U.S. patent application number 12/916294 was filed with the patent office on 2011-04-28 for partially loaded antibodies and methods of their conjugation.
This patent application is currently assigned to Seattle Genetics, Inc.. Invention is credited to Stephen C. Alley, Michael Sun, Michael Torgov.
Application Number | 20110097322 12/916294 |
Document ID | / |
Family ID | 34919495 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110097322 |
Kind Code |
A1 |
Alley; Stephen C. ; et
al. |
April 28, 2011 |
PARTIALLY LOADED ANTIBODIES AND METHODS OF THEIR CONJUGATION
Abstract
A protein containing one or more activatable groups, e.g., an
antibody, is subjected to partial or complete reduction of one or
more such bonds to form reactive groups; the resulting protein is
reacted with a drug which is reactive with some of the reactive
groups, such as certain radiometals, chelating agents, and toxins,
so as to form a conjugate useful in, e.g., in vitro diagnosis, in
vivo imaging, and therapy.
Inventors: |
Alley; Stephen C.; (Seattle,
WA) ; Torgov; Michael; (Santa Monica, CA) ;
Sun; Michael; (Bothell, WA) |
Assignee: |
Seattle Genetics, Inc.
Bothell
WA
|
Family ID: |
34919495 |
Appl. No.: |
12/916294 |
Filed: |
October 29, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10591743 |
Aug 13, 2008 |
7837980 |
|
|
PCT/US05/07239 |
Mar 2, 2005 |
|
|
|
12916294 |
|
|
|
|
60549476 |
Mar 2, 2004 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/178.1 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 33/00 20180101; A61P 19/02 20180101; A61P 33/02 20180101; A61P
37/08 20180101; A61P 25/00 20180101; A61P 33/12 20180101; A61P
17/06 20180101; A61P 31/10 20180101; A61P 35/02 20180101; A61P 3/00
20180101; A61K 47/6803 20170801; A61P 31/00 20180101; A61P 43/00
20180101; A61P 31/08 20180101; A61P 7/00 20180101; A61P 11/02
20180101; A61P 39/00 20180101; A61K 47/6849 20170801; A61K 2039/505
20130101; A61P 27/02 20180101; A61P 33/10 20180101; A61P 21/04
20180101; A61P 31/04 20180101; A61P 31/22 20180101; A61P 17/00
20180101; Y02A 50/412 20180101; A61P 19/04 20180101; A61P 29/00
20180101; A61P 37/06 20180101; A61P 1/16 20180101; A61P 31/12
20180101; A61P 35/00 20180101; A61P 37/02 20180101; A61P 1/04
20180101; A61P 5/14 20180101; Y02A 50/30 20180101; A61P 3/10
20180101; A61P 25/28 20180101; A61P 9/00 20180101; A61P 11/06
20180101; C07K 16/2878 20130101; A61P 31/18 20180101; A61P 37/00
20180101; A61P 7/06 20180101 |
Class at
Publication: |
424/133.1 ;
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 37/02 20060101
A61P037/02; A61P 31/00 20060101 A61P031/00 |
Claims
1-75. (canceled)
76. A method for the treatment of cancer, immune disease,
autoimmune disease or infectious disease in a patient, comprising
administering to the patient an amount of a modified protein, which
is an antibody, comprising: points of conjugation for a cytotoxic
or cytostatic agent, wherein at least one point of conjugation for
the cytotoxic or cytostatic agent on the antibody can be readily
assigned, and wherein less than all possible points of conjugation
are available for conjugation to the cytotoxic or cytostatic
agent.
77. A method for the treatment of cancer, immune disease,
autoimmune disease or infectious disease in a patient, comprising
administering to the patient an amount of a modified protein, which
is a partially loaded, modified protein, comprising: a binding
region for interaction with a binding partner; at least two points
of conjugation having a similar accessibility or activability for
conjugation of a drug or label by chemical means; at least two
drugs or labels, each drug or label covalently linked to one point
of conjugation; wherein less than all of the possible points of
conjugation having a similar accessibility or activability are
linked to a drug or label.
78. The method of claim 77, wherein: the modified protein is an
antibody; the binding region is an antigen-binding domain of the
antibody; the points of conjugation are thiol groups; and the
antibody comprises at least one interchain disulfide bond.
Description
CONTINUITY
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/549,476, filed Mar. 2, 2004, the disclosure of
which is incorporated by reference herein in its entirety.
BACKGROUND
[0002] The present invention is directed to modified proteins
having at least one point of conjugation with, for example, a drug
resulting in specific isomers of the protein-drug conjugate, and to
methods for such conjugation resulting in the specific isomers. The
invention is further directed to antibodies to which cytotoxic
agents and/or cytostatic agents can be conjugated resulting in
specific isomers and methods for their conjugation.
[0003] Monoclonal antibodies (mAbs) are a valuable weapon in the
battle against cancer. mAbs are also used in the treatment of
immune disorders. To further advance the use of mAb-based therapies
for cancer and immune disorders, a number of novel approaches have
been explored. One approach is to increase the cytotoxic potential
of mAbs against tumor cells by attaching cell-killing payloads.
Molecules such as protein toxins, radionuclides, and anti-cancer
drugs have been conjugated to certain mAbs to generate
immunotoxins, radioimmunoconjugates, and antibody-drug conjugates
(ADCs), respectively.
[0004] Factors which have previously been considered in developing
ADCs have included the choice of antibody, and optimizing the
potency of the drug component, the stability of the linker, and the
method by which the drug was covalently attached to the mAb. The
common convention for producing ADCs conjugated through the
disulfide bonds has been by reducing all inter-chain disulfide
bonds of an antibody and reacting all the reduced mAb thiols with a
compound capable of interaction with all the reduced thiols,
forming uniformly-substituted ADCs with 8 drugs/mAb, i.e. "fully
loaded," without the ability to obtain specificity for certain site
of conjugation.
[0005] For example, the antigen CD30 is highly expressed on cancers
such as Hodgkin's disease (HD) and anaplastic large cell lymphomas
(ALCL). This expression of CD30, coupled with limited expression on
normal cells, makes it an attractive target for ADC therapy. The
chimeric mAb directed to CD30, cAC10, has antitumor activity
against HD both in vitro and in subcutaneous and disseminated SCID
mouse xenograft models. The anti-tumor activity of cAC10 was
enhanced by generating fully loaded ADCs in which all eight of the
interchain thiols were linked to derivatives of the cytotoxic agent
auristatin E as the drug component. These ADCs were highly
effective in murine xenograft models at well-tolerated doses.
[0006] Because the convention in the production of ADCs has been to
fully load them with drug, it was not previously appreciated that
partially-loaded ADCs could have the same or greater therapeutic
efficacy. Further, methods did not exist which could take into
consideration that other substitution patterns on antibodies could
produce equal or better therapeutic efficacy with equal or lower
toxicity. These and other limitations and problems of the past are
solved by the present invention.
BRIEF SUMMARY
[0007] The present invention provides protein-drug conjugates and
methods of making protein-drug and protein-labeled conjugates. Also
provided are proteins having points of conjugation for receiving a
drug or label. The conjugates can be used therapeutically,
diagnostically (e.g., in vitro or in vivo), for in vivo imaging,
and for other uses.
[0008] Generally, partially loaded, modified protein having
assignable conjugation points are provided. The modified proteins
generally include a binding region for interaction with a binding
partner and at least two points of conjugation, each point of
conjugation covalently linked a drug or label. Typically, less that
all possible points of conjugation having a similar accessibility
or activability are linked to a drug or label. The modified protein
can be, for example, an antibody, a receptor, a receptor ligand, a
hormone, a cytokine, or the like. The points of conjugation can be,
for example, amino groups, vicinal hydroxyl groups, hydroxyl
groups, carboxyl groups, or thiol groups. The protein can be, for
example, a receptor, a receptor ligand, a hormone, a cytokine, or
the like.
[0009] In further embodiments, a method of preparing a conjugate of
a protein having one or more disulfide bonds, and a drug reactive
with free thiols is provided. The method generally includes
partially reducing the protein with a reducing agent; and
conjugating the drug reactive with free thiols to the partially
reduced protein. In yet another embodiment, a method of preparing a
conjugate of a protein having one or more disulfide bonds, and a
drug reactive with free thiols, is provided. The method generally
includes fully reducing the protein with a reducing agent;
partially reoxidizing the protein with a reoxidizing agent; and
conjugating the drug reactive with free thiols to the antibody.
[0010] In some embodiments, a partially-loaded antibody is
provided. The antibody includes an antigen binding region, at least
one interchain disulfide bond, and at least two drugs or labels,
each drug or label conjugated to an interchain thiol. The points of
the conjugation of the drug or label optionally are readily
assignable. In an example, the antibody can have at least four
cytotoxic or cytostatic drugs, each drug conjugated to an
interchain thiol. In certain embodiments, the antibody has the
configuration of species 4A, 4B, 4C, 4D, 4E or 4F.
[0011] The partially-loaded antibody can be, for example, a murine,
humanized, human or chimeric antibody. The drug can be, for
example, a cytotoxic or cytostatic agent such as, for example,
MMAF, MMAE or AFP. Also provided are pharmaceutical compositions
comprising partially loaded antibodies.
[0012] In anther embodiment, antibodies are provided having at
least one point of conjugation for a cytotoxic or cytostatic agent,
wherein the point of conjugation for the cytotoxic or cytostatic
agent on the antibody can be readily assigned. On the antibody,
less than all possible points are conjugation are available for
conjugation to the cytotoxic or cytostatic agent. The points of
conjugation can be, for example, interchain thiols. The points of
conjugation can be, for example, at least one of species 4A through
4F.
[0013] A composition of modified antibodies having assignable
conjugation points is also provided. The composition can have, for
example, at least two, at least four, at least 6 at least 7, at
least 10 or more species of modified antibody. In one example, each
species can have at least one specified conjugation pairs having
two interchain thiols, and at least one interchain disulfide bond.
The antibody species can have, for example, 4A, 4B, 4C, 4D, 4E
and/or 4F. In further examples, the specified conjugation pair can
be at a constant light-constant heavy interchain disulfide bond
and/or at a constant heavy-constant heavy interchain disulfide
bond. The specified conjugation pair can be proximal to the
N-terminal end of the hinge region and/or proximal to the
C-terminal end of the hinge region. In another example, the
specified conjugation pair is at the constant light-constant heavy
interchain sulfide bond and at the hinge region located closer to
the N-terminal end of the modified antibody, or at the constant
light-constant heavy interchain disulfide bond and at the hinge
region located closer to the C-terminal end of the modified
antibody.
[0014] Each species of antibody optionally can include at least two
specified conjugation pairs at the constant light-constant heavy
interchain disulfide bonds or at least two specified conjugation
pairs at the hinge region interchain disulfide bonds. The
composition optionally can further include a pharmaceutically
acceptable carrier.
[0015] In yet another embodiment, a partially loaded antibody is
provided. The antibody includes at least one antigen-binding
domain, at least two reactive group on the antibody, and at least
two drugs or labels, each drug or label conjugated to a reactive
group to form a point of conjugation. The points of conjugation for
the drug or label are readily assignable.
[0016] In yet other embodiments, a method of reducing and
conjugating a drug to an antibody resulting in selectivity in the
placement of the drug is provided. The method generally includes
fully reducing the antibody with a reducing agent, treating the
fully reduced antibody with limiting amounts of a reoxidizing agent
to reform at least one interchain disulfide bond of the antibody,
such that at least two interchain thiols remain; and conjugating
the drug to the interchain thiols. The reoxidizing agent can be,
for example, 5,5'-dithio-bis-2-nitrobenzoic acid,
4,4'-dithiodipyridine, 2,2'-dithiodipyridine, sodium tetrathionate
or iodosobenzoic acid. The drug can be, for example, a cytotoxic or
cytostatic agent or an immunosuppressive agent. In some examples,
the drug can be a minor grove binder, AEB, AEVB, MMAF, MMAE or AFP.
The reducing agent can be, for example, DTT or TCEP.
[0017] In related embodiments, a method of reducing antibody
interchain disulfide bonds and conjugating a drug to the resulting
interchain thiols resulting in selectivity in the placement of the
drugs on the antibody is provided. The method generally includes
fully reducing the antibody with a reducing agent to form
interchain thiols; partially reoxidizing the antibody with a
reoxidizing agent to reform at least one interchain disulfide bond;
and conjugating the drug to the interchain thiols. The reoxidizing
agent can be, for example, 5,5'-dithio-bis-2-nitrobenzoic acid,
4,4'-dithiodipyridine, 2,2'-dithiodipyridine, sodium tetrathionate,
or iodosobenzoic acid. The reducing agent can be, for example, DTT
or TCEP. The drug can be, for example, MMAF, MMAE, or AFP. The
partially reoxidized antibody optionally can be purified prior to
conjugation.
[0018] In yet other related embodiments, a method of reducing
antibody interchain disulfide bonds and conjugating a drug to the
resulting interchain thiols to selectively locate drugs on the
antibody is provided. The method generally includes partially
reducing the antibody with a reducing agent to form at least two
interchain thiols; and conjugating the drug to the interchain
thiols of the partially reduced antibody. In an example, the
antibody is partially reduced with a limiting concentration of a
reducing agent in a buffer with a chelating agent. The drug can be
conjugated, for example, by cooling the antibody solution and
dissolving the drug in a cold solvent and mixing with the antibody
solution. The antibody and drug solution are incubated for a period
of time sufficient to form a partially loaded antibody-drug
conjugate(s). The reaction can be quenched with a quenching the
excess drug with a thiol-containing reagent. The conjugate can be
further purified. In a specific example, the antibody is partially
reduced for about 1 hour at about 37.degree. C. The reduced
antibody can be cooled, for example, to about 0.degree. C. The
antibody and drug solution can be incubated, for example, for about
30 minutes at about 0.degree. C.
[0019] The thiol-containing reagent can be, for example, cysteine
or N-acetyl cysteine. The reducing agent can be, for example, DTT
or TCEP. The buffer can be, for example, a sodium borate solution
and the chelating agent is dethylenetriaminepentaacetic acid. The
chelating agent also can be, for example,
ethylenetriaminepentaacetic acid or EDTA. The solvent can be, for
example, acetonitrile, alcohol or DMSO. The drug can be, for
example, a cytotoxic or a cytostatic agent.
[0020] In some embodiments, the reduced antibody can be purified
prior to conjugation, using for example, column chromatography,
dialysis, or diafiltration. The column used in column
chromatography can be, for example, a desalting column, such as a
PD-10 column. Alternatively, the reduced antibody is not purified
after partial reduction and prior to conjugation.
[0021] The conjugate can be purified using, for example, column
chromatography, dialysis, or diafiltration. The column used in
column chromatography can be, for example, a desalting column, such
as a PD-10 column.
[0022] In yet another embodiment, a method of producing an antibody
with selective conjugation of drug is provided. The method
generally includes fully reducing the antibody for a period of time
sufficient to produce interchain thiols, as determined by, for
example, DTNB titration, by adding a large excess of a reducing
agent and incubating the solution at about 37.degree. C. for about
30 minutes; purifying the antibody; partially reoxidizing the
antibody using an oxidizing agent to form at least one interchain
disulfide bond by cooling the reduced antibody to 0.degree. C.;
treating the reduced and cooled antibody with 1.5 to 2.5 molar
equivalents of the oxidizing agent; mixing the solution by
inversion; allowing the solution to incubate at about 0.degree. C.
for about 10 minutes; purifying the partially reoxidized antibody;
conjugating the drug to the interchain thiols of the partially
reoxidized antibody to form a conjugated antibody; and purifying
the conjugated antibody.
[0023] The reducing agent can be, for example, DTT or TCEP. The
partially-reduced antibody optionally can be purified, for example,
using column chromatography, dialysis, or diafiltration. The column
used in column chromatography can be, for example, a desalting
column such as as a PD-10 column. The conjugated antibody can be
purified, for example, using column chromatography, dialysis, or
diafiltration. The column used in column chromatography can be, for
example, a desalting column such as as a PD-10 column. The
reoxidizing agent can be, for example,
5,5'-dithio-bis-2-nitrobenzoic acid, 4,4'-dithiodipyridine,
2,2'-dithiodipyridine, sodium tetrathionate, or iodosobenzoic
acid.
[0024] The invention will best be understood by reference to the
following detailed description of the specific embodiments, taken
in conjunction with the accompanying drawings. The discussion below
is descriptive, illustrative and exemplary and is not to be taken
as limiting the scope defined by any appended claims.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0025] FIG. 1 shows the "E4" isomers (isomers with four drugs
attached per antibody) of a cartoon antibody. The interchain
disulfide bonds are shown as solid lines between the heavy-heavy
chains of the antibody or the heavy-light chains of the antibody.
The drugs and their points of conjugation to the antibody are shown
as circles. The fragments generated under non-reducing ("Non-red")
and reducing ("Red") conditions are shown below each isomer (with
the number of drugs per fragment in parentheses).
[0026] FIG. 2 shows a chromatogram of the elution profile of a
hydrophobic interaction chromatography (HIC) analysis of
vcMMAE-cAC10 conjugate prepared by one aspect of method 2a: partial
DTNB reoxidation, PD-10 purification, and vcMMAE conjugation. "E0",
"E2", "E4", "E6" and "E8" refer to the isomers of the cAC10
antibody with 0, 2, 4, 6 and 8 MMAF molecules attached per
antibody, respectively.
[0027] FIG. 3 shows a HIC chromatogram for another aspect of method
2b: one-pot DTNB reoxidation and vcMMAE conjugation. E0", "E2",
"E4", "E6" and "E8" refer to the isomers of the cAC10 antibody with
0, 2, 4, 6 and 8 MMAF molecules attached per antibody,
respectively. Pure E4 was collected from about 34-38 min (indicated
by arrow).
[0028] FIG. 4 shows bar graph comparison of the percent composition
of antibody-drug conjugates for even drug loaded species from
methods 1, 2a, and 2b. For each species, antibody-drug conjugates
were prepared by the DTT partial reduction (left bar), DTNB
reoxidation (middle bar) or one pot DTNB reoxidation (right bar)
conjugation methods.
[0029] FIG. 5 shows a bioanalyzer trace for E4 material collected
as indicated in the HIC chromatogram in FIG. 3 (method 2b). "L"
indicates free light chains. "H" indicates free heavy chains. "HL"
indicates associated heavy-light chains. "HH" indicates associated
heavy-heavy chains. "HHL" indicates associated heavy-heavy and
heavy-light chains.
[0030] FIG. 6 shows PLRP analysis of material collected from the
HIC chromatogram in FIG. 3 (method 2b). "L0" and "L1" indicate a
light chain with no or one drug molecule attached, respectively.
"H0", "H1", "H2" and "H3" indicate a heavy chain with zero, one,
two or three drug molecules attached.
[0031] FIG. 7 shows a cartoon representation of the major conjugate
species obtained by reduction, full or partial, of the inter-chain
disulfide bonds followed by conjugation with a drug. Full reduction
and conjugation produces primarily the fully-loaded species with 8
drugs per antibody, whereas partial reduction and conjugation can
lead to the generation of all the shown species. There is only one
isomer each for the 0 and 8 drug-loaded species, whereas the 2, 4
and 6 drug-loaded species contain 3, 4 and 3 isomers, respectively.
(Referring to FIG. 1, note that species 4A is a mirror image of
species 4C and species 4B is a mirror image of species 4D. In FIG.
7, species 4A and 4C are referred to as 4A, and species 4B and 4D
are referred to as species 4B.) The interchain disulfide bonds are
shown as solid lines between the heavy-heavy chains or the
heavy-light chains of the antibody. The drugs and their point of
conjugation to the antibody are shown as circles.
[0032] FIG. 8 is a process flow diagram for one aspect of a
"Partial Reduction" conjugation process using DTT to produce E4
mixed isomers (E4M). The pH of the cAC10 antibody is adjusted to
7.5 with sodium phosphate dibasic and EDTA is added to a final
concentration of 5 mM. The antibody solution is then heated to
37.degree. C. To partially reduce the antibody, 2.95 molar
equivalents of DTT is added to the antibody solution and allowed to
reduce for 105 min at 37.degree. C. After reduction, the antibody
solution is cooled down to 2-8.degree. C. and the excess DTT
removed by constant-volume ultrafiltration/-diafiltraton (UF/DF) to
obtain the reduced, purified cAC10. A sample of the reduced,
purified cAC10 is taken and the thiol concentration, the antibody
concentration, and the thiol-to-antibody molar ratio determined by
A280 and DTNB tests. A slight excess of the drug-linker vcMMAE
(typically 2-15% excess in the form of a DMSO solution) is then
added into the antibody solution to start the conjugation reaction.
The conjugation reaction is allowed to proceed for 30 min at
2-8.degree. C. to obtain the crude E4M. At the end of the
conjugation reaction, any excess vcMMAE drug-linker is quenched by
reacting with a large excess of cysteine for 15 min at 2-8.degree.
C. to obtain the quenched, crude E4M. Buffer-exchange and removal
of free drug and other small-molecule species is performed by
constant-volume UF/DF (typically 6-10 diavolumes) to obtain the E4M
drug substance.
[0033] FIG. 9 is a process flow diagram for another aspect of a
"Partial Reduction" conjugation process using TCEP, in which an
intermediate purification step is not used, to produce E4 mixed
isomers (E4M). The pH of the cAC10 antibody is adjusted to 7.5 with
sodium phosphate dibasic and EDTA is added to a final concentration
of 5 mM. The antibody solution is then heated to 37.degree. C. To
partially reduce the antibody, 2.20 molar equivalents of TCEP is
added to the antibody solution and allowed to reduce for 105 min at
37.degree. C. A sample of the reduction reaction is taken and the
thiol concentration, the antibody concentration, and the
thiol-to-antibody molar ratio determined by A280 and DTNB tests.
After reduction, the antibody solution is cooled down to
2-8.degree. C. A slight excess of the drug-linker vcMMAE (typically
2-15% excess in the form of a DMSO solution) is then added into the
antibody solution to start the conjugation reaction. The
conjugation reaction is allowed to proceed for 20 min at
2-8.degree. C. to obtain the crude E4M. At the end of the
conjugation reaction, any excess vcMMAE drug-linker is quenched by
reacting with a large excess of N-acetyl cysteine for 20 min at
2-8.degree. C. to obtain the quenched, crude E4M. Buffer-exchange
and removal of free drug and other small-molecule species is
performed by constant-volume UF/DF (typically 6-10 diavolumes) to
obtain the E4M drug substance.
[0034] FIG. 10 shows a graph of the internalization of
cAC10-conjugated antibody by CD30.sup.+ Karpas-299 cells. The cells
were combined with 1 .mu.g/mL of fluorescently-labeled cAC10 and
serial dilutions of either cAC10, cAC10-E2, cAC10-E4, or cAC10-E8
from 20 .mu.g/mL to 9 ng/mL. After incubation of the cells with the
antibody, the labeled cells were washed with staining media, and
the fluorescence was measured. The normalized fluorescence
intensities were plotted versus mAb concentration as described in
Example 8.
[0035] FIGS. 11A and 11B show graphs of the internalization of
cAC10 and cAC10-conjugated antibodies by CD30.sup.+ cells: A)
Karpas-299 and B) L540cy cells were incubated with serial dilutions
of cAC10 and E2, E4 and E8 species of cAC10 ADCs. Following a
96-hour incubation with the samples, [.sup.3H]-TdR was added and
its incorporation was measured. The radioactivity of the treated
samples was normalized to the untreated controls and plotted versus
concentration.
[0036] FIGS. 12A and 12B show in vivo efficacy cAC10 and
cAC10-conjugated antibodies in SCID mice bearing subcutaneous
xenografts. FIG. 12A shows the results with SCID mice bearing
Karpas-299 subcutaneous tumors injected with cAC10-E2 at 0.5 mg/kg
or 1.0 mg/kg every four days for four injections. cAC10-E4 and
cAC10-8 were dosed at either 0.25 or 0.5 mg/kg every four days for
four injections. FIG. 12B shows the results with SCID mice with
Karpas-299 subcutaneous tumors were treated with a single dose of
E2, E4 or E8 at 1.0 mg/kg.
[0037] FIGS. 13A-D show hydrophobic interaction chromatography HPLC
traces of (A) E4 mix; (B) E2 pure made by preparative HIC; (C) E4
pure made by preparative HIC; and (D) E6 pure made by preparative
HIC, respectively. Samples were made by DTT partial reduction
followed by MMAE conjugation. Chromatograms were normalized to the
height of the tallest peak in each chromatogram. Injections were 50
.mu.L of 5-10 mg/mL cAC10-vcMMAE in PBS mixed with 50 .mu.L of 2.0
M NaCl and 50 mM sodium phosphate pH 7. Separations were performed
at 30.degree. C.
[0038] FIG. 14 show PLRP-S HPLC traces of (A) E4 mix made by
partial DTT reduction followed by MMAE conjugation (top trace); (B)
E4 mix made by DTNB partial reoxidation followed by MMAE
conjugation (second trace from top); (C) E4 pure made by partial
DTT reduction followed by MMAE conjugation and purified by
preparative HIC (second trace from bottom); and (D) E4 pure made by
partial DTNB reoxidation followed by MMAE conjugation and purified
by preparative HIC (bottom trace). Injections were 20 .mu.L of 1
mg/mL cAC10-vcMMAE treated with 20 mM DTT for 15 min at 37.degree.
C. Separations were performed at 80.degree. C.
[0039] FIG. 15 shows Bioanalyzer (capillary electrophoresis) traces
of (A) E4 mix made by partial DTT reduction followed by MMAE
conjugation (top trace); (B) E4 mix made by DTNB partial
reoxidation followed by MMAE conjugation (second trace from top);
(C) E4 pure made by partial DTT reduction followed by MMAE
conjugation and purified by preparative HIC (second trace from
bottom); and (D) E4 pure made by partial DTNB reoxidation followed
by MMAE conjugation and purified by preparative HIC (bottom trace).
Samples were prepared under non-reducing conditions as directed by
the manufacturer.
DETAILED DESCRIPTION
[0040] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
I. Definitions
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art pertinent to the methods and compositions
described. As used herein, the following terms and phrases have the
meanings ascribed to them unless specified otherwise.
[0042] The term "drug" as used herein means an element, compound,
agent, or molecular entity, including, e.g., a pharmaceutical,
therapeutic, or pharmacologic compound. Drugs can be natural or
synthetic or a combination thereof. A "therapeutic drug" is an
agent that exerts a therapeutic (e.g., beneficial) effect on cancer
cells or immune cells (e.g., activated immune cells), either alone
or in combination with another agent (e.g., a prodrug converting
enzyme in combination with a prodrug). Typically, therapeutic drugs
useful in accordance with the methods and compositions described
herein are those that exert a cytotoxic, cytostatic, or
immunosuppressive effect. In certain embodiments, a drug is not a
radioactive element.
[0043] "Cytotoxic agent," in reference to the effect of an agent on
a cell, means killing of the cell.
[0044] "Cytostatic agent" means an inhibition of cell
proliferation.
[0045] The term "polypeptide" refers to a polymer of amino acids
and its equivalent and does not refer to a specific length of a
product; thus, "peptides" and "proteins" are included within the
definition of a polypeptide. Also included within the definition of
polypeptides are "antibodies" as defined herein. A "polypeptide
region" refers to a segment of a polypeptide, which segment may
contain, for example, one or more domains or motifs (e.g., a
polypeptide region of an antibody can contain, for example, one or
more CDRs). The term "fragment" refers to a portion of a
polypeptide typically having at least 20 contiguous or at least 50
contiguous amino acids of the polypeptide. A "derivative" includes
a polypeptide or fragment thereof having conservative amino acid
substitutions relative to a second polypeptide; or a polypeptide or
fragment thereof that is modified by covalent attachment of a
second molecule such as, e.g., by attachment of a heterologous
polypeptide, or by glycosylation, acetylation, phosphorylation, and
the like. Further included are, for example, polypeptide analogs
containing one or more analogs of an amino acid (e.g., unnatural
amino acids and the like), polypeptides with unsubstituted
linkages, as well as other modifications known in the art, both
naturally and non-naturally occurring. Polypeptide analogs include,
for example, protein mimetics and bombesin.
[0046] The term "antibody" as used herein refers to (a)
immunoglobulin polypeptides and immunologically active portions of
immunoglobulin polypeptides, i.e., polypeptides of the
immunoglobulin family, or fragments thereof, that contain an
antigen binding site that immunospecifically binds to a specific
antigen, or (b) conservatively substituted derivatives of such
immunoglobulin polypeptides or fragments that immunospecifically
bind to the antigen. Antibodies are generally described in, for
example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1988).
[0047] An "antibody derivative" as used herein means an antibody,
as defined above, that is modified by covalent attachment of a
heterologous molecule such as, e.g., by attachment of a
heterologous polypeptide, or by glycosylation, acetylation or
phosphorylation not normally associated with the antibody, and the
like.
[0048] The term "monoclonal antibody" refers to an antibody that is
derived from a single cell clone, including any eukaryotic or
prokaryotic cell clone, or a phage clone, and not the method by
which it is produced. Thus, the term "monoclonal antibody" as used
herein is not limited to antibodies produced through hybridoma
technology.
[0049] The term "interchain disulfide bond," in the context of an
antibody, refers to a disulfide bond between two heavy chains, or a
heavy and a light chain.
[0050] The term "interchain thiol" refers to a thiol group of an
antibody heavy or light chain that can participate in the formation
of an interchain disulfide bond.
[0051] A protein is referred to as "fully-loaded" when all points
of conjugation of a particular type and/or of similar reactivity
are conjugated to drugs, resulting in a homogeneous population of
protein-drug conjugate. A protein is referred to as
"partially-loaded" when only some of the possible points of
conjugation of a particular type and/or of a similar reactivity are
conjugated to drugs, resulting in formation of a certain isomer or
isomers of the protein-drug conjugate.
[0052] The term "isolated," in the context of a molecule or
macromolecule (e.g., an antibody) is one which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with the desired use (e.g.,
diagnostic or therapeutic) of the molecule, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous
solutes. In some embodiments, an isolated molecule or macromolecule
will be purified (1) to greater than 95%, or greater than 99%, by
weight of the molecule or macromolecule as determined by, for
example, the Lowry or Bradford methods, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated molecules and
macromolecules include the molecule and macromolecule in situ
within recombinant cells since at least one component of the
antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0053] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine having the general formula shown immediately following:
##STR00001##
[0054] The abbreviation "MMAE" refers to monomethyl auristatin E
having the general formula shown immediately following:
##STR00002##
[0055] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleunine-dolaproine-phenylalanine having the
general formula shown immediately following:
##STR00003##
[0056] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid. The
abbreviation "AEVB" refers to an ester produced by reacting
auristatin E with benzoylvaleric acid.
[0057] The phrase "pharmaceutically acceptable salt," as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a molecule or macromolecule. Acid addition salts can be
formed with amino groups. Exemplary salts include, but are not
limited, to sulfate, citrate, acetate, oxalate, chloride, bromide,
iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'
methylene bis-(2-hydroxy 3-naphthoate))salts. A pharmaceutically
acceptable salt may involve the inclusion of another molecule such
as an acetate ion, a succinate ion or other counterion. The
counterion may be any organic or inorganic moiety that stabilizes
the charge on the parent compound. Furthermore, a pharmaceutically
acceptable salt may have more than one charged atom in its
structure. Instances where multiple charged atoms are part of the
pharmaceutically acceptable salt can have multiple counter ions.
Hence, a pharmaceutically acceptable salt can have one or more
charged atoms and/or one or more counterion.
[0058] "Pharmaceutically acceptable solvate" or "solvate" refer to
an association of one or more solvent molecules and a molecule or
macromolecule. Examples of solvents that form pharmaceutically
acceptable solvates include, but are not limited to, water,
isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,
and ethanolamine.
II. Polypeptides, Proteins, Antibodies
[0059] The present invention provides protein-drug conjugates and
methods of making protein-drug conjugates. Also provided are
proteins having points of conjugation for receiving a drug. The
protein-drug conjugates can be used therapeutically, diagnostically
(e.g., in vitro or in vivo), for in vivo imaging, and for other
uses.
[0060] Various classes of proteins can be conjugated, including
antibodies, enzymes, glycosylated proteins, lectins, various
biological receptors, protein hormones, and other proteins that can
serve as a binding agent for a binding partner. The proteins
contain at least one reactive site, such as a disulfide bond, amino
group, hydroxyl group or carboxyl group, where conjugation of a
drug to the protein can occur.
[0061] The reactive site is accessible and capable of activation,
such as by chemical or means. In some embodiments, the protein to
be chemically activated for conjugation purposes is one containing
disulfide bonds non-essential for the intended use of the protein,
and/or one which would not interfere with the protein (such as but
not limited to causing degradation of the protein or interfere with
binding or other functions (e.g., effector function.)). In such a
protein, a disulfide bond is present as a result of the oxidation
of the thiol (--SH) side groups of two cysteine residues. These
residues may lie on different polypeptide chains, or on the same
polypeptide chain. As a result of the oxidation, a disulfide bond
(--S--S--) is formed between the beta carbons of the original
cysteine residues. After reduction, the residues are termed often
interchangeably half-cystines and cystine. Treatment of the
disulfide bond with a reducing agent causes reductive cleavage of
the disulfide bonds to leave free thiol groups. Examples of
proteins containing disulfide bonds include antibodies, many
enzymes, certain hormones, and certain receptors.
[0062] In some embodiments, the disulfide bond can be naturally
occurring. In some embodiments, a sulfhydryl group(s) can also be
chemically introduced into a protein (e.g., an antibody). Suitable
methods for introducing sulfhydryl groups include chemical means
(e.g., using a thiolating agent such as 2-IT), or using recombinant
DNA technology. For example, cysteine residues can be introduced
into a protein by mutagenesis of a nucleic acid encoding the
protein. See generally Sambrook et al., Molecular Cloning, A
Laboratory Manual, 3rd ed., Cold Spring Harbor Publish., Cold
Spring Harbor, N.Y. (2001); Ausubel et al., Current Protocols in
Molecular Biology, 4th ed., John Wiley and Sons, New York (1999);
which are incorporated by reference herein.) Sulfhydryl groups can
be introduced into a protein, for example, within the polypeptide
or at the carboxy-terminus.
[0063] In some embodiments, the protein is an antibody. Such an
antibody may be used in in vitro or in vivo diagnosis, in vivo
imaging, or therapy of diseases or conditions with distinctive
antigens. The basic unit of an antibody structure is a complex of
four polypeptides--two identical low molecular weight ("light")
chains and two identical high molecular weight ("heavy") chains,
linked together by both non-covalent associations and by disulfide
bonds. Different antibodies will have anywhere from one to five of
these basic units. The antibody may be represented schematically as
a "Y". Each branch of the "Y" is formed by the amino terminal
portion of a heavy chain and an associated light chain. The base of
the "Y" is formed by the carboxy terminal portions of the two heavy
chains. The node of the "Y" is referred to as the hinge region.
[0064] Five human antibody classes (IgG, IgA, IgM, IgD and IgE),
and within these classes, various subclasses (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.,
are recognized on the basis of structural differences, such as the
number of immunoglobulin units in a single antibody molecule, the
disulfide bridge structure of the individual units, and differences
in chain length and sequence. The class and subclass of an antibody
is its isotype.
[0065] The antibody can be an intact antibody or an antigen-binding
antibody fragment such as, for example, a Fab, a F(ab'), a
F(ab').sub.2, a Fd chain, a single-chain Fv (scFv), a single-chain
antibody, a disulfide-linked Fv (sdFv), a fragment comprising
either a V.sub.L or V.sub.H domain, or fragments produced by a Fab
expression library. Antigen-binding antibody fragments, including
single-chain antibodies, can comprise the variable region(s) alone
or in combination with the entirety or a portion of the following:
hinge region, C.sub.H1, C.sub.H2, C.sub.H3, C.sub.H4 and C.sub.L
domains. Also, antigen-binding fragments can comprise any
combination of variable region(s) with a hinge region, C.sub.H1,
C.sub.H2, C.sub.H3, C.sub.H4 and C.sub.L domains. In some
embodiments, an antibody fragment comprises at least one domain, or
part of a domain, that includes interchain disulfide bonds.
[0066] Typically, the antibodies are human, rodent (e.g., mouse and
rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or
chicken. As used herein, "human" antibodies include antibodies
having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries,
from human B cells, or from animals transgenic for one or more
human immunoglobulin, as described infra and, for example in U.S.
Pat. Nos. 5,939,598 and 6,111,166. The antibodies may be
monospecific, bispecific, trispecific, or of greater
multispecificity.
[0067] In some embodiments, the constant domains have effector
function. The term "antibody effector function(s)," or AEC, as used
herein refers to a function contributed by an Fc domain(s) of an
Ig. Such function can be effected by, for example, binding of an Fc
effector domain(s) to an Fc receptor on an immune cell with
phagocytic or lytic activity or by binding of an Fe effector
domain(s) to components of the complement system. Typically, the
effect(s) mediated by the Fe-binding cells or complement components
result in inhibition and/or depletion of the CD70 targeted cell.
The effector function can be, for example, "antibody-dependent
cellular cytotoxicity" or ADCC, "antibody-dependent cellular
phagocytosis" or ADCP, "complement-dependent cytotoxicity" or CDC.
In other embodiments, the constant domain lack one or more effector
functions.
[0068] The antibodies may be directed against antigen of interest,
such as medical and/or therapeutic interest. For example, the
antigen can be one associated with pathogens (such as but not
limited to viruses, bacteria, fungi, and protozoa), parasites,
tumor cells, or particular medical conditions. In the case of a
tumor-associated antigen (TAA), the cancer may be of the immune
system, lung, colon, rectum, breast, ovary, prostate gland, head,
neck, bone, or any other anatomical location. Antigens of interest
include, but are not limited to, CD30, CD40, Lewis Y, and CD70. In
some embodiments, the antigen is CD2, CD20, CD22, CD33, CD38, CD40,
CD52, HER2, EGFR, VEGF, CEA, HLA-DR, HLA-Dr10, CA125, CA15-3,
CA19-9, L6, Lewis X, alpha fetoprotein, CA 242, placental alkaline
phosphatase, prostate specific antigen, prostatic acid phosphatase,
epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4,
anti-transferrin receptor, p97, MUC1-KLH, gp100, MARTI, IL-2
receptor, human chorionic gonadotropin, mucin, P21, MPG, and Neu
oncogene product.
[0069] Some specific useful antibodies include, but are not limited
to, BR96 mAb (Trail et al. (1993), Science 261:212-215), BR64
(Trail et al. (1997), Cancer Research 57:100-105), mAbs against the
CD 40 antigen, such as S2C6 mAb (Francisco et al. (2000) Cancer
Res. 60:3225-3231), and mAbs against the CD30 antigen, such as AC
10 (Bowen et al. (1993) J. Immunol. 151:5896-5906). Many other
internalizing antibodies that bind to tumor specific antigens can
be used, and have been reviewed (see, e.g., Franke et al. (2000),
Cancer Biother Radiopharm. 15:459-76; Murray (2000), Semin Oncol.
27:64-70; Breitling et al., Recombinant Antibodies, John Wiley, and
Sons, New York, 1998). The disclosures of these references are
incorporated by reference herein.
[0070] The term "tumor-specific antigen" as used herein will be
understood to connote an antigen characteristic of a particular
tumor, or strongly correlated with such a tumor. However,
tumor-specific antigens are not necessarily unique to tumor tissue,
however, i.e., that antibodies to them may cross-react with
antigens of normal tissue. Where a tumor-specific antigen is not
unique to tumor cells, it frequently occurs that, as a practical
matter, antibodies binding to tumor-specific antigens are
sufficiently specific to tumor cells to carry out the desired
procedures without unwarranted risk or interference due to
cross-reactions. Many factors contribute to this practical
specificity. For example, the amount of antigen on the tumor cell
may greatly exceed the amount of the cross-reactive antigen found
on normal cells, or the antigen on the tumor cells may be more
effectively presented. Therefore the term "tumor-specific antigen"
relates herein to a specificity of practical utility, and is not
intended to denote absolute specificity or to imply an antigen is
unique to the tumor.
[0071] The antibody may be a polyclonal antibody or a monoclonal
antibody. When the subject is a human subject, the antibody may be
obtained by immunizing any animal capable of mounting a usable
immune response to the antigen. The animal may be a mouse, rat,
goat, sheep, rabbit or other suitable experimental animal. The
antigen may be presented in the form of a naturally occurring
immunogen, or a synthetic immunogenic conjugate of a hapten and an
immunogenic carrier. In the case of a monoclonal antibody, antibody
producing cells of the immunized animal may be fused with
"immortal" or "immortalized" human or animal cells to obtain a
hybridoma which produces the antibody. If desired, the genes
encoding one or more of the immunoglobulin chains may be cloned so
that the antibody may be produced in different host cells, and if
desired, the genes may be mutated so as to alter the sequence and
hence the immunological characteristics of the antibody produced.
Human monoclonal antibodies may be made by any of numerous
techniques known in the art (e.g., Teng et al. (1983), Proc. Natl.
Acad. Sci. USA. 80, 7308-7312; Kozbor et al. (1983) Immunology
Today 4, 72-79; and Olsson et al. (1982), Meth. Enzymol. 92,
3-16).
[0072] The antibody can be, for example, a murine, a chimeric,
humanized, or fully human antibody produced by techniques
well-known to one of skill in the art. Recombinant antibodies, such
as chimeric and humanized monoclonal antibodies, comprising both
human and non-human portions, which can be made using standard
recombinant DNA techniques, are useful antibodies. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine monoclonal and human immunoglobulin constant
regions. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and
Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein
by reference in their entirety.) Humanized antibodies are antibody
molecules from non-human species having one or more complementarity
determining regions (CDRs) from the non-human species and a
framework region from a human immunoglobulin molecule. (See, e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety.) Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in International
Publication No. WO 87/02671; European Patent Publication No.
184,187; European Patent Publication No. 171496; European Patent
Publication No. 173494; International Publication No. WO 86/01533;
European Patent Publication No.12,023; Berter et al. (1988),
Science 240:1041-1043; Liu et al. (1987), Proc. Natl. Mad. Sci. USA
84:3439-3443; Liu et al. (1987), J. Immunol. 139:3521-3526; Sun et
al. (1987), Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987), Cancer. Res. 47:999-1005; Wood et al. (1985), Nature
314:446-449; and Shaw et al. (1988), J. Natl. Cancer Inst.
80:1553-1559; Morrison (1985), Science 229:1202-1207; Oi et al.
(1986), BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al.
(1986), Nature 321:552-525; Verhoeyan et al. (1988), Science
239:1534; and Beidler et al. (1988), J. Immunol. 141:4053-4060;
each of which is incorporated herein by reference in its
entirety.
[0073] Completely human antibodies can be produced, for example,
using transgenic mice that are incapable of expressing endogenous
immunoglobulin heavy and light chains genes, but which can express
human heavy and light chain genes. The transgenic mice are
immunized in the normal fashion with a selected antigen or a
portion thereof. Monoclonal antibodies directed against the antigen
can be obtained using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA, IgM and IgE
antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol.
13:65-93). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., U.S. Pat. Nos.
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; each of
which is incorporated herein by reference in its entirety. Other
human antibodies can be obtained commercially from, for example,
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose,
Calif.).
[0074] Completely human antibodies that recognize a selected
epitope also can be generated using a technique referred to as
"guided selection." In this approach a selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a completely human antibody recognizing the same
epitope. See, e.g., Jespers et al. (1994), Biotechnology
12:899-903. Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom and Winter (1991), J. Mol. Biol. 227:381; Marks et al.
(1991), J. Mol. Biol. 222:581; Quan and Carter (2002), "The rise of
monoclonal antibodies as therapeutics." In Anti-IgE and Allergic
Disease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker,
New York, N.Y., Chapter 20, pp. 427-469.
[0075] The antibody can also be a bispecific antibody. Methods for
making bispecific antibodies are known in the art. Traditional
production of full-length bispecific antibodies is based on the
coexpression of two immunoglobulin heavy chain-light chain pairs,
where the two chains have different specificities (Milstein et al.,
1983, Nature 305:537-539). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of
which only one has the correct bispecific structure. Similar
procedures are disclosed in International Publication No. WO
93/08829, and in Traunecker et al. (1991), EMBO J.
10:3655-3659.
[0076] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. It is preferred to have the first heavy-chain
constant region (C.sub.H1) containing the site necessary for light
chain binding, present in at least one of the fusions. Nucleic
acids with sequences encoding the immunoglobulin heavy chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide
fragments in embodiments when unequal ratios of the three
polypeptide chains used in the construction provide the optimum
yields. It is, however, possible to insert the coding sequences for
two or all three polypeptide chains in one expression vector when
the expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0077] In an embodiment of this approach, the bispecific antibodies
have a hybrid immunoglobulin heavy chain with a first binding
specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. This asymmetric structure facilitates the separation
of the desired bispecific compound from unwanted immunoglobulin
chain combinations, as the presence of an immunoglobulin light
chain in only one half of the bispecific molecule provides for a
facile way of separation (International Publication No. WO
94/04690) which is incorporated herein by reference in its
entirety.
[0078] For further details for generating bispecific antibodies
see, for example, Suresh et al. (1986), Methods in Enzymology
121:210; Rodrigues et al. (1993), J. Immunology 151:6954-6961;
Carter et al. (1992), Bio/Technology 10:163-167; Carter et al.
(1995), J. of Hematotherapy 4:463-470; Merchant et al. (1998),
Nature Biotechnology 16:677-681. Using such techniques, bispecific
antibodies can be prepared for use in the treatment or prevention
of disease.
[0079] Bifunctional antibodies are also described in European
Patent Publication No. EPA 0 105 360. As disclosed in this
reference, hybrid or bifunctional antibodies can be derived either
biologically, i.e., by cell fusion techniques, or chemically,
especially with cross-linking agents or disulfide-bridge forming
reagents, and may comprise whole antibodies or fragments thereof.
Methods for obtaining such hybrid antibodies are disclosed for
example, in International Publication WO 83/03679, and European
Patent Publication No. EPA 0 217 577, both of which are
incorporated herein by reference.
[0080] In other embodiments, the antibody is a fusion protein of an
antibody, or a functionally active fragment thereof, for example in
which the antibody is fused via a covalent bond (e.g., a peptide
bond), at either the N-terminus or the C-terminus to an amino acid
sequence of another protein (or portion thereof, preferably at
least 10, 20 or 50 amino acid portion of the protein) that is not
the antibody. Preferably, the antibody or fragment thereof is
covalently linked to the other protein at the N-terminus of the
constant domain.
[0081] In yet other embodiments, the protein can be a fusion
protein of the binding portion of a non-antibody molecule fused via
a covalent bond to the antibody heavy and/or light chain constant
region domain, optionally including a hinge region. Such a fusion
protein optionally can include at least one, typically at least
two, interchain disulfide bonds. For example, the fusion protein
can include the C.sub.H1 and C.sub.L regions, and a hinge
region.
III. Activation Methods
[0082] In general, a drug can be coupled to a protein or other
suitable molecule at an activatable site. Suitable activatable
sites include conjugation points such as thiol groups, amino groups
(e.g., the epsilon amino group of lysine residues or at the
N-terminus of proteins), vicinal hydroxyl groups (1,2-diols) (e.g.,
oxidized carbohydrates) and carboxyl groups (e.g., the C-terminus
of proteins, aspartic acid and glutamic acid residues, and
carbohydrate, such as sialic acid residues).
[0083] A drug can be coupled directly to a conjugation point. For
example, a drug can be attached by alkylation of the s-amino group
of antibody lysines, reductive amination of oxidized carbohydrate
or reaction with a hydrazide, transesterification between hydroxyl
and carboxyl groups, amidation at amino groups or carboxyl groups,
and conjugation to thiols (e.g., interchain thiols) or introduced
thiols by, for example, alkylating lysines with 2-iminothiolane
(Traut's reagent). Suitable methods conjugating drugs to
conjugation points are disclosed in, for example, Current Protocols
in Protein Science (John Wiley & Sons, Inc.), Chapter 15
(Chemical Modifications of Proteins) (the disclosure of which is
incorporated by reference herein in its entirety.)
[0084] A drug also can be coupled indirectly via another molecule,
such as a linker. For example, a drug also can be conjugated via a
maleimide group coupled to a sulthydryl group in, for example, but
not limited to, the hinge region of an antibody. Antibody
conjugates can be made by reacting a maleimide-derivatized form of
the drug with the antibody. More specifically, antibody conjugates
can be made by reducing an antibody to produce the reduced
antibody, producing an amine drug, derivatizing the amine drug with
maleimide to produce a maleimide-derivatized drug, and reacting the
maleimide-derivatized drug with the antibody.
[0085] In an exemplary embodiment, an IgG.sub.1 such as cAC10
possesses many disulfide bonds, only four of which are interchain.
Because the four interchain disulfide bonds are clustered in the
highly-flexible hinge region and much more solvent-accessible than
other (intra-chain) disulfide bonds, reduction with an excess of,
for example, a reducing agent, such as but not limited to
dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine (TCEP), or
2-Mercaptoethanol, breaks all four bonds and generates eight
cysteines (i.e., containing the free thiol group). Conjugation of
all eight cysteines with the drug-linker generates a fully-loaded
conjugate with approximately eight drugs per antibody, as shown in
see FIG. 7.
[0086] The present invention surprisingly demonstrates that the
biological properties of ADCs can be improved with antibodies
having an average of 2, 2.5, 4 or 6 drugs per antibody, which
yields lower toxicity while maintaining the efficacy of fully
loaded conjugates, i.e. conjugates having 8 drugs per antibody. The
therapeutic window (concentration of drug-antibody conjugate where
toxicity is first seen divided by the lowest efficacious dose) of
partially-drug loaded conjugates is larger than antibodies with 8
drugs. There are a number of ways to conjugate the 8 cysteines with
4 drugs, yielding a large number of potential drug loaded species
(9 total, with 0 through 8 drugs per antibody, see FIGS. 1 and 7).
For those antibodies that have 4 drugs, there are 6 possible ways
to distribute the 4 drugs, yielding 6 isomers (See FIGS. 1 and 7).
The homogeneity of the 8 drug loaded species is lost when 4 drugs
per antibody is desired. For antibodies with 2 or 6 drugs per
antibody, there are three possible ways to distribute the drugs on
the molecules.
[0087] Methods to produce partially loaded ADCs (e.g., with 4 (E4)
rather than 8 drugs per antibody) include the following: method 1
("partial reduction") partial reduction of the antibody by a
reducing agent such as but not limited to DTT or TCEP followed by
conjugation, and method 2 ("full reduction and reoxidation") full
reduction of the antibody with a reducing agent such as but not
limited to DTT or TCEP, followed by partial reoxidation of the
antibody with a reoxidizing agent (for example but not limited to
5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), 4,4'-dithiodipyridine,
2,2'-dithiodipyridine, sodium tetrathionate, or iodosobenzoic acid)
and finally conjugation. In the full reduction and reoxidation
method, there are two aspects: 2a purification after DTNB
reoxidation, and 2b, no purification after DTNB reoxidation (one
pot reoxidation and drug conjugation). These methods yield
different percent of different species (e.g., for E4 from 25 to
40%) and also yield different isomeric mixtures of the possible
species. Encompassed in the disclosure are hybrids and variations
of the above methods which would be known to one of skill in the
art.
[0088] As an example for antibody drug conjugates, FIG. 1 shows the
6 possible E4 species (referred to as 4A through 4F species) that
can be generated during a conjugation reaction. Species 4A-D are
not individually distinguishable by certain analytic methods;
however, they can be distinguished from both 4E and 4F.
[0089] In one embodiment of method 1 of partial reduction,
conjugates with, for example, 4 drugs per antibody can be made by
full reduction of the antibody with, for example but not limited to
DTT, to yield 8 antibody cysteines followed by conjugation to 4
equivalents of drug. This leads to a mixture where antibodies have
from 0 to 8 drugs. Alternatively, if the antibody is reduced by
limiting quantities of, for example DTT, such that an average of
only 2 of the 4 disulfides are reduced (liberating 4 cysteines)
followed by complete drug conjugation, only even drug loaded
species (0, 2, 4, 6, and 8 drugs per antibody) will be formed. This
reduces the complexity of the mixture, which can be further reduced
by purification to isolate these different drug loaded species.
[0090] In some embodiments, certain potential points of conjugation
on a protein can be selectively activated. This selective
activation allows for ready assignability of the conjugation
site(s) of a drug on the protein. For example, treatment of an
antibody (e.g., cAC10) with limiting amounts of the strong reducing
agents DTT or TCEP results in the selective reduction of the
heavy-light chain disulfides. In another example, full DTT
reduction of an antibody followed by partial reoxidation using a
strong thiol oxidizing agent such as DTNB results in selective
reoxidation of the heavy-light chain hinge disulfides, leading to
drug predominantly conjugated on the heavy chain in the hinge
region. The isomer populations of E2 and E6 produced by both of
these methods can approach 90% isomeric homogeneity.
[0091] Following conjugation of the drug to the antibody, the
conjugated drug-antibody species can be separated. In some
embodiments, the conjugated antibody species can be separated based
on the characteristics of the antibody, the drug and/or the
conjugate. For example, hydrophobic interaction chromatograph (HIC)
has been successful in isolating and separating species
corresponding to 0, 2, 4, 6, and 8 drugs per antibody. The yields
of each of these drug loaded isomers by method 1 is close to what
would be expected by a statistical distribution. The 4 drug loaded
species is typically 30% of the total material.
[0092] Analytical methods have been developed to determine drug
loading and the location of the drugs on the antibody (see also
infra). Characterization of the pure 4 drug loaded conjugates
prepared by partial DTT reduction by Bioanalyzer (capillary
electrophoresis) and HPLC on a crosslinked divinylbenzene column
(PLRP) revealed that the drugs are predominantly located on
cysteines that originally made disulfides between the heavy and
light chains of the antibody. The specificity of the drug location,
where one isomer is favored over the other five isomers, unlike the
convention, is unexpected.
[0093] In another embodiment, method 2, full reduction with partial
reoxidation, to prepare drug-antibody conjugates with, for example,
4 drugs, the antibody was fully reduced with, for example but not
limited to, DTT and then treated with limiting amounts of, for
example but not limited to, DTNB to reform some of the disulfides
such that 4 antibody cysteine thiols remained. These cysteines were
conjugated to drug and analyzed by the methods described herein.
The yield of 4 drug loaded antibodies in the mixture increased to
as much as 40%, and once purified the location of the drug favored
placement on the cysteines that originally made disulfides between
the heavy chains in the hinge region. Both the yield of 4 drug
loaded antibodies and the selectivity compared to common
convention, which favors a different isomer from certain partial
reduction methods, are unexpected. Using various chemical means,
drug location within the antibody can be readily assigned for the
production of different isomers.
[0094] If reduction is controlled by addition of limiting amounts
of reducing agent, partial reduction occurs in which, on average,
less than four inter-chain disulfide bonds are broken per antibody.
Because all four inter-chain disulfide bonds are highly exposed,
reduction proceeds through various pathways and produces
partially-reduced antibody composed of a mixture of species with 0,
2, 4, 6, or 8 cysteines. Conjugation of partially-reduced antibody,
therefore can generate a mixture of conjugates with 0, 2, 4, 6, or
8 drugs per antibody, as shown in FIGS. 1 and 7. Depending on the
extent of partial reduction, the drug-load distribution (i.e., the
percent of 0, 2, 4, 6, or 8 drug-loaded species) changes.
[0095] Partial reduction not only produces a mixture containing
species with variable number of drugs per antibody, it also creates
further heterogeneity as a result of the multiple locations of drug
attachment. FIG. 7 shows that there is more than one isomer
possible for the 2, 4, and 6 drug-loaded species.
[0096] Following conjugation of the drug to a protein, the
conjugated drug-protein species can be separated. For example, in
some embodiments, the conjugated antibody species can be separated
based on the characteristics of the antibody, the drug and/or the
conjugate. For example, hydrophobic interaction chromatograph (HIC)
has been successful in isolating and separating species
corresponding to 0, 2, 4, 6, and 8 drugs per antibody.
IV. Analytical Methods
[0097] Various analytical methods can be used to determine the
yields and isomeric mixtures of the conjugates. For example, in one
embodiment HIC is the analytical method used to determine yields
and isomeric mixtures from resultant conjugates (e.g., for E4
conjugates). This technique is able to separate antibodies loaded
with various numbers of drugs. The drug loading level can be
determined based on the ratio of absorbances, e.g., at 250 nm and
280 nm. For example, if a drug can absorb at 250 nm while the
antibody absorbs at 280 nm. The 250/280 ratio therefore increases
with drug loading. Using the conjugation methods described herein,
generally antibodies with even numbers of drugs were observed to be
conjugated to the antibody since reduction of disulfides yields
even numbers of free cysteine thiols. FIGS. 2 and 3 show HIC
separations for cAC10-vcMMAE produced by methods 2a and 2b,
respectively. FIG. 4 shows the percent composition for the various
substitutions from these chromatograms as well as from method 1.
Method 1 yields about 30% E4, while method 2b yields about 40%
E4.
[0098] HIC can also be used preparatively at milligram to grain
levels to purify E4 from a mixture of substitution levels. Pure E4
from FIG. 3 (collection time of 34-38 min indicated) was obtained
and analyzed by two methods to determine the isomeric E4 mixture.
First, an Agilent Bioanalyzer was used, which denatures noncovalent
interactions and separates based on protein mass, yielding the
following antibody components in order of elution: light chain (L),
heavy chain (H), heavy-light (HL), heavy-heavy (HH),
heavy-heavy-light (HHL), and heavy-heavy-light-light (HHLL). The
smaller species are formed when disulfides are reduced and the free
thiols conjugated to vcMMAE.
[0099] FIG. 1 also describes which antibody components will be
observed from denaturation of the various E4 isomers. As can be
seen in FIG. 5, pure E4 prepared by method 2b is dominated by HL,
with a small amount of L and HHL. This result can be explained by
the presence of mostly species 4F (exclusively yields HL) with some
species 4A-D (yielding HHL and L). Interestingly, the same analysis
of cAC10-vcMMAE made by method 1 yields approximately equal amounts
of L, HL, and HH, which would be consistent with a mixture of
mostly species 4E and some species 4F.
[0100] Another embodiment of an analytical tool is chromatography
on a reversed-phase PLRP column; the column support is composed of
crosslinked divinylbenzene, rather than a typical reversed phase
column built on a silica support which can nonspecifically retain
proteins. This denaturing and reductive technique cleanly separates
the 6 species consisting of light chain with 0 and 1 drug (L0 and
L1) and heavy chain with 0 through 3 drugs (HO through H3). FIG. 1
shows the drug loading levels that can be observed for the various
E4 species. Pure E4 from method 2b was separated by PLRP in FIG. 6.
Unmodified light chain (L0) and heavy chain with two drugs (H2) are
the species expected from 4F, while L1 and H1 are expected from
4A-D. Together with the Bioanalyzer, these data are consistent with
method 1 producing about a 2:1 mixture of 4E to 4F while method 2b
produces 2:1 4F to 4A-D. Thus using different chemical conditions,
both the E4 yield and distribution of E4 isomers is significantly
different between method 1 and 2b.
V. Compound Capable of Conjugation to Protein.
[0101] A protein may be conjugated with any drug of interest,
including a cytostatic agent or cytotoxic agent, an
immunosuppressive agent, a toxin, a chelate, a compound, a
molecule, a radionucleotide, or the like.
[0102] Cytotoxic Agents and Cytostatic Agents
[0103] Cytotoxic and cytostatic drugs include antibiotics (e.g.,
adriamycin), antitumor agents such as auristatins and auristatin
derivatives, methotrexate, mitomycin C, daunorubicin, doxorubicin,
and vinblastine, 5-fluorouracil DNA minor grove binders, DNA
replication inhibitors, alkylating agents (e.g., platinum complexes
such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear
platinum complexes and carboplatin), antiparasitic agents (e.g.,
pentamidine isethionate), anthracyclines, antifolates,
antimetabolites, chemotherapy sensitizers, duocarmycins,
etoposides, fluorinated pyrimidines, ionophores, lexitropsins,
nitrosoureas, platinols, pre-forming compounds, purine
antimetabolites, puromycins, radiation sensitizers, steroids,
taxanes, topoisomerase inhibitors, vinca alkaloids, antimicrobial
agents, antimicrotubule agents, or the like. When an antibody is
conjugated to such a drug, it serves to direct the drug to the
sites where the corresponding antigen occurs. Other agents and
drugs which can be coupled to antibody are known, or can be easily
ascertained, by those of skill in the art.
[0104] Individual cytotoxic or cytostatic agents include, for
example, an androgen, anthramycin (AMC), asparaginase,
5-azacytidine, azathioprine, bleomycin, busulfan, buthionine
sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065,
chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine,
cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin
(formerly actinomycin), daunorubicin, decarbazine, docetaxel,
doxorubicin, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil,
gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan,
lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine,
methotrexate, mithramycin, mitomycin C, mitoxantrone,
nitroimidazole, paclitaxel, plicamycin, procarbizine,
streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan,
vinblastine, vincristine, vinorelbine, VP-16 and VM-26.
[0105] Other cytotoxic agents include, for example, dolastatins
(see infra) DNA minor groove binders such as the enediynes (e.g.,
calicheamicin) and lexitropsins (see, also U.S. Pat. No.
6,130,237), duocarmycins, taxanes (e.g., paclitaxel and docetaxel),
puromycins, CC-1065, SN-38, topotecan, morpholino-doxorubicin,
rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A, B or D, estramustine, cryptophysins,
cemadotin, maytansinoids, discodermolide, eleutherobin, and
mitoxantrone.
[0106] In certain embodiments, the cytotoxic agent is a
chemotherapeutic such as, for example, doxorubicin, melphalan,
vinca alkaloids, methotrexate, mitomycin C or etoposide. In
addition, potent agents such as CC-1065 analogues, calicheamicin,
maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can
be linked to proteins.
[0107] In specific embodiments, the cytotoxic or cytostatic agent
is auristatin E (also known in the art as dolastatin-10) or a
derivative thereof. Typically, the auristatin E derivative is,
e.g., an ester formed between auristatin E and a keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid
or benzoylvaleric acid to produce AEB and AEVB, respectively. Other
typical auristatin derivatives include AFP, MMAF, and MMAE. The
synthesis and structure of auristatin E and its derivatives are
described in U.S. patent application Ser. No. 09/845,786 (U.S.
Patent Application Publication No. 20030083263) and Ser. No.
10/001,191; International Patent Application No. PCT/US03/24209:
International Patent Application No. PCT/US02/13435: and U.S. Pat.
Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;
5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;
5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;
4,879,278; 4,816,444; and 4,486,414.
[0108] In certain embodiments, the cytotoxic or cytostatic agent is
an anti-tubulin agent. Examples of anti-tubulin agents include, but
are not limited to, taxanes (e.g., Taxol.RTM. (paclitaxel),
Taxotere.RTM. (docetaxel)), T67 (Tularik), vinca alkyloids (e.g.,
vincristine, vinblastine, vindesine, and vinorelbine), and
dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other
antitubulin agents include, for example, baccatin derivatives,
taxane analogs (e.g., epothilone A and B), nocodazole, colchicine
and colcimid, estramustine, cryptophysins, cemadotin,
maytansinoids, combretastatins, discodermolide, and
eleutherobin.
[0109] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine or DM-1
(ImmunoGen, Inc.; see also Chari et al. (1992), Cancer Res.
52:127-131).
[0110] In some embodiments, the therapeutic agent is not a
radioisotope.
[0111] In some embodiments, the cytotoxic or immunosuppressive
agent is an antimetabolite. The antimetabolite can be, for example,
a purine antagonist (e.g., azothioprine or mycophenolate mofetil),
a dihydrofolate reductase inhibitor (e.g., methotrexate),
acyclovir, gangcyclovir, zidovudine, vidarabine, ribavarin,
azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine,
iododeoxyuridine, poscarnet, or trifluridine.
[0112] In other embodiments, the cytotoxic or immosuppressive agent
is tacrolimus, cyclosporine or rapamycin. In further embodiments,
the cytotoxic agent is aldesleukin, alemtuzumab, alitretinoin,
allopurinol, altretamine, amifostine, anastrozole, arsenic
trioxide, bexarotene, bexarotene, calusterone, capecitabine,
celecoxib, cladribine, Darbepoetin alfa, Denileukin diftitox,
dexrazoxane, dromostanolone propionate, epirubicin, Epoetin alfa,
estramustine, exemestane. Filgrastim, floxuridine, fludarabine,
fulvestrant, gemcitabine, goserelin, idarubicin, ifosfamide,
imatinib mesylate, Interferon alfa-2a, irinotecan, letrozole,
leucovorin, levamisole, meclorethamine or nitrogen mustard,
megestrol, mesna, methotrexate, methoxsalen, mitomycin C, mitotane,
nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate,
pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman,
plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase,
Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide,
testolactone, thioguanine, toremifene, tretinoin, uracil mustard,
vairubicin, vinblastine, vincristine, vinorelbine and
zoledronate.
[0113] In some embodiments, the agent is an immunosuppressive
agent. The immunosuppressive agent can be, for example,
gancyclovir, tacrolimus, cyclosporine, rapamycin, cyclophosphamide,
azathioprine, mycophenolate mofetil or methotrexate. Alternatively,
the immunosuppressive agent can be, for example, a glucocorticoid
(e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g.,
prednisone or dexamethasone).
[0114] In some embodiments, the immunosuppressive agent is an
anti-inflammatory agent, such as arylcarboxylic derivatives,
pyrazole-containing derivatives, oxicam derivatives and nicotinic
acid derivatives. Classes of anti-inflammatory agents include, for
example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and
leukotriene receptor antagonists.
[0115] Suitable cyclooxygenase inhibitors include meclofenamic
acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen,
fenoprofen, ibuprofen, indomethacin, ketoprofen, nabumetone,
naproxen, sulindac, tenoxicam, tolmetin, and acetylsalicylic
acid.
[0116] Suitable lipoxygenase inhibitors include redox inhibitors
(e.g., catechol butane derivatives, nordihydroguaiaretic acid
(NDGA), masoprocol, phenidone, Ianopalen, indazolinones,
naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox
inhibitors (e.g., hydroxythiazoles, methoxyalkylthiaiazoles,
benzopyrans and derivatives thereof, methoxytetrahydropyran,
boswellic acids and acetylated derivatives of boswellic acids, and
quinolinemethoxyphenylacetic acids substituted with cycloalkyl
radicals), and precursors of redox inhibitors.
[0117] Other suitable lipoxygenase inhibitors include antioxidants
(e.g., phenols, propyl gallate, flavonoids and/or naturally
occurring substrates containing flavonoids, hydroxylated
derivatives of the flavones, flavonol, dihydroquercetin, luteolin,
galangin, orobol, derivatives of chalcone,
4,2',4'-trihydroxychalcone, ortho-aminophenols, N-hydroxyureas,
benzofuranols, ebselen and species that increase the activity of
the reducing selenoenzymes), iron chelating agents (e.g.,
hydroxamic acids and derivatives thereof, N-hydroxyureas,
2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,
catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic
acid and 4-(omega-arylalkyl)phenylalkanoic acids),
imidazole-containing compounds (e.g., ketoconazole and
itraconazole), phenothiazines, and benzopyran derivatives.
[0118] Yet other suitable lipoxygenase inhibitors include
inhibitors of eicosanoids (e.g., octadecatetraenoic,
eicosatetraenoic, docosapentaenoic, eicosahexaenoic and
docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1),
PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic,
15-monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic
acids, and leukotrienes B5, C5 and D5), compounds interfering with
calcium flows, phenothiazines, diphenylbutylamines, verapamil,
fuscoside, curcumin, chlorogenic acid, caffeic acid,
5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide,
lonapalen, esculin, diethylcarbamazine, phenantroline, baicalein,
proxicromil, thioethers, diallyl sulfide and di-(1-propenyl)
sulfide.
[0119] Leukotriene receptor antagonists include calcitriol,
ontazolast, Bayer Bay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo
Denmark ETH-615, Lilly LY-293111, Ono ONO-4057, Terumo TMK-688,
Boehringer Ingleheim BI-RM-270, Lilly LY 213024, Lilly LY 264086,
Lilly LY 292728, Ono ONO LB457, Pfizer 105696, Perdue Frederick PF
10042, Rhone-Poulenc Rorer RP 66153, SmithKline Beecham SB-201146,
SmithKline Beecham SB-201993, SmithKline Beecham SB-209247, Searle
SC-53228, Sumitamo SM 15178, American Home Products WAY 121006,
Bayer Bay-o-8276, Warner-Lambert CI-987, Warner-Lambert
CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283, MacroNex
MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, Ono
ONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,
Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698,
Shionoogi S-2474, Searle SC-41930, Searle SC-50505, Searle
SC-51146, Searle SC-52798, SmithKline Beecham SK&F-104493, Leo
Denmark SR-2566, Tanabe T-757 and Teijin TEI-1338.
[0120] Toxins
[0121] Toxins are usefully conjugated to antibodies specific for
antigens associated with tumor, parasite or microbial cells. The
toxin may be from, e.g., a plant (e.g., ricin or abrin), animal
(e.g., a snake venom), or microbial (e.g., diphtheria or tetanus
toxin).
[0122] Besides antibodies, the drugs or toxins may be conjugated to
other carrier proteins, such as albumin.
[0123] Conjugation of Drugs to Protein
[0124] The drug has, or is modified to include, a group reactive
with a conjugation point on the protein. For example, a drug can be
attached by alkylation (e.g., at the .epsilon.-amino group of
antibody lysines or the N-terminus of protein), reductive amination
of oxidized carbohydrate, transesterification between hydroxyl and
carboxyl groups, amidation at amino groups or carboxyl groups, and
conjugation to thiols. For a examples of chemistries that can be
used for conjugation, see, e.g., Current Protocols in Protein
Science (John Wiley & Sons, Inc.), Chapter 15 (Chemical
Modifications of Proteins) (the disclosure of which is incorporated
by reference herein in its entirety.)
[0125] For example, when chemical activation of the protein results
in formation of free thiol groups, the protein may be conjugated
with a sulfhydryl reactive agent. In one aspect, the agent is one
which is substantially specific for free thiol groups. Such agents
include, for example, malemide, haloacetamides (e.g., iodo, bromo
or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl
ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g.,
iodide, bromide or chloride), vinyl sulfone and pyridyithio.
[0126] Sulfhydryl Reactive Agents
[0127] Sulfyhydryl reactive agents include alpha-haloacetyl
compounds such as iodoacetamide, maleimides such as
N-ethylmaleimide, mercury derivatives such as
3,6-bis-(mercurimethyl)dioxane with counter ions of acetate,
chloride or nitrate, and disulfide derivatives such as disulfide
dioxide derivatives, polymethylene bismethane thiosulfonate
reagents and crabescein (a fluorescent derivative of fluorescein
containing two free sulfhydryl groups which have been shown to add
across disulfide bonds of reduced antibody).
[0128] Alpha-haloacetyl compounds such as iodoacetate readily react
with sulfhydryl groups to form amides. These compounds have been
used to carboxymethylate free thiols. They are not strictly SH
specific and will react with amines. The reaction involves
nucleophilic attack of the thiolate ion resulting in a displacement
of the halide. The reactive haloacetyl moiety, X--CH.sub.2 CO--,
has been incorporated into compounds for various purposes. For
example, bromotrifluoroacetone has been used for F-19
incorporation, and N-chloroacetyliodotyramine has been employed for
the introduction of radioactive iodine into proteins.
[0129] Maleimides such as N-ethylmaleimide are considered to be
fairly specific to sulfhydryl groups, especially at pH values below
7, where other groups are protonated. Thiols undergo Michael
reactions with maleimides to yield exclusively the adduct to the
double bond. The resulting thioether bond is very stable. They also
react at a much slower rate with amino and imidazoyl groups. At pH
7, for example, the reaction with simple thiols is about 1,000 fold
faster than with the corresponding amines. The characteristic
absorbance change in the 300 nm region associated with the reaction
provides a convenient method for monitoring the reaction. These
compounds are stable at low pH but are susceptible to hydrolysis at
high pH. See generally Wong, Chemistry of Protein Conjugation and
Cross-linking; CRC Press, Inc., Boca Raton, 1991: Chapters 2 and
4).
[0130] A molecule (such as a drug) which is not inherently reactive
with sulfhydryls may still be conjugated to the chemically
activated proteins by means of a bifunctional crosslinking agent
which bears both a group reactive with the molecule of interest and
a sulfhydryl reactive group. This agent may be reacted
simultaneously with both the molecule of interest (e.g., through an
amino, carboxy or hydroxy group) and the chemically activated
protein, or it may be used to derivatize the molecule of interest
to form a partner molecule which is then sulfhydryl reactive by
virtue of a moiety derived from the agent, or it may be used to
derivatize the chemically activated protein to make it reactive
with the molecule of interest.
[0131] Linkers
[0132] The drug can be linked to a protein by a linker. Suitable
linkers include, for example, cleavable and non-cleavable linkers.
A cleavable linker is typically susceptible to cleavage under
intracellular conditions. Suitable cleavable linkers include, for
example, a peptide linker cleavable by an intracellular protease,
such as lysosomal protease or an endosomal protease. In exemplary
embodiments, the linker can be a dipeptide linker, such as a
valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys)
linker. Other suitable linkers include linkers hydrolyzable at a pH
of less than 5.5, such as a hydrazone linker. Additional suitable
cleavable linkers include disulfide linkers.
[0133] A linker can include a group for linkage to the protein. For
example, linker can include an amino, hydroxyl, carboxyl or
sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g.,
iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro),
halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides
(e.g., iodide, bromide or chloride), vinyl sulfone and
pyridyithio). See generally Wong, Chemistry of Protein Conjugation
and Cross-linking; CRC Press, Inc., Boca Raton, 1991.
[0134] In certain embodiments, the antibody or protein drug
conjugate can be of the following formula:
##STR00004##
or pharmaceutically acceptable salts or solvates thereof.
[0135] wherein:
[0136] Ab is an antibody or other protein,
[0137] A is a stretcher unit,
[0138] a is 0 or 1,
[0139] each W is independently a linker unit,
[0140] w is an integer ranging from 0 to 12,
[0141] Y is a spacer unit, and
[0142] y is 0, 1 or 2,
[0143] p ranges from 1 to about 20, and
[0144] D is a drug, a label or other molecule.
[0145] z is the number of potential conjugation sites on the
protein, wherein p<z.
In other embodiments, p can be, for example, 2, 4, 8, 10, 12, 16,
25, or more.
[0146] A stretcher unit can is capable of linking a linker unit to
an antibody or other protein. The stretcher unit has a functional
group that can form a bond with a functional group of the antibody
or other protein. Useful functional groups include, but are not
limited to, sulfhydryl (--SH), amino, hydroxyl, carboxy, the
anomeric hydroxyl group of a carbohydrate, and carboxyl.
[0147] The linker unit is typically an amino acid unit, such as for
example a dipeptide, tripeptide, tetrapeptide, pentapeptide,
hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,
undecapeptide or dodecapeptide unit. The linker unit can be
cleavage or non-cleavable inside the cell.
[0148] A spacer unit, if present, links a linker unit to the drug.
alternately, a spacer unit can link a stretcher unit to a drug
moiety when the linker unit is absent. The spacer unit can also
link a drug to an antibody or protein when both the linker unit and
stretcher unit are absent.
VI. Conjugates and Their Uses
[0149] In vitro Immunodiagnosis.
[0150] In one embodiment, a protein (e.g., an antibody) is
conjugated to a detectable label for use in in vitro
immunodiagnosis. The label may be a radiolabel, fluorophore, or
enzyme which is directly or indirectly conjugatable to conjugation
point (e.g., a free thiol group) of the chemically activated
antibody. The sample may be of clinical (e.g., blood, urine, semen,
or cerebrospinal fluid, or a solid tissue or organ) or non-clinical
(e.g., soil, water, food) nature. The assay may be qualitative or
quantitative, and in any desired format, including sandwich and
competitive formats. Numerous immunoassay formats, labels,
immobilization techniques, etc., are disclosed in the following
publications, hereby incorporated by reference herein: O'Sullivan
(1976), Annals Clin. Biochem. 16:221-240; McLaren (1981), Med. Lab.
Sci. 38:245-51; Ollerich (1984), J. Clin. Chem. Clin. Biochem.
22:895-904; Ngo and Lenhoff (1982), Mol. Cell. Biochem.,
44:3-12.
[0151] Immunoimaging.
[0152] An immunoconjugate may also be used for in vivo
immunoimaging. For this purpose, the protein (e.g., an antibody) is
labeled by means which permit external visualization of its
position or location within a subject or part thereof, such as an
organ. Typically, an immunoimaging agent will be an antibody
labeled directly (as with Technetium) or indirectly (as with
chelated Indium) with a suitable radioisotope. After injection into
the patient, the location of the conjugate may be tracked by a
detector sensitive to particles emitted by the radiolabel, e.g., a
gamma-scintillation camera in the case of a gamma emitter.
[0153] Immunotherapy.
[0154] For immunotherapy, a protein can be conjugated to suitable
drug, such as a cytotoxic or cytostatic agent, an immunosuppressive
agent, a radioisotope, a toxin, or the like. The conjugate can be
used for inhibiting the multiplication of a tumor cell or cancer
cell, causing apoptosis in a tumor or cancer cell, or for treating
cancer in a patient. The conjugate can be used accordingly in a
variety of settings for the treatment of animal cancers. The
conjugate can be used to deliver a drug to a tumor cell or cancer
cell. Without being bound by theory, in some embodiments, the
conjugate binds to or associates with a cancer-cell or a
tumor-associated antigen, and the conjugate and/or drug can be
taken up inside a tumor cell or cancer cell through
receptor-mediated endocytosis. The antigen can be attached to a
tumor cell or cancer cell or can be an extracellular matrix protein
associated with the tumor cell or cancer cell. Once inside the
cell, one or more specific peptide sequences within the conjugte
(e.g., in a linker) are hydrolytically cleaved by one or more
tumor-cell or cancer-cell-associated proteases, resulting in
release of the drug. The released drug is then free to migrate
within the cell and induce cytotoxic or cytostatic or other
activities. In some embodiments, the drug is cleaved from the
antibody outside the tumor cell or cancer cell, and the drug
subsequently penetrates the cell, or acts at the cell surface.
[0155] Thus, in some embodiments, the conjugate or other protein
binds to the tumor cell or cancer cell. In some embodiments, the
conjugate binds to a tumor cell or cancer cell antigen which is on
the surface of the tumor cell or cancer cell. In other embodiments,
the conjugate binds to a tumor cell or cancer cell antigen which is
an extracellular matrix protein associated with the tumor cell or
cancer cell.
[0156] The specificity of the protein for a particular tumor cell
or cancer cell can be important for determining those tumors or
cancers that are most effectively treated. For example, antibodies
having an anti-CD30 or an anti-CD40 antibody or other binding
protein can be useful for treating hematologic malignancies.
[0157] Other particular types of cancers that can be treated with
the protein-drug conjugates include, but are not limited to, solid
tumors, including but not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, retinoblastoma, blood-borne
cancers (including but not limited to: acute lymphoblastic leukemia
"ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia "AML", acute
promyelocytic leukemia "APL", acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia "CML",
chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple
myeloma), acute and chronic leukemias (e.g., lymphoblastic,
myelogenous, lymphocytic, and myelocytic leukemias), and Lymphomas
(e.g., Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,
Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera). The proteins provide conjugation-specific tumor
or cancer targeting.
[0158] Multi-Modality Therapy For Cancer
[0159] As discussed above, cancers, including, but not limited to,
a tumor, metastasis, or other disease or disorder characterized by
uncontrolled cell growth, can be treated or prevented by
administration of a protein-drug conjugate.
[0160] In other embodiments, methods for treating or preventing
cancer are provided, including administering to a patient in need
thereof an effective amount of a conjugate and a chemotherapeutic
agent. In some embodiments, the chemotherapeutic agent is that with
which treatment of the cancer has not been found to be refractory.
In some embodiments, the chemotherapeutic agent is that with which
the treatment of cancer has been found to be refractory. The
conjugate can be administered to a patient that has also undergone
an treatment, such as surgery for treatment for the cancer. In
another embodiment, the additional method of treatment is radiation
therapy.
[0161] In an exemplary embodiment, the protein-drug conjugate is
administered concurrently with the chemotherapeutic agent or with
radiation therapy. In another exemplary embodiment, the
chemotherapeutic agent or radiation therapy is administered prior
or subsequent to administration of the protein-drug conjugate, in
one aspect at least an hour, five hours, 12 hours, a day, a week, a
month, in further aspects several months (e.g., up to three
months), prior or subsequent to administration of the
conjugate.
[0162] A chemotherapeutic agent can be administered over a series
of sessions. Any one or a combination of the chemotherapeutic
agents listed below can be administered. With respect to radiation,
any radiation therapy protocol can be used depending upon the type
of cancer to be treated. For example, but not by way of limitation,
x-ray radiation can be administered; in particular, high-energy
megavoltage (radiation of greater that 1 MeV energy) can be used
for deep tumors, and electron beam and orthovoltage x-ray radiation
can be used for skin cancers. Gamma-ray emitting radioisotopes,
such as radioactive isotopes of radium, cobalt and other elements,
can also be administered.
[0163] Additionally, methods of treatment of cancer with a
protein-drug conjugate are provided as an alternative to
chemotherapy or radiation therapy, where the chemotherapy or the
radiation therapy has proven or can prove too toxic, e.g., results
in unacceptable or unbearable side effects, for the subject being
treated. The animal being treated can, optionally, be treated with
another cancer treatment such as surgery, radiation therapy or
chemotherapy, depending on which treatment is found to be
acceptable or bearable.
[0164] The protein-drug conjugate can also be used in an in vitro
or ex vivo fashion, such as for the treatment of certain cancers,
including, but not limited to leukemias and lymphomas, such
treatment involving autologous stem cell transplants. This can
involve a multi-step process in which the animal's autologous
hematopoietic stem cells are harvested and purged of all cancer
cells, the animal's remaining bone-marrow cell population is then
eradicated via the administration of a high dose of a conjugate
with or without accompanying high dose radiation therapy, and the
stem cell graft is infused back into the animal. Supportive care is
then provided while bone marrow function is restored and the animal
recovers.
[0165] Multi-Drug Therapy For Cancer
[0166] Methods for treating cancer include administering to a
patient in need thereof an effective amount of an a protein-drug
conjugate and another therapeutic agent that is an anti-cancer
agent are disclosed. Suitable anticancer agents include, but are
not limited to, methotrexate, taxol, L-asparaginase,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,
cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,
camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
vincristine, vinorelbine, paclitaxel, and docetaxel.
[0167] The anti-cancer agent includes, but is not limited to, a
drug such as an alkylating agents such as a nitrogen mustard (e.g.,
cyclophosphamide, ifosfamide, trofosfamide, chlorambucil,
melphalan), nitrosoureas (e.g., carmustine (BCNU), lomustine
(CCNU)), alkylsulphonates (e.g., busulfan, treosulfan), triazenes
(e.g., decarbazine), Platinum containing compounds (e.g.,
cisplatin, carboplatin); plant alkaloids, such as vinca alkaloids
(e.g., vincristine, vinblastine, vindesine, vinorelbine), taxoids
(e.g., paclitaxel, docetaxol); DNA topoisomerase inhibitors such as
epipodophyllins (e.g., etoposide, teniposide, topotecan,
9-aminocamptothecin, camptothecin, crisnatol, mitomycins (e.g.,
mitomycin C); anti-metabolites such as anti-folates such as DHFR
inhibitors (e.g., methotrexate, trimetrexate), IMP dehydrogenase
inhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR) and
ribonucleotide reductase inhibitors (e.g., hydroxyurea,
deferoxamine), pyrimidine analogs such as uracil analogs
(5-fluorouracil, floxuridine, doxifluridine, ratitrexed), cytosine
analogs (e.g., cytarabine (ara C), cytosine arabinoside,
fludarabine), and purine analogs (e.g., mercaptopurine,
thioguanine); hormonal therapies, such as receptor antagonists,
such as anti-estrogens (e.g., tamoxifen, raloxifene, megestrol),
LHRH agonists (e.g., goscrclin, leuprolide acetate), and
anti-androgens (e.g., flutamide, bicalutamide; retinoids/deltoids
such as vitamin D3 analogs (e.g., EB 1089, CB 1093, KH 1060),
photodynamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine,
photosensitizer Pc4, demethoxy-hypocrellin A (2BA-2-DMHA)),
cytokines (e.g, interferon-.alpha., interferon-.gamma., tumor
necrosis factor), as well as other drugs, such as gemcitabine,
velcade, revamid, thalamid, isoprenylation inhibitors (e.g.,
lovastatin), dopaminergic neurotoxins (e.g.,
1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.,
staurosporine), actinomycin (e.g., actinomycin D, dactinomycin),
bleomycin, bleomycin A2, bleomycin B2, peplomycin), anthracyclines
(daunorubicin, Doxorubicin (adriamycin), idarubicin, epirubicin,
pirarubicin, zorubicin, mtoxantrone), MDR inhibitors (e.g.,
verapamil), and Ca.sup.2+ ATPase inhibitors (e.g.,
thapsigargin)
[0168] Treatment Of Autoimmune Diseases
[0169] The protein-drug conjugates are useful for killing or
inhibiting the replication of a cell that produces an autoimmune
disease or for treating an autoimmune disease. The conjugates can
be used accordingly in a variety of settings for the treatment of
an autoimmune disease in a patient. The conjugates can be used to
deliver a drug to a target cell. Without being bound by theory, in
one embodiment, the conjugates associate with an antigen on the
surface of a target cell, and the conjugate is then taken up inside
a target-cell through receptor-mediated endocytosis. Once inside
the cell, one or more specific peptide sequences (e.g., within a
linker) are enzymatically or hydrolytically cleaved, resulting in
release of a drug. The released drug is then free to migrate in the
cytosol and induce cytotoxic or cytostatic activities. In an
alternative embodiment, the drug is cleaved from the conjugate
outside the target cell, and the drug subsequently penetrates the
cell.
[0170] In some embodiments, the protein-drug conjugate binds to an
autoimmune antigen. In one aspect, the antigen is on the surface of
a cell involved in an autoimmune condition. In some embodiments, an
antibody binds to an autoimmune antigen which is on the surface of
a cell. In an exemplary embodiment, an antibody binds to activated
lymphocytes that are associated with the autoimmune disease state.
In a further embodiment, the conjugates kill or inhibit the
multiplication of cells that produce an autoimmune antibody
associated with a particular autoimmune disease.
[0171] Particular types of autoimmune diseases that can be treated
with the protein-drug conjugate include, but are not limited to,
Th2 lymphocyte related disorders (e.g., atopic dermatitis, atopic
asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,
systemic sclerosis, and graft versus host disease); Th1 lymphocyte
related disorders (e.g., rheumatoid arthritis, multiple sclerosis,
psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's
disease, primary biliary cirrhosis, Wegener's granulomatosis and
tuberculosis); and activated B lymphocyte related disorders (e.g.,
systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid
arthritis and type I diabetes). Other autoimmune diseases include,
but are not limited to, active chronic hepatitis, Addison's
disease, allergic alveolitis, allergic reaction, allergic rhinitis,
Alport's Syndrome, anaphlaxis, ankylosing spondylitis,
anti-phosholipid syndrome, arthritis, ascariasis, aspergillosis,
atopic allergy, atropic dermatitis, atropic rhinitis, Behcet's
disease, Bird-Fancier's Lung, bronchial asthma, Caplan's syndrome,
cardiomyopathy, Celiac disease, Chagas' disease, chronic
glomerulonephritis, Cogan's Syndrome, cold agglutinin disease,
congenital rubella infection, CREST syndrome, Crohn's disease,
cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid
lupus, Dressler's syndrome, Eaton-Lambert syndrome, echovirus
infection, encephalomyelitis, endocrine opthalmopathy, Epstein-Barr
virus infection, equine heaves, erythematosis, Evan's syndrome,
Felty's syndrome, fibromyalgia, Fuch's cyclitis, gastric atrophy,
gastrointestinal allergy, giant cell arteritis, glomerulonephritis,
goodpasture's syndrome, graft v. host disease, Graves' disease,
Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia,
Henoch-Schonlein Purpura, idiopathic adrenal atrophy, idiopathic
pulmonary fibritis, IgA nephropathy, inflammatory bowel disease,
insulin-dependent diabetes mellitus, juvenile arthritis, juvenile
diabetes mellitus (Type I), Lambert-Eaton syndrome, laminitis,
lichen planus, lupoid hepatitis, lupus, lymphopenia, Meniere's
disease, mixed connective tissue disease, multiple sclerosis,
myasthenia gravis, pernicious anemia, polyglandular syndromes,
presenile dementia, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, psoriatic arthritis, Raynauds phenomenon,
recurrent abortion, Reiter's syndrome, rheumatic fever, rheumatoid
arthritis, Sampter's syndrome, schistosomiasis, Schmidt's syndrome,
scleroderma, Shulman's syndrome, Sjorgen's syndrome, stiff-man
syndrome, sympathetic ophthalmia, systemic lupus erythematosis,
Takayasu's arteritis, temporal arteritis, thyroiditis,
thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, Type
B insulin resistance, Type I diabetes mellitus, ulcerative colitis,
uveitis, vitiligo, Waldenstrom's macroglobulemia, and Wegener's
granulomatosis.
[0172] Multi-Drug Therapy of Autoimmune Diseases
[0173] Methods for treating an autoimmune disease are also
disclosed that include administering to a patient in need thereof
an effective amount of a protein-drug conjugate alone or in
combination another therapeutic agent known for the treatment of an
autoimmune disease. The anti-autoimmune disease agent can include,
but is not limited to, the following: cyclosporine, cyclosporine A,
mycophenylate mofetil, sirolimus, tacrolimus, enanercept,
prednisone, azathioprine, methotrexate, cyclophosphamide,
aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone,
dexamethasone, chlorambucil, DHEA, danazol, bromocriptine,
meloxicam and infliximab.
[0174] Treatment of Infectious Diseases
[0175] The protein-drug conjugates are useful for killing or
inhibiting the multiplication of a cell that produces an infectious
disease or for treating an infectious disease. The conjugates can
be used accordingly in a variety of settings for the treatment of
an infectious disease in a patient. The ADCs can be used to deliver
a drug to a target cell. In one embodiment, the antibody binds to
the infectious disease cell. In some embodiments, the conjugate
kills or inhibit the multiplication of cells that produce a
particular infectious disease. Particular types of infectious
diseases that can be treated with the conjugates include, but are
not limited to, the following: bacterial diseases, such as
diphtheria, pertussis, occult bacteremia, urinary tract infection,
gastroenteritis, cellulites, epiglottitis, tracheitis, adenoid
hypertrophy, retropharyngeal abcess, impetigo, ecthyma, pneumonia,
endocarditis, septic arthritis, pneumococcal, peritonitis,
bactermia, meningitis, acute purulent meningitis, urethritis,
cervicitis, proctitis, pharyngitis, salpingitis, epididymitis,
gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella,
typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis,
tularemia, cholera, bubonic plague, tetanus, necrotizing enteritis,
and actinomycosis; mixed anaerobic infections, such as syphilis,
relapsing fever, leptospirosis, Lyme disease, rat bite fever,
tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial
pneumonia, trachoma, and inclusion conjunctivitis; systemic fungal
diseases such as histoplamosis, coccidiodomycosis, blastomycosis,
sporotrichosis, cryptococcsis, systemic candidiasis, aspergillosis,
mucormycosis, mycetoma, and chromomycosis; rickettsial diseases
such as typhus, Rocky Mountain Spotted Fever, ehrlichiosis, Eastern
Tick-Borne Rickettsioses, rickettsialpox, Q fever and
bartonellosis; parasitic diseases such as malaria, babesiosis,
African sleeping sickness, Chagas' disease, leishmaniasis, Dum-Dum
fever, toxoplasmosis, meningoencephalitis, keratitis, entamebiasis,
giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis,
microsporidiosis, ascariasis, whipworm infection, hookworm
infection, threadworm infection, ocular larva migrans, trichinosis,
Guinea worm disease, lymphatic Filariasis, loiasis, River
Blindness, canine heartwoun infection, schistosomiasis, swimmer's
itch, Oriental lung fluke, Oriental liver fluke, fascioliasis,
fasciolopsiasis, opisthorchiasis, tapeworm infections, hydatid
disease, and alveolar hydatid disease; viral diseases such as
measles, subacute sclerosing panencephalitis, common cold, mumps,
rubella, roseola, Fifth Disease, chickenpox, respiratory syncytial
virus infection, croup, bronchiolitis, infectious mononucleosis,
poliomyelitis, herpangina, hand-foot-and-mouth disease, Bornholm
disease, genital herpes, genital warts, aseptic meningitis,
myocarditis, pericarditis, gastroenteritis, acquired
immunodeficiency syndrome (AIDS), human immunodeficiency virus
(HIV), Reye's syndrome, Kawasaki syndrome, influenza, bronchitis,
viral "Walking" pneumonia, acute febrile respiratory disease, acute
pharyngoconjunctival fever, epidemic keratoconjunctivitis, Herpes
Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), shingles,
cytomegalic inclusion disease, rabies, progressive multifocal
leukoencephalopathy, kuru, fatal familial insomnia,
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease,
tropical spastic paraparesis, western equine encephalitis,
California encephalitis, St. Louis encephalitis, Yellow Fever,
Dengue, lymphocytic choriomeningitis, Lassa fever, hemorrhagic
fever, Hantvirus pulmonary syndrome, Marburg virus infections,
Ebola virus infections and smallpox.
[0176] Multi-Drug Therapy of Infectious Diseases
[0177] Methods for treating an infectious disease are disclosed as
including administering to a patient in need thereof a protein-drug
conjugate alone or in combination with another therapeutic agent
that is an anti-infectious disease agent. The anti-infectious
disease agent can be, but not limited to, the following:
.beta.-lactam antibiotics, such as penicillin G, penicillin V,
cloxacilliin, dicloxacillin, methicillin, nafcillin, oxacillin,
ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin,
mezlocillin, piperacillin and ticarcillin; aminoglycosides such as
amikacin, gentamicin, kanamycin, neomycin, netihnicin, streptomycin
and tobramycin; macrolides such as azithromycin, clarithromycin,
erythromycin, lincomycin and clindamycin; tetracyclines such as
demeclocycline, doxycycline, minocycline, oxytetracycline and
tetracycline; quinolones such as cinoxacin, and nalidixic acid;
fluoroquinolones such as ciprofloxacin, enoxacin, grepafloxacin,
levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin
and trovafloxicin; polypeptides such as bacitracin, colistin and
polymyxin B; sulfonamides such as sulfisoxazole, sulfamethoxazole,
sulfadiazine, sulfamethizole and sulfacetamide; and other
antibacterial agents, such as trimethoprim, sulfamethazole,
chloramphenicol, vancomycin, metronidazole, quinupristin,
dalfopristin, rifampin, spectinomycin and nitrofurantoin; and
antiviral agents, such as general antiviral agents such as
idoxuradine, vidarabine, trifluridine, acyclovir, famcicyclovir,
pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin,
amantadine, rimantadine, cidofovir; antisense oligonucleotides,
immunoglobulins and interferons; and drugs for HIV infection such
as tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine,
stavudine, lamivudine, nevirapine, delavirdine, saquinavir,
ritonavir, indinavir and nelfinavir.
VII. Pharmaceutical Compositions
[0178] In in vivo use, generally, whether for immunoimaging, for
immunotherapy or by other uses, the conjugate is introduced into a
subject. The composition can comprise a single isomer, or one or
more partially-loaded isomers, of the conjugate. For example, if
the protein is an antibody, the composition can comprise a single
E2, E4 or E6 isomer, a mixture of selected E2, E4 or E6 isomers,
all E2, E4 or E6 isomers alone, or a mixture of E2, E4 and E6
isomers. In some embodiments, a composition containing a certain
isomer(s) can be substantially free of other isomers. In this
context, "substantially free" means the composition contains less
than about 20%, less than about 10%, less than about 5% less than
about 2% or less than about 1% of the other isomers.
[0179] The compositions can be in any form that allows for the
composition to be administered to a patient. For example, the
composition can be in the form of a solid, liquid or gas (aerosol).
Typical routes of administration include, without limitation, oral,
topical, parenteral, sublingual, rectal, vaginal, ocular,
intra-tumor, and intranasal. Parenteral administration includes
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques. In one aspect, the compositions
are administered parenterally. In yet another aspect, the conjugate
or compositions are administered intravenously.
[0180] The conjugate can be introduced by injection. Typically, the
conjugate is administered intravascularly (intravenously or
intraarterially) or intrathetically, often by infusion. In
addition, in appropriate cases the conjugate may be introduced
subcutaneously, submucosally, intramuscularly, intracranially, or
by other accepted routes of drug administration.
[0181] In other embodiments, the composition includes an effective
amount of a conjugate and a pharmaceutically acceptable carrier or
vehicle. Such compositions are suitable for veterinary or human
administration.
[0182] Pharmaceutical compositions can be formulated so as to allow
a conjugate to be bioavailable upon administration of the
composition to a patient. Compositions can take the form of one or
more dosage units, where for example, a tablet can be a single
dosage unit, and a container of a conjugate in injectable form can
hold a plurality of dosage units.
[0183] Materials used in preparing the pharmaceutical compositions
can be non-toxic in the amounts used. It will be evident to those
of ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of animal (e.g., human), the particular form of the
conjugate, the manner of administration, and the composition
employed.
[0184] The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) can be gaseous or particulate, so as to
provide an aerosol composition useful in, e.g., inhalatory
administration.
[0185] When intended for oral administration, the composition is
preferably in solid or liquid form, where semi-solid, semi-liquid,
suspension and gel forms are included within the forms considered
herein as either solid or liquid.
[0186] As a solid composition for oral administration, the
composition can be formulated into a powder, granule, compressed
tablet, pill, capsule, chewing gum, wafer or the like. Such a solid
composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins; disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon
dioxide; sweetening agents such as sucrose or saccharin, a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring, and a coloring agent.
[0187] When the composition is in the form of a capsule, e.g., a
gelatin capsule, it can contain, in addition to materials of the
above type, a liquid carrier such as polyethylene glycol,
cyclodextrin or a fatty oil.
[0188] The composition can be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid can be
useful for oral administration or for delivery by injection. When
intended for oral administration, a composition can comprise one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
[0189] The liquid compositions, whether they are solutions,
suspensions or other like form, can also include one or more of the
following: sterile diluents such as water for injection, saline
solution, preferably physiological saline, Ringer's solution,
isotonic sodium chloride, fixed oils such as synthetic mono or
digylcerides which can serve as the solvent or suspending medium,
polyethylene glycols, glycerin, cyclodextrin, propylene glycol or
other solvents; antibacterial agents such as benzyl alcohol or
methyl paraben; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
A parenteral composition can be enclosed in ampoule, a disposable
syringe or a multiple-dose vial made of glass, plastic or other
material. Physiological saline is an exemplary adjuvant. An
injectable composition is preferably sterile.
[0190] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0191] The amount of the conjugate that is effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. The dosage ranges for the
administration of the disclosed protein-drug conjugates are those
large enough to produce the desired effect in which the symptoms of
the condition or disorder are ameliorated. The dosage should not be
so large as to cause adverse side effects, such as unwanted
cross-reactions, anaphylactic reactions, and the like. In addition,
in vitro or in vivo assays can optionally be employed to help
identify optimal dosage ranges.
[0192] The precise dose to be employed in the compositions will
also depend on the age, condition, sex and extent of the disease in
the patient, route of administration, and the seriousness of the
disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
[0193] The compositions comprise an effective amount of a conjugate
such that a suitable dosage will be obtained. Typically, this
amount is at least about 0.01% of a conjugate by weight of the
composition. When intended for oral administration, this amount can
be varied to range from about 0.1% to about 80% by weight of the
composition. In one aspect, oral compositions can comprise from
about 4% to about 50% of the conjugate by weight of the
composition. In yet another aspect, present compositions are
prepared so that a parenteral dosage unit contains from about 0.01%
to about 2% by weight of the conjugate.
[0194] For intravenous administration, the composition can comprise
from about 0.01 to about 100 mg of a conjugate per kg of the
animal's body weight. In one aspect, the composition can include
from about 1 to about 100 mg of a conjugate per kg of the animal's
body weight. In another aspect, the amount administered will be in
the range from about 0.1 to about 25 mg/kg of body weight of the
conjugate.
[0195] Generally, the dosage of an conjugate administered to a
patient is typically about 0.01 mg/kg to about 2000 mg/kg of the
animal's body weight. In one aspect, the dosage administered to a
patient is between about 0.01 mg/kg to about 10 mg/kg of the
animal's body weight. In another aspect, the dosage administered to
a patient is between about 0.1 mg/kg and about 250 mg/kg of the
animal's body weight. In yet another aspect, the dosage
administered to a patient is between about 0.1 mg/kg and about 20
mg/kg of the animal's body weight. In yet another aspect the dosage
administered is between about 0.1 mg/kg to about 10 mg/kg of the
animal's body weight. In yet another aspect, the dosage
administered is between about 1 mg/kg to about 10 mg/kg of the
animal's body weight.
[0196] The conjugates can be administered by any convenient route,
for example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.). Administration can be systemic or local.
Various delivery systems are known, e.g., encapsulation in
liposomes, microparticles, microcapsules, capsules, etc., and can
be used to administer a conjugate or composition. In certain
embodiments, more than one conjugate or composition is administered
to a patient.
[0197] In specific embodiments, it can be desirable to administer
one or more conjugates or compositions locally to the area in need
of treatment. This can be achieved, for example, and not by way of
limitation, by local infusion during surgery; topical application,
e.g., in conjunction with a wound dressing after surgery; by
injection; by means of a catheter; by means of a suppository; or by
means of an implant, the implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. In one embodiment, administration can be by
direct injection at the site (or former site) of a cancer, tumor or
neoplastic or pre-neoplastic tissue. In another embodiment,
administration can be by direct injection at the site (or former
site) of a manifestation of an autoimmune disease.
[0198] In certain embodiments, it can be desirable to introduce one
or more conjugates or compositions into the central nervous system
by any suitable route, including intraventricular and intrathecal
injection. Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0199] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant.
[0200] In yet another embodiment, the conjugates can be delivered
in a controlled release system, such as but not limited to, a pump
or various polymeric materials can be used. In yet another
embodiment, a controlled-release system can be placed in proximity
of the target of the conjugates, e.g., the brain, thus requiring
only a fraction of the systemic dose (see, e.g., Goodson, in
Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)). Other controlled-release systems discussed in the
review by Langer (Science 249:1527-1533 (1990)) can be used.
[0201] In some embodiments, a protein conjugate can be combined
with a carrier to form a compostion. The term "carrier" refers to a
diluent, adjuvant or excipient, with which a conjugate is
administered. Such pharmaceutical carriers can be liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. The carriers can be saline, gum acacia,
gelatin, starch paste, talc, keratin, colloidal silica, urea, and
the like. In addition, auxiliary, stabilizing, thickening,
lubricating and coloring agents can be used. In one embodiment,
when administered to a patient, the conjugate or compositions and
pharmaceutically acceptable carriers are sterile. Water is an
exemplary carrier when the conjugate are administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers also include
excipients such as starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The compositions,
if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents.
[0202] The compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use. Other examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0203] In an exemplary embodiment, the conjugate is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to animals, particularly
human beings. Typically, the carriers or vehicles for intravenous
administration are sterile isotonic aqueous buffer solutions. Where
necessary, the compositions can also include a solubilizing agent.
Compositions for intravenous administration can optionally comprise
a local anesthetic such as lignocaine to ease pain at the site of
the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where a conjugate is to be administered
by infusion, it can be dispensed, for example, with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the conjugate is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
[0204] Compositions for oral delivery can be in the font), of
tablets, lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs, for example. Orally
administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
driving compound are also suitable for orally administered
compounds. In these later platforms, fluid from the environment
surrounding the capsule is imbibed by the driving compound, which
swells to displace the agent or agent composition through an
aperture. These delivery platforms can provide an essentially zero
order delivery profile as opposed to the spiked profiles of
immediate release formulations. A time-delay material such as
glycerol monostearate or glycerol stearate can also be used.
[0205] The compositions can be intended for topical administration,
in which case the carrier may be in the form of a solution,
emulsion, ointment or gel base. If intended for transdermal
administration, the composition can be in the form of a transdermal
patch or an iontophoresis device. Topical formulations can comprise
a concentration of a conjugate of from about 0.05% to about 50% w/v
(weight per unit volume of composition), in another aspect, from
0.1% to 10% w/v.
[0206] The composition can be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the conjugate.
[0207] The composition can include various materials that modify
the physical form of a solid or liquid dosage emit. For example,
the composition can include materials that form a coating shell
around the active ingredients. The materials that form the coating
shell are typically inert, and can be selected from, for example,
sugar, shellac, and other enteric coating agents. Alternatively,
the active ingredients can be encased in a gelatin capsule.
[0208] The compositions can consist of gaseous dosage units, e.g.,
it can be in the form of an aerosol. The term aerosol is used to
denote a variety of systems ranging from those of colloidal nature
to systems consisting of pressurized packages. Delivery can be by a
liquefied or compressed gas or by a suitable pump system that
dispenses the active ingredients.
[0209] Whether in solid, liquid or gaseous form, the present
compositions can include a pharmacological agent used in the
treatment of cancer, an autoimmune disease or an infectious
disease.
VIII. Pharmokinetics
[0210] In vivo characterization of the toxicity and efficacy of
purified 4 drug conjugates of cAC10-vcMMAE in mice has been
performed and are discussed in more detail in the Examples (see
e.g., Examples 8 and 9). Briefly, these studies have shown that
mixtures with an average of 4 drugs per antibody are equally
efficacious as those conjugates with 8 drugs (single dose of 1
mg/Kg for both), while being less toxic (MTD of 100 mg/Kg for 4
drugs per antibody versus 50 mg/Kg for 8 drugs per antibody). The
purified material with 4 drugs per antibody from the DTT method is
similar in efficacy and toxicity, while purified material with 4
drugs per antibody from the DTNB method has a slightly higher MTD
of 120 mg/Kg while being efficacious at half the dose of the other
conjugates (0.5 mg/Kg).
[0211] Loading cAC10 with two, four, or eight drugs per antibody
had no effect on the binding to the target antigen CD30. The in
vitro potency of the cAC10-ADCs was directly dependent on drug
loading, and thus the total MMAE exposure.
[0212] cAC10-E4 demonstrated comparable anti-tumor activity to
cAC10-E8 in a Karpas-299 xenograft model at the same dose of
antibody, half the MMAE dose. Based on the in vitro finding that
potency was directly related to drug loading, the equivalent in
vivo anti-tumor activity of cAC10-E4 and cAC10-E8 was
unanticipated. Investigation of the pharmacokinetics of the ADCs
revealed that clearance was directly related to the drug loading of
the ADCs and exposure (Area Under the Curve--AUC) was inversely
related to drug loading. The AUC of cAC10-E4 was 3-fold higher than
cAC10-E8. The larger AUC of cAC10-E4 compared to cAC10-E8 was
apparently sufficient to compensate for the reduced potency,
leading to equivalent efficacy. Attempts to improve efficacy by
decelerating the plasma elimination half-life to augment AUCs have
been accomplished by methods including the construction of albumin
fusion proteins for interferon-.alpha. and liposomal delivery of
the anti-cancer drug Lurtotecan. Unlike these examples where the
objective was to lengthen the plasma half-life, the enhanced
exposure of cAC10-E4 was a valuable consequence of reducing MMAE
loading.
[0213] As disclosed in Example 8, dosing cAC10-E2 with 1.0
mg/kg/dose q4dx4 yielded ten out of ten cures. While cAC10-E2 did
not demonstrate equivalent anti-tumor activity compared to cAC10-E4
at the same mAb dose, the dose of cAC10-E2 to achieve equivalent
anti-tumor activity compared to cAC10-E4 is probably less than
two-fold, based on the in vivo efficacy experiments. Similar to
cAC10-E4, the improved exposure of cAC10-E2 may play a significant
part in compromising the lower in vitro potency.
[0214] To maximize the therapeutic potential of cAC10-Val-Cit-MMAE
ADCs, a high therapeutic index is needed. Reducing the amount of
MMAE molecules per mAb from eight to four enhanced the therapeutic
index from 100 to 200. Given steep dose-response curves of
chemotherapeutic reagents a two-fold difference in therapeutic
index may be significant in terms of the overall clinical
implications with regards to toxicities.
[0215] By reducing the quantity of MMAE from eight to four
molecules per mAb, there was a decrease of in vitro activity, yet a
demonstrated equivalent anti-tumor activity in vivo. While a
further reduction in drug loading to two MMAE molecules per
antibody further reduced the in vitro activity, cAC10-E2 had
equivalent or better efficacy than cAC10-E4 and cAC10-E8 at double
the dose in a multi-dose setting. The therapeutic window was
increased two-fold by reducing drug loading from eight MMAE
molecules to four, and at the very least maintained with a further
reduction to two drugs per antibody. There is considerable value in
optimizing drug substitution of ADCs.
EXAMPLES
Example 1 of Method 1
[0216] cAC10 was partially reduced with limited concentration of
DTT as follows: cAC10 (8 mg/mL or 53.8 .mu.M) was treated with 3.5
molar equivalents of DTT (188.4 .mu.M; Sigma) in 0.05 M sodium
borate pH 8, 0.05 M NaCl, and 1 mM diethylene-triaminepentaacetic
acid (DTPA; Aldrich) for 1 h at 37.degree. C. The reduced antibody
was then purified by desalting on a PD-10 column (Amersham
Biosciences). The PD-10 column was equilibrated with 25 mL of
phosphate buffered saline (PBS) pH 7.4 (GIBCO) with 1 mM DTPA
(PBSD), 1 mL of the above solution applied to the column, the
column washed with 1.8 mL of PBSD, and the column eluted with 1.4
mL of PBSD. The protein concentration was quantitated using an
absorbance value of 1.58 at 280 nm for a 1.0-mg/mL solution, and
the molar concentration determined using a molecular weight of
150,000 g/mol. The concentration of antibody-cysteine thiols
produced was determined by titrating with
5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB; Pierce), typically
resulting in slightly higher than 4 antibody-cysteine thiols per
antibody using this method.
[0217] The drug vcMMAE was then conjugated to reduced cAC10 as
follows: reduced cAC10 (typically 30 .mu.M antibody and 120 .mu.M
antibody-cysteine thiols final concentration) was first cooled to
0.degree. C. vcMMAE was dissolved in cold acetonitrile and rapidly
mixed with the antibody solution. The final acetonitrile
concentration was 20%, while the final drug concentration was 135
to 150 .mu.M (4.5 to 5 molar equivalents, which is a slight excess
over the antibody-cysteine thiols). This solution was allowed to
incubate for 30 min at 0.degree. C., the excess vcMMAE quenched
with cysteine (1 mM final concentration), and the conjugate
purified using a PD-10 column as described above.
Example 2 of Method 1
[0218] cAC10 with 4 vcMMAE per antibody (E4 mix) was prepared with
limiting amounts of DTT as follows: cAC10 was treated with 3.25
molar equivalents of DTT in 0.025 M sodium borate pH 8, 0.025 M
NaCl, 1 mM DTPA for 2 h at 37.degree. C. This mixture was diluted 5
fold with water and applied to a hydroxyapatite column (Macroprep
ceramic type I 40 .mu.m, BioRad, Hercules, Calif.) at a flow rate
of 10 mL/min. The column size was 1 mL per 10 mg of cAC10. The
column was previously equilibrated with 5 column volumes of 0.5 M
sodium phosphate pH 7, 10 mM NaCl and 5 column volumes of 10 mM
sodium phosphate pH 7, 10 mM NaCl. Following application, the
column was washed with 5 column volumes of 10 mM sodium phosphate
pH 7, 10 mM NaCl and then eluted with 100 mM sodium phosphate pH 7,
10 mM NaCl. DTPA was added to 1 mM following elution. The protein
concentration was quantitated using an absorbance value of 1.58 at
280 nm for a 1.0-mg/mL solution, and the molar concentration
determined using a molecular weight of 148,449 g/mol. The
concentration of antibody-cysteine thiols produced was determined
by titrating with DTNB, typically resulting in 4.0 to 4.5 thiols
per antibody.
[0219] Reduced cAC10 was alkylated with a slight excess of vcMMAE
over antibody-cysteine thiols (1.1 molar equivalents). To keep the
vcMMAE soluble, 10% DMSO was present in the final reaction mixture.
Alternatively, the vcMMAE could be kept in a solution comprising 5%
by volume of an alcohol, such as ethanol and isopropyl alcohol. The
alkylation reaction was performed at 0.degree. C. for 30 min.
Cysteine (1 mM final) was used to quench any unreacted vcMMAE.
cAC10-vcMMAE was purified by hydroxyapatite chromatography as
described above. Following elution, the buffer was changed to
phosphate buffered saline (Invitrogen, Carlsbad, Calif.) using
Amicon (Millipore, Bedford, Mass.) Ultrafree 30K cutoff spin
concentration devices. The protein concentration was quantitated
using an absorbance value of 1.62 at 280 nm for a 1.0-mg/mL
solution.
Example 3 of Method 2a
[0220] cAC10 was fully reduced by adding a large excess of DTT. The
final reaction concentrations were 8 mg/mL cAC10, 0.05 M sodium
borate pH 8, 0.05 M NaCl, 10 mM DTT, and 1 mM DTPA. This solution
was incubated at 37.degree. C. for 30 min and the antibody purified
by desalting on a PD-10 column as described above. Slightly more
than 8 antibody-cysteine thiols as determined by DTNB titration
were produced using these conditions.
[0221] Partial reoxidation was achieved using DTNB as an oxidizing
agent. Reduced cAC10 (typically 30 .mu.M) was cooled to 0.degree.
C. and then treated with 1.5 to 2.5 molar equivalents of DTNB (45
to 75 .mu.M final concentration; the highest yields of E4 were
obtained using 2.0 equivalents). The solution was rapidly mixed by
inversion and allowed to incubate at 0.degree. C. for 10-20 min.
The extent of reaction can be observed since the released TNB.sup.-
is yellow. Typically, the reaction appeared to be complete within a
few seconds. Cysteine was added (1 mM final concentration) to
ensure that all TNB was present as TNB.sup.- rather than in mixed
disulfides with antibody cysteines. The antibody was then purified
on a PD-10 column or a hydroxylapatite column as described above.
Typically 4 antibody-cysteine thiols were observed by DTNB
titration following this partial reoxidation procedure. The vcMMAE
drug was finally conjugated to these antibody-cysteine thiols and
purified by PD-10 as described above for method 1.
Example 4 of Method 2b
[0222] Fully reduced cAC10 was prepared as described above for
method 2a. Fully reduced cAC10 (typically 30 .mu.M) was cooled to
0.degree. C. and then treated with 1.5 to 2.5 equivalents of DTNB
(45 to 75 .mu.M final concentration). The solution was rapidly
mixed by inversion and allowed to incubate at 0.degree. C. for 10
min. Without further purification, the partially reoxidized cAC10
was then rapidly mixed with 5 equivalents vcMMAE dissolved in cold
acetonitrile. As with method 1, the final concentration of
acetonitrile was 20%. In the conjugation reaction, the cAC10 final
concentration was 24 .mu.M (96 .mu.M antibody-cysteine thiols or 4
per antibody) and the final vcMMAE concentration was 120 .mu.M (5
molar equivalents). This solution was incubated for 30 min at
0.degree. C. before quenching with cysteine and purifying by PD-10
as described above.
[0223] Preparative purification of E4 mix by hydroxyapatite. The
buffer of cAC10 (25 mM sodium citrate pH 6.5, 250 mM NaCl, and
0.02% Tween-80) was changed to PBS using several 15 mL Amicon
Ultrafree 30K cutoff spin concentration devices. 1.09 g of cAC10 in
PBS was fully reduced with DTT in a final volume of 89 mL as
follows: cAC10 (82.3 .mu.M) was treated with 10 mM DTT in 0.025 M
sodium borate pH 8, 0.025 M NaCl for 1 h at 37.degree. C. This
mixture was diluted to 250 mL with water and applied to a 70 mL
hydroxyapatite column (Macroprep ceramic type I 40 .mu.m, BioRad)
at a flow rate of 10 mL/min. The column was previously equilibrated
with 5 column volumes of 0.5 M sodium phosphate pH 7, 10 mM NaCl
and 5 column volumes of 10 mM sodium phosphate pH 7, 10 mM NaCl.
Following application, the column was washed with 5 column volumes
of 10 mM sodium phosphate pH 7, 10 mM NaCl and then eluted with 100
mM sodium phosphate pH 7, 10 mM NaCl.
[0224] Fully reduced cAC10 was reoxidized with DTNB as follows:
eluted material from above (6.02 mg/mL or 40.2 .mu.M, 1.02 g in 170
mL) was cooled to 0.degree. C. and then treated with 2.0
equivalents of DTNB (10 mM stock) for 20 min. Without further
purification, reoxidized cAC10 was conjugated to vcMMAE. Cold cAC10
(31.9 .mu.M final) was treated with 5 equivalents of vcMMAE (159.5
.mu.M final) dissolved in DMSO (20% final) in a final volume of 214
mL. After 40 min at 0.degree. C., 1.07 mL of 100 mM cysteine was
added to quench any unreacted vcMMAE and the mixture was diluted to
750 mL with water. The conjugate was purified on a hydroxyapatite
column as described above for the DTT reduction. The recovered
cAC10-vcMMAE E4 mix (0.99 g, or 91% overall yield based on cAC10)
was concentrated and the buffer changed to PBS using several 15 mL
Amicon Ultrafree 30K cutoff spin concentration devices.
[0225] Preparative purification of pure E4 by HIC was performed on
a 45 mL Toyopearl phenyl 650M HIC column at a flow rate of 10
mL/min at ambient temperature. Solvent A was 2 M NaCl and 50 mM
sodium phosphate pH 7. Solvent B was 80% v/v 50 mM sodium phosphate
pH 7 and 20% v/v acetonitrile. The column was previously
equilibrated with 5 column volumes of solvent A. Up to 400 mg of
cAC10-vcMMAE E4 mix purified by hydroxyapatite (above) was mixed
with 1 volume of 4 M NaCl and 50 mM sodium phosphate pH 7 and
applied to the column. E0 was not retained by the column. The
different drug loaded species were eluted by sequential step
gradients: E2 was eluted with 35% solvent B, E4 was eluted with 70%
solvent B, E6 was eluted with 95% solvent B, and E8 was eluted with
100% solvent B. Purified E4 was concentrated and the buffer changed
to PBS using several 15 mL Amicon Ultrafree 30K cutoff spin
concentration devices, yielding 235 mg of pure E4 from two 400 mg
purifications. Purity analysis by analytical HIC (below) showed E4
purity greater than 90%.
Example 5 of Method 1
[0226] TCEP limited reduction followed by alkylation without
intermediate purification was accomplished by treating cAC10 with
2.75 molar equivalents of TCEP in 0.025 M sodium borate pH 8, 0.025
M NaCl, 1 mM DTPA for 2 h at 37.degree. C. See also FIG. 9 The
mixture was then cooled to 0.degree. C., and partially reduced
cAC10 was alkylated with vcMMAE as described above. cAC10-vcMMAE
was desalted using PD-10 columns (Amersham Biosciences, Piscataway,
N.J.) equilibrated with phosphate buffered saline. (Partial
reduction can also be performed with an intermediate purification
step of the partially reduced antibody, as shown in FIG. 8.)
[0227] The samples used to determine the kinetics of isomer
distribution were prepared as follows: cAC10 was reduced with 3.0
equivalents of DTT in 50 mM sodium phosphate pH 7.5 and 5 mM EDTA
at 37.degree. C. At the indicated time points, samples were
removed, quenched with an equal volume of 200 mM sodium citrate pH
5, and purified using PD-10 columns equilibrated with phosphate
buffered saline containing 5 mM EDTA. Reduced cAC10 was treated
with vcMMAE as previously described and purified using PD-10
columns equilibrated with phosphate buffered saline.
[0228] Purification of E2, E4, and E6 pure by HIC was performed on
a Toyopearl phenyl 650M HIC column (Tosoh Biosciences,
Montgomeryville, Pa.) at a flow rate of 10 mL/min at ambient
temperature. The column size was 1 mL per 7.5 mg of cAC10-vcMMAE.
Solvent A was 2.0 M NaCl and 50 mM sodium phosphate pH 7. Solvent B
was 80% v/v 50 mM sodium phosphate pH 7 and 20% v/v acetonitrile.
The column was previously equilibrated with 5 column volumes of
solvent A. cAC10-vcMMAE was mixed with 0.67 volume of 5 M NaCl (2.0
M final) and applied to the column. E0 was not retained by the
column. The different drug loaded species were eluted by sequential
step gradients: E2 was eluted with 35% solvent B, E4 was eluted
with 70% solvent B, E6 was eluted with 95% solvent B, and E8 was
eluted with 100% solvent B. Purified E4 was concentrated and the
buffer changed to phosphate buffered saline using Ainicon Ultrafree
30K cutoff spin concentration devices.
Example 6 of Analytical Methods
[0229] Drug loading was determined by measuring the ratio of the
absorbance at 250 and 280 nm (A250/280). The number of vcMMAE per
cAC10 has been empirically determined to be
(A250/280-0.36)/0.0686.
[0230] The conjugates were analyzed for percent E4 purity by
hydrophobic interaction chromatography (HIC) using a Tosoh
Biosceince Ether-5PW column (part 08641) at a flow rate of 1 mL/min
and a column temperature of 30.degree. C. Solvent A was 50 mM
sodium phosphate pH 7 and 2 M NaCl. Solvent B was 80% 50 mM sodium
phosphate pH 7, 10% 2-propanol, and 10% acetonitrile. Isocratic 0%
B for 15 min, a 50-min linear gradient from 0 to 100% B, a 0.1-min
linear gradient from 100 to 0% B, and isocratic 0% B for 14.9 min.
Injections (typically 90-100 .mu.L) were 1 volume of purified
vcMMAE-cAC10 conjugate (concentration of at least 3 mg/mL) and 1
volume of 50 mM sodium phosphate pH 7 and 4 M NaCl.
[0231] The ADC's, including pure E4 from HIC chromatography, were
analyzed under denaturing and non-reducing conditions using an
Agilent Bioanalyzer. A protein 200 chip was used under denaturing
but nonreducing conditions as described by the manufacturer.
Briefly, 4 .mu.L of 1 mg/mL cAC10-vcMMAE was mixed with 2 .mu.L of
nonreducing loading buffer and heated to 100.degree. C. for 5 min.
Water (84 .mu.L) was added and 6 .mu.L of this mixture was loaded
into each well of the chip.
[0232] Pure E4 was finally analyzed on a PLRP-S column (Polymer
Laboratories). The flow rate was 1 mL/min and the column
temperature was 65.degree. C. Solvent A was 0.05% trifluoroacetic
acid in water and solvent B was 0.04% trifluroacetic acid in
acetonitrile. Isocratic 25% B for 3 min, a 15-min linear gradient
to 50% B, a 2-min linear gradient to 95% B, a 1-min linear gradient
to 25% B, and isocratic 25% B for 2 min. Injections were 10 .mu.L
of cAC10-vcMMAE previously reduced with 20 mM DTT at 37.degree. C.
for 20 min to cleave the interchain disulfides.
[0233] The ADC's also were analyzed under denaturing and reducing
conditions on a PLRP-S column (Polymer Laboratories) (2.1.times.150
mm, 8 .mu., 1000 .ANG.). The flow rate was 1 mL/min and the column
temperature was 80.degree. C. Solvent A was 0.05% trifluoroacetic
acid in water and solvent B was 0.04% trifluroacetic acid in
acetonitrile. Isocratic 25% B for 3 min, a 25-min linear gradient
to 50% B, a 2-min linear gradient to 95% B, a 1-min linear gradient
to 25% B, and isocratic 25% B for 2 min. Injections were 10-20
.mu.L of I mg/ml cAC10-vcMMAE previously reduced with 20 mM DTT at
37.degree. C. for 15 min to cleave the remaining interchain
disulfides. The mole fraction of each chain was determined using
the following molar extinction coefficients: light chain with 0
vcMMAE: 30,160 M.sup.-1 cm.sup.-; light chain with 1 vcMMAE: 31,660
M.sup.-1 cm.sup.-1; heavy chain with 0 vcMMAE: 86,915 M.sup.-1
cm.sup.-1; heavy chain with 1 vcMMAE: 88,415 M.sup.-1 cm.sup.-1;
heavy chain with 2 vcMMAE: 89,915 M.sup.-1 cm.sup.-1; heavy chain
with 3 vcMMAE: 91,415 M.sup.-1 cm.sup.-1.
[0234] The isomeric distribution for E2 and E6 was determined using
solely PLRP-S HPLC data. For E2 isomer A (for these analyses,
"isomer A" refers to both isomers 2A and 2B of FIG. 7), the mole
fraction of light chain with 0 vcMMAE (L0) is equal to the mole
fraction of heavy chain with 1 vcMMAE (H1), while for E2 isomer C,
the mole fraction of light chain with 0 and 1 vcMMAE and the mole
fraction of heavy chain with 0 and 1 vcMMAE are all equal. Since
only light chain with 1 vcMMAE (L1) and heavy chain with 0 vcMMAE
(HO) contribute to the percentage of isomer C, the percent isomer C
can be expressed as follows:
% C=2L1+2H0 (1)
[0235] The percent of isomer A is assumed to be 100-% C. Small
amounts (less than 3% total) of heavy chain with 2 or 3 vcMMAE are
often observed in the PLRP-S HPLC data. These are probably due to
contaminating E4 or E6 in the E2 sample. For the purposes of
calculating the percent of E2 isomers A and C, the sum of the mole
percent of L0, L1, H1, and H2 was set to 100%.
[0236] Similarly, for E6 isomer A (for these analyses, "isomer A"
refers to both isomers 6A and 6B of FIG. 7), the mole fraction of
H2 is equal to the mole fraction of L1, while for E6 isomer C, the
mole fractions of L0, L1, H2, and H3 are equal. Since only L0 and
H3 contribute to the percentage of isomer C, the percent isomer C
can be expressed as follows:
% C=2L0+2H3 (2)
The percent of isomer A is assumed to be 100-% C. As with E2, the
sum of the mole percent of L0, L1, H2, and H3 was set to 100%.
[0237] The percentages of the E4 isomers cannot be obtained solely
from the PLRP-S HPLC data because there is not a unique solution.
At least one isomer needs to be fixed before PLRP data can be used
to solve for the other two isomers. The mole percent of HHL, HH,
and HL were determined from Bioanalyzer data using the following
molecular weights: 124,720.8 (HHL), 100,992.6 (HH), and 74,224.5
(HL) g/mol. The Bioanalyzer uses the fluorescence of bound dye for
instrument readout, and it is assumed that HHL, HH, and HL bind the
dye equally per unit molecule weight, although it is unlikely that
this assumption is true. To minimize the error that would result
from this assumption, only isomer E4A was calculated from
Bioanaylzer data using the HHL, HH, and HL peak areas as follows
(the HL peak area is divided by 2 since each antibody would produce
2 HL if the heavy-heavy chain disulfides were cleaved):
% A = H H L 124720.8 H H L 124720.8 + H H 100992.6 + H L 2 *
74224.5 ( 3 ) ##EQU00001##
[0238] PLRP-S HPLC data was then used to solve for the remaining
contribution of E4 isomers E and F using the following
formulas:
% E=H1+L1-0.5% A (4)
% F=H2+L0-0.5% A (5)
[0239] E4 isomer A contributes equally to the populations of L0,
L1, H1, and H2, and the H1 and L1 contributions of isomer A (half
of its total contribution) must be subtracted from the total
observed amount of H1 and L1 to give the remaining amount of H1 and
L1 that must be due to the presence of isomer E. A similar
subtraction for the contribution of H2 and L0 for isomer A will
yield the amount present due to isomer F. As with E2 and E6, the
sum of the mole percent of L0, L1, H1, and H2 was set to 100%.
Example 7 of Strategies for Partial Loading of Protein
[0240] Two different strategies were used to prepare partially drug
loaded ADCs. First, partial reduction of cAC10 with limiting
amounts of DTT or TCEP yields fewer than 8 antibody cysteines.
About 3.25 and 2.75 equivalents of DTT and TCEP, respectively, will
cleave 2 interchain disulfide bonds to yield an average of 4 cAC10
cysteines per antibody (a mixture of 0, 2, 4, 6, and 8
antibody-cysteines). The amount of reducing agent can be
empirically determined: cBR96 requires only 2.1 equivalents of DTT
or TCEP to yield 4 antibody cysteines, while murine IgG1 antibodies
can often by extremely resistant to reduction (data not shown). An
advantage of using TCEP rather than DTT is that phosphines react
poorly with maleimides, and any remaining reducing agent does not
have to be removed before adding vcMMAE. Excess DTT readily reacts
with vcMMAE and would compete with antibody-cysteines for the drug.
Following antibody reduction, treatment of antibody cysteines with
a slight molar excess of vcMMAE (1.1 molar equivalents per
cysteine) yields cAC10 with an average drug loading of 4 MMAE per
antibody (E4 mix).
[0241] Alternatively, cAC10 can be fully reduced with 10 mM DTT and
then partially reoxidized with DTNB. This reoxidiation process is
very efficient, requiring 2.0 equivalents of DTNB to reoxidize 8
antibody cysteines to 4. Treatment of this reoxidized antibody with
a thiol such as cysteine does not liberate any bound
thionitrobenzoic acid, suggesting that the reoxidized cysteines are
in the form of antibody disulfides rather than mixed TNB-cysteine
disulfides. The analytical methods described below also show the
presence of antibody disulfides. The remaining antibody cysteines
can be conjugated to vcMMAE as described above to yield E4 mix.
[0242] To determine the isomeric population of each of the drug
loaded species, E2, E4, and E6, are separated and isolated,
yielding E2, E4, and E6 pure. FIG. 13A shows a hydrophobic
interaction (HIC) HPLC trace of E4 mix made by DTT partial
reduction. All of the even drug loaded species can be separated
from each other, and small amounts of odd drug loaded species can
be seen in the trough between the even species. The drag loading of
these species can be assigned by inspection of the UV spectra of
the peaks. The PABA group in the drug linker has a maximum
absorbance near 248 mu, while the antibody has a minimum absorbance
at the same wavelength. Using the drug and antibody extinction
coefficients at 248 and 280 nm, the number of drugs per antibody
can be assigned for the starting ADC mixture and each of the
observed peaks (Hamblett et al. (2004), Clin Cancer Res 10:
7063-70).
[0243] Table 1 shows the percentages of the even drug loaded
species prepared by DTT partial reduction, TCEP partial reduction,
and partial DTNB reoxidation. The DTNB partial reduction method
yields a slightly higher percentage of E4 (38%) than the partial
reduction methods (30% for DTT and 33% for TCEP). This comes at the
expense of mainly E6 and E8, which total about 34% for DTT partial
reduction and 31% for TCEP partial reduction, while only 24% for
DTNB partial reoxidation. The odd drug loaded species not shown on
the table and account for 7-10% of the total material.
TABLE-US-00001 TABLE 1 Percent composition of E4 mixture..sup.a
Production method E0 E2 E4 E6 E8 DTT partial 9 .+-. 2 20 .+-. 3 30
.+-. 1 24 .+-. 3 10 .+-. 3 reduction TCEP partial 8 .+-. 1 20 .+-.
3 33 .+-. 2 22 .+-. 2 9 .+-. 1 reduction DTNB partial 10 .+-. 4 18
.+-. 3 38 .+-. 2 20 .+-. 4 4 .+-. 2 reoxidiation .sup.aHIC-HPLC
chromatograms were integrated for percent composition. Values are
plus or minus standard deviation for 4 (DTT partial reduction), 3
(TCEP partial reduction), or 6 (DTNB partial reoxidation) separate
batches. The contributions from odd species are not shown, causing
the total to be less than 100%.
[0244] This HIC-HPLC method can be used to isolate a few milligrams
of E2, E4, and E6 pure. Alternatively, preparative HIC using step
gradients can be used to isolate hundreds of milligrams of E2, E4,
and E6 pure, as shown in FIGS. 13B, C, D. The purity of these
materials, with respect to their drug loading levels, is at least
95%.
[0245] These purified materials were subjected to two analytical
methods to determine the distribution of the drugs on the antibody
(see Example 6). First, reducing and denaturing HPLC on a PLRP-S
column was used to determine the number of drugs per antibody
chain. Pretreatment of the ADC with an excess of DTT breaks the
remaining interchain disulfides and allows separation of light
chain with 0 or 1 drugs (L0 and L1) from heavy chain with 0, 1, 2,
or 3 drugs (H0, H1, H2, and H3) (FIG. 4). Second, non-reducing and
denaturing capillary electrophoresis allows separation of antibody
chains with the remaining interchain disulfides intact, resulting
in 6 potential species: L, H, HL, HH, HHL, and HHLL (FIG. 15).
[0246] Quantitation of the species observed by PLRP-S HPLC and
capillary electrophoresis allows assignment of the isomeric
populations. FIGS. 1 and 7 illustrate the antibody fragments and
the number of associated drugs for each of the isomers. The
isomeric populations of E2 and E6 can easily be determined by
PLRP-S HPLC alone or capillary electrophoresis alone because each
isomer yields a unique pattern. For instance, only isomer E2C
yields L1 and H0 under denaturing and reducing conditions, while
E2A only yields L0 and H1, and under denaturing and non-reducing
conditions isomer E2A yields HHLL while E2C yields L and HHL. For
E4, neither PLRP-S HPLC nor capillary electrophoresis alone is
sufficient to calculate the isomeric populations, so the two
methods must be used in combination to determine the composition.
Table 2 shows the percent composition for each of these isomers.
PLRP-S HPLC data was used exclusively for calculating the isomeric
composition of the E2 and E6 isomers using Equations 1 and 2 (see
Example 6). Capillary electrophoresis was used to calculate the
amount of E4A using Equation 3, and PLRP-S HPLC was used to
calculate the amount of E4B and E4C using Equations 4 and 5 which
subtract out the contribution of E4A (see Example 6).
TABLE-US-00002 TABLE 2 Composition of isomeric population of
purified E2, E4, and E6. Production method E2A.sup.a E2C.sup.a
E4A.sup.b E4E.sup.c E4F.sup.c E6A.sup.d E6C.sup.d DTT partial 8 92
10 59 31 2 98 reduction DTNB partial 77 23 17 8 75 4 96 reoxidation
AET pH 5 partial 17 83 13 46 41 2 98 reduction .sup.aDetermined
from PLRP-S HPLC data using Equation 1. .sup.bDetermined from
Bioanalyzer data using Equation 3. .sup.cDetermined from PLRP-S
HPLC data using Equations 4 and 5. .sup.dDetermined from PLRP-S
HPLC data using Equation 2.
[0247] The data in Table 2 is striking because the production
method significantly effects the location of the drugs, suggesting
that the antibody disulfides can be selectively reduced. Partial
DTT reduction yields 92% isomer E2C, which results from reduction
of one of the heavy-light chain disulfides, 59% isomer E4E, which
results from the reduction of both heavy-light chain disulfides,
and 98% isomer E6C, which results from reduction of both
heavy-heavy chain disulfides and one heavy-light chain disulfide.
Isomers with one heavy-heavy chain disulfide reduced are in the
extreme minority. On the other hand, partial DTNB reoxidation
yields almost the opposite result for E2 and E4 isomers, 77% isomer
E2A and 75% MA, where one heavy-heavy chain disulfide is intact,
and the same result for E6, 96% E6C. Acidic reduction with AET
yields an isomer population that is very similar to DTT partial
reduction, and favors cleavage of the heavy-light chain
disulfides.
[0248] The kinetics of the isomer distribution for DTT partial
reduction is shown in Table 3. cAC10 was reduced with 3.0
equivalents of DTT and samples were periodically removed and
alkylated with vcMMAE. E2, E4, and E6 pure were obtained by
HIC-HPLC, and the isomer distribution was determined by PLRP-S HPLC
and Bioanaylzer. The isomer compositions are identical over the
course of the experiment, covering 10 to 120 min of reduction time
and a total drug loading of 1.3 to 3.9 drugs per antibody. These
results show that the DTT partial reduction isomer populations
shown in Table 2, prepared by reducing cAC10 for 2 h with a
limiting amount of DTT, are representative of the isomeric
population over the entire course of the reduction reaction.
TABLE-US-00003 TABLE 3 Kinetics of isomer distribution for DTT
partial reduction. Time (min).sup.a Drugs/mAb.sup.b E2A.sup.c
E2C.sup.c E4A.sup.d E4E.sup.e E4F.sup.e E6A.sup.f E6C.sup.f 10 1.3
12 88 9 63 28 N/D N/D 20 2.1 9 91 7 65 29 7 93 35 2.7 9 91 7 63 31
6 94 55 3.3 9 91 7 63 30 8 92 80 3.6 9 92 7 61 32 6 94 120 3.9 11
90 8 61 31 7 93 .sup.aReduction time. Once reduced, all antibodies
were treated with vcMMAE for identical times. .sup.bDetermined by
HIC-HPLC. .sup.cDetermined from PLRP-S HPLC data using Equation 1.
.sup.dDetermined from Bioanalyzer data using Equation 3.
.sup.eDetermined from PLRP-S HPLC data using Equations 4 and 5.
.sup.fDetermined from PLRP-S HPLC data using Equation 2. N/D, not
determined. At this time point, very little E6 was produced and
this material was not sufficient for determining the isomer
population.
TABLE-US-00004 TABLE 4 In vitro binding and cytotoxicity of
cAC10-vcMMAE. ADC Binding IC.sub.50 (.mu.g/mL).sup.a Karpas 299
IC.sub.50 (ng/mL).sup.b E0 (cAC10) 2.70 .+-. 1.91 N/D E2 mix DTT
N/D 11.4 .+-. 2.4 E2 pure DTT 3.57 .+-. 2.41 13.8 .+-. 3.6 E2 mix
DTNB N/D 11.7 .+-. 4.5 E2 pure DTNB 2.02 .+-. 1.22 13.2 .+-. 2.7 E4
mix DTT N/D 3.4 .+-. 1.2 E4 pure DTT 7.76 .+-. 3.93 4.8 .+-. 0.7 E4
mix DTNB N/D 5.0 .+-. 0.0 E4 pure DTNB 7.69 .+-. 4.42 4.3 .+-. 0.9
E8 6.53 .+-. 3.09 2.7 .+-. 0.2 .sup.aBinding to Karpas 299, in
.mu.g of antibody component/mL, determined from 4-7 independent
measurement plus or minus the standard deviation. N/D, not
determined. .sup.bIn vitro cytotoxicity, in ng of antibody
component/mL, determined from 3 independent measurements plus or
minus the standard deviation. N/D, not determined. cAC10 alone
displays poor potency against Karpas 299.
[0249] Table 4 lists the results of in vitro binding and
cytotoxicity experiments that were performed for ADCs of several
drug loading levels. E2 and E4 mix as well as E2 and E4 pure from
DTT partial reduction and DTNB partial reoxidation were tested. The
fully loaded conjugate with 8 drugs per antibody was the most
cytotoxic, with an IC50 value on the CD30 positive Karpas 299 cell
line of 2.7 ng/mL (calculated based on the weight of the antibody).
The ADCs with 4 drugs per antibody were slightly less cytotoxic,
with IC50 values between 3.4 and 5.0 ng/mL, and the ADCs with 2
drugs per antibody were the least cytotoxic, with IC50 values
between 11.4 and 13.8 ng/mL. The chemistry used to produce the ADCs
did not show any significant differences in the cytotoxicity, nor
were there significant differences between the mixtures and the
HIC-purified ADCs. The in vitro cytotoxicity appears to depend only
on the total dose of drug. Binding to CD30 positive cells was very
similar for E0, E2, and E4, with E8 being slightly impaired,
demonstrating that conjugation does not interfere with antigen
binding. The in vitro cytotoxicities of the ADCs (measuring the
antibody component) show the expected trend: the larger the number
of drugs, the lower the IC50 value. Within the error of the
experiment, the location of the drugs does not appear to influence
the in vitro cytotoxicity.
Example 8 of Drug Loading Effects on Antitumor Activity of
Monoclonal Antibody Drug Conjugate
[0250] Cells and reagents. CD30-positive ALCL line Karpas-299 was
obtained from the Deutsche Sammlung von Mikroorganism and
Zellkulturen GmbH (Braunschweig, Germany). L540cy, a derivative of
the HD line L540 adapted to xenograft growth, was graciously
provided by Dr. Harald Stein (Institut fur Pathologie, Univ.
Veinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin,
Germany). Cell lines were grown in RPMI-1640 media (Life
Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal
bovine serum.
[0251] Construction and purification of cAC10-Val-Cit-MMAE ADCs.
Briefly, cAC10 with 8 drugs per antibody was produced by cAC10 was
mixed with dithiothreitol (DTT) at 37.degree. C. for 30 min, and
the buffer was exchanged by elution through Sephadex G-25 resin
with PBS containing 1 mM diethylenetriaminepentaacetic acid (DTPA).
PBS containing 1 mM DTPA (PBS/D) was added to the reduced mAb
(final concentration 2.5 mg/mL). A 9.5 molar excess of
maleimidocaproyl-Val-Cit-MMAE, referred to as Val-Cit-MMAE, was
added to the reduced antibody at 4.degree. C. for 1 h and the
conjugation reaction was quenched by adding a 20-fold excess of
cysteine. The reaction mixture was concentrated by centrifugal
ultrafiltration and buffer-exchanged through Sephadex G25
equilibrated in PBS at 4.degree. C. The conjugate was then filtered
through a 0.2 micron filter under sterile conditions.
[0252] The generation of cAC10 ADCs with two and four MMAE
molecules per antibody involved a partial reduction followed by
reaction with Val-Cit-MMAE. The antibody cAC10 (10 mg/ml) was
partially reduced by addition of DTT to a final DTT:mAb molar ratio
of 3.0 followed by incubation at 37.degree. C. for .about.2 h. The
reduction reaction was then chilled to .about.10.degree. C. and the
reduced cAC10 purified away from excess DTT via diafiltration.
Following diafiltration, the thiol concentration in the
partially-reduced cAC10 was determined by the DTNB (Elhnan's)
assay; in this manner, an average of about 2 disulfide bonds were
reduced, thus exposing about 4 reduced Cys:mAb. To conjugate all of
the reduced Cys, Val-Cit-cMMAE was added to a final
Val-Cit-MMAE:reduced Cys molar ratio of about 1.15. The conjugation
reaction was carried out in the presence of 15% v/v of DMSO and
allowed to proceed at about 10.degree. C. for about 30 min.
Following the conjugation reaction, excess free Cys (2 moles of Cys
per mole of Val-Cit-MMAE) was added to quench unreacted
Val-Cit-MMAE to produce the Cys-Val-Cit-MMAE adduct. The Cys
quenching reaction was allowed to proceed at about 10.degree. C.
for about 30 min. The Cys-quenched reaction mixture was purified
and buffer-exchanged into PBS by diafiltration to obtain the
partially loaded cAC10-Val-Cit-MMAE.
[0253] Preparative HIC fractionation. All chromatographic steps
were performed at room temperature. A 1.6.times.25 cm column
(.about.50 ml) was packed with Toyopearl Phenyl-650M HIC resin
(Tosoh Bioscience, Montgomeryville, Pa.) and equilibrated with
>5 column volumes of Buffer A (50 mM sodium phosphate, 2 M NaCl,
pH 7.0) at a flow rate of 15 ml/min. To prepare the sample for
loading onto the column, 39 ml of partially loaded cAC10-vcMMAE
(12.9 mg/ml) was blended with 39 ml of Buffer A' (50 mM sodium
phosphate, 4 M NaCl, pH 7.0). The sample was loaded onto the column
at 10 ml/min; all subsequent steps were performed at a flow rate of
10 ml/min. Following sample loading, the column was washed with
Buffer A until an A.sub.280 baseline was achieved. cAC10-E2 was
eluted and collected with a step gradient consisting of 65% Buffer
A/35% Buffer B (80% v/v 50 mM sodium phosphate, pH 7.0, 20% v/v
acetonitrile). After baseline was again achieved, cAC10-E4 was
eluted and collected with a step gradient consisting of 30% Buffer
A/70% Buffer B. Both cAC10-E2 and cAC10-E4 peaks were collected to
.about.20% of their respective peak heights. The fractions of
interest were buffer exchanged into PBS using Ultrafree-15
centrifugal filter devices with a molecular weight cutoff of 30 kDa
(Millipore, Billerica, Mass.).
[0254] Analysis of conjugates. Analysis of the conjugates was
accomplished by HIC-HPLC using an Ether-5PW column (Tosoh
Bioscience, Montgomeryville, Pa.). The method consisted of a linear
gradient from 100% Buffer A to 100% Buffer C (80% v/v 50 mM sodium
phosphate, pH 7.0, 10% v/v acetonitrile, 10% v/v isopropanol) in 50
min. The flow rate was set at 1 ml/min, the temperature was set at
30.degree. C., and detection was followed at both 248 and 280 nm.
The identity of unmodified cAC10 and cAC10-E8 peaks was confirmed
by injection of cAC10 and cAC10-E8 standards. Because the antibody
and drug have distinct absorbance maxima (.omicron..sub.max=280 and
248 nm, respectively), it was possible to identify peaks
corresponding to cAC10 conjugates with 2, 4, and 6 drugs per
antibody by overlaying peak spectra.
[0255] In vitro characterization of cAC10-Val-Cit-MMAE ADCs.
Competition binding was performed on the ADCs to determine if the
conjugation or presence of drug affected the antigen binding. To
compare saturation binding of mAb and ADC, 5.times.10.sup.5
Karpas-299 cells were combined with serial dilutions of cAC10,
cAC10-E2, cAC10-E4, or cAC10-E8 in the presence of 1 .mu.g/ml cAC10
labeled with Alexa Fluor 488 (Molecular Probes, Eugene, Oreg.) in
staining medium for 30 min on ice and washed twice with ice cold
staining medium. Labeled cells were examined by a Fusion microplate
reader (Perkin-Elmer, Boston, Mass.). Sample data were
background-corrected and reported as the percent of maximum
fluorescence as calculated by the sample fluorescence divided by
the fluorescence of cells stained with 1 .mu.g/mL cAC10-Alexa
Fluor.RTM. 488 alone.
[0256] The growth inhibitory activities of cAC10 conjugates were
determined by measuring DNA synthesis. Conjugates were incubated
with CD30.sup.+ Karpas-299 or L540cy cells or CD30.sup.- WSU-NHL
cells. After a 92 h incubation with cAC10 or cAC10 ADCs cells were
labeled with [.sup.3H]-thymidine, 0.5 .mu.Ci/well, for 4 h at
37.degree. C. Cells were harvested onto filters using a harvester,
mixed with scintillation fluid and the radioactivity was measured
with a Topcount scintillation counter (Packard Instruments,
Meriden, Conn.). The percent untreated was plotted versus
concentration for each molecule to determine the IC.sub.50 (defined
as the mAb concentration that gave 50% inhibition of DNA
synthesis).
[0257] Xenograft models of human ALCL. To establish a subcutaneous
disease model of ALCL 5.times.10.sup.6 Karpas-299 cells were
implanted into the right flank of CB-17 SOD mice (Harlan,
Indianapolis, Ind.). Therapy with ADCs was initiated when the tumor
size in each group of 6-10 animals averaged approximately 50-100
mm.sup.3. Treatment consisted of either a single injection or
multiple i.v. injections using the schedule of one injection every
4 days for 4 injections (q4dx4). Tumor volume was calculated using
the formula (length.times.width.sup.2)/2. A tumor that decreased in
size such that it was impalpable was defined as a complete
regression (CR). A complete regression that lasted for .gtoreq.10
tumor doubling times was defined as a cure. Tumor growth inhibition
(TGI) was calculated when tumors in the control group reached
750-1000 mm.sup.3 in size as follows:
TGI = 1 - ( Mean tumor volume of treated group ) ( Mean tumor
volume of control group ) .times. 100 ##EQU00002##
[0258] Maximum tolerated dose. Groups of three BALB/c mice (Harlan,
Indianapolis, Ind.) were injected with 30-60 mg/kg of cAC10-E8,
60-120 mg/kg of cAC10-E4, or 200-250 mg/kg of cAC10-vcMMAE2 via the
tail vein to determine the single dose maximum tolerated dose
(MTD). Mice were monitored daily for 14 days, and both weight and
clinical observations were recorded. Mice that developed
significant signs of distress were sacrificed in accordance with
ACUC guidelines. The maximum tolerated dose was defined as the
highest dose which did not cause a serious overt toxicities or
greater than 20 percent weight loss within two weeks of injection
in any of the animals.
[0259] Pharmacokinetics. The pharmacokinetics of cAC10, cAC10-E2,
cAC10-E4, and cAC10-E8 were evaluated in SOD mice. SCID mice (n=3)
were administered 10 mg/kg of test material (based on the antibody
component) by tail vein injection. Blood samples were collected
from each mouse via the saphenous vein at 1 h, 4 h, 1 d, 2 d, 4 d,
7 d, 14 d, 21 d, 28 d, 35 d, 42 d, and 49 days post injection.
Blood was collected into heparin coated tubes followed by
centrifugation (14,000.times.g, 3 min) to isolate plasma. Plasma
concentrations of cAC10 and ADCs were measured by ELISA.
[0260] Briefly, the ELISA consisted of the following steps: plate
coat, block, sample binding, secondary mAb, TMB, and acid stop.
After each step the wells were washed with wash buffer (PBS, 0.05%
Tween-20, pH=7.4) three times. In the plate coat step anti-cAC10
mAb was coated onto 96-well plates at 2 .mu.g/mL in carbonate
buffer (0.1 M carbonate/bicarbonate, pH=9.6) at 4.degree. C.
overnight. Following the plate coat, blocking buffer (PBS, 1% BSA,
0.05% Tween-20) was added and incubated at room temperature for 1
h. Next, 100 .mu.L of standard or diluted plasma sample was added
to triplicate wells for 1 h at room temperature. The secondary step
consisted of a mouse anti-human IgG-HRP conjugate (Southern
Biotech, Birmingham, Ala.) incubated for 1 h. Subsequently, 100
.mu.L of 3,3',5,5'-tetramethylbenzidine (Sigma, St. Louis, Mo.) was
added to each well and upon color development the reaction was
stopped with 100 .mu.L of 1 N sulfuric acid. Optical density was
measured using a VMax Kinetic Microplate reader (Molecular Devices,
Sunnyvale, Calif.) at 450 nm and a blank at 630 nm.
Non-compartmental pharmacokinetic parameters were calculated with
WinNonlin (Pharsight, Mountain View, Calif.).
[0261] In vitro characterization. Competition binding experiments
were performed to evaluate if conjugation of MMAE to cAC10
interfered with the CD30 binding capability of the ADCs. CD30.sup.+
Karpas-299 cells were incubated with 1 pg/mL of fluorescently
labeled cAC10 combined with serial dilutions of unlabeled antibody,
cAC10-E2, cAC10-E4, or cAC10-E8. As shown in FIG. 10, each of the
ADC variants effectively competed with fluorescently labeled cAC10
equivalent to unlabeled cAC10. Thus, conjugation with MMAE did not
reduce antigen binding.
[0262] The in vitro cytotoxic activities of the ADCs were evaluated
by a [.sup.3H]-TdR incorporation assay with CD30.sup.+ Karpas-299
and L540cy cells and CD30.sup.- WSU-NHL cells. cAC10-E8
demonstrated significant activity against the Karpas-299 cells with
an IC.sub.50 of 1.0 ng/mL (FIG. 11a). Decreasing the amount of drug
in half to four MMAE molecules per mAb (cAC10-E4) increased the
IC.sub.50 to 2.9 ng/mL. Halving the drug loading again further
increased the IC.sub.50 to 6.2 ng/mL with cAC10-E2. Against the HD
line L540cy the ADCs had a similar trend with 10.sub.50 values of
1.4, 4.5, and 9.2 ng/mL for cAC10-E8, cAC10-E4, and cAC10-E2,
respectively (FIG. 11b). Selectivity of the
[0263] ADCs was evaluated with CD30.sup.- WSU-NHL cell line which
were insensitive to all cAC10-ADCs with IC.sub.50 values >1000
ng/ml (data not shown).
[0264] Xenograft models of human ALCL. The effect of drug loading
on in vivo anti-tumor activity was evaluated with a Karpas-299
subcutaneous xenograft models. Therapy was administered every
fourth day for a total of 4 injections (q4dx4) starting when tumor
volumes reached 50-100 mm.sup.3. Using this schedule, it was
previously found that cAC10-E8 at 1 mg/kg produced 100% CRs, at the
same dose cAC10-E4 obtained 100% CRs (data not shown). With the
goal of comparing the activity of the ADCs, lower doses were used
for cAC10-E4 and cAC10-E8. Cohorts of mice bearing subcutaneous
Karpas-299 xenografts were treated with multiple doses of cAC10-E2
(0.5 mg/kg/dose or 1 mg/kg/dose), cAC10-E4 (0.25 or 0.5
mg/kg/dose), or cAC10-E8 (0.25 or 0.5 mg/kg/dose). Table 5 displays
a summary of the efficacy results.
TABLE-US-00005 TABLE 5 Table 5: Anti-tumor activity of cAC10-E2,
cAC10- E4, cAC10-E4-Mixture, and cAC10-E8 in a subcutaneous
Karpas-299 xenograft model. Dose Complete Schedule ADC (mg/kg)
Regressions Cures TGI q4dx4 cAC10-E2 0.5 0/10 0/10 68% 1.0 10/10
10/10 97% cAC10-E4 0.25 1/10 1/10 56% 0.50 5/10 3/10 91% cAC10-E8
0.25 0/10 0/10 47% 0.50 6/10 6/10 90% x1 cAC10-E2 1.0 4/6 4/6 96%
cAC10-E4 1.0 6/6 5/6.sup.a 100% cAC10-E8 1.0 5/6 5/6 100% x1
cAC10-E4 1.0 9/10 7/10 99.2% cAC10-E4- 1.0 8/10 8/10 98.4% Mixture
Animals were treated with ADCs once tumor volumes reached 50-100
mm.sup.3. Doses were given once (x1) or four times every four days
(q4dx4). The number of complete regressions, cures and tumor growth
inhibition (TGI) are reported. .sup.aone mouse with a cure was
found dead on Day 72 with no sign of tumor mass.
[0265] While cAC10-E8 had twice the amount of MMAE as cAC10-E4 at
the same mAb dose, they were equally effective at both dose levels
(FIG. 12A). At the 0.5 mg/kg/dose, out of the ten animals treated
with cAC10-E4 five achieved complete regressions (CRs) and cAC10-E8
induced six of ten CRs. Untreated tumors reached a mean tumor
volume of 986 mm.sup.3 19 days following implant. Tumor growth
inhibition of cAC10-E4 was 91% compared to 90% for cAC10-E8. At
0.25 mg/kg/dose both of these ADCs induced a similar delay in tumor
growth compared to untreated control animals but no complete
regressions. cAC10-E2 at 1.0 mg/kg/dose, a dose which contained the
same amount of MMAE as cAC10-E4 at 0.5 mg/kg/dose and cAC10-E8 at
0.25 mg/kg, induced 10/10 cures. The effect on tumor growth with
cAC10-E2 at 0.5 mg/kg/dose was comparable to that seen with
cAC10-E4 and cAC10-E8 at 0.25 mg/kg/dose generating a TGI of 68%. A
physical mixture of the drug MMAE with cAC10, equivalent to
cAC10-E8 at the 0.5 mg/kg dose, produced only a slight delay in
tumor growth compared to untreated, highlighting that the linkage
of drug to antibody is critical for achieving anti-tumor
activity.
[0266] Single dose treatment of cAC10-E2, cAC10-E4, and cAC10-E8 in
this same model were then compared at 1.0 mg/kg (FIG. 12B). Five of
six animals treated with cAC10-E8 achieved cures. Of the six
animals treated with cAC10-E4 all achieved complete regressions
with five cures out to 108 days, the end of the study, one animal
was found dead on day 72 with no sign of tumor mass. Even though
cAC10-E2 at 1.0 mg/kg contained half as much MMAE as cAC10-E4 four
of six mice achieved CRs. The control group consisting of 1 mg/kg
of cAC10 plus 0.037 mg/kg free MMAE, equivalent to the amount of
drug contained in 1 mg/kg of cAC10-E8, had little effect on tumor
growth compared to untreated mice.
[0267] The initial conjugation of the partially-loaded ADC resulted
in a mixture of species containing 0-8 drugs/mAb. To evaluate the
activity of this mixture (cAC10-E4-Mixture) single dose anti-tumor
activity of cAC10-E4-Mixture was compared to the purified cAC10-E4
at 1.0 mg/kg. Similar to the previous single dose experiment nine
of ten mice treated with cAC10-E4 achieved CRs. Complete regression
were generated in eight of ten mice treated with cAC10-E4-Mixture,
with an average molar ratio of 4.0. Although it contained a
population of ADCs with different drug loadings the partially
loaded cAC10-E4-Mixture demonstrated equivalent anti-tumor activity
to the purified cAC10-E4.
[0268] Maximum tolerated dose and therapeutic window. The
single-dose tolerability of cAC10-E2, cAC10-E4, and cAC10-E8 was
evaluated in BALB/c mice with three per group. The maximum
tolerated dose (MTD) was defined as the highest dose that did not
induce greater than 20% weight loss or severe signs of distress or
overt toxicities in any of the animals. For cAC10-E8, mice were
dosed at 10 mg/kg intervals from 30-60 mg/kg. At a dose of 50
mg/kg, mice had a maximum weight loss of 14% six days after
injection, after which the weight loss recovered. A dose of 60
mg/kg induced 23% weight loss six days post injection in one
animal. With cAC10-E4 at 100 mg/kg, mice reached a maximum weight
loss of approximately 10%. At 120 mg/kg of cAC10-E4 one animal
displayed signs of significant distress and 17% weight loss and the
animal was euthanized. Mice treated with cAC10-E2 at doses up to
250 mg/kg, the highest dose tested, experienced a maximum weight
loss of 10.5% 6 days post injection, with no signs of distress.
Based on our observations, the MTD of cAC10-E2 was at least 250
mg/kg, cAC10-E4 was 100 mg/kg, and cAC10-E8 was 50 mg/kg.
Therapeutic index was defined as ratio of the single dose MTD to
the multi-dose efficacious dose, yielding 100 for cAC10-E8, 200 for
cAC10-E4, and at least 250 for cAC10-E2.
[0269] Pharmacokinetics. SCID mice were administered with cAC10,
cAC10-E2, cAC10-E4, and cAC10-E8 to determine how drug loading
effects pharmacokinetics. Table 6 illustrates the pharmacokinetic
parameters established by non-compartmental analysis.
TABLE-US-00006 TABLE 6 Pharmacokinetic parameters of cAC10 and
cAC10 ADCs in SCID mice at a dose of 10 mg/kg. t1/2 AUC CL Vz Name
days (.mu.g-day/mL) (mL/day/kg) (mL/kg) cAC10 16.7 2638 3.8 91
cAC10-E2 16.9 2313 4.4 107 cAC10-E4 14.0 1689 6.0 121 cAC10-E8 14.9
520 19.2 414 The half-life (t1/2), area under the curve (AUC),
clearance (CL), volume of distribution (Vz), and area under the
curve from injection to day 14 (AUC t(0-14d)) were calculated using
non-compartmental analysis.
[0270] The time-concentration curves of cAC10, cAC10-E2, cAC10-E4,
and cAC10-E8 followed bi-exponential declines. The terminal
half-lives were 16.7, 16.9, 14.0 and 14.7 days, respectively and
thus, did not directly correlate with drug loading. However, the
exposure of ADCs as determined by the AUC increased as drug loading
decreased, ranging from 2638 .mu.g-day/mL for unmodified cAC10 to
520 .mu.g-day/mL for cAC10-E8. Conversely, the clearance values
increased from 3.8 mL/day/kg for cAC10 to 4.4, 6.0 and 19.2
mL/day/kg for cAC10-E2, cAC10-E4, and cAC10-E8, respectively.
Similarly, the volume of distribution was found to directly
correlate to drug loading.
Example 9 of Further Study of In Vivo Efficacy
[0271] In vivo efficacy experiments were performed using the Karpas
299 CD30 positive cell line and are shown in Table 7. Subcutaneous
Karpas-299 tumors were grown in C.B.-17 SCID mice, with the test
articles administered when tumors reached approximately 100 mm3
(length.times.width.sup.2). Animals were separated into groups of
5-10 animals, and each group was injected with ADC intravenously.
Doses were made in 2-fold serial dilutions (0.5, 1.0 2.0 mg/Kg) and
tumors that regressed to an unmeasurable size were defined as
complete remissions. The dose that yielded .gtoreq.80% complete
remissions over several experiments (3-8, with 5-15 animals per
dose except E6P, which was a single group of 4 animals) was
assigned as the efficacious dose. For all the ADCs tested, the
efficacious dose was 1 mg/Kg, despite the fact that the amount of
injected drug component changed with the drug loading. Within the
precision of the experiment (2-fold serial dilutions), the isomeric
distribution of the drugs did not influence the efficacy.
[0272] To evaluate the tolerability of the ADCs BALB/c mice (parent
strain of the C.B.-17 SCID) were administered with ADCs. Animal
weights were measured and clinical observations were recorded over
a 14 day period. The MTD was assigned as the highest single dose
administered to a BALB/c mouse that did not result in weight loss
.gtoreq.20% or show signs of distress. For E8, doses were 40, 50
and 60 mg/Kg, for E4, doses were 80, 100 and 120 mg/Kg, and for E2,
doses were 200 and 250 mg/Kg. As observed previously (see Example
8), the absolute drug loading level did influence the MTD, with
higher drug loading levels having lower MTD values. For E4 pure
made by DTNB partial reoxidation, the MID was slightly higher than
for E4 pure made by the DTT partial reduction (120 versus 100
mg/Kg).
TABLE-US-00007 TABLE 7 Mouse in vivo efficacy on CD30.sup.+ Karpas
299 cells and MTD for cAC10-vcMMAE. ADC Efficacious dose
(mg/Kg).sup.a MTD (mg/Kg).sup.a E2P DTT 1 >250 E4P DTT 1 100 E4P
DTNB 1 120 E6P DTT 1 80 E8 1 50 .sup.aIn vivo doses were based on
mg of antibody component per Kg of body weight.
[0273] No license is expressly or implicitly granted to any patent
or patent applications referred to or incorporated herein. The
discussion above is descriptive, illustrative and exemplary and is
not to be taken as limiting the scope defined by any appended
claims.
[0274] Various references, including patent applications, patents,
and scientific publications, are cited herein, the disclosures of
each of which is incorporated herein by reference in its
entirety.
* * * * *