Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system

Article abstract of DOI:10.1016/j.bmcl.2009.03.145

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody-drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile

Full text of DOI:10.1016/j.bmcl.2009.03.145

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2650–2653
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel immunoconjugates comprised of streptonigrin and
17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker
system
*
Patrick J. Burke , Brian E. Toki, David W. Meyer, Jamie B. Miyamoto, Kim M. Kissler, Martha Anderson,
Peter D. Senter, Scott C. Jeffrey
Seattle Genetics, Inc., 21823 30th Drive SE, Bothell, WA 98021, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies
(mAbs), forming antibody–drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The
drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a
valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine
functional groups present in both drugs. The resulting ADCs release drug following internalization into
antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6
(anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The
streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro
and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for
further research, and that the novel linker system used to make them is well-suited for the conjugation
of cytotoxic agents containing electron-deficient amine functional groups.
Received 5 March 2009
Revised 26 March 2009
Accepted 30 March 2009
Available online 5 April 2009
Keywords:
Antibody-drug conjugate
Streptonigrin
Geldanamycin
Antitumor
Cancer
Anti-CD30
Anti-CD70
Ó 2009 Elsevier Ltd. All rights reserved.
Cytotoxic
Cathepsin B
cAC10
h1F6
Targeted delivery
Self-immolative spacer
Immunoconjugate
Conjugate
There is a great deal of interest in the use of mAbs that bind spe-
cifically to antigens overexpressed on cancer cell populations for
the targeted delivery of cytotoxic agents.1,2 The strategy seeks to
combine the exquisite targeting specificity of mAbs with the
potent cytotoxicity of small-molecule antitumor agents. In contrast
to small molecule drugs, which are often rapidly cleared from sys-
temic circulation, mAb therapeutics typically remain in circulation
for many days to weeks.1,2 Therefore, in addition to tumor target-
ing, ADCs benefit from enhanced exposure relative to free drugs.
Early work has elucidated the importance of drug linker circulation
stability, efficient on-target drug release, antitumor agent potency,
and a clean antigen expression profile as important parameters for
the success of the ADC strategy.2,3 Various different drug linkers
have been used for conjugation to mAbs, including hydrazones,
disulfides, and peptides, which are cleaved to release drug under
acidic, reducing, and proteolytic conditions, respectively.1,3 Our
experience with the auristatin drug class has led us toward
amine-containing drugs, which are amenable to conjugation via
dipeptide-PAB-carbamate linkages.4 We have also shown that phe-
nolic drugs can be stably attached to mAbs through a dipeptide-
PAB-ether linker and that drug is efficiently released upon proteol-
ysis.5 Monoclonal antibodies empowered with drugs attached
through dipeptide linkers have been characterized by a high degree
of circulation stability.4,6
We are interested in all classes of cytotoxic drugs that may be
suitable for empowering mAbs. In an effort to expand our ADC
platform, we sought to evaluate streptonigrin and 17-amino-
geldanamycin as potential ADC chemotypes (Scheme 1A). Strep-
tonigrin (1) is a quinoline quinone antitumor agent originally iso-
lated in 1959 from Streptomyces flocculus.7 The cytotoxic
mechanism is thought to involve metal chelation and redox cycling
leading to the production of reactive oxygen species and subse-
quent DNA strand breaks, culminating in cell death.8,9 The
* Corresponding author. Tel.: +1 425 527 4766; fax: +1 425 527 4109.
E-mail address: pburke@seagen.com (P.J. Burke).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.145

P. J. Burke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2650–2653
2651
to Herceptin in a mouse xenograft.17 Potential liabilities with this
conjugation strategy include the lack of a mechanism for drug re-
lease and the use of an analog (4) with inferior potency relative to
the parent compound (2).
In contrast to the conjugation strategies described above, our
approach involves the incorporation of a conditionally-stable
dipeptide drug linker to achieve release of the free drug upon inter-
nalization of ADC into the lysosomes of the targeted cells (Scheme
1B). The Val-Ala-PAB drug linker system is designed to provide cir-
culation stability and efficient drug release in the presence of lyso-
somal proteases.18 One unique aspect of this drug linker approach
is that electron-deficient, aryl amine-containing drugs are linked
through a benzyl amine functional group to the mAb, instead of
the commonly used carbamate moiety.
The streptonigrin drug linker was synthesized as shown in
Scheme 2. Commercially available streptonigrin (1) was alkylated
with Fmoc-Ala-para-aminobenzyl bromide in the presence of Hu-
nig’s base to provide 5 in 44% yield. Chemoselectivity of alkylation
was established by NOESY, in which an NOE was observed between
the benzyl methylene and a D-ring aromatic proton. This would
not be observed if alkylation had occurred at the D-ring phenol
or the A-ring vinylogous amide. Deprotection of the Fmoc group,
followed by coupling with Fmoc-Val-OSu, afforded dipeptide inter-
mediate 7. Another round of Fmoc deprotection and coupling with
the NHS ester of 6-maleimidocaproic acid (MC-OSu) provided drug
linker 9. It is worth noting that attempts to make the analogous
PAB-carbamate linked constructs were unsuccessful, presumably
due to the low nucleophilicity of the electron deficient aryl amine
in the C-ring. This is in contrast to drugs containing electron-rich,
primary or secondary aliphatic amines that readily react with
O
A.
O
A
O
R
O
17
MeO
H2N
B
N
H
N
C
CO2H
N
O
H3CO
H2N
Me
OH
H3CO
OH
O
D
O
OMe
NH2
OMe
R = OCH3, geldanamycin (2)
R = NH2, 17-amino-GA (3)
R = NH(CH2)3NH2, (4)
streptonigrin (1)
drug
HN
B.
mAb
O
O
O
H
N
N
N
H
N
H
O
O
proteolytic
cleavage site
lysosomal
proteases
drug
HN
1,6
free drug 1 or 3
elimination
H2N
PAB adduct
Scheme 1. (A) Structure of cytotoxic agents, (B) design of conditionally-stable
streptonigrin and 17-amino-geldanamycin immunoconjugates.
O
O
MeO
H2N
MeO
H2N
N
CO2H
N
CO2H
N
N
compound was found to be highly potent in vitro and efficacious
in vivo for the treatment of various lymphomas and carcinomas.
However, in 1977 the NCI discontinued clinical investigations
due to severe and unpredictable toxicities,9 perhaps making this
drug a suitable candidate for targeted delivery. Geldanamycin
(GA), a benzoquinoid ansamycin produced by actinomycete Strep-
tomyces hydroscopicus, exerts cytotoxic activity through binding to
hsp90, leading to the accelerated degradation of signal-transducing
protein tyrosine kinases and, ultimately, cell death.10,11 Although a
highly potent antitumor agent, severe toxicity and formulation
challenges have precluded the clinical development of GA.12 Gel-
danamycin (2) lacks a suitable functional group, such as an amine,
phenol, or thiol, with which to devise a circulation-stable conjuga-
tion strategy. However, structure activity relationships have estab-
lished that 17-amino-GA (3) retains the cellular potency of GA.12,13
Analogs of STN and GA have been used to prepare ADCs previ-
ously. The C-ring NHS ester of streptonigrin was conjugated to sol-
vent-exposed antibody lysines to form an ADC.14,15 The conjugate
was shown to be active against a hepatocarcinoma cell line15 and
Ehrlich carcinoma cells14 in vitro. To the best of our knowledge,
in vivo data was never reported for this conjugate. One potential
liability with this approach is that the conjugation strategy in-
cludes no provision for the release of the free drug. A Herceptin–
GA immunoconjugate has also been described.12,16 The drug pay-
load in this case was 17-(3-aminopropyl)-GA (4) attached directly
to Herceptin with a putatively non-cleavable maleimidobutyryl
linker. Despite the fact that 17-amino-GA (3) is more potent than
17-(3-aminopropyl)-GA (4), analog 4 was employed because the
electron deficient amino group (a vinylogous amide) in 3 was
deemed not suitable for conjugation.12 The Herceptin-GA conju-
(a)
O
O
H2N
Me
OH
HN
Me
OH
O
H
N
OMe
R
N
H
OMe
OMe
OMe
streptonigrin (1)
5, R = Fmoc
6, R = H
(b)
O
O
MeO
N
CO2H
H2N
O
N
(c)
(d)
HN
Me
OH
H
N
R
N
H
N
H
OMe
O
OMe
7, R = Fmoc
8, R = H
(b)
O
O
MeO
N
CO2H
H2N
O
N
HN
Me
OH
O
O
H
N
N
N
H
N
OMe
H
O
O
OMe
9
Scheme 2. Synthesis of a streptonigrin drug linker. Reagents and conditions: (a)
Fmoc-Ala-PAB-Br, DIPEA, DMF, rt, 18 h, 44%; (b) DMF:piperidine (4:1), quant.; (c)
Fmoc-Val-OSu, DIPEA, DMF, rt, 88%; (d) MC-OSu, DIPEA, DMF, rt, 86%.
gate was moderately active in vitro, with IC50 = 40
lg ADC/mL or
580 nM in terms of GA,12 and displayed enhanced efficacy relative

2652
P. J. Burke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2650–2653
para-nitrophenyl carbonate reagents to form PAB-carbamate based
drug linkers.18,19
and renal cell carcinoma. The GA drug linker (12) was conjugated
to h1F6. Drug linkers were conjugated through reduction of mAb
interchain disulfides and subsequent alkylation of the cysteine thi-
ols with the drug linker maleimide.4,24 The mAbs were alkylated at
a level of 4 drugs/mAb to minimize aggregation, as determined by
size exclusion chromatography. SEC analysis indicated that cAC10-
9 (3.8 drugs/mAb) and h1F6-9 (4.0 drugs/mAb) were 89% and 96%
monomeric, respectively. h1F6-12 (4.0 drugs/mAb) was 95% mono-
meric. Excess drug linker was removed by purification of the con-
jugates via gel filtration.
The preparation of a 17-amino-GA drug linker was achieved as
shown in Scheme 3. The 17-methoxy group in GA was exchanged20
via addition/elimination with Fmoc-Val-Ala-para-aminobenzyl-
amine in the presence of Hunig’s base, providing intermediate 10
in 50% yield. Subsequent Fmoc deprotection provided free amine
11, which was then subjected to diethyl cyanophosphonate-medi-
ated coupling with 6-maleimidocaproic acid (MC–OH) to give drug
linker 12 in 33% yield over two steps.
Prior to the preparation of ADCs, it was desirable to ensure that
the 1,6-elimination of the PAB adducts (Scheme 1B) would occur
following protease cleavage at the dipeptide. Drug linkers 9 and
12 were quenched at the maleimide and subjected to incubation
with papain, a general cysteine protease, or cathepsin B, a lyso-
somal protease, to effect quantitative cleavage of the Ala-PAB amide
bond. The reactions21 were followed by LC–MS which showed that
in both cases the quenched drug linkers had been quantitatively
converted to free drugs 1 and 3 within 30 minutes following treat-
ment with papain and cathepsin B, respectively. No intermediate
PAB-STN or PAB-17-amino-GA adducts were observed.
The ADCs were assayed for in vitro cytotoxic activity and spec-
ificity (Table 1). The STN conjugates displayed potency and immu-
nologic specificity on Karpas 299 (anaplastic large cell lymphoma),
L540cy (Hodgkin lymphoma), and Caki-1 (renal cell carcinoma)
cell lines. Karpas 299 and L540cy are CD30 positive and CD70 neg-
ative, while Caki-1 is CD30 negative and CD70 positive. Therefore,
h1F6-9 serves as an IgG specificity control conjugate for Karpas
299 and L540cy, while cAC10-9 serves as the specificity control
for Caki-1. The STN ADCs displayed antigen-specific potencies
across the panel with the IC50 values ranging from 2.1 to 17 nM
(in terms of the drug component). Immunological specificity was
assessed with non-binding conjugates, which had a small fraction
of the activity compared to binding conjugates. The 17-amino-GA
ADC was inactive (IC50 in terms of drug component >240 nM) on
The STN drug linker (9) was conjugated to chimeric AC10,22 spe-
cific for CD30 on hematologic malignancies, and to humanized
1F6,23 which recognizes the CD70 antigen on hematologic tumors
O
O
H
N
H
N
O
H
H3CO
H3CO
R
N
H
O
O
N
O
N
H
(a)
O
N
H
H3CO
O
H3CO
OH
O
H3CO
O
OH
O
NH2
10, R = Fmoc
O
geldanamycin (2)
(b)
NH2
11, R = H
O
O
N
H
H
N
N
N
H
O
H
O
O
N
(c)
O
O
N
H
O
H3CO
12
H3CO
OH
O
O
NH2
Scheme 3. Synthesis of a 17-amino-geldanamycin drug linker. Reagents and conditions: (a) Fmoc-Val-Ala-PAB–NH2, DIPEA, pyridine, 50 °C, 7 h, 50%; (b) CH2Cl2:piperidine
(4:1); (c) MC–OH, DEPC, DIPEA, CH2Cl2, rt, 33% (two steps).
Table 1
Characterization and in vitro potency of the STN conjugates
Test article
Antigen
Monomer (%)
Drugs/mAb
IC50 (nM)
Karpas 299a CD30+/CD70À
L540cya CD30+/CD70À
Caki-1a CD30À/CD70+
cAC10-9
h1F6-9
1
CD30
CD70
—
89
96
—
3.8
4.0
—
3.3
2.1
>270b
17
0.4
>270b
0.4
>270b
0.8
a
Cells were treated with test articles for 96 h and cell viability was assessed by resazurin conversion. The IC50 values represent the concentrations (nM) of the drug
component of the antibody–drug conjugates.
b
IC50 values are greater than the highest concentration tested.

P. J. Burke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2650–2653
2653
1000
900
800
700
600
500
400
300
200
100
0
Untreated
cAC10-9, 3 mg/kg
cAC10-9, 10 mg/kg
IgG-9, 10 mg/kg
n = 5/group
schedule: single dose, ip
treatment
0
5
10
15
20
25
30
35
40
45
DAYS POST TUMOR IMPLANT
Figure 1. Single dose efficacy of 4-loaded streptonigrin immunoconjugate in a subcutaneous L540cy Hodgkin lymphoma xenograft model.
7. Rao, K. W.; Cullen, W. P. In Antibiotics Annual; Welch, H., Marti-Ibanez, F., Eds.;
Antibiotica: New York, 1959–1960. p. 950.
8. Bringmann, G.; Reichert, Y.; Kane, V. V. Tetrahedron 2004, 60, 3539.
9. Harding, M. H.; Long, G. V. Curr. Med. Chem. 1997, 4, 405.
10. DeBoer, C.; Meulman, P. A.; Wnuk, R. J.; Peterson, D. J. Antibiot. 1970, 23,
442.
11. Neckers, L.; Schulte, T. W.; Mimnaugh, E. Invest. New Drugs 1999, 17, 361.
12. Mandler, R.; Wu, C.; Sausville, E. A.; Roettinger, A. J.; Newman, D. J.; Ho, D. K.;
King, C. R.; Yang, D.; Lippman, M. E.; Landolfi, N. F.; Dadachova, E.; Brechbiel, M.
W.; Waldmann, T. A. J. Natl. Cancer Inst. 2000, 92, 1573.
13. Schnur, R. C.; Corman, M. L.; Gallaschun, R. J.; Cooper, B. A.; Dee, M. F.; Doty, J.
L.; Muzzi, M. L. al. e. J. Med. Chem. 1995, 38, 3813.
14. Komissarenko, S. V.; Penezina, O. P.; Fomovskaya, G. N.; Tolstikov, V. V.;
Preobrazhenskaya, M. N. Int. J. Immunopharmac. 1994, 16, 1053.
15. Liu, Y. P.; Wu, J. B. Yao Xue Xue Bao 1992, 27, 498.
16. Mandler, R.; Dadachova, E.; Brechbiel, J. K.; Waldmann, T. A.; Brechbiel, M. W.
Bioorg. Med. Chem. Lett. 2000, 10, 1025.
17. Mandler, R.; Kobayashi, H.; Hinson, E. R.; Brechbiel, M. W.; Waldmann, T. A.
Cancer Res. 2004, 64, 1460.
18. Jeffrey, S. C.; Nguyen, M. T.; Andreyka, J. B.; Meyer, D. L.; Doronina, S. D.; Senter,
P. D. Bioorg. Med. Chem. Lett. 2006, 16, 358.
a panel of CD70 positive cell lines (data not shown) and was ex-
cluded from further evaluation. While free 17-amino-GA (3) is a
potent small molecule, the lack of potency of the ADC may be
due to many reasons including drug metabolism in lysosomes
and altered intracellular trafficking.
In light of the encouraging in vitro data for the STN conjugates,
in vivo experiments were performed in an L540cy mouse xenograft
model.25 SCID mice were implanted with L540cy cells and treated
with test articles on day 9 when the tumors had reached nominally
100 mm3. Mice were treated with one injection (delivered ip) of
cAC10-9 at 3 mg/kg or 10 mg/kg. Controls consisted of an untreated
group and non-binding IgG control group treated with h1F6-9 at
10 mg/kg. As seen in Figure 1, an immunologically specific, dose-
dependent response was observed. Mice treated with 3 and
10 mg/kg cAC10-9 experienced delay in tumor outgrowth, while
the non-binding ADC was less active. The observed activity of the
non-binding IgG control is perhaps a consequence of non-specific
conjugate uptake. Both doses were tolerated (<20% weight loss
and no outward signs of toxicity), but 50 mg/kg of cAC10-9 exceeded
the maximum tolerated dose. Thus, the STN ADC described here is
not as well-tolerated as an analogous cAC10-auristatin immunocon-
jugate that utilizes a dipeptide benzylcarbamate linker.26
In summary, ADCs were prepared with potent cytotoxic agents
utilizing a dipeptide-PAB-amine linker system. Upon protease-
mediated dipeptide hydrolysis, the linker efficiently releases drugs
such as STN (1) and 17-amino-GA (3) in chemically unmodified
form, suggesting that the 1,6-elimination is rapid. Applications
for this linker technology were demonstrated with STN (1) ADCs,
which were mainly monomeric (P89%) with 4 drugs/mAb, and
were active and immunologically specific in vitro and in vivo. Thus,
the dipeptide-PAB-amine linker is well-suited for mAb conjugation
and efficient protease-mediated drug release.
19. Doronina, S. D.; Mendelsohn, B. A.; Bovee, T. D.; Cerveny, C. G.; Alley, S. C.;
Meyer, D. L.; Oflazoglu, E.; Toki, B. E.; Sanderson, R. J.; Zabinski, R. F.; Wahl, A.
F.; Senter, P. D. Bioconjugate Chem. 2006, 17, 114.
20. Mandler, R.; Kobayashi, H.; Davis, M. Y.; Waldmann, T. A.; Brechbiel, J. K.
Bioconjugate Chem. 2002, 13, 786.
21. A. Papain reaction:
A 10 mM DMSO stock solution of drug linker 9 was
quenched with 2 equiv of N-acetylcysteine. The quenched linker was treated
with papain from papaya latex (Sigma–Aldrich, St. Louis, MO, USA) at a final
concentration of 1 mg/mL (21 units/mg) in a pH 7 phosphate buffer. The final
concentration of drug linker was 0.31 mM. The progress of the reaction was
followed by LC–MS with monitoring for masses corresponding to the quenched
drug linker (exact mass = 1137.4), the PAB-STN adduct (exact mass = 611.2),
and streptonigrin (exact mass = 506.1). By 30 min the quenched drug linker
had been quantitatively converted to free streptonigrin as determined by the
appearance of one new peak in the chromatogram characterized by masses
(ES+) m/z = 507.0 (M+H)+, and (ESÀ) m/z = 505.1 [MÀH]À. B. Cathepsin
B
reaction: Dubowchik, G. M.; Firestone, R. A. Bioorg. Med. Chem. Lett. 1998, 8,
3341.
22. Wahl, A. F.; Klussman, K.; Thompson, J. D.; Chen, J. H.; Francisco, L. V.; Risdon,
G.; Chace, D. F.; Siegall, C. B.; Francisco, J. A. Cancer Res. 2002, 62, 3736.
23. Law, C.-L.; Gordon, K. A.; Toki, B. E.; Yamane, A. K.; Hering, M. A.; Cerveny, C. G.;
Petroziello, J. M.; Ryan, M. C.; Smith, L.; Simon, R.; Sauter, G.; Oflazoglu, E.;
Doronina, S. D.; Meyer, D. L.; Francisco, J. A.; Carter, P. J.; Senter, P. D.; Copland,
J. A.; Wood, C. G.; Wahl, A. F. Cancer Res. 2006, 66, 2328.
References and notes
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