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Cancer Patent Abstract
The present invention is based on the discovery that the administration
of at least one immunoconjugate and at least one chemotherapeutic
agent provides an unexpectedly superior treatment for cancer. The
present invention is directed to compositions comprising at least
one immunoconjugate and at least one chemotherapeutic agent and
to methods of treating cancer using at least one immunoconjugate
and at least one chemotherapeutic agent. The present invention also
provides methods of modulating the growth of selected cell populations,
such as cancer cells, by administering a therapeutically effective
amount of at least one chemotherapeutic agent and at least one immunoconjugate.
Cancer Patent Claims
What is claimed is:
1. A pharmaceutical composition comprising a synergistic combination
of at least one chemotherapeutic agent and at least one immunoconjugate;
wherein the immunoconjugate comprises at least one maytansinoid
compound linked to a monoclonal antibody or fragment thereof; wherein
the monoclonal antibody or fragment thereof binds to an antigen
expressed by a cancer cell, and wherein the chemotherapeutic agent
is a taxane compound, an epothilone compound, a platinum compound,
an epipodophyllotoxin compound, a camptothecin compound, or a mixture
of two or more thereof.
2. The pharmaceutical composition of claim 1, wherein the chemotherapeutic
agent is a taxane compound, a platinum compound, an epipodophyllotoxin
compound, a camptothecin compound, or a mixture of two or more thereof.
3. The pharmaceutical composition of claim 1, wherein the chemotherapeutic
agent is paclitaxel, docetaxel, epothilone A, epothilone B, epothilone
C, epothilone D, epothilone E, epothilone F, cisplatin, carboplatin,
oxaliplatin, iproplatin, ormaplatin, tetraplatin, etoposide, teniposide,
camptothecin, topotecan, irinotecan, 9-aminocamptothecin, or a mixture
of two or more thereof.
4. The pharmaceutical composition of claim 1, wherein the chemotherapeutic
agent is paclitaxel, cisplatin, etoposide, docetaxel, topotecan,
or a mixture of two or more thereof.
5. The pharmaceutical composition of claim 1, wherein the monoclonal
antibody or fragment thereof binds to a CD56 antigen.
6. The pharmaceutical composition of claim 1, wherein the monoclonal
antibody or fragment thereof is at least one of Fv, Fab, Fab' or
F(ab').sub.2.
7. The pharmaceutical composition of claim 1, wherein the monoclonal
antibody or fragment thereof is humanized N901.
8. The pharmaceutical composition of claim 1, wherein the monoclonal
antibody or fragment thereof is humanized C242.
9. The pharmaceutical composition of claim 1, wherein the immunoconjugate
comprises at least one maytansinoid compound of formula (IV): ##STR00009##
wherein is Z.sub.0 is H or SR; R is methyl, linear alkyl, branched
alkyl, cyclic alkyl, simple or substituted aryl or heterocyclic;
t is 1, 2 or 3; Y.sub.0 is chlorine or hydrogen; and X.sub.3 is
hydrogen or methyl.
10. The pharmaceutical composition of claim 9, wherein Z.sub.0
is H; t is 2; Y.sub.0 is chlorine; and X.sub.3 is methyl.
11. The pharmaceutical composition of claim 1, wherein the immunoconjugate
is of the formula: ##STR00010## wherein MAb is a monoclonal antibody
or fragment thereof that binds to an antigen expressed by the cancer
cell.
12. A pharmaceutical composition comprising a synergistic combination
of at least one chemotherapeutic agent and at least one immunoconjugate;
wherein the chemotherapeutic agent is a taxane compound, an epothilone
compound, a platinum compound, an epipodophyllotoxin compound, a
camptothecin compound, or a mixture of two or more thereof; and
wherein the immunoconjugate is: ##STR00011## wherein MAb is a monoclonal
antibody or fragment thereof that binds to an antigen expressed
by a cancer cell.
13. A pharmaceutical composition comprising a synergistic combination
of (i) at least one chemotherapeutic agent selected from the group
consisting of paclitaxel, docetaxel, cisplatin, etoposide, topotecan
and irinotecan and (ii) an immunoconjugate comprising a maytansinoid
and a humanized monoclonal antibody selected from the group consisting
of N901 and C242.
14. The pharmaceutical composition of claim 13, wherein the maytansinoid
is a compound of formula (IV): ##STR00012## wherein Z.sub.0 is H
or SR; wherein R is methyl, linear alkyl, branched alkyl, cyclic
alkyl, simple or substituted aryl or heterocyclic; t is 1, 2 or
3; Y.sub.0 is chlorine or hydrogen; and X.sub.3 is hydrogen or methyl.
15. A pharmaceutical composition comprising a synergistic combination
of (i) at least one chemotherapeutic agent selected from the group
consisting of paclitaxel, docetaxel, cisplatin, etoposide, topotecan
and irinotecan and (ii) an immunoconjugate comprising a maytansinoid
and a humanized monoclonal antibody or fragment thereof that binds
to an antigen expressed by a small cell lung cancer cell, a non
small cell lung cancer cell or a colorectal cancer cell.
16. The pharmaceutical composition of claim 15, wherein the maytansinoid
is a compound of formula (IV): ##STR00013## wherein Z.sub.0 is H
or SR; wherein R is methyl, linear alkyl, branched alkyl, cyclic
alkyl, simple or substituted aryl or heterocyclic; t is 1, 2 or
3; Y.sub.0 is chlorine or hydrogen; and X.sub.3 is hydrogen or methyl.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention is based on the discovery that the administration
of at least one immunoconjugate and at least one chemotherapeutic
agent provides an unexpectedly superior treatment for cancer. The
present invention is directed to compositions comprising at least
one immunoconjugate and at least one chemotherapeutic agent and
to methods of treating cancer using a therapeutically effective
amount of at least one immunoconjugate and at least one chemotherapeutic
agent. The present invention is also directed to methods of modulating
the growth of selected cell populations using a therapeutically
effective amount of at least one chemotherapeutic agent and at least
one immunoconjugate.
BACKGROUND OF THE INVENTION
Of all lung cancer cases diagnosed in the United States every year,
20-25% are small cell lung cancer (SCLC). Current treatments for
small cell lung cancer include surgery, radiation treatment, and
chemotherapeutic agents, such as paclitaxel or a combination of
etoposide and cisplatin. Despite these treatment options, there
is only a 1-5% survival rate after 5 years in patients who have
clinically evident metastatic disease upon diagnosis. Glisson et
al, Journal of Clinical Oncology, 17(8):2309-2315 (August 1999).
Pre-clinical studies reveal that small cell lung cancers can also
be treated with an immunoconjugate comprising a monoclonal antibody
and a maytansinoid. Liu et al, Proceedings of the American Association
for Cancer Research, 38:29 (abstract 190) (1997). In this study,
the maytansinoid was DM1, and the monoclonal antibody was humanized
N901. Humanized monoclonal antibody N901 targets CD56, which is
expressed on substantially all small cell lung cancers.
There is a need in the art for new and more effective methods for
treating cancer. The present invention is directed to these, as
well as other, important ends.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that the use of
at least one chemotherapeutic agent and at least one immunoconjugate
produces unexpectedly superior results in the treatment of cancer.
The present invention describes methods of treating cancer in a
patient in need thereof by administering to the patient a therapeutically
effective amount of at least one chemotherapeutic agent and at least
one immunoconjugate. The chemotherapeutic agent can be any known
in the art including, for example, taxane compounds, compounds that
act via taxane mechanisms, platinum compounds, epipodophyllotoxin
compounds, camptothecin compounds, or any combination thereof. The
immunoconjugate can comprise a cell binding agent and at least one
therapeutic agent for killing selected cell populations. The cell
binding agent is preferably a monoclonal antibody or a fragment
thereof, and the therapeutic agent for killing selected cell populations
is preferably an anti-mitotic agent, such as a maytansinoid, a Vinca
alkaloid, a dolastatin, or a cryptophycin. In particularly preferred
embodiments, the immunoconjugate comprises the maytansinoid DM1
and humanized N901 monoclonal antibody. The chemotherapeutic agent
and immunoconjugate can be administered separately or as components
of the same composition.
The present invention also describes methods of modulating the
growth of selected cell populations, such as cancer cells, by administering
a therapeutically effective amount of at least one chemotherapeutic
agent and at least one immunoconjugate. The chemotherapeutic agent
can be any known in the art including, for example, taxane compounds,
compounds that act via taxane mechanisms, platinum compounds, epipodophyllotoxin
compounds, camptothecin compounds, or any combination thereof. The
immunoconjugate can comprise a cell binding agent and at least one
therapeutic agent for killing selected cell populations. The cell
binding agent is preferably a monoclonal antibody or a fragment
thereof, and the therapeutic agent for killing selected cell populations
is preferably an anti-mitotic agent, such as a maytansinoid, a Vinca
alkaloid, a dolastatin, or a cryptophycin. In particularly preferred
embodiments, the immunoconjugate comprises the maytansinoid DM1
and humanized N901 monoclonal antibody. The chemotherapeutic agent
and immunoconjugate can be administered separately or as components
of the same composition.
The present invention also describes compositions comprising at
least one chemotherapeutic agent and at least one immunoconjugate.
The chemotherapeutic agent can be any known in the art including,
for example, taxane compounds, compounds that act via taxane mechanisms,
platinum compounds, epipodophyllotoxin compounds, camptothecin compounds,
or any combination thereof. The immunoconjugate can comprise a cell
binding agent and at least one therapeutic agent for killing selected
cell populations. The cell binding agent is preferably a monoclonal
antibody or a fragment thereof, and the therapeutic agent for killing
selected cell populations is preferably an anti-mitotic agent, such
as a maytansinoid, a Vinca alkaloid, a dolastatin, or a cryptophycin.
In particularly preferred embodiments, the immunoconjugate comprises
the maytansinoid DM1 and humanized N901 monoclonal antibody. The
composition can comprise a pharmaceutically acceptable carrier,
excipient or diluent.
These and other aspects of the present invention are described
in detail herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows maytansine (1a) and maytansinol (1b).
FIG. 2 shows the synthesis of disulfide-containing derivatives
of N-methyl-L-alanine.
FIG. 3 shows the synthesis of disulfide- and thiol-containing maytansinoids
which can be linked to cell binding agents via a disulfide or any
other sulfur-containing link such as thioether or thioester links.
The synthesis starts with the intermediates of FIG. 2.
FIG. 4a shows the synthesis of disulfide- and thiol-containing
derivatives of N-methyl-L-cysteine.
FIG. 4b shows the synthesis of disulfide- and thiol-containing
maytansinoids from the intermediates of FIG. 4a that can be conjugated
to cell binding agents via a disulfide or any other sulfur-containing
link such as thioether or thioester links.
FIG. 5 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huN901-DM1, (iii) paclitaxel, and (iv) the combination of huN901-DM1
and paclitaxel, against small cell lung cancer xenografts in SCID
mice.
FIG. 6 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huN901-DM1, (iii) the combination of cisplatin and etoposide,
and (iv) the combination of huN901-DM1, cisplatin and etoposide,
against small cell lung cancer xenografts in SCID mice.
FIG. 7 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huN901-DM1, (iii) docetaxel, and (iv) the combination of huN901-DM1
and docetaxel, against small cell lung cancer xenografts in SCID
mice.
FIG. 8 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huN901-DM1, (iii) topotecan, and (iv) the combination of huN901-DM1
and topotecan, against small cell lung cancer xenografts in SCID
mice.
FIG. 9 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huC242-DM1, (iii) paclitaxel, and (iv) the combination of huC242-DM1
and paclitaxel, against human lung adenocarcinoma xenografts in
SCID mice.
FIG. 10 is a graph comparing the anti-tumor activity of (i) a control,
(ii) huC242-DM1, (iii) CPT-11 (also called irinotecan), and (iv)
the combination of huC242-DM1 and CPT-11, against human colon cancer
xenografts in SCID mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the unexpected discovery that
the administration of at least one chemotherapeutic agent and at
least one immunoconjugate produces superior results in the treatment
of cancer. Appropriate chemotherapeutic agents and immunoconjugates
are described herein.
The immunoconjugates of the present invention comprise at least
one therapeutic agent for killing selected cell populations linked
to a cell binding agent. The therapeutic agent for killing selected
cell populations is preferably an anti-mitotic agent. Anti-mitotic
agents, which are known in the art, kill cells by inhibiting tubulin
polymerization and, therefore, microtubule formation. Any anti-mitotic
agent known in the art can be used in the present invention, including,
for example, maytansinoids, Vinca alkaloids, dolastatins, cryptophycins,
and/or any other agent that kills cells by inhibiting tubulin polymerization.
Preferably, the anti-mitotic agent is a maytansinoid.
Maytansinoids that can be used in the present invention, to produce
the modified maytansinoid capable of being linked to a cell binding
agent, are well known in the art and can be isolated from natural
sources according to known methods or prepared synthetically according
to known methods. Preferred maytansinoids are those described, for
example, in U.S. Pat. No. 5,208,020, the disclosure of which is
incorporated by reference herein in its entirety.
Suitable maytansinoids include maytansinol and maytansinol analogues.
Examples of suitable maytansinol analogues include those having
a modified aromatic ring and those having modifications at other
positions. Specific examples of suitable analogues of maytansinol
having a modified aromatic ring include: C-19-dechloro (U.S. Pat.
No. 4,256,746) (prepared by LAH reduction of ansamitocin P-2); C-20-hydroxy
(or C-20-demethyl)+/-C-19-dechloro (U.S. Pat. Nos. 4,361,650 and
4,307,016) (prepared by demethylation using Streptomyces or Actinomyces
or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (--OCOR),
+/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using
acyl chlorides).
Specific examples of suitable analogues of maytansinol having modifications
of other positions include: C-9-SH (U.S. Pat. No. 4,424,219) (prepared
by the reaction of maytansinol with H.sub.2S or P.sub.2S.sub.5);
C-14-alkoxymethyl(demethoxy/CH.sub.2OR) (U.S. Pat. No. 4,331,598);
C-14-hydroxymethyl or acyloxymethyl (CH.sub.2OH or CH.sub.2OAc)
(U.S. Pat. No. 4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy
(U.S. Pat. No. 4,364,866) (prepared by the conversion of maytansinol
by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)
(isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos.
4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol
by Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared
by the titanium trichloride/LAH reduction of maytansinol).
In order to link the maytansinoid to the cell binding agent, the
maytansinoid must be modified, and a linking group can be used.
Suitable linking groups are known in the art and include, for example,
disulfide groups, thioether groups, acid labile groups, photolabile
groups, peptidase labile groups and esterase labile groups. Preferred
are disulfide groups and thioether groups.
The linking group is part of a chemical moiety that is covalently
bound to the maytansinoid through conventional methods. In a preferred
embodiment, the chemical moiety can be covalently bound to the maytansinoid
via an ester linkage.
Many positions on maytansinoids are useful as the linkage position,
depending upon the type of link. For example, for forming an ester
linkage, the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with hydroxy,
and the C-20 position having a hydroxy group are all expected to
be useful. The C-3 position is preferred and the C-3 position of
maytansinol is especially preferred. Also preferred is an N-methyl-alanine-containing
C-3 ester and an N-methyl-cysteine-containing C-3 ester of maytansinol
or its analogues.
The synthesis of esters of maytansinol having a linking group is
described in U.S. Pat. No. 5,208,020. While the synthesis of esters
of maytansinol having a linking group is described herein in terms
of thiol and disulfide linking groups, one of skill in the art will
understand that other linking groups can also be used with the invention,
as can other maytansinoids.
The synthesis of maytansinoid derivatives can be described by reference
to FIGS. 1, 2, 3, 4a and 4b, where disulfide-containing maytansinoid
esters are prepared by condensing maytansinol 1b with freshly prepared
N-methyl-L-alanine or N-methyl-L-cysteine derivatives containing
a disulfide group.
.omega.-Mercapto-carboxylic acids of varying chain lengths are
converted into their respective methyl-dithio, e.g., 3a-3d (where
n=1-10, including branched and cyclic aliphatics), or aryl-dithio,
e.g., 4a-4b, derivatives by reacting them with methyl methanethiolsulfonate
or aryldisulfides, such as diphenyldisulfide and ring substituted
diphenyldisulfides and heterocyclic disulfides such as 2,2-dithiopyridine.
The carboxylic acids are activated and then reacted with N-methyl-L-alanine
to form the desired carboxylic acid compounds, e.g., 5a-5f, for
condensation with maytansinol 1b.
Esterification of maytansinol 1b or an analogue with the carboxylic
acids 5a-5f gives the disulfide-containing maytansinoids 6a-6f.
Cleavage of the disulfide group in 6a-6f with dithiothreitol gives
the thiol-containing maytansinoids 7a-7c, which are readily linked
via disulfide or thioether links to cell binding agents. N-methyl-L-alanine
can be prepared as described in the literature (Fu et al, J. Amer.
Chem. Soc., 75:1953); or is obtainable commercially (Sigma Chemical
Company).
In another embodiment, N-methyl-cysteine or N-methylhomocysteine
can be converted to the respective disulfide derivatives 8 (n=1
and 2, respectively) which are then acylated to yield the desired
carboxylic acids 9 (n=1 and 2, respectively). Maytansinol is then
esterified with 9 (n=1) to give disulfide-containing ester 10. Reduction
of 10a with dithiothreitol as described for 7b produces the thiol-containing
maytansinoid 11 which can be conjugated to cell binding agents.
N-methyl-cysteine can be prepared as described in Undheim et al,
Acta Chem. Scand., 23:3129-3133 (1970).
More specifically, maytansinol 1b is derived from maytansine 1a
or other esters of maytansinol by reduction such as with lithium
aluminum hydride. (Kupchan et al, J. Med. Chem., 21:31-37 (1978);
U.S. Pat. No. 4,360,462). It is also possible to isolate maytansinol
from the microorganism Nocardia (U.S. Pat. No. 4,151,042). Maytansinol
is then converted to the different ester derivatives, 6a to 6f and
10, using a suitable agent such as dicyclohexylcarbodiimide (DCC)
and catalytic amounts of zinc chloride (U.S. Pat. Nos. 4,137,230
and 4,260,609; Kawai et al, Chem. Pharm. Bull., 32:3441-3951 (1984)).
The two diastereomeric products containing the D and L-aminoacyl
side chains result. The diastereomeric maytansinoid esters are readily
separated by preparative TLC on silica gel. For example, using Analtech
GF plates (1000 microns) and developing with 6% methanol in chloroform
yields distinct banding: the desired bands are scraped off the plate
and the products extracted with ethyl acetate (Kupchan, J. Med.
Chem., 21:31-37 (1978) and U.S. Pat. No. 4,360,462).
Reduction of the disulfide-containing maytansinoids to the corresponding
mercapto-maytansinoids 7a, 7b, 7c and 11, is achieved by treatment
with dithiothreitol (DTT) and purification by HPLC using a Waters
radialpak C-18 column and eluting with a linear gradient of 55%
to 80% acetonitrile in H.sub.2O over 10 minutes at a flow rate of
1.5 ml/min.
When analogues of maytansinol are used as the starting material
to give analogous disulfide-containing maytansinoid esters, the
analogues are prepared before reacting them with the N-methyl-L-alanine
or N-methyl-L-cysteine derivatives.
One example of N-methyl-alanine-containing maytansinoid derivatives
useful in the present invention is represented by formula (I):
##STR00001## wherein
Z.sub.0 represents H or SR, wherein R represents methyl, linear
alkyl, branched alkyl, cyclic alkyl, simple or substituted aryl
or heterocyclic;
p represents an integer of 1 to 10; and
"may" represents a maytansinoid.
In a preferred embodiment of the compound of formula (I), Z.sub.0
represents SR, R represents methyl, and p represents an integer
of 2.
Another example of N-methyl-alanine-containing maytansinoid derivatives
useful in the present invention is represented by formula (II):
##STR00002## wherein
R.sub.1 and R.sub.2, which may be the same or different, represents
H, CH.sub.3 or CH.sub.2CH.sub.3;
Z.sub.1 represents H or SR.sup.3, wherein R.sup.3 represents methyl,
linear alkyl, branched alkyl, cyclic alkyl, simple or substituted
aryl, or heterocyclic:
m represents 0, 1, 2 or 3; and
"may" represents a maytansinoid.
Another example of N-methyl-alanine-containing maytansinoid derivatives
useful in the present invention is represented by formula (III):
##STR00003## wherein:
Z.sub.2 represents H or SR.sub.4, wherein R.sub.4 represents methyl,
linear alkyl, branched alkyl cyclic alkyl, simple or substituted
aryl, or heterocyclic;
n represents an integer of 3 to 8; and
"may" represents a maytansinoid.
Yet another example of N-methyl-alanine-containing maytansinoid
derivatives useful in the present invention is represented by formula
(IV):
##STR00004## wherein:
Z.sub.0 represents H or SR, wherein R represents methyl, linear
alkyl, branched alkyl, cyclic alkyl, simple or substituted aryl
or heterocyclic:
t represents 1, 2 or 3;
Y.sub.0 represents Cl or H; and
X.sub.3 represents H or CH.sub.3.
A specific example of N-methyl-cysteine-containing maytansinoid
derivatives useful in the present invention is represented by formula
(V):
##STR00005## wherein:
Z.sub.3 represents H or SR.sub.5, wherein R.sub.5 represents methyl,
linear alkyl, branched alkyl, cyclic alkyl, simple or substituted
aryl, or heterocyclic;
o represents 1, 2 or 3;
p represents 0 or an integer of 1 to 10; and
"may" represents a maytansinoid.
Another specific example of N-methyl-cysteine-containing maytansinoid
derivatives useful in the present invention is represented by formula
(VI):
##STR00006## wherein:
Z.sub.3 represents H or SR.sub.5, wherein R.sub.5 represents methyl,
linear alkyl, branched alkyl, cyclic alkyl, simple or substituted
aryl or heterocyclic;
o represents 1, 2, or 3;
q represents 0 or an integer of 1 to 10;
Y.sub.0 represents Cl or H; and
X.sub.3 represents H or CH.sub.3.
Examples of linear alkyls include methyl, ethyl, propyl, butyl,
pentyl, and hexyl. Examples of branched alkyls include isopropyl,
isobutyl, sec-butyl, tert-butyl, isopentyl, and 1-ethyl-propyl.
Examples of cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl,
and cyclohexyl. Examples of simple aryls include phenyl, and naphthyl.
Examples of substituted aryls include aryls such as those described
above substituted with alkyl groups, with halogens, such as Cl,
Br, F, nitro groups, amino groups, sulfonic acid groups, carboxylic
acid groups, hydroxy groups, and alkoxy groups. Examples of heterocyclics
are compounds wherein the heteroatoms are selected from O, N and
S, and include pyrrollyl, pyridyl, furyl, and thiophene.
Vinca alkaloids that can be used in the present invention, to produce
the modified Vinca alkaloids capable of being linked to a cell binding
agent, are well known in the art. Such Vinca alkaloids include,
for example, those described in Cancer Principles and Practice in
Oncology, 4th Ed., DeVita et al, eds., J. B. Lippincott Company,
Philadelphia Pa. (1993) and by Morris et al, J. Clin. Oncol., 16:1094-1098
(1998), the disclosures of which are incorporated herein by reference
in their entirety. Exemplary Vinca alkaloids include vincristine,
vinblastine, vindesine, navelbine (vinorelbine), and the like. Other
Vinca alkaloids that can be used in the present invention include
those described, for example, in U.S. Pat. Nos. 5,369,111, 4,952,408,
5,395,610, 4,522,750, 5,888,537, 5,891,724, 5,795,589, 4,172,077,
5,714,163, 5,436,243, 3,932,417, 5,869,620, 5,795,575, 5,780,446,
5,676,978, 5,604,237, 5,171,217, 4,831,038, 4,828,831, 4,765,972,
4,375,432, 4,309,415, 5,939,455, 5,874,402, 5,767,260, 5,763,733,
5,728,687, 5,716,928, 5,660,827, 5,541,232, 5,346,897, 5,220,016,
5,208,238, 5,190,949, 4,479,957, 4,160,767, 4,159,269, 4,096,148,
RE 30,561, RE 30,560, U.S. Pat. Nos. 5,935,955, 5,922,340, 5,886,025,
5,866,679, 5,863,538, 5,855,866, 5,817,321, 5,783,178, 5,776,427,
5,767,110, 5,753,507, 5,723,625, 5,698,178, 5,686,578, 5,667,764,
5,654,287, 5,646,124, 5,635,515, 5,635,218, 5,606,017, 5,597,830,
5,595,756, 5,583,052, 5,561,136, 5,547,667, 5,543,152, 5,529,076,
5,491,285, 5,482,858, 5,455,161, 5,430,026, 5,403,574, 5,399,363,
5,397,784, 5,387,578, 5,364,843, 5,300,282, 5,182,368, 5,162,115,
5,147,294, 5,108,987, 5,100,881, 5,047,528, 5,030,620, 5,004,593,
4,946,833, 4,931,468, 4,923,876, 4,801,688, 4,737,586, 4,667,030,
4,617,305, 4,578,351, 4,476,026, 4,399,069, 4,279,817, 4,208,414,
4,199,504, 4,070,358, 4,029,663, 3,965,254, 3,954,773, 3,944,554,
3,887,565, 6,120,800, 6,071,947, 6,071,930, 6,069,146, 6,063,911,
5,994,367, 5,962,216, and 5,945,315, the disclosures of which are
incorporated by reference herein in their entirety.
The Vinca alkaloids can be linked to cell binding agents, such
as antibodies, via acid-labile hydrazide links by methods described
by, for example, Laguzza et al, J. Med. Chem., 32:548-555 (1989),
Schrappe et al, Cancer Res., 52:3838-3844 (1992), and Apelgren et
al, Cancer Res., 50:3540-3544 (1990), the disclosures of which are
incorporated by reference herein in their entirety. A preferable
method is to link the Vinca alkaloids to a cell binding agent via
disulfide bonds. The carboxy ester at the C-3 position of vinblastine,
vincristine and navelbine can be hydrolzyed to the corresponding
carboxylic acid using standard chemical methods. In vindesine, the
carboxamide group at C-3 can be hydrolyzed to the free carboxy group.
The free carboxy group in each of the Vinca alkaloids can be converted
to an amide compound containing a terminal disulfide group by reaction
with a protected cysteamine (e.g., methydithiocysteamine) in the
presence of a coupling agent such as dicyclohexyl-carbodidimide
(DCC) or ethyl dimethylamin-propylcarbodiimide (EDC). The resulting
disulfide containing Vinca alkaloid is reduced with a reducing agent,
such as dithiothreitol, to provide a thiol-containing compound.
The thiol-containing Vinca alkaloid can be coupled to a cell-binding
agent via disulfide exchange as described herein for the preparation
of antibody-maytansinoid conjugates.
Dolastatins that can be used in the present invention, to produce
the modified dolastatins capable of being linked to a cell binding
agent, are well known in the art. Such dolastatins include, for
example, those described by Pitot et al, Clin. Cancer Res., 5:525-531
(1999) and Villalona-Calero et al, J. Clin. Oncol., 16:2770-2779
(1998), the disclosures of which are incorporated by reference herein
in their entirety. Exemplary dolastatins include dolastatin 10,
dolastatin 15, and the like. Other dolastatins that can be used
in the present invention include those described, for example, in
U.S. Pat. Nos. 5,945,543, 5,939,527, 5,886,147, 5,886,025, 5,883,120,
5,856,324, 5,840,699, 5,831,002, 5,821,222, 5,807,984, 5,780,588,
5,767,237, 5,750,713, 5,741,892, 5,665,860, 5,663,149, 5,654,399,
5,635,483, 5,626,864, 5,599,902, 5,554,725, 5,530,097, 5,521,284,
5,504,191, 5,502,032, 5,410,024, 5,410,024, 5,378,803, 5,352,804,
5,138,036, 5,091,368, 5,076,973, 4,986,988, 4,978,744, 4,879,278,
4,816,444, 4,486,414, 4,414,205, 6,103,913, 6,103,698, 6,096,757,
6,034,065, 6,020,495, 6,017,890, 6,004,934, 5,985,837, 5,965,700,
and 5,965,537, the disclosures of which are incorporated by reference
herein in their entirety.
The synthetic scheme described for dolastatin 10 by Pettit et al,
J. Am. Chem. Soc., 111:5463-5465 (1989), the disclosure of which
is incorporated by reference herein in its entirety, can be followed,
with minor modification, to provide a thiol-containing dolastatin
that can be linked via disulfide bonds to a cell binding agent,
such as an antibody. The phenylalanine moiety in the dolphenine
residue in the C-terminal of dolastatin 10 is replaced by a methyldithio-substituent
containing amino acid. Thus, tyrosine can be converted into an ether
by reaction with a commercially available dibromoalkane, such as
1,3-dibromobutane, using standard chemical methods. The resulting
bromo compound is reacted with potassium thioacetate, followed by
hydrolysis, to give a thiol-containing tyrosine. Conversion is achieved
as described by Pettit, supra. The thiol-containing dolastatin can
be coupled to a cell binding agent via disulfide exchange as described
herein for the preparation of an antibody-maytansinoid conjugate.
Cryptophycins that can be used in the present invention, to produce
the modified cryptophycins capable of being linked to a cell binding
agent, are well known in the art. Such cryptophycins include, for
example, those described by Smith et al, Cancer Res., 54:3779-3783
(1994), Panda et al, Proc. Natl. Acad. Sci., 95:9313-9318 (1998),
and Bai et al, Cancer Res., 56:4398-4406 (1996), the disclosures
of which are incorporated by reference herein in their entirety.
Exemplary cryptophycins include cryptophycin 52, cryptophycin 1,
and the like. Other cryptophycins that can be used in the present
invention include those described, for example, in Great Britain
Patent No. 2220657; European Patent Nos. 870506, 870501, 861838,
861839, 792875 and 870510; U.S. Pat. Nos. 6,103,913, 6,046,177,
6,020,512, 6,013,626, 5,977,387, 5,955,423, 5,952,298, 5,945,315,
5,886,025, and 5,833,994; and WIPO Publication Nos. 98/38178, 98/38164,
98/08829, 98/08506, 98/08505, 97/31632, 97/08334, 97/07798, 98/09601,
97/23211, 98/46581, 98/38158, 98/09988, 98/09974, 98/08812, and
98/09955, the disclosures of which are incorporated herein by reference
in their entirety.
The aromatic methoxy group in the cryptophycins can be hydrolyzed
by standard chemical or enzymatic methods to give the phenolic derivative.
The phenol group can be converted into an ether by reaction with
a commercially available dibromoalkane, such as 1,3-dibromobutane,
using standard chemical methods. The resulting bromo compound is
reacted with potassium thioacetate, followed by hydrolysis, to give
a thiol-containing cryptophycin. The thiol-containing cryptophycin
can be coupled to a cell binding agent via disulfide exchange as
described herein for the preparation of antibody-maytansinoid conjugates.
Disulfide-containing and mercapto-containing maytansinoid (or Vinca
alkaloid or dolastatin or cryptophycin) drugs of the invention can
be evaluated for their ability to suppress proliferation of various
unwanted cell lines using in vitro methods generally accepted in
the art as being predictive of in vivo activity. For example, cell
lines such as the human epidermoid carcinoma line KB, the human
breast tumor line SKBR3 and the Burkitt's lymphoma line Namalwa
can easily be used for the assessment of cytotoxicity of these compounds.
Cells to be evaluated can be exposed to the compounds for 24 hours
and the surviving fractions of cells measured in direct assays by
known methods. IC.sub.50 values can then be calculated from the
results of the assays.
The effectiveness of the immunoconjugates as therapeutic agents
depends on the careful selection of an appropriate cell binding
agent. Cell binding agents may be of any kind presently known, or
that become known, and include peptides and non-peptides. Generally,
these can be antibodies (especially monoclonal antibodies), lymphokines,
hormones, growth factors, nutrient-transport molecules (such as
transferrin), or any other cell binding molecule or substance.
More specific examples of cell binding agents that can be used
include: monoclonal antibodies; fragments of antibodies such as
Fv, Fab, Fab', and F(ab').sub.2 (Parham, J. Immunol., 131:2895-2902
(1983); Spring et al, J. Immunol., 113:470-478 (1974); Nisonoff
et al, Arch. Biochem. Biophys., 89:230-244 (1960)); interferons
(e.g., .alpha., .beta., .gamma.); lymphokines such as IL2, IL3,
IL-4, IL-6; hormones such as insulin, TRH (thyrotropin releasing
hormone), MSH (melanocyte-stimulating hormone), steroid hormones
such as androgens and estrogens; growth factors and colony-stimulating
factors such as EGF, TGF-.alpha., G-CSF, M-CSF and GM-CSF (Burgess,
Immunology Today, 5:155-158 (1984)); and transferrin (O'Keefe et
al, J. Biol. Chem., 260:932-937 (1985)).
Monoclonal antibody techniques allow for the production of extremely
specific cell binding agents in the form of specific monoclonal
antibodies. Particularly well known in the art are techniques for
creating monoclonal antibodies produced by immunizing mice, rats,
hamsters or any other mammal with the antigen of interest such as
the intact target cell, antigens isolated from the target cell,
whole virus, attenuated whole virus, and viral proteins such as
viral coat proteins. Sensitized human cells can also be used.
Selection of the appropriate cell binding agent is a matter of
choice that depends upon the particular cell population that is
to be targeted, but in general monoclonal antibodies are preferred
if an appropriate one is available.
For example, the monoclonal antibody J5 is a murine IgG.sub.2a
antibody that is specific for the Common Acute Lymphoblastic Leukemia
Antigen (CALLA) (Ritz et al, Nature, 283:583-585 (1980)) and can
be used if the target cells express CALLA such as in the disease
of acute lymphoblastic leukemia. Similarly, the monoclonal antibody
anti-B4 is a murine IgG.sub.1, that binds to the CD19 antigen on
B cells (Nadler et al, J. Immunol., 131:244-250 (1983)) and can
be used if the target cells are B cells or diseased cells that express
this antigen such as in non-Hodgkin's lymphoma or chronic lymphoblastic
leukemia.
Additionally, GM-CSF which binds to myeloid cells can be used as
a cell binding agent to diseased cells from acute myelogenous leukemia.
IL-2 which binds to activated T-cells can be used for prevention
of transplant graft rejection, for therapy and prevention of graft-versus-host
disease, and for treatment of acute T-cell leukemia. MSH which binds
to melanocytes can be used for the treatment of melanoma.
Cancers of the breast and testes can be successfully targeted with
estrogen (or estrogen analogues) or androgen (or androgen analogues),
respectively, as cell binding agents.
In a preferred embodiment, the antibody or fragment thereof is
one that is specific for lung cancer, preferably small cell lung
cancer. An antibody or fragment thereof that is specific for small
cell lung cancer can be determined by methods described in the art,
such as by Doria et al, Cancer 62:1939-1945 (1988). Preferably,
the antibody or fragment thereof binds to an epitope on the CD56
antigen, which is expressed on substantially all small cell lung
cancers. For example, N901 is an IgG1 murine monoclonal antibody
(also called anti-N901) that is reactive with CD56, which is expressed
on tumors of neuroendocrine origin, such as small cell lung cancer.
See Griffin et al, J. Immunol. 130:2947-2951 (1983), and Roguska
et al, Proc. Natl. Acad. Sci. USA, 91:969-973 (1994), the disclosure
of which are incorporated by reference herein in their entirety.
Preferred antibodies or fragments thereof that are specific for
small cell lung cancers include, but are not limited to, N901, NKH-1,
Leu-7, anti-Leu-7, and the like (Doria et al, Cancer 62:1939-1945
(1988); Kibbelaar et al, Journal of Pathology, 159:23-28 (1989)).
Other suitable antibodies or fragments thereof for use in the present
invention include, for example, S-L 3-5, S-L 4-20, S-L 7-3, S-L11-14,
TFS-4, MOC-1, MOC-21, MOC-31, MOC-32, MOC-52, 123A8, 123C3, UJ13A,
B10/B12, SWA4, SWA20, SWA21, SWA22, SWA23, LAM-8, 534F8, 703D4704A1
and SM1, which are further described in Table I of Chapter 3 of
the Proceedings of the First International Workshop on Small Cell
Lung Cancer Antigens (London 1987), published in Lung Cancer, 4:15-36
(1988), the disclosures of which are incorporated by reference herein
in their entirety. In a most preferred embodiment, the antibody
is N901, or a fragment thereof, that binds to an epitope on the
CD56 antigen, such as Fv, Fab, Fab' and F(ab').sub.2. The monoclonal
antibody or fragment thereof can be any other antibody that binds
to the CD56 antigen with the same specificity as N901. "Same
specificity" means that the antibody or fragment thereof can
bind to the same antigen as demonstrated by a competitive binding
assay with N901.
Another preferred antibody or fragment thereof that is useful in
the present invention is C242 (commercially available from CanAg
Diagnostics AB, Sweden). C242 is also described in U.S. Pat. No.
5,552,293, the disclosure of which is incorporated by reference
herein in its entirety.
In other preferred embodiments, the antibodies described herein
are humanized antibodies or fragments thereof because humanized
antibodies or fragments thereof are not expected to elicit an immune
response in humans. Generally, antibodies can be humanized through
the application of different humanization technologies described,
for example, in U.S. Pat. Nos. 5,225,539, 5,585,089, and 5,639,641,
the disclosures of which are incorporated by reference herein in
their entirety. The preparation of different versions of humanized
N901, is described, for example, by Roguska et al Proc. Natl. Acad.
Sci. USA, 91:969-973 (1994), and Roguska et al, Protein Eng., 9:895:904
(1996), the disclosures of which are incorporated by reference herein
in their entirety. To denote a humanized antibody, the letters "hu"
or "h" appear before the name of the antibody. For example,
humanized N901 is also referred to as huN901 or hN901.
Conjugates of the maytansinoid derivatives of the invention and
a cell binding agent can be formed using any techniques presently
known or later developed. The maytansinoid ester can be modified
to yield a free amino group and then linked to an antibody or other
cell binding agent via an acid-labile linker, or a photolabile linker.
The maytansinoid ester can be condensed with a peptide and subsequently
linked to a cell binding agent to produce a peptidase-labile linker.
The maytansinoid ester can be treated to yield a primary hydroxyl
group, which can be succinylated and linked to a cell binding agent
to produce a conjugate that can be cleaved by intracellular esterases
to liberate free drug. Most preferably, the maytansinoid esters
are treated to create a free or protected thiol group, and then
one or many disulfide or thiol-containing maytansinoid derivatives
are covalently linked to the cell binding agent via disulfide bond(s).
Representational conjugates of the invention are antibody/maytansinoid
derivatives, antibody fragment/maytansinoid derivatives, epidermal
growth factor (EGF)/maytansinoid derivatives, melanocyte stimulating
hormone (MSH)/maytansinoid derivatives, thyroid stimulating hormone
(TSH)/maytansinoid derivatives, estrogen/maytansinoid derivatives,
estrogen analogue/maytansinoid derivatives, androgen/maytansinoid
derivatives, androgen analogue/maytansinoid derivatives.
Maytansinoid conjugates of antibodies, antibody fragments, protein
hormones, protein growth factors and other proteins are made in
the same way. For example, peptides and antibodies can be modified
with crosslinking reagents such as N-succinimidyl 3-(2-pyridyldithio)propionate,
N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), 4-succinimidyl-oxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluen-
e (SMPT), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2-iminothiolane,
or acetylsuccinic anhydride by known methods (U.S. Pat. No. 4,563,304;
Carlsson et al, Biochem. J., 173:723-737 (1978); Blattler et al,
Biochem., 24:1517-1524 (1985); Lambert et al, Biochem., 22:3913-3920
(1983); Klotz et al, Arch. Biochem. Biophys., 96:605 (1962); and
Liu et al, Biochem., 18:690 (1979), Blakey and Thorpe, Antibody,
Immunoconjugates and Radiopharmaceuticals, 1:1-16 (1988); Worrell
et al, Anti-Cancer Drug Design, 1:179-184 (1986), the disclosures
of which are incorporated by reference herein in their entirety).
The cell binding agent containing free or protected thiol groups
thus-derived is then reacted with a disulfide- or thiol-containing
maytansinoid to produce conjugates. The conjugates can be purified
by HPLC or by gel filtration.
Similarly, for example, estrogen and androgen cell binding agents,
such as estradiol and androstenediol, can be esterified at the C-17
hydroxy group with an appropriate disulfide-containing carboxylic
acid, e.g., dicyclohexylcarbodiimide, as a condensing agent. Examples
of such carboxylic acids that can be used are 3-(2-pyridyldithio)propanoic
acid, 3-methyldithiopropanoic acid, 3-phenyldithio-propanoic acid,
and 4-(2-pyridyldithio)pentanoic acid. Esterification of the C-17
hydroxy group can also be achieved by reaction with an appropriately
protected thiol group-containing carboxylic acid chloride, such
as 3S-acetylpropanoyl chloride. Other methods of esterification
can also be used as described in the literature (Haslam, Tetrahedron,
36:2400-2433 (1980)). The protected or free thiol-containing androgen
or estrogen can then be reacted with a disulfide or thiol-containing
maytansinoid to produce conjugates. The conjugates can be purified
by column chromatography on silica gel or by HPLC.
Preferably monoclonal antibody or cell binding agent/maytansinoid
conjugates are those that are joined via a disulfide bond, as discussed
above, that are capable of delivering maytansinoid molecules. Such
cell binding conjugates are prepared by known methods such as modifying
monoclonal antibodies with succinimidyl pyridyl-dithiopropionate
(SPDP) or SPP (Carlsson et al, Biochem. J., 173:723-737 (1978)).
The resulting thiopyridyl group is then displaced by treatment with
thiol-containing maytansinoids to produce disulfide linked conjugates.
Alternatively, in the case of the aryldithio-maytansinoids, the
formation of the cell binding conjugate is effected by direct displacement
of the aryl-thiol of the maytansinoid by sulfhydryl groups previously
introduced into antibody molecules. Conjugates containing 1 to 10
maytansinoid drugs linked via a disulfide bridge are readily prepared
by either method.
More specifically, a solution of the dithiopyridyl modified antibody
at a concentration of 2.5 mg/ml in 0.05 M potassium phosphate buffer
and 0.05 M sodium chloride, at pH 6.5 containing 2 mM EDTA is treated
with the thiol-containing maytansinoid (1.7 molar equivalent/dithiopyridyl
group). The release of pyridine-2-thione from the modified antibody
is monitored spectrophotometrically at 343 nm. The reaction is allowed
to proceed up to 18 hours. The antibody-maytansinoid conjugate is
purified and freed of unreacted drug and other low molecular weight
material by gel filtration through a column of Sephacryl S-300.
The number of maytansinoids bound per antibody molecule can be determined
by measuring the ratio of the absorbance at 252 nm and 280 nm. An
average of 1-10 maytansinoid molecules/antibody molecule can be
linked via disulfide bonds by this method.
Antibody-maytansinoid conjugates with non-cleavable links can also
be prepared. The antibody can be modified with crosslinking reagents
such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS), sulfo-MBS or succinimidyl-iodoacetate, as described in the
literature, to introduce 1-10 reactive groups (Yoshitake et al,
Eur. J. Biochem., 101:395-399 (1979); Hashida et al, J. Applied
Biochem., 56-63 (1984); and Liu et al, Biochem., 18:690-697 (1979)).
The modified antibody is then reacted with the thiol-containing
maytansinoid derivative to produce a conjugate. The conjugate can
be purified by gel filtration through a Sephacryl S-300 column.
The modified antibodies are treated with the thiol-containing maytansinoid
(1.25 molar equivalent/maleimido group). The mixtures are incubated
overnight at about 4.degree. C. The antibody-maytansinoid conjugates
are purified by gel filtration through a Sephadex G-25 column. Typically,
an average of 1-10 maytansinoids per antibody are linked.
A preferred method is to modify antibodies with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC) to introduce maleimido groups followed by reaction of the
modified antibody with a thiol-containing maytansinoid to give a
thioether-linked conjugate. Again conjugates with 1 to 10 drug molecules
per antibody molecule result.
As described herein, the present invention is based on the unexpected
discovery that the use of at least one immunoconjugate and at least
one chemotherapeutic agent produces superior results in treating
cancer. Any chemotherapeutic agent known in the art can be used
in combination with the immunoconjugate of the present invention
to achieve the unexpectedly superior results described and demonstrated
herein. Preferably, the chemotherapeutic agent is a taxane compound,
a compound that acts via a taxane mechanism, a platinum compound,
an epidophyllotoxin compound, a camptothecin compound, and/or any
combination thereof. As is known in the art, platinum compounds
and epidophyllotoxin compounds are generally used together for treating
cancer.
In one embodiment, the present invention provides methods of treating
cancer and/or modulating the growth of selected cell populations
(e.g., cancer cells) by administering at least one immunoconjugate
and at least one taxane compound. In another embodiment, the present
invention provides methods of treating cancer and/or modulating
the growth of selected cell populations (e.g., cancer cells) by
administering at least one immunoconjugate and at least one compound
that acts via a taxane mechanism. In another embodiment, the present
invention provides methods of treating cancer and/or modulating
the growth of selected cell populations (e.g., cancer cells) by
administering at least one immunoconjugate, and at least one platinum
compound. In another embodiment, the present invention provides
methods of treating cancer and/or modulating the growth of selected
cell populations (e.g., cancer cells) by administering at least
one immunoconjugate, at least one platinum compound, and at least
one epidophyllotoxin compound. In another embodiment, the present
invention provides methods of treating cancer and/or modulating
the growth of selected cell populations (e.g., cancer cells) by
administering at least one immunoconjugate and at least one camptothecin
compound. In another embodiment, the present invention provides
methods of treating cancer and/or modulating the growth of selected
cell populations (e.g., cancer cells) by administering at least
one immunoconjugate and at least one compound that is capable of
inhibiting DNA topoisomerase I. In yet another embodiment, the present
invention provides methods of treating cancer and/or modulating
the growth of selected cell populations (e.g., cancer cells) by
administering at least one immunoconjugate, at least one taxane
compound, and at least one platinum compound. In yet another embodiment,
the present invention provides methods of treating cancer and/or
modulating the growth of selected cell populations (e.g., cancer
cells) by administering at least one immunoconjugate, at least one
taxane compound, at least one platinum compound, and, optionally,
at least one epidophyllotoxin compound. In yet another embodiment,
the present invention provides methods of treating cancer and/or
modulating the growth of selected cell populations (e.g., cancer
cells) by administering at least one immunoconjugate, at least one
camptothecin compound, at least one platinum compound and, optionally,
at least one epidophyllotoxin compound. In yet another embodiment,
the present invention provides methods of treating cancer and/or
modulating the growth of selected cell populations (e.g., cancer
cells) by administering at least one immunoconjugate, at least one
compound that acts via a taxane mechanism and at least one camptothecin
compound. One skilled in the art will appreciate that the methods
described in the present invention encompass administering at least
one immunoconjugate with one or more chemotherapeutic agents selected
from the group consisting of taxane compounds, compounds that act
through a taxane mechanism, platinum compounds, epidophyllotoxin
compounds, camptothecin compounds and compounds that can inhibit
DNA topoisomerase I. In the methods of the present invention, the
immunoconjugate and chemotherapeutic agent can be administered simultaneously,
about the same time, or at different times, or can be components
of a single composition.
Taxane compounds prevent the growth of cancer cells by affecting
cell structures called microtubules, which play an important role
in cell functions. In normal cell growth, microtubules are formed
when a cell starts dividing. Once the cell stops dividing, the microtubules
are broken down or destroyed. Taxane compounds stop the microtubules
from breaking down, such that the cancer cells become clogged with
microtubules so that they cannot grow and divide.
Taxane compounds are known in the art and include, for example,
paclitaxel (available as TAXOL.RTM. from Bristol-Myers Squibb, Princeton,
N.J.), docetaxel (available as TAXOTERE.RTM. from Aventis), and
the like. Other taxane compounds that become approved by the U.S.
Food and Drug Administration (FDA) or foreign counterparts thereof
are also preferred for use in the methods and compositions of the
present invention. Other taxane compounds that can be used in the
present invention include those described, for example, in 10th
NCI-EORTC Symposium on New Drugs in Cancer Therapy, Amsterdam, page
100, Nos. 382 and 383 (Jun. 16-19, 1998); and U.S. Pat. Nos. 4,814,470,
5,721,268, 5,714,513, 5,739,362, 5,728,850, 5,728,725, 5,710,287,
5,637,484, 5,629,433, 5,580,899, 5,549,830, 5,523,219, 5,281,727,
5,939,567, 5,703,117, 5,480,639, 5,250,683, 5,700,669, 5,665,576,
5,618,538, 5,279,953, 5,243,045, 5,654,447, 5,527,702, 5,415,869,
5,279,949, 5,739,016, 5,698,582, 5,478,736, 5,227,400, 5,516,676,
5,489,601, 5,908,759, 5,760,251, 5,578,739, 5,547,981, 5,547,866,
5,344,775, 5,338,872, 5,717,115, 5,620,875, 5,284,865, 5,284,864,
5,254,703, 5,202,448, 5,723,634, 5,654,448, 5,466,834, 5,430,160,
5,407,816, 5,283,253, 5,719,177, 5,670,663, 5,616,330, 5,561,055,
5,449,790, 5,405,972, 5,380,916, 5,912,263, 8,808,113, 5,703,247,
5,618,952, 5,367,086, 5,200,534, 5,763,628, 5,705,508, 5,622,986,
5,476,954, 5,475,120, 5,412,116, 5,916,783, 5,879,929, 5,861,515,
5,795,909, 5,760,252, 5,637,732, 5,614,645, 5,599,820, 5,310,672,
RE 34,277, U.S. Pat. Nos. 5,877,205, 5,808,102, 5,766,635, 5,760,219,
5,750,561, 5,637,723, 5,475,011, 5,256,801, 5,900,367, 5,869,680,
5,728,687, 5,565,478, 5,411,984, 5,334,732, 5,919,815, 5,912,264,
5,773,464, 5,670,673, 5,635,531, 5,508,447, 5,919,816, 5,908,835,
5,902,822, 5,880,131, 5,861,302, 5,850,032, 5,824,701, 5,817,867,
5,811,292, 5,763,477, 5,756,776, 5,686,623, 5,646,176, 5,621,121,
5,616,739, 5,602,272, 5,587,489, 5,567,614, 5,498,738, 5,438,072,
5,403,858, 5,356,928, 5,274,137, 5,019,504, 5,917,062, 5,892,063,
5,840,930, 5,840,900, 5,821,263, 5,756,301, 5,750,738, 5,750,562,
5,726,318, 5,714,512, 5,686,298, 5,684,168, 5,681,970, 5,679,807,
5,648,505, 5,641,803, 5,606,083, 5,599,942, 5,420,337, 5,407,674,
5,399,726, 5,322,779, 4,924,011, 5,939,566, 5,939,561, 5,935,955,
5,919,455, 5,854,278, 5,854,178, 5,840,929, 5,840,748, 5,821,363,
5,817,321, 5,814,658, 5,807,888, 5,792,877, 5,780,653, 5,770,745,
5,767,282, 5,739,359, 5,726,346, 5,717,103, 5,710,099, 5,698,712,
5,683,715, 5,677,462, 5,670,653, 5,665,761, 5,654,328, 5,643,575,
5,621,001, 5,608,102, 5,606,068, 5,587,493, 5,580,998, 5,580,997,
5,576,450, 5,574,156, 5,571,917, 5,556,878, 5,550,261, 5,539,103,
5,532,388, 5,470,866, 5,453,520, 5,384,399, 5,364,947, 5,350,866,
5,336,684, 5,296,506, 5,290,957, 5,274,124, 5,264,591, 5,250,722,
5,229,526, 5,175,315, 5,136,060, 5,015,744, 4,924,012, 6,118,011,
6,114,365, 6,107,332, 6,072,060, 6,066,749, 6,066,747, 6,051,724,
6,051,600, 6,048,990, 6,040,330, 6,030,818, 6,028,205, 6,025,516,
6,025,385, 6,018,073, 6,017,935, 6,011,056, 6,005,138, 6,005,138,
6,005,120, 6,002,023, 5,998,656, 5,994,576, 5,981,564, 5,977,386,
5,977,163, 5,965,739, 5,955,489, 5,939,567, 5,939,566, 5,919,815,
5,912,264, 5,912,263, 5,908,835, and 5,902,822, the disclosures
of which are incorporated by reference herein in their entirety.
Other compounds that can be used in the invention are those that
act through a taxane mechanism. Compounds that act through a taxane
mechanism include compounds that have the ability to exert microtubule-stabilizing
effects and cytotoxic activity against rapidly proliferating cells,
such as tumor cells or other hyperproliferative cellular diseases.
Such compounds include, for example, epothilone compounds, such
as, for example, epothilone A, B, C, D, E and F, and derivatives
thereof. Other compounds that act through a taxane mechanism (e.g.,
epothilone compounds) that become approved by the FDA or foreign
counterparts thereof are also preferred for use in the methods and
compositions of the present invention. Epothilone compounds and
derivatives thereof are known in the art and are described, for
example, in U.S. Pat. Nos. 6,121,029, 6,117,659, 6,096,757, 6,043,372,
5,969,145, and 5,886,026; and WO 97/19086, WO 98/08849, WO 98/22461,
WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02514, WO 99/03848,
WO 99/07692, WO 99/27890, and WO 99/28324, the disclosures of which
are incorporated herein by reference in their entirety.
Other compounds that can be used in the invention include platinum
compounds such as, for example, cisplatin (available as PLATINOL.RTM.
from Bristol-Myers Squibb, Princeton, N.J.), carboplatin (available
as PARAPLATIN.RTM. from Bristol-Myers Squibb, Princeton, N.J.),
oxaliplatin (available as ELOXATINE.RTM. from Sanofi, France), iproplatin,
ormaplatin, tetraplatin, and the like. Other platinum compounds
that become approved by the FDA or foreign counterparts thereof
are also preferred for use in the methods and compositions of the
present invention. Platinum compounds that are useful in treating
cancer are known in the art and are described, for example in U.S.
Pat. Nos. 4,994,591, 4,906,646, 5,902,610, 5,053,226, 5,789,000,
5,871,710, 5,561,042, 5,604,095, 5,849,790, 5,705,334, 4,863,902,
4,767,611, 5,670,621, 5,384,127, 5,084,002, 4,937,262, 5,882,941,
5,879,917, 5,434,256, 5,393,909, 5,117,022, 5,041,578, 5,843,475,
5,633,243, 5,178,876, 5,866,169, 5,846,725, 5,646,011, 5,527,905,
5,844,001, 5,832,931, 5,676,978, 5,604,112, 5,562,925, 5,541,232,
5,426,203, 5,288,887, 5,041,581, 5,002,755, 4,946,954, 4,921,963,
4,895,936, 4,686,104, 4,594,238, 4,581,224, 4,250,189, 5,829,448,
5,690,905, 5,665,771, 5,648,384, 5,633,016, 5,460,785, 5,395,947,
5,256,653, 5,132,323, 5,130,308, 5,106,974, 5,059,591, 5,026,694,
4,992,553, 4,956,459, 4,956,454, 4,952,676, 4,895,935, 4,892,735,
4,843,161, 4,760,156, 4,739,087, 4,720,504, 4,544,759, 4,515,954,
4,466,924, 4,462,998, 4,457,926, 4,428,943, 4,325,950, 4,291,027,
4,291,023, 4,284,579, 4,271,085, 4,234,500, 4,234,499, 4,200,583,
4,175,133, 4,169,846, 5,922,741, 5,922,674, 5,922,302, 5,919,126,
5,910,102, 5,876,693, 5,871,923, 5,866,617, 5,866,615, 5,866,593,
5,864,024, 5,861,139, 5,859,034, 5,855,867, 5,855,748, 5,849,770,
5,843,993, 5,824,664, 5,821,453, 5,811,119, 5,798,373, 5,786,354,
5,780,478, 5,780,477, 5,776,925, 5,770,593, 5,770,222, 5,747,534,
5,739,144, 5,738,838, 5,736,156, 5,736,119, 5,723,460, 5,697,902,
5,693,659, 5,688,773, 5,674,880, 5,670,627, 5,665,343, 5,654,287,
5,648,362, 5,646,124, 5,641,627, 5,635,218, 5,633,257, 5,632,982,
5,622,977, 5,622,686, 5,618,393, 5,616,613, 5,612,019, 5,608,070,
5,595,878, 5,585,112, 5,580,888, 5,580,575, 5,578,590, 5,575,749,
5,573,761, 5,571,153, 5,563,132, 5,561,136, 5,556,609, 5,552,156,
5,547,982, 5,542,935, 5,525,338, 5,519,155, 5,498,227, 5,491,147,
5,482,698, 5,469,854, 5,455,270, 5,443,816, 5,415,869, 5,409,915,
5,409,893, 5,409,677, 5,399,694, 5,399,363, 5,380,897, 5,340,565,
5,324,591, 5,318,962, 5,302,587, 5,292,497, 5,272,056, 5,258,376,
5,238,955, 5,237,064, 5,213,788, 5,204,107, 5,194,645, 5,182,368,
5,130,145, 5,116,831, 5,106,858, 5,100,877, 5,087,712, 5,087,618,
5,078,137, 5,057,302, 5,049,396, 5,034,552, 5,028,726, 5,011,846,
5,010,103, 4,985,416, 4,970,324, 4,936,465, 4,931,553, 4,927,966,
4,912,072, 4,906,755, 4,897,384, 4,880,832, 4,871,528, 4,822,892,
4,783,452, 4,767,874, 4,760,155, 4,687,780, 4,671,958, 4,665,210,
4,645,661, 4,599,352, 4,594,418, 4,593,034, 4,587,331, 4,575,550,
4,562,275, 4,550,169, 4,482,569, 4,431,666, 4,419,351, 4,407,300,
4,394,319, 4,335,087, 4,329,299, 4,322,391, 4,302,446, 4,287,187,
4,278,660, 4,273,755, 4,255,417, 4,255,347, 4,248,840, 4,225,529,
4,207,416, 4,203,912, 4,177,263, 4,151,185, 4,140,707, 4,137,248,
4,115,418, 4,079,121, 4,075,307, 3,983,118, 3,870,719, RE 33,071,
U.S. Pat. Nos. 6,087,392, 6,077,864, 5,998,648, and 5,902,610, the
disclosures of which are incorporated by reference herein in their
entirety.
As is known in the art, platinum compounds are preferably used
in combination with at least one epipodophyllotoxin compound, including,
for example, etoposide (also known as VP-16) (available as VEPESID.RTM.
from Bristol-Myers Squibb, Princeton, N.J.), teniposide (also known
as VM-26) (available as VUMON.RTM. from Bristol-Myers Squibb, Princeton,
N.J.), and the like. Other epipodophyllotoxin compounds that become
approved by the FDA or foreign counterparts thereof are also preferred
for use in the methods and compositions of the present invention.
Other epipodophyllotoxin compounds that can be used in the present
invention include those described, for example, in U.S. Pat. Nos.
3,524,844, 5,643,885, 5,066,645, 5,081,234, 5,891,724, 5,489,698,
5,821,348, 5,571,914, 4,997,931, 4,547,567, 5,536,847, 5,326,753,
5,120,862, 5,011,948, 4,895,727, 4,795,819, 4,644,072, 5,688,926,
5,676,978, 5,660,827, 5,395,610, 5,346,897, 5,208,238, 5,190,949,
5,086,182, 4,965,348, 4,958,010, 4,874,851, 4,866,189, 4,853,467,
4,728,740, 4,716,221, 5,935,955, 5,863,538, 5,855,866, 5,776,427,
5,747,520, 5,739,114, 5,622,960, 5,606,060, 5,605,826, 5,541,223,
5,459,248, 5,455,161, 5,364,843, 5,300,500, 5,041,424, 5,036,055,
5,034,380, 4,935,504, 4,916,217, 4,912,204, 4,904,768, 4,900,814,
4,888,419, 4,567,253, RE 35,524, U.S. Pat. Nos. 6,107,284, 6,063,801,
and 6,051,230, the disclosures of which are incorporated herein
by reference in their entirety.
Other compounds that can be used in the present invention include
camptothecin compounds. Camptothecin compounds are capable of inhibiting
DNA topoisomerase I. Camptothecin compounds include camptothecin,
derivatives of camptothecin and analogs of camptothecin. Camptothecin
compounds are known in the art and include, for example, camptothecin,
topotecan (available as HYCAMTIN.RTM. from SmithKline Beecham Pharmaceuticals),
CPT-11 (also called irinotecan), 9-aminocamptothecin, and the like.
Other camptothecin compounds (or other compounds that can inhibit
DNA topoisomerase I) that become approved by the FDA or foreign
counterparts thereof are also preferred for use in the methods and
compositions of the present invention. Other camptothecin compounds
that can be used in the present invention include those described
in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem.,
23:554 (1980); J. Med. Chem., 30:1774 (1987); European Patent Application
Nos. 0 418 099, 0 088 642, and 0 074 770; and U.S. Pat. Nos. 5,633,016,
5,004,758, 4,604,463, 4,473,692, 4,545,880, 4,513,138, 4,399,276,
6,121,451, 6,121,278, 6,121,277, 6,121,275, 6,121,263, 6,107,486,
6,100,273, 6,096,336, 6,093,721, 6,063,801, 6,046,209, 6,040,313,
6,034,243, 6,028,078, 5,998,426, 5,990,120, 5,985,888, 5,981,542,
5,972,955, 5,968,943, 5,958,937, 5,955,467, 5,948,797, 5,935,967,
5,932,709, 5,932,588, 5,922,877, 5,916,897, 5,916,896, 5,910,491,
5,900,419, 5,892,043, 5,889,017, 5,880,133, 5,859,023, 5,859,022,
5,856,333, 5,843,954, 5,840,899, 5,837,673, 5,834,012, 5,807,874,
5,801,167, 5,786,344, 5,773,522, 5,767,142, 5,744,605, 5,734,056,
5,731,316, 5,726,181, 5,677,286, 5,674,874, 5,674,873, 5,670,500,
5,633,177, 5,652,244, 5,646,159, 5,633,260, 5,614,628, 5,604,233,
5,602,141, 5,597,829, 5,559,235, 5,552,154, 5,541,327, 5,525,731,
5,496,952, 5,475,108, 5,468,859, 5,468,754, 5,459,269, 5,447,936,
5,446,047, 5,401,747, 5,391,745, 5,364,858, 5,340,817, 5,244,903,
5,227,380, 5,200,524, 5,191,082, 5,180,722, 5,162,532, 5,122,606,
5,122,526, 5,106,742, 5,061,800, 5,053,512, 5,049,668, 5,004,758,
4,981,968, 4,943,579, 4,939,255, 4,914,205, 4,894,456, RE 32,518,
U.S. Pat. Nos. 4,604,463, 4,513,138, 4,473,692, 4,399,282, 4,399,276,
and 4,031,098, the disclosures of which are incorporated by reference
herein in their entirety.
The immunoconjugates and chemotherapeutic agents of the present
invention can be administered in vitro, in vivo and/or ex vivo to
treat patients and/or to modulate the growth of selected cell populations
including, for example, cancer of the lung, breast, colon, prostate,
kidney, pancreas, brain, bones, ovary, testes, and lymphatic organs;
autoimmune diseases, such as systemic lupus, rheumatoid arthritis,
and multiple sclerosis; graft rejections, such as renal transplant
rejection, liver transplant rejection, lung transplant rejection,
cardiac transplant rejection, and bone marrow transplant rejection;
graft versus host disease; viral infections, such as CMV infection,
HIV infection, and AIDS; and parasite infections, such as giardiasis,
amoebiasis, schistosomiasis, and the like. Preferably, the immunoconjugates
and chemotherapeutic agents of the invention are administered in
vitro, in vivo and/or ex vivo to treat cancer in a patient and/or
to modulate the growth of cancer cells, including, for example,
cancer of the lung, breast, colon, prostate, kidney, pancreas, brain,
bones, ovary, testes, and lymphatic organs; more preferably lung
cancer or colon cancer. In a most preferred embodiment, the lung
cancer is small cell lung cancer (SCLC).
"Modulating the growth of selected cell populations"
includes inhibiting the proliferation of selected cell populations
(e.g., SCLC cells, NCI N417 cells, SW-2 cells, NCI-H441 cells, HT-29
cells, and the like) from dividing to produce more cells; reducing
the rate of increase in cell division as compared, for example,
to untreated cells; killing selected cell populations; and/or preventing
selected cell populations (such as cancer cells) from metastasizing.
The growth of selected cell populations can be modulated in vitro,
in vivo or ex vivo.
In the methods of the present invention, the immunoconjugates and
chemotherapeutic agents can be administered in vitro, in vivo, or
ex vivo separately or as components of the same composition. The
immunoconjugates and chemotherapeutic agents can be used with suitable
pharmaceutically acceptable carriers, diluents, and/or excipients,
which are well known, and can be determined by one of skill in the
art as the clinical situation warrants. Examples of suitable carriers,
diluents and/or excipients include: (1) Dulbecco's phosphate buffered
saline, pH about 6.5, which would contain about 1 mg/ml to 25 mg/ml
human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5%
(w/v) dextrose.
The compounds and compositions described herein may be administered
in appropriate form, preferably parenterally, more preferably intravenously.
For parenteral administration, the compounds or compositions can
be aqueous or nonaqueous sterile solutions, suspensions or emulsions.
Propylene glycol, vegetable oils and injectable organic esters,
such as ethyl oleate, can be used as the solvent or vehicle. The
compositions can also contain adjuvants, emulsifiers or dispersants.
The compositions can also be in the form of sterile solid compositions
which can be dissolved or dispersed in sterile water or any other
injectable sterile medium.
The "therapeutically effective amount" of the chemotherapeutic
agents and immunoconjugates described herein refers to the dosage
regimen for inhibiting the proliferation of selected cell populations
and/or treating a patient's disease, and is selected in accordance
with a variety of factors, including the age, weight, sex, diet
and medical condition of the patient, the severity of the disease,
the route of administration, and pharmacological considerations,
such as the activity, efficacy, pharmacokinetic and toxicology profiles
of the particular compound used. The "therapeutically effective
amount" can also be determined by reference to standard medical
texts, such as the Physicians Desk Reference 1999 (53rd Ed.), the
disclosure of which is incorporated by reference herein in its entirety.
The patient is preferably an animal, more preferably a mammal, most
preferably a human. The patient can be male or female, and can be
an infant, child or adult.
Examples of suitable protocols of immunoconjugate administration
are as follows. Immunoconjugates can be given daily for about 5
days either as an i.v. bolus each day for about 5 days, or as a
continuous infusion for about 5 days. Alternatively, they can be
administered once a week for six weeks or longer. As another alternative,
they can be administered once every two or three weeks. Bolus doses
are given in about 50 to about 400 ml of normal saline to which
about 5 to about 10 ml of human serum albumin can be added. Continuous
infusions are given in about 250 to about 500 ml of normal saline,
to which about 25 to about 50 ml of human serum albumin can be added,
per 24 hour period. Dosages will be about 10 .mu.g to about 1000
mg/kg per person, i.v. (range of about 100 ng to about 10 mg/kg).
About one to about four weeks after treatment, the patient can receive
a second course of treatment. Specific clinical protocols with regard
to route of administration, excipients, diluents, dosages, and times
can be determined by the skilled artisan as the clinical situation
warrants.
The present invention also provides pharmaceutical kits comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compounds and/or compositions of the present
invention, including, one or more immunoconjugates and one or more
chemotherapeutic agents. Such kits can also include, for example,
other compounds and/or compositions, a device(s) for administering
the compounds and/or compositions, and written instructions in a
form prescribed by a governmental agency regulating the manufacture,
use or sale of pharmaceuticals or biological products.
EXAMPLES
The following examples are for purposes of illustration only, and
are not intended to limit the scope of the invention or claims.
Example 1
The maytansinoid DM1 was conjugated to the humanized monoclonal
antibody N901.
Ansamitocin P-3, provided by Takeda (Osaka, Japan) was converted
to the disulfide-containing maytansinoid DM1, as described herein
and in U.S. Pat. No. 5,208,020, the disclosure of which is incorporated
by reference herein in its entirety.
##STR00007##
##STR00008##
Humanized N901 is an antibody that binds to the CD56 antigen expressed
on all human small cell lung cancer (SCLC) tissues, neuroblastomas
and carcinoid tumors (Doria et al, Cancer 62:1839-1845 (1988); Kibbelaar
et al, Eur. J. Cancer, 27:431-435 (1991);Rygaard et al, Br. J. Cancer,
65:573-577 (1992)).
Humanized N901 was modified with N-succinimidyl-4-[2-pyridyldithio]-pentanoate
(SPP) to introduce dithiopyridyl groups. Alternatively, N-succinimidyl-3-[2-pyridyldithio]-propanoate
(SPDP) can be used. The antibody (8 mg/mL) in 50 mM potassium phosphate
buffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM) was treated
with SPP (5.6 molar equivalents in ethanol). The final ethanol concentration
was 5% (v/v). After 90 minutes at ambient temperature, the reaction
mixture was gel filtered through a Sephadex G25 column equilibrated
in the above buffer. Antibody-containing fractions were pooled and
the degree of modification was determined by treating a sample with
dithiothreitol and measuring the change in absorbance at 343 nm
(release of 2-mercaptopyridine with .epsilon..sub.343 nm=8,080 M.sup.-1).
Recovery of the antibody was typically 90%, with 4.8 pyridyldithio
groups linked per antibody molecule.
The modified antibody was diluted with 50 mM potassium phosphate
buffer, 6.5, containing NaCl (50 mM) and EDTA (2 mM) to a final
concentration of 2.5 mg/mL. DM1 (1.7 eq.) in dimethylacetamide (DMA,
3% v/v in final reaction mixture) was then added to the modified
antibody solution. The reaction proceeded at ambient temperature
under argon for 20 hours.
The reaction mixture was then loaded on to a Sephacryl S300 gel
filtration column equilibrated with phosphate-buffered saline (PBS,
pH 6.5). The major peak comprised monomeric hu901-DM1. The number
of DM1 drug molecules linked per antibody molecule was determined
by measuring the absorbance at 252 nm and 280 nm and found to be
3.9 DM1 molecules per antibody molecule.
Example 2
In this experiment, a low, non-curative dose of huN901-DM1 was
used with an optimal does of paclitaxel (Sigma Chemical Co., St.
Louis, Mo.). SCID mice (7 animals per group) were inoculated subcutaneously
with NCI N417 cells (8.times.10.sup.6 cells/animal). After the tumors
were well-established (average tumor size was approximately 100
mm.sup.3), one group of animals was treated with huN901-DM1 at a
DM1 dose of 75 .mu.g/kg/d.times.5, administered i.v. everyday. A
second group of animals was treated with paclitaxel at a dose of
10 mg/kg/d.times.5, administered by i.p. everyday. A third group
of animals was treated with huN901-DM1 and paclitaxel using the
same dose and schedule used for the individual agents. A fourth,
control group of animals was left untreated. Tumor size was measured
as described by Liu et al, Proc. Natl. Acad. Sci., 93:8618-8623
(1996). Animals were also monitored for weight loss as an indicator
of signs of toxicity.
The results of the experiment are shown in FIG. 5. In the control
group of animals, the tumors grew rapidly to a size of about 900
mm.sup.3 by Day 28 post-tumor inoculation. In animals treated with
either huN901-DM1 or paclitaxel, there was a modest anti-tumor effect
with a tumor growth delay of 4 days in each case. In the animals
treated with huN901-DM1 and paclitaxel, the tumors disappeared with
complete regression lasting 58 days. Importantly, there was no evidence
of toxicity in the animals. These data demonstrate that treatment
with huN901-DM1 and paclitaxel has an unexpectedly superior (e.g.,
synergistic) anti-tumor effect.
Example 3
In this experiment, a low, non-curative dose of huN901-DM1 was
used with an optimal dose of cisplatin (Sigma Chemical Co., St.
Louis, Mo.) and etoposide (Sigma Chemical Co., St. Louis, Mo.).
SCID mice (7 animals per group) were inoculated subcutaneously with
NCI N417 cells (8.times.10.sup.6 cells/animal). After the tumors
were well-established (average tumor size was approximately 100
mm.sup.3), one group of animals was treated with huN901-DM1 at a
DM1 dose of 75 .mu.g/kg/d.times.5, administered i.v. everyday. A
second group of animals was treated with cisplatin (at a dose of
2 mg/kg/d.times.3, administered by i.v. ever other day) and etoposide
(at a dose of 8 mg/kg/d.times.3, administered every other day).
A third group of animals was treated with huN901-DM1, cisplatin
and etoposide using the same dose and schedule used for a the individual
agents. A fourth, control group of animals was left untreated. Tumor
size was measured as described by Liu et al, Proc. Natl. Acad. Sci.,
93:8618-8623 (1996). Animals were also monitored for weight loss
as an indicator of signs of toxicity.
The results of the experiment are shown in FIG. 6. In the control
group of animals, the tumors grew rapidly to a size of about 900
mm.sup.3 by Day 28 post-tumor inoculation. In animals treated with
either huN901-DM1 or cisplatin and etoposide, there was a modest
anti-tumor effect with a tumor growth delay of 4 days in each case.
In the animals treated with huN901-DM1, cisplatin and etoposide,
there was a tumor growth delay of 12 days, which is 50% longer than
what one would expect for an additive anti-tumor effect of the individual
compounds. Importantly, there was no evidence of toxicity in the
animals. These data demonstrate that treatment with huN901-DM1,
cisplatin, and etoposide has an unexpectedly superior (e.g., synergistic)
anti-tumor effect.
Example 4
The anti-tumor effect of a combination of a low dose of huN901-DM1
and docetaxel (available as TAXOTERE.RTM. from Aventis) was evaluated
in an established subcutaneous xenograft model of small cell lung
cancer. SCID mice (24 animals) were inoculated with human small
cell lung cancer SW-2 cells (8.times.10.sup.6 cells/animal) injected
subcutaneously into the right flank of the mice. When the tumors
reached about 100 mm.sup.3 in size (10 days after tumor cell inoculation),
the mice were randomly divided into four groups (6 animals per group).
The first group of mice was treated with docetaxel (5 mg/kg.times.5,
q2d) administered i.v. A second group of animals was treated with
huN901-DM1 (DM1 dose of 75 .mu.g/kg.times.5, qd) administered i.v.
The third group of mice received a combination of docetaxel and
huN901-DM1, using the same doses and schedules as in groups 1 and
2. A control group of animals received phosphate-buffered saline
(PBS) using the same schedule as the animals in group 2. Tumor growth
was monitored by measuring tumor size twice per week. Tumor size
was calculated with the formula: length.times.width.times.height.times.1/2.
The change in tumor size is shown in FIG. 7. In the control group
of animals, tumors grew rapidly to about 1000 mm.sup.3 in 26 days.
Treatment with docetaxel alone, or a low dose of huN901-DM1 alone,
resulted in tumor growth delays of 8 days and 20 days, respectively.
In contrast, treatment with the combination of docetaxel and huN901-DM1
showed a remarkable anti-tumor effect resulting in complete tumor
regression in all the treated animals. In 3 out of 6 animals in
this treatment group, the tumor was eradicated--resulting in cures
lasting greater than 200 days. In the remaining 3 animals in this
group, there was a tumor growth delay of 52 days, which is 24 days
longer than the calculated additive effect. Thus, the combination
of docetaxel and huN901-DM1 shows an unexpectedly superior (e.g.,
synergistic) anti-tumor effect in this human SCLC xenograft model.
Example 5
The anti-tumor effect of a combination of a low dose of huN901-DM1
and topotecan (available as HYCAMTIN.RTM. from SmithKline Beecham
Pharmaceuticals), one of the approved drugs for the treatment of
small cell lung cancer (SCLC) in humans, was evaluated in an established
subcutaneous xenograft model of SCLC. SCID mice (24 animals) were
inoculated with human small cell lung cancer SW-2 cells (8.times.10.sup.6
cells/animal) injected subcutaneously into the right flank of the
mice. When the tumors reached about 80 mm.sup.3 in size, the mice
were randomly divided into four groups (6 animals per group). The
first group of mice was treated with topotecan (1.4 mg/kg.times.5,
qd) administered i.v. A second group of animals was treated with
huN901-DM1 (DM1 dose of 100 .mu.g/kg.times.5, qd) administered i.v.
The third group of mice received a combination of topotecan and
huN901-DM1, using the same doses and schedules as in groups 1 and
2. A control group of animals received phosphate-buffered saline
(PBS) using the same schedule as the animals in group 2. Tumor growth
was monitored by measuring tumor size twice per week. Tumor size
was calculated using the formula: length.times.width.times.height.times.1/2.
The change in tumor size is shown in FIG. 8. In the control group
of animals, tumors grew to about 800 mm.sup.3 in 44 days. Treatment
with topotecan alone resulted in tumor growth delays of 12 days.
Treatment with a low dose of huN901-DM1 alone resulted in a tumor-growth
delay of 34 days in 3 out of 6 animals. The remaining 3 animals
in this group had complete tumor regressions. Treatment with the
combination of topotecan and huN901-DM1 showed a remarkable anti-tumor
effect resulting in complete tumor regression in 5 out of the 6
treated animals. These animals were tumor-free on day 78, the last
measurement point. Thus, the combination of topotecan and huN901-DM1
is unexpectedly superior (e.g., synergistic) when compared to the
single agents in this human SCLC xenograft model.
Example 6
The anti-tumor effect of a combination of a low dose of huC242-DM1
(manufactured by ImmunoGen, Inc. following the procedures described
in U.S. Pat. No. 5,208,020, the disclosure of which is incorporated
by reference herein in its entirety, and also described in Example
1) and paclitaxel (Sigma Chemical Co., St. Louis, Mo.) was evaluated
in an established subcutaneous xenograft model of non-small cell
lung cancer. SCID mice (24 animals) were inoculated with human lung
adenocarcinoma NCI-H441 cells (8.times.10.sup.6 cells/animal), injected
subcutaneously into the right flank of the mice. When the tumors
reached about 125 mm.sup.3 in size (4 days after tumor cell inoculation),
the mice were randomly divided into four groups (6 animals per group).
The first group of mice was treated with paclitaxel (15 mg/kg.times.5,
q2d) administered i.p. A second group of animals was treated with
huC242-DM1 (DM1 dose of 75 .mu.g/kg.times.5, qd) administered i.v.
The third group of mice received a combination of paclitaxel and
huC242-DM1, using the same doses and schedules as in groups 1 and
2. In the combination group, the huC242-DM1 conjugate was administered
2 hours after the paclitaxel. A control group of animals received
phosphate-buffered saline (PBS) using the same schedule as the animals
in group 2. Tumor growth was monitored by measuring tumor size twice
per week. Tumor size was calculated using the formula: length.times.width.times.height.times.1/2.
The change in tumor size is shown in FIG. 9. In the control group
of animals, tumors grew rapidly to about 1000 mm.sup.3 in 32 days.
Treatment with paclitaxel alone, resulted in a small tumor growth
delay of 4 days. Treatment with huC242-DM1 resulted in shrinkage
of the tumor, but none of the 6 treated animals showed complete
tumor regression. Treatment with a combination of paclitaxel and
huC242-DM1 showed a greater anti-tumor effect resulting in complete
tumor regression, with 3 out of the 6 animals showing no evidence
of tumor. The remaining 3 animals in this group showed a significant
shrinkage in the tumor. Thus, the combination of paclitaxel and
huC242-DM1 is unexpectedly superior (e.g., synergistic) in this
human SCLC lung adenocarcinoma xenograft model.
Example 7
The anti-tumor effect of a combination of a low dose of huC242-DM1
(manufactured by ImmunoGen, Inc. following the methods described
in U.S. Pat. No. 5,208,020, the disclosure of which is incorporated
by reference herein in its entirety, and also described in Example
1) and paclitaxel (Sigma Chemical Co., St. Louis, Mo.) was evaluated
in an established subcutaneous xenograft model of non-small cell
lung cancer. SCID mice (32 animals) were inoculated with human colon
cancer HT-29 cells (8.times.10.sup.6 cells/animal), injected subcutaneously
into the right flank of the mice. When the tumors reached about
80 mm.sup.3 in size, the mice were randomly divided into four groups
(8 animals per group). The first group of mice was treated with
CPT-11 (50 mg/kg.times.2, q3d) administered i.v. The second group
of animals was treated with murine C242-DM1 (DM1 dose of 75 .mu.g/kg.times.5,
qd) administered i.v. The third group of mice received a combination
of CPT-11 and C242-DM1, using the same doses and schedules as in
groups 1 and 2. A control group of animals received phosphate-buffered
saline (PBS) using the same schedule as the animals in group 2.
Tumor growth was monitored by measuring tumor size twice per week.
Tumor size was calculated using the formula: length.times.width.times.height.times.1/2.
The change in tumor size is shown in FIG. 10. In the control group
of animals, tumors grew rapidly to about 1000 mm.sup.3 in 31 days.
Treatment with CPT-11 alone resulted in a small tumor growth delay
of 6 days. Treatment with C242-DM1 resulted in a delay in tumor
growth of 22 days. Treatment with a combination of CPT-11 and C242-DM1
showed an unexpectedly superior anti-tumor effect resulting in a
tumor growth delay of 38 days, which is 10 days longer than the
calculated additive effect. Thus, the combination of CPT-11 and
C242-DM1 is unexpectedly superior (e.g., synergistic) in this human
colon cancer xenograft model.
Each of the patents and publications cited in the specification
is incorporated by reference herein in its entirety.
Various modifications of the invention, in addition to those described
herein, will be apparent to one skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. |