 and 6
induced potent and near-complete degradation of the ER protein when assessed via an
immunofluorescence (IF) readout in parental MCF7 breast cancer cells as well as in MCF7
cells that had been engineered to express high levels of the HER2 cell-surface receptor.21
A
control compound which incorporated an epimer of the hydroxy-proline moiety present in 6
(compound 7) exhibited attenuated but measurable ER degradation activity relative to the
latter molecule (Table 1 and Figure S1, note differences in Max Degradation outcomes). This
result was consistent with proper recognition of VHL by degraders such as 6 being a
requirement for potent and extensive degradation effects. The observed degradation
properties exhibited by 7 likely resulted from binary complex formation with ER due to the
endoxifen ligand contained within its chemical structure. Consistent with this hypothesis,
endoxifen itself (compound 9) displayed ER degradation effects in the IF assessment that
were comparable to those exhibited by compound 7 (Max Degradation = approximately 50%;
Table 1 and Figure S1).
Table 1. Degradation of the estrogen receptor alpha by SERDs 2, 5, and 6 and associated
control compounds in MCF7-neo/HER2 and parental MCF7 cell lines. Time point = 4 h.
MCF7-neo/HER2
MCF7 Parental
ER DC50
ER % Max
ER DC50
ER % Max
Cmpd
(nM)a
Degradationb
(nM)a
Degradationb
0.45 ± 0.01
0.68 ± 0.13
2.7 ± 0.71
5.2 ± 0.01
131 ± 2.1
1.6 ± 0.01
87
87
92
54
75
49
2
5
6
7
8
9
0.38 ± 0.07
1.6 ± 0.12
4.9 ± 0.57
7.9 ± 0.71
305 ± 103
1.1 ± 0.37
84
85
92
57
76
48
aDC50 indicates the concentration achieving 50% degradation of ER.
bMaximum reduction of ER protein levels relative to DMSO-treated controls.
6

Figure 3. Structures of chimeric degraders 5-8.
OR
NC
O
N
N
O
O
O
O
N
O
H
O
N
NH
O
O
5
8
R = OH
R = CH2Ph
HN
OH
OH
O
*
N
N
O
O
O
N
H
N
O
O
N
O
H
S
6
7
* = (R)
* = (S)
OH
H
N
O
9
Legend. The red portions depict the ER-binding regions of the compounds. The purple
sections indicate the fragments that recognize an E3 ligase (5 and 8 = XIAP, 6 and 7 = VHL).
The structure of endoxifen (9) is also shown.
The concentration-dependent ER degradation activity of compound 6 was also
confirmed via western blot analysis in MCF7-neo/HER2 cells (Figure S3). However, the
ER-degradation effects observed via IF for compounds 7 and 9 were not apparent in the
western blot assessments (Figure S3). Related discrepancies between IF and western ER-
7

degradation outcomes were previously observed for relatively weak and/or incomplete
degraders (e.g., tamoxifen).22 These differences may result from ER conformational and/or
subcellular localization changes induced by the weak ligands that limit/prevent access of the
ER detection antibody in the intracellular environment during IF assessments (related
access problems would not exist for western blot analysis since the cells are lysed and the
ER protein is fully denatured).22 A second control compound which contained a benzylated
phenol moiety in the ER-binding region exhibited significantly weaker DC50 values relative
to the parent molecule and thereby illustrated the importance of the free phenol in
maintaining biological potency (compare compound 8 with 5; Table 1).
As a prelude to preparing antibody conjugates capable of delivering compounds 5 and
6, we first assessed their stability toward human liver lysosomes to ensure that they were
robust enough to survive exposure to the intracellular lysosomal environment following
antibody-mediated delivery.23 As shown in Table S1, both molecules displayed acceptable
stability properties in these in vitro assessments (T1/2 >24 h), although some hydrolysis of an
amide linkage present in 6 was observed. We then transformed chimeric degrader 5 into the
linker-drug molecule 10 in preparation for synthesizing the desired conjugates (Figure 4).
Compound 10 contains a Valine-Citrulline-para-amino-benzyloxy moiety (Val-Cit-PAB)
linker that can undergo protease-mediated cleavage in lysosomes following antibody-directed
intracellular delivery to efficiently release a variety of attached payloads (Scheme S1).24 In
addition, the maleimide present in linker-drug 10 rapidly reacts with engineered Cys residues
on the surface of monoclonal antibodies to afford homogeneous conjugation products (Figure
4).25,26 Accordingly, we first attempted to prepare a conjugate derived from 10 and a HER2-
targeting antibody in which engineered Cys residues were introduced at the LC-K149 site
[target drug-antibody ratio (DAR) = 2.0].27 This mAb location affords maleimide-derived
ADCs that are highly stable in vivo (i.e., do not appreciably undergo retro-Michael-related
8

deconjugation).25,28 However, although the desired conjugate could be synthesized, it
exhibited extensive self-aggregation behavior that precluded its isolation in quantities and
purities sufficient for subsequent biological profiling. We suspected that such undesired
properties resulted from the highly hydrophobic nature of linker-drug 10 that was not well-
tolerated on the surface of the antibody.29
Figure 4. Structures of linker-drugs 10 and 11 derived from chimeric degrader 5 and
construction of associated ADCs.
OH
NC
O
N
N
O
O
O
O
N
O
H
O
N
NH
O
O
O
O
O
O
H
N
N
N
N
N
H
O
H
O
O
O
HN
10
H2N
O
O
O
O
O
H
N
N
N
N
H
H
O
O
NC
NH
O
N
N
O
O
NH2
O
O
N
O
H
O
N
NH
O
O
11
HN
10
HER2- -lc
O
or
10
11
+
S
SH
11
HER2- -lc
2
N
R
11
B7H4- -lc
O
11
HER2- -hc
2
Legend. Blue fragments depict the linker portions of the molecules employed for mAb
attachment.
9

Table 2. Degradation of the alpha estrogen receptor by unconjugated HER2 mAb, HER2
antibody-conjugates, and various control antibody-conjugates in MCF7-neo/HER2 and
parental MCF7 cells. Time point = 72 h.
MCF7-neo/HER2
MCF7 Parental
ER Max
ER Max
Conjugatea
DARb
Sitec
ER DC50
ER DC50
Degrad.
(%)e
ND
81
Degrad.
(%)e
ND
71
(μg/mL)d
(μg/mL)d
HER2-10-lcf
HER2-11-lc
B7H4-11-lc
HER2-12
CD22-12
HER2-13
HER2-14
CD22-14
HER2-mAb
NAg LC-K149
ND
ND
22 ± 3.4
43 ± 16
2.0
2.3
5.9
5.7
5.9
5.6
5.9
NA
LC-K149 0.11 ± 0.001
LC-K149
multi
multi
multi
multi
multi
NA
50 ± 23
0.04 ± 0.007
0.51 ± 0.094
0.05 ± 0.016
0.03 ± 0.002
4.2 ± 0.078
0.04
40
99
90
87
94
70
43
62
95
93
51
95
91
9
0.23 ± 0.07
0.48 ± 0.16
0.70 ± 0.068
0.09 ± 0.013
1.6 ± 0.035
>100
aUnless otherwise indicated, all HER2 conjugates were prepared using the 4D5 mAb variant
that recognizes HER2 extracellular domain region IV.
bDrug-antibody ratio.
cDAR2 ADCs were prepared via conjugation to Cys engineered at the indicated mAb
location (LC = light chain, HC = heavy chain); DAR6 conjugates were all prepared via
simultaneous derivatization of engineered Cys at the LC-K149, HC-L174, and HC-Y373
mAb locations.
dDC50 indicates the concentration achieving 50% degradation of ER.
eMaximum reduction of ER protein levels relative to DMSO-treated controls.
fConjugate was prepared using the 7C2 mAb variant that recognizes HER2 extra cellular
domain region I. This alternate mAb variant is not anticipated to significantly alter
aggregation outcomes relative to the 4D5 format.
gSignificant aggregation was observed during the conjugation process (28%). The
aggregated material could not be separated from the monomer conjugate via various
purification techniques.
NA = not applicable.
ND = not determined.
10

As one solution to this aggregation challenge, we prepared compound 11 in which the
Val-Cit-PAB moiety was attached to compound 5 via an ether linkage to the phenol that was
present in the ER-binding portion of the chimeric degrader. The electron withdrawing
substituents located para to this phenol moiety (olefin substituted with additional aromatic
rings) should appropriately polarize that functional group such that efficient release of 5
would follow protease-mediated cleavage of the Val-Cit-PAB linker present in 11 (Scheme
S2).30 In addition, the alternate linker connection approach employed with compound 11 did
not modify the basic amine present in 5 (as was the case with compound 10). Accordingly,
protonation of this moiety at physiological pH should enhance the aqueous solubility of 11
relative to 10 and thereby reduce the aggregation potential of conjugates prepared using the
former linker-drug molecule.
With the above strategies in mind, a HER2-targeting DAR2 conjugate was
successfully prepared from compound 11 that displayed manageable aggregation properties
(Table 2, HER2-11-lc). Encouragingly, the conjugate exhibited potent reduction in ER
protein levels when assessed using MCF7-neo/HER2 cells, and this activity was well-
separated from that displayed by both a B7H4-targeting control conjugate prepared from the
same linker-drug (B7H4-11-lc) and an unconjugated HER2-targeting mAb (Table 2, Figure
S2A).31 The HER2-11-lc ADC also exhibited attenuated ER degradation activity when
tested using parental MCF7 cells that did not strongly express the HER2 receptor (Table 2,
Figure S2B). Collectively, these outcomes were consistent with the antibody-mediated
delivery of compound 5 via the HER2-targeting conjugate to the MCF7-neo/HER2 cells. The
data also supported efficient intracellular release of 5 by the employed cleavable linker, since
catabolites containing O-benzyl-substituted endoxifen moieties should exhibit significantly
attenuated ER degradation properties (c.f., compound 8, Table 1).
11

In preparation for possible in vivo pharmacodynamic and/or efficacy studies utilizing
the HER2-11-lc ADC, we first assessed its stability in a mouse pharmacokinetic experiment.
Unfortunately, as a result of unexpected biotransformation of the attached chimeric degrader,
the conjugate was surprisingly unstable during in vivo circulation in mouse (Figure S4).
Specifically, a critical amide linkage present in linker-drug 11 was observed to undergo near-
complete biotransformation after 4 days following initial intravenous administration of the
HER2-11-lc ADC to mice (Figures S4). Although we could not confirm the structure(s) of
the biotransformation products with certainty (Figure S5), we suspected that the described
modifications were likely to significantly impair the ability of the corresponding degrader to
interact with the intended E3 ligase. Accordingly, we were concerned that the noted
instability would complicate the interpretation of subsequent in vivo pharmacodynamic
and/or efficacy experiments conducted with conjugates such as HER2-11-lc, and we therefore
shifted our studies to the exploration of ADCs that did not incorporate 5 in some form.
With these new goals in mind, we prepared linker-drugs 12 and 13 respectively from
compound 6 and its corresponding hydroxy-proline epimer 7 (Figure 5). These molecules
contain a methanethiosulfonyl (MTS) disulfide moiety that efficiently reacts with Cys
residues on the surface of engineered monoclonal antibodies and thereby connects the linker-
drugs to the biologics via new disulfide bonds (Figure 5).32 The resulting disulfide-based
conjugates should release the attached degrader payloads following internalization into
targeted cells, disulfide reduction, and subsequent linker self-immolation (Scheme S3).33 In
addition, linker-drugs 12 and 13 utilize carbonate functional groups to connect the disulfide
linkers to the associated payloads as opposed to the carbamate moieties that are frequently
employed in ADC linker-drug designs.34 Accordingly, a methyl group was incorporated
adjacent to the carbonate groups of 12 and 13 in order to protect those moieties from
unwanted hydrolysis during circulation in vivo.
12

Figure 5. Structures of linker-drugs 12 and 13 derived from chimeric degrader 6 and epimer
7 and construction of associated ADCs.
OH
O
O
O
O
S
S
O
O
*
N
O
N
O
O
N
H
N
H
O
O
N
O
S
12
13
* = (R)
* = (S)
12
HER2-
or
12
13
+
S
SH
12
CD22-
6
S
OR
13
HER2-
6
Legend. Green fragments depict the linker portions of the molecules employed for mAb
attachment.
As shown in Table 2 and Figure 5, several antibody-drug conjugates were
successfully prepared using linker-drugs 12 and 13. Since the latter molecules were
significantly less hydrophobic than linker-drugs 10 and 11 described above, 12 and 13 could
be conjugated to mAbs bearing six engineered surface Cys residues without encountering
significant aggregation issues [target drug-antibody ratio (DAR) = 6.0]. The higher drug
loading of the new conjugates relative to the entities described earlier in this work would
likely afford increased intracellular concentrations of the corresponding chimeric degraders
following ADC-mediated delivery.35 As with the DAR2 conjugates described earlier in this
work, the locations of the introduced Cys residues (LC-K149, HC-L174, and HC-Y373)27 all
afforded disulfide-linked conjugates that exhibited relatively good in vivo stability
properties.28
13

The HER2-targeting conjugate derived from linker-drug 12 exhibited strong ER
degradation activity when assessed using the MCF7-neo/HER2 cell line that was much more
extensive than effects observed for the corresponding unconjugated HER2 mAb (Table 2,
compare HER2-12 with HER2-mAb, Figure S2A). Weaker ER degradation was noted for a
related control conjugate (CD22-12, Table 2, Figure S2A), although the potency difference
relative to the HER2-12 ADC was not as dramatic as that observed previously for conjugate
pairs derived from linker-drug 11 (c.f., HER2-11-lc and B7H4-11-lc, Table 2, Figure S2A).
These IF outcomes observed for HER2-12, CD22-12, and the unconjugated HER2 mAb in
the MCF7-neo/HER2 cell line were qualitatively reproduced in separate western blot
experiments (Figure S3). A relatively small activity difference was also detected when the
HER2-12 conjugate was tested using MCF7 parental cells as compared to the MCF7-
neo/HER2 outcomes (Table 2, Figure S2B). The degradation data obtained with HER2-12
and CD22-12 suggested that the disulfide linker present in the conjugates underwent partial
cleavage in the cell-culture media during the course of the in vitro experiments. Similar in
vitro instability was previously noted for conjugates derived from related disulfide-containing
linker-drugs.21 Importantly however, these legacy ADCs exhibited reasonably good in vivo
stability properties and also displayed large separations between targeted and non-targeted
pharmacodynamic effects in xenograft tumor models.21 We were therefore encouraged by
(and not concerned with) the in vitro activity profiles displayed by HER2-12 and CD22-12.
Somewhat surprisingly, control conjugate HER2-13 displayed ER degradation
properties in MCF7-neo/HER2 cells that were similar to those exhibited by the HER2-12
ADC (Table 2, Figure S2A; these results were also observed qualitatively via western blot,
Figure S3). In contrast, greater activity differences between the two conjugates were noted
when they were tested in MCF7 cells that did not express high levels of the HER2 receptor
(Table 2, Figure S2B). The latter results were consistent with (1) a nominal level of ER
14

degradation activity associated with HER2-13 in MCF7 cells due to the presence of the
endoxifen ER binding fragment in the released degrader 7 (compare compounds 7 and 9 in
Table 1 and Figure S1B) and (2) enhanced activity of HER2-12 relative to HER2-13 due to
additional VHL-mediated ER degradation effects (compare compounds 6 and 7 in Table 1
and Figure S1B). The stronger ER degradation activity observed for HER2-13 in the
MCF7-neo/HER2 experiments relative to parental MCF7 cells presumably results from the
combination of endoxifen-derived activity and the (relatively small) HER2-mAb ER
alterations that are exclusively observed in the former line (c.f., Table 1, Figure S2A). Due to
these combined effects, it was difficult to assess the efficiency of intracellular release of
degrader 6 from HER2-12 in the MCF7-neo/HER2 experiments.
We also explored an alternate method to attach 6 to various antibodies that did not
involve disulfide-based linkers. As shown in Figure 6 below, we utilized a pyrophosphate di-
ester moiety to derivatize the secondary hydroxyl group present in 6 and thereby connect the
chimeric degrader molecule with a maleimide that could be employed for conjugation
purposes.
The corresponding antibody-drug conjugates should undergo sequential
phosphodiesterase and phosphatase-mediated hydrolysis following lysosomal antibody
catabolism to release 6 (Scheme S4). Related pyrophosphate di-esters were described by
others and were utilized to construct ADCs bearing glucocorticoid payloads.36 However, one
of these earlier reports also described the failure of phosphatase enzymes to efficiently
hydrolyze a secondary glucocorticoid phosphate ester that was generated following initial
pyrophosphate di-ester cleavage.36a Accordingly, prior to synthesizing linker-drug 14, we
first confirmed via assessment of a simple model system that the corresponding secondary
phosphate moiety present in the expected intracellular catabolite was cleaved in a simulated
lysosomal environment (transformation of 15 to 16, Figure 7, see Table S2 and Supporting
Information for additional details).
15

Figure 6. Structure of alternate linker-drug 14 derived from chimeric degrader 6 and
construction of associated ADCs.
O
O
O
N
O
O
OH
N
P
H
O
O
O
P
O
O
O
N
O
N
O
O
N
H
N
O
O
N
O
H
S
14
O
14
+
14
HER2-
S
SH
6
N
R
14
CD22-
O
6
Legend. Brown fragments depict the linker portion of compound 14 employed for mAb
attachment.
Figure 7. Conversion of model compound 15 to 16 via phosphate ester hydrolysis.
O
OH
P
O
OH
OH
O
O
N
N
N
N
H
H
N
H
N
O
O
N
N
O
O
H
S
S
15
16
16

Encouragingly, a DAR6 HER2-targeting conjugate prepared using linker-drug 14
displayed strong ER degradation activity in MCF7-neo/HER2 cells that was clearly
separated from the activities of the corresponding anti-CD22 ADC and the unconjugated
HER2 mAb (Table 2, Figure S2A, compare HER2-14 with CD22-14 and HER2-mAb).
These results were consistent with the antigen-selective delivery of compound 6 to the
engineered MCF7 cells via the HER2-targeting conjugate. The data also suggested that
efficient intracellular release of 6 occurred via the anticipated phosphodiesterase cleavage
mechanism (c.f., Scheme S4). However, since we did not prepare and test a pyrophosphate
di-ester control conjugate derived from compound 7, we cannot rule out that the ER
degradation activity observed for HER2-14 results from a combination of endoxifen ligand
and HER2-mAb effects. Somewhat surprisingly, the HER2-14 conjugate exhibited relatively
strong ER degradation activity when assessed using parental MCF7 cells that did not over-
express the surface receptor (Table 2, Figure S2B). The origin of this biological activity is
not known with certainty, but it may involve intrinsic low-level HER2 expression on the
parental cells whose effects are enhanced with the DAR6 ADC relative to the previously
tested DAR2 conjugate (compare HER2-11-lc with HER2-14, Table 2).
Similar to our characterization of HER2-11-lc, we conducted preliminary in vivo
assessments of the HER2-12 and HER2-14 conjugates in mice to evaluate their stability and
pharmacokinetic properties. As shown in Figure S6, both conjugates were moderately stable
in mice with each retaining approximately 80% of the original DAR value after 72 h in
circulation. For each conjugate, the majority of DAR loss was associated with deconjugation
of the corresponding linker-drugs: disulfide exchange in the case of HER2-12 and retro-
Michael transformation of HER2-14. Importantly, cleavage of the carbonate moiety present
in HER2-12 was not observed to a significant extent. This favorable outcome suggested that
the 1,2-dimethyl-containing linker present in HER2-12 accomplished its intended purpose of
17

protecting the carbonate from unwanted hydrolysis during circulation in vivo. Related
cleavage of the HER2-14 pyrophosphate moiety was also not appreciably observed in
accordance with prior literature reports.36
Sparse (two-point) pharmacokinetic data were also obtained for HER2-12 and HER2-
14 in these mouse experiments and the results are depicted in Figure S7. These limited data
were consistent with lower exposures (especially at 72 h) being associated with the two
conjugates relative to the unconjugated HER2 mAb. Related exposure differences between
other conjugates bearing multiple copies of hydrophobic payloads and the corresponding
unconjugated antibodies have been described in the literature.37 Additional (more extensive)
pharmacokinetic data are required to fully characterize the clearance and distribution
properties of conjugates such as HER2-12 and HER2-14.
The prospect of modulating intracellular proteins levels via rationally designed
chimeric degrader molecules is rapidly transforming the biological and medicinal chemistry
fields. Such entities offer multiple advantages over traditional small-molecule inhibitors
(e.g., the ablation of target protein scaffolding properties), that may enhance their ability to
more strongly impact biological pathways. Given their typical modular composition in which
independently optimized target and ligase recognition elements are attached to each other via
a chemical spacer, chimeric degraders frequently exhibit molecular and/or physiochemical
properties well outside parameters currently associated with orally bioavailable
compouds.10a,38 These shortcomings may also compromise other in vivo delivery approaches
employed with such compounds. For example, as shown in Table S4, both compounds 5 and
6 exhibited extremely poor in vitro DMPK properties (e.g., solubility, permeability, and
metabolic stability). Consistent with these observations, both molecules also displayed poor
in vivo performance when administered to mice orally or intravenously (Table S4, Figure
18

S8).39 These unfavorable DMPK outcomes influenced our desire to explore alternate
chimeric degrader delivery options including the conjugation of 5 and 6 to antibodies.
In this report, we describe a new application of antibody-drug conjugate technologies
to effectively deliver chimeric ER degrader molecules to targeted cells that expands on our
related work involving BET-degrading entities.12 Highlights of this disclosure include: (1)
the antigen-dependent delivery to MCF7-neo/HER2 cells of two distinct chimeric degrader
entities (compounds 5 and 6) using three independent ADC linker modalities, (2) strong
evidence for efficient intracellular degrader release from the benzyl-ether linker contained in
HER2-11-lc following antibody-mediated delivery of the conjugate, and (3) preliminary data
suggesting that the described linkers can afford in vivo stability properties that may enable
their use in pharmacodynamic and/or efficacy experiments. Based on these outcomes, it is
our expectation that this disclosure will drive interest in exploring the potential of degrader-
antibody-conjugates to broadly impact medicinal chemistry and biological research activities.
.
Acknowledgements
The authors thank Dr. Jason Zbieg for helpful discussions regarding the described research.
19

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25

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26

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39. We did not explore alternate formulations and/or delivery approaches (IP/SC) that might
improve the in vivo DMPK outcomes described for compounds 5 and 6.
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