Tumor-specific novel taxoid-monoclonal antibody conjugates

Article abstract of DOI:10.1021/jm025540g

Taxoids bearing methyldisulfanyl(alkanoyl) groups for taxoid-antibody immunoconjugates were designed, synthesized and their activities evaluated. A highly cytotoxic C-10 methyldisulfanylpropanoyl taxoid was conjugated to monoclonal antibodies recognizing

Full text of DOI:10.1021/jm025540g

5620
J . Med. Chem. 2002, 45, 5620-5623
mors2,3 and showed encouraging potency and selectivity
Tu m or -Sp ecific Novel
Ta xoid -Mon oclon a l An tibod y Con ju ga tes
in preclinical models.
Paclitaxel and docetaxel have brought about signifi-
cant impact on the current cancer chemotherapy mainly
because of their unique mechanism of action8 but
seriously suffer from the lack of tumor specificity and
multidrug resistance (MDR). Thus, it is beneficial to
develop immunoconjugates of these drugs. Indeed, two
research groups have recently reported paclitaxel-mAb
conjugates9,10 as potential tumor-specific anticancer
agents. However, one of the conjugates was only tested
in vitro10 and the other showed only limited efficacy in
vivo.9 It is clear from the current understanding of the
requirements for effective immunoconjugates that the
cytotoxicity level of paclitaxel or docetaxel is not suf-
ficient as the cytotoxic component of the conjugate for
human clinical use.1 In addition, those conjugates are
anticipated to be inactive against tumors expressing
MDR phenotype.
On the basis of our structure-activity relationship
study of taxoids,11,12 we have developed a series of highly
potent second-generation taxoids.13-18 Most of these
taxoids exhibited 2-3 orders of magnitude higher
potency than that of paclitaxel and docetaxel against
drug-resistant cell lines expressing MDR phenotypes.
One of these second-generation taxoids, SB-T-110131
(IDN5109; BAY59-8862), exhibited excellent pharma-
cological profile in preclinical studies and is currently
undergoing phase II human clinical trials sponsored by
Bayer Corporation. Accordingly, in principle we should
be able to develop novel chemotherapeutic agents with
high potency and exceptional tumor specificity by link-
ing these second-generation taxoids with mAb’s. We
report here our promising preliminary results on the
design, synthesis, and evaluation of mAb-taxoid im-
munoconjugates.
Iwao Ojima,*,† Xudong Geng,† Xinyuan Wu,†
Chuanxing Qu,† Christopher P. Borella,†
Hongsheng Xie,‡ Sharon D. Wilhelm,‡
Barbara A. Leece,‡ Laura M. Bartle,‡
Victor S. Goldmacher,‡ and Ravi V. J . Chari‡
Department of Chemistry, State University of New York at
Stony Brook, Stony Brook, New York 11794-3400, and
ImmunoGen, Inc., 128 Sidney Street,
Cambridge, Massachusetts 02139
Received May 14, 2002
Abstr a ct: Taxoids bearing methyldisulfanyl(alkanoyl) groups
for taxoid-antibody immunoconjugates were designed, syn-
thesized and their activities evaluated. A highly cytotoxic C-10
methyldisulfanylpropanoyl taxoid was conjugated to mono-
clonal antibodies recognizing the epidermal growth factor
receptor (EGFR) expressed in human squamous cancers. These
conjugates were shown to possess remarkable target-specific
antitumor activity in vivo against EGFR-expressing A431
tumor xenografts in severe combined immune deficiency mice,
resulting in complete inhibition of tumor growth in all the
treated mice.
Current cancer chemotherapy is based on the premise
that rapidly proliferating tumor cells are more likely to
be killed by cytotoxic drugs. Unfortunately, the differ-
ence in activity of current drugs against tumor tissues
in comparison to healthy tissues is relatively small. The
amount of a drug required to achieve a clinically
effective level of cell kill often causes severe damage to
actively propagating nonmalignant cells such as cells
of the gastrointestinal tract and bone marrow, resulting
in a variety of undesirable side effects.1 Therefore, it is
very important to develop new chemotherapeutic agents
with improved tumor specificity.
The discovery of antigens that are particularly over-
expressed on the surface of cancer cells suggests that
by using certain antibodies to selectively “mark” tumor
cells, malignant tissues could be distinguished from
normal tissues.1 Monoclonal antibodies (mAb’s), which
have shown high binding specificity for tumor-specific
antigens, could fulfill this task. In fact, these mAbs could
be used as vehicles to deliver cytotoxic drugs selectively
to tumor cells.1-3 A drug-mAb conjugate would target
the tumor cells by binding to the antigens on their
surfaces. The conjugate is then internalized and releases
the original cytotoxic agent in its active form.4-6 This
type of immunoconjugates can be categorized as a
“tumor-activated prodrug” (TAP).1 Ideally, a TAP should
be stable during circulation (no premature release of the
drug) and should not bind to normal tissue cells.
The practical efficacy of such immunoconjugates
heavily depends on the nature of the cytotoxic agents
as well as the tumor specificity of mAb’s. An mAb-
calcheamicin conjugate “Mylotarg” has been approved
for clinical use.7 Maytansinoids and CC-1065 analogues
have been conjugated to mAb’s for treatment of tu-
Design of m Ab-Ta xoid Con ju ga tes. Use of an
appropriate linker between a taxoid and an mAb is
crucial for the efficacy of the resulting immunoconju-
gate. It is required that the linker be stable for an
extended period of time upon storage and also in
circulation in vivo, while it is readily cleavable inside
cancer cells. Among possible linker units reported, we
chose to employ a disulfide linker unit because of its
favorable characteristics.1-3 It is expected that the mAb
component of the conjugate binds to the specific antigens
on tumor surfaces and the whole conjugate is internal-
ized via endocytosis. The disulfide bond is then cleaved
by an intracellular thiol such as glutathione1 to release
taxoid in its active form.
To synthesize a mAb-taxoid conjugate, both a taxoid
and a mAb need to be modified to form a disulfide
linkage by disulfide-thiol exchange reaction. Since the
necessary modification of mAb had been worked out in
one of these laboratories, the critical issue was to find
highly potent second-generation taxoids modified with
a sulfhydrylalkanoyl group, which would be the actual
cytotoxic agent in the target cancer cells. Because the
logical precursor (or synthon) for the sulfhydrylalkanoyl
group is the methyldisulfanyl (MDS) alkanoyl group, we
decided to synthesize MDS-alkanoyltaxoids. It has been
* To whom the correspondence should be addressed: Phone +1-631-
632-7947. Fax: +1-631-632-7942. E-mail: iojima@notes.cc.sunysb.edu.
† State University of New York at Stony Brook.
‡ ImmunoGen, Inc.
10.1021/jm025540g CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5621
Sch em e 2. Syntheses of C-7-MDS-propanoyltaxoids 11a
shown that the number of tumor-associated antigens on
the cancer cell surface is limited (estimated to be
typically 105 molecules/cell). Thus, the cytotoxic agents
that can be effectively used in these conjugates must
have an IC50 value of 10-10-10-11 M against target
cancer cells.1 Two of the second-generation taxoids
possess cytotoxicity in the required range.13 Thus, these
two taxoids were chosen for modification with an MDS-
alkanoyl group.
Since incorporation of an MDS-alkanoyl group into
these taxoids may well affect the cytotoxicity of the
resulting taxoids, an SAR study was necessary to
determine the optimal position for the introduction of
an MDS-alkanoyl group. Thus, we have synthesized new
taxoids bearing an MDS-alkanoyl group at the C-10,
C-7, C-2′ and C-2 positions, and their cytotoxicity was
assayed.
a
(i) 0.1 N HCl in EtOH, overnight (a 88%, b 85%); (ii) 7a (5
equiv), DIC (10 equiv), DMAP, CH2Cl2, 2 h (a 85%, b 88%); (iii)
HF-pyridine, CH3CN, pyridine, overnight (a 86%, b 90%).
Sch em e 3. Synthesis of 2′-MDS-propanoyltaxoid 12
Syn th eses of MDS-a lk a n oylta xoid s. The syntheses
of 10-MDS-alkanoyltaxoids are shown in Scheme 1. We
planned the syntheses of taxoids using the â-lactam
ring-opening coupling protocol11,12,19,20 with appropri-
ately modified baccatins. However, we found that the
introduction of 3-MDS-propanoyl group at the C-10
position of taxoids is not a trivial matter. After the
attempted acylation of 7-TES-10-deacetylbaccatin with
3-MDS-propanoyl chloride/LiHMDS failed, we chose the
acetyl group as the protecting group for 10-OH, which
could be selectively removed after the attachment of the
C-13 isoserine moiety. As Scheme 1 shows, 7-TES-
baccatin (3)17,18,20,21 was coupled with enantiopure â-lac-
tam 413-15,17,18 to give taxoid 522 in excellent yield. After
several attempts, we found that the 10-Ac group of
taxoid 5 could be cleanly cleaved using a procedure
reported by Georg et al.23 with modifications. Hydrazi-
nolysis of 5 using hydrazine‚hydrate in EtOH afforded
10-deacetyltaxoid 6 in high yield. Subsequently, 6 was
coupled with acids 7a and 7b,24 followed by removal of
the silyl groups by HF-pyridine to give 10-MDS-taxoids,
9a and 9b, respectively, in high overall yields.
Sch em e 4. 10-(3-MDS-benzoyl)taxoid 13 and
2-(3-MDS-benzoyl)taxoid 14
Eva lu a tion of Biologica l Activities. (a ) In Vitr o
Cytotoxicity Assa y. Cytotoxicity of new taxoids thus
synthesized was assayed against A431 (epidermoid),
A549 (non-small-cell lung), MCF7 (breast), and MCF7-
MDR (breast) cancer cell lines. Their parent taxoids
were also assayed for comparison. The IC50 values of
these taxoids are summarized in Table 1. As Table 1
Sch em e 1. Syntheses of C-10-MDS-alkanoyltaxoidsa
Ta ble 1. In Vitro Cytotoxicity of Taxoids
a
IC50
,
nM
taxoid
A431b
A549c
MCF7
MCF7-MDR
paclitaxel
docetaxel
1
2
3.0 (3.6d)
(1.0d)
0.1
1.7d
299d
235d
2.2d
1.0d
0.09
0.2
0.18d
0.09d
0.6
12.5d
9a
9b
11a
11b
12
13
14
0.5
0.7
2.0
>3.0
>3.0
>3.0
>3.0
0.8
0.8
0.9
>3.0
>3.0
a
The concentration of compound that inhibits 50% of the growth
of cancer cell line after 72 h of drug exposure. b Human epidermoid
d
carcinoma. c Non-small-cell lung carcinoma. See ref 13.
a
shows, the 10-MDS-alkanoyltaxoids 9a and 9b retain
subnanomolar IC50 values, making each of them very
promising as a cytotoxic component for taxoid-mAb
conjugates.
(i) LiHMDS (1.5 equiv), 4 (1.5 equiv), THF, -40 °C, 40 min;
(ii) N2H4‚H2O, EtOH, 3 h; (iii) 7a or 7b (10 equiv), DIC (11 equiv,
DMAP, CH2Cl2, overnight; (iv) HF-pyridine, pyridine, CH3CN,
overnight.
Synthesis of 7- and 2′-MDS-alkanoyltaxoids 11 and
12 are shown in Schemes 2 and 3. In a similar manner,22
we have synthesized two more analogues 13 and 14
shown in Scheme 4 (see Supporting Information for
detailed synthetic procedure).
Previous SAR studies of taxoids have shown that the
C-7 position is well tolerated for modifications.26 To our
surprise, taxoids 11a and 11b, bearing an MDS-pro-
panoyl group at the C-7 position, exhibit compromised
cytotoxicity. Interestingly, when the MDS-propanoyl

5622 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Letters
group is attached to 2′-OH, the resulting taxoid 12 is
much less active against the A431 cell line while
maintaining its potency against the A549 cell line.
Taxoids 13 and 14, bearing a 3-MDS-benzoyl group at
C-10 and C-2, respectively, show substantial loss of
activity.
KS78-9a , were thus prepared. A conjugate of 9a with
monoclonal antibody mN901 that does not bind to EGFR
was also prepared in a similar manner for comparison.
(c) In Vitr o Cytotoxicity Assa ys. In vitro cytotox-
icity was determined in a clonogenic assay27 after
continuous exposure of the cells to the conjugates. It is
expected that antigen-expressing cancer cells could only
be “targeted” by an immunoconjugate bearing an mAb
specific to the antigen. In fact, mN901-9a exhibits no
cytotoxicity against the A431 cell line, expressing EGFR.
In sharp contrast, KS78-9a shows high potency (IC50
) 1.5 nM) against the same A431 cell line (see Sup-
porting Information for details). It should be noted that
the addition of an excess of unconjugated anti-EGFR
antibody, e.g., KS-61 at 3 × 10-8 M to the KS-61-9a
conjugate, abolished its cytotoxicity against A-431 cells,
indicating that cytotoxicity depended on the specific
binding of the conjugate to the antigen on cells and that
the preparation of conjugate had little unconjugated
taxoid. If there were free taxoid present, addition of
unconjugated antibody should not have blocked the
cytotoxic effect.
These results demonstrate that the binding of anti-
EGFR mAb-taxoid conjugate to EGFR is highly spe-
cific. Moreover, it is strongly indicated that the immu-
noconjugate KS78-9a generates highly cytotoxic agent
15 upon binding to EGFR, followed by internalization
and the subsequent cleavage of the disulfide linkage.
(d ) In Vivo Tu m or Gr ow th In h ibition Assa ys.
The antitumor activities of two anti-EGFR mAb-taxoid
conjugates, KS61-9a and KS77-9a , were evaluated
against human tumor xenografts in severe combined
immune deficiency (SCID) mice (Figure 1). Each mouse
Thus, the SAR study has clearly demonstrated that
cytotoxicity can be retained when an MDS-propanoyl
group is attached to the C-10 position of a taxoid. This
is an important finding, which is totally unexpected.
(b) Con ju ga tion of Ta xoid w ith m Ab. On the basis
of the in vitro study described above, 9a was selected
for linking to mAb’s to form immunoconjugates.
The epidermal growth factor receptor (EGFR) is
known to be overexpressed in several human squamous
cancers such as head, neck, lung, and breast cancers.
Murine monoclonal antibodies directed against the
human EGFR were used as the tumor-targeting moi-
eties in immunoconjugates. Three such immunoglobulin
G class monoclonal antibodies, KS61 (IgG2a), KS77
(IgG1), and KS78 (IgG2a), were linked to 9a via disul-
fide bonds. The preparation of mAb-9a conjugates is
illustrated in Scheme 5. Taxoid 9a was treated with
Sch em e 5. Preparations of mAb-Taxoid Conjugatesa
a
(i)dithiothreitol (DTT); (ii) N-succinimidyl 4-(2-pyridyldithio)-
pentanoate (SPP, 10 equiv in ethanol), 50 mM potassium phos-
phate buffer, pH 6.5, NaCl (50 mM), EDTA (2 mM), 90 min; (iii)
50 mM potassium phosphate buffer, pH 6.5, NaCl (50 mM), EDTA
(2 mM), 15 (1.7 equiv per dithiopyridyl group in EtOH), 24 h.
dithiothreitol to generate HS-taxoid 15 bearing a free
thiol functionality. The anti-EGFR mAb was modified
with N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP)
to attach 4-pyridyldithio (PDT) pentanoyl groups. Re-
covery of the antibody was about 90%, with four to five
PDT-propanoyl groups linked per antibody molecule on
average. Then, the modified mAb was conjugated with
HS-taxoid 15 (2 equiv in aqueous EtOH) at pH 6.5 in a
buffer solution. The mAb-taxoid conjugates were puri-
fied by gel filtration over Sephacryl S300, which sepa-
rated aggregates from monomeric species, and only the
fractions corresponding to the monomeric conjugates
were collected. Recovery of the conjugate was 65-70%
(see Supporting Information for detailed procedures).
Preliminary MALDI-TOF analyses of the KS77-9a
conjugate (peaked at m/z 151 784 with the half peak
width of 3,650) in comparison with KS77 (peaked at m/z
148 500) strongly support that four to five taxoids, on
average, are attached to the mAb. The final formulation
of the conjugate in phosphate-buffer saline (PBS) con-
tained 20% propylene glycol and 0.1% Tween 80 (v/v).
Three immunoconjugates, KS61-9a , KS77-9a , and
F igu r e 1. Antitumor activity of anti-EGFR mAb-taxoid
conjugates against A-431 xenografts in SCID mice.
was inoculated with 1.5 × 106 A431 human squamous
cancer cells, and the tumors were allowed to grow for
11 days to an average size of 100 mm3 (range of 54-
145 mm3). The mice were then randomly divided into
four groups. The first group received KS61-9a conju-
gate (10 mg/kg, qd × 5, administered iv). The second
group received KS77-9a conjugate in the same manner.
The third group received free taxoid 9a (0.24 mg/kg,
qd × 5, iv) at the same dose as that present in the
conjugate. A control group of mice received phosphate-
buffered saline using the same treatment schedule as
in groups 1-3.

Letters
The weights of the mice and tumors sizes were
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5623
cheamicin conjugate for treatment of acute myeloid leukemia.
Bioconjugate Chem. 2002, 13, 47-58.
measured twice weekly, and the tumor volumes were
calculated with the formula 1/2(length × width ×
height). The results are shown in Figure 1. The tumors
in the control group of mice grew to a size of nearly 1000
mm3 in 31 days. Treatment with free taxoid 9a showed
no therapeutic effect, and the tumors in this group grew
at essentially the same rate as in the untreated control
group of mice. In contrast, both anti-EGFR mAb-taxoid
conjugates showed remarkable antitumor activity, re-
sulting in complete inhibition of tumor growth in all the
treated animals for the duration of the experiment.
Necropsy on day 75, followed by histopathological
examination, showed residual calcified material at the
tumor site but no evidence of tumor cells. These data
also indicate that mAb-mediated delivery of the taxoid
using tumor-selective mAbs leads to more pronounced
antitumor activities than systemic drug treatment.
However, further experiments that include nonbinding
control mAb conjugates are required to determine if the
effects are due to immunologically specific drug delivery.
Notably, the doses of antibody-taxoid conjugates used
are nontoxic to the mice, as demonstrated by the
absence of any weight loss (see Supporting Information).
Con clu sion . The results described above clearly
indicate that the TAP approach incorporating second
generation taxoids to mAbs highly specific to the antigen
on tumor cell surfaces is very promising for producing
potential chemotherapeutic agents with few side effects.
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