Antibody conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) for targeted cancer chemotherapy

Article abstract of DOI:10.1021/jm800719t

CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.

Full text of DOI:10.1021/jm800719t

6916
J. Med. Chem. 2008, 51, 6916–6926
Antibody Conjugates of 7-Ethyl-10-hydroxycamptothecin (SN-38) for Targeted Cancer
Chemotherapy
Sung-Ju Moon,† Serengulam V. Govindan,*,† Thomas M. Cardillo,† Christopher A. D’Souza,† Hans J. Hansen,† and
David M. Goldenberg‡
Immunomedics, Inc., Morris Plains, New Jersey 07950, Garden State Cancer Center at the Center for Molecular Medicine and Immunology,
BelleVille, New Jersey 07109
ReceiVed June 16, 2008
CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38
(7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase
the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted
therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in
the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation.
Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38
derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and
cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16
and 17 were found promising for further development.
Introduction
complexation with human serum albumin.11,12 The pharmaco-
logical activity of CPTs depends on the presence of the intact
hydroxylactone moiety.12 Early findings in the CPT area showed
that derivatization of 20-hydroxyl group of CPT considerably
minimized the undesirable lactone ring opening under physi-
ological conditions.13
Erratic and patient-variable conversion of CPT-11 to the
active drug and the complex in vivo metabolism14 of both CPT-
11 and SN-38 result in reduced bioavailability of the active drug.
These considerations have spawned considerable interest in
utilizing SN-38 itself in water-soluble forms other than CPT-
11. This then led to the attachment of parent CPT or 10-hydroxy-
CPT, and later on SN-38, to hydrophilic polymers such as
polyglutamic acid, poly HPMA, or polyethylene glycol.15-18
A liposomal formulation of SN-38 was also evaluated.19 In other
approaches, the 10-hydroxyl group of the molecule was utilized
in the form of an ether or an ester, with further attachment to
dextran or to a peptide, respectively.20,21
We embarked on a project to target SN-38 selectively to
tumor sites using tumor-selective mAbs so as to increase both
solubility and therapeutic index. SN-38 is a potent drug,22 with
IC50 values in the nanomolar range in a number of tumor cell
lines. There is considerable current interest in targeted therapies
of cancer. The use of tumor-selective mAbs as carriers of
therapeutic radionuclides or chemotherapy drugs or protein
toxins has been investigated extensively in view of the op-
portunity for “patient-friendly” treatments. Several recent
reviews describe the scope of these approaches.23-26 An
advantage to using SN-38 in the antibody conjugate format is
that the drug’s in vivo pharmacology is well established.
In this report, we describe the syntheses of bifunctional SN-
38 derivatives and in vitro characteristics of their antibody
conjugates. Selected conjugates were also evaluated as thera-
peutic agents in a number of preclinical models of solid
tumors;27,28 the preclinical therapy data will be detailed
elsewhere.
SN-38 is the active drug form of the clinically used anticancer
agent, CPT-11 (irinotecan, 7-ethyl-10-{4-[1-piperidino]-1-
piperidino}carbonyoxycamptothecin), and belongs to the class
of 20(s)-camptothecin (CPTa) group of compounds that act as
potent topoisomerase I inhibitors.1-3 SN-38 is 2-3 orders of
magnitude more potent than CPT-11 in vitro.4 The latter has
shown antitumor activity clinically in colorectal, lung, cervical,
and ovarian cancers.5,6
The in vivo conversion of CPT-11 to SN-38 by human liver
carboxylesterase is inefficient.7 In the liver, SN-38 is further
transformed to its ꢀ-glucuronide, SN-38G. Once excreted into
bile, SN-38G is reconverted to SN-38 by intestinal ꢀ-glucu-
ronidase,8 resulting in severe delayed diarrhea in patients
undergoing the CPT-11 treatment.9 Oxidative degradations,
mediated by cytochrome P450, give rise to metabolites, some
of which are poorer substrates than CPT-11 for human car-
boxylesterase.10 Further, at physiological pH, CPTs exist in
equilibrium with the corresponding lactone opened form, with
the carboxylate form possessing ∼10% of the potency of intact
lactone form; this equilibrium is further shifted to the inactive
carboxylate form due to the stabilization of the latter by
* To whom correspondence should be addressed. Phone: 973-605-8200.
Fax: 973-605-1340. E-mail: sgovindan@immunomedics.com.
†
Immunomedics, Inc.
Garden State Cancer Center at the Center for Molecular Medicine and
‡
Immunology.
a Abbreviations: AcOH, acetic acid; BOC, tert-butoxycarbonyl; CLn-
SN-38 (n ) none or 1-5), cross-linker-attached SN-38 versions; CEACAM5,
carcinoembryonic antigen-related cell adhesion molecule 5; CPT, camp-
tothecin; DCA, dichloroacetic acid; DCC, dicyclohexylcarbodiimide; DIEA,
diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, dimeth-
ylformamide; DMSO, dimethylsulfoxide; D-PBS, Dulbecco’s phosphate
buffered saline; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline;
EGP-1, epithelial glycoprotein-1; Fmoc, fluorenylmethoxycarbonyl; HPMA,
hydroxypropyl methacrylamide; mAb, monoclonal antibody; MC, male-
imidocaproyl; MMT, monomethoxytrityl; NHS, N-hydroxysuccinimide;
PAB, p-aminobenzyl; PEG, polyethylene glycol; sc, subcutaneous; SE-
HPLC, size-exclusion HPLC; SMCC, succinimidyl 4-(N-maleimidometh-
yl)cyclohexane-1-carboxylate; TBAF, tetrabutylammonium fluoride; TB-
DMS, tert-butyldimethylsilyl chloride; TCEP, tris(2-carboxyethyl) phosphine;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; tR, retention time.
Results and Discussion
Synthetic Chemistry: The Initial Approach. We originally
set out to prepare a bifunctional SN-38, 3 (“CL-SN-38”), using
10.1021/jm800719t CCC: $40.75
2008 American Chemical Society
Published on Web 10/22/2008

Antibody Conjugates of SN-38
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6917
Scheme 1. SN-38, Variously Derivatized at 10 and 20 Positions
instituted, followed by immediate column chromatography to
separate the product from DMAP and unreacted triphosgene.
The best yield of the isolated product, 2, was ∼ 30%, and in
most runs, the product was obtained in low and variable yields,
sometimes as low as 10%, despite attempted optimization. In
the final step, the BOC group was removed by treating with
TFA for 2-5 min and isolating the product, 3 (“CL-SN-38”),
by solvent removal and precipitation with diethyl ether. The
20-carbonate was concomitantly deprotected to some extent, but
the level of SN-38 so formed was limited to e20% by carrying
out the TFA reaction for e5 min. The level of free SN-38 was
further reduced to e10% by additional purification. The product,
3, was used as such for mAb conjugations because underivatized
SN-38 was removable during purification.
Conjugates of CL-SN-38 were prepared from humanized anti-
CEACAM5 mAb, hMN-14 (labetuzumab), humanized anti-
EGP-1 mAb, hRS7, and humanized anti-CD22 mAb, hLL2
(epratuzumab), used as control, and were found to exhibit
significant and selective therapeutic effects in nude mice bearing
human colonic and lung carcinoma xenografts.27 Briefly, in a
lung metastatic model of human colon carcinoma in nude mice,
therapy with CL-SN-38 conjugate of the specific humanized
anti-CEACAM5 mAb, hMN-14, increased median survival
2-fold to 74 days compared to treatments with equidoses of the
conjugate of nontargeting control antibody, mixture of antibody
and SN-38, or nontreatment control (P < 0.005). In the therapy
of sc Calu-3 human lung adenocarcinoma xenografts in nude
mice, the CL-SN38 conjugate of the specific, rapidly internal-
izing, humanized anti-EGP-1 mAb, hRS7, produced “cures” in
80% of animals (no visible tumor) out to 110 days; in this
experiment, treatment with equidose mixture of nontargeting
control conjugate and nontreatment resulted in an 8.3-fold and
a 14-fold increase in mean tumor volumes, respectively, on day
61.
Scheme 2. Synthesis of the Bifunctional SN-38, CL-SN-38 (3)
While this approach helped establish the utility of mAb-SN-
38 conjugates in preclinical models, it had certain shortcomings
that precluded further development, namely: (1) The yield at
the carbonate formation stage was low and irreproducible, and
(2) solubility problem caused turbidity or precipitation during
conjugations, resulting in significant protein loss. Even with 15%
v/v of DMF or DMSO as cosolvent, low salt buffer, and gradual
addition of the solution of the drug to reduced antibody, the
protein recovery was only ∼ 20% in many instances. By
replacing maleimidocaproyl with a defined-PEG in the cross-
linker, the solubility problem was overcome at the mAb
conjugation stage (not shown). Still, the synthesis was impracti-
cal in the key aspects of yield, reproducibility, and scale-up.
For our program, we needed a robust and higher-yield synthetic
design.
On the basis of the hypothesis that the yield at the carbonate-
forming step was hampered by the damage to the maleimide
functional group, a substrate with Fmoc-protected version of
the cross-linker without the maleimide group, namely Fmoc-
Phe-Lys(MMT)-PABOH, was briefly examined for reacting with
1b. While the carbonate formation proceeded in ∼60% yield,
subsequent Fmoc deprotection liberated a free amino group
under basic conditions that were incompatible with the lactone
group of SN-38. Both the yield and the quality of the product
were unsatisfactory.
the sequence shown in Scheme 2. The synthesis was modeled
on an approach described for a camptothecin (CPT) derivative
wherein the lone 20-hydroxyl group of CPT was converted into
a carbonate using a cross-linker that contained an intracellularly
cleavable dipeptide, Phe-Lys, and a maleimide.29 For application
to SN-38, the more reactive phenolic hydroxyl group at the 10
position had to be protected, with deprotection performed after
the carbonate formation at the 20-hydroxyl position. The
maleimide-containing bifunctional linker precluded protection
of the 10-hydroxyl as a silyl derivative because fluoride-
mediated deprotection was found to affect the maleimide group.
The phenolic group was protected as the BOC derivative after
determining that this group could be removed selectively in a
short-duration TFA treatment.
BOC-SN-38, 1a,18 was converted to its 20-O-chloroformate,
1b, using triphosgene and DMAP (Scheme 1), and the latter
was reacted in situ for <5 min with the known linker, MC-
Phe-Lys(MMT)-PABOH30 (Scheme 2). Initial experiments
involving longer duration of reaction gave intractable products.
We hypothesized that the maleimide functional group was
incompatible with the conditions used in the SN-38-20-O-
chloroformate formation, resulting in low yields. Thus, a short-
duration reaction (<5 min) at the carbonate-formation step was
Azide-Acetylene Cycloaddition Route. An azide-incorpo-
rated linker, containing the PABOH moiety, was conceptualized
to overcome the synthetic hurdle in forming the 20-carbonate,
with the subsequent Cu(I)-mediated “click” cycloaddition31
enabling the introduction of the maleimide group. To this end,

6918 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Chart 1. Structures of Some Reagents and Structural Residues
Moon et al.
dipeptide in the linker in addition to the ester or carbonate, we
prepared a series of bifunctional SN-38 derivatives CL1-SN-
38 through CL5-SN-38 that differed in the nature of the linker
at the 20-position and the presence of the cleavable peptide.
Chart 2 gives the structures of the various bifunctional SN-38
products made.
Preparations Using the Modified Approach. We first
prepared “CL1-SN-38”, 7 (Scheme 3). In this, SN-38 was first
derivatized as 20-O-glycinate, 1d, by a three-step procedure of
conversion to BOC-SN-38, ester formation using BOC-glycine,
and removal of BOC groups along the lines originally reported
for 10-hydroxycamptothecin.16 The glycinate, 1d, was further
transformed to the azido derivative, 6, which in turn was
subjected to click cycloaddition with 4 to obtain CL1-SN-38,
7. The glycinate linkage, as in this product, has been used in
the design of some water-soluble versions of CPT and SN-38.16,18
Scheme 4 shows the preparation of “CL2-SN-38”, 16.
Although we continued with the “BOC” protection for the 10-
hydroxyl group, it was imperative to find an alternate protecting
group so as to avoid the impractical 5-min deprotection as well
as the yield reduction in the final step. The click chemistry
approach enabled the silyl protecting group to be examined.
Accordingly, we prepared the TBDMS derivative of SN-38, 1g,
transformed it to the azide-appended SN-38, 11, and cleaved
off the silyl group to afford 12. Click cycloaddition, followed
by mild deprotection using dichloroacetic acid, furnished CL2-
SN-38. Separating the carbonate-forming step from the male-
imide introduction conferred certain robustness in yield repro-
ducibility. Yields were generally in the range of 70-80% at
the carbonate formation and the click cycloaddition steps in a
number of preparations. Furthermore, the final product, after
removing the protecting groups, could be readily purified by
flash chromatography.
The 10-carbonate variant, 17, designated as CL2-SN-38(Et),
was designed as the CL2-SN-38 equivalent. The phenolic
carbonate in this derivative was expected to cleave readily under
physiological conditions based on a report on the differential
cleavage rates of 10-carbonate vs 20-carbonate in 10,20-
biscarbonate derivatives of 10-hydroxycamptothecin.32 The
original rationale for preparing 17 was to avoid the use of the
BOC protecting group. Unexpectedly, this derivative enhanced
the stability of the conjugate, as discussed in a later section.
Scheme 5 depicts a bifunctional SN-38, 21 (“CL3-SN-38”),
possessing an ester linkage at the 20-position as well as the
cleavable dipeptide, Phe-Lys. This product has structural
similarities to both CL1-SN-38 and CL2-SN-38 and was
designed as an improved version of both of these structures.
Lysosomal cleavage of the peptide bond will liberate SN-
38-20-O-glycinate from the conjugate, which can undergo ester
hydrolysis chemically or enzymatically in lysosome or cytosol.
SN-38 derivative 25 (“CL4-SN-38”; Scheme 6), which has
a carbonate linking at the 20 position, does not have the “Phe-
Lys” cleavable peptide but possesses the PABO residue, and
the latter is attached to the PEG azide. Its synthesis is
straightforward. A simpler carbonate, 28 (Scheme 7), was
prepared by directly attaching O-(2-azidoethyl)heptaethyleneg-
lycol to the chloroformate 1b; the resultant product 26 was
coupled to 4, and the click chemistry product 27 was deprotected
with TFA to obtain 28. In principle, BOC protecting group of
Schemes 6 and 7 can be replaced with TBDMS to further
improve yields.
the commercially available O-(2-azidoethyl)-O′-(N-diglycolyl-
2-aminoethyl)heptaethyleneglycol, 5, was used as a linker to
introduce the azido group on to SN-38; the acetylenic counter-
part for the click chemistry, 4, was prepared by reacting
succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC) with propargylamine. The provision of a short PEG
moiety in the linker addressed the aqueous solubility issue.
Structures of the relevant reagents used in the modified approach
are shown in Chart 1. For simplicity, various azide- and
maleimide-containing SN-38 derivatives are uniformly indicated
with “A” and “M” descriptors, respectively, whose structures
are also given in Chart 1.
At the outset, our goal was to prepare a bifunctional SN-38
that contained an intracellularly cleavable dipeptide, akin to that
in “CL-SN-38” (3) and other drug conjugates.29,30 Thus, the
derivative “CL2-SN-38” (16) was prepared as an alternative to
3. The choice of SN-38 as the drug for targeted therapy and
the requirement for the liberation of the intact drug from the
conjugate dictated that the hydroxyl group be derivatized as an
ester or a carbonate. The utility of the stable, intracellularly
cleavable dipeptide in both 3 and 16 must therefore be viewed
in the context of the 20-carbonate (or ester) that is also a
cleavable bond. To determine the putative need for the cleavable
Azide-acetylene cycloaddition reaction was carried out using
5 mol % of cupric sulfate and 50 mol % of sodium ascorbate
in the preparation of CL1-SN-38. In other derivatives, in view

Antibody Conjugates of SN-38
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6919
Chart 2. Structures of Bifunctional SN-38 Derivatives
Scheme 3. Synthesis of the Bifunctional SN-38, CL1-SN-38 (7)
near-quantitative conversion in the cycloaddition reaction, within
30 min in most cases, as documented by HPLC analyses of
reaction mixtures.
Characterizations of the intermediates and the final products
were facilitated by comparisons with the well-separated 1H
NMR signals in the spectra of simpler SN-38 derivatives 1a,
1e, and 1g, the acetylenic reagent 4, and the cross-linkers 8
and 22.
Antibody Conjugations. Interchain disulfides of mAbs were
reduced with DTT or TCEP, generating 7-8 thiol groups on
the antibody, as determined by Ellman’s assay.33 The reduced
mAbs were conjugated to maleimide-containing biunctional SN-
38 derivatives. The conjugations were done at a slightly acidic
pH of 6.5 for a short duration of 20 min. Table 1 shows the
SN-38 substitutions in representative conjugate preparations. The
average SN-38/mAb molar substitution ratio (MSR) was
calculated from determinations of SN-38 concentration by
absorbance at 366 nm (correlated to standards) and mAb
concentration by absorbance at 280 nm, the latter corrected for
SN-38 absorbance at 280 nm. MALDI mass spectra of the
reduced mAb and of the SN-38 conjugates showed a series of
fragmented peaks corresponding to half IgG and light and heavy
chains, with molecular ion peaks frequently being too weak to
of sluggish conversion, 1-2 mol equiv of cuprous bromide were
used. Use of a 3-fold excess of the acetylenic reagent, 4, led to

6920 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Moon et al.
Scheme 6. Synthesis of the Bifunctional SN-38, CL4-SN-38
Scheme 4. Synthesis of the Bifunctional SN-38, CL2-SN-38
(16) and CL2-SN-38(Et) (17)
(25)
Scheme 7. Synthesis of the Bifunctional SN-38, CL5-SN-38
(28)
Scheme 5. Synthesis of the Bifunctional SN-38, CL3-SN-38
(21)
Drug Release from Conjugates. Figure 2 and Table 2
describe the stabilities of hMN-14 conjugates of the SN-38
derivatives in buffer and human serum. The procedure was
adapted from the reported method for quantifying SN-38 in sera
of patients undergoing the CPT-11 treatment.34 The hMN-14
conjugate of CL1-SN-38 (7), with the glycinate linkage, was
the least stable under physiological conditions in vitro; this was
counterintuitive, as the ester was expected to be more stable
than a carbonate. The hMN-14 conjugates of CL5-SN-38 (28)
CL2-SN-38 (16), CL2-SN-38 (Et) (17), and CL4-SN-38 (25),
all containing carbonate at the 20-position, with variations in
the linkers, were more stable than the conjugate of 7. This
suggested that perhaps hydrophobicity and/or steric hindrance
due to the linker conferred a certain stability. This effect might
be operative in view of the attachment of the drug in the
interchain regions of the antibody, and perhaps such an effect
might not be evident in conjugates involving drug attachments
to the lysine groups of mAb and the exposure to the more
hydrophilic milieu. The most stable conjugate was the hMN-
observe; these fragmentations complicate the determinations of
mean drug/mAb molar substitutions by this method.
Figure 1 shows the comparative HPLCs of a humanized anti-
CEACAM mAb, hMN-14, and its CL2-SN-38 conjugate with
a mean drug substitution of 6.1.

Antibody Conjugates of SN-38
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6921
Table 1. Representative SN-38/mAb MSRa
mAbb
conjugate
MSR
hMN-14c
hMN-14-7d
hMN-14-16d
hMN-14-17d
hMN-14-21d
hMN-14-25d
hMN-14-28d
7.7
6.8
6.3
5.5
4.1
6.9
hRS7e
hRS7-7
hRS7-16
5.3
6.3
hPAM4f
hLL2g
hPAM4-16
hLL2-7
hLL2-16
hLL2-17
5.7
7.4
6.4
5.5
hA20h
hA20-16
6.1
a MSR, molar substitution ratio. b Monoclonal antibody. c Humanized
anti-CEACAM5 mAb, hMN-14 (labetuzumab). d Bifunctional SN-38
derivatives were: CL1-SN-38 (7), CL2-SN-38 (16), CL2-SN-38[Et](17),
CL3-SN-38 (21), CL4-SN-38(25), and CL5-SN-38 (28). e Humanized anti-
EGP-1 mAb, hRS7. f Humanized anti-MUC-1 mAb, hPAM4. g Humanized
anti-CD22 mAb, hLL2 (epratuzumab). h Humanized anti-CD20 mAb, hA20
(veltuzumab).
Figure 2. Kinetics of SN-38 release from various hMN-14-SN-38
conjugates incubated in PBS, pH 7.4 (top) or human serum (bottom)
at 37 °C. Y-axis displays the ratios of the reverse-phase HPLC peak
areas due to SN-38 and the fixed amount of internal standard
(10-hydroxycamptothecin) extracted from the incubates. Conjugates:
hMN-14-[CL1-SN-38 (7)], 9; hMN-14-[CL2-SN-38 (16)], 2; hMN-
14-[CL2-SN-38-10-CO2Et) (17)], 1; hMN-14-[CL3-SN-38 (21)],
[; hMN-14-[CL4-SN-38 (25)], ×; and hMN-14-[CL5-SN-38 (28)],
b. With hMN-14-21 conjugate, only buffer-stability was determined.
The plots were generated using the Prism software and the equation
for one-phase exponential association [Y ) Ymax(1 - exp(-KX))]. The
half-lives, determined as 0.69/K where K is rate constant, are given in
Table 2.
Table 2. In Vitro Stability of hMN-14-SN-38 Conjugates at 37 °C
half-life
conjugate
PBS (h)
human serum (h)
hMN-14-7
hMN-14-16
hMN-14-17
hMN-14-21
hMN-14-25
hMN-14-28
8.6
30.1
98.4
48.8
27.6
18.7
10.8
36.2
65.9
n/da
32.2
14.4
a n/d, not determined.
Table 3. In Vitro Binding on LoVo Human Colon Adenocarcinoma
Cell Line
expt no.
1
material
SN-38 MSRa
Kd (nM)
95% CIb
hMN-14
1.4
1.4
1.4
1.5
1.2-1.7
0.6-2.2
1.2-1.7
1.1-1.9
hMN-14-7
hMN-14-16
hMN-14-21
7.1
4.0
5.5
Figure 1. Size-exclusion HPLC of unmodified hMN-14 (top) and of
hMN-14-CL2-SN-38 conjugate with a drug molar substitution of 6.1
(bottom). Unmodified hMN-14 was detected by absorbance at 280 nm.
The HPLC profile of the conjugate was detected at the absorbance
wavelength of SN-38, namely 360 nm; thus, the peak eluting near the
antibody position corresponds to antibody substituted with SN-38.
Abbreviation: tR, retention time.
2
hMN-14
hMN-14-17
hMN-14-28
2.1
2.3
2.4
1.1-3.0
1.2-3.4
1.8-3.7
6.2
6.9
a MSR, molar substitution ratio. b CI, confidence interval.
speculation of the role of hydrophobicity/steric hindrance in
drug-release kinetics.
14 conjugate of 17; its in vitro half-life of 66 h in human serum
resembles that of conjugates with cross-linkers containing the
acid-sensitive hydrazone bond.35,36 Conjugates of both CL3-
SN-38 (21) and CL1-SN-38 (7) contain glycinate at the 20
position, but their different linker compositions dictate their
differential stabilities. A 5-fold greater stability, in buffer, for
the conjugate of 21 vs the conjugate of 7 lends credence to the
In Vitro Bindings and Cytotoxicities. Table 3 details binding
data obtained using five SN-38 conjugates of hMN-14 and LoVo
human colon adenocarcinoma cell line. The cell bindings (Kd)
are similar to the unmodified mAb in these determinations. Cell-
bindings of the conjugates of 7, 16, and 21 and the humanized
anti-EGP-1 mAb, hRS7, in the pancreatic adenocarcinoma cell

6922 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Moon et al.
Table 4. In Vitro Cytotoxicity on LoVo Human Colon Adenocarcinoma
Cell Line (96 h Exposure)
DCL (Charlottetown, PE, Canada). SN-38 was purchased from
SINOVA (Bethesda, MD). All nonaqueous reactions were per-
formed using anhydrous solvents, unless otherwise stated, under
positive pressure of argon. In reaction work up, organic extracts
were dried with anhydrous sodium sulfate. 1H NMR spectra at 500
MHz and 13C NMR spectra at 125.7 MHz were recorded on a
Varian INOVA NMR spectrometer (Varian Inc., Palo Alto, CA)
at Rutgers University Chemistry Department (Newark, NJ). Chemi-
cal shifts were referenced to tetramethylsilane at 0 ppm. Exact mass
determinations by high-resolution mass spectra were performed at
Scripps Center for Mass Spectrometry (La Jolla, CA) using an
Agilent LC/MSD TOF instrument, a flow rate of 0.3 mL/min of
methanolic sample solution, and a capillary voltage of 3500 V.
Purifications were performed by flash chromatography on 230-400
mesh silica gel according to well-established procedures using
dichloromethane-methanol gradient elutions (usually 0-8% metha-
nol, with up to 18% methanol in final preparations that generate
TFA or DCA salt of lysine side chain amino group) unless otherwise
stated. Analytical HPLC was carried out on a Waters Nova-pak
HR C18 column (6 µm, 60 Å, 7.8 mm × 300 mm), fitted with a
guard column and an in-line dual absorbance detector set at 254
and 360 nm and with gradient elution carried out by method 1 or
method 2 using buffers A and B as follows: 100% A changing to
100% B over 10 min (linear) at a flow rate of 3 mL/min, changing
to 100% B at a flow rate of 4.5 mL/min at 10.1 min and remaining
isocratic at this flow rate for 5 min (the isocratic elution with 100%
B was performed for 10 or 15 min for relatively lipophilic
intermediates). Method 1: buffer A was 0.3% aq ammonium acetate,
pH 4.43; buffer B was 9:1 v/v acetonitrile: buffer A. Method 2:
buffer A was 0.3% aq ammonium acetate, pH 4.43; buffer B was
9:1 v/v methanol:buffer A. Analytical samples of some of the
intermediates were procured by HPLC using method 1. Monoclonal
antibodies were obtained from Immunomedics’ production labo-
ratories. Size-exclusion HPLCs of antibody conjugates were carried
out on an analytical Bio-Sil 250 size-exclusion column, in series
with a guard column (both from Bio-Rad laboratories, Hercules,
CA), using 0.2 M sodium phosphate buffer (pH 6.8) as the mobile
phase at a flow rate of 1 mL/min with in-line UV (280 or 360 nm)
detection. Cell lines were obtained from American Type Culture
Collection (ATCC, Manassas, VA).
expt no.
1
material
SN-38 MSRa
IC50 (nM)
95% CIb
free SN-38
3.2
4.1
5.3
9.5
2.6-4.0
3.4-5.0
4.7-5.9
8.4-10.8
hMN-14-7
hMN-14-16
hMN-14-21
7.1
4.0
5.5
2
free SN-38
hMN-14-17
hMN-14-28
2.4
5.2
5.8
1.9-3.1
4.6-5.9
5.1-6.6
6.2
6.9
a MSR, molar substitution ratio. b CI, confidence interval.
line, CaPan-1, were likewise similar to that of unmodified hRS7
(not shown). Table 4 shows the in vitro cytotoxicities of the
hMN-14 conjugates. The IC50 for the conjugate of 7 is not
significantly different from that of SN-38. The IC50 values for
conjugates of 16, 17, and 28 are somewhat higher than that of
SN-38 (5-6 nM vs 2-3 nM for SN-38) but are still in a single
digit nM range similar to that for the free drug. The IC50 value
for the conjugate of 21 was the highest (IC50: 9.5 nM), in this
cell line, among the SN-38 derivatives tested.
The Choice of the Bifunctional SN-38 Derivative. As with
the CL-SN-38 conjugate, the CL1-SN-38 and CL2-SN-38
conjugates of hMN-14 mAb exhibited significant and selective
efficacies in the lung metastatic model of human colon
carcinoma in nude mice.28 Therapy with the specific CL1-SN-
38 conjugate increased the median survival by 1.7-fold versus
treatment with a nontargeting control conjugate, while the
specific CL2-SN-38 conjugate increased the median survival
3-fold versus treatment with the control conjugate. Specific
tumor growth controls were also observed using CL2-SN-38
conjugates of other mAbs in other tumor models.28 In vitro
stability is one criterion to consider in the choice of the specific
conjugate, but therapeutic efficacy depends on other factors. For
instance, the in vitro cytotoxicity of hMN-14-CL3-SN-38 was
inferior to that of hMN-14-CL1-SN-38 conjugate (Table 4),
despite the stability advantage for the former. This may be due
to the generation of SN-38-20-O-glycinate from hMN-14-21
conjugate after intracellular processing and the opportunity for
intramolecular lactone ring opening before ester hydrolysis.
In ongoing experiments, the conjugates of 16 and 17 are seen
to be similar in therapeutic efficacies, and a choice between
the two must await more detailed comparisons.
In TFA-mediated deprotection of “BOC” and “MMT” groups
in SN-38 derivatives, a TFA deprotection mixture consisting of 2
mL of TFA, 0.5 mL of dichloromethane, 0.12 mL of anisole, and
0.06 mL of water was used and is simply referred to as “treatment
with TFA” in the relevant experiments. In a similar manner, in
instances where only “MMT” needed to be removed, the milder
DCA-mediated deprotection was carried out using a mixture of 0.5
mL of DCA, 5 mL of dichloromethane, 0.05 mL of anisole, and
0.025 mL of water, and the deprotection is referred to as “treatment
with DCA”.
Conclusion
The versatile click cycloaddition approach provided a facile
access to a series of bifunctional SN-38 derivatives suitable for
antibody conjugation. The provision of a short PEG spacer in
the cross-linker facilitated aqueous solubility. Several antibody
conjugates could be made with mean drug substitutions in the
range of 5-7. Detailed stability determinations in biological
media indicated a correlation between conjugate stability and
the apparent hydrophobicity of the cross-linker-attached SN-
38. The conjugates were found to maintain antigen bindings in
in vitro assays involving specific cell lines and were found
cytotoxic in a single-digit nanomolar range. On the basis of
extensive preclinical studies, it is our intent to further focus on
the conjugates of CL2-SN-3828 and CL2-SN-38(Et).
10-O-tert-Butoxycarbonyl-SN-38-20-O-chloroformate (1b). In
a typical example, 1a18 (0.358 g, 0.073 mmol), DMAP (0.266 g,
0.218 mmol), and triphosgene (0.0095 g, 0.032 mmol) were taken
in an Eppendorf vial and the reaction was initiated by adding
dichloromethane (1.5 mL). The mixture turned to clear light-yellow
solution within a minute. The formation of the chloroformate was
monitored by quenching an aliquot with anhydrous methanol and
comparing the TLC mobility with that of 1a. The formation of the
chloroformate was complete within a few minutes, and the product
was used as such after 7 min from the start-up of the reaction. In
larger-scale runs, the reagents were scaled-up proportionally. 20-
Chloroformates derived from other 10-substituted SN-38 derivatives
were prepared similarly. In all instances, the chloroformates were
reacted in situ with relevant cross-linkers.
MC-Phe-Lys-PABOCO-20-O-SN-38 (3; CL-SN-38). In one run,
the chloroformate (1b), generated from BOC-SN-38 (1a, 0.0227
g, 0.046 mmol), was reacted in situ with the known reagent, MC-
Phe-Lys(MMT)-PABOH,30 (0.0487 g, 0.056 mmol) for a short
duration, typically under 5 min. The reaction mixture was then
purified by flash chromatography using methanol-dichloromethane
gradient to obtain the carbonate, 2 (off-white solid, 0.022 g, 34.5%).
Experimental Section
General Procedures. All chemicals were purchased from Sigma-
Aldrich company (St. Louis, MO) unless otherwise stated. Suc-
cinimidyl 6-maleimidocaproate and SMCC were purchased from
Molecular Biosciences (Boulder, CO). The PEG derivatives were
obtained from EMD chemicals (San Diego, CA) and BioVectra

Antibody Conjugates of SN-38
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6923
In one run, 2 (0.0316 g, 0.0229 mmol) was treated with TFA for
a few min, typically <5 min, and the product was recovered by
precipitation with diethyl ether. TFA treatment was repeated two
times additionally, each for a short duration of less than 5 min,
and the title product was obtained as the TFA salt in 91.7% purity
(cream solid, 0.0215 g, 83.7%). This level of purity was judged
adequate for exploratory mAb conjugations. Analytical sample of
3 was prepared by HPLC under the condition described in General
Procedures.
SN-38-20-O-glycinate HCl Salt (1d). BOC-SN-38 (1a, 0.322
g, 0.65 mmol) was dissolved in dichloromethane (10 mL) and
reacted, under stirring, with BOC-glycine (0.23 g, 1.31 mmol),
diisopropylcarbodiimide (0.202 mL, 0.165 g, 1.31 mmol), and
DMAP (0.096 g, 0.79 mmol) overnight at ambient temperature.
Product 1c was isolated by flash chromatography (pale-yellow
powder; yield: 0.48, 94%). TFA deprotection of 1c (0.4 g), followed
by precipitation in ethyl ether, yielded the TFA salt, which was
dissolved in ethanol (5 mL) and a drop of concentrated HCl, and
the solvent was evaporated to obtain 1d (yellow powder; yield:
quantitative).
cooled in ice bath. EEDQ (0.211 g, 0.853 mmol) was then added,
and the clear solution was stirred under argon overnight, with the
bath temperature rising to ambient temperature. Solvent removal
followed by flash chromatography using the general condition or
ethyl acetate-methanol (100:0-90:10) gradient elution furnished
the title product (gum; yield: 0.56 g, 60%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-Phe-Lys(M-
MT) PABOCO-20-O-SN-38-10-O-tert-butoxycarbonyl]heptaeth-
yleneglycol (9). Reagent 8 (0.341 g; 0.283 mmol) was coupled to
1b; the latter was generated from 1a (0.12 g; 0.244 mmol) according
to the general procedure for chloroformate formation. The product
was isolated by flash chromatography (gum; yield: 0.294 g, 70%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-Phe-Lys(M-
MT) PABOCO-20-O-SN-38-10-O-tert-butyldimethylsilyl]hepta-
ethyleneglycol (11). The TBDMS derivative 1g (0.540 g, 1.065
mmol) was converted to the corresponding 20-O-chloroformate,
1h, according to the general procedure described previously, which
was reacted in situ with the cross-linker 8 (1.20 g, 0.994 mmol)
for 20 min. Flash chromatography furnished the title product (pale-
yellow foam; yield: 1.339 g, 77.5%).
10-O-Ethoxycarbonyl-SN-38 (1e). To a stirred suspension of SN-
38 (0.52 g, 1.327 mmol) in DMF (5 mL), at 4 °C, was added
ethylchloroformate (0.252 mL, 0.287 g, 2.645 mmol) and DIEA
(0.462 mL, 0.342 g, 2.65 mmol); the cooling bath was removed
and the clear solution was stirred at room temperature for 30 min.
The product was purified by flash chromatography (off-white solid;
yield: 0.586 g, 95%).
10-O-tert-Butyldimethylsilyl-SN-38 (1g). SN-38 (0.63 g, 1.607
mmol) and tert-butyldimethylsilyl chloride (0.58 g, 3.85 mmol) were
dissolved in DMF (15 mL), and DIEA (0.84 mL, 0.622 g, 4.81
mmol) was added. The reaction mixture was stirred for 2 h, solvent
and DIEA were removed, and the residue was purified by flash
chromatography to obtain 1g (yellow powder, yield: 0.79 g, 97%).
Anal. (C28H34N2O5Si) C, H, N.
4-(N-Maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxa-
mide (4). To SMCC (1.131 g, 3.383 mmol), taken in dichlo-
romethane (25 mL) was added propargylamine (0.25 mL, 0.2 g,
3.63 mmol) and DIEA (0.59 mL, 0.4366 g, 3.378 mmol) and stirred
for 1 h at ambient temperature under argon. The reaction mixture
was diluted with dichloromethane (100 mL), washed with 1N HCl
and brine (100 mL each), and dried. The crude product was purified
by flash chromatography using dichloromethane-methanol gradient
elution (100:0-98:2) to obtain the title compound (colorless
powder; yield: 0.844 g, 91%). Anal. Calcd for C15H18N2O3: C,
65.68; H, 6.61; N, 10.21. Found: C, 65.13; H, 6.72; N, 10.04
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-Gly-20-O-SN-
38]heptaethyleneglycol (6). O-(2-Azidoethyl)-O′-(N-diglycolyl-2-
aminoethyl)heptaethyleneglycol (5; 0.6 g, 1.083 mmol) was acti-
vated with DCC (0.228 g, 1.105 mmol), NHS (0.127 g, 1.103
mmol), and catalytic amount of DMAP (0.01 g, 0.008 mmol) in
dichloromethane (20 mL) for 30 min at ambient temperature. To
this was added solid SN-38-20-O-glycinate (1d; 0.7 g, 1.264
mmol) and DIEA (0.22 mL, 1.26 mmol). After stirring for 1 h, the
precipitated urea was filtered and the filtrate was concentrated and
purified by flash chromatography to obtain 6 (oil; yield: 1.4 g).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Gly-20-
O-SN-38]heptaethyleneglycol (7; CL1-SN38). A solution of the
azide precursor 6 (0.208 g, 0.211 mmol) in DMSO (0.5 mL) was
added to the acetylenic reagent 5 (0.173 g, 0.631 mmol) in DMSO
(1.5 mL), followed by 1 mL of water, 0.05 M aqueous cupric sulfate
(0.21 mL, 0.011 mmol), and 0.5 M aqueous sodium ascorbate (0.21
mL, 0.105 mmol). The somewhat cloudy solution was stirred at
ambient temperature for 1 h. Solvents were removed by bulb-to-
bulb distillation, and the title product was purified by flash
chromatography (gum; yield: 0.173 g, 65%).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Phe-
Lys(MMT)-PABOCO-20-O-SN-38-10-O-tert-butoxycarbonyl-
]heptaethyleneglycol (13). The azide, 9 (0.255 g, 0.148 mmol), and
the acetylenic reagent, 4 (0.101 g, 0.369 mmol), were mixed in
DMSO (3 mL) and water (1 mL). Solid cuprous bromide (0.042 g,
0.294 mmol) was added to the clear solution, and the heterogeneous
mixture was stirred for 30 min. Solvents were removed, and the
crude product was purified by flash chromatography to obtain 13
(yellowish gum; yield: 0.233 g, 79%).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Phe-
Lys(MMT)-PABOCO-20-O-SN-38]heptaethyleneglycol (15). The
azide 11 (1.339 g, 0.77 mmol) was dissolved in dichloromethane
(50 mL) and treated with 1 M solution of tetrabutylammonium
fluoride in THF (1.9 mL, 1.9 mmol) and acetic acid (0.231 mL,
3.8 mmol). The reaction mixture was stirred for 20 min, diluted
with dichloromethane (200 mL), washed twice with half-saturated
sodium chloride, and dried. Solvent removal furnished the desily-
lated product 12 (gum), which was used as such in the next step.
Intermediate 12 and the acetylenic reagent 4 (0.52 g, 1.90 mmol)
were dissolved in DMSO (8 mL) and water (2 mL); to this was
added cuprous bromide (0.22 g, 1.54 mmol). The reaction mixture
was stirred for 80 min and then diluted with dichloromethane. The
organic extract was washed twice with half-saturated aq sodium
chloride (100 mL) and dried. The title product was obtained by
flash chromatography (gum; yield: 0.84 g; 58% in 2 steps).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Phe-
Lys-PABOCO-20-O-SN-38]heptaethyleneglycol (16; CL2-SN-38).
The product 13 (0.14 g, 0.07 mmol) was subjected to TFA treatment
for <5 min and purified on a short column of silica gel (230-400
mesh) using 5-18% methanol-dichloromethane gradient elution.
The product, 16, “CL2-SN-38”, was isolated as the TFA salt (gum;
yield: 0.097 g; 80%). The same product was also obtained from
15 (0.584 g, 0.307 mmol) by deprotection with DCA, followed by
ether precipitation (yellowish-green glassy solid; yield: 0.58 g,
95%).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Phe-
Lys-PABOCO-20-O-SN-38-10-O-ethoxycarbonyl]heptaethyle-
neglycol (17). This product was prepared from 1e in a sequence of
three steps of carbonate formation, click chemistry, and MMT
deprotection under conditions used for preparing 16, except that
the final deprotection of MMT group was accomplished by exposure
to DCA. The bis carbonate 10 (gum; yield: 88%) was subjected to
click cycloaddition with 4 to obtain 14 (gum; yield: 76%).
Deprotection of 14 (0.111 g) with DCA furnished the title
compound, 17, after precipitation of crude reaction product in ethyl
ether, followed by flash chromatography (glassy solid; yield: 0.0883
g, 86%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-Phe-Lys(M-
MT) PABOH]heptaethyleneglycol (8). O-(2-Azidoethyl)-O′-(N-
diglycolyl-2-aminoethyl)heptaethyleneglycol (5; 0.430 g, 0.776
mmol) and the known intermediate Phe-Lys(MMT)-PABOH (0.52
g, 0.776 mmol) were dissolved in dichloromethane (20 mL) and

6924 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Moon et al.
Activated Carbonate, 18. The azido compound 8 (0.27 g, 0.22
mmol) was activated with bis(nitrophenyl)carbonate (0.204 g, 0.67
mmol) and DIEA (0.039 mL, 0.22 mmol) in dichloromethane (10
mL) for 3 days at ambient temperature. Flash chromatography
furnished the activated carbonate, 18 (gum; yield: 0.25 g, 69%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-Phe-Lys(MMT)-
PABOCO-Gly-20-O-SN-38]heptaethyleneglycol (19). SN-38-20-
O-glycinate 1d (0.028 g; 0.058 mmol) was coupled to 18 (0.08 g,
0.058 mmol) in DMF (1 mL) and DIEA (0.025 mL, 0.14 mmol).
After 4 h of stirring, solvent was removed and the crude product
was purified by flash chromatography to obtain 19 (foam; yield:
0.052 g, 54%).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-Phe-
Lys-PABOCO-Gly-20-O-SN-38]heptaethyleneglycol (21; CL3-
SN-38). The azide 19 (0.05 g, 0.03 mmol) and the acetylenic reagent
4 (0.024 g, 0.087 mmol) were mixed in 2 mL of 1:1 DMSO-water.
Solid cuprous bromide (0.0045 g, 0.031 mmol) was added, and
the heterogeneous mixture was stirred for 1 h. The crude product
was precipitated by dilution with water and purified by flash
chromatography to obtain 20 as gummy material (yield: 0.041 g,
76%). The product was subjected to short-duration treatment with
TFA and purified by precipitation with ethyl ether to obtain the
title product, 21 (glassy solid; yield: 0.031 g, 90%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-PABOH]hep-
taethyleneglycol (22). O-(2-azidoethyl)-O′-(N-diglycolyl-2-amino-
ethyl)heptaethyleneglycol (5; 0.207 g, 0.374 mmol), p-aminobezyl
alcohol (0.055 g, 0.447 mmol), and EEDQ (0.137 g, 0.554 mmol)
were dissolved in dichloromethane (4 mL) and stirred at room
temperature overnight. Solvent was removed, and the residue was
washed with ethyl ether. The crude product was purified by flash
chromatography (gum; yield: 0.207 g, 83%).
O-(2-Azidoethyl)-O′-[(N-diglycolyl-2-aminoethyl)-PABOCO-20-
O-SN-38-10-O-tert-butoxycarbonyl]heptaethyleneglycol (23). Prod-
uct 22 (0.190 g, 0.288 mmol) was reacted with 1b; the latter was
generated from 1a (0.122 g, 0.248 mmol) according to the general
procedure. The title product was obtained by flash chromatography
(gum; yield: 0.14 g, 48% yield).
O-{2-[(1,2,3-Triazolyl)-4-(4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-PAB-
OCO-20-O-SN-38-10-O-tert-butoxycarbonyl]heptaethyleneg-
lycol (24). The azide precursor 23 (0.14 g; 0.119 mmol) and the
acetylenic reagent 4 (0.1 g, 0.364 mmol) were mixed in DMSO (3
mL) and water (1 mL). Solid cuprous bromide (0.05 g, 0.348 mmol)
was added, and the heterogeneous mixture was stirred for 10 min.
More water (0.5 mL) was added, and the reaction was continued
for 30 min. Solvents were removed, and the crude product was
purified by flash chromatography (gum; 0.135 g, 84% yield).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-[(N-diglycolyl-2-aminoethyl)-PAB-
OCO-20-O-SN-38]heptaethyleneglycol (25; CL4-SN-38). Product
24 (0.143 g) was treated with TFA for <5 min. After removal of
TFA and solvent, the product was purified by flash chromatography
(glassy solid; yield, as TFA salt: 0.1 g, 75%).
O-(2-Azidoethyl)-O′-(10-O-tert-butoxycarbonyl-SN-38-20-O-
carbonyl)heptaethyleneglycol (26). The chloroformate 1b, prepared
from 1a (0.213 g; 0.433 mmol), was reacted with O-(2-azidoeth-
yl)heptaethyleneglycol (0.205 g, 0.518 mmol) and the reaction
mixture was purified by flash chromatography to obtain the azido
derivative (yellow glassy solid; yield: 0.312 g, 79%). Analysis:
Calcd for C44H59N5O16 ·3H2O: C, 54.55%; H, 6.72%; N, 7.23%.
Found: C, 54.24; H, 6.12; N, 6.98%.
crude product was purified by flash chromatography to furnish the
title product (glassy solid; yield: 0.04 g, 87%).
Conjugation of Maleimide-Containing SN-38 Substrates to
Mildly Reduced Antibodies. The anti-CEACAM5 humanized mAb,
hMN-14 (labetuzumab), the anti-CD22 humanized mAb, hLL2
(epratuzumab), the anti-CD20 humanized mAb, hA20 (veltuzumab),
the anti-EGP-1 humanized mAb, hRS7, and anti-MUC1 humanized
mAb, hPAM4, were used in these studies. Each antibody was
reduced with 64-fold excess of DTT in 40 mM PBS, pH 7.4,
containing 5.4 mM EDTA, at 37 °C (bath) for 45 min. The reduced
product was purified on centrifuged size-exclusion column and
buffer-exchanged with 75 mM sodium acetate-1 mM EDTA, pH
6.5. The thiol content was determined by Ellman’s assay and was
in the 7-8 SH/IgG range. Alternatively, the antibodies were reduced
with TCEP in 75 mM sodium acetate buffer, pH 6.5, followed by
in situ conjugation. The reduced mAb was reacted with ∼10-15-
fold molar excess of bifunctional SN-38 using DMSO (10% v/v)
as cosolvent and incubated for 20 min at ambient temperature. The
conjugate was purified by centrifugal SEC, passage through a
Biobead hydrophobic column, and finally by ultrafiltration-diafil-
tration. The product was assayed for SN-38 concentration by
absorbance at 366 nm that was correlated with standards, while
the protein concentration was deduced from absorbance at 280 nm
and corrected for spillover of SN-38 absorbance at this wavelength.
From these, average SN-38/mAb molar substitution ratios were
calculated. The purified conjugates were formulated with the
addition of excipients (25 mM trehalose and 0.01% polysorbate
80), lyophilized, and stored in a -20 °C freezer.
In Vitro Hydrolytic and Serum Stabilities. Solutions of the
conjugates (10 mg/mL, 0.027 mL), freshly reconstituted from
lyophilizates, were added to 0.873 mL of 40 mM PBS, pH 7.4, or
human serum and incubated at 37 °C. At periodic intervals, aliquots
were withdrawn, and a known amount of 10-hydroxycamptothecin
was added as an internal standard. After precipitating the protein
with acetonitrile, the dissociated SN-38 was extracted with chlo-
roform. Fixed volumes of the extracts were analyzed by reversed-
phase HPLC, quantifying for SN-38 by fluorescence detection of
HPLC peaks. SN-38/internal standard peak ratios were correlated
with SN-38 standard curve, the latter generated by plotting SN-
38/internal standard peak ratios as a function of SN-38 concentra-
tion. The kinetic plots for SN-38 release were generated with the
standard Prism software. Equation for one phase exponential
association, with plots starting at zero and ascending to Ymax with
a rate constant K, was used to calculate half-lives as 0.69/K.
Binding Study. LumiGLO Chemiluminescent Substrate System
(KPL, Gaithersberg, MD) was used to detect the binding of hMN-
14-immunoconjugate to LoVo cells. Briefly, cells were plated into
a 96-well plate (1 × 105cells/well) and incubated overnight. The
media in the wells were replaced with fresh media (50 µL/well).
Each immunoconjugate was serially diluted, and additions of 50
µL aliquots into triplicate wells yielded a concentration range of
20-0.0098 µg/mL. Plates were incubated for 1.5 h at 4 °C,
centrifuged for 5 min at 600g, and the media was poured out and
the plate was blotted dry. The cells were washed once with ice-
cold media (200 µL/well) and centrifuged and blotted dry as before.
Each well then received 100 µL of HRP-conjugated goat-antihuman
secondary antibody (Jackson Immunoresearch, West Grove, PA)
at a 1:20000 dilution in media. Control wells received only cells
plus secondary antibody. The plate was incubated for 1 h at 4 °C
in the dark and was centrifuged and blotted dry as before. The cells
were washed twice with ice-cold media and once with ice-cold
D-PBS. LumiGLO reagent (100 µL/well) was added to all wells,
and the plate was read for luminescence using an Envision plate
reader (Perkin-Elmer, Boston MA). Data were analyzed by
nonlinear regression to determine the equilibrium dissociation
constant (Kd).
O-{2-[(1,2,3-Triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-
carboxamidomethyl]]ethyl}-O′-(SN-38-20-O-carbonyl)heptaethyl-
eneglycol (28; CL5-SN-38). The azido derivative 26 (0.105 g, 0.115
mmol) was reacted with reagent 4 (0.09 g, 0.328 mmol) and cuprous
bromide (0.047 g, 0.328 mmol) in a mixture of 1:1 v/v DMSO and
water (2 mL) for 30 min. DMSO was removed by bulb-bulb
distillation, and the crude product was subjected to flash chroma-
tography to obtain 27 (glassy solid; yield: 0.110 g, 81%). The latter
intermediate (0.05 g) was subjected to TFA deprotection, and the
Growth Inhibition. In vitro cytotoxicities of the immunoconju-
gates were determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium dye re-
duction assay (MTS dye reduction assay; Promega, Madison, WI).
Briefly, LoVo cells were harvested with cell dissociation buffer

Antibody Conjugates of SN-38
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6925
tabolism of irinotecan (CPT-11). Clin. Cancer Res. 2001, 7, 2182–
2194.
(Invitrogen Corp., Grand Island, NY) and plated into 96-well plates
(2 × 103 cells per well). Each lyophilized vial of immunoconjugate
was reconstituted with sterile media to make a final concentration
of 1500 nM in SN-38 equivalents. From these stock vials, various
1:5 dilutions of each were made such that adding 20 µL per well
would yield final concentrations ranging from 250 to 0.004 nΜ of
SN-38 equivalents. Free SN-38, used as positive control, was
likewise diluted from 250 to 0.004 nΜ in 1:5 serial dilutions.
Unconjugated hMN-14 IgG (negative control) was diluted to match
the same protein dose as the immunoconjugates (6 µg/mL to 0.015
ng/mL). After all the reagents were added, the plates were placed
in a 37 °C/5% CO2 incubator for 96 h. MTS dye was the added to
the plates (20 µL/well). The plates were further incubated for 4 h,
and then read at 490 nm. Dose-response curves were generated
from the mean of triplicate determinations and IC50 values were
calculated using PrismPad software (Advanced Graphics Software,
Encinitas, CA).
(15) Greenwald, R. B.; Pendri, A.; Conover, C.; Gilbert, C.; Yang, R.;
Xia, J. Drug delivery systems. 2. Camptothecin 20-O-poly(ethylene
glycol) ester transport forms. J. Med. Chem. 1996, 39, 1938–1940.
(16) Singer, J. W.; De, V. P.; Bhatt, R.; Tulinsky, J.; Klein, P.; Li, C.;
Milas, L.; Lewis, R. A.; Wallace, S. Conjugation of camptothecins
to poly-(L-glutamic acid. Ann. N.Y. Acad. Sci. 2000, 922, 136–150.
(17) Zamai, M.; VandeVen, M.; Farao, M.; Gratton, E.; Ghiglieri, A.;
Castelli, M. G.; Fontana, E.; D’Argy, R.; Fiorino, A.; Pesenti, E.;
Suarato, A.; Caiolfa, V. R. Camptothecin poly[n-(2-hydroxypropyl)
methacrylamide] copolymers in antitopoisomerase-I tumor therapy:
intratumor release and antitumor efficacy. Mol. Cancer Ther. 2003,
2, 29–40.
(18) Zhao, H.; Rubio, B.; Sapra, P.; Wu, D.; Reddy, P.; Sai, P.; Martinez,
A.; Gao, Y.; Lozanguiez, Y.; Longley, C.; Greenberger, L. M.;
Horak, I. D. Novel prodrugs of SN-38 using multiarm poly(ethylene
glycol) linkers. Bioconjugate Chem. 2008, 19, 849–859.
(19) Zhang, J. A.; Xuan, T.; Parmar, M.; Ma, L.; Ugwu, S.; Ali, S.;
Ahmad, I. Development and characterization of a novel liposome-
based formulation of SN-38. Int. J. Pharm. 2004, 270, 93–107.
(20) Okuno, S.; Harada, M.; Yano, T.; Yano, S.; Kiuchi, S.; Tsuda, N.;
Sakamura, Y.; Imai, J.; Kawaguchi, T.; Tsujihara, K. Complete
regression of xenografted human carcinomas by camptothecin analogue-
carboxymethyl dextran conjugate (T-0128). Cancer Res. 2000, 60,
2988–2995.
Acknowledgment. This work was supported in part by SBIR
grant CA114802-01A1 (PI: S.V.G.) from the National Cancer
Institute of the NIH. We thank Agatha Sheerin, Roberto Arrojo,
and Fatma Tat for expert technical support.
(21) Meyer-Losic, F.; Nicolazzi, C.; Quinonero, J.; Ribes, F.; Michel,
M.; Dubois, V.; de, C. C.; Boukaissi, M.; Chene, A. S.; Tranchant,
I.; Arranz, V.; Zoubaa, I.; Fruchart, J. S.; Ravel, D.; Kearsey, J.
DTS-108, a novel peptidic prodrug of SN-38: in vivo efficacy and
toxicokinetic studies. Clin. Cancer Res. 2008, 14, 2145–2153.
(22) Shimizu, Y.; Nagata, H.; Kikuchi, Y.; Umezawa, S.; Hasumi, K.;
Yokokura, T. Cytotoxic agents active against mucinous adenocar-
cinoma of the ovary. Oncol. Rep. 1998, 5, 99–101.
Supporting Information Available: H-1 and C-13 NMR data
and HRMS data for target compounds, a table listing the purity
based on HPLC, HPLC traces for target compounds, and elemental
analysis for 1g. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(23) Sharkey, R. M.; Goldenberg, D. M. Targeted therapy of cancer: new
prospects for antibodies and immunoconjugates. CA Cancer J. Clin.
2006, 56, 226–243.
(1) Potmesil, M. Camptothecins: from bench research to hospital wards.
Cancer Res. 1994, 54, 1431–1439.
(2) Sawada, S.; Okajima, S.; Aiyama, R.; Nokata, K.; Furuta, T.;
Yokokura, T.; Sugino, E.; Yamaguchi, K.; Miyasaka, T. Synthesis
and antitumor activity of 20(S)-camptothecin derivatives: carbam-
ate-linked, water-soluble derivatives of 7-ethyl-10-hydroxycamp-
tothecin. Chem. Pharm. Bull. (Tokyo) 1991, 39, 1446–1450.
(3) Rivory, L. P.; Bowles, M. R.; Robert, J.; Pond, S. M. Conversion of
irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamp-
tothecin (SN-38), by human liver carboxylesterase. Biochem. Phar-
macol. 1996, 52, 1103–1111.
(4) Kawato, Y.; Aonuma, M.; Hirota, Y.; Kuga, H.; Sato, K. Intracel-
lular roles of SN-38, a metabolite of the camptothecin derivative
CPT-11, in the antitumor effect of CPT-11. Cancer Res. 1991, 51,
4187–4191.
(24) Govindan, S. V.; Griffiths, G. L.; Hansen, H. J.; Horak, I. D.;
Goldenberg, D. M. Cancer therapy with radiolabeled and drug/
toxin-conjugated antibodies. Technol. Cancer Res. Treat. 2005, 4,
375–391.
(25) Wu, A. M.; Senter, P. D. Arming antibodies: prospects and
challenges for immunoconjugates. Nat. Biotechnol. 2005, 23, 1137–
1146.
(26) Chari, R. V. Targeted cancer therapy: confering specificity to cytotoxic
drugs. Acc. Chem. Res. 2008, 41, 98–107.
(27) Govindan, S. V.; Cardillo, T. M.; D’Souza, C. A.; Nair, A.; Sheerin,
A.; Singh, S.; Hansen, H. J.; Goldenberg, D. M. Therapy of human
colonic and lung cancer xenograftswith SN-38 conjugates of anti-
CEACAM5 and anti-EGP-1 humanized monoclonal antibodies.
Proceedings of the 2007 AACR-NCI-EORTC International Confer-
ence on Molecular Targets and Cancer Therapeutics, San Francisco,
CA, October 22-26, 2007 , abstract C287.
(28) Govindan, S. V.; Moon, S.-J.; Cardillo, T. M.; Gold, D. V.; Hansen,
H. J.; Goldenberg, D. M. Antibody-targeted chemotherapy of human
colonic, lung, and pancreatic cancer xenografts with conjugates of the
topoisomerase I inhibitor, SN-38. Proceedings of the 6th International
Symposium on Targeted Anticancer Therapies, Bethesda, MD, March
20-22, 2008, abstract B06.
(29) Walker, M. A.; Dubowchik, G. M.; Hofstead, S. J.; Trail, P. A.;
Firestone, R. A. Synthesis of an immunoconjugate of camptothecin.
Bioorg. Med. Chem. Lett. 2002, 12, 217–219.
(30) Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.; Hofstead,
S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.; Trail, P. A. Cathepsin
B-labile dipeptide linkers for lysosomal release of doxorubicin from
internalizing immunoconjugates: model studies of enzymatic drug
release and antigen-specific in vitro anticancer activity. Bioconjugate
Chem. 2002, 13, 855–869.
(31) Kolb, H. C.; Sharpless, K. B. The growing impact of click chemistry
on drug discovery. Drug DiscoVery Today 2003, 8, 1128–1137.
(32) He, X.; Lu, W.; Jiang, X.; Cai, J.; Zhang, X.; Ding, J. Synthesis
and biological evaluation of bis and monocarbonate prodrugs of
10-hydroxycamptothecins. Bioorg. Med. Chem. 2004, 12, 4003–4008.
(33) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959,
82, 70–77.
(34) de Bruijn, P.; de Jonge, M. J.; Verweij, J.; Loos, W. J.; Nooter, K.;
Stoter, G.; Sparreboom, A. Femtomole quantitation of 7-ethyl-10-
hydroxycamptothecine (SN-38) in plasma samples by reversed-phase
high-performance liquid chromatography. Anal. Biochem. 1999, 269,
174–178.
(5) Rougier, P.; Bugat, R. CPT-11 in the treatment of colorectal cancer:
Clinical efficacy and safety profile. Semin. Oncol. 1996, 23 (Suppl.
3), 34–41.
(6) Saijo, N. Preclinical and clinical trials of topoisomerase inhibitors.
Ann. N.Y. Acad. Sci. 2000, 922, 92–99.
(7) Satoh, T.; Hosokawa, M.; Atsumi, R.; Suzuki, W.; Hakusui, H.;
Nagai, E. Metabolic activation of CPT-11,7-ethyl-10-[4-(1-piperi-
dino)-1- piperidino]carbonyloxycamptothecin, a novel antitumor
agent, by carboxylesterase. Biol. Pharm. Bull. 1994, 17, 662–664.
(8) Sparreboom, A.; de Jonge, M. J.; de, B. P.; Brouwer, E.; Nooter,
K.; Loos, W. J.; van Alphen, R. J.; Mathijssen, R. H.; Stoter, G.;
Verweij, J. Irinotecan (CPT-11) metabolism and disposition in
cancer patients. Clin. Cancer Res. 1998, 4, 2747–2754.
(9) Gupta, E.; Lestingi, T. M.; Mick, R.; Ramirez, J.; Vokes, E. E.;
Ratain, M. J. Metabolic fate of irinotecan in humans: correlation
of glucuronidation with diarrhea. Cancer Res. 1994, 54, 3723–3725.
(10) Rivory, L. P.; Riou, J. F.; Haaz, M. C.; Sable, S.; Vuilhorgne, M.;
Commercon, A.; Pond, S. M.; Robert, J. Identification and
properties of a major plasma metabolite of irinotecan (CPT-11)
isolated from the plasma of patients. Cancer Res. 1996, 56, 3689–
3694.
(11) Burke, T. G.; Mi, Z. The structural basis of camptothecin interactions
with human serum albumin: impact on drug stability. J. Med. Chem.
1994, 37, 40–46.
(12) Giovanella, B. C.; Harris, N.; Mendoza, J.; Cao, Z.; Liehr, J.;
Stehlin, J. S. Dependence of anticancer activity of camptothecins
on maintaining their lactone function. Ann. N.Y. Acad. Sci. 2000,
922, 27–35.
(13) Zhao, H.; Lee, C.; Sai, P.; Choe, Y. H.; Boro, M.; Pendri, A.; Guan,
S.; Greenwald, R. B. 20-O-acylcamptothecin derivatives: evidence
for lactone stabilization. J. Org. Chem. 2000, 65, 4601–4606.
(14) Mathijssen, R. H.; van Alphen, R. J.; Verweij, J.; Loos, W. J.; Nooter,
K.; Stoter, G.; Sparreboom, A. Clinical pharmacokinetics and me-
(35) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.;
Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.;

6926 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Moon et al.
Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer,
D. L.; Senter, P. D. Development of potent monoclonal antibody
auristatin conjugates for cancer therapy. Nat. Biotechnol. 2003, 21,
778–784.
Mountain, A.; Flowers, D. A.; Bernstein, I. Gemtuzumab ozogamicin,
a potent and selective anti-CD33 antibody-calicheamicin conjugate
for treatment of acute myeloid leukemia. Bioconjugate Chem. 2002,
13, 47–58.
(36) Hamann, P. R.; Hinman, L. M.; Hollander, I.; Beyer, C. F.; Lindh,
D.; Holcomb, R.; Hallett, W.; Tsou, H. R.; Upeslacis, J.; Shochat, D.;
JM800719T