Minor groove binder antibody conjugates employing a water soluble β-glucuronide linker

Article abstract of DOI:10.1016/j.bmcl.2007.01.071

The minor groove binder β-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli β-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5 drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble β-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.

Full text of DOI:10.1016/j.bmcl.2007.01.071

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2278–2280
Minor groove binder antibody conjugates employing a water
soluble b-glucuronide linker
Scott C. Jeffrey,* Minh T. Nguyen, Ruth F. Moser, Damon L. Meyer,
Jamie B. Miyamoto and Peter D. Senter
Seattle Genetics Inc., 21823 30th Drive S.E., Bothell, WA 98021, USA
Received 21 December 2006; revised 16 January 2007; accepted 18 January 2007
Available online 27 January 2007
Abstract—The minor groove binder b-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a sub-
strate for Escherichia coli b-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs
cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug
1. The ADCs were largely monomeric at intermediate loading levels (4–5 drug/mAb), in contrast to highly aggregated p-amin-
obenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with
cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble
b-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.
Ó 2007 Elsevier Ltd. All rights reserved.
Recently, we reported the development of a novel anti-
O
O
OH
O
O
OH
body-drug conjugate (ADC) linker system in which the
enzyme b-glucuronidase releases free drug through the
cleavage of a b-glucuronide glycosidic bond (Scheme
1).1 The linker was an extension of the doxorubicin anti-
body dependent enzyme prodrug therapy (ADEPT)
CO2H
O
O
Drug
OH
HO
HO
OH
O
O
O
O
O
HO
HN
mAb
HN
HO
O
HO2C
HO
`
agent 2 reported by Desbene and coworkers, which effi-
O
ciently released doxorubicin upon activation by a b-glu-
curonidase fusion protein.2 This prodrug construct was
modified to allow for attaching drugs to monoclonal
antibodies (mAbs). ADCs of 2-pyrrolinodoxorubicin,
and monomethyl auristatin E and F were constructed
and found to be highly potent and immunologically spe-
cific both in vitro and in vivo.1 The b-glucuronide linker
provided for facile drug release and was highly stable in
rat plasma. Importantly, the resulting ADCs were non-
aggregated and monomeric even when heavily loaded
(8 drugs/mAb) with hydrophobic anticancer drugs.
HO
O
β-glucuronidase
HO
HN
2
O
O
O
Drug
CO2H
HO
HO
O
1,6-elimination
drug release
HO
OH
HN
HO
mAb
O
Scheme 1.
The cyclopropyl indole minor groove binders (MGB)
alkylate double-stranded DNA in a sequence selective
manner resulting in single-strand breaks and subsequent
cell death.3–5 These compounds, including their seco ha-
lide derivatives, have been viewed as attractive mole-
cules for mAb targeted delivery based on their
synthetic tractability, mechanism of action, and potent
cytotoxic activity. Our work in this area has focused
on the amino cyclopropylbenz[e]indole (amino CBI)
seco-chloride 1 in which dipeptide-based drug linkers
were designed to stabilize the drug while attached to
Keywords: Aggregation; Antibody; Anti-CD30; Anti-CD70; b-Glucu-
ronidase; b-Glucuronide; cAC10; Cancer; Conjugate; Delivery; Drug;
Drug-linker; Minor groove binder; h1F6; Linker; Monoclonal; Rele-
ase; Targeted.
*
Corresponding author. Tel.: +1 425 527 4738; fax: +1 425 527
4109; e-mail: sjeffrey@seagen.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.071

S. C. Jeffrey et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2278–2280
2279
the mAb and liberate drug under the action of a prote-
olytic enzyme such as cathepsin B once delivered inside
the cancer cell.6,7 Others have published in this area
using both peptide and hydrazone-based linker
strategies.8,9
ment of 5 with lithium hydroxide removed the acetates
and Fmoc protecting group, and saponified the methyl
ester.2 Finally, the free amine was capped by 6 to afford
the bromoacetamide drug-linker 3. The b-glucuronide
linker is an effective solubilizing element for 1; drug-
linker 3 was soluble at 1 mM in phosphate buffered sal-
ine, pH 7.4, whereas the free drug 1 had a solubility of
only 5 lM in phosphate buffered saline, pH 7.4, contain-
ing 10% DMSO.
Two key challenges in working with this class of mole-
cules are their propensities to cause ADC aggregation,
due presumably to their planar hydrophobic structures
and their inherent instability. It has been found that
functionalizing the activating heteroatom (N or O) as
a carbamate, amide or ether renders the molecules inac-
tive while attached to the mAb. Several linker strategies
were developed to address aggregation, all of which em-
ployed a polyethylene glycol unit as part of the solution.
Here, we describe a follow up investigation using the b-
glucuronide linker, specifically the preparation of ADCs
of the hydrophobic MGB 1, with a goal of developing
active ADCs and understanding the impact a soluble
b-glucuronide linker might have on ADC aggregation.
Compound 3 was determined to be a substrate for Esch-
erichia coli b-glucuronidase (EC 3.2.1.31) using an
HPLC/MS based assay1. Compound 1 was the sole re-
leased drug. No deglucuronidated intermediate could
be detected, suggesting that once the b-glucuronide
group is cleaved, drug release via 1,6-elimination is fast.
In the absence of b-glucuronidase, incubation of 3 for
24 h in buffer gave no measurable drug release.
ADCs of 3 were prepared with chimeric AC10 (IgG1
against CD30 antigen) and humanized 1F6 (IgG1
against the CD70 antigen). The CD30 antigen resides
on activated B cells and is a marker for both Hodgkin’s
lymphoma and anaplastic large cell lymphoma
(ALCL).11 The CD70 antigen is overexpressed on renal
cell carcinoma (RCC) lines as well as some hematologic
malignancies.12–16 mAb interchain disulfides were par-
tially reduced using stoichiometric quantities of tris(2-
carboxyethyl) phosphine to give on average approxi-
mately 4 free thiols/mAb as previously described.17,18
The reduced mAbs were purified via gel filtration and
conjugated to 3 at pH 8. An excess of drug (1.4 drug/thi-
ol) was required to fully consume all free thiols and the
resulting ADCs were purified by cation exchange
chromatography.
The b-glucuronide drug-linker 3 was prepared in three
steps from 16,10 and the protected b-glucuronide inter-
mediate 4 (Scheme 2).1 Reaction of 4 with diphosgene
and capture of the resulting chloroformate with 1 pro-
ceeded to give a modest yield of the carbamate 5. Treat-
O
O
Cl
Cl
N
N
O
(a)
O
HO
NH2
HN
O
NHFmoc
O
1
O
NHFmoc
O
O
N
Analysis of the conjugates by size exclusion chromatog-
raphy revealed that they were largely monomeric. The
cAC10-3 ADC contained 4.8 drugs/mAb and was 79%
monomeric while the h1F6-3 contained 4.4 drugs/mAb
and was 85% monomeric (Table 1). Increasing mAb
loading to 8 drugs resulted in high levels of aggregation
(70% for h1F6-3). The results with the glucuronides
compare favorably to our previous results7 with the
dipeptide-based (valine–citrulline) p-aminobenzyl carba-
mates (PABC) MGB-linker in preventing ADC aggrega-
tion. The valine–citrulline PABC of 1, when conjugated
to reduced cAC10 with 4.5 available thiols/mAb, was
only 32% monomeric. Finally, we note that the conjuga-
tion conditions have not yet been fully optimized. Such
studies, which would require analyses of several param-
eters such as reaction time, temperature and pH, as well
as the effects of concentration and stoichiometry, could
lead to even lower levels of aggregate formation than
those reported here.
H
N
H3CO2C
AcO
O
O
H
H3CO2C
AcO
O
OAc
4
OAc
5
AcO
AcO
(b), (c)
O
O
N
O
Br
6
O
O
Cl
N
O
O
HN
O
O
O
N
O
H
In vitro cytotoxicity studies demonstrated that the
ADCs were potent and selective for antigen positive cells
(Table 1). The cAC10-3 ADC was highly potent against
the CD30 positive lines Karpas 299, L428, and L540cy
with IC50 values comparable with those of free drug 1.
The cytotoxic activity of the h1F6-3 against the CD70
positive cell line Caki-1 also was equivalent to 1. These
HO2C
HO
O
N
H
Br
OH
3
HO
Scheme 2. Reagents and conditions: (a) diphosgene (2.0 equiv),
pyridine, CH2Cl2, 1, 37%; (b) LiOH–H2O, CH3OH/H2O (1:1); (c) 6,
DIPEA, DMF, 21% two steps.

2280
S. C. Jeffrey et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2278–2280
Table 1. Characterization and in vitro cytotoxicity of conjugates and free drug
Cytotoxic
agent
Antigen
Drug
loading
Percent
monomer
Karpas 299a
(CD30+)
L428a
L540 Cya
(CD30+)
WSU-NHLa
Caki-1a
(CD70+)
(CD30+, CD70À)
(CD30À)
cAC10-3
1F6-3
1
CD30
CD70
4.8
4.4
—
79
85
—
0.4
—
0.4
0.9
—
>32b
—
—
>29b
0.1
0.6
0.7
1.2
0.5
—
a Cells were treated with the test agents for 96 h and viability was determined by reduction of resazurin. The IC50 values indicated are the
concentrations (nM) of the drug component of the ADC.
b No activity at highest concentration tested.
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data suggest that the drug is efficiently delivered to the
cell. The cAC10-3 ADC gave good selectivity for anti-
gen positive cells versus the antigen negative line
WSU-NHL, and the non-binding control h1F6-3 was
significantly less active against the CD30 positive line
L428. The in vitro profile of the b-glucuronide ADCs
of 1 with respect to both cytotoxic activity and specific-
ity is comparable to that of the dipeptide reagents previ-
ously disclosed.7
Investigations by our group and others have demon-
strated the importance of linker composition for devel-
oping clinically viable ADCs.18–20 Linker stability and
drug release kinetics are parameters inherent to the link-
er construct and profoundly impact ADC safety and
efficacy. In addition, the linker can have a marked influ-
ence on aggregation, particularly when hydrophobic
drugs are employed. The data presented here and previ-
ously suggest that the b-glucuronide linker, with its high
aqueous solubility, long plasma half-life, and facile drug
release, is a complementary alternative to PABC dipep-
tide, disulfide, and hydrazone-based linkers.21–24 The b-
glucuronide linker, when used to tether the hydrophobic
amino CBI 1, significantly reduced aggregation relative
to PABC dipeptide-based ADCs. ADCs of 3 were
immunologically specific and displayed high cytotoxic
activity against Hodgkin’s lymphoma lines L428 and
L540cy, the ALCL line Karpas 299, and RCC line
Caki-1. These results provide strong justification for fur-
ther developing this promising agent by investigating its
efficacy in preclinical in vivo models for human cancer.
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