Site-Specific Tandem Knoevenagel Condensation-Michael Addition to Generate Antibody-Drug Conjugates

Article abstract of DOI:10.1021/acsmedchemlett.6b00253

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported.

Full text of DOI:10.1021/acsmedchemlett.6b00253

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Letter
A Site-Specific Tandem Knoevenagel Condensation-Michael Addition to
Generate Antibody-Drug Conjugates with Increased Drug-to-Antibody Ratios
Romas A. Kudirka, Robyn M. Barfield, Jesse M. McFarland, Penelope Marie Drake,
Adam Carlson, Stefanie Bañas, Wesley Zmolek, Albert W. Garofalo, and David Rabuka
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00253 • Publication Date (Web): 30 Aug 2016
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A Site-Specific Tandem Knoevenagel Condensation-Michael Addi-
tion to Generate Antibody-Drug Conjugates
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Romas A. Kudirka, Robyn M. Barfield, Jesse M. McFarland, Penelope M. Drake, Adam Carlson,
Stefanie Bañas, Wes Zmolek, Albert W. Garofalo, and David Rabuka*
Catalent Biologics West, 5703 Hollis St., Emeryville CA, 94530, USA
KEYWORDS: Antibody-drug conjugate (ADC), site-specific bioconjugation, bivalent drugs, tandem reactions
ABSTRACT: Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of func-
tionally-enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition
of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-
Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a
second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild
conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional liga-
tions reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs)
with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the
site-specific
ADCs
are
reported.
Functionally-enhanced proteins are of growing promise
1
,2
in the therapeutic arena. The lynch pin holding the field
together is the ligation chemistry that unites the protein
with its payload. Historically, protein modification has
relied on the functionalization of native residues, such as
Glu, Asp, Lys and Cys. However, the abundance of these
residues within a protein results in nonspecific and pro-
3
miscuous conjugation of the payload of interest. In order
to access new and expanded utilities, new conjugation
chemistries must be developed to broaden the available
conjugation chemistry tool kit, especially outside the
realm of native amino acids, the use of which often results
in stochastic mixtures of labeled proteins.
Antibody-drug conjugates (ADCs) are a field that will
directly benefit from the intense exploration of new con-
jugation techniques. ADCs combine the targeted speci-
ficity and long circulating half-life of an antibody with the
lethal potency of a cytotoxic chemotherapy. First-
generation ADCs are prepared using conventional ligation
chemistries resulting in heterogeneous mixtures contain-
ing average drug-to-antibody ratios (DARs) ranging from
Scheme 1. Overview of the tandem-Knoevenagel
condensation-Michael addition ligation using a py-
razolone nucleophile. A) Generation of a DAR 4 site-
specific ADC via first addition of pyrazolone via
Knoevenagel condensation, then subsequent second
addition of pyrazolone via Michael addition. B) Pre-
viously published Trapped-Knoevenagel Ligation C)
Double addition of pyrazolone to reducing sugar via
a tandem Knoevenagel condensation-Michael addi-
tion.
4
zero to eight. These mixtures contain unique chemical
entities that exhibit differing potencies, pharmacokinetics
5
(PK), and toxicities. The complexities in activity, analysis,
and manufacturing associated with ADCs synthesized by
conventional ligation have created a strong push towards
using site-specific chemistries that promise homogeneous
entities with well-defined, monodisperse DARs and pay-
load placement.
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Site-specific bioconjugation can be implemented with a Knovenagel Condensation-Michael Addition (TKM). We
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bioorthogonal handle on which chemistry can be per-
formed. Aldehydes are an attractive option for handles
then examined the impact of payload placement at three
different sites on the antibody.
2
due to their ability to selectively react with nucleophiles
in the presence of diverse sets of functionality. Our group
utilizes an aldehyde in the context of the formylglycine
(
fGly) amino acid residue. The fGly is produced through
the highly selective oxidation of a cysteine residue found
within a specific pentapeptide consensus sequence by
6
formylglycine-generating enzyme (FGE). The fGly-
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containing protein can then be modified using aldehyde-
7,8
specific chemistries (Scheme 1A).
The value of site-specific chemistry and the connec-
tions used to create the bioconjugates is becoming more
apparent as it has been shown that ADCs generated using
these methods possess increased therapeutic indices and
9,10
increased therapeutic activities.
In addition to these
significant advances, there is mounting literature that
demonstrates that payload placement is an increasingly
11-13
important design parameter.
A key design component of an ADC is the average DAR.
5
In a seminal publication, Hamblett et al. showed that the
in vivo efficacy of a DAR 4 species was equal to that of a
DAR 8 species due to improved PK. Recent evidence has
shown that even DAR 8 species, if sufficiently hydrophilic,
can exhibit potent in vivo efficacy and good clearance
1
4
properties. This observation has opened the door to a
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3
reexamination of higher DAR species especially in the
context of targets with low antigen copy number where
delivery of more drug/antibody could potentially enhance
therapeutic outcomes.
The potency effects of DAR 4 species in conjunction
with the compelling site-specific ADC literature suggests
that it would be of significant interest to examine site-
specific ADCs with higher average DARs. While there
exist means to form homogeneous DAR 4 species using
the interchain disulfides of a native antibody, the payload
placement is restricted to cysteine locations that do not
significantly interfere with endogenous cysteines involved
Scheme 2. Synthesis of building blocks and conjuga-
tion construct. A) Synthetic route to pyrazolone acid
construct (3). B) Synthetic route to pyrazolone con-
jugation construct (8).
1
5
in disulfide bonding. Ideally, one would vary the site at
which the payloads are placed allowing for insight into
structure-activity relationships.
Previously, we presented a trapped-Knoevenagel liga-
tion where a stabilized carbanion originating from a pyra-
zolone reacts with an aldehyde to form an olefin. This
olefin is then subject to Michael addition via a pendant
There are at least two straight-forward approaches to
generate DAR 4 species in a site-specific manner. First,
incorporation of two separate conjugation sites, which
collectively would allow for a DAR 4 species with the pay-
1
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thiol (Scheme 1B). Work performed by Honda et al.
Scheme 1C) showed that when pyrazolones react with
(
1
3
reducing sugars under mild conditions, a double addition
product is formed via an initial Knoevenagel condensa-
tion followed by rapid Michael addition by a second pyra-
zolone moiety. The Michael addition is proposed to be
faster than the Knoevenagel condensation, as only double
addition products are formed. The extensive work done
on carbohydrate derivitization using pyrazolones under
mild conditions emboldened us to try using this pyrazo-
lone double addition as an aldehyde-based conjugation
chemistry. We postulated that we could harness this rap-
id second addition via a tandem Knovenagel Condensa-
tion-Michael Addition to form homogeneous site-specific
ADCs with a DAR of 4 under physiological conditions.
load located at two distinct amino acid residue positions.
Second, chemical synthesis of a linker that contains two
cleavable payloads attached to a single conjugatable
group, which would allow for well-defined, monodisperse
DAR 4 species at one amino acid residue (tag-site). We
envisioned a third, less obvious approach employing a
tandem process based on two additions of linker-drug to
the same aldehyde using the same nucleophile as shown
in Scheme 1A.
In this paper, we present a simple and mild one-pot
conjugation chemistry that allows access to well-defined,
monodisperse and site-specific DAR 4 ADCs via a tandem
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In order to achieve a DAR 4 ADC, it is important to use
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a cleavable construct because a non-cleavable double ad-
dition adduct would result in a DAR 2 ADC with the pay-
load being a bivalent drug. With this in mind we chose to
synthesize compound 8, which would release compound 5
as our cytotoxic payload. As shown in Figure S1, the va-
line-citrulline dipeptide is cleaved by cathepsin B releas-
ing the p-aminobenzylcarbamate (PABC) self-immolative
spacer, which undergoes a 1,6-elimination to liberate car-
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bon dioxide and cytotoxic compound 5.
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The pyrazolone acid 2 was synthesized in two steps
from ethyl acetoacetate and the ethyl hydrazinoacetate
8
hydrochloride salt. In two steps, this was converted to
compound 3 (Scheme 2A). The maytansinoid payload was
synthesized starting from previously reported compound
4
, which was coupled to Fmoc-N-methyl-γ-aminobutyric
acid followed by deprotection to afford compound 5 in
3% yield. Amine 5 was then reacted with the commer-
7
cially available peptide 6 and the resulting carbamate was
treated with piperidine to afford free amine 7 in 59% yield
over two steps. Coupling of this to the pyrazolone acid 3
in 48% yield provided the desired pyrazolone construct 8
containing the cleavable maytansine moiety (Scheme 2B).
As there have been several papers highlighting the im-
1
0-13,20
portance of site-specificity with ADCs,
we sought to
explore this new conjugation technique in that context. In
order to assess the impact of payload placement, we used
three differentially-tagged antibodies bearing aldehydes
either at the heavy chain C-terminus (CT), in the CH1
domain (CH1), or at the hinge region (H) (Figure 1A). The
antibodies all had the same variable region, which was
directed against an undisclosed epitope found on liquid
tumors. The fGly-containing antibodies were conjugated
to compound 8 at 15 mg/mL and 8.3 drug/antibody equiv-
alents for 16 h at 37 °C in 50 mM NaCitrate/50 mM NaCl,
pH 7.2 or 6.0 in 5% DMA and 0.085% Triton X-100. There
was no observable loss of protein or increase in aggregate
under the conjugation conditions. At pH 6.0, the fastest
initial pyrazolone addition occurred at the CT, followed
by the CH1 position, and finally the hinge region (Figure
S2A). The same trend existed for the rate of the second
addition of the pyrazolone via Michael addition (Figure
S2B). Presumably, this trend is a function of sterics at the
site of the aldehyde. The more accessible CT has the fast-
est rate of addition and all second additions are slowed
due to the increased steric density resulting from the first
addition. Interestingly, at all conjugation sites the rates of
first and second addition were retarded slightly at pH 7.2
relative to pH 6.0 (Figure S2A and S2B). Mass spectromet-
ric analysis confirmed the double addition of compound 8
on the tagged antibody heavy chain (Figure S3). Addition-
ally, a control experiment showed that no reaction oc-
curred on an untagged antibody (Figure S4).
Figure 1. Site-specific DAR 4 ADCs generated using the TKM
ligation display high potency in in vitro and in vivo tumor
models. A) ADCs with payloads placed at CH1 (blue), CT
(
red), and Hinge (green). B) In vitro cytotoxicity. Cells were
treated with varying concentrations of maytansine (black),
compound 5 (purple), antigen-targeted ADCs conjugated at
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the CH1 (blue), CT (red), or Hinge (green) positions, and an
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isotype control ADC (gold). Error bars = S.D.; n= 9. C and D)
The in vivo efficacy of ADCs bearing payloads at the CH1
(blue), CT (red), or hinge (green) positions was compared to
the vehicle-treated negative control group. Mean tumor vol-
ume ((C), Error bars = S.E.M.) and survival (D) are shown.
The three ADCs were examined for in vitro potency
against an antigen-positive cell line (antigen copy number
~
10,000-40,000/cell); free maytansine was included as a
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positive control, and an isotype anitbody conjugated to
compound 8 at the CT was included as a negative control.
As shown in Figure 1B, all antigen-targeting ADCs exhib-
ited potent dose-dependent toxicity with IC50 values of 74,
6
6, and 61 pM (antibody) for the CT-, CH1-, and hinge-
tagged constructs, respectively, as compared to 207 pM
for the natural product maytansine. Compound 5 dis-
played no activity. The low IC50 values demonstrate the
efficient internalization of the ADC and the effective re-
lease of the cleavable payload. The isotype control exhib-
ited no effect on cell growth at the doses administered,
highlighting the antigen specific response and the chemi-
cal stability of the TKM ligation linkage. We did not ob-
serve meaningful differentiation among the three payload
placements with respect to in vitro activity
Next, we examined the TKM ADCs in an in vivo efficacy
study in mice bearing the antigen-expressing WSU-
DLCL2 xenograft. The ADCs were dosed intravenously at
1
0 mg/kg every four days for a total of four doses. ADCs
bearing the payload at the CH1, the hinge (H), and the CT
position exhibited 77, 73, and 60% tumor growth inhibi-
tion, respectively, as compared to the vehicle control
group at day 15 (Figure 1C). After the last dose at day 12,
the tumors in mice treated with CT-tagged ADC (red)
began to regrow immediately, whereas the tumors in the
mice dosed with the other ADCs did not begin to regrow
for another 10 days. This disparity is reflected in the sur-
vival curves (Figure 1D) and the resulting tumor growth
delay (TGD) values: 115, 106, and 57% TGD for groups
treated with ADCs conjugated at the CH1, hinge, or CT
sites, respectively.
7,8
Until recently, oximes were the default conjugation
technique used with carbonyl-labeled proteins. The major
drawbacks of oxime ligation are the slow rate of reaction
and the low pH requirement (pH 4.6) for the conjugation
to occur. This limits the oxime ligations utility, as not all
Figure 2. Pharmacokinetic stability of the site-specific DAR 4
ADCs varies with placement of the payload. Rats were dosed
with 3 mg/kg of site-specific ADC conjugated to the (A) CH1,
1
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proteins are stable under these conditions. While there
have been advancements in oxime formation catalysts
(
B) CT, or (C) Hinge sites.
1
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that shift the pH closer to neutral, the oxime is subject
7
to hydrolysis and has limited serum stability. The TKM
In an effort to understand the in vivo efficacy differ-
ligation is performed under physiological conditions in
citrate buffer (pH 7.2) and creates a C-C bond that is not
subject to hydrolysis.
ences observed among the payload locations, we con-
ducted a PK study in rats. Previous data from our group
has shown that payload conjugation to an inserted alde-
hyde tag need not markedly change the basic PK proper-
12
ties of an antibody. The total antibody half-life for the
CT DAR 4 ADC was the shortest at 4.1 days, while the CH1
and H were markedly better at 12.0 and 11.7 days, respec-
tively. The two payload locations that resulted in the
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strongest in vivo efficacy, CH1 and H, also were the most
stable in circulation, with total ADC half-lives of 5.8 and
.2 days, respectively. Regarding the difference between
in high spatial concentration. We envision that this
unique ligation chemistry, which affords ready access to
bivalent molecules, will prove valuable both for ADC gen-
eration and to the bioconjugation community as a whole.
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conjugate and antibody half-lives, a small molecule model
system showed that the newly formed C-C bonds were
stable at 37 °C, pH 7.4 over four days (Figure S5), indicat-
ing that this connection likely persisted in vivo. By con-
trast, we have previously observed hydrolysis of may-
ASSOCIATED CONTENT
Supporting Information
1
2
tansine to release maytansinol; this may account for the
differentiation between total antibody and total ADC.
The Supporting Information includes five figures, experi-
mental details and characterization data for new compounds.
The Supporting Information is available free of charge on the
ACS Publications website.
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In a demonstration of the importance of site placement,
the CT conjugate had an ADC half-life of only 1.2 days
(
Figure 2). The rapid clearance of this conjugate could
explain its relatively poor in vivo efficacy, and could be a
consequence of placing four hydrophobic maytansine
payloads close to one another, thus drastically increasing
the local hydrophobicity of the molecule. Intriguingly, the
HIC retention times, which are a proxy for hydrophobi-
AUTHOR INFORMATION
Corresponding Author
* David.Rabuka@Catalent.com.
city of the overall construct, correspond closely to the PK
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Here, we have developed a unique new bioconjugation
technique that allows access to well-defined, monodis-
perse and site-specific DAR 4 ADCs via a tandem
Knoevenagel condensation-Michael addition, where the
pyrazolone nucleophile reacts with an aldehyde to form a
reactive enone. This Michael acceptor is then subject to
attack by a second molecule of the same pyrazolone to
form the double addition species. This ligation occurs
under neutral pH in the absence of catalyst, minimizing
perturbation of essential amino acid residues in the varia-
ble region of the antibody (e.g., deamidation of aspara-
gine residues that might occur with maleidimide chemis-
tries). We have examined this new TKM ligation in the
context of site-specific payload placement of DAR 4
ADCs. Three different DAR 4 ADCs were synthesized with
the payloads placed at the CT, CH1, and hinge regions. In
vivo efficacy and PK data revealed that the location of the
payload affects both PK and efficacy. These observations
are corroborated by studies from Strop et al. showing that
site-specific high DAR species prepared via multiple tag-
sites have poor PK when hydrophobic payloads are placed
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