U.S. patent application number 14/096767 was filed with the patent office on 2014-04-03 for anti-integrin immunoconjugates, methods and uses.
This patent application is currently assigned to Immunogen, Inc.. The applicant listed for this patent is Immunogen, Inc., Janssen Biotech, Inc.. Invention is credited to Godfrey Amphlett, Qiming Chen, Robert J. Lutz, Rita M. Steeves, Mohit Trikha.
Application Number | 20140093523 14/096767 |
Document ID | / |
Family ID | 36578407 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140093523 |
Kind Code |
A1 |
Chen; Qiming ; et
al. |
April 3, 2014 |
Anti-Integrin Immunoconjugates, Methods and Uses
Abstract
The invention relates to conjugates of anti-integrin specific
antibodies with cytotoxic compounds, the synthesis, selection, and
use of such conjugates for use in cancer therapy or other diseases
mediated by cell proliferation, cell migration, or inflammation and
which pathology involves angiogenesis or neovascularization of new
tissue. In addition the invention relates to combination therapy of
such diseases wherein the treatment comprises use of said
conjugates in combination with one or more other treatment
modalities including but not limited to: chemotherapy, surgery or
radiation therapy. The preferred conjugates contain maytansinoid
compounds linked to the antibody by a disulfide linkage, and
preferred chemotherapeutic agents are doxorubicin, a taxane, a
camptothecin, a podophyllotoxin, a nucleoside analog, or a
pyrimidine analog.
Inventors: |
Chen; Qiming; (Collegeville,
PA) ; Trikha; Mohit; (San Mateo, CA) ; Lutz;
Robert J.; (Wayland, MA) ; Steeves; Rita M.;
(Stoneham, MA) ; Amphlett; Godfrey; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunogen, Inc.
Janssen Biotech, Inc. |
Cambridge
Radnor |
MA
PA |
US
US |
|
|
Assignee: |
Immunogen, Inc.
Cambridge
MA
Janssen Biotech, Inc.
Radnor
PA
|
Family ID: |
36578407 |
Appl. No.: |
14/096767 |
Filed: |
December 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11290249 |
Nov 30, 2005 |
8603483 |
|
|
14096767 |
|
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|
|
60634445 |
Dec 9, 2004 |
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Current U.S.
Class: |
424/181.1 ;
530/391.9 |
Current CPC
Class: |
A61P 17/10 20180101;
A61P 27/02 20180101; A61P 35/02 20180101; A61K 47/6803 20170801;
C07K 2317/73 20130101; A61P 17/00 20180101; A61P 17/08 20180101;
A61P 9/00 20180101; A61P 43/00 20180101; A61P 27/06 20180101; A61K
47/6849 20170801; A61K 2039/505 20130101; A61P 17/06 20180101; A61P
35/00 20180101; A61P 9/10 20180101; C07K 2317/21 20130101; A61P
29/00 20180101; A61P 19/02 20180101; C07K 16/2839 20130101 |
Class at
Publication: |
424/181.1 ;
530/391.9 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. An antibody-drug conjugate of the formula: ##STR00002## wherein
the antibody is a monoclonal antibody, or antigen-binding fragment
thereof, that competes with CNTO 95 for binding human .alpha.V
integrin; and wherein the maytansinol is esterified at C-3; R.sub.1
and R.sub.2 are Me; X.sub.1 and X.sub.2 are H; n is 2; p is 2; and
m is 3-4, and the pharmaceutically acceptable salts and esters
thereof.
2. The antibody conjugate of claim 1, wherein the antibody binds an
epitope not shared by one or more of the antibodies m7E3 IgG;
anti-.alpha.V.beta.3, clone LM609; anti-.alpha.V.beta.5, clone
P1F6; anti-.beta.3; or anti-alphaV, clone VNR139.
3. The antibody conjugate of claim 1, wherein the antibody, or
antigen-binding fragment thereof, has the epitopic specificity of
CNTO 95.
4. The antibody conjugate of claim 1, wherein the antibody, or
antigen-binding fragment thereof, comprises the six complementarity
determining region (CDR) amino acid sequences of CNTO 95 as shown
in SEQ ID NOs: 1-6, or six CDR amino acid sequences with
conservative sequence modifications to one or more of the sequences
as shown in SEQ ID NOs: 1-6.
5. The antibody conjugate of claim 1, wherein the antibody, or
antigen-binding fragment thereof, comprises the six CDRs of CNTO 95
as shown in SEQ ID NOs: 1-6.
6. The antibody-drug conjugate of claim 1, wherein the antibody is
a humanized or chimeric antibody.
7. A method of production of an antibody-drug conjugate according
to claim 1, comprising the steps: (a) introducing one or more free
or protected thiol groups into the antibody by treating the
antibody with a bifunctional coupling reagent represented by one of
the following structural formulas: ##STR00003## to form an
activated antibody; (b) reacting the activated antibody with DM4 to
form the antibody-drug conjugate; and (c) recovering the
antibody-drug conjugate.
8. A method of treatment of cancer in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of a conjugate according to claim 1, wherein the cancer
expresses .alpha.V integrin on its cell surface.
9. The method of claim 8, wherein the cancer is melanoma, lung
cancer, or colon cancer.
10. A method for inhibiting the growth of cancer cells in a mammal
in need thereof comprising administering to the mammal a monoclonal
antibody conjugate according to claim 1 in an amount effective to
inhibit the growth of said cancer cells in said mammal, wherein the
cancer expresses .alpha.V integrin on its cell surface.
11. The method of claim 10, wherein the cancer is melanoma, lung
cancer, or colon cancer.
12. The method according to claim 9, in which the antibody
conjugate is administered intravenously.
13. The method according to claim 12, in which the antibody
conjugate is administered in the amount of from about 0.05 mg/kg to
about 12.0 mg/kg body weight.
14. The method according to claim 10, in which the mammal is a
human.
15. A pharmaceutical composition comprising a conjugate according
to claim 1 and a pharmaceutically acceptable carrier, diluent, or
excipient.
16. An article of manufacture comprising the conjugate composition
of claim 1 and a container, and further comprising a package insert
or label indicating that the composition can be used to treat a
disease characterized by cells expressing .alpha.V integrin.
17. An article of manufacture according to claim 16, wherein the
container comprises a package insert that indicates that the
composition can be used to treat cancer.
18. An antibody conjugate according to claim 1, wherein at least
one of the heavy chain variable region sequences of the antibody
comprises the heavy chain variable region sequence of CNTO 95 as
shown in SEQ ID NO: 7.
19. An antibody conjugate according to claim 1, wherein at least
one of the light chain variable region sequences of the antibody
comprises the light chain variable region sequence of CNTO 95 as
shown in SEQ ID NO: 8.
20. An antibody conjugate according to claim 1, wherein at least
one of the heavy chain variable region sequences of the antibody
comprises the heavy chain variable region sequence of CNTO 95 as
shown in SEQ ID NO: 7 and at least one of the light chain variable
region sequences of the antibody comprises the light chain variable
region sequence of CNTO 95 as shown in SEQ ID NO: 8.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/290,249, filed Nov. 30, 2005, which claims the benefit of
U.S. Provisional Application No. 60/634,445, filed on Dec. 9,
2004.
[0002] The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates to conjugates of tumor specific
antibodies with cytotoxic compounds. The preferred conjugates
contain maytansinoid compounds linked to an anti-integrin antibody
by a disulfide linkage.
[0005] 2. Background of the Invention
[0006] There have been numerous attempts to improve the efficacy of
antineoplastic drugs by conjugating such drugs to monoclonal
antibodies (Mabs) against tumor-associated antigens in order to
elevate local concentration of the drug by targeted delivery to the
tumor. Conversely, the potential for antibodies to actually destroy
tumor cells is limited to those antibodies directed to blocking
proliferative stimuli, such as the growth factors EGF and Her-2 by
blocking the ligand binding to the receptors or blocking signaling
to of the receptors (ErbB1 and ErbB2) or those that elicit effector
functions (ADCC or CDC). Therefore, a product combining the
specificity of a Mab with the killing potential of a metabolic
poison has been sought. Examples of the former are doxorubicin
conjugated Mab BR96 (Braslawsky, et al. Cancer Immunol Immunother
33:367-374, 1991) and pseudomonas exotoxin fused to anti-growth
factor antibodies or fragments (Kreitment, et al., Internat. J.
Immunopharm. 14(3):465-72, 1992).
[0007] These attempts have encountered unforeseen limitations, such
as the requirement for relatively high intracellular concentrations
of the toxin compared to the number of external binding sites per
cell. If the number of tumor-associated antigens on the cancer cell
surface is estimated to be 10.sup.5 molecules/cell, the cytotoxic
agents that can be effectively used in these conjugates must have
an IC.sub.50 value of 10.sup.-10-10.sup.-11 M against target cancer
cells. (Chari, R. V. J. Adv. Drug Delivery Rev. 1998, 31, 89-104).
Secondly, the drug must either be released upon binding to the
target and penetrate the cell or the entire construct must be
transported into the cell and toxin cleaved or otherwise activated
there.
[0008] Some of these drawbacks can be solved to a greater or lesser
extent by using a highly potent drug conjugated to an internalizing
antibody and using a chemical bond which has enhanced lability
under intracellular conditions. Chari et al (Cancer Res.
52:127-131, 1992; Liu et al., Proc. Natl. Acad. Sci. USA
93:8618-8623, 1996; U.S. Pat. No. 5,208,020) developed antibody
conjugates wherein the antibody is linked to a maytansinoid via a
disulfide linkage.
[0009] Maytansinoids are plant derived anti-fungal and anti-tumor
agents. The isolation of three ansa macrolides from ethanolic
extracts of Maytenus ovatus and Maytenus buchananii was first
reported by S. M. Kupchan et al. and is the subject of U.S. Pat.
No. 3,896,111 along with demonstration of their anti-leukemic
effects in murine models at the microgram/kg dose range.
Maytansinoids, however, have unacceptable toxicity, causing both
central and peripheral neuropathies, and side effects: particularly
nausea, vomiting, diarrhea, elevations of hepatic function tests
and, less commonly, weakness and lethargy. Therefore, it has been a
focus of research for some time to find the correct targeting
moiety along with a suitable chemical process to form a
maytansine-antibody conjugate with acceptable half-life of
degradation.
[0010] In contrast to the high cytotoxicity of free maytansinoid,
an antibody conjugate has a toxicity which is several orders of
magnitude lower on antigen-negative cells compared to
antigen-positive cells. The linkage by disulfide bonding has the
advantage that these bonds are readily cleaved inside the target
cells by intracellular glutathione, releasing highly toxic free
drug. This approach has been applied to antibodies against
tumor-associated antigens, for example the C242-DM1 conjugate (Liu
et al., Proc. Natl. Acad. Sci. USA 93:8618-8623, 1996), and
HuN901-DM1 (Chari et al., 2000). However, the application of these
conjugates is restricted due to the limited expression of the
respective target antigens.
[0011] There is, therefore, still the need to improve this approach
by using antibodies targeted to the more highly expressed
tumor-associated antigens, and optionally, antigens highly
expressed during the proliferative and metastatic stages of the
malignancy, thus allowing for a natural concentration of toxin to
the most virulent cells.
Anti-Integrin Monoclonal Antibodies
[0012] Considerable evidence shows that progressive tumor growth is
dependent upon angiogenesis, the formation of new blood vessels, to
provide tumors with nutrients and oxygen, to carry away waste
products and to act as conduits for the metastasis of tumor cells
to distant sites (Gastl et al., Oncol. 54:177-184). Recent studies
have further defined the roles of integrins in the angiogenic
process. During angiogenesis, a number of integrins that are
expressed on the surface of activated endothelial cells regulate
critical adhesive interactions with a variety of ECM proteins to
regulate distinct biological events such as cell migration,
proliferation and differentiation. Specifically, the closely
related but distinct integrins .alpha.V.beta.3 and .alpha.V.beta.5
have been shown to mediate independent pathways in the angiogenic
process. An antibody generated against .alpha.V.beta.3 blocked
basic fibroblast growth factor (bFGF) induced angiogenesis, whereas
an antibody specific to .alpha.V.beta.5 inhibited vascular
endothelial growth factor (VEGF) induced angiogenesis (Eliceiri, et
al., J. Clin. Invest. 103: 1227-1230 (1999); Friedlander et al.,
Science 270: 1500-1502 (1995)). Therefore, integrins and especially
the alpha V subunit containing integrins, are reasonable
therapeutic targets for diseases that involve angiogenesis such as
disease of the eye and neoplastic disease, tissue remodeling such
as restenosis, and proliferation of certain cells types
particularly epithelial and squamous cell carcinomas.
[0013] Antibody Drug Conjugates
[0014] Conjugates of cell binding agents with the highly cytotoxic
maytansine has been described (U.S. Pat. Nos. 5,208,020 and
5,416,064; R. V. J. Chari et al., 1992 Cancer Res. 52:127-131).
Certain reagents or reactants such as N-hydroxysuccinimidyl esters
(NHS) for reaction with protein amine groups have been developed
for use in forming drug-protein conjugates. Reagents of this type
were generally described by Carlsson et al. (Biochem J. 173: 723,
1978 and in U.S. Pat. No. 4,149,003. Nitro-pyridyl linker reagents
for maytansine conjugation to Mabs and other proteins are disclosed
in WO2004/016801.
[0015] In the above referenced processes, the cell binding agents
are modified with a bifunctional agent such as
N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to introduce an
active disulfide moiety. Reaction with a thiol-containing cytotoxic
drug provides a conjugate in which the cell binding agent, such as
a monoclonal antibody, and drug are linked via disulfide bonds. It
was found that the C-3 hydroxyl position could be modified without
loss of activity, in fact, certain esters were found to have
enhanced cell killing activity (See Cassady, et al. Chem Pharm Bull
52(1): 1-26, 2004 for a review). U.S. Pat. Nos. 5,208,020 and
5,416,064 specifically teach the use of the activated maytansol
ester of N-methyl-N-(3-methyldithiopropanoyl)-L-alanine. The
maytansoid moiety from this reaction, and which is released upon
reductive cleavage of the disulfide bond, has been designated DM1
[N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine,
CAS Reg. No. 139504-50-0]. Thus, all the conjugates prepared using
the methyldithiolated form of DM1 retain an unsubstituted methylene
carbon adjacent to the disulfide bond on the drug side of the
conjugate (FIG. 1).
[0016] In order to enhance the in vivo stability of this disulfide
link, it is important to provide sterically hindered disulfide
bonds as has been noted previously (Thorpe, et al. Cancer Research
47:5924-31, 1987). This objective can be achieved by using
cross-linkers that bear one or two methyl substituents on the
carbon atom adjacent to the disulfide bond or using activated drugs
bearing at least one substituent on the alpha-carbon atom adjacent
the sulfhydryl or disulfide substituent.
[0017] While the problems of targeted delivery are now clearly
recognized, finding a suitable combination of antibody specificity
and affinity, conjugation chemistry, and toxin is unpredictable. It
is the object of the present invention to provide novel antibody
maytansine conjugates wherein the antibody is directed to cell
surface antigens sufficient in number to deliver a cytocidal dose
of a maytansinoid and which conjugate has appropriate chemical and
biologic stability to provide a therapeutically effective rate of
release when administered to a subject.
SUMMARY OF THE INVENTION
[0018] It is the object of the present invention to provide novel
antibody maytansine conjugates wherein the antibody is directed to
cell surface antigens sufficient in number to deliver a cytocidal
dose of a maytansinoid and where the antibody is known to be
internalized by the cell after binding the target antigen. In a
specific embodiment, the conjugates comprise a disulfide bond which
has been engineered through substitution of the adjacent methylene
carbons to provide a therapeutically effective rate of release when
administered to a subject. In a specific embodiment, the invention
relates to an antibody-drug conjugate comprising: an antibody that
binds to human alphaV integrin subunit conjugated to a cytotoxic
agent with an IC50 of 10.sup.-9 M or less, wherein the
antibody-drug conjugate exerts a cytotoxic or cytostatic effect on
an alpha V integrin expressing cancer cell line. In this
embodiment, the antibodies of the invention are specific for at
least one alphaV subunit of a heterodimeric integrin receptor, such
as an alphaVbeta1, alphaVbeta3, alphaVbeta5, alphaVbeta6, or
alphaVbeta8 heterodimeric integrin protein or fragment thereof. The
preferred conjugates contain maytansinoid compounds linked to the
antibody by a disulfide linkage and the antibody is capable of
binding vitronectin and fibrogen.
[0019] In one aspect, the antibody conjugates of the invention are
represented by the Formula
[C-L].sub.m-A I
where A is a human alphaV integrin subunit specific antibody,
wherein said antibody is capable of being internalized by the cell
expressing said alphaV subunit; C is a cytotoxin with a IC.sub.50
of 10.sup.-9 M or less; and L is a linking group which binds the
antibody and cytotoxin and further comprises a bond cleavable by
components of the intracellular environment; and m represents the
average number of cytotoxin molecules linked to the antibody and is
an integer from 1-10, specifically from 3-4. The cytotoxin may be
selected from the group consisting of maytansinoids,
calicheamicins, epothilones, discodermolide, eleuthrobins,
dolastatins, cryptophycins, camptothecins, rhizoxin (CAS reg. no.
90996546), or taxane derivatives and such other compounds that
exhibit half maximal inhibition (IC50 or GI50) of on tumor cell
growth at 10.sup.-9 M or less.
[0020] In an aspect of the first object of the invention, the
anti-alphaV integrin antibody-maytansinoid conjugate comprises any
protein or peptide containing molecule that comprises an antibody
that competes for binding to alpha-V subunit of a heterodimeric
human integrin receptor with the monoclonal antibody CNTO 95. In
one embodiment, the antibody comprises at least a portion of a
complementarity determining region (CDR) of a heavy or light chain
or a ligand binding portion thereof derived from the antibody
designated CNTO 95, in combination with a heavy chain or light
chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that can be
incorporated into the antibody with the CDR. The antibody CNTO 95
described herein is a human anti-alphaV antibody derived from
immunization of a transgenic mouse containing genes for the
expression of human immunoglobulins. Thus, in one embodiment, the
invention is directed to antibodies containing at least one CDR
region or variable region derived from the CNTO 95 antibody. In a
preferred embodiment the antibody is CNTO 95.
[0021] In another aspect of the invention, the
antibody-maytansinoid conjugate comprises a maytansinol ester which
is released upon cleavage of a bond linking the cytotoxin, C, to
the linker, L by components of the intracellular environment. In
one embodiment, the maytansinoid is esterified at C-3, C-14, C-15,
or C-20 with an acylated amino acid where the acyl group bears a
protected sulfhydryl group, wherein the carbon atom of the acyl
group adjacent the protected sulfhydryl group has one or two
substituents, said substituents being CH.sub.3, C.sub.2H.sub.5,
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched
or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl, or heterocyclic aromatic,
heterocycloalkyl radical, or H; and wherein the acyl group has a
linear chain length of at least two carbon atoms between the
carbonyl functionality and the sulfur atom. In a preferred
embodiment, the maytansinoid is a 3-maytansinol ester and the
acylated amino acid group bears 0, 1 or 2 methyl groups on the
carbon atom adjacent to the protected sulfhydryl. In a preferred
embodiment, the esterified maytansinol is selected from
N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine
(DM1, CAS Reg. No. 139504-50-0),
N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-1-oxopentyl)-maytansine
(DM3), and
N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine
(DM4).
[0022] It is a second object of the invention to provide
anti-integrin antibody-maytansinoid conjugate compounds useful for
treatment of human proliferative diseases caused by abnormal
proliferation and characterized by neovascularization. In a
particularly preferred embodiment, the compounds of the invention
are used in a method of treating cancer including, breast, colon,
rectal, lung, prostate, kidney, liver, pancreatic, esophageal,
stomach, endometrial, ovarian, cervical, or bone. The compounds of
the invention may be used alone or in combination with other agents
in the prevention or therapy of primary cancers or the prevention
or therapy of metastatic disease.
[0023] In another method of the second object of the invention
relates to the combined use of anti-integrin antibody maytansinoid
conjugate compounds with chemotherapeutic agents in methods of
cancer treatment. The preferred conjugates contain maytansinoid
compounds linked to the antibody by a disulfide linkage, and
preferred chemotherapeutic agents are doxorubicin, a taxane, a
camptothecin, a podophyllotoxin, a nucleoside analog, or a
pyrimidine analog.
[0024] In a third object of the invention, the
antibody-maytansinoid conjugate is prepared in a process whereby
the antibody is reacted with a bispecific chemical linker reagent,
such as an N-succinimidyl-(2-pyridylthio)alkanoate, and
subsequently reacted with a pre-activated maytansinoid whereby
disulfide exchange occurs to yield a hindered disulfide linkage
between the antibody and the maytansinoid.
[0025] In another aspect of the invention the antibody-maytansinoid
conjugate is prepared using a maytansinol ester wherein the acyl
moiety bears a protected sulfhydryl group. In one embodiment, the
maytansinoid is esterified at C-3, C-14, C-15, or C-20 with an
acylated amino acid where the acyl group bears a protected
sulfhydryl group, wherein the carbon atom of the acyl group
adjacent the protected sulfhydryl group has one or two
substituents, said substituents being selected from: CH.sub.3,
C.sub.2H.sub.5, linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10
carbon atoms, phenyl, substituted phenyl, a heterocyclic aryl
moiety, a heterocycloalkyl moiety, or H; and wherein the acyl group
has a linear chain length of at least two carbon atoms between the
carbonyl functionality and the sulfur atom. In a preferred
embodiment, the maytansinoid is a 3-maytansinol ester and the
acylated amino acid group bears 0, 1 or 2 methyl groups on the
carbon atom adjacent the protected sulfhydryl. In a preferred
embodiment, the esterified maytansinol is selected from
N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine
(DM1, CAS Reg. No. 139504-50-0),
N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-1-oxopentyl)-maytansine
(DM3), and
N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine
(DM4).
[0026] In a another aspect of the invention, the anti-alphaV
integrin antibody-maytansinoid conjugate is prepared by essentially
a single step of reacting a maytansinoid bearing a reactive ester
with an anti-integrin antibody not previously chemically activated.
The reactive ester of the maytansinoid may be a N-succinimidyl,
N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl,
2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl,
3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl ester.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1. shows the chemical structure of thiolated maytansine
amides with preferred species; DM1, DM3 and DM4 noted.
[0028] FIG. 2 shows the chemical structures and their chemical
acronyms of preferred bifunctional linker reagents of the
invention.
[0029] FIG. 3 is a schematic showing the synthetic method of
preparing antibody-maytansinoid conjugates on the invention.
[0030] FIG. 4 is a graph showing the change volume over time of a
human melanoma tumor in nude mice and the effect of administering
CNTO 95. Mice were inoculated subcutaneously with A375.S2 cells
(3.times.10.sup.6), and dosing with CNTO 95 or control was
initiated three days later. Mice were treated with CNTO 95 or
vehicle three times per week at a dose of 10 mg/kg i.p. Each data
point is the mean tumor volume from 10 tumor-bearing animals
(.+-.SEM). CNTO 95 given three times per week significantly
inhibited growth of tumors when compared to control treated animals
at day 26 (P=0.0005).
[0031] FIG. 5 is a graph showing the change volume over time of a
human melanoma tumor in nude rats and the effect of administering
CNTO 95. Rats were inoculated subcutaneously with A375.S2 cells
(3.times.10.sup.6), and therapy with CNTO 95 or control was
initiated three days later. Rats were treated with CNTO 95 or
vehicle once per week at a dose of 10 mg/kg i.v. Each data point is
the mean tumor volume from 9 tumor-bearing animals (.+-.SEM).
[0032] FIG. 6 is a graph showing the growth of A375.S2 human
melanoma cells over time in nude mice. Tumor volumes are expressed
as mean+/-SEM (n=9 or 10). The arrows indicate intravenous drug
injections. The asterisk indicates that one non-responding animal
was sacrificed since its tumor volume was over 1500 mm3.
[0033] FIG. 7 is a graph showing the growth of human A375.S2
melanoma cells in athymic nude rats. On day 14, when the average
tumor volumes reached 250 mm.sup.3, the animals were randomly
grouped and the first dose was administered. All animals were
sacrificed on day 35. Tumor volumes were expressed as mean+/-SEM
(n=9 or 10). The arrows indicate the days of intravenous drug
administration.
[0034] FIG. 8 is a graph showing the changing total body weight
over time in tumor bearing mice injected on day 7 after tumor
implantation and again every 7 days.times.5 with 3, 6, or mg/kg
CNTO 364; on day 7 and 14 for 25 mg/kg CNTO 364 or F105-DM1, and on
day 7, 14 and 35 with 15 mg/kg CNTO364.
[0035] FIG. 9 is a graph showing the change in tumor volume over
time in the same animals as in FIG. 8.
[0036] FIG. 10 is a graph showing the individual tumor volumes for
all of the animals in the groups as described in FIG. 8.
[0037] FIG. 11 is a graph of mean body weight+/-SEM (n=6) over time
for nude rats bearing subcutaneous human A549 human lung carcinoma
tumors and treated with CNTO 364 at 15 mg/kg or with control
treatments. The arrows indicate the times of intravenous drug
injections.
[0038] FIG. 12 is a graph showing the growth of human A549 human
lung carcinoma tumors in female athymic rats. CNTO 364 (15 mg/kg)
treatment regressed established A549 human lung carcinoma tumors in
female athymic rats.
[0039] FIG. 13 is a scatter plot showing individual tumor weights
at the termination of the study of growth of human A549 human lung
carcinoma tumors in female athymic rats treated with 15 mg/kg
CNTO364 or control substances. The horizontal lines indicate the
median of each study group.
[0040] FIGS. 14A &B are graphs showing the change in mean tumor
volume over time in rats bearing HT29 human colon tumor cells and
treated with CNTO 364 (CNTO95-SPP-DM1), CNTO 365
(CNTO95-SSNPB-DM4), and CNTO 366 (CNTO95-SSNPP-DM4). A. PBS control
and irrelevant antibody, F105, conjugated to the thiolated
maytansines using the same process and reagents and injected on day
7 and 21 with 10 mg/kg. B. PBS control and conjugated antibodies as
described injected at 20 mg/kg on day 7 and 21 except the CNTO365
group which received a single injection on day 7.
[0041] FIGS. 15A & B are graphs showing the mean change in body
weight in rats bearing HT29 tumors as described in FIG. 14.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0042] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0043] The term "alphaV integrin", "alphaV subunit integrin", and
"alphaV subunit containing integrin" are used interchangeably
herein to mean alphaV transmembrane glycoprotein subunits of a
functional integrin heterodimer and include all of the variants,
isoforms and species homologs of alphaV. Accordingly, antibodies of
the invention may, in certain cases, cross-react with alphaV from
species other than human, or other proteins which are structurally
related to human alphaV (e.g., human alphaV homologs). In other
cases, the antibodies may be completely specific for human alphaV
and not exhibit species or other types of cross-reactivity.
[0044] As used herein, an "antibody" includes whole antibodies and
any antigen binding fragment or a single chain thereof. Thus the
antibody includes any protein or peptide containing molecule that
comprises at least a portion of an immunoglobulin molecule, such as
but not limited to at least one complementarity determining region
(CDR) of a heavy or light chain or a ligand binding portion
thereof, a heavy chain or light chain variable region, a heavy
chain or light chain constant region, a framework (FR) region, or
any portion thereof, or at least one portion of a binding protein,
which can be incorporated into an antibody of the present
invention. An antibody could be murine, human, humanized, or
chimeric.
[0045] The "antigen binding fragment" or portion thereof, includes
single chain antibodies and fragments thereof. Functional fragments
include antigen-binding fragments that bind to a mammalian alpha-V
subunit. Examples of binding fragments encompassed within the term
"antigen binding portion" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH, domains; (ii) a F(ab).sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH,
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment in which the VH and
VL domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv). Such single chain antibodies are also intended to
be encompassed within the term "antigen-binding portion" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those with skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies. Such fragments can be produced by enzymatic cleavage,
synthetic or recombinant techniques, as known in the art and/or as
described herein.
[0046] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. Conformational and nonconformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents. The
term "native conformational epitope" or "native protein epitope"
are used interchangeably herein, and include protein epitopes
resulting from conformational folding of the integrin molecule
which arise when amino acids from differing portions of the linear
sequence of the integrin molecule come together in close proximity
in 3-dimensional space. Such conformational epitopes are
distributed on the extracellular side of the plasma membrane.
[0047] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from or closely matching human germline immunoglobulin sequences.
The human antibodies of the invention may include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo). Thus as used herein, the
term "human antibody" refers to an antibody in which substantially
every part of the protein (e.g., CDR, framework, C.sub.L, C.sub.H
domains (e.g., C.sub.H1, C.sub.H2, C.sub.H3), hinge, (V.sub.L,
V.sub.H)) is substantially similar to a human germline antibody.
Human antibodies have been classified into groupings based on their
amino acid sequence similarities, see e.g.
http://people.cryst.bbk.ac.uk/.about.ubcg07s/. Thus, using a
sequence similarity search, an antibody with similar linear
sequence can be chosen as a template to create "humanized
antibodies".
[0048] "Humanization" (also called Reshaping or CDR-grafting) is
now a well-established technique for reducing the immunogenicity of
monoclonal antibodies (mAbs) from xenogencic sources (commonly
rodent) and for improving the effector functions (ADCC, complement
activation, C1q binding). The engineered mAb is engineered using
the techniques of molecular biology, however simple CDR-grafting of
the rodent complementarity-determining regions (CDRs) into human
frameworks often results in loss of binding affinity and/or
specificity of the original mAb. In order to humanize an antibody,
the design of the humanized antibody includes variations such as
conservative amino acid substitutions in residues of the CDRs, and
back substitution of residues from the rodent mAb into the human
framework regions (backmutations). The positions can be discerned
or identified by sequence comparison for structural analysis or by
analysis of an homology model of the variable regions' 3D
structure.
[0049] The process of affinity maturation has most recently used
phage libraries to vary the amino acids at chosen positions.
Similarly, many approaches have been used to choose the most
appropriate human frameworks in which to graft the rodent CDRs. As
the datasets of known parameters for antibody structures increases,
so does the sophistication and refinement of these techniques.
Consensus or germline sequences from a single antibody or fragments
of the framework sequences within each light or heavy chain
variable region from several different human mAbs can be used.
Another approach to humanization is to modify only surface residues
of the rodent sequence with the most common residues found in human
mAbs and has been termed "resurfacing" or "veneering". Known human
Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;
www.atcc.org/phage/hdb.html; www.kabatdatabase.com/top.html;
www.antibodyresource.com/onlinecomp.html;
www.appliedbiosystems.com; www.biodesign.com; antibody.bath.ac.uk;
http://www.unizh.ch/.about.antibody/;
www.cryst.bbk.ac.uk/.about.ubcg07s; Kabat et al., Sequences of
Proteins of Immunological Interest, U.S. Dept. Health (1983), each
entirely incorporated herein by reference.
[0050] "Chimeric antibodies" are those antibodies that retain
distinct domains, usually the variable domain, from one species and
the remainder from another species; e.g. mouse-human chimeras.
[0051] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. In one embodiment, the
human monoclonal antibodies are produced by a hybridoma which
includes a B cell obtained from a transgenic nonhuman animal, e.g.,
a transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene fused to an immortalized
cell. However, generally, the antibody encoding sequences are
cloned and inserted into a host cell or a production cell line.
[0052] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, CHO lines or a mouse
myeloma SP/0 derived cell line.
[0053] The term "recombinant human antibody", as used herein,
includes all human or humanized antibodies that are prepared,
expressed, created or isolated by recombinant means, such as (a)
antibodies isolated from an animal (e.g., a mouse) that is
transgenic or transchromosomal for human immunoglobulin genes or a
hybridoma prepared therefrom, (b) antibodies isolated from a host
cell transformed to express the antibody, e.g., from a
transfectoma, (c) antibodies isolated from a recombinant,
combinatorial human antibody library, and (d) antibodies prepared,
expressed, created or isolated by any other means that involve
splicing of human immunoglobulin gene sequences to other DNA
sequences.
[0054] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to alphaV is substantially free of
antibodies that specifically bind antigens other than alphaV). An
isolated antibody that specifically binds to an epitope, isoform or
variant of human AlphaV may, however, have cross-reactivity to
other related antigens, e.g., from other species (e.g., alphaV
species homologs). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0055] As used herein, "specific binding" refers to antibody
binding to a predetermined antigen. Typically, the antibody binds
with a dissociation constant (K.sub.D) of 10-7 M or less, and binds
to the predetermined antigen with a K.sub.D that is at least
twofold less than its K.sub.D for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen.
[0056] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes.
Abbreviations
[0057] Abs antibodies, polyclonal or monoclonal
[0058] aV integrin subunit alpha V
[0059] b3 integrin subunit beta 3
[0060] bFGF basic fibroblast growth factor
[0061] HUVEC human umbilical vein endothelial
[0062] IFN interferon
[0063] Ig immunoglobulin
[0064] IgG immunoglobulin G
[0065] Mab monoclonal antibody
[0066] NPB=N-succinimidyl-5-nitro-(2-pyridyldithio)butyrate
[0067] SMCC=succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate
[0068] SMNP=N-succinimidyl
4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate
[0069] SMPT=4-succinimidyloxycarbonyl-(2-pyridyldithio)toluene
[0070] SPDB=N-succinimidyl-4-(2-pyridyldithio)butyrate
[0071] SPDP=N-succinimidyl-3-(2-pyridyldithio)propionate
[0072] SPP=N-succinimidyl-4-(2-pyridylthio)pentanoate
[0073] SP=N-succinimidyl 4-(2-pyridyl)
[0074] SS=sulfosuccinimidyl
[0075]
SSNPP=sulfosuccinimidyl-N-succinimidyl-4-(5-nitro-2-pyridyldithio)p-
entanoate
[0076] VEGF vascular endothelial growth factor
2. Compositions
[0077] A. Antibody Conjugates of the Invention
[0078] The antibody conjugates of the invention are represented by
the Formula
[C-L].sub.m-A I
where A is a human alphaV integrin subunit specific antibody,
wherein said antibody is capable of being internalized by the cell
expressing said alphaV subunit; C is a cytotoxin with a IC.sub.50
of 10.sup.-9 M or less; and L is linking group which binds the
antibody and cytotoxin and further comprises a bond cleavable by
components of the intracellular environment; and m represents the
average number of cytotoxin molecules linked to the antibody and is
an integer from 1-5, preferably, 3-4. The cytotoxin may be selected
from the group consisting of maytansinoids, calicheamicins,
epothilones, discodermolide, eleuthrobins, dolastatins,
cryptophycins, camptothecins, rhizoxin (CAS reg. no. 90996546), or
taxane derivatives and such other compounds that exhibit half
maximal inhibition (IC50 or GI50) of on tumor cell growth at
10.sup.-9 M or less.
[0079] Linkers comprising intracellularly cleavable bonds include
acid-labile linkages such as cis-aconityl linkages, esters,
acid-sensitive hydrazone linkages, lysosomally degradable peptide
linkers, hydrolase cleavable linkers, peptidase or protease
specific linkers, and disulfide (sulphydryl) linkers (see Dyba, M.,
et al. 2004 Curr Pharm Design 10:2311-2334 for a review). By being
capable of more rapid or selective cleavage under intracellular
conditions versus the conditions predominating in, for example, the
circulation, the linker imparts further specificity and safety to
the overall pharmacodynamics of the conjugate. Disulfide linkages
are particularly preferred because of the favorable reduction
potential within the cellular compartments as well as inducible
redox enzyme activation (Saito, G. et al. Adv. Drug Delivery Rev
2003 55:199-215). In one embodiment of the invention, the bond is
between a sulfur atom present in the antibody molecule, e.g. in the
side chain of a cysteine residue, and another sulfur atom present
in the toxic compound. In another embodiment, the linking moiety
consists of one or more atoms or chemical groups.
[0080] Another major consideration in chemically linking a biologic
molecule, such as a recombinant protein, to a chemical compound is
that the derivatization chemistry may, and in most cases will,
yield a new molecular entity which may have heretofore unknown
biologic properties. Thus, it should be understood that the
products of physiological cleavage should be designed to yield the
intended derivatives with biological activity. The maytansinoids of
the invention including DM1, DM3, DM4 and others as shown and
described in FIG. 1 retain biological activity.
[0081] The anti-alphaV integrin antibody-maytansinoid conjugates of
the invention are prepared by chemically linking an anti-alphaV
antibody to a maytansinoid molecule without significantly reducing
the biological activity of the antibody and providing a
maytansinoid, which when released under physiological conditions,
retains its cytotoxic potential. Examples of suitable maytansinoids
are esters of maytansinol and maytansinol analogues including but
not limited to those having a modified aromatic ring and those
having modifications at C-19, C-20, or C-14, or C-15, or C-4,5
deoxy. Preferred are maytansinol C-3 esters. Particularly preferred
maytansinoids are derivatives of N-methyl-alanine esters of
maytansinol (N.sup.2'-deacetyl-maytansine). Particularly preferred
conjugates comprise a disulfide linkage, which when cleaved by
reduction, releases a corresponding maytansinoid bearing a free
thiol. Thiol containing maytansinoids of the preferred type are
shown in FIG. 1:
N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine
(DM1, CAS Reg. No. 139504-50-0),
N.sup.2'-deacetyl-N.sup.2'-(4-mercapto-1-oxopentyl)-maytansine
(DM3), and
N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine
(DM4).
[0082] Conjugates of the antibody molecules of the invention and
toxic compound can be formed using any techniques presently known
or later developed. For example, the cytotoxic compound can be
modified to yield a free amino group and then linked to the
antibody molecule via an acid-labile linker, or a photolabile
linker. The toxic compound can be condensed with a peptide and
subsequently linked to an antibody molecule to produce a
peptidase-labile linker. The toxic compound can be treated to yield
a primary hydroxyl group, which can be succinylated and linked to
an antibody molecule to produce a conjugate that can be cleaved by
intracellular esterases to liberate free drug.
[0083] In order to create the disulfide linkage between antibody A
and the cytotoxin C, preferably, the toxic compound is treated to
create a free or protected thiol group, and then one or many
disulfide or thiol containing toxic compounds are covalently linked
to the antibody molecule via disulfide bond(s). The disulfide bond
need not be formed directly with a free thiol of the antibody but
can be formed by derivatization of any reactive group within the
antibody to introduce a site for disulfide exchange, for example,
as by coupling a bifunctional linker to free amine groups in the
antibody. For example, antibody molecules can be modified with
crosslinking reagents such as N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP),
4-succinimidyl-oxycarbonyl-a-methyl a-(2-pyridyldithio)-toluene
(SMPT), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB),
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-5-(2-pyridyldithio)pentanoate, 2-iminothiolane (IT),
or acetylsuccinic anhydride by known methods.
[0084] The anti-alphaV integrin antibody-cytotoxin conjugates of
the invention are thus represented by formula II, where maytansinol
is esterified at C-3, and the antibody is a anti-alphaV integrin
subunit antibody; R.sub.1, R.sub.2, X.sub.1 and X.sub.2 are
independently H, Me, C.sub.2H.sub.5, linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or a
heterocyclic aryl moiety, or a heterocycloalkyl moiety; n is 1-5; p
is 1-5; and m is 1 to 10.
##STR00001##
[0085] In a preferred embodiment, the linker moiety is a
4-thiopentanoate derived from SPP, or 4-thiopentanoate. The
antibody molecule containing free or protected thiol groups thus
derived, is then reacted with a disulfide- or thiol-containing
toxic compound to produce conjugates. The conjugates can be
purified by HPLC or by gel filtration.
[0086] B. Anti-Alpha-V Subunit Antibodies of the Invention
[0087] In addition to binding AlphaV, the human antibodies or
antigen binding fragments or portions thereof as those described
above may be selected for their retention of other functional
properties of antibodies of the invention, such as: [0088] 1)
binding to live cells expressing human alphaV; [0089] 2) preventing
live cell binding to matrix proteins; [0090] 3) binding to human
alphaV with a KD of 10.sup.-8 M or less (e.g., 10.sup.-9 M or
10.sup.-10 M or less); [0091] 4) exhibiting calcium-independent
binding to alphaV; [0092] 5) binding to a unique epitope on alphaV
or belonging to a unique complementation group of antibodies
binding to alphaV; [0093] 6) inhibition of angiogenesis in vitro or
in vivo; or [0094] 7) reduction of tumor mass or prevention of
tumor growth in vivo.
[0095] In another aspect of the invention, the structural features
of an human anti-alpha V antibodies of the invention, CNTO 95, are
used to create structurally related human anti-Alpha V antibodies
that retain at least one functional property of the antibodies of
the invention, such as binding to AlphaV. More specifically, one or
more CDR regions of CNTO 95 can be combined recombinantly with
known human framework regions and CDRs to create additional,
recombinantly-engineered, human anti-Alpha V antibodies of the
invention.
[0096] In a preferred embodiment, the antibody for use in the
anti-alphaV antibodies conjugates described herein is a human
anti-alpha V antibody derived from immunization of a transgenic
mouse containing genes for the expression of human immunoglobulins.
Preparation of the antibody is described in detail in PCT
publication no. WO 02/12501 and in U.S. Publication No.
2003/040044, both incorporated by reference herein. The antibody
includes any protein or peptide containing molecule that comprises
at least a portion of a complementarity determining region (CDR) of
a heavy or light chain or a ligand binding portion thereof derived
from the antibody designated "CNTO 95" (see PCT publication no. WO
02/12501 and U.S. Publication No. 2003/040044), in combination with
a heavy chain or light chain variable region, a heavy chain or
light chain constant region, a framework region, or any portion
thereof, that can be incorporated into an antibody.
[0097] Preferably, the CDR1, 2, and/or 3 of the engineered
antibodies described above comprise the exact amino acid
sequence(s) as those of the fully human Mab designated CNTO 95,
Gen0101, CNTO 95, C371A generated by immunization of a transgenic
mouse as disclosed herein. However, the ordinarily skilled artisan
will appreciate that some deviation from the exact CDR sequences of
CNTO 95 may be possible while still retaining the ability of the
antibody to bind Alpha V effectively (e.g., conservative
substitutions). In a particular embodiment, the antibody or
antigen-binding fragment can have an antigen-binding region that
comprises at least a portion of at least one heavy chain CDR (i.e.,
CDR1, CDR2 and/or CDR3) having the amino acid sequence of the
corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 1, 2, and/or
3). In another particular embodiment, the antibody or
antigen-binding portion or variant can have an antigen-binding
region that comprises at least a portion of at least one light
chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acid
sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS:
4, 5, and/or 6) of the light chain of CNTO95. In a preferred
embodiment the three heavy chain CDRs and the three light chain
CDRs of the antibody or antigen-binding fragment have the amino
acid sequence of the corresponding CDR of mAb CNTO 95. Accordingly,
in another embodiment, the engineered antibody may be composed of
one or more CDRs that are, for example, 90%, 95%, 98% or 99.5%
identical to one or more CDRs of CNTO 95. Anti-alpha-V subunit
antibodies of the present invention can include, but are not
limited to, at least one portion, sequence or combination selected
from 5 to all of the contiguous amino acids of at least one of SEQ
ID NOS:1, 2, 3, 4, 5, 6. An anti-alpha-V subunit antibody can
further optionally comprise a polypeptide of at least one of
70-100% of the contiguous amino acids of at least one of SEQ ID
NOS: 7, 8. For example, the amino acid sequence of a light chain
variable region can be compared with the sequence of SEQ ID NO: 8,
or the amino acid sequence of a heavy chain CDR3 can be compared
with SEQ ID NO: 7.
[0098] As disclosed and claimed herein, the sequences set forth in
SEQ ID NOs. 1-8 include "conservative sequence modifications", i.e.
amino acid sequence modifications which do not significantly affect
or alter the binding characteristics of the antibody encoded by the
nucleotide sequence or containing the amino acid sequence. Such
conservative sequence modifications include amino acid
substitutions, additions and deletions. Modifications can be
introduced into SEQ ID NOs: 1-8 or to the nucleic acids encoding
them by standard techniques known in the art, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Conservative amino acid
substitutions include ones in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a human anti-Alpha V antibody is
preferably replaced with another amino acid residue from the same
side chain family.
[0099] At least one antibody of the invention binds at least one
specified epitope specific to at least one alphaV subunit protein,
subunit, fragment, portion or any combination thereof. The at least
one epitope can comprise at least one antibody binding region that
comprises at least one portion of said protein, which epitope is
preferably comprised of at least one extracellular, soluble,
hydrophillic, external or cytoplasmic portion of said protein. The
at least one specified epitope can comprise any combination of at
least one amino acid sequence of at least 1-3 amino acids to the
entire specified portion of contiguous amino acids of the SEQ ID
NO: 9.
[0100] As previously stated, the invention also relates to
antibodies, antigen-binding fragments, immunoglobulin chains and
CDRs comprising amino acids in a sequence that is substantially the
same as an amino acid sequence described herein. Preferably, such
antibodies or antigen-binding fragments and antibodies comprising
such chains or CDRs can bind human alpha-V subunit with high
affinity (e.g., K.sub.D less than or equal to about 10.sup.-9 M).
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0101] Amino acids in an anti-alpha-V subunit antibody of the
present invention that are essential for function can be identified
by methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15;
Cunningham and Wells, Science 244:1081-1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity, such as, but not limited to at least one
alpha-V subunit neutralizing activity. Sites that are critical for
antibody binding can also be identified by structural analysis such
as crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos, et al., Science 255:306-312 (1992)).
3. Methods of Preparation of the Conjugates
[0102] The starting compound, maytansinol, as used in the
production of compounds DM1, DM3 and DM4 and related activated
maytansinoids (FIG. 1) according to this invention can be prepared
from maytansine a natural C-3 ester isolated from natural sources
(Kupchan et al., J. Amer. Chem. Soc. 97, 5294 (1975)) by reductive
cleavage. The reagent lithium trimethoxyaluminum hydride in
tetrahydrofuran at -40.degree. C. is particularly useful for this
step. Other natural maytansinoid esters may also be advantageously
produced by cultivating microorganisms, which belongs to the genus
Nocardia (U.S. Pat. No. 4,151,042) or Actinosynnema spp. that have
been engineered to produce maytansinol, maytanacine or C-3
maytansinol esters such as maytansinol propionate in the culture
broth and extracting the compounds from the culture broth for
further purification. There are many linking groups known in the
art for making antibody maytansinoid conjugates, including, for
example disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile peptide linkers, or esters
which may be acid labile or esterase cleavable.
[0103] As taught in U.S. Pat. No. 5,208,020; esterification of
maytansinol or an analogue with the carboxylic acids containing a
methyldithio group or other protected thio group, including, for
example, N-methyl-N-[3-(methyldithio)-1-oxopropyl]-L-alanine
produce the corresponding disulfide-containing maytansinoids. In
the case where two diastereomeric products containing the D- and
L-acyl side chains result, the diastereomeric maytansinoid esters
are readily separated by methods known in the art and the less
desirable D-alanyl analog isomer product reduced to recover
maytansinol as taught in WO03096782. Reductive cleavage of the
disulfide group with dithiothreitol gives the corresponding
thiol-containing maytansinoid, which is readily linked via
disulfide or thioether linkages to cell binding agents.
Thiol-maytansinoids can by purification by HPLC using a C18 column
in the reverse phase mode eluting with a gradient of
water-acetonitrile.
[0104] Bifunctional Coupling Reagents.
[0105] It is known in the preparation of conjugates of two
substances, of which at least one comprises a protein or a
polypeptide, to use bifunctional agents in order to couple the
components of the conjugate covalently, amino groups in the
conjugated molecules normally being utilized for the conjugating
reaction. Bifunctional protein coupling agents include
N-succinimidyl-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters such as
dimethyl adipimidate.HCl, active esters such as disuccinimidyl
suberate, aldehyes such as glutaraldehyde, bis-azido compounds suc
has bis(p-axidobenzoyl)hexanediamine, bis-diazonium derivatives
such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates
(such as tolyene 2,6-diisocyanate), and bis active fluorine
compounds such as 1,5-difluoro-2,4-dinitrobenzene). SPDP is among
the most frequently used reagent for this purpose and many other
N-succinimidyl-(2-pyridyldithio)-,
N-succinimidyl-(5-nitro-2-pyridyldithio)- or
N-succinimidyl-(4-pyridyldithio)-short chain alkane acids have
proved useful. FIG. 2 shows the structures of commonly used
bifunctional linkers and their acronyms.
[0106] Conjugation of Activated Antibody to Thiolated
Maytansinoid
[0107] The preparation of CNTO 95-Maytansinoid conjugates followed
the method described previously described (Chari et al., Cancer
Res. 52: 127-131, 1992 and U.S. Pat. No. 5,208,020) and as outlined
in FIG. 3. In this procedure, antibody is modified with
bifunctional linker at a ratio of linker to antibody in the range
of 5 to 10:1 to introduce dithiopyridyl groups onto the antibody
amino acid side chains. The activated antibody is separated from
residual linker by G25 gel filtration chromatography. The linker
antibody ratio after the purification is less than 5 to 10:1 and
typically in the range of 3 to 5:1 and is measured by absorbance at
252 nm and 280 nm. The activated thiol-maytansinoid is added at
molar excess to that of the measured linker. Following the
conjugation, the mixture is again purified by G25 size exclusion
chromatography to yield bulk product.
[0108] In the alternative, the anti-integrin antibody maytansinoid
conjugate is prepared by essentially a single step of reacting a
maytansinoid bearing a reactive ester with anti-integrin antibody
not previously chemically activated. The reactive ester of the
maytansinoid may be a N-succinimidyl, N-sulfosuccinimidyl,
N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl,
2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or
3-carboxy-4-nitrophenyl ester. The method is described in
publication WO2002098883, the contents of which are incorporated
herein by reference.
4. Methods of Using the Conjugates of the Invention
[0109] The antibodies of the invention may be administered to a
subject in need thereof to prevent, treat, manage or ameliorate a
cancer or one or more symptoms thereof. The antibodies of the
invention may also be administered in combination with one or more
other therapies, preferably therapies useful for the prevention,
management or treatment of cancer (including, but not limited to
the prophylactic or therapeutic agents listed hereinbelow) to a
subject in need thereof to prevent, treat, manage or ameliorate a
cancer or one or more symptoms thereof. In a specific embodiment,
the invention provides a method of preventing, treating, managing
or ameliorating cancer or one or more symptoms thereof, said method
comprising administering to a subject in need thereof a dose of a
prophylactically or therapeutically effective amount of a
formulation comprising the anti-alphaV antibody conjugates of the
invention. In another embodiment, the invention provides a method
of preventing, treating or ameliorating cancer or one or more
symptoms thereof, said method comprising administering to a subject
in need thereof a dose of a prophylactically or therapeutically
effective amount of anti-alphaV antibody conjugates of the
invention in conjunction with a prophylactically or therapeutically
effective one or more therapies (e.g., surgery, radiation therapy,
or administration of therapeutic agents other than anti-alphaV
antibody conjugates). The antibody conjugates of the invention may
be used as a first, second, third or fourth line cancer treatment.
The invention provides methods for treating or ameliorating one or
more symptoms of a cancer in a subject. Further, the invention
provides methods for preventing the recurrence of cancer in
patients that have been treated and have no disease activity by
administering an anti-alphaV antibody conjugate of the
invention.
[0110] Cancers that can be treated by the methods encompassed by
the invention include, but are not limited to, neoplasms, tumors,
metastases, or any disease or disorder characterized by
uncontrolled cell growth. The cancer may be a primary or metastatic
cancer. The cancerous cells may or may not express alphaV subunit
integrins. In a preferred embodiment, the cancer that is being
managed, treated or ameliorated in accordance with the methods of
the invention is a cancer expressing integrin alphaV subunit and
has metastasized to the another site or organ within the body of
the patient or has the potential to metastasize. Specific examples
of cancers that can be treated by the methods encompassed by the
invention include, but are not limited to, cancer of the head,
neck, eye, mouth, throat, esophagus, chest, bone, lung, colon,
rectum, stomach, prostate, breast, ovaries, kidney, liver,
pancreas, and brain. Additional cancers include, but are not
limited to, the following: leukemias such as but not limited to,
acute leukemia, acute lymphocytic leukemia, acute myelocytic
leukemias such as myeloblastic, promyelocytic, myelomonocytic,
monocytic, erythroleukemia leukemias and myelodysplastic syndrome,
chronic leukemias such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's lymphoma; myelomas such as
multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma,
plasma cell leukemia, solitary plasmacytoma and extramedullary
plasmacytoma; Waldenstrom's macroglobulinemia; bone cancer and
connective tissue sarcomas such as bone sarcoma, myeloma bone
disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, Paget's
disease of bone, malignant giant cell tumor, fibrosarcoma of bone,
chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma
(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,
liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma,
synovial sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including adenocarcinoma and
intraductal carcinoma, and papillary breast cancer; adrenal cancer
including pheochromocytoma and adrenocortical carcinoma; thyroid
cancer; pancreatic cancer; pituitary cancers; eye cancers not
limited to ocular melanoma, choroidal melanoma, cilliary body
melanoma, and retinoblastoma; vaginal cancers; vulvar cancer;
cervical cancers; uterine cancers not limited to endometrial
carcinoma and uterine sarcoma; ovarian cancers; esophageal and
other head and neck cancers such as but not limited to, squamous
cancer, adenocarcinoma, mucoepidermoid carcinoma, adenosquamous
carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma,
and oat cell (small cell) carcinoma; stomach cancers; colon
cancers; rectal cancers; liver cancers such as hepatocellular
carcinoma and hepatoblastoma, gallbladder cancers;
cholangiocarcinomas; lung cancers such as non-small cell lung
cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers, choriocarcinoma (yolk-sac tumor), prostate
cancers; penal cancers; oral cancers not limited to squamous cell
carcinoma; basal cancers; salivary gland cancers; renal cell cancer
and other kidney cancers; and bladder cancers not limited to
transitional cell carcinoma (for a review of such disorders, see
DeVita, V. T., Hellman, S., & Rosenberg, S. A. Cancer:
Principles and practice of oncology. Philadelphia: J. B. Lippincott
Company; 6th Edition, 2001). Pre-malignant conditions may also be
treated by the methods and compositions of the invention. Such
cancers may include, but not be limited to, follicular lymphomas,
carcinomas with p53 mutations, hormone dependent tumors of the
breast, prostate and ovary, and precancerous lesions such as
familial adenomatous polyposis, and myelodysplastic syndromes.
[0111] In a preferred embodiment, the cancer that is being
prevented, managed, treated or ameliorated in accordance with the
method of the invention is selected from prostate cancer, breast
cancer, bone cancer, melanoma, lung cancer and ovarian cancer. In
another embodiment, the cancer that is being prevented, managed,
treated or ameliorated in accordance with the methods of the
invention is selected from metastatic tumors including, but not
limited to, tumors that have or may metastasize to the bone
(non-limiting examples are prostate, breast and lung cancers that
have metastasized or have the potential to metastasize to the
bone), tumors that have or may metastasize to the lung, tumors that
have or may metastasize to the brain, and tumors that have or may
metastasize to other organs or tissues of a subject.
Anti-Cancer Therapies
[0112] Any agent or therapy (e.g., chemotherapies, radiation
therapies, hormonal therapies, and/or biological therapies or
immunotherapies) which is known to be useful, or which has been
used or is currently being used for the prevention, treatment,
management or amelioration of cancer or one or more symptoms
thereof can be used in combination with an anti-alphaV antibody
conjugate of the invention in accordance with the invention
described herein. Therapeutic or prophylactic agents include, but
are not limited to, peptides, polypeptides, proteins, fusion
proteins, nucleic acid molecules, small molecules, mimetic agents,
synthetic drugs, inorganic molecules, and organic molecules.
Examples of the classes of such agents (i.e., anti-cancer agents)
include, but are not limited to, cytotoxins, angiogenesis
inhibitors, and immunomodulatory agents and agents used to provide
relief from pain or to offset the deleterious effects of one or
more therapeutic agents (e.g. bisphosphonate use to reduce the
hypercalcemia effects of glucocorticoids).
[0113] Biologic immunomodulatory agents include: anti-T cell
receptor antibodies such as anti-CD3 antibodies (e.g. Nuvion
(Protein Design Labs), OKT3 (Johnson & Johnson), or anti-CD20
antibodies Rituxan (IDEC)), anti-CD52 antibodies (e.g. CAMPATH 1H
(Ilex)), anti-CD11a antibodies (e.g. Xanelim (Genentech));
anti-cytokine or anti-cytokine receptor antibodies and antagonists
such as anti-IL-2 receptor antibodies (Zenapax (Protein Design
Labs)), anti-IL-6 receptor antibodies (e.g. MRA (Chugai)), and
anti-IL-12 antibodies (CNTO 275 (Centocor)), anti-TNFalpha
antibodies (Remicade (Centocor)) or TNF receptor antagonist (Enbrel
(Immunex)), anti-IL-6 antibodies (BE8 (Diaclone) and CNTO328
(Centocor)), and antibodies that immunospecifically bind to
tumor-associated antigens (e.g., trastuzimab (Genentech).
[0114] Angiogenesis inhibitors (i.e., anti-angiogenic agents)
include, but are not limited to, angiostatin (plasminogen
fragment); antiangiogenic antithrombin III; angiozyme.
Bisphosphonates include, but are not limited to, alendronate,
clodronate, etidronate, ibandronate, pamidronate, risedronate,
tiludronate, and zoledronate.
[0115] Specific examples of anti-cancer agents which can be used in
accordance with the methods of the invention include, but not
limited to: 5-fluoruracil; acivicin; aldesleukin; altretamine;
aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase; azacitidine; azetepa; azotomycin; batimastat;
bicalutamide; bleomycin sulfate; brequinar sodium; bropirimine;
busulfan; carboplatin; carmustine; carubicin hydrochloride;
carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;
cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;
dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;
dexonnaplatin; dezaguanine; dezaguanine mesylate; diaziquone;
docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene;
droloxifene citrate; dromostanolone propionate; duazomycin;
edatrexate; eflomithine hydrochloride; enloplatin; enpromate;
epipropidine; epirubicin hydrochloride; erbulozole; esorubicin
hydrochloride; estramustine; estramustine phosphate sodium;
etanidazole; etoposide; etoposide phosphate; fazarabine;
fenretinide; floxuridine; fludarabine phosphate; fluorouracil;
fluorocitabine; fosquidone; fostriecin sodium; gemcitabine;
gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;
ifosfamide; ilmofosine; interleukin II (including recombinant
interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;
interferon alpha-m; interferon alpha-n3; interferon beta-I a;
interferon gamma-I b; iproplatin; irinotecan hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate; liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; mechlorethamine hydrochloride; megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; ormaplatin;
paclitaxel; pegaspargase; porfromycin; prednimustine; procarbazine
hydrochloride; puromycin; rogletimide; safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur,
talisomycin; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate;
trimetrexate glucuronate; triptorelin; uracil mustard; uredepa;
vapreotide; verteporfn; vinblastine sulfate; vincristine sulfate;
vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate
sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine
sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin;
zorubicin hydrochloride.
[0116] The invention also encompasses administration of an antibody
of the invention in combination with radiation therapy comprising
the use of x-rays, gamma rays and other sources of radiation to
destroy the cancer cells. In preferred embodiments, the radiation
treatment is administered as external beam radiation or teletherapy
wherein the radiation is directed from a remote source. In other
preferred embodiments, the radiation treatment is administered as
internal therapy or brachytherapy wherein a radioactive source is
placed inside the body close to cancer cells or a tumor mass.
[0117] In specific embodiments, patients with breast cancer are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with the administration of a prophylactically or
therapeutically effective amount of one or more other agents useful
for breast cancer therapy including but not limited to:
doxorubicin, epirubicin, the combination of doxorubicin and
cyclophosphamide (AC), the combination of cyclophosphamide,
doxorubicin and 5-fluorouracil (CAP), the combination of
cyclophosphamide, epirubicin and 5-fluorouracil (CEF), or other
agents such as Herceptin, tamoxifen, paclitaxel or taxotere.
[0118] In specific embodiments, patients with prostate cancer are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with the administration of a prophylactically or
therapeutically effective amount of one or more other agents useful
for prostate cancer therapy including but not limited to
external-beam radiation therapy; interstitial implantation of
radioisotopes of i.e., rhenium, palladium, or iridium; leuprolide
or other LHRH agonists; non-steroidal antiandrogens (flutamide,
nilutamide, bicalutamide), steroidal antiandrogens (cyproterone
acetate), the combination of leuprolide and flutamide, estrogens
such as DES, ethinyl estradiol; low-dose prednisone, or other
chemotherapy regimens reported to produce subjective improvement in
symptoms and reduction in PSA level.
[0119] In specific embodiments, patients with ovarian cancer are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with a prophylactically or therapeutically effective
amount of one or more other agents useful for ovarian cancer
therapy including but not limited to: intraperitoneal radiation
therapy, such as .sup.32P therapy; total abdominal and pelvic
radiation therapy, cisplatin, the combination of paclitaxel (Taxol)
or docetaxel (Taxotere) and cisplatin or carboplatin, the
combination of cyclophosphamide and cisplatin, the combination of
cyclophosphamide and carboplatin, the combination of 5-FU and
leucovorin, etoposide, liposomal doxorubicin, gemcitabine,
ifosfamide, hexamethylmelamine (HMM), or topotecan.
[0120] In specific embodiments, patients with tumor metastatic to
bone are administered a prophylactically or therapeutically
effective amount of an anti-alphaV antibody conjugate of the
invention in combination with a prophylactically or therapeutically
effective amount of one or more other agents useful for bone
metastatic tumor therapy including but not limited to: agents or
therapies used in treatment of underlying malignancy such as
hornone inhibitors for prostate or breast cancer metastasized to
bone, radiotherapy or chemoradiotherapy with bone-seeking
radioisotopes of metals (strontium-89 and samarium-153), and
bisphosphonates (e.g. palmidronate or alendronate).
[0121] Cancer therapies and their dosages, routes of administration
and recommended usage are known in the art and have been described
in such literature as the Physician's Desk Reference (57th ed.,
2003) now available through the internet by subscription from
PDR.RTM. Electronic Library, Thomson Micromedex, Greenwood Village,
Colo. (Edition 2004).
Inflammatory Disorder Treatment
[0122] The anti-alphaV antibody conjugates of the invention may be
administered to a subject in need thereof to prevent, manage, treat
or ameliorate an inflammatory disorder or one or more symptoms
thereof. The antibodies of the invention may also be administered
in combination with one or more other therapies, preferably
therapies useful for the prevention, management, treatment or
amelioration of an inflammatory disorder (including, but not
limited to the prophylactic or therapeutic agents listed in
hereinbelow) to a subject in need thereof to prevent, manage, treat
or ameliorate an inflammatory disorder or one or more symptoms
thereof.
[0123] The inflammatory disorders that can be treated by the
methods encompassed by the invention include, but are not limited
to, asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentiated spondyloarthropathy,
undifferentiated arthropathy, arthritis, osteoarthritis,
spondyloarthropathies (e.g. psoriatic arthritis, ankylosing
spondylitis, Reiter's Syndrome (reactive arthritis), inflammatory
osteolysis, Wilson's disease and chronic inflammation resulting
from chronic viral or bacteria infections. In addition, autoimmune
disorders are associated with an inflammatory pathology.
[0124] Anti-Inflammatory Therapies
[0125] The present invention provides methods of preventing,
managing, treating or ameliorating an inflammatory disorder or one
or more symptoms thereof, said methods comprising administering to
a subject in need thereof an anti-alphaV antibody conjugate of the
invention and one or more therapies (e.g. prophylactic or
therapeutic agents other than antibodies or antibody fragments that
immunospecifically bind to alphaV integrins. Any agent or therapy
which is known to be useful, or which has been used or is currently
being used for the prevention, management, treatment or
amelioration of an inflammatory disorder or one or more symptoms
thereof can be used in combination with an anti-alphaV antibody
conjugate of the invention in accordance with the invention
described herein. Examples of such agents include, but are not
limited to, immunomodulatory agents, anti-angiogenic agents,
anti-inflammatory agents and TNFalpha antagonists.
[0126] Specific examples of immunomodulatory agents which can be
administered in combination with an anti-alphaV antibody conjugate
of the invention to a subject with an inflammatory disorder
include, methothrexate, leflunomide, cyclophosphamide, cytoxan,
nuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g.
FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,
steroids, mycophenolate mofetil, rapamycin (sirolimus),
leflunamide, anti-T cell receptor antibodies (e.g. Orthoclone OKT3
(Johnson & Johnson), Nuvion (Protein Design Labs), or anti-CD20
antibodies (Rituxan (IDEC)), anti-CD52 antibodies (e.g. CAMPATH 1H
(flex)), anti-IL-2 receptor antibodies (e.g. Zenapax (Protein
Design Labs)), anti-IL6 (CNTO 328, Centocor) or anti-IL-6 receptor
antibodies (MRA, Chugai), and anti-IL-12 antibodies (CNTO1275,
Centocor), anti-IFN antibodies, anti-TNF antibodies, anti-IL-1
antibodies and IL-1alpha/beta antagonists.
[0127] Examples of TNFalpha antagonists which can be administered
in combination with a anti-alphaV antibody conjugates of the
invention to a subject with an inflammatory disorder include
proteins, polypeptides, peptides, fusion proteins, antibodies (and
antigen-binding fragments thereof) such as antibodies that
immunospecifically bind to TNFalpha, nucleic acid molecules (e.g.
antisense molecules or triple helices), organic molecules,
inorganic molecules, and small molecules that block, reduce,
inhibit or neutralizes the function, activity and/or expression of
TNFalpha. Examples of TNFalpha antagonists include: infliximab
(REMICADE; Centocor), D2E7 (HUMARA; Abbott Laboratories/Knoll
Pharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known
as HUMICADE and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.),
TNF-R1 (Amgen), etanercept (ENBREL; Immunex), and inhibitors of
other members of the TNFR superfamily of receptors. Other TNF
antagonists encompassed by the invention include, but are not
limited to, IL-10, which is known to block TNFalpha production and
anti-p38 MAPK agents.
[0128] Non-limiting examples of anti-inflammatory agents which can
be administered in combination with an anti-alphaV antibody
conjugate of the invention to a subject with an inflammatory
disorder include non-steroidal anti-inflammatory drugs (NSAIDs),
steroidal anti-inflammatory drugs, beta-agonists, anticholingeric
agents, and methyl xanthines. Examples of NSAIDs include, but are
not limited to, aspirin, ibuprofen, celecoxib (CELEBREX),
diclofenac (VOLTAREN), etodolac (IODINE), fenoprofen (NALFON),
indomethacin (INDOCIN), ketoralac (TORADOL), oxaprozin (DAYPRO),
nabumentone (RELAFEN), sulindac (CLINORIL), tolmentin (TOLECTIN),
rofecoxib (VIOXX), naproxen (ALEVE, NAPROSYN), ketoprofen (ACTRON)
and nabumetone (RELAFEN). Such NSAIDs function by inhibiting a
cyclooxygenase enzyme (e.g. COX-1 and/or COX-2). Examples of
steroidal anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON), cortisone,
hydrocortisone, prednisone (DELTASONE), prednisolone, and
triamcinolone.
[0129] In specific embodiments, patients with osteoarthritis are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with other agents or therapies useful for
osteoarthritis prevention, treatment, management or amelioration
including but not limited to: analgesics such as acetaminophen,
phenacetin; and tramadol, NSAIDs such as aspirin, diflunisal,
diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen,
ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone,
naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and
tolmetin; cyclooxygenase (Cox)-2-specific inhibitors (CSIs) such as
celecoxib and rofecoxib; intra- or periarticular injection of a
depot preparations of, for example, glucocorticoids or
biopharmaceuticals, and intra-articular injection of hyaluronic
acid. The an anti-alphaV antibody conjugate of the invention can
also be used in combination with other nonpharmacologic measures in
prevention, treatment, management and amelioration of
osteoarthritis including but not limited to: irrigation of the
osteoarthritic joint, reduction of joint loading; application of
heat or cold to the affected joint; capsaicin cream; exercise and
other physical therapies, and joint replacement surgery.
[0130] In specific embodiments, patients with rheumatoid arthritis
are administered a prophylactically or therapeutically effective
amount of an anti-alphaV antibody conjugate of the invention in
combination with other agents or therapies useful in prevention,
treatment, management and amelioration of rheumatoid arthritis
include NSAIDs, analgesics, and CSIs as discussed for
osteoarthritis. In addition, other therapies may be used
concurrently, prior to, or subsequently to administration an
anti-alphaV antibody of the invention such as monthly pulses with
high-dose glucocorticoids, or intraarticular glucocorticoids;
disease-modifying antirheumatic drugs (DMARDs) including
methotrexate, gold compounds (e.g. Auranofin), D-penicillamine, the
antimalarials (e.g. chloroquine), and sulfasalazine; TNFalpha
neutralizing agents such as etanercept and infliximab;
immunosuppressive and cytotoxic agents not limited to,
azathioprine, leflunomide, cyclosporine, and cyclophosphamide; and
surgical interventions such as arthroplasties, total joint
replacement, reconstructive hand surgery, open or arthroscopic
synovectomy, and early tenosynovectomy of the wrist; external
interventions such as a variety of orthotic and assistive devices,
and other physical therapies: and dietary supplements such as
increasing intake of omega-3 fatty acids (such as eicosapentaenoic
acid).
[0131] In specific embodiments, patients with chronic obstructive
pulmonary disease (COPD) are administered a prophylactically or
therapeutically effective amount of an anti-alphaV antibody
conjugate of the invention in combination with other agents or
therapies useful in prevention, treatment, management and
amelioration of COPD including but not limited to: bronchodilators
including but not limited to, short- and long-acting
beta-adrenergic agonists such as albuterol, pirbuterol,
terbutaline, and metaproterenol, oral sustained-release albuterol
and inhaled salmeterol; anticholinergics such as ipratropium
bromide, and theophylline and its derivatives; glucocorticoids;
oxygen; lung transplantation; lung volume reduction surgery;
endotracheal intubation, ventilation support; yearly influenza
vaccine and pneumococcal vaccination; exercise; and smoking
cessation.
[0132] In specific embodiments, patients with pulmonary fibrosis
are administered a prophylactically or therapeutically effective
amount of an anti-alphaV antibody conjugate of the invention in
combination with an effective amount of one or more other agents
useful for pulmonary fibrosis therapy including but not limited to:
oxygen; corticosteroids; cytotoxic drugs (cyclophosphamide or
azathioprine); bronchodilators e short- and long-acting
beta-adrenergic agonists, anticholinergics, and theophylline and
its derivatives); and antihistamines (diphenhydramine and
doxylamine).
[0133] In specific embodiments, patients with asthma are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with an effective amount of one or more other agents
useful for asthma therapy including but not limited to: adrenergic
stimulants (examples include but not limited to, catecholamines,
e.g., epinephrine, isoproterenol, and isoetharine; resorcinols,
e.g. metaproterenol, terbutaline, and fenoterol; and saligenins,
e.g. salbutamol; methylxanthines including theophylline and its
various salts; anticholinergics including atropine sulfate, akopine
methylnitrate, and ipratropium bromide; glucocorticoids; mast cell
stabilizing agents cromolyn sodium and nedocromil sodium;
leukotriene modifiers Zileuton, zafirlukast and montelukast;
immunosuppressant agents including methotrexate; and
acetylcysteine.
[0134] In specific embodiments, patients with allergy are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with an effective amount of one or more other agents
useful for allergy therapy including but not limited to: cromolyn;
antihistamines; sympathomimetic drugs (both alpha-adrenergic and
beta-adrenergic drugs); theophylline and its derivatives;
glucocorticoids; and immune desensitization treatments with
allergen injections.
Autoimmune Disorder Treatment
[0135] The anti-alphaV antibody conjugate of the invention may be
administered to a subject in need thereof to prevent, manage, treat
or ameliorate an autoimmune disorder or one or more symptoms
thereof. The anti-alphaV antibody conjugate of the invention may
also be administered in combination with one or more other
therapies, preferably therapies useful for the prevention,
management or treatment of an autoimmune disorder (including, but
not limited to the prophylactic or therapeutic agents listed in
hereinbelow) to a subject in need thereof to prevent, manage, treat
or ameliorate an autoimmune disorder or one or more symptoms
thereof. In a specific embodiment, the invention provides a method
of preventing, managing, treating or ameliorating an autoimmune
disorder or one or more symptoms thereof, said method comprising
administering to a subject in need thereof a dose of a
prophylactically or therapeutically effective amount of a liquid
formulation of the invention. In another embodiment, the invention
provides a method of preventing, managing, treating or ameliorating
an autoimmune disorder or one or more symptoms thereof, said method
comprising administering to a subject in need thereof a dose of a
prophylactically or therapeutically effective amount of a liquid
formulation of the invention and a dose of a prophylactically or
therapeutically effective amount of one or more therapies (e.g:,
prophylactic or therapeutic agents) other than antibodies or
antibody fragments that immunospecifically bind to alphaV
integrins.
[0136] The invention provides methods for managing, treating or
ameliorating an autoimmune disorder or one or more symptoms thereof
in a subject refractory to conventional therapies for such an
autoimmune disorder, said methods comprising administering to said
subject a dose of a prophylactically or therapeutically effective
amount of the antibodies of the invention. The invention also
provides methods for managing, treating or ameliorating an
autoimmune disorder or one or more symptoms thereof in a subject
refractory to existing single agent therapies for such an
autoimmune disorder, said methods comprising administering to said
subject a dose of a prophylactically or therapeutically effective
amount of an anti-alphaV antibody conjugate of the invention and a
dose of a prophylactically or therapeutically effective amount of
one or more therapies (e.g. prophylactic or therapeutic agents)
other than antibodies or antibody fragments that immunospecifically
bind to alphaV integrin. The invention also provides methods for
managing, treating or ameliorating an autoimmune disorder or one or
more symptoms thereof by administering an anti-alphaV antibody
conjugate of the invention in combination with any other treatment
to patients who have proven refractory to other treatments but are
no longer on these treatments. The invention also provides
alternative methods for the management or treatment of an
autoimmune disorder where another therapy has proven or may prove
too toxic, i.e., results in unacceptable or unbearable side
effects, for the subject being treated.
[0137] Particularly, the invention provides alternative methods for
the management or treatment of an autoimmune disorder where the
patient is refractory to other therapies. Further, the invention
provides methods for preventing the recurrence of an autoimmune
disorder in patients that have been treated and have no disease
activity by administering an anti-alphaV antibody conjugate of the
invention.
[0138] In autoimmune disorders, the immune system triggers an
immune response when there are no foreign substances to fight and
the body's normally protective immune system causes damage to its
own tissues by mistakenly attacking self. There are many different
autoimmune disorders which affect the body in different ways. For
example, the brain is affected in individuals with multiple
sclerosis, the gut is affected in individuals with Crohn's disease,
and the synovium, bone and cartilage of various joints are affected
in individuals with rheumatoid arthritis. As autoimmune disorders
progress, destruction of one or more types of body tissues,
abnormal growth of an organ which may be accompanied by
neovascularization of said organ or tissue, or changes in organ
function may result. The autoimmune disorder may affect only one
organ or tissue type or may affect multiple organs and tissues.
Organs and tissues commonly affected by autoimmune disorders
include red blood cells, blood vessels, connective tissues,
endocrine glands (e.g. the thyroid or pancreas), muscles, joints,
and skin. Examples of autoimmune disorders that can be treated by
the methods of the invention include, but are not limited to,
alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune diseases of the adrenal
gland, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune oophoritis and orchitis, autoimmune thrombocytopenia,
Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac
sprue-dermatitis, chronic fatigue immune dysfunction syndrome
(CFIDS), chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold
agglutinin disease, Crohn's disease, discoid lupus, essential mixed
cryoglobulinemic, fibromyalgia-fibromyositis, glomerulonephritis,
Graves' disease, Guillain-Barre, Hashimoto's thyroiditis,
idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura
(ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus
erthematosus, Meniere's disease, mixed connective tissue disease,
multiple sclerosis, type 1 or immune-mediated diabetes mellitus,
myasthenia gravis, pemphigus vulgaris, pernicious anemia,
polyarteritis nodosa, polychondritis, polyglandular syndromes,
polymyalgia rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal arteristis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's
granulomatosis.
[0139] Autoimmune therapies and their dosages, routes of
administration and recommended usage are known in the art and have
been described in such literature as the Physician's Desk Refererce
(56th ed., 2002 and 57th ed., 2003).
[0140] The present invention provides methods of preventing,
managing, treating or ameliorating an autoimmune disorder or one or
more symptoms thereof, said methods comprising administering to a
subject in need thereof an anti-alphaV antibody conjugate of the
invention and one or more therapies (e.g., prophylactic or
therapeutic agents) other than antibodies or antibody fragments
that immunospecifically bind to alphaV integrins. Any agent or
therapy which is known to be useful, or which has been used or is
currently being used for the prevention, management, treatment or
amelioration of an autoimmune disorder or one or more symptoms
thereof can be used in combination with an anti-alphaV antibody
conjugate of the invention in accordance with the invention
described herein. Examples of such agents include, but are not
limited to, immunomodulatory agents, anti-inflammatory agents and
TNFalpha antagonists. Specific examples of immunomodulatory agents,
anti-inflammatory agents and TNFalpha antagonists which can be used
in combination with an anti-alphaV antibody conjugate of the
invention for the prevention, management, treatment or amelioration
of an autoimmune disorder are disclosed herein above.
[0141] In specific embodiments, patients with multiple sclerosis
(MS) are administered a prophylactically or therapeutically
effective amount of an anti-alphaV antibody conjugate of the
invention in combination with other agents or therapies useful in
prevention, treatment, management and amelioration of MS including
but not limited to: IFN-beta1b (Betaseron) and IFN-alpha2a
(Avonex); glatiramer acetate (Copaxone); mitoxantrone;
methotrexate; cyclophosphamide; intravenous immunoglobulin;
glucocorticoids; methylprednisolone; 2-chlorodeoxyadenosine
(cladribine); baclofen (orally or intrathecally via an indwelling
catheter); cyclobenzaprine hydrochloride; clonazepam; clonidine
hydrochloride; carbamazepine; gabapentin; amitriptyline; primidone;
ondansetron; isoniazid; oxybutynin; tolterodine; propantheline;
bethanecol; terazosin hydrochloride; sildenafil citrate;
amantadine; pemoline; high dose vitamins; calcium orotate;
gancyclovir; antibiotic; and plasma exchange.
[0142] In specific embodiments, patients with psoriasis are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody conjugate of the invention in
combination with other agents or therapies useful in prevention,
treatment, management and amelioration of psoriasis including
topical steroid-containing preparations; tar (Estar, Psorigel,
Fototar cream); topical vitamin D analogues such as calcipotriene
ointment; ultraviolet light with or without psoralen; methotrexate;
cyclosporine; sulfasalazine; and synthetic retinoids.
[0143] In specific embodiments, patients with Crohn's disease are
administered a prophylactically or therapeutically effective amount
of an anti-alphaV antibody of the invention in combination with
other agents or therapies useful in prevention, treatment,
management and amelioration of Crohn's disease including but not
limited to: antidiarrheals (loperamide, diphenoxylate with
atropine, cholestyramine or colestipol); antispasmodics
(propantheline, dicyclomine, or hyoscyamine); 5-aminosalicylic acid
agents (sulfasalazine, mesalamine (Asacol) and its slow release
form (Pentasa); corticosteroids; the immunomodulatory drugs useful
in rheumatic diseases--azathioprine, mercaptopurine, cyclosporine,
and methotrexate; antibiotics; TNF inhibitors including enteracept
and infliximab; immunosuppressive agents including tacrolimus,
mycophenolate mofetil, and thalidomide; nutritional therapies;
enteral therapy with elemental diets (e.g., Vivonex for 4 weeks);
and total parenteral nutrition.
[0144] In specific embodiments, patients with lupus erythematosus
are administered a prophylactically or therapeutically effective
amount of an anti-alphaV antibody of the invention in combination
with other agents or therapies useful in prevention, treatment,
management and amelioration of lupus erythematosus including but
not limited to: antimalarials (including but not limited to,
hydroxychloroquine); glucocorticoids (e.g., low dose, high dose, or
high-dose intravenous pulse therapy can be used); immunosuppressive
and immunomodulatory agents including cyclophosphamide,
chlorambucil, and azanthioprine, methotrexate and mycophenolate
mofetil; androgenic steroids (including but not limited to
danazol); and anticoagulants (including but not limited to
warfarin).
[0145] Non-Malignant or Immunological-Related Cell-Proliferative
Diseases
[0146] The conjugates of the invention are also useful for treating
non-malignant proliferative diseases and, especially those
involving angiogenesis. Angiogenesis is know to be a contributing
factor in number of pathological conditions in addition to the
ability of tumors to grow and metastasize, disorders of the eye
including retinopathies, and disorders of the skin including
psoriasis and Kaposi's Sarcoma. Representative examples of such
non-tumorigenic angiogenesis-dependent diseases include corneal
neovascularization, hypertrophic scars and keloids, proliferative
diabetic retinopathy, rheumatoid arthritis, arteriovenous
malformations (discussed above), atherosclerotic plaques and
ischemic heart disease, delayed wound healing, hemophilic joints,
nonunion fractures, Osler-Weber syndrome, psoriasis, emphigus
vulgaris, Behcet's syndrome, acute respiratory distress syndrome
(ARDS), pyogenic granuloma, scleroderma, tracoma, menorrhagia
(discussed above) and vascular adhesions.
4. Pharmaceutical Formulations
[0147] The invention provides for stable formulations of the
anti-alphaV-maytansinoid conjugates, which is preferably an aqueous
phosphate buffered saline or mixed salt solution, as well as
preserved solutions and formulations containing a preservative as
well as multi-use preserved formulations suitable for
pharmaceutical or veterinary use, comprising at least one
anti-alphaV-maytansinoid conjugate in a pharmaceutically acceptable
formulation.
[0148] Preferred preservatives include those selected from the
group consisting of phenol, m-cresol, p-cresol, o-cresol,
chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl,
butyl and the like), benzalkonium chloride, benzethonium chloride,
sodium dehydroacetate and thimerosal, or mixtures thereof.
[0149] At least one anti-alphaV-maytansinoid conjugate in either
the stable or preserved formulations or solutions described herein,
can be administered to a patient in accordance with the present
invention via a variety of delivery methods including SC or IM
injection; transdermal, pulmonary, transmucosal, implant, osmotic
pump, cartridge, micro pump, or other means appreciated by the
skilled artisan, as well-known in the art.
4. Pharmaceutical Formulations
[0150] In a preferred method of administering CNTO 95-maytansinoid,
the drug substance is given intravenously from a previously
installed catheter equipped with an infusion bag. CNTO
95-mertansine is supplied in 20-ml single-use vials by ImmunoGen,
Inc. (Cambridge, Mass.). Each vial contains protein at a
concentration of from 0.05 to about 2.0 mg/ml in a buffered
solution (pH 6.5.+-.0.5) comprised essentially of monobasic
potassium phosphate (0.57 mg/ml), monobasic sodium phosphate
monohydrate (0.20 mg/ml), dibasic sodium phosphate (0.555 mg/ml),
and sodium chloride (8.16 mg/ml) in purified water, USP. The drug
product is prefiltered twice upon instilling the dose volume into
the infusion bag by passing it through a low protein-binding 5-.mu.
filter and is administered to patients through an inline 0.22 .mu.m
filter within 8 h of preparation. After infusion, the i.v. line
should be flushed with fluid to ensure delivery of the full drug
dose.
[0151] The based on previous experience in human patients with
Mab-maytansoid conjugates, given by the intravenous method, doses
of ranging from 22 to 295 mg/m.sup.2 can be given every three weeks
(J Clin Oncol. 21:211-222, 2003).
6. Articles of Manufacture
[0152] The invention includes an article of manufacture containing
materials useful for the treatment of the disorders described above
comprising an anti-alphaV-maytansinoid conjugate, a container and a
label or package insert on or associated with the container. The
article of manufacture preferably contains at least one vial
comprising a solution of at least one anti-alphaV-maytansinoid
conjugate with the prescribed buffers and/or preservatives,
optionally in an aqueous diluent, wherein said packaging material
comprises a label that indicates that such solution can be held
over a period of time. The invention may comprise an article of
manufacture, comprising packaging material, a first vial comprising
lyophilized at least one anti-alphaV-maytansinoid conjugate, and a
second vial comprising an aqueous diluent of prescribed buffer or
preservative, wherein said packaging material comprises a label
that instructs a practitioner or patient how to reconstitute the at
least one anti-alphaV-maytansinoid conjugate in the aqueous diluent
to form a solution.
[0153] Suitable containers include, for example, bottles, vials,
syringes, etc. The containers may be formed from a variety of
materials such as glass or plastic. The container may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle).
[0154] At least one active agent in the composition is an
anti-alphaV antibody-maytansinoid conjugate. The label or package
insert indicates that the composition is used for treating the
condition of choice, such as cancer. The package insert herein may
indicate that the antibody or composition is used to treat cancer
that does not respond, or respond poorly, to treatment with the
standard of care as outlined herein for specific diseases and
diagnoses. In other embodiments, the package insert may indicate
that the antibody-maytansinoid conjugate or composition can be used
also to treat metastatic cancer, prostate cancer, breast cancer or
colorectal cancer.
[0155] While having described the invention in general terms, the
embodiments of the invention will be further disclosed in the
following examples.
Example 1
Production and Characterization of Monoclonal Antibody CNTO95
[0156] Preparation of the anti-alpha V integrin antibody CNTO95 is
described in detail in PCT publication no. WO 02/12501 and in U.S.
Publication No. 2003/040044, both incorporated by reference herein.
Specifically, the human Mab CNTO 95 was generated by immunizing
(CBA/J.times.C57/BL6/J, GenPharm International) F2 hybrid mice with
.alpha..sub.v.beta..sub.3 integrin purified from human placenta.
The antibody is composed of human variable and IgG1 kappa constant
regions. The method of making and the desirable characteristics of
CNTO95 have been previously described in WO0212501 and Trikha, et
al. 2004, Int. J. Cancer 110 (3): 326-335.
[0157] Transgenic mice from GenPharm International express human
immunoglobulins but not mouse IgM or Ig.kappa. were used. These
mice contain human sequence transgenes that undergo V(D)J joining,
heavy-chain class switching and somatic mutation to generate a
repertoire of human sequence immunoglobulins (Taylor et al.,
International Immunology 6:579-591 (1993)). The light chain
transgene is derived in part from a yeast artificial chromosome
clone that includes nearly half of the germline human V.kappa.
region. In addition to several VH genes, the heavy-chain (HC)
transgene encodes both human .mu. and human .gamma.1 (Lonberg et
al., Nature 368:856-859 (1994)) and/or .gamma.3 constant regions. A
mouse derived from the HC012 genotypic lineage was used in the
immunization and fusion process to generate an anti-alphaV
monoclonal antibody used in the preparation of a conjugate of the
invention.
[0158] Human placenta (disrupted using a meat grinder) or M21 human
melanoma cells expressing the .alpha.V.beta.3 integrin were
extracted with OTG (Octylthioglucoside Pierce) in buffered saline
as described (WO0212501). These preparations were emulsified with
an equal volume of complete Freund's adjuvant and used to immunize
15 to 17 week old surgically castrated male mouse (GenPharm, Foster
City, Calif.) on days 0 and 14 and in incomplete Freunds on days
28, 48, and 56. Three days later splenocytes were harvested from a
mouse showing a titer of 1:1280 against alphaVbeta3 using a solid
phase EIA format. Fusion was carried out at a 1:1 ratio of murine
myeloma cells (SP2/0) to viable spleen cells. Hybridoma
supernatants were screened using the EIA microplate assay or EIA
capture assay and selected antibody producing lines expanded and
retested for the desired properties.
[0159] ELISA analysis confirmed that purified antibody from two
hybridomas, C371A (also called Mab CNTO 95) and C372A, bind
alphaVbeta3 in a concentration-dependent manner. Fifty percent
binding is achieved at 0.07 and 0.7 .mu.g/mL for C372A and CNTO 95
respectively. In the same assay, the anti-alphaIIbeta3 antibody,
c7E3 IgG, demonstrated fifty-percent maximal binding at 0.07
.mu.g/mL.
[0160] To ascertain the unique specificity of CNTO95 competition
binding (or complementation) assays were performed using of the
following murine antibodies: m7E3 IgG, anti-alphaVbeta3 (clone
LM609, Chemicon), anti-alphaVbeta 5 (clone P1F6, Gibco), anti-beta3
(Chemicon, AMAC), or anti-alphaV (clone VNR139, Gibco) antibodies.
These results demonstrated that CNTO 95 binds an epitope not shared
by the other antibodies tested.
[0161] The binding affinity values for purified integrins were
compared to binding to receptors expressed on various cell lines
using 125-I CNTO 95. A375S2 and M21 cells express both
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5, HT-29
cells express .alpha..sub.v.beta.5. For comparison, other Mabs
capable of binding integrins were used. The Kd was calculated from
a saturation binding curve in each case using multiple replicates
of multiple lots of antibody. On .alpha..sub.v.beta.3 coated
plates, the CNTO 95 mean K.sub.D was 2.1.+-.1.33.times.10.sup.-10
M; and the mean abciximab Kd was 2.5.+-.1.46.times.10.sup.-10 M.
The CNTO 95 mean K.sub.D on .alpha..sub.v.beta..sub.5 was
2.5.+-.1.04.times.10.sup.-11 M. Abciximab showed no binding and no
dose-response on .alpha..sub.v.beta..sub.5 coated plates.
[0162] As shown in FIG. 4, dosing with CNTO 95 inhibited growth of
human melanoma tumors in nude mice. At day 26, CNTO 95 inhibited
tumor growth by approximately 80% compared to tumors from
control-treated animals. In this model, CNTO 95 does not interact
with host angiogenic vessels since it does not bind mouse
integrins, suggesting that blockade of human tumor-expressed
integrins alone can inhibit tumor growth in mice independently of
antiangiogenic effects For rat studies, female nude rats, aged 6-7
weeks, were purchased from Harlan.
[0163] Twenty rats were inoculated s.c. with A375.S2 cells
(3.times.10.sup.6) in the flank region (day 1). On day 4, rats were
randomly assigned to 2 groups. One group was injected i.v. with
CNTO 95 (10 mg/kg in PBS), while the other group received an
isotype-matched control IgG (10 mg/kg). Dosing was continued weekly
thereafter until day 46 (total of 6 doses). Tumors were measured by
calipers twice a week and tumor volumes calculated by the formula
(length.times.width2)/2. Body weights were also recorded
weekly.
[0164] In the rat, CNTO 95 is capable of blocking both rat
angiogenic integrins and human tumor cell-expressed integrins.
Weekly treatment of tumor-bearing nude rats with CNTO 95 at 10
mg/kg reduced tumor growth compared to the isotype-matched human
IgG control MAb (FIG. 5). By day 46, treatment with CNTO 95
resulted in significant reduction in final tumor size compared to
control-treated nude rats (p=0.0007).
[0165] In summary, CNTO 95 is a fully human MAb, which binds
members of the alphaV family of integrins with unique specificity,
avidity and activity as demonstrated by multiple functional assays
showing that it neutralizes the biologic effects of the integrin
receptors alphaVbeta3 and alphaVbeta5 in vitro and in vivo. CNTO 95
inhibited adhesion, migration, proliferation and invasion of both
tumor and endothelial cells in vitro and demonstrated that binding
and blocking multiple alphaV integrin receptors was more effective
than blocking of a single integrin alone. In addition, CNTO 95
inhibited angiogenesis and tumor growth in vivo.
[0166] Growth of human melanoma tumors was significantly reduced by
blockage of tumor cell integrins in the mouse model or by combined
blockage of tumor cell and host angiogenic integrins in the rat
model, highlighting the potential importance of targeting multiple
cellular targets for antitumor efficacy.
[0167] Results of in vitro models demonstrate that CNTO 95 has
potent antiangiogenic properties, inhibiting endothelial cell
adhesion, proliferation, migration and capillary sprouting.
[0168] In addition, CNTO 95 blocked angiogenesis stimulated by both
bFGF and M21 melanoma cells in the rat Matrigel model and by bFGF
in a primate angiogenesis model. CNTO 95 displayed potent
antiangiogenic effects in both a rodent model and a novel nonhuman
primate model in cynomulgus monkeys.
[0169] In addition to blocking integrins on angiogenic endothelium,
the ability to inhibit integrin function on tumor cells themselves
reduced the growth of tumors. A number of alphaV integrins have
been suggested to play critical roles in tumor cell biology. In a
mouse xenograft model where CNTO 95 does not cross-react with host
integrins, treatment with CNTO 95 significantly inhibited the
growth of v3/5-positive melanoma tumors.
[0170] One of the most important features of CNTO 95 is its fully
human nature. Because it is fully human, CNTO 95 may be less likely
to cause immune responses in patients. Furthermore, because CNTO 95
is able to bind not only alphaVbeta3 and alphaVbeta5 but also other
alphaV integrins, such as alphaVbeta 6 and alphaVbeta1, it has the
potential to inhibit multiple integrin-mediated events.
Example 2
Preparation of CNTO 95-Maytansine Conjugates
[0171] Antibody conjugates of thiolated maytansines were prepared
for further biological testing starting using bifunctional linkers
as described.
[0172] CNTO 95 antibody was supplied by Centocor for conjugation.
CNTO 95 was supplied at approximately 20 mg/ml (260 mg) total. The
antibody was dialysed into Buffer A (50 mM KPi, 50 mM NaCl, 2 mM
EDTA pH6.5), then brought to 8 mg/ml in 95% Buffer A, 5% ETOH. The
antibody was modified with 6.5 fold molar excess of SPP to
introduce the linker for drug conjugation, forming CNTO 95-SS-Py
where S-Py is 2-mercaptopyridine. Residual SPP was removed by G25
gel filtration chromatography. The linker to Ab ratio was measured
as 4.7. The Ab-SS-Py conjugate was modified with 1.7 fold molar
excess of DM1 (MW=737.5 g/mole) to linker, using an antibody
concentration of 3.2/mlin 97% Buffer A, 3% dimethylacetamide.
Following conjugation, the conjugate was rechromatographed on G25
using PBS, pH 6.5 as the buffer. The resulting conjugate contained
3.2 moles of DM1 per mole of CNTO-95: [Ab]=2.59 mg/ml, [DM1]=38.3
microgm/ml. Calculations were based on absorbance readings at 252
and 280 nm of the filtered material and using extinction
coefficients: Ab=224,000 M.sup.-1cm.sup.-1 at 280 nm, DM1=5700
M.sup.-1cm.sup.-1 at 280 nm, Ab=82,880 M.sup.-1cm.sup.-1 at 252 nm
and DM1=26,790 M.sup.-1cm.sup.-1 at 252 nm.
[0173] The product was analyzed by nonreducing SDS-PAGE, SEC HPLC,
and by binding affinity to alphaVbeta3 and alphaVbeta5 protein by
ELISA. By PAGE, the product was predominantly a band around 160 kDa
with a fainter lower molecular weight band also visible. By SEC
HPLC analysis, the fraction of the conjugate eluting a monomer
(18.8.degree.) was 96% and about 4% of the conjugate eluted as a
higher molecular weight species (16.2.degree.). Binding affinity
was calculated by graphing the absorbance v concentration giving an
apparent Kd of 3.0 e-11 M for CNTO95 and Kd of 3.5 e-11 M for the
conjugate on alphaVbeta5. Both species gave an apparent Kd of
approximately 3.0 e-9 on alphaVbeta3.
[0174] Other batches of CNTO95-SPP-DM1 and CNTO 95 conjugated to
DM4 and an irrelevant antibody which targets a non-human antigen,
F105, conjugated similarly were prepared in an analogous manner
using bifunctional linkers as shown in FIG. 2 and as outlined in
FIG. 3. The characterisitics of these preparations are given
hereinbelow:
[0175] Preparation of CNTO95-SPP-DM1 (CNTO 364).
[0176] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM1 with SPP linker as follows: 270.6 mg CNTO95 was conjugated to
3.7 mg of DM1, the resulting 98 ml of conjugate was stored at
2.degree. C. to 8.degree. C. at 2.74 mg/mL in PBS at pH 6.5. The
DM1 concentration was determined to 37.6 .mu.g/mL by absorbance.
Therefore, the ratio of DM1 per mole of CNTO95 is 2.98 (1 microgram
of DM1 is equivalent to 68.9 microgm of conjugated CNTO95
antibody). By HPLC the preparation was 96.3% monomer with 0.59%
free drug.
[0177] Preparation of CNTO95-SPP-DM1 (CNTO 364).
[0178] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM1 with SPP linker as follows: 104 mg CNTO95 was conjugated to
1.82 mg of DM1, the resulting 26 ml of conjugate was stored at
2.degree. C. to 8.degree. C. at 3.65 mg/mL of conjugated
CNTO95-SPP-DM1 antibody in PBS at pH 6.5. The DM1 concentration was
determined to 70.07 .mu.g/mL by absorbance. Therefore, the
preparation contains a ratio of 3.80 moles of DM1 per mole of
CNTO95 (1 .mu.g of DM1 is equivalent to 57.2 .mu.g of conjugated
CNTO95 antibody). By HPLC the preparation was 93.4% monomer with
1.61% free drug.
[0179] Preparation of CNTO95-SPP-DM1 (CNTO 364).
[0180] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM1 with SPP linker as follows: 228 mg CNTO95 conjugated to 4.13 mg
of DM1, the resulting 102 ml of conjugate was stored at 2.degree.
C. to 8.degree. C. at 2.24 mg/mL of conjugated CNTO95-SPP-DM1
antibody in PBS at pH 6.5. The DM1 concentration was determined to
40.53 .mu.g/mL by absorbance. Therefore, the preparation contains a
ratio of 3.93 moles of DM1 per mole of CNTO95 (1 .mu.g of DM1 is
equivalent to 55.3 .mu.g of conjugated CNTO95 antibody). By HPLC
the preparation was 94.7% monomer with 1.00% free drug.
[0181] Preparation of CNTO 95-SSNPB-DM4 (CNTO 365).
[0182] Monoclonal antibody CNTO 95 was conjugated to Maytansinoid
DM4 with SSNPB linker as follows: 121 mg CNTO 95 was conjugated to
2.18 mg of DM4, the resulting 34 ml conjugate was stored at
2.degree. C. to 8.degree. C. at 3.25 mg/mL in PBS at pH 6.5. The
DM4 concentration was determined to 64.11 microgm/mL by absorbance.
Therefore the ratio of DM4 per mole of antibody is 3.57 (1 microgm
of DM4 is equivalent to 55.6 microgm of conjugated CNTO95
antibody). By HPLC the preparation was 95.4% monomer with 3.23%
free drug.
[0183] Preparation of CNTO95-SSNPP-DM4 (CNTO 366).
[0184] Monoclonal antibody CNTO 95 was conjugated to Maytansinoid
DM4 with SSNPP linker as follows: 101 mg CNTO 95 was conjugated to
1.45 mg of DM4, the resulting 30 ml conjugate was stored at
2.degree. C. to 8.degree. C. at 3.07 mg/mL antibody in PBS at pH
6.5. The DM4 concentration was determined to 48.39 microgm/mL by
absorbance. Therefore, the preparation has 2.95 moles of DM4 per
mole of CNTO95 (1 .mu.g of DM4 is equivalent to 69.5 .mu.g of
conjugated CNTO95 antibody). By HPLC the preparation was 85.9%
monomer with 1.18% free drug.
[0185] Preparation of CNTO95-SPDB-DM4 (CNTO 365).
[0186] Monoclonal antibody CNTO 95 was conjugated to Maytansinoid
DM4 with SPDB linker as follows: 228.5 mg CNTO95 conjugated to 4.37
mg of DM4, the resulting 104.5 ml conjugate was stored at 2.degree.
C. to 8.degree. C. at 2.19 mg/mL of conjugated CNTO95-SPDB-DM4
antibody in PBS at pH 6.5. The DM4 concentration was determined to
41.84 microgm/mL by absorbance. Therefore, the preparation has 3.92
moles of DM4 per mole of CNTO95 (1 .mu.g of DM4 is equivalent to
52.3 .mu.g of conjugated CNTO95 antibody). By HPLC the preparation
was 93.6% monomer with 0.55% free drug.
[0187] Preparation of CNTO95-SPDB-DM4 (CNTO 365).
[0188] Monoclonal antibody CNTO 95 was conjugated to Maytansinoid
DM4 with SPDB linker as follows: 309 mg CNTO95 conjugated to 5.4 mg
of DM4, the resulting 130.7 ml conjugate was stored at 2.degree. C.
to 8.degree. C. at 2.36 mg/mL of conjugated CNTO95-SPDB-DM4
antibody in PBS at pH 6.5. The DM4 concentration was determined to
41.2 microgm/mL by absorbance. Therefore, the preparation has 3.57
moles of DM4 per mole of CNTO95 (1 .mu.g of DM4 is equivalent to
57.5 .mu.g of conjugated CNTO95 antibody). By HPLC the preparation
was 93.8% monomer with 0.40% free drug.
[0189] Preparation of CNTO95-SPP-DM1 (CNTO 364).
[0190] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM1 with SPP linker as follows: 270.6 mg CNTO95 was conjugated to
3.7 mg of DM1, the resulting conjugate was stored at 2.degree. C.
to 8.degree. C. at 2.74 mg/mL in PBS at pH 6.5. The DM1
concentration was determined to 37.6 .mu.g/mL by absorbance.
Therefore the preparation contains a ratio of 2.98 DM1 per mole of
CNTO95 (1 of DM microgm 1 is equivalent to 68.9 microgm of
conjugated CNTO95 antibody). By HPLC the preparation was 96.3%
monomer with 0.59% free drug.
[0191] Preparation of CNTO95-SPP-DM1 (CNTO 364).
[0192] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM1 with SPP linker as follows: 245 mg F105 conjugated to 4.28 mg
of DM1, the resulting 91.5 ml conjugate was stored at 2.degree. C.
to 8.degree. C. at 2.68 mg/mL of conjugated F105-SPP-DM1 antibody
in PBS at pH 6.5. The DM1 concentration was determined to 46.76
microgm/mL by absorbance. Therefore the preparation contains a
ratio of 3.79 moles of DM1 per mole of F105 (1 .mu.g of DM1 is
equivalent to 57.3 .mu.g of conjugated F105 antibody). By HPLC the
preparation was 90.3% monomer with 2.44% free drug.
[0193] Preparation of CNTO95-SPP-DM4 (CNTO 366)
[0194] Monoclonal antibody CNTO95 was conjugated to Maytansinoid
DM4 with SPP linker as follows: 76.7 mg CNTO95 conjugated to 1.10
mg of DM4, the resulting 35 ml of conjugate was stored at 2.degree.
C. to 8.degree. C. at 2.19 mg/mL of conjugated CNTO95-SPP-DM4
antibody in PBS at pH 6.5. The DM4 concentration was determined to
31.5 microgm/mL by absorbance. Therefore the preparation contains a
ratio of 2.96 moles of DM4 per mole of CNTO95 (1 .mu.g of DM4 is
equivalent to 69.4 .mu.g of conjugated CNTO95 antibody). By HPLC
the preparation was 97.3% monomer with 1.14% free drug.
[0195] Preparation of F105-SSNPB-DM4.
[0196] Monoclonal antibody F105 was conjugated to Maytansinoid DM4
with SSNPB linker as follows: 106 mg F105 was conjugated to 1.84 mg
of DM4, the resulting 25 ml conjugate was stored at 2.degree. C. to
8.degree. C. at 3.85 mg/mL in PBS at pH 6.5. The DM4 concentration
was determined to 73.45 .mu.g/mL by absorbance. Therefore, the
ratio of DM4 per mole of antibody is 3.57 (1 microgm of DM4 is
equivalent to 57.5 microgm of conjugated F105 antibody). By HPLC
the preparation was 85.1% monomer with 1.92% free drug.
[0197] Preparation of F105-SSNPB-DM4.
[0198] Monoclonal antibody F105 was conjugated to Maytansinoid DM4
with SSNPB linker as follows: 106 mg F105 was conjugated to 1.84 mg
of DM4, the resulting 30 ml conjugate was stored at 2.degree. C. to
8.degree. C. at 3.46 mg/mL in PBS at pH 6.5. The DM4 concentration
was determined to 57.93 .mu.g/mL by absorbance. Therefore the ratio
of DM4 per mole of antibody is 3.32 (1 microgm of DM1 is equivalent
to 65.4 microgm of conjugated F105 antibody). By HPLC the
preparation was 88.8% monomer with 1.85% free drug.
[0199] Preparation of F105-SSNPP-DM4.
[0200] Monoclonal antibody F105 was conjugated to Maytansinoid DM4
with SSNPP linker as follows: 105 mg F105 conjugated to 1.76 mg of
DM4, the resulting 28 ml of conjugate was stored at 2.degree. C. to
8.degree. C. at 3.41 mg/mL in PBS at pH 6.5. The DM4 concentration
was determined to 62.87 .mu.g/mL by absorbance. Therefore, the
preparation contains 3.45 moles of DM4 per mole of F105 (1 .mu.g of
DM4 is equivalent to 59.4 .mu.g of conjugated F105 antibody). By
HPLC the preparation was 85.9% monomer with 3.75% free drug.
[0201] Preparation of F105-SPDB-DM4.
[0202] Monoclonal antibody F105 was conjugated to Maytansinoid DM4
with SPDP linker as follows: 230.5 mg F105 conjugated to 4.12 mg of
DM4, the resulting 104.5 ml of conjugate was stored at 2.degree. C.
to 8.degree. C. at 2.21 mg/mL in PBS at pH 6.5. The DM4
concentration was determined to 39.41 microgm/mL by absorbance.
Therefore, the preparation contains 3.66 moles of DM4 per mole of
F105 (1 .mu.g of DM4 is equivalent to 56.0 .mu.g of conjugated F105
antibody). By HPLC the preparation was 89.2% monomer with 0.65%
free drug.
[0203] In summary, the following species were synthesized for
further analysis (Table 6).
TABLE-US-00001 TABLE 6 Methyl Groups Neighboring the Disulfide in
the Product Maytansinol Activating (Mab side:Drug Compound Drug
Reagent Mab Activating Reagent side) CNTO364 DM1 N-methyl, N-(1-
SPP or SSNPP 1:0 dithiomethyl-3-carboxy- propyl)alanine CNTO365 DM4
N-methyl, N-(1- SPDB or SSNPB 0:2 dithiomethyl-2-methyl-4-
carboxy-n-butyl)alanine CNTO366 DM4 N-methyl, N-(1- SPP or SSNPP
1:2 dithiomethyl-2-methyl-4- carboxy-n-butyl)alanine F105-DM1 DM1
N-methyl, N-(1- SPP or SSNPP 1:0 dithiomethyl-3-carboxy-
propyl)alanine F105-DM4 DM4 N-methyl, N-(1- SPDB or SSNPB 0:2
dithiomethyl-2-methyl-4- carboxy-n-butyl)alanine F105-DM4 DM4
N-methyl, N-(1- SPP or SSNPP 1:2 dithiomethyl-2-methyl-4-
carboxy-n-butyl)alanine
Example 3
CNTO 95-Maytansine Conjugate Binding to Tumor Cells
[0204] The ability and affinity of CNTO95-Maytansinoid conjugate
binding to living cells was tested.
[0205] Materials and Methods. CNTO 95, Centocor lot #95-VF30A03-1,
20 mg/ml in PBS; CNTO 364 (CNTO 95-SPP-DM1), ImmunoGen lot
#1806-164, 37.6 mg/ml of DM1, 2.74 mg/ml of conjugated antibody,
Endotoxin level <0.1 EU/mg; CNTO 365 (CNTO 95-SPDB-DM4),
ImmunoGen lot #2020-78, 41.2 mg/ml of DM4, 2.36 mg/ml of conjugated
antibody, Endotoxin level <0.1 EU/mg; CNTO 366 (CNTO95-SPP-DM4),
ImmunoGen lot #2020-48, 3.1.5 mg/ml of DM4, 2.19 mg/ml of
conjugated antibody, Endotoxin level <0.1 EU/mg.
[0206] Cells: HT29 human colon carcinoma and A549 human lung
carcinoma cells were from ATCC and maintained in alphaMEM
supplemented with 10% fetal bovine serum (FBS). A2780 human ovarian
carcinoma cells were obtained from National Cancer Institute. A2780
cells were cultured in RPMI 1640 medium containing 10% FBS. Cells
were harvested, rinsed, suspended in serum free DMEM, and
sequentially incubated for 60 minutes on ice with serial diluted
CNTO 95, CNTO 364, CNTO 365 and CNTO 366 and FITC-labeled
anti-human antibody (10 mg/ml). Absence of primary antibody or
substitution of primary antibody with isotype matched antibody
served as negative controls. Cells were immediately analyzed with a
FACS Scan II flow cytometer (Becton Dickinson, Mountain View,
Calif.). Data was analyzed with GraphPad Prism software using
non-liner regression to determine the concentration at 50% maximal
binding (Table 7). The effective binding constant was changed less
than two-fold in most cases.
TABLE-US-00002 TABLE 7 EC50 (mg/ml) Compound HT29 A549 A2780 CNTO
95 0.14 0.18 0.17 CNTO 364 0.19 0.27 0.27 CNTO 365 0.21 0.34 0.27
CNTO 366 0.29 0.42 0.30
Example 4
Cytoxicity of CNTO 95-Maytansine Conjugates to Tumor Cells
[0207] The ability of CNTO95-Maytansinoid conjugates to kill tumor
cells over time was tested in vitro. CNTO 364 (CNTO 95-SPP-DM1),
ImmunoGen lot #1806-164, 37.61 g/ml of DM1, 2.74 mg/ml of
conjugated antibody, Endotoxin level <0.1 EU/mg. CNTO 365 (CNTO
95-SPDB-DM4), ImmunoGen lot #2020-78, 41.2 .mu.g/ml of DM4, 2.36
mg/ml of conjugated antibody, Endotoxin level <0.1 EU/mg. CNTO
366 (CNTO95-SPP-DM4), ImmunoGen lot #2020-48, 31.5 .mu.g/ml of DM4,
2.19 mg/ml of conjugated antibody, Endotoxin level <0.1
EU/mg.
[0208] Human HT29 human colon carcinoma and human non-small cell
lung carcinoma cells A549 (ATCC) were cultured in .alpha.MEM
supplemented with 10% FBS at 37.degree. C. in the presence of 5%
CO.sub.2. Cells were seeded into white 96-well tissue culture
plates (5000 cells/well) in culture medium and incubated for 16
hrs. Serial dilutions of immunoconjugates were added to each
appropriate wells (0-20 .mu.g/ml). Tissue culture plates were
incubated at 37.degree. C. for 96 hrs.
[0209] ATPLIte assay was performed according manufacturer's
instruction. Data was analyzed with GraphPad Prism software using
non-liner regression (Table 8) to determine the concentration at
half maximal cell number as measured by luminosity.
TABLE-US-00003 TABLE 8 EC50 (.mu.g/ml) Immunoconjugate HT29 A549
CNTO 364 1.0 12 CNTO 365 0.24 0.3 CNTO 366 1.0 1.5
Example 5
CNTO 95-DM1 Treatment of Rats Bearing Human Melanoma-Derived
Tumors
[0210] The efficacy of CNTO 364 compared to CNTO95 against advanced
s.c. A375.S2 human melanoma cells was investigated.
[0211] CNTO 95-DM1 was prepared by ImmunoGen, Inc. Lot #1716-74B,
stock concentration: 2.59 mg/ml. 5 mg/kg of CNTO 95-DM1 is
equivalent to 74 .mu.g/kg of DM1. CNTO 95 was from Centocor, Lot
#5380-027, stock concentration=20 mg/ml. Human IgG-DM1: ChromPure
human IgG was from Jackson ImmunoResearch Laboratories. Human
IgG-DM1 was prepared by ImmunoGen, Lot #1762-50, stock
concentration 2.8 mg/ml. 5 mg/kg of this conjugate is equivalent to
74 .mu.g/kg of DM1. Maytansine: ImmunoGen, Lot #1710-121, stock
concentration=16.38 .mu.g/ml in PBS, pH 6.5. The stock solution of
Maytansine was diluted with PBS to 15 and 7.5 .mu.g/ml. PBS:
ImmunoGen, pH 6.5. A375.S2 human melanoma cells were purchased from
ATCC and subpassaged and stored in frozen aliquots at Centocor Cell
Biology Services.
[0212] Nine-week-old athymic nude rats were subcutaneously
inoculated with A375.S2 human melanoma cells. On day 14, when
average tumor volumes reached to 250-300 m.sup.3, animals were
randomized to groups of 9/10 and treatment initiated. CNTO 95-DM1
and appropriate control compounds were intravenously injected
(three injection every other day in the first week followed by one
injection per week for two weeks on days 11, 14, 16, 21 & 28.
Tumor sizes and body weights were recorded. FIG. 6 shows the change
in tumor volumes over time for human melanoma in nude mice. Tumor
volumes are expressed as mean+/-SEM (n=9 or 10). The arrows
indicate intravenous drug injections. The asterisk that one
non-responding animal was sacrificed since its tumor volume was
over 1500 mm.sup.3. All animals were sacrificed on day 35. Tumor
volumes were expressed as mean+/-SEM (n=9 or 10). The arrows
indicate intravenous drug administration. CNTO95-DM1 at 5 mg/kg
blocked tumor growth and reduced the average tumor volume whereas
CNTO95 at 10 mg/kg had no effect in this experiment.
[0213] In a second experiment in rats, the average tumor volumes
reached to 250 mm.sup.3 on day 14. Animals were randomly grouped
and the first dosing was intravenously administered on day 14.
Subsequent injections were given on D 16 and 18 of the same week
and then once per week thereafter on D 23 and D 31. All animals
were sacrificed on day 35. CNTO 95-DM1 5 mg/kg caused complete
regression of A375.S2 human melanoma xenografts in female athymic
rats (FIG. 7). Control compounds including PBS, free CNTO 95,
irrelevant antibody-DM1 conjugate, free maytansine, and free CNTO
95 plus free maytansine did not show any significant effects.
Example 6
CNTO 95-DM1 Treatment of Rats Bearing Human Colon Carcinoma-Derived
Tumors
[0214] The efficacy of CNTO 364 compared to CNTO95 against advanced
s.c. HT29 human colon carcinoma was investigated.
[0215] CNTO 364, ImmunoGen lot #1806-50, protein concentration 2.53
mg/ml, concentration of DM1 41.8 mg/ml, ratio of DM1 to CNTO 95 3.6
mole of DM1 per mole of CNTO 95. PBS or antibody F105-DM1 were used
as controls F105-DM1; ImmunoGen lot #1806-44, protein concentration
2.2 mg/ml, concentration of DM1 38.3 mg/ml, ratio of DM1 to F105
3.8 mole of DM1 per mole of F105. All test articles have been
tested for endotoxin contamination and LAL values are below 1.0
EU/mg. The HT29 human colon carcinoma cell line, which expresses
avb3, avb5, and avb6 integrins, was obtained from Centocor's cell
bank. This cell line was determined to be free from mycoplasma and
bacterial agents. Cells were cultured in aMEM supplemented with 10%
FBS, 1% pyruvate, and 1% MEM non-essential amino acid in the
presence of 5% CO2 at 37.degree. C.
[0216] Seventy female athymic rats obtained from Harlan
Laboratories (Indianapolis, Ind.) were used in this study. Rats
were injected with 5.times.10.sup.6 HT29 cells subcutaneously (0.2
ml of 25.times.10.sup.6 cells/ml) on the rear flank area (dorsal
side, approximately 0.5 inches caudal to the last rib and 0.5
inches from the backbone). All rats were monitored daily (work
days) for the appearance of palpable tumor. The animals were
stratified by individual tumor volume into seven groups, each
containing 9 animals (Table 9). The mean starting tumor volume for
all groups was between 250-260 mm3.
TABLE-US-00004 TABLE 9 CNTO 364 Group N (mg/kg) Days of Dosing 1)
PBS 9 0 7, 14, 21, 28, and 35 2) F105-DM1 9 25 7 and 14 3) CNTO 364
9 3 7, 14, 21, 28, and 35 4) CNTO 364 9 6 7, 14, 21, 28, and 35 5)
CNTO 364 9 10 7, 14, 21, 28, and 35 6) CNTO 364 9 15 7, 14, and 35
7) CNTO 364 9 25 7 and 14
[0217] On the day of grouping (Day 0), animals were weighed and
intravenously injected with control antibody F105-DM1 at 25 mg/kg
or CNTO 364 at 3, 6, 10, 15 or 25 mg/kg. All test and control
articles were given in a volume of 1 ml/100 gm of body weight. CNTO
364 at 3, 6, and 10 mg/kg groups was administered i.v. on a
q7d.times.5 schedule. CNTO 364 at 15 mg/kg was dosed on day 7, 14,
and 35. CNTO 364 at 25 mg/kg and F105-DM1 at 25 mg/kg was dosed on
days 7 and day 14. The latter two groups were euthanized because of
significant body weight loss (more than 10% from day 0) in
accordance with the facility's IACUC guidelines (FIG. 8).
[0218] Tumor volume measurements were recorded twice weekly. Tumors
were measured with calipers in two dimensions (length and width) in
millimeters (mm). Tumor volume (mm3) was calculated using the
formula V=(length.times.width.times.width)/2. Statistics were
performed with GraphPad Prism software using unpaired t-test:
[0219] CNTO 364 inhibited the growth of established colon tumors in
a dose-dependent manner (FIG. 9). CNTO 364 at 10 mg/kg on
q7d.times.5 dosing schedule produced 3 complete tumor regressions
and 2 partial regressions out of 9 animals (FIG. 10). Treatment
with CNTO 364 at 15 mg/kg on day 7, 14 and 35 produced 4 complete
regressions and 4 partial regressions in the 9 tumor bearing
animals. The PBS control group was terminated on 35 post tumor cell
implantation (mean tumor volume over 5000 mm3). At that time the
difference in tumor volumes between the CNTO 364 at 10 mg/kg and 15
mg/kg treated groups versus PBS control had a P value of <0.0001
using a two-tailed unpaired t test.
[0220] Two consecutive injections for both CNTO 364 at 25 mg/kg and
F105-DM1 at 25 mg/kg were toxic and produced unacceptable body
weight loss (FIG. 8). However, a single injection of CNTO 364 at 25
mg/kg completely regressed advanced HT29 human colon carcinoma
tumors with mean tumor volume of above 4000 mm3 (not shown)
Example 7
CNTO 95-DM1 Treatment of Rats Bearing Human Lung Carcinoma-Derived
Tumors
[0221] This study was conducted to evaluate the in vivo efficacy of
a CNTO 95-DM1 conjugate, in female athymic rats bearing A549 human
lung carcinoma. The CNTO 95 was prepared at Centocor, (Malvern,
Pa.) and the conjugation of DM1 performed by ImmunoGen (Cambridge,
Mass.). CNTO 364 is (CNTO95-SPP-DM1) was as described in Example
1.
[0222] Human lung carcinoma cell line A549 (ATCC) were cultured in
MEMalpha containing 10% FBS and cells were prepared in serum free
.alpha.MEM for subcutaneous implantation in athymic rats. The rear
flank region of female athymic rats (6 weeks of age Harlan
Laboratory, Indianapolis, Ind.) were implanted with
5.times.10.sup.6 cells subcutaneously (0.2 ml of 25.times.10.sup.6
cells/ml) on the rear flank area (dorsal side, approximately 0.5
inches caudal to the last rib and 0.5 inches from the backbone).
When mean tumor volumes reached to 250 mm.sup.3, animals were
stratified into dosage groups with similar average tumor volumes
(Table 10) and dosed intravenously on days 17 and 29 after tumor
cell injection.
TABLE-US-00005 TABLE 10 Group Drug Dose on Day 17 and 29 1 PBS N/A
2 F105-DM1 15 mg/kg 3 CNTO 364 15 mg/kg 4 CNTO 95 alone 15 mg/kg 5
CNTO 95 + 15 mg/kg + 260 microgm/kg Maytansine 6 Maytansine alone
260 microgm/kg
[0223] Tumor volume measurements were recorded twice weekly. Tumors
were measured with electronic Vernier calipers in two dimensions
(length and width) in millimeters (mm). Tumor volume (mm) was
calculated using the formula V=(length.times.width.times.width)/2.
Tumor volumes are expressed as mean+/-SEM (n=6). These results were
plotted over time, FIG. 12, where the arrows indicate the time of
intravenous drug injections. Using body weight as a indicator for
tolerability (FIG. 11) also shows that the CNTO 364 dosing schedule
used in this study was well tolerated by animals, producing only a
3% body weight transient loss in initial body weight after the
first dosing.
[0224] One-way analysis of variance (ANOVA) with Bonferroni test
was performed with GraphPad Prism 4 software (GraphPad Software,
Inc., San Diego, Calif.) using a 95% confidence interval. Referring
to FIG. 12: Group 1, PBS; group 2, F105-DM1 at 15 mg/kg; group 3,
CNTO 364 at 15 mg/kg; group 4, CNTO 95 alone at 15 mg/kg; group 5,
CNTO 95 at 15 mg/kg plus maytansine at 260 mg/kg; group 6,
maytansine alone at 260 mg/kg. P value was determined by one-way
analysis of variance (ANOVA) with the Bonferroni test for multiple
comparisons. *P<0.05, CNTO 364 v.s. F105-DM1, CNTO 95 plus
maytansine or maytansine alone; **P<0.01, CNTO 364 v.s. CNTO 95
alone; ***P<0.001, CNTO 364 v.s. PBS
[0225] Conjugation of DM1 to CNTO 95, a large molecule, might
change the pharmacokinetic properties of DM1 by prolonging the
half-life of DM1 in vivo. To exclude this possibility, an
irrelevant antibody F105-DM1 immunoconjugate was included in this
study to determine if the activity of CNTO 95-DM1 conjugate was
CNTO 95-dependent. Since CNTO 95 is an anti-angiogenic and
anti-tumor compound and DM1 is a cytotoxic agent, it is possible
that the anti-tumor activity was due to the simple additive effects
of free CNTO 95 and free DM1. Therefore, free CNTO 95, free CNTO 95
plus free maytansine, and free maytansine dosed groups were
included as controls. As shown in FIG. 13, CNTO 364 at 15 mg/kg
eliminated six out of six tumors A549 lung tumor xenograft on the
q.times.12d.times.2 dosing schedule.
[0226] One complete tumor regression in the F105-DM1 group and two
in the CNTO 95 plus maytansine group were observed. These results
demonstrate the superiority of the CNTO 95-DM1 immunoconjugate,
CNTO 364, in A549 lung tumor regression. Animals treated with
CNTO364 on this dosing regimen showed a transient skin toxicity and
body weight loss, no overt signs of severe toxicity.
Example 8
Comparison of Conjugate Structures in a Human Colon Carcinoma Tumor
Models
[0227] An advanced tumor model using subcutaneously implanted HT29
human colon tumor-derived cells in immunocompromised rats was
chosen to examine tolerability and potency of various linkages used
to prepare immunoconjugates of CNTO 95.
[0228] CNTO 95-SPP-DM1 (CNTO 364), CNTO 95-SSNPB-DM4 (CNTO 365),
CNTO 95-SSNPP-DM4 (CNTO 366), and the Mab F105 equivalents of these
were prepared by ImmunoGen (Cambridge, Mass.). One hundred female
athymic rats (4-6 weeks of age) were obtained from Harlan
Laboratories.
[0229] Human HT29 (ATCC) were cultured in aMEM supplemented with
10% FBS at 37.degree. C. in the presence of 5% CO.sub.2. Cells were
prepared at a concentration of twenty-five million cells per ml in
serum free aMEM for inoculation. Female athymic rats were
inoculated with 5.times.10.sup.6 HT29 cells subcutaneously (0.2 ml
of 25.times.10.sup.6 cells/ml) on the rear flank area (dorsal side,
approximately 0.5 inches caudal to the last rib and 0.5 inches from
the backbone). All rats were monitored twice per week for the
appearance of tumor. The animals were stratified into 13 groups, 6
animals per group based on a mean tumor volume for each group of
approximately 250 mm.sup.3. On the day of grouping (Day 7) each
group received its initial dosing as listed in Table 11. Doses of
immunoconjugates were calculated based on the contents of DM1 or
DM4 in each conjugate. The L and H represent 175 and 350 .mu.g/kg
of DM1 or DM4, respectively. All test articles were given in a
volume of 1 ml/100 gm of body weight. Except for group 11, which
received only one dose on day 7, all other groups were dosed on day
7 and day 21. Changes in tumor volumes were used as indicators of
potency (FIGS. 14A & B) and changes in body weight (FIGS. 15A
& B) were used to monitor tolerability. All measurements are
expressed as the group mean+/-SEM (n=6).
TABLE-US-00006 TABLE 11 Ab conjugate administered Dose of DMx Group
No (mg/kg) microgm/kg 1. PBS 0 0 2. F105-SPP-DM1 11.5 DM1: 175 3.
F105-SPP-DM1 23 DM1: 350 4. F105-SSNPB- 10 DM4: 175 DM4 5.
F105-SSNPB- 20 DM4: 350 DM4 6. F15-SSNPP-DM4 10.5 DM4: 175 7.
F105-SSNPP- 21 DM4: 350 DM4 8. CNTO 364 10 DM1: 175 9. CNTO 364 20
DM1: 350 10. CNTO 365 10 DM4: 175 11. CNTO 365 20 DM4: 350 12. CNTO
366 12 DM4: 175 13. CNTO 366 24 DM4: 350
[0230] Animals injected with CNTO 365 and F105-SSNPB-DM4 at 350
microgm/kg of DM4 lost more than 10% of original body weight after
the first dosing as shown in FIG. 14B.
[0231] Therefore, dosing was discontinued in these two groups after
one injection. The >10% body weight loss was transient and the
animals recovered within 10 days of cessation of treatment. The
remaining groups were dosed both on Day 7 and Day 21 and no
significant body weight loss was observed except for the high dose
CNTO 364 group, in which animals lost above 10% body weight.
Significant weight loss had not been seen in other experiments
using CNTO 364 on this dosing schedule. As shown in FIG. 14B,
single injection of CNTO 365 at high dose caused complete
regression of established sc human HT29 colon carcinoma in 4 out of
6 animals. CNTO 364 at high dose (350 microgm/kg of DM1) and CNTO
365 at low dose (175 microgm/kg of DM4) also regressed preformed
colon tumors (2 out of 6 animals in each group respectively) or
significantly inhibited the growth of HT29 colon tumors. However,
CNTO 364 low dose (175 microgm/kg of DM1) and CNTO 366 at both
doses did not have any significant effect on tumor sizes. These
results suggest that CNTO 365 has better potency and efficacy than
CNTO 364 and CNTO 366 when administered iv on a q14d.times.2
schedule in this tumor xenograft model.
[0232] It will be clear that the invention can be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0233] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
Sequence CWU 1
1
915PRTHomo sapiens 1Arg Tyr Thr Met His1 5 217PRTHomo sapiens 2Val
Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15 Gly310PRTHomo sapiens 3Glu Ala Arg Gly Ser Tyr Ala Phe Asp Ile1
5 10 411PRTHomo sapiens 4Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
Ala1 5 10 57PRTHomo sapiens 5Asp Ala Ser Asn Arg Ala Thr1 5
68PRTHomo sapiens 6Gln Gln Arg Ser Asn Trp Pro Pro1 5 7119PRTHomo
sapiens 7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15 Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Phe Asp Gly Ser
Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr65 70 75 80 Leu Gln Val Asn
Ile Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Ala Arg Gly Ser Tyr Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110
Thr Met Val Thr Val Ser Ser 115 8108PRTHomo sapiens 8Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 100 105 91048PRTHomo sapiens 9Met Ala Phe Pro Pro
Arg Arg Arg Leu Arg Leu Gly Pro Arg Gly Leu1 5 10 15 Pro Leu Leu
Leu Ser Gly Leu Leu Leu Pro Leu Cys Arg Ala Phe Asn 20 25 30 Leu
Asp Val Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly Ser Tyr 35 40
45 Phe Gly Phe Ala Val Asp Phe Phe Val Pro Ser Ala Ser Ser Arg Met
50 55 60 Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro
Gly Ile65 70 75 80 Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp Ser
Ser Thr Arg Arg 85 90 95 Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly
Asn Arg Asp Tyr Ala Lys 100 105 110 Asp Asp Pro Leu Glu Phe Lys Ser
His Gln Trp Phe Gly Ala Ser Val 115 120 125 Arg Ser Lys Gln Asp Lys
Ile Leu Ala Cys Ala Pro Leu Tyr His Trp 130 135 140 Arg Thr Glu Met
Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu145 150 155 160 Gln
Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Gln Asp 165 170
175 Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp
180 185 190 Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser
Phe Tyr 195 200 205 Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu
Ile Val Ser Lys 210 215 220 Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr
Asn Asn Gln Leu Ala Thr225 230 235 240 Arg Thr Ala Gln Ala Ile Phe
Asp Asp Ser Tyr Leu Gly Tyr Ser Val 245 250 255 Ala Val Gly Asp Phe
Asn Gly Asp Gly Ile Asp Asp Phe Val Ser Gly 260 265 270 Val Pro Arg
Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly 275 280 285 Lys
Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met Ala Ala 290 295
300 Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp
Tyr305 310 315 320 Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp
Arg Gly Ser Asp 325 330 335 Gly Lys Leu Gln Glu Val Gly Gln Val Ser
Val Ser Leu Gln Arg Ala 340 345 350 Ser Gly Asp Phe Gln Thr Thr Lys
Leu Asn Gly Phe Glu Val Phe Ala 355 360 365 Arg Phe Gly Ser Ala Ile
Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly 370 375 380 Phe Asn Asp Ile
Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys385 390 395 400 Gly
Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ala Val 405 410
415 Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met Pro Pro
420 425 430 Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys
Asn Gly 435 440 445 Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp
Arg Ala Ile Leu 450 455 460 Tyr Arg Ala Arg Pro Val Ile Thr Val Asn
Ala Gly Leu Glu Val Tyr465 470 475 480 Pro Ser Ile Leu Asn Gln Asp
Asn Lys Thr Cys Ser Leu Pro Gly Thr 485 490 495 Ala Leu Lys Val Ser
Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp 500 505 510 Gly Lys Gly
Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu Leu Leu 515 520 525 Leu
Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu 530 535
540 Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser Arg
Gly545 550 555 560 Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu
Arg Asp Glu Ser 565 570 575 Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr
Ile Phe Met Glu Tyr Arg 580 585 590 Leu Asp Tyr Arg Thr Ala Ala Asp
Thr Thr Gly Leu Gln Pro Ile Leu 595 600 605 Asn Gln Phe Thr Pro Ala
Asn Ile Ser Arg Gln Ala His Ile Leu Leu 610 615 620 Asp Cys Gly Glu
Asp Asn Val Cys Lys Pro Lys Leu Glu Val Ser Val625 630 635 640 Asp
Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr 645 650
655 Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu
660 665 670 Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val
Val Arg 675 680 685 Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe
Lys Thr Glu Asn 690 695 700 Gln Thr Arg Gln Val Val Cys Asp Leu Gly
Asn Pro Met Lys Ala Gly705 710 715 720 Thr Gln Leu Leu Ala Gly Leu
Arg Phe Ser Val His Gln Gln Ser Glu 725 730 735 Met Asp Thr Ser Val
Lys Phe Asp Leu Gln Ile Gln Ser Ser Asn Leu 740 745 750 Phe Asp Lys
Val Ser Pro Val Val Ser His Lys Val Asp Leu Ala Val 755 760 765 Leu
Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Ile Phe 770 775
780 Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr Glu
Glu785 790 795 800 Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu
Arg Asn Asn Gly 805 810 815 Pro Ser Ser Phe Ser Lys Ala Met Leu His
Leu Gln Trp Pro Tyr Lys 820 825 830 Tyr Asn Asn Asn Thr Leu Leu Tyr
Ile Leu His Tyr Asp Ile Asp Gly 835 840 845 Pro Met Asn Cys Thr Ser
Asp Met Glu Ile Asn Pro Leu Arg Ile Lys 850 855 860 Ile Ser Ser Leu
Gln Thr Thr Glu Lys Asn Asp Thr Val Ala Gly Gln865 870 875 880 Gly
Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu Ser Glu 885 890
895 Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu Lys Ile
900 905 910 Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile
Leu Tyr 915 920 925 Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn
Lys Glu Asn Gln 930 935 940 Asn His Ser Tyr Ser Leu Lys Ser Ser Ala
Ser Phe Asn Val Ile Glu945 950 955 960 Phe Pro Tyr Lys Asn Leu Pro
Ile Glu Asp Ile Thr Asn Ser Thr Leu 965 970 975 Val Thr Thr Asn Val
Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val 980 985 990 Pro Val Trp
Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu 995 1000 1005
Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg Val
1010 1015 1020 Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln
Pro His Glu1025 1030 1035 1040 Asn Gly Glu Gly Asn Ser Glu Thr
1045
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References