Expanded utility of the β-glucuronide linker: ADCs that deliver phenolic cytotoxic agents

Article abstract of DOI:10.1021/ml100039h

The β-glucuronide linker has been used for antibody-drug conjugates (ADCs) to deliver amine-containing cytotoxic agents. The linker is stable in circulation, hydrophilic and provides ADCs that are highly active in vitro and in vivo. To extend the utility of the β-glucuronide linker toward phenol-containing drugs, an N,N′-dimethylethylene diamine self-immolative spacer was incorporated with the linker for release of the potent cytotoxic phenol psymberin A. Exposure of the drug-linker to β-glucuronidase resulted in facile drug release. The corresponding ADCs were active and immunologically selective against CD30-positive L540cy and CD70-positive Caki-1 cell lines.

Full text of DOI:10.1021/ml100039h

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Expanded Utility of the β-Glucuronide Linker:
ADCs That Deliver Phenolic Cytotoxic Agents
Scott C. Jeffrey,*,† Jef De Brabander,‡ Jamie Miyamoto,† and Peter D. Senter†
†Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, and ‡Department of Biochemistry and Simmons
Comprehensive Cancer Center, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard,
Dallas, Texas 75390-9038
ABSTRACT The β-glucuronide linker has been used for antibody-drug conju-
gates (ADCs) to deliver amine-containing cytotoxic agents. The linker is stable in
circulation, hydrophilic and provides ADCs that are highly active in vitro and
in vivo. To extend the utility of the β-glucuronide linker toward phenol-containing
drugs, an N,N0-dimethylethylene diamine self-immolative spacer was incorporated
with the linker for release of the potent cytotoxic phenol psymberin A. Exposure of
the drug-linker to β-glucuronidase resulted in facile drug release. The correspond-
ing ADCs were active and immunologically selective against CD30-positive L540cy
and CD70-positive Caki-1 cell lines.
KEYWORDS β-Glucuronide linker, antibody-drug conjugates, phenolic cytotoxic
agents, N,N0-dimethylethylene diamine, CD30-positive L540cy cell line, CD70-
positive Caki-1 cell line
ntibody-drug conjugates (ADCs) consist of three
primary components: a monoclonal antibody that
targets a tumor antigen with high tumor expression,
Our goal was to modify the β-glucuronide linker to con-
tain a dimethylethylene diamine (DMED) self-immolative
spacer12 forstable linkage and facile release of 3. Drug release
would involve enzymatic deglucuronidation, 1,6-elimination,
decarboxylation, and cyclization of the DMED carbamate to
liberate free phenol (Scheme 1). Toward this end, the glucur-
onide para-nitrophenyl (pNP) carbonate 4 was reacted with
excess DMED to readily afford the stable coupling partner 5
(Scheme 2). Psymberin (3) was activated with pNPcarbonate
to give the phenolic carbonate 6. The coupling of 5 and 6 gave
a modest yield of 7. Saponification of the glucuronide acetate
and methyl ester groups was accomplished concomitantly
with Fmoc removal, and coupling to maleimidocaproyl-N-
hydroxy succinimide ester provided the final drug-linker 8.
To confirm the reactivity of 8 toward enzymatic cleavage,4
the linker was treated with cysteine (Scheme 3) to form the
cysteine-succinimide adduct 9 (80 μM), which was then
exposed to β-glucuronidase (Escherichia coli, 36 μg/mL) at
37 °C. After 10 min, inspection of the reaction mixture by
liquid chromatography-mass spectrometry (Figure 2) de-
monstrated that 9 had been almost completely consumed.
The specific activity for enzymatic hydrolysis is 0.21 μmol/
min/mg, which is consistent with a previous measurement
on an auristatin-based drug-linker.4 The products were 3 and
the residual linker components, specifically the diastereo-
meric cysteine adduct 10. The absence of any detectible
intermediate carbamate 11 demonstrates that self-immola-
tion of the DMED intermediate 11 to release 3 was rapid.
A
a cytotoxic agent, and a linker system that releases drug
from the antibody upon internalization into cancer cells.
When a linker system with high circulation stability and
tumor specificity is used, tolerability and efficacy can be
significantly augmented.1-3 In an effort to develop safe and
effective ADCs, we have focused on linker systems meet-
ing these criteria. We have reported on ADCs employing the
β-glucuronide linker (Figure 1) with several drug classes
including auristatins,4 a highly potent doxorubicin deri-
vative,5 CBI minor groove binders,6 and camptothecin
analogues.7 The β-glucuronide linker is readily cleaved by
β-glucuronidase, an enzyme present in lysosomes and tu-
mor interstitium.8,9 The linker is highly stable in circulation
and hydrophilic, which allows the use of hydrophobic drugs
for ADCs that otherwise would lead to high degrees of
aggregation.6,10
Potent cytotoxic agents with basic amine residues are
attractive molecules for targeted delivery using the β-
glucuronide linker because the amine can be used for linker
attachment. We were interested in extending the applica-
tion of the β-glucuronide linker beyond amines to phenol-
containing drugs, since the phenol functional group is a
common element in many anticancer drugs. For example,
the camptothecin derivative SN38 (1) and duocarmycins
such as 2 possess phenol residues. Another phenol-contain-
ing drug, psymberin (aka irciniastatin A, 3), is a potent
anticancer molecule with subnanomolar cytotoxic activity
(Table 1).11 The treatment of cells with 3 leads to apoptosis
through an undefined mechanism.
Received Date: February 19, 2010
Accepted Date: May 11, 2010
Published on Web Date: June 14, 2010
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Figure 1. Examples of ADCs with the β-glucuronide linker.
Scheme 1. Release Mechanism from β-Glucuronide Linker
Table 1. IC50 Values of Psymberin (3) and β-Glucuronide Drug-
Linker 8 Conjugatesa
Drug-linker 8 was conjugated to the monoclonal anti-
bodies cAC10 (anti-CD30 mAb) and h1F6 (anti-CD70 mAb)
via reduced interchain disulfides. An average loading of 5.4
drugs/mAb was achieved, and the conjugates were >95%
monomeric. Exposure to the CD30-expressing cell line
L540cy (Hodgkin's lymphoma) to cAC10-8 over a 96 h
period gave an IC50 value of 0.15 nM (Table 1). The cell killing
was immunologically selective as exposure to the CD30-
negative cell line Caki-1 (renal cell carcinoma) gave an IC50
Caki-1
(CD70þ/CD30-)
L540cy
(CD70-/CD30þ)
psymberin (3)
0.3
62
0.7
0.15
58.6
cAC10-8 (5.4 dr/mAb)
h1F6-8 (5.4 dr/mAb)
0.8
a Values are listed in nM.
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Scheme 2a
a Reagents and conditions: (a) DMED (10 equiv), CH2Cl2, 61%. (b) pNP carbonate, DIPEA, THF, 49%. (c) Compound 5 and 6, CH2Cl2, 42%. (d) LiOH
(9 equiv), CH3OH, THF, H2O. (e) MC-OSu, DMF, DlPEA, 62% two steps.
Scheme 3a
a Reagents and conditions: (a) Cysteine. (b) β-Glucuronidase (E. coli), 30 min.
value of 62 nM. Conversely, the h1F6-8 ADC showed good
activity on the CD70-expressing line Caki-1 but significantly
reduced activity against the CD30-negative line L540cy.
Unconjugated 3 was highly potent on these cell lines. The
selectivity observed over the 96 h exposure assay suggests a
high degree of stability, which is consistent with previous
findings with the β-glucuronide linker.7
ADCs continue to demonstrate their value in the clinic,3,13-16
and as a consequence, investigations into new chemotypes
and linkers for ADCs will continue. The favorable properties
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Figure 2. HPLC analysis of β-glucuronidase cleavage of psymber-
in drug linker 9. Peaks were observed using a diode array detector.
of the β-glucuronide linker, including its hydrophilicity, sta-
bility in circulation, tumor selectivity, and efficient drug
release inside tumor cells elevate this linker as an appealing
component for new ADC development and chemotype
screening. The expansion of its utility for the release of a
phenol-containing cytotoxic such as psymberin (3) has been
demonstrated in this communication and will be an avenue
of continued investigation in our laboratory.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 425-527-4738. Fax: 425-527-4109. E-mail: sjeffrey@
seagen.com.
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