Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity

Article abstract of DOI:10.1002/cmdc.201900497

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

Full text of DOI:10.1002/cmdc.201900497

Accepted Article
Title: Antibody Conjugation of a Chimeric BET Degrader Enables In
Vivo Activity
Authors: Thomas H. Pillow, Pragya Adhikari, Robert A. Blake, Jinhua
Chen, Geoffrey Del Rosario, Gauri Deshmukh, Isabel
Figueroa, Karen E. Gascoigne, Amrita V. Kamath, Susan
Kaufman, Tracy Kleinheinz, Katherine R. Kozak, Brandon
Latifi, Douglas D. Leipold, Chun Sing Li, Ruina Li, Melinda M.
Mulvihill, Aimee O’Donohue, Rebecca K. Rowntree, Jack D.
Sadowsky, John Wai, Xinxin Wang, Cong Wu, Zijin Xu, Hui
Yao, Shang-Fan Yu, Donglu Zhang, Richard Zang, Hongyan
Zhang, Hao Zhou, Xiaoyu Zhu, and Peter Dragovich
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: ChemMedChem 10.1002/cmdc.201900497
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Antibody Conjugation of a Chimeric BET Degrader Enables
In Vivo Activity
Thomas H. Pillow,[a] Pragya Adhikari,[a] Robert A. Blake,[a] Jinhua Chen,[b] Geoffrey Del Rosario,[a]
Gauri Deshmukh,[a] Isabel Figueroa,[a] Karen E. Gascoigne,[a] Amrita V. Kamath,[a] Susan Kaufman,[a]
Tracy Kleinheinz,[a] Katherine R. Kozak,[a] Brandon Latifi,[a] Douglas D. Leipold,[a] Chun Sing Li,[b]
Ruina Li,[a] Melinda M. Mulvihill,[a] Aimee O’Donohue,[a] Rebecca K. Rowntree,[a] Jack D.
Sadowsky,[a] John Wai,[b] Xinxin Wang,[b] Cong Wu,[a] Zijin Xu,[b] Hui Yao,[b] Shang-Fan Yu,[a]
Donglu Zhang,[a] Richard Zang,[a] Hongyan Zhang,[b] Hao Zhou,[b] Xiaoyu Zhu,[b] Peter S.
Dragovich*[a]
[a]
Dr. T. H. Pillow (https://orcid.org/0000-0001-7300-1002), P. Adhikari, Dr. R. A. Blake, G. Del Rosario, G. Deshmukh, Dr. I. Figueroa, Dr. K. E. Gascoigne,
Dr. A. V. Kamath, S. Kaufman, T. Kleinheinz, Dr. K. R. Kozak (https://orcid.org/0000-0003-3751-7225), B. Latifi, D. D. Leipold (https://orcid.org/0000-0003-
2116-4277), R. Li, Dr. M. M. Mulvihill (https://orcid.org/0000-0002-0057-1204), A. O’Donohue, Dr. R. K. Rowntree, Dr. J. D. Sadowsky, Dr. C. Wu, S.-F. Yu,
Dr. D. Zhang, Dr. R. Zang, Dr. P. S. Dragovich (https://orcid.org/0000-0001-7372-2862)
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
E-mail: dragovich.peter@gene.com
[b]
Dr. J. Chen, Dr. C. S. Li, Dr. J. Wai, X. Wang, Z. Xu, H. Yao, Dr. H. Zhang, H. Zhou, Dr. X. Zhu
Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China
Supporting information for this article is given via a link at the end of the document.
Abstract: The ability to selectively degrade proteins with bifunctional
small molecules has the potential to fundamentally alter therapy in a
variety of diseases. However, the relatively large size of these
chimeric molecules often results in challenging physico-chemical
properties (e.g., low aqueous solubility) and poor pharmacokinetics
which may complicate their in vivo applications. We recently
discovered an exquisitely potent chimeric BET degrader (GNE-987)
which exhibited picomolar cell potencies but also demonstrated low
in vivo exposures. In an effort to improve the pharmacokinetic
properties of this molecule, we discovered the first degrader-
antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody
via a novel linker. A single IV dose of the conjugate afforded
sustained in vivo exposures that resulted in antigen-specific tumor
regressions. Enhancement of a chimeric protein degrader with poor
in vivo properties through antibody conjugation thereby expands the
utility of directed protein degradation as both a biological tool and a
therapeutic possibility.
AML clinical trials, and results from these explorations are
beginning to emerge.[7,8]
Introduction
Bromodomain-containing protein 4 (BRD4) is a member of the
bromodomain and extra-terminal (BET) family of proteins (BRD2,
3, 4, and T) and functions as an epigenetic “reader” of
acetylated histone lysine residues via its two bromodomain
motifs (BD1 and BD2).[1,2]
Disruption of BRD4-histone
interactions is currently viewed as an attractive strategy for the
development of novel anti-cancer agents, and many BET
inhibitors have been designed, characterized, and advanced to
clinical-stage studies in recent years.[3,4] Encouragingly, clinical
efficacy was observed in early-stage trials that explored the use
of a BET inhibitor to treat acute myeloid leukemia (AML)
patients.[5] However, potentially complicating side effects (e.g.,
thrombocytopenia) were also observed in these and other
clinical studies[6] of BET inhibitors suggesting that further
exploration of the compound class was warranted. Accordingly,
additional BRD4/BET inhibitors have since been advanced into
Figure 1. Chimeric BET degraders discussed in this work.
Recently, chimeric molecules which form ternary complexes
between BRD4 and components of various E3 ubiquitin ligase
complexes [e.g., VHL (Von Hippel-Lindau tumor suppressor),[9]
CRBN (cereblon),[10] XIAP (X-linked inhibitor of apoptosis
protein),[11] MDM2 (mouse double minute 2 homolog)[12]] were
shown to induce potent degradation of BRD4 and (sometimes)
other BET family proteins via ubiquitination and subsequent
destruction in the cellular proteasome.[13] These chimeric
1
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Figure 2. Activity of compounds 1 (orange), 2 (purple), 3 (blue), and 4 (red) in various in vitro biological assessments. a) BRD4 degradation in EOL-1 cells, b)
EOL-1 cell viability, c) HL-60 cell viability, d) MYC expression inhibition (MV-4-11 cells). DC50 and IC50 values are reported as means from a minimum of two
determinations; graphs show a representative experiment and error bars reflect the standard deviation of duplicate points. See supporting information for
experimental details.
degraders offer possible therapeutic advantages relative to
simple BET inhibitors including the potential to operate
catalytically,[14,15] the ablation of target protein scaffolding
properties,[14,15] and the ability to exhibit broader transcriptional
impacts compared to related inhibitors that do not degrade the
protein.[16] Two extensively-characterized BRD4/BET degraders
that were previously disclosed in the scientific literature are
shown in Figure 1 (MZ1,[17] structure 1 and ARV-771,[9] structure
2). These molecules contain portions of the well-known BET
inhibitor JQ1,[18] and each binds to VHL as a means to induce
ubiquitination of the targeted BRD4 protein. In the first part of
this report, we describe the characterization of a highly-active
chimeric BET degrader that also engages VHL but which
incorporates an alternate and more potent BET inhibitor moiety.
In the second portion of this work, we detail the conjugation of
this new entity to engineered monoclonal antibodies as a novel
means to deliver the molecule to tumors in vivo.
examined in the same EOL-1 experiment. The dramatically
improved BRD4 degradation properties of 3 relative to MZ1 (1)
were also apparent in western blot assessments of EOL-1 cells
that were treated with the two compounds (Figure S1;
degradation of the BRD2 and BRD3 BET family proteins was
also observed). Picomolar potency of compound 3 was similarly
detected when the molecule was tested in viability assessments
employing EOL-1 and HL-60 AML cell lines (Figures 2b and 2c).
Degraders
1 and 2 were also active in these viability
experiments but once again were significantly less potent
relative to 3. In addition, extremely potent cell viability activity
that was dramatically improved relative to degrader 1 was
observed for compound 3 when the molecules were tested
against a larger panel of AML cell lines (Table S1). Consistent
with the mechanism of action of the BRD4/BET degrader
compounds, the described cell viability outcomes correlated well
with the ability of the various degraders to suppress MYC
expression using either a transcription-based assay performed
in the MV-4-11 cell line (Figure 2d) or a western blot assessment
conducted with EOL-1 cells (Figure S1; i.e., 3 was significantly
more potent than 1 or 2).
Results and Discussion
We subsequently synthesized and characterized compound 4
to determine what portions of the biological activities described
above for degrader 3 were due to its BET degradation activity
(as opposed to its ability to simply bind to and inhibit the target
BRD4 protein). Compound 4 incorporated an epimer of the
hydroxy-proline moiety present in 3 that was expected to
abrogate binding to VHL and thus impair the protein degradation
As shown in Figure 2a,
compound 4 exhibited negligible BRD4 degradation activity
when it was tested in the EOL-1 cell line. The absence of BRD4
As part of our BET degrader discovery efforts, we synthesized
and profiled compound 3 (GNE-987, Figure 1). In particular, we
formed a chimeric degrader using VHL-binding moiety present in
MZ1 (compound 1) along with
a potent BET inhibitor
fragment.[19] As shown in Figure 2a, compound 3 exhibited
extremely potent (DC50 value = 0.03 nM) and near-complete (Sinf
value = 99%) BRD4 degradation activity when tested in the
EOL-1 AML cell line. The BRD4 degradation activity of 3 was at
least an order of magnitude more potent than that observed for
chimeric degraders 1 and 2 when the latter molecules were
properties of the compound.[9]
2
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Figure 3. Activity of compounds 1 (orange), 2 (purple), 3 (blue), and 4 (red) in various in vitro biological assessments. a) Binding to BRD4 BD1, b) Binding to
BRD4 BD2, c) Binding to VHL. IC50 values are reported as means from a minimum of two determinations; graphs show a representative experiment and error
bars reflect the standard deviation of duplicate points. See supporting information for experimental details.
degradation properties for 4 resulted in weaker observed activity
for the compound relative to degrader 3 in the EOL-1 and HL-60
cell viability assessments (Figures 2b and 2c) as well as in the
MYC transcription assay (Figure 2d). Interestingly, the cell
viability potencies of degradation-inactive compound 4 observed
in these characterizations were similar to those displayed by the
active BET degrader 2 (Figures 2b and 2c). In addition,
compound 4 displayed a more potent anti-MYC IC50 value
relative to 1 and 2 although its maximum degree of anti-MYC
activity was modestly (~20%) attenuated compared to
compounds that were capable of BRD4/BET degradation
(degraders 1, 2, and 3; Figures 2a and 2d). Similar differences
in anti-MYC maximal response values were noted by others
when comparing BET degraders with simple BET inhibitors.[10,20]
Collectively, these outcomes demonstrated that (1) the
degradation-inactive compound 4 which contained the dihydro-
pyrrolopyridinone BET binding fragment could exhibit potencies
similar to JQ1-derived degraders 1 and 2 in multiple biological
assessments, and (2) significant cell viability and anti-MYC
potency improvements were associated with the BET
degradation activity of compound 3.
(Figure 3c). The disparate VHL binding outcomes for degraders
1 and 3 was somewhat surprising since the compounds share a
common VHL binding moiety. The VHL binding properties of the
two molecules are likely influenced by the remainder of their
chimeric structures thereby resulting in the different measured
binding properties. Compound 4 (the GNE-987 hydroxy-proline
epimer) bound to the BRD4 BD1 and BD2 bromodomains in a
manner that was similar to 3 (Figures 3a and 3b). However, as
expected based on its inability to degrade BRD4, the former
molecule was inactive in the VHL binding assessment (Figure
3c). We suspected that the poor VHL affinity exhibited by
compound 4 would also negatively impact its ability to form a
ternary complex with VHL and the BRD4 protein. We therefore
characterized the potential of all the chimeric degraders
described in this work to generate such ternary entities.
Surface plasmon resonance (SPR) techniques were
employed to assess the stabilities of various degrader-BRD4-
VHL entities as determined by the corresponding dissociation
half-life (t1/2
) values measured for the ternary structures.
Related SPR techniques were utilized by others to similarly
characterize such degrader-BRD4-VHL complexes, and the
ternary complex stabilities described for compound 1 below
were consistent with previously reported values.[21] As shown in
Figures 4a-4c, the ternary structure formed between compound
3, the BRD4 BD1 domain, and VHL exhibited a half-life that was
dramatically longer than the t1/2 values measured for the
corresponding complexes formed by degraders 1 and 2 in
related experiments. For reasons that are currently not
understood, less dramatic differences in ternary complex
stabilities were observed for the three compounds when the
SPR assessments were conducted using the BRD4 BD2 domain
in place of BD1 (Figures 4d-4f). An inverse rank-order of the
compounds was also noted in the BD2 assessments with
To better understand the potent degradation activity exhibited
by compound 3, we characterized its affinity for the BRD4 and
VHL proteins using various techniques. As shown in Figures 3a
and 3b,
3
bound equipotently to the BD1 and BD2
bromodomains of BRD4 with low nanomolar affinities that were
considerably more potent than the outcomes observed for
degraders 1 and 2. The improved BRD4 affinity of the
dihydropyrrolopyridinone-containing
fragment
present
in
compound 3 relative to the alternate BET binding moiety
incorporated in degraders 1 and 2 presumably contributed to
these binding differences. The affinity of compound 3 for VHL
was slightly less than that observed for degraders 1 and 2
3
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Figure 4. Characterization of ternary complexes formed between indicated chimeric degrader (compound 1, 2, or 3), indicated BRD4 domain (BD1 or BD2), and
VHL via SPR: data (black), Fit (red). Reported t1/2 values represent the averages of 3 independent measurements (a, c, d, f) or 1 independent measurement (b,
e). Compounds 1, 2, and 4 were tested using co-injection methods and compound 3 was tested using sequential injections; see supporting information for
experimental details.
degrader 1 exhibiting the longest half-life (although this value
was significantly shorter than that observed for compound 3 in
the BD1-based assay). As expected based on its lack of affinity
for the VHL protein (c.f., Figure 3c), no ternary complex was
observed for compound 4 (data not shown). We currently
suspect that the long t1/2 exhibited by compound 3 in the BD1-
VHL SPR assessment was responsible for the exquisite
biological potencies displayed by the molecule. Consistent with
this hypothesis, related extension of ternary complex stability
(i.e., half-life) was observed by others to increase the rate of
BRD4 degradation relative to other BET family members.[21]
Given the extremely potent BRD4 degradation and cell
viability properties of compound 3, we were very interested in
profiling the compound in a variety of in vivo experiments.
However, the physiochemical characteristics of the molecule
(mw = 1096, clogD7.4 = 5.6, TPSA = 228, kinetic water solubility
= < 1 µM) suggested that its oral bioavailability was likely to be
very low.[22] These same characteristics also created concerns
that attempts to administer the compound by alternate non-oral
routes to animals (e.g., intraperitoneal, sub-cutaneous,
conjugation of 3 to monoclonal antibodies might provide an
alternate delivery option for the degrader molecule.[23-25] We
envisioned that overcoming the challenges of attaching such
chimeric degraders to antibodies would enable the creation of
degrader-antibody conjugates and potentially offer a novel
solution to delivery issues encountered with the chimeric
molecules in both in vitro and in vivo settings.
As shown in Figure 5, we derivatized compound 3 with a small
disulfide-containing linker via a carbonate moiety attached to the
hydroxy-proline fragment present in the molecule to produce
linker-drug 5. Although this modification inactivates the chimeric
degrader, release of 3 was expected to occur following antibody-
mediated intracellular delivery of the related conjugate, disulfide
reduction, and subsequent linker self-immolation (Scheme S1).
We also prepared the analogous linker-drug from degrader 4
(compound 6) via a similar derivatization strategy. These
entities contain methanethiosulfonyl (MTS) disulfide moieties
which were found to efficiently react with Cys residues on the
surface of engineered monoclonal antibodies and thereby
connect the linker-drugs to the biologics via new disulfide bonds
(Figure 5).[26] In addition, conjugates bearing related disulfide-
based linkers were found to efficiently release attached cytotoxic
payloads following internalization into targeted cells (Scheme
S1).[27,28] Importantly, however, these studies utilized carbamate
functional groups to connect the disulfide linkers to the
intravenous) would be problematic as well.
Subsequent
experiments confirmed that 3 exhibited unfavorable in vitro
DMPK characteristics (Table S2) as well as poor in vivo
pharmacokinetic properties following IV or oral administration to
mice (Figure S2). Accordingly, we wished to explore whether
4
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associated payloads as opposed to the carbonate moieties
the CLL1-targeting ADC (CLL1-5) exhibited significantly stronger
HL-60 efficacy outcomes relative to the HER2-targeting control
conjugate (HER2-5, Figure 6a). This result was consistent with
the antigen-dependent delivery of compound 3 to the AML
tumors via the CLL1-targeted ADC. Similar dose-dependent
antitumor activity and even more profound antigen-dependent
delivery effects were observed when CLL1-5 and HER2-5 were
assessed via single IV administrations in mice bearing EOL-1
depicted in linker-drugs
5
and 6.
Accordingly, prior to
synthesizing and testing the desired degrader-antibody
conjugates, we were somewhat concerned regarding potential
enzymatic and chemical hydrolysis liabilities associated with the
carbonate functionality.
AML xenografts (Figure 6b).
In a separate experiment,
measurable levels of 3 were detected in EOL-1 tumors four days
following IV administration of CLL1-5 to xenografted mice
(Figure S3). The measured intratumor amounts of 3 correlated
well with the administered doses of CLL1-5 as well as with the
EOL-1 tumor growth inhibition activity displayed by the
conjugate (compare Figures 6b and S3; CLL1-5 dose levels 1.5,
5.0, and 10 mg/kg). The described relationships between
xenograft efficacy and intratumor degrader concentrations for
the CLL1-5 degrader-antibody conjugate paralleled related
outcomes observed for several other previously-studied ADCs
bearing cytotoxic payloads.[33] Importantly, both CLL1-5 and
HER2-5 conjugates were well tolerated in all of these in vivo
studies with minimal reductions detected in the associated
mouse body-weights (Figures S4 and S5).
To confirm that the efficacy observed in the in vivo studies
resulted from intratumor BET degradation by the delivered
compound 3 and not simply its highly potent inhibition properties,
we prepared another CLL1-targeting ADC derived from
compound 4 (CLL1-6; Figure 5, Table S4). As demonstrated in
Figures 2a, 3a, and 3b above, compound 4 retained the potent
BRD4 binding activity of 3 but did not meaningfully degrade the
protein. Subsequent experiments demonstrated that CLL1-6
afforded minimal in vivo activity in the HL-60 AML xenograft
model when the conjugate was tested at two separate dose
levels (both of which enabled CLL1-5 to give strong efficacy
outcomes; Figure 7a). These results supported a CLL1-5
mechanism of action in which BET degradation induced by
compound 3 following antibody-mediated delivery was primarily
responsible for the observed antitumor activity. Consistent with
this mechanism, significant BET degradation was observed via
western blot analysis of EOL-1 tumors four days following IV
administration of the CLL1-5 conjugate in a parallel group of
xenografted mice (Figure S6). Importantly, the weak activity of
CLL1-6 also demonstrated that the mAb component of CLL1-5
was not responsible for the potent efficacy displayed by the
conjugate in the HL-60 model.
We also determined whether conjugation of 3 enhanced the
molecule’s ability to provide favorable xenograft outcomes.
Accordingly, we assessed the unconjugated compound in the
EOL-1 tumor model via administration of a single IV dose (0.4
mg/kg). The amount of unconjugated 3 employed in this
experiment was equivalent to that associated with a 10 mg/kg
dose of the corresponding CLL1-5 conjugate (note the large
molecular weight differences between compound 3 and the
CLL1-5; 1096 and 156,690 g/mole, respectively). As shown in
Figure 7b, administration of 3 in unconjugated form afforded no
efficacy in the EOL-1 model. In contrast, the matched dose of
the CLL1-5 conjugate (10 mg/kg) exhibited strong antitumor
activity in the same experiment (Figure 7b). These outcomes
clearly demonstrated the ability of antibody conjugation to
enable the in vivo activity of the chimeric degrader molecule and
thereby significantly enhance its efficacy performance relative
Figure 5. Linker-drugs 5 and 6 respectively derived from
compounds 3 and 4 and corresponding degrader-antibody
conjugates. Green fragments depict the linker portions of the
molecules employed for mAb attachment purposes.
As depicted in the lower portion of Figure 5, several antibody-
drug conjugates (ADCs) were successfully prepared using
linker-drugs 5 and 6 (see also Table S4). In spite of the
relatively high lipophilicity of the linker-drugs, it was possible to
attach six of each entity to Cys-engineered mAbs which targeted
the CLL1 [C-type lectin-like molecule-1; also known as C-type
lectin domain family 12 member A (CLEC12A)] and HER2 cell
surface receptors without encountering significant aggregation
issues [drug-antibody ratio (DAR) = ~6.0]. The preparation of
these conjugates represents the first published example of
employing our site-specific engineered Cys antibody conjugation
technology to generate DAR6 entities. The higher drug loading
of these conjugates could afford increased intracellular
concentrations of the corresponding chimeric degraders
following ADC-mediated delivery relative to more-traditional
DAR2 entities. The CLL1 antigen was known to be highly
expressed on the surface of AML cells, and CLL1-targeting
ADCs bearing cytotoxic payloads were previously shown to
respectively inhibit the growth of AML cell lines and tumor
models in vitro and in vivo.[29,30] In contrast, the HER2 receptor
was not expressed on AML cells, and conjugates derived from
mAbs which targeted this antigen were therefore anticipated to
serve as non-targeted controls in AML-related experiments.
Importantly, the locations of the engineered Cys residues
employed to construct the Figure
5
degrader-antibody
conjugates (LC-K149, HC-L174, and HC-Y373)[31] afforded
disulfide-linked ADCs that exhibited favorable in vivo stability
properties.[32]
Single intravenous (IV) administration of the CLL1-5 ADC to
mice bearing HL-60 AML xenografts afforded dose-dependent
tumor growth inhibition activity that was well-separated from the
vehicle control (Figure 6a). At a matched dose level of 5 mg/kg,
5
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Figure 6. In vivo efficacy of degrader-mAb-conjugates in the a) HL-60 and b) EOL-1 xenograft models. Green traces: CLL1-5; blue
traces: HER2-5, black traces: vehicle control. Numbers next to traces indicate doses of each conjugate (in mg/kg) that were
administered once IV at the day 0 time point. Group mean tumor volumes (+/- SEM) are plotted over the duration of study
(n=5/group). See supporting information for experimental details.
to results obtained with the unconjugated compound. It is not
this property likely contributed to the favorable efficacy
known at this time whether improved pharmacokinetic properties, outcomes observed with the conjugate. In addition, single IV
increased intratumor delivery, and/or other in vivo alterations
were responsible for the conjugation-related enhancements of
administration of the unconjugated CLL1 antibody at both the 5
and 10 mg/kg dose levels resulted in no EOL-1 in vivo efficacy
(Figure 7b). These important results confirmed that the efficacy
observed for CLL1-5 in the EOL-1 model did not result from the
mAb portion of the conjugate.
compound 3 in vivo activity.
However, the stability and
pharmacokinetic analyses conducted below with CLL1-5
supported a prolonged in vivo exposure of conjugated 3, and
Figure 7. In vivo efficacy of degrader-mAb-conjugates in the a) HL-60 and b) EOL-1 xenograft models. Green traces: CLL1-5;
purple traces: CLL1-6, blue traces: unconjugated CLL1-mAb, black trace (dotted line): unconjugated compound 3; black trace
(solid line): vehicle control. Numbers next to traces indicate doses of each conjugate or unconjugated degrader (in mg/kg) that were
administered once IV at the day 0 time point. All conjugates and unconjugated compounds were well-tolerated in these experiments
with minimal body-weight loss observed (Figures S7a and S7b). Group mean tumor volumes (+/- SEM) are plotted over the duration
of study (n=5/group). See supporting information for experimental details.
6
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To assess the in vivo stability profiles of the degrader-
antibody conjugates, we obtained mouse plasma samples on
days 1, 4, and 7 following single IV administration of CLL1-5 at
10 mg/kg in a pharmacokinetic assessment involving non-tumor-
bearing mice. Subsequent analysis using affinity-capture LC-
MS[34] revealed that CLL1-5 was surprisingly stable in vivo in the
mouse with only a small amount of linker-drug carbonate moiety
cleavage representing the major biotransformation events
(Figure S8a). Removal of the Fc portion of the conjugate (which
contains the HC-Y373 linker-drug attachment site) as part of
these analyses afforded stability profiles that were similar to
those obtained for the intact ADC (Figure S8b). This result
suggested that the observed carbonate hydrolysis occurred in
linker-drugs that were positioned at the other two mAb
attachment locations (LC-K149 and HC-L174). Importantly, loss
of entire linker-drug entities via cysteine-mediated disulfide
exchange was not detected in any of these analyses. Such
stability outcomes paralleled observations made with related
disulfide-based cytotoxic ADCs in which a single methyl group
resided adjacent to the linker-drug/mAb disulfide bond (c.f.,
Figure 5).[27] Encouragingly, the collective in vivo stability
properties of the CLL1-5 conjugate resulted in >70% of the
originally attached linker-drugs remaining connected to the mAb
and untransformed after 7 days of circulation in mice (Figure
S8c).
A separate 21-day experiment was conducted in non-tumor-
bearing mice to characterize the pharmacokinetic properties of
the CLL1-5 conjugate. As shown in Figure S9 and Table S3, the
ADC exhibited total antibody pharmacokinetic behaviors in mice
that were nearly indistinguishable from an unconjugated control
antibody (similarly engineered with Cys residues located at the
LC-K149, HC-L174, and HC-Y373 sites) that was examined in
the same assessment. As expected for IgG1-derived biologics,
both CLL1-5 and the control antibody showed bi-exponential
pharmacokinetic profiles with long half-lives and slow clearance
values. These studies provided assurance that attachment of
six copies of the somewhat hydrophobic linker-drug 5 to the
engineered CLL1-targeting antibody did not significantly alter the
pharmacokinetic properties of the resulting conjugate relative to
the unconjugated mAb.
The preparations of CLL1-5 and related ADCs described in
this work represent the first syntheses of degrader-antibody
conjugates to be reported in the scientific literature. The
associated biological characterizations demonstrate the ability of
such entities to deliver bioactive, VHL-targeting chimeric BET
degrader payloads to tumors in vivo in an antigen-dependent
manner following IV administration. Although multiple instances
of degrader-based antitumor efficacy experiments have been
reported to date,[35-38] the majority of the chimeric molecules
examined in these studies do not contain a VHL binding
fragment in their chemical structures (for some notable
exceptions, see references 9, and 39-42). The rarity of such
examples may reflect non-ideal physicochemical and/or
pharmacokinetic properties imparted to the degrader
compounds by the relatively large peptidomimetic fragments
typically utilized to engage the VHL protein (mw >400; see
structures 1-4 in Figure 1).[37]
As demonstrated in this work, degrader-antibody conjugates
can be made to mitigate potential in vivo delivery challenges
associated with VHL-targeting degrader compounds and thereby
exhibit dramatically enhanced in vivo antitumor efficacy
outcomes relative to the unconjugated degrader molecules.
Antibody conjugation may thus speed the in vivo evaluation of
such entities (as well as degraders which engage other ligase
complexes) by bypassing the need to optimize the unconjugated
molecules’ pharmacokinetic performance characteristics where
any structural changes might compromise existing desirable
biological properties (e.g., potency and/or selectivity).
In
addition, many of the degrader-based in vivo assessments
described in the literature employ administration routes that may
be difficult to translate to routine human use (e.g., intraperitoneal
dosing). Conjugation of chimeric degraders to antibodies now
provides an IV administration option for these entities with the
potential for relatively infrequent dosing.
Further experimentation is needed to determine the generality
of the mAb attachment and conjugation strategies detailed in
this work. Additional studies are also required to assess
whether degrader-antibody conjugates that target non-CLL1
antigens and/or deliver chimeric degraders of proteins other than
BET family members can function appropriately in related in vitro
and in vivo assessments.
However, the strong efficacy
outcomes associated with CLL1-5 coupled with the conjugate’s
favorable in vivo pharmacokinetic and stability profiles should
spur exploration into overcoming the challenges associated with
similar entities as a means to effectively deliver additional
chimeric protein degrader molecules in vivo.
Conclusions
This work describes the discovery of a novel and highly active
chimeric BET degrader which incorporates a potent BET
binder/inhibitor, a VHL-binding fragment, and a ten methylene
spacer moiety (compound 3, GNE-987). The new molecule
exhibited extremely potent BET degradation properties (pM DC50
values) in cell-based experiments along with similarly dramatic
in vitro impacts on cell viability and suppression of MYC
expression. However, compound 3 exhibited unfavorable in
vitro DMPK characteristics as well as poor in vivo
pharmacokinetic properties following IV or oral administration to
mice. Attachment of 3 to a CLL1-targeting antibody using a
novel disulfide-based carbonate linker provided a degrader-
antibody conjugate (CLL1-5) that displayed dose and antigen-
dependent activity in multiple in vivo xenograft efficacy
experiments. Importantly, the CLL1-5 conjugate also exhibited
favorable in vivo stability and pharmacokinetic properties
following IV administration to mice.
Experimental Section
Full experimental details associated with compound synthesis, conjugate
preparation, and biological testing are provided in the supporting
information.
Acknowledgements
We thank Christine Yu for purification of the human VHL domain
and Elongin B,C protein constructs employed in this work.
7
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10.1002/cmdc.201900497
ChemMedChem
FULL PAPER
D. H. Albert, Y. Shen, W. M. Kati, K. F. McDaniel, Bioorg. Med. Chem.
Lett. 2018, 28, 1804 ‒ 1810.
Conflict of interest
[20] K.-H. Chan, M. Zengerle, A. Testa, A. Ciulli, J. Med. Chem. 2018, 61,
504 – 513.
The authors declare no conflict of interest.
[21] M. Roy, S. Winkler, S. J. Hughes, C. Whitworth, M. Galant, W. Farnaby,
K. Rumpel, A. Ciulli, ACS Chem. Biol. 2019, 14, 361 ‒ 368.
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[23] A. Beck, L. Goetsch, C. Dumontet, N. Corvaïa, Nat. Rev. Drug Discov.
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Keywords: antibodies, antibody-drug conjugate, BRD4,
chimeric protein degrader, drug delivery
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8
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10.1002/cmdc.201900497
ChemMedChem
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Entry for the Table of Contents
mAb-Mediated delivery of protein degraders. A novel strategy for attaching chimeric degrader payloads to antibodies was
developed using a self-immolative disulfide linker. A conjugate prepared using this methodology displayed strong antigen-dependent
efficacy in xenograft tumor models. The corresponding unconjugated degrader was inactive in vivo when administered at a matched
dose.
9
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