U.S. patent application number 15/636460 was filed with the patent office on 2017-10-19 for methods for screening antibodies.
This patent application is currently assigned to SEATTLE GENETICS, INC.. The applicant listed for this patent is SEATTLE GENETICS, INC.. Invention is credited to Dennis Benjamin, Robert Lyon, Maureen Ryan.
Application Number | 20170298118 15/636460 |
Document ID | / |
Family ID | 44542524 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170298118 |
Kind Code |
A1 |
Lyon; Robert ; et
al. |
October 19, 2017 |
METHODS FOR SCREENING ANTIBODIES
Abstract
The invention provides methods for making antibody conjugates
for use in antibody screening assays and antibody conjugates
produced by the claimed methods.
Inventors: |
Lyon; Robert; (Sammamish,
WA) ; Benjamin; Dennis; (Redmond, WA) ; Ryan;
Maureen; (Bellevue, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEATTLE GENETICS, INC. |
Bothell |
WA |
US |
|
|
Assignee: |
SEATTLE GENETICS, INC.
Bothell
WA
|
Family ID: |
44542524 |
Appl. No.: |
15/636460 |
Filed: |
June 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13581236 |
Aug 24, 2012 |
9725500 |
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PCT/US2011/026534 |
Feb 28, 2011 |
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15636460 |
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61309725 |
Mar 2, 2010 |
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61323433 |
Apr 13, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5014 20130101;
C07K 1/04 20130101; C07K 16/00 20130101; A61K 47/6803 20170801;
G01N 33/533 20130101; A61P 35/00 20180101; C07K 2317/24
20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 1/04 20060101 C07K001/04; G01N 33/50 20060101
G01N033/50; G01N 33/533 20060101 G01N033/533 |
Claims
1. A method for making antibody conjugates for use in antibody
screening assays, the method comprising the steps of: (a) providing
a first and second antibody-containing sample, wherein the first
and second antibody-containing samples vary with respect to
antibody quantity and antibody sequence provided that substantially
all of the antibody present in the first sample is of the same
sequence and substantially all of the antibody present in the
second sample is of the same sequence, wherein the antibodies of
the first and second samples have sulfhydryl groups introduced into
said antibodies; (b) immobilizing the antibodies on solid support
to provide a first and second sample comprising immobilized
antibodies; (c) fully reducing the interchain disulfide bonds and
introduced sulfhydryl groups of the immobilized antibodies to
provide a first sample comprising reduced immobilized antibodies
and second sample comprising reduced immobilized antibodies; (d)
reacting the reduced immobilized antibodies with capping agent,
drug or drug-linker, and optionally a detection agent to provide
immobilized antibody conjugates, wherein the capping agent, drug or
drug-linker, and optional detection agent selectively react with
reactive thiols, the capping agent, drug or drug-linker, and
optional detection agent are provided in molar excess, and the
ratio of capping agent, drug or drug linker, and optional detection
agent is selected so as to achieve a desired level of drug loading;
and (e) eluting the antibody conjugates to provide a first and
second antibody drug conjugate composition.
2. The method of claim 1 wherein the antibody in the first and
second antibody-containing samples have the same number of
introduced sulfhydryl groups and the reduced immobilized antibodies
are contacted with the same ratio of capping agent, drug or
drug-linker, and optional detection agent.
3. The method of claim 1 wherein the antibody in the first and
second antibody-containing samples have different number of
introduced sulfhydryl groups and the reduced immobilized antibodies
are contacted with a different ratio of capping agent, drug or
drug-linker, and optional detection agent.
4. The method of claim 1 wherein the reduced immobilized antibodies
are reacted with a detection agent.
5. The method of claim 4 wherein the detection agent is a
fluorophore and the resultant antibody conjugates compositions have
an average fluorophore loading of about 3 fluorophores per
antibody.
6. The method of claim 1 wherein following elution of the antibody
conjugates, the actual or relative quantity of antibody present in
the antibody conjugate compositions is determined.
7. The method of claim 1 wherein the antibody present in the
antibody-containing samples prior to immobilization is impure.
8. The method of claim 1 wherein the quantity of antibody present
in the antibody-containing samples prior to immobilizing,
reduction, conjugation, and elution is not known.
9. The method of claim 1 wherein the antibody-containing samples
independently have from 1 .mu.g to 100 .mu.g of antibody present in
each of the sample.
10. The method of claim 9 wherein the antibody-containing samples
independently have from 1 .mu.g to 50 .mu.g of antibody present in
each of the samples.
11. The method of claim 10 wherein the antibody containing samples
independently have from 1 .mu.g to 20 .mu.g of antibody present in
each of the samples.
12. The method of claim 1 wherein the antibody in the first
antibody-containing sample and the antibody in the second
antibody-containing sample are of the same species.
13. The method of claim 1 wherein the capping agent, drug or
drug-linker and detection agent comprise a maleimide group.
14. The method of claim 13 wherein the drug-linker loading between
samples is substantially uniform.
15. The method of claim 14 wherein the antibody conjugate
compositions have an average drug-linker loading of about 2
drug-linkers per antibody.
16. The method of claim 1 wherein the antibody-containing samples
are cell culture supernatant samples.
17. The method of claim 16 wherein the cell culture supernatant is
unpurified hybridoma cell culture supernatant.
18. The method of claim 16 wherein the cell culture supernatant is
unpurified CHO cell culture supernatant.
19. The method of claim 16 wherein the cell culture media used for
providing the antibody-containing samples was substantially IgG
depleted.
20. The method of claim 16 wherein the solid support is added to
the cell culture supernatant.
21. A method for making antibody conjugates for use in high
throughput screening assays comprising the steps of: (a) providing
a plurality of samples of unpurified hybridoma supernatant
comprising unquantified antibody produced from a plurality of
hybridoma clones, wherein the plurality of samples vary with
respect to antibody quantity and antibody sequence provided that,
in a majority of the plurality of the samples, substantially all of
the antibody present in each sample is from a single hybridoma
clone wherein the antibodies of the plurality of samples have
sulfhydryl groups introduced into said antibodies; (b) immobilizing
the unquantified antibodies on solid support to provide a plurality
of samples comprising immobilized antibodies; (c) fully reducing
the intrachain disulfide and introduced sulfhydryl groups of the
immobilized antibodies to provide a plurality of samples comprising
reduced immobilized antibodies; (d) reacting the reduced
immobilized antibodies with capping agent, drug or drug-linker, and
a detection agent to provide immobilized antibody conjugates,
wherein the capping agent, drug or drug-linker, and detection agent
selectively react with reactive thiols, the capping agent, drug or
drug-linker, and optional detection agent are provided in molar
excess, and the ratio of capping agent, drug or drug linker and
detection agent is selected so as to achieve a desired level of
drug loading; and (e) eluting the immobilized antibody conjugates
from the solid supports to provide a plurality of antibody
conjugate compositions.
22. The method of claim 21 wherein the cell culture media used for
production of the antibody-containing samples was substantially IgG
depleted.
23. The method claim 21 wherein there is from 1 .mu.g to 100 .mu.g
of antibody present in each sample of hybridoma supernatant.
24. The method claim 23 wherein there is from 1 .mu.g to 50 .mu.g
of antibody present in each sample of hybridoma supernatant.
25. The method of claim 24 wherein there is from 1 .mu.g to 20
.mu.g of antibody present in each sample of hybridoma
supernatant.
26. The method of claim 21 wherein the detection agent is a
fluorescent label.
27. The method of claim 21 wherein the capping agent, drug or
drug-linker and detection agent comprise a maleimide group.
28. The method of claim 21 wherein the drug-linker loading between
samples is substantially uniform.
29. A method for preparing antibody conjugates, the method
comprising the steps of: (a) providing a plurality of
antibody-containing samples that vary with respect to antibody
quantity and antibody sequence provided that, in a majority of the
plurality of the antibody-containing samples, substantially all of
the antibody present in a single sample is of the same sequence;
(b) immobilizing the antibodies on solid support to provide a
plurality of samples comprising immobilized antibodies; (c) fully
reducing the interchain disulfide bonds and introduced sulfhydryl
groups of the immobilized antibodies to provide a plurality of
samples comprising reduced immobilized antibodies, (d) reacting the
reduced immobilized antibodies with capping agent, drug or
drug-linker, and optionally a detection agent to provide a
plurality of samples comprising immobilized antibody conjugates,
wherein the capping agent, drug or drug-linker, and optional
detection agent selectively react with reactive thiols, the capping
agent, drug or drug-linker, and optional detection agent are
provided in molar excess, and the ratio of capping agent, drug or
drug linker; and optional detection agent is selected so as to
achieve a desired level of drug loading; and (e) eluting the
antibody conjugates to provide a plurality of antibody conjugate
compositions.
30. A method for selecting an antibody for use in an antibody drug
conjugate, the method comprising the steps of: (a) providing a
plurality of antibody containing samples that vary with respect to
antibody quantity and antibody sequence provided that, in a
majority of the plurality of the antibody-containing samples,
substantially all of the antibody present in a single sample is of
the same sequence; (b) immobilizing the antibodies on a solid
support to provide a plurality of samples comprising immobilized
antibodies; (c) fully reducing the intrachain disulfide bonds and
introduced sulfhydryl groups of the immobilized antibodies to
provide a plurality of samples comprising reduced immobilized
antibodies; (d) reacting the reduced immobilized antibodies with
capping agent, drug or drug-linker, and optionally a detection
agent to provide a plurality of samples comprising immobilized
antibody conjugates, wherein the capping agent, drug or
drug-linker, and optional detection agent selectively react with
reactive thiols, the capping agent, drug or drug-linker, and
optional detection agent are provided in molar excess, and the
ratio of capping agent, drug or drug linker; and optional detection
agent is selected so as to achieve a desired level of drug loading;
(e) eluting the antibody conjugates to provide a plurality of
antibody conjugate compositions comprising free antibody
conjugates; (f) assaying for an activity of the antibody
conjugates; (g) determining the actual or relative quantity of
antibody present in the antibody conjugate compositions; (h)
selecting the antibody providing antibody drug conjugates with the
greatest activity based on the results of the assay and the actual
or relative quantity of antibody present in the antibody conjugate
compositions.
31. The method of claim 30, wherein the activity is cytotoxicity.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/581,236 filed Aug. 24, 2012, which is the
national stage filing under 35 U.S.C. .sctn.371 of International
Application No. PCT/US2011/026534 filed Feb. 28, 2011, which claims
the benefit of U.S. Provisional App. No. 61/309,725 filed Mar. 2,
2010 and U.S. Provisional App. No. 61/323,433 filed Apr. 13, 2010,
each of which is incorporated by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The activity of antibody-drug conjugates (ADCs) on cancer
cells can be affected by a multitude of factors, such as binding
affinity, rate of internalization, subcellular trafficking, and
efficient drug release within the target cell population.
Consequently, the properties of an ideal antibody for drug delivery
are not necessarily the same as those for a therapeutic
unconjugated antibody. Furthermore, indirect assays involving the
use of secondary antibodies to screen for optimal ADCs can be
misleading, since crosslinking on the cell surface can lead to
altered downstream events, and the affinity of the secondary
antibody constrains the dynamic range of the assay. When seeking
candidate antibodies directed against a novel antigen for ADC
therapy, it is therefore most desirable to screen a large antibody
panel in the form of ADCs and evaluate their cytotoxic activities,
since these results provide a direct measurement of parameters that
can affect cytotoxic activity. However, when dealing with microgram
quantities of a large number of antibodies as is typical of an
antibody discovery campaign, the yields from conventional
conjugation methodologies are limiting. A need exists for improved
methods of screening antibodies for use as ADCs. This present
invention addresses this and other needs.
SUMMARY OF THE INVENTION
[0003] The invention provides methods for making antibody
conjugates for use in antibody screening assays and antibody
conjugates produced by the claimed methods.
[0004] In some embodiments, the methods comprise the steps of
providing a first and second antibody-containing sample wherein the
first and second antibody-containing sample vary with respect to
antibody quantity and antibody sequence provided that substantially
all of the antibody present in the first sample is of the same
sequence and substantially all of the antibody present in the
second sample is of the same sequence; immobilizing the antibodies
on a solid support to provide a first and second sample comprising
immobilized antibodies; fully reducing the reducible disulfide
bonds of the immobilized antibodies to provide a first sample
comprising reduced immobilized antibodies and second sample
comprising reduced immobilized antibodies, wherein the reduction is
selective for the reducible disulfide bonds; reacting the reduced
immobilized antibodies with capping agent, drug or drug-linker, and
optionally a detection agent to provide immobilized antibody
conjugates, wherein the capping agent, drug or drug-linker, and
optional detection agent selectively react with reactive thiols,
the capping agent, drug or drug-linker, and optional detection
agent are provided in molar excess, and the ratio of capping agent,
drug or drug linker, and optional detection agent is selected so as
to achieve a desired level of drug loading; and eluting the
antibody conjugates to provide a first sample of free antibody
conjugates and second sample of free antibody conjugates.
[0005] In some embodiments, the methods comprise the steps of
providing a plurality of samples of unpurified hybridoma
supernatant comprising unquantified antibody produced from a
plurality of hybridoma clones, wherein the plurality of samples
vary with respect to antibody quantity and antibody sequence
provided that, in a majority of the plurality of the samples,
substantially all of the antibody present in each sample is from a
single hybridoma clone; immobilizing the unquantified antibodies on
a solid support to provide a plurality of samples comprising
immobilized antibodies; fully reducing the interchain disulfides of
the immobilized antibodies to provide a plurality of samples
comprising reduced immobilized antibodies; reacting the reduced
immobilized antibodies with capping agent, drug or drug-linker, and
a detection agent to provide immobilized antibody conjugates,
wherein the capping agent, drug or drug-linker, and detection agent
selectively react with reactive thiols, the capping agent, drug or
drug-linker, and optional detection agent are provided in molar
excess, and the ratio of capping agent, drug or drug linker and
detection agent is selected so as to achieve a desired level of
drug loading; and eluting the antibody conjugates from the solid
supports to provide a plurality of antibody conjugate
compositions.
[0006] In some embodiments, the methods comprise the steps of
providing a plurality of antibody containing samples that vary with
respect to antibody quantity and antibody sequence provided that,
in a majority of the plurality of the antibody-containing samples,
substantially all of the antibody present in a single sample is of
the same sequence; immobilizing the antibodies on a solid support
to provide a plurality of samples comprising immobilized
antibodies; fully reducing the reducible disulfide bonds of the
immobilized antibodies to provide a plurality of samples comprising
reduced immobilized antibodies, wherein the reduction is selective
for reducible disulfide bonds; reacting the reduced immobilized
antibodies with capping agent, drug or drug-linker, and optionally
a detection agent to provide a plurality of samples comprising
immobilized antibody conjugates, wherein the capping agent, drug or
drug-linker, and optional detection agent selectively react with
reactive thiols, the capping agent, drug or drug-linker, and
optional detection agent are provided in molar excess, and the
ratio of capping agent, drug or drug linker; and optional detection
agent is selected so as to achieve a desired level of drug loading;
and eluting the antibody conjugates to provide a plurality of
antibody conjugate compositions comprising free antibody
conjugates.
[0007] In some embodiments, the methods comprise the steps of
providing a plurality of antibody containing samples that vary with
respect to antibody quantity and antibody sequence provided that,
in a majority of the plurality of the antibody containing samples,
substantially all of the antibody present in a single sample is of
the same sequence; immobilizing the antibodies on a solid support
to provide a plurality of samples comprising immobilized
antibodies; fully reducing the reducible disulfide bonds of the
immobilized antibodies to provide a plurality of samples comprising
reduced immobilized antibodies, wherein the reduction is selective
for reducible disulfide bonds; reacting the reduced immobilized
antibodies with capping agent, and a detection agent to provide a
plurality of samples comprising immobilized antibody conjugates,
wherein the capping and detection agent selectively react with
reactive thiols, the capping agent, and detection agent are
provided in molar excess, and the ratio of capping agent and
detection agent is selected so as to achieve a desired level of
detection agent and/or capping agent loading; and eluting the
antibody conjugates to provide a plurality of antibody conjugate
compositions comprising free antibody conjugates.
[0008] In some embodiments, the methods comprise the steps of
providing a plurality of antibody containing samples that vary with
respect to antibody quantity and antibody sequence provided that,
in a majority of the plurality of the antibody-containing samples,
substantially all of the antibody present in a single sample is of
the same sequence; immobilizing the antibodies on a solid support
to provide a plurality of samples comprising immobilized
antibodies; fully reducing the reducible disulfide bonds of the
immobilized antibodies to provide a plurality of samples comprising
reduced immobilized antibodies, wherein the reduction is selective
for reducible disulfide bonds; reacting the reduced immobilized
antibodies with capping agent, drug or drug-linker, and optionally
a detection agent to provide a plurality of samples comprising
immobilized antibody conjugates, wherein the capping agent, drug or
drug-linker, and optional detection agent selectively react with
reactive thiols, the capping agent, drug or drug-linker, and
optional detection agent are provided in molar excess, and the
ratio of capping agent, drug or drug linker; and optional detection
agent is selected so as to achieve a desired level of drug loading;
eluting the antibody conjugates to provide a plurality of antibody
conjugate compositions comprising free antibody conjugates;
assaying for an activity of the antibody conjugates; and selecting
an antibody of the basis of the outcome of the assay.
[0009] In some embodiments, the methods comprise the steps of
providing a plurality of antibody containing samples that vary with
respect to antibody quantity and antibody sequence provided that,
in a majority of the plurality of the antibody containing samples,
substantially all of the antibody present in a single sample is of
the same sequence; immobilizing the antibodies on a solid support
to provide a plurality of samples comprising immobilized
antibodies; fully reducing the reducible disulfide bonds of the
immobilized antibodies to provide a plurality of samples comprising
reduced immobilized antibodies, wherein the reduction is selective
for reducible disulfide bonds; reacting the reduced immobilized
antibodies with capping agent, and a detection agent to provide a
plurality of samples comprising immobilized antibody conjugates,
wherein the capping and detection agent selectively react with
reactive thiols, the capping agent, and detection agent are
provided in molar excess, and the ratio of capping agent and
detection agent is selected so as to achieve a desired level of
detection agent and/or capping agent loading; eluting the antibody
conjugates to provide a plurality of antibody conjugate
compositions comprising free antibody conjugates; assaying for an
activity of the antibody conjugates; and selecting an antibody of
the basis of the outcome of the assay.
[0010] These and other aspects of the present invention may be more
fully understood by reference to the following detailed
description, non-limiting examples of specific embodiments, and the
appended figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. This figure provides an overlay of hydrophobic
interaction chromatograms of a murine IgG1 in its unconjugated form
(dashed), fully reduced and conjugated with mcMMAF in solution
(heavy solid line), and fully reduced and conjugated with mcMMAF
while immobilized on Protein G sepharose (light solid line).
[0012] FIG. 2. This figure illustrates the mole fraction of mcMMAF
in an exemplary reaction mixture comprising mcMMAF and N-ethyl
maleimide necessary in order to achieve a select drug loading on a
murine IgG1 murine immobilized on protein G and fully reduced with
excess tris(2-carboxyethyl)phosphine.
[0013] FIG. 3. The figure provides a sample PLRP chromatogram of an
antibody drug conjugate illustrating the distribution of mcMMAF and
NEM on the heavy and light chains of the antibody. The
hydrophobicity of the drug results in later retention times for
species with more drug; the number of drugs for each species is
indicated.
[0014] FIG. 4. This figure demonstrates the fluorescence output of
drug-Alexa Fluor.RTM. 647 conjugates as a function of fluorophore
loading. The number of fluorophores per antibody is plotted on the
x axis and fluorescence is plotted on the y axis. Fluorescence
increases rapidly to a maximum value when loading is about 2.5 to 3
fluorophores per antibody, then decreases with further loading.
[0015] FIG. 5. This figure provides the ratio of the absorbance at
650 nm to 280 nm plotted as a function of Alexa Fluor.RTM. 647
loading level in mixed fluorophore-mcMMAF antibody conjugates.
[0016] FIG. 6. This figure demonstrates the consistency of Alexa
Fluor.RTM. 647 loading across 65 samples. The fluorophore loading
was determined by obtaining the 650 nm/280 nm absorbance ratio of
each antibody conjugate sample and referring back to FIG. 5 to
determine the fluorophore loading associated with the absorbance
ratio.
[0017] FIG. 7. This figure provides a PLRP chromatogram of an
mcMMAF-AF647-NEM mixed conjugate. The antibody has 5 reducible
disulfides. This figure provides an overlay of two analytical
wavelengths. The 280 nm wavelength represented by a light solid
line detects all of the peaks containing protein and the 620 nm
wavelength represented by a heavy solid line detects all of the
peaks containing at least one Alexa Fluor.RTM. 647.
[0018] FIG. 8. This figure illustrates the consistency of mcMMAF
loading across 34 samples.
[0019] FIG. 9. This figure provides a PLRP chromatogram of an
mcMMAF-AF647-NEM mixed conjugate. The antibody has 6 reducible
disulfides (e.g., a murine IgG2b). The 280 nm wavelength is
represented by a light solid line and the 620 nm wavelength is
represented by a heavy solid line.
[0020] FIG. 10. This figure provides an exemplary scheme for
plate-based solid phase synthesis of ADCs.
[0021] FIG. 11. This figure provides an exemplary scheme for
application of solid phase conjugation technology to the discovery
of ADCs with desirable properties.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0022] 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 or (b) conservatively
substituted derivatives of such immunoglobulin polypeptides or
fragments that immunospecifically bind to a target antigen. The
antibodies (including antibody fragments) for use in the present
invention contain (i) an antigen binding site that
immunospecifically binds to a target antigen, (ii) at least one
reducible disulfide bond (e.g., interchain disulfide bond) and
(iii) a domain capable of reversibly binding to a solid phase. In
some embodiments, an antibody will comprise a full length Fc region
and binding to the solid phase will be through the Fc region. In
some embodiments, an antibody will comprise one or more Fc domains
of an antibody and binding to the solid phase will be through the
one or more Fc domains. In some embodiments, the domain capable of
reversibly binding to a solid phase will not be a Fc region, but
will be a domain engineered on the antibody, such as, for example,
an affinity tag. The term antibody includes antibodies that are
non-fucosylated or have reduced core fucosylation. Antibodies are
generally described in, for example, Harlow & Lane, Antibodies:
A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).
The basic unit of an intact 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.
The class and subclass of an antibody is its isotype. Antibodies
can be, for example, in their natural tetrameric form (2 light
chains and 2 heavy chains) and can be of any of the known isotypes
IgG, IgA, IgM, IgD and IgE and their subtypes, for example, human
IgG1, IgG2, IgG3, IgG4 and mouse IgG1, IgG2a, IgG2b, and IgG3. The
antibodies are preferably monoclonal.
[0023] In the context of an antibody, the term "reducible disulfide
bond" refers to a disulfide bond that is (i) reducible while the
antibody is reversibly bound to a solid support, and (ii) reducible
under mild reducing conditions. Mild reducing conditions are those
conditions that generally do not cause any substantial denaturation
of the antibody and generally do not affect the antigen binding
affinity of the antibody. An example of mild reducing conditions is
reduction under aqueous conditions at near neutral pH with a weak
reducing agent. An example of weak reducing agents are TCEP
(tris(2-carboxyethyl)phosphine) and DTT (dithiothreitol).
Accordingly, one example of mild reducing conditions is reduction
in an excess of TCEP or DTT at a temperature of about 5.degree. C.
to about 37.degree. C. and a pH of from about 5 to 8. Because
organic cosolvents can substantially denature proteins, if organic
cosolvents are to be used in the denaturation and/or subsequent
conjugation steps, it should be a minimal amount of cosolvents
(e.g., less than 20%, preferably less than 15%, 10%, or even 5%)
such that substantial denaturation of the antibody does not occur.
Typically, the reducible disulfide bonds are those that are solvent
accessible, i.e., not buried within the folded domains of the
antibody. (The skilled artisan will understand that when reducing
the reducible disulfide bonds of a population of antibodies within
a sample according to the methods described herein, there may be a
minor amount of antibodies that do become irreversibly denatured
(e.g., generally less than 10%, even less than 5% or 3%)).
Typically, in an antibody, 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
(interchain), or on the same polypeptide chain (intrachain). As a
result of the oxidation, a disulfide bond (--S--S--) is formed
between the beta carbons of the original cysteine residues.
Treatment of the disulfide bond with a reducing agent causes
reductive cleavage of the disulfide bonds to generate two free
thiol groups, i.e., reactive thiols. In some embodiments, the
reducible disulfide bond is naturally occurring. In some aspects,
the term "reducible disulfide bond" refers to the naturally
occurring interchain disulfide bonds of an antibody. In some
embodiments, a sulfhydryl group(s) is chemically introduced into
the antibody. Suitable methods for introducing sulfhydryl groups
include recombinant DNA technology. Sulfhydryl groups can be
introduced into an antibody, for example, within the antibody or at
the carboxy-terminus. Because it is preferable that the methods
described herein do not interfere with the antigen binding activity
of the resultant antibody conjugates, it is preferable that
introduced sulfhydryl groups be introduced at a site other than the
antigen binding site of the antibody. Preferably introduced
sulfhydryl groups are introduced at a site other than the heavy or
light chain variable regions, e.g., preferably in the constant
region of an antibody. In some embodiments, a cysteine residue is
engineered into an antibody. The sulfhydryl group of the cysteine
will typically form a disulfide bond that can then be reduced using
the methods described herein.
[0024] In the context of a fusion protein, the term "reducible
disulfide bond" refers to a disufide bond of a fusion protein that
is (i) reducible while the fusion protein is reversibly bound to a
solid support, and (ii) reducible under mild reducing conditions.
For a fusion protein to be of use in the present methods, it should
remain generally intact following reduction of the reducible
disulfide bond(s). An example of mild reducing conditions is
reduction under aqueous conditions at near neutral pH with a weak
reducing agent. In some preferred embodiments, the reducible
disulfide bond will be in the Ig domain of the fusion protein. In
some embodiments, the disulfide bond is naturally occurring and
refers to the naturally occurring interchain disulfide bonds of the
Ig domain of the fusion protein. In some embodiments, a sulfhydryl
group(s) is chemically introduced into Ig domain of the fusion
protein.
[0025] 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" is not
limited to antibodies produced through hybridoma technology.
[0026] The term "Fc region" refers to a constant region of an
antibody, e.g., a C.sub.H1-hinge-C.sub.H2-C.sub.H3 domain,
optionally having a C.sub.H4 domain, or a conservatively
substituted derivative of such an Fc region.
[0027] The term "Fc domain" refers to the constant region domain of
an antibody, e.g., a C.sub.H1, hinge, C.sub.H2, C.sub.H3 or
C.sub.H4 domain, or a conservatively substituted derivative of such
an Fc domain.
[0028] An "antigen" is a molecule to which an antibody specifically
binds.
[0029] A "cytotoxic agent" refers to an agent that has a cytotoxic
and/or cytostatic effect on a cell. A "cytotoxic effect" refers to
the depletion, elimination and/or the killing of a target cell(s).
A "cytostatic effect" refers to the inhibition of cell
proliferation.
[0030] 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 of an antibody.
[0031] As used herein, "free antibody conjugates" refers to
antibody conjugates that are not immobilized on a solid support,
e.g., antibodies that have been released from a solid support.
[0032] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine having the general formula shown immediately following:
##STR00001##
[0033] The abbreviation "MMAE" refers to monomethyl auristatin E
having the general formula shown immediately following:
##STR00002##
[0034] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleunine-dolaproine-phenylalanine having the
general formula shown immediately following:
##STR00003##
[0035] 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.
[0036] 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.
General
[0037] A method of directly screening antibodies on the basis of
their performance as ADCs or as unconjugated antibodies (i.e.,
naked antibodies) has been invented. A labeling technique has been
developed that is insensitive to the concentration of antibody
present in a sample and applicable to small amounts of antibody
allowing for a comparison of the activities of individual
antibodies of a heterogenous population of antibodies.
[0038] The screening assay is useful for identifying antibodies
with desired characteristics. The antibodies can be generated
though any technique known in the art for generating antibodies
provided that the antibodies to be screened comprise (i) an antigen
binding site that immunospecifically binds to a specific antigen,
(ii) at least one reducible disulfide bond (e.g., interchain
disulfide bond) and (iii) a domain capable of binding to a solid
phase.
[0039] In one aspect of the invention, a plurality of
antibody-containing samples are provided. The phrase "a plurality
of samples" refers to two or more samples. Because the methods
provided herein are ideally suited for high throughput screening,
in one aspect of the invention, the methods are performed
simultaneously on at least tens or at least hundreds of samples.
One of the strengths of the methods provided herein is that a
comparison between antibodies can be made even though the
antibody-containing samples may not contain the same quantity of
antibody. Accordingly, in one aspect, the samples vary with respect
to antibody quantity and with respect to antibody sequence. For
example, in one aspect, a first sample will comprise a first
antibody at a first quantity and a second sample will comprise a
second antibody at a second quantity. The first and second
quantities will vary and the first and second antibodies will vary.
In embodiments wherein it is desirable to compare antibodies that
target the same antigen, the antibodies will immunospecifically
bind to the same antigen. For purpose of clarification, the phrase
"wherein the plurality of samples vary with respect to antibody
quantity and antibody sequence" does not require that all of the
samples within a plurality of samples vary with respect to antibody
quantity and antibody sequence, only that there is certain level of
heterogeneity between samples. Although there is a variance in
antibody sequence (e.g., a first sample will contain a different
antibody than a second sample), it is preferable that a single
sample contain one antibody, i.e., that the antibody present in a
single sample is of the same sequence. The phrase "substantially
all of the antibody present in a single sample is of the same
sequence" reflects the preference that a single sample contain one
antibody with the recognition that, in some samples, there may be
some contamination with another antibody. Preferably, in those
samples that have some contamination with another antibody, there
is less than 30%, preferably less than 20%, preferably less than
15%, more preferably less than 10%, and even more preferably less
than 5%, less than 4%, or less than 3% of contamination with
another antibody. In preferred embodiments, the majority of
antibody-containing samples (greater that 50% of samples and even
more preferably greater than 60%, greater than 70%, greater than
75%, or even greater than 80% of the samples) in a plurality of
antibody-containing samples contain one antibody with no or minor
amounts of contamination with another antibody (e.g., less than
15%, preferably even less than 10% or less than 5% contamination
with another antibody). In some preferred embodiments, a majority
of the antibody-containing samples will comprise antibodies that
immunospecifically bind to the same antigen.
[0040] The antibodies to be screened using the present methods can
be targeted to any antigen. In exemplary embodiments, an antibody
to be screened by the present methods will immunospecifically bind
to an antigen selected from CD19, CD20, CD21, CD22, CD30, CD33,
CD38, CD40, CD70, CD74, CD83, CD133, CD138, CD200, or CD276. In
other embodiments, the antibody will immunospecifically bind to
BMPR1B, LAT1 (SLC7A5), STEAP1, MUC16, MUC1, megakaryocyte
potentiating factor (MPF), Napi3b, Sema 5b, PSCA hlg, ETBR
(Endothelin type B receptor), STEAP2, TrpM4, CRIPTO, CD21, CD79a,
CD79b, FcRH2, HER2, HER3, HER4, NCA, MDP, IL20R.alpha., Brevican,
Ephb2R, ASLG659, PSCA, PSMA, TMPRSS2, TMPRSS4, GEDA, BAFF-R, CXCRS,
HLA-DOB, P2X5, CD72, LY64, FCRH1, VEGF, PLAC1, VEGFR1, VEGFR2, or
IRTA2. In other embodiments, the antibody will immunospecifically
bind to CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19,
CD23, CD25, CD28, CD29, CD30, CD32, CD4OL, CD51, CD52, CD54, CD56,
CD70, CD80, CD123, CD133, CD138, CD147, CD227, or CD276. In other
embodiments, the antibody will immunospecifically bind to IL-1,
IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15,
IL-18, or IL-23. In other embodiments, the antibody will
immunospecifically bind to a protein from the solute carrier family
of proteins (e.g., solute carrier family 44, member 4 (protein
encoded by SLC44A4 gene) or solute carrier family 34, member 2
(protein encoded by the SLC34A2 gene)); LIV-1 (protein encoded by
SLC39A6 gene); protein from the SLAM family of proteins (e.g., SLAM
family members 1, 2, 3, 4, 5, 6, 7, 8 or 9); protein from the mucin
family of proteins (e.g., MUC1, MUC2, MUC3, MUC4, MUCS, MUC6, MUC7,
MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, or MUC16);
protein from the STEAP family of proteins (e.g., STEAP1, STEAP2,
STEAP3 or STEAP4); a protein from the tumor necrosis factor
receptor family (e.g., TNF-RI, TNF-RII, DR1, DR2, DR3, DR4, DRS);
MN protein; mesothelin protein; protein encoded by the Slitrk
family of proteins (e.g., SLITRK1, SLITRK2, SLITRK3, SLITRK4,
SLITRKS, or SLITRK6), or a protein encoded by the GPNMB gene.
[0041] The antibody-containing samples can be generated in many
different ways. There are many techniques known in the art for
generating antibodies. For example, antibodies that are useful in
the present methods can be produced by recombinant expression
techniques, phage display technique, from hybridomas, from
myelomas, from other antibody expressing mammalian cells, and from
combinations thereof. Antibodies to be used in the present
invention can be of any species (e.g., human, murine, rat) and can
be of mixed species, e.g., chimeric. Antibodies to be used in the
present invention can comprise full length variable regions or
fragments thereof.
[0042] A variety of mammalian cells and cell lines can be utilized
to express an antibody. For example, mammalian cells such as
Chinese hamster ovary cells (CHO) (e.g., DG44, Dxbll, CHO-K, CHO-K1
and CHO-S) can be used. In some embodiments, human cell lines are
used. Suitable myeloma cell lines include SP2/0 and IR983F and
human myeloma cell lines such as Namalwa. Other suitable cells
include human embryonic kidney cells (e.g., HEK293), monkey kidney
cells (e.g., COS), human epithelial cells (e.g., HeLa), PERC6,
Wil-2, Jurkat, Vero, Molt-4, BHK, and K6H6. Other suitable host
cells include YB2/0 cells.
[0043] Any antibody generating techniques can be used to generate
the antibody-containing samples described herein provided that the
antibodies generated can be immobilized on a solid support and
contain at least one reducible disulfide bond. In some embodiments,
the antibody will be generated by a method known in the art and
will be modified in order to place it in condition for use in the
present methods. For example, antibodies generated by phage display
or other methods can be modified to contain an affinity tag and/or
can be reformatted to express a Fc region. For an overview of phage
display technology for producing antibodies, see Schmitz et al.,
2000, Placenta 21, supplement A, S106-112. See also Lightwood et
al., 2006, Journal of Immunological Methods 316, 133-143.
[0044] In some aspects, the antibodies to be assayed are generated
using well known hybridoma techniques. For example, in some
embodiments, the host cells are from a hybridoma. Hybridoma
techniques are generally discussed in, for example, Harlow et al.,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 2nd ed., 1988); and Hammerling, et al., In Monoclonal
Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y.,
1981). Antibodies can also be generated using immortal or
conditionally immortal cell lines other than hybridoma cell lines,
including, for example, antibodies generated from conditionally
immortal cell lines from H-2K.sup.b-tsA58 mice (Pasqualinie and
Arap, PNAS, 2004, 101(1), 257-259). These technologies can be used
to generate fully rodent, chimeric rodent-human, or human
antibodies. For example, for an overview of a technology for
producing human antibodies from immunized transgenic mice using
hybridoma technology, see Lonberg and Huszar, 1995, Int. Rev.
Immunol. 13:65-93.
[0045] In addition, companies such as Amgen, Inc. (Thousand Oaks,
Calif.) and BMS (Princeton, N.J.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that referenced above. Completely human antibodies can
be produced 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, e.g.,
all or a portion of a target polypeptide. 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).
[0046] The present methods do not require a purification step prior
to antibody immobilization on a solid support. In some aspects, the
antibody provided in the antibody-containing sample is not
purified. In some embodiments, unpurified cell culture supernatant
or unpurified conditioned media is provided as the
antibody-containing sample. For example, in some embodiments
wherein hybridoma technology is used to generate antibodies, the
antibody-containing samples are unpurified hybridoma supernatant
samples. In some aspects, the supernatant samples vary with respect
to antibody quantity and antibody sequence. It is preferable that a
single hybridoma supernatant sample contain antibody from a single
hybridoma clone, although antibody-containing samples can contain
contamination with other antibodies. Methods of picking clonal
populations from hybridomas are known in the art as are methods of
generating hybridoma supernatant. For example, in one aspect, newly
fused hybridomas are plated in semi-solid media (e.g,.
methylcellulose) with a selective medium (e.g., a medium that
promotes the survival and proliferation of hybridoma cells and the
elimination of non-fused B cells and myeloma cells. Examples of
such a medium include one containing hypoxanthine, aminopterin and
thymidine). Clonal IgG-producing colonies are selected and placed
in invididual wells containing media to support cell line expansion
and antibody production. The resulting hybridoma supernatant can be
assayed by the present methods. In another aspect, hybridoma cells
are cloned using a limited dilution approach. In some embodiments,
prior to immobilization and conjugation, the unpurified hybridoma
culture supernatants are screened for the presence of antibodies
with desired antigen specificity. In some embodiments, from about 1
ml to about 5 mls of hybridoma supernatant is provided.
[0047] In some embodiments, the unpurified cell culture supernatant
is other than hybridoma supernatant, e.g., CHO cell culture
supernatant (e.g., DG44, Dxb11, CHO-K1 and CHO-S cell lines), or
other cell culture supernatant.
[0048] In some embodiments, the antibody in the antibody-containing
samples is produced in culture media lacking endogenous IgG, and,
in particular, culture media lacking bovine IgG. In some
embodiments, the culture medium is depleted of endogenous IgG prior
to use (see, for example, example 8). Suitable culture media
include those containing, for example, salts, carbon source (e.g.,
sugars), nitrogen source, amino acids, trace elements, antibiotics,
selection agents, and the like, as required for growth.
Commerically available media as well as commercially available
cloning media, including IgG depleted cloning media can be used.
The culture conditions, such as temperature, pH, and the like, will
be apparent to the ordinarily skilled artisan.
[0049] The present methods use a solid support for conjugation of
the antibodies to a desired chemical entity. Because the present
methods are performed in solid phase and not in solution, the
present methods can be performed with samples that contain very
small amounts (e.g., 1 to 500 .mu.g) of antibody. In some
embodiments, there will be from 1 .mu.g to 100 .mu.g, from 1 .mu.g
to 50 .mu.g, from 1 .mu.g to 20 .mu.g, from 3 .mu.g to 100 .mu.g,
from 3 .mu.g to 50 .mu.g, from 3 .mu.g to 20, from 5 .mu.g to 100
.mu.g, from 5 .mu.g to 50 .mu.g, from 5 .mu.g to 20 .mu.g of
antibody present in a single sample. In one aspect, at least one of
the samples in a plurality of samples will have from 1 .mu.g to 100
.mu.g, from 1 .mu.g to 50 .mu.g, from 1 .mu.g to 20 .mu.g, from 3
.mu.g to 100 .mu.g, from 3 .mu.g to 50 .mu.g, from 3 .mu.g to 20,
from 5 .mu.g to 100 .mu.g, from 5 .mu.g to 50 .mu.g, from 5 .mu.g
to 20 .mu.g of antibody present.
[0050] A solid support refers to an insoluble, functionalized
material to which the antibodies can be reversibly attached, either
directly or indirectly, allowing them to be separated from unwanted
materials, for example, excess reagents, contaminants, and
solvents. Examples of solid supports include, for example,
functionalized polymeric materials, e.g., agarose, or its bead form
Sepharose.RTM., dextran, polystyrene and polypropylene, or mixtures
thereof; compact discs comprising microfluidic channel structures;
protein array chips; pipet tips; membranes, e.g., nitrocellulose or
PVDF membranes; and microparticles, e.g., paramagnetic or
non-paramagnetic beads. In some embodiments, an affinity medium
will be bound to the solid support and the antibody will be
indirectly attached to solid support via the affinity medium. In
one aspect, the solid support comprises a protein A affinity medium
or protein G affinity medium. A "protein A affinity medium" and a
"protein G affinity medium" each refer to a solid phase onto which
is bound a natural or synthetic protein comprising an Fc-binding
domain of protein A or protein G, respectively, or a mutated
variant or fragment of an Fc-binding domain of protein A or protein
G, respectively, which variant or fragment retains the affinity for
an Fc-portion of an antibody.
[0051] The present methods comprise a step of immobilizing antibody
on a solid support to provide immobilized antibodies. In some
embodiments, the solid support will have the capacity to bind more
antibody than the amount present in the antibody-containing sample
or, in other words, the amount of antibody bound to the solid
support following the immobilization step will be less than the
capacity of the solid support. Because the samples generally vary
with respect to antibody quantity, there will be corresponding
variability in the amount of immobilized antibody from one sample
as compared to another.
[0052] In some other embodiments, it might be desirable to limit
the quantity of bound antibody and the solid support will only have
the capacity to bind up to a certain amount of antibody (e.g., up
to 5 .mu.g, up to 10 .mu.g, or up to 15 .mu.g of protein). In these
embodiments, although there will be a limit as to the maxium amount
of antibody that can be bound to the solid support, there may still
be variability in the amount of immobilized antibody in one sample
as compared to another. This is because one or more of the samples
might contain a small quantity of antibody, less than the maximum
loading capacity of the solid support. One approach for preparing a
solid support that has limited capacity for binding antibody is to
make a very low-capacity resin such that a larger volume of resin
slurry (20 uL for example) contains only enough capacity to bind 5
ug of antibody. An alternative approach is to reduce the effective
capacity of a resin by diluting the resin with an appropriate
volume of non-functionalized resin. For example, a protein
G-sepharose resin with a binding capacity of 20 ug/uL could be
converted to a mixed resin with an effective binding capacity of
0.5 ug/uL by mixing 1 part of protein G-sepharose with 40 parts
unfunctionalized sepharose. In performing such a resin dilution, in
some embodiments, the diluent will be a resin which is constructed
from the same base material as the affinity resin, has pore sizes
small enough to exclude antibodies, and lacks any surface
functionality which may interact with antibodies or the chemical
reagents used to prepare antibody conjugates.
[0053] In some aspects of the invention, antibodies are immobilized
on a solid support by the step of applying an antibody-containing
sample to a solid support. If desired, a washing step can be
performed following immobilization to separate the immobilized
antibodies from the cell culture supernatant or other components of
the antibody-containing samples.
[0054] Once the antibodies are immobilized on the solid support, a
reduction step is performed in order to fully reduce the reducible
disulfide bonds of the immobilized antibodies and to generate
reactive thiols. The antibodies are reduced under conditions that
are favorable to a complete reduction of the reducible disulfide
bonds. Typically, the antibodies are reduced with an excess of
reducing agent in order to ensure a substantially complete
reduction of the reducible disulfide bonds. By the phrase "fully
reducing the reducible disulfide bonds of the antibody" it is meant
that substantially all (e.g., greater than 70%, preferably greater
than 80%, even more preferably greater than 85%, 90%, or 95%) of
the antibodies in a sample are fully reduced as to their reducible
disulfide bonds. In other words, for a substantial amount of the
antibodies in a sample, all of the antibodies' reducible disulfide
bonds will be cleaved during the reduction step. For example, if
the antibodies in a sample have 4 reducible disulfide bonds, after
the reduction step, all 4 reducible disulfide bonds of a
substantial amount of the antibodies will be cleaved to generate 8
reactive thiols. The reduction is one that is selective for
reducible disulfide bonds. By the phrase "selective for reducible
disulfide bonds" it is meant that the reducible disulfide bonds are
substantially the only bonds that are reduced. In some embodiments
of the invention, the reducible disulfide bonds are the naturally
occurring interchain disulfides of the antibody, the antibodies are
reduced under conditions that are favorable to a complete reduction
of the naturally occurring interchain disulfides, and the reduction
is one that is selective for the naturally occurring interchain
disulfides. By the phrase "selective for interchain disulfides" it
is meant that interchain disulfides are selectively reduced. In
other words, the interchain disulfides are substantially the only
bonds that are reduced. Because the antibodies are contacted with
an excess of reducing agent and the reducing agent is selective for
the reducible disulfide bonds, the generation of reactive thiols
per antibody will generally be independent of the quantity of
antibody in the sample.
[0055] In one aspect, the reducing agent used in the reduction step
is TCEP (tris(2-carboxyethyl)phosphine) and the TCEP is added at an
excess for 30 minutes at room temperature. For example, 250 uL of a
10 mM solution of TCEP at pH 7.4 will readily reduce the interchain
disulfides of 1 to 100 ug of antibody in 30 minutes at room
temperature. Other reducing agents and conditions, however, can be
used. Examples of other reducing agents include DTT
(dithiothreitol), mercaptoethanol and mercaptoethylamine. Examples
of reaction conditions include temperatures from 5.degree. C. to
37.degree. C. over a pH range of 5 to 8. Conjugation of the
resulting antibody thiols and analysis by hydrophobic interaction
or reveresed-phase chromatography (for examples, see FIGS. 1 and 3
respectively) provides an indicator of the extent of disulfide
reduction achieved under various reducing conditions. Following the
reduction, a washing step can be performed in order to remove
reducing agent and any other components that may have
nonspecifically attached to the solid support during the antibody
capture step, for example, culture media components.
[0056] In some aspects of the present invention, although the
samples will vary with respect to antibody quantity and antibody
sequence, the majority of antibodies will not vary substantially
with respect to the number of reducible disulfide bonds. For
example, in some embodiments, substantially all of the antibody
contained in the first and second sample will have the same amount
of reducible disulfide bonds. In some such embodiments, the
reducible disulfide bonds will be interchain disulfides. If the
antibody in the first and second sample have 4 interchain disulfide
bonds (e.g., human IgG1), after reduction, the reduced immobilized
antibodies in both samples will each have 8 reactive thiols. This
level of reduction to 8 reactive thiols per antibody is independent
of the quantity of antibody in the samples due to the excess of
reducing agent, the selectivity of the reduction step, and the
uniform number of reducible bonds on each antibody. Similarly, if
the antibody in the first and second sample have 5 interchain
disulfide bonds, after reduction, the reduced immobilized
antibodies in both samples will each have 10 reactive thiols. This
level of reduction to 10 reactive thiols per antibody is also
independent of the quantity of antibody due to the excess of
reducing agent, the selectivity of the reduction step, and the
uniform number of reducible disulfides on each antibody. In some
embodiments wherein a panel of murine antibodies is being screened,
e.g., a panel of murine antibodies from hybridomas, a majority of
the antibodies will be either one of the major murine isoforms IgG1
and IgG2a. Murine IgG1 and IgG2a isoforms both contain 5 interchain
disulfide bonds and after reduction, each antibody will have 10
reactive thiols. Accordingly, a majority of the antibodies will
have the same amount of reducible disulfide bonds. Although the
majority of isoforms in these embodiments may be IgG1 and IgG2a,
other isoforms may be present as well. For example, murine IgG2b,
murine IgG2c and murine IgG3 isoforms may be present as well. In
instances where murine IgG2b isoforms are present, the reduction of
these antibodies will generate 12 reactive thiols as IgG2b isoforms
have 6 interchain disulfide bonds. In some embodiments, transgenic
mice will be used for antibody production and the mice can be
genetically engineered to produce antibodies having a certain
isotype as well as antibodies having human IgG isotypes. In some
such embodiments, the mice can be engineered to only express
specific isotypes. In some embodiments, the mice can be engineered
to only express only one isotype or one or two major isotypes.
[0057] Following the reduction step, the antibodies are loaded with
the desired chemical entity (in other words, conjugated to the
desired chemical entity). The selection of the chemical entities to
be used depends in part on the purpose of the assay. In some
embodiments, the antibodies will be screened for the purpose of
selecting an antibody for use as an ADC. In these embodiments, it
is desirable for the antibodies to be conjugated to a drug. The
antibodies can be conjugated directly to the drug or indirectly via
a linker. The drug and drug-linker can be any drug or drug-linker
that is effective for use as an ADC and that is thiol reactive. By
the phrase "thiol reactive" it is meant that the chemical entity
will react with a reactive thiol generated by reduction of a
reducible disulfide bond and will form a covalent bond thereto.
Thiol reactive drugs and drug-linkers include those drugs or
drug-linkers that aren't naturally thiol reactive but have been
derivatized with a thiol reactive agent to render them thiol
reactive. The conditions used for conjugation are such that the
drug will selectively react with a reactive thiol (either directly
or through its linker). Examples of thiol reactive groups that are
highly selective for reactive thiols include, for example,
maleimides, such as N-ethylmaleimide. 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. At pH 7, for example, the reaction with simple thiols
is about 1,000 fold faster than with the corresponding amines
Reactions of thiols with maleimides are very rapid at room
temperature at pH 7.4, and 30 minutes is adequate to ensure
complete reaction without risking conjugation of the maleimide to
amine groups. Accordingly, in some embodiments, the drug will be
linked to the antibody via a maleimide group. Other reactive groups
that are highly selective for reactive thiols include, for example,
iodoacetamides, vinyl sulfones, and aziridines.
[0058] In some embodiments, it will be desirable to fully load an
antibody with drug. In such embodiments, the desirable drug loading
level will be equal to the number of reactive thiols per antibody.
For example, in some such embodiments, the desired drug loading
will be 10 drugs per antibody and the number of reactive thiols per
antibody will be 10. In some embodiments wherein a drug loading
level which is equal to the number of reactive thiols is desired,
the thiol reactive drug or drug-linker will be provided in
sufficient excess to the immobilized antibodies in order to react
with all of the available reactive thiols. Because the reaction is
set up such that the drugs and drug-linkers to be used in this step
are thiol reactive and the conditions used are selective for
conjugation to reactive thiols, the drug or drug-linker selectively
reacts with the reactive thiols (i.e., the drug and drug linkers do
not substantially react with other sites on the antibody, including
for example, other amino acids (e.g, lysine residues)). Because of
this selectivity, it is possible to control the drug loading and to
design the experiment such that there will be a substantial uniform
drug loading between samples. By the phrase "substantial uniform
drug loading between samples" it is meant that the average drug
loading between samples is substantially the same or, in other
words, the average number of drug molecules per antibody in sample
one will be substantially the same as the average number of drug
molecules per antibody in sample two. Some variance in drug loading
can be expected but generally it will be within a variance of about
25%, preferably within a variance of 20% or even 10%. Accordingly,
in some embodiments where a majority of the samples contain
antibodies of the murine IgG1 and IgG2a subtypes, if a thiol
reactive drug or drug-linker is added to the samples in sufficient
excess to react with all of the available reactive thiols, there
will be an average of 10 drug molecules per antibody in the
majority of samples. Because these samples have a substantial
uniform drug loading, once eluted, the concentration of the
purified ADCs can be determined by methods known in the art, e.g.,
spectrophotometric methods, and their activities can be compared to
determine which antibodies are more or less active in an assay.
This comparison can be performed even if the antibodies to be
compared are provided at variable concentrations and in some
embodiments, at unknown variable concentrations. A comparison
between antibodies provided at unknown variable concentration is
aided by the ability to substantially uniformly load them with drug
or drug-linker.
[0059] In some embodiments, it is not desirable to fully load an
antibody with drug or drug-linker. In some such embodiments, if a
lower drug loading level is desired, the immobilized antibodies can
be reacted with both a drug or drug-linker and a thiol capping
agent. The term "thiol capping agent" is used herein to refer to an
agent which selectively blocks a reactive thiol. The drug or
drug-linker and thiol capping agent will be provided in a ratio of
drug or drug-linker to thiol capping agent which results in the
desired drug loading. Like the drug or drug-linker, the capping
agent will be highly selective for reactive thiols. Thiol reactive
capping agents include those capping agents that aren't naturally
thiol reactive but have been derivatized with a thiol reactive
agent to render them thiol reactive. Examples of thiol capping
agents that can be used include maleimide capping agents such as,
for example, N-ethyl maleimide. Other capping agents include, for
example, iodoacetamide and iodoacetic acid. In some embodiments,
both the drug or drug-linker and thiol capping agent have the same
type of thiol reactive agent. For example, in some preferred
embodiments, if the drug is to be linked to the antibody via a
maleimide group, the capping agent will also be linked to the
antibody via the same type of maleimide group. This helps ensure
that the relative reaction rates of the drug-linker and capping
agent are similar. Preferably, there will be no more than about a
100-fold different in the relative reaction rates, more preferably
no more than 10-fold, and even more preferably no more than 5-fold
difference in the relative reaction rates.
[0060] In one aspect, the ratio of capping agent and drug or
drug-linker chosen will be dependent on the desired level of drug
loading. In some embodiments, the ratio of drug linker or drug to
capping agent provided to the immobilized antibodies will be
reflected in the ratio at which these reagents are conjugated to
the antibodies. In embodiments when the drug or drug-linker and
capping agent are provided in molar excess, the ratio of drug
linker or drug to capping agent provided will be reflected in the
ratio at which these reagents are conjugated to the antibodies if
the intrinisic thiol reaction rates of these two components are the
same. For example, if a reaction mixture to be used for conjugation
has a 1:1 mixture of drug linker or drug to capping agent, in
embodiments where the instrinsic thiol reactive rates of the drug
or drug-linker and capping agent are the same and a majority of the
samples comprise antibodies having 5 reducible disulfide bonds
(e.g., antibodies of the murine IgG1 and IgG2a subtypes), following
the reaction, there will be an average of 5 drug molecules per
antibody and 5 capping agents per antibody in the majority of
samples. It has been observed, however, by the present inventors
that the instrinsic thiol reaction rates of the drug or drug-linker
and capping agent are generally not the same, and consequently, if
the drug or drug-linker and capping agent are provided in excess,
the ratio at which the drug or drug-linker and capping agent are
provided to the samples comprising immobilized antibodies will not
be same ratio at which they are conjugated to the antibodies. In
such embodiments, the appropriate ratio of drug or drug-linker to
capping agent can be determined experimentally in order to achieve
the desired level of drug loading. Notably, as long as the drug or
drug-linker and capping agent are provided in excess (generally, an
excess of at least about 3-fold) and the antibodies present in the
samples have substantially the same number of reducible disulfide
bonds, the ratio will produce consistent (i.e., substantially
uniform) levels of drug loading across samples regardless of
quantity of antibody present on the solid support.
[0061] In some preferred embodiments, the conjugation reaction of
the antibody to the drug or drug-linker and capping agent will be
under kinetic control, not thermodynamic control. For example,
under conditions in which the total moles of drug or drug-linker
and capping agent provided to a sample containing the immobilized
antibodies is equal to or less than the number of moles of reactive
thiol in the sample, then the ratio of drug or drug-linker and
capping agent provided to the sample comprising the immobilized
reduced antibodies will be reflected in the actual conjugation
ratio of the antibodies to drug or drug-linker and capping agent.
Such a conjugation reaction can be said to be under thermodynamic
control. For example, if 100 pmoles of a murine IgG1 antibody
(about 15 ug) were reduced with excess reducing agent to produce 10
thiols per antibody, then 1 nmole of reactive thiol would be
present. If a 1:1 mixture of drug or drug-linker to capping agent
were prepared such that the concentration of each was 0.5 mM and
the total concentration was 1 mM, the addition of 1 uL of this
solution to the reduced antibody would present a total of 1 nmole
of drug or drug-linker and capping agent. Assuming the drug or drug
linker and capping agent and thiol reaction is a highly favorable
one (for example, both of the drug or drug-linker and capping
agents are maleimido derivatives), the conjugate prepared by this
procedure would have a 1:1 mixture of the two compounds (the
thiol-maleimide reaction is highly favorable and thermodynamics
would effectively drive this reaction to completion). This would be
true even if one of the compounds reacted at a substantially faster
rate than the other. In embodiments where it is desirable for a
plurality of sampes to be uniformly loaded, this approach would
generally require that the quantity of antibody present in each of
the samples be known. Moreover, in embodiments where there is
variability in the amount of antibody between samples (such as a
panel of antibodies from hybridomas), it would require a great deal
of effort to tailor the quantity of drug or drug linker and capping
agent to be added to each sample in order to arrive at samples that
are substantially uniformly loaded. In embodiments where the
quantity of antibody in the samples is unknown and/or there is
variability between samples, it is generally preferable to
manipulate the reaction so that it is under kinetic control and
accordingly, to provide the antibody-containing samples with an
excess of total drug or drug-linker and capping agent.
[0062] In some embodiments of the present invention, the chemical
entities to be conjugated to the reactive thiols of the reduced
antibodies will be provided in molar excess (molar excess as to the
reactive thiols). In these embodiments, if the drug or drug-linker
reacts more quickly with a reactive thiol than the capping agent,
the drug or drug-linker will be disproportionately represented on
the final conjugate. This is because the drug or drug linker and
capping agent are effectively competing with each other to react
with a limiting number of available reactive thiols. If the drug or
drug linker and capping agent are present at equal concentrations
in the reaction solution, they will only be conjugated at equal
concentrations if their reaction rates are the same. By altering
the composition of a reaction mixture such that the concentrations
of the drug or drug linker and capping agent are not equal, the
ratio at which they react with the available thiols can be
controlled. For example, a slow-reacting drug or drug linker will
be disproportionately underrepresented on a conjugate prepared with
a 1:1 mixture with a faster reacting capping agent. By changing the
ratio to 2:1 in favor of the slower reacting drug or drug-linker,
its representation on the conjugate will be increased. Thus, by the
modulation of the ratio of drug or drug linker and capping agent
provided to the samples comprising the immobilized reduced
antibodies, a desired ratio of drug or drug linker and capping
agent on the final conjugate can be achieved. Under conditions in
which the total drug or drug linker and capping agent is present in
excess relative to the available reactive thiols, their
distribution on the final product will be independent of the
starting thiol quantity. In this manner, a plurality of samples can
be substantially uniformly loaded even when the quantity of
antibody in the samples is unknown and/or there is variability
between samples. In some embodiments, an appropriate volume of drug
or drug-linker and capping agnet is provided to the samples such
than a molar excess of about 2 fold (and ,even more preferably, a
molar exces of 3-fold or more) of total reactants relative to total
thiols is present. If the quantity of antibody in the samples is
unknown, each sample can be treated as if it has the maximum amount
of antibody. For many of the samples, significantly less than the
maximum amount will be present and the excess will be greater than
2 fold. This provision of excess reactants having a set ratio
allows for variable quantities of antibodies across a panel to be
treated with a large, fixed quantity of total drug or drug linker
and capping agent to produce a panel of conjugates with comparable
loading of each drug or drug linker and capping agent present. The
fact that equal treatment of samples results in comparable levels
of loading, regardless of the quantity of antibody initially
present in the sample, makes this method convenient for
high-throughput applications in which large numbers of antibodies
are conjugated.
[0063] As discussed herein, although it is preferable that a
majority of the samples to be assayed do not vary with respect to
the number of reducible disulfide bonds present on the antibodies
contained therein, in some embodiments, there will be some
variation. In some embodiments, despite the variation, the samples
will be treated with the same ratio of chemical entities. When
interpreting the data, the skilled artisan will recognize that a
certain subset of the samples differed in the amount of reducible
disulfide bonds. If desired, the skilled artisan can determine the
antibody isotype prior to or post conjugation to aid in data
interpretation.
[0064] In some embodiments, prior to the conjugation step, standard
methods can be used to determine the antibody isotype in each of
the samples and therefore, the number of reducible disulfide bonds
per antibody in each of the samples. In some such embodiments,
samples that contain antibodies having the same number of reducible
disulfide bonds will be contacted with a reaction mixture having
one ratio of drug or drug linker to capping agent to arrive at a
desired drug loading and samples that contain antibodies having a
differing number of reducible disulfide bonds will be contacted
with a reaction mixture having a different ratio of drug or drug
linker to capping agent to arrive at that same desired drug
loading. For example, in some embodiments, if the desired average
drug loading is 4, samples that contain antibodies of murine IgG1
and IgG2a (10 reactive thiols per antibody when fully reduced) will
all be contacted with a reaction mixture having a ratio of drug or
drug linker to capping agent to arrive at an average drug loading
of 4 and average capping agent loading of 6. Samples that contain
antibodies of murine IgG2b (12 reactive thiols per antibody when
fully reduced) will be contacted with a reaction mixture having a
different ratio of drug or drug linker to capping agent (e.g., a
higher fraction of capping agent) to arrive at the same average
drug loading of 4. In other embodiments, although there may be
variation between isotypes and number of interchain disulfides, it
will be accepted that there will be some variation in loading and
all of the samples will receive the same ratio of drug or drug
linker to capping agent.
[0065] In some embodiments, prior to the conjugation step and
following the reduction step, there will be a partial reoxidation
step. For example, in some embodiments, the reducible disulfide
bonds will consist of naturally occurring interchain disulfide
bonds as well as disulfide bonds formed from introduced sulfhydryl
groups. In some of these embodiments, it will be desirable to
conjugate the selected chemical entities to the introduced
sulfhydryls but not to the sulfhydryl groups of the naturally
occurring interchain disulfide bonds. In these embodiments,
following the complete reduction of the reducible disulfide bonds,
there can be a partial reoxidation step to reoxidize the naturally
occurring interchain disulfide bonds leaving the introduced
sulfhydryls available for binding to the desired chemical entities.
Reoxidation of the native disulfides can be achieved, for example,
by treatment of the reduced antibodies with a large molar excess of
dehydroascorbic acid at pH 6.5, with the reaction allowed to
proceed for 1 hour at room temperature.
[0066] In any of the embodiments described herein, instead of, or
in addition to the capping agent, a detection agent is provided for
conjugation. The detection agents can be, for example, primary
labels or secondary labels. In some embodiments, the detection
agent will be one that is detected directly. In other embodiments,
the detection agent will be one that is detected indirectly. In
some embodiments, the detection agent will be, for example, any
thiol reactive label that can be used for antibody quantiation
and/or as a reporter for a binding assay or any other desirable
assay. Thiol reactive labels include those labels that aren't
naturally thiol reactive but have been derivatized with a thiol
reactive agent to render them thiol reactive. In some embodiments,
the same type of thiol reactive agent will be used to link the
various chemical entities (detection agent and/or drug or
drug-linker and/or capping agent) to the antibody. In some
embodiments, the detection agent will be a radioactive compound, a
chemiluminescent agent, a fluorescent agent, or a chromogen. In
some embodiments, the detection agent will be a fluorescent
molecule such as a fluorophore. In some embodiments, the detection
agent will be biotin. In one aspect, the detection agent will be a
fluorophore and the fluorophore will be derivatized with a
maleimide group in order to make it thiol reactive. The teachings
described herein can be used to assess the preferred loading level
of a select detection agent. In some embodiments, a fluorophore is
used as the detection agent and the fluorophore is loaded at an
average loading of about 2.5 to about 3 fluorophores per antibody.
Examples 3 and 4 provide exemplary descriptions of how to tailor
the ratio of chemical entities in order to achieve a desired drug
and/or fluorophore loading level.
[0067] The present invention encompasses embodiments wheren the
antibodies are screened not for the purpose of selecting an
antibody for use as an ADC but for the purpose of selecting an
antibody for use as an unconjugated antibody. In these embodiments,
immobilized antibodies will be contacted with a detection agent and
capping agent at a selected ratio and there will be no use of drug
or drug-linker. Using the teachings described herein, including the
teachings of examples 3 and 4, the appropriate ratio of detection
agent to capping agent can be determined.
[0068] After contacting the reduced antibodies with the appropriate
amount and type of chemical entities (selection of the chemical
entities will be dependent, for example, on whether it is desired
to screen antibodies as unconjugated antibodies or ADCs; whether it
is desired to have a full drug loading or partial drug loading; and
whether it is desired to include a detection agent in the mix) and
allowing sufficient time for completion of the reaction (e.g., 30
minutes for maleimide-containing chemical entities), it is
desirable to perform a washing step in order to remove any
unreacted materials. Subsequently, the immobilized antibody
conjugates can be eluted from the solid support to provide antibody
conjugate compositions. Methods of eluting proteins from solid
supports are known in the art and the skilled practitioner will be
able to select an appropriate buffer for elution. For example, in
embodiments, where the solid support comprises protein A or protein
G resin, the antibody conjugates can be eluted with standard low pH
buffers for elution from protein A or protein G columns.
[0069] In some embodiments of the invention, the methods described
herein for making antibody conjugates will result in a plurality of
antibody drug conjugate compositions having substantially uniform
drug loading (the skilled artisan will understand that there may be
some outliers depending on the uniformity of number of reducible
disulfide bonds across samples). In these embodiments, because of
the substantially uniform drug loading between samples, the
relative characteristics of antibodies in a first and second sample
can be compared. This comparison can be performed even though the
antibodies to be compared were provided at variable concentrations
and, in some embodiments, at unknown variable concentrations. A
comparison between the antibodies of unknown and variable
concentration is made easier with the ability to substantially
uniformly load them with drug or drug-linker.
[0070] Methods for determining drug loading are known in the art.
One method that is used herein is high-performance liquid
chromatography on a polystyrene divinylbenzene copolymer, e.g., a
reversed-phase PLRP.TM. column. This denaturing technique can
cleanly separate the variously loaded light chain and heavy chain
species. Hydrophobic interaction chromatography (HIC) can also be
used as an analytical method used to determine isomeric mixtures
from resultant conjugates. The drug loading level can be determined
based on a ratio of absorbances, e.g., at 250 nm and 280 nm. See,
for example, U.S. Publication No. 20090010945.
[0071] In some embodiments, following elution of the antibody
conjugates, activity assays and/or other assays will be performed
in order to characterize the antibody conjugates. In some
embodiments, cell binding, affinity, and/or cytoxicity assays will
be performed. Many methods of determining whether an ADC binds a
target of interest or exerts a cytotoxic effect on a cell are known
to those of skill in the art, and can be used in the present
methods. For example, cell viability assays can be used to
determine the cytotoxic effect of an ADC on a cell. See, for
example, U.S. Pat. Nos. 7,659,241 and 7,498,298, each of which is
incorporated herein in its entirety and for all purposes, for
exemplary cell binding and cytotoxicity assays.
[0072] In some embodiments, following elution of the antibody
conjugates, it will be desirable to determine the quantity of
antibody or antibody conjugate in the antibody conjugate
compositions. In some embodiments, it will be desirable to
determine the actual quantity of antibody or antibody conjugate in
a sample. In other embodiments, it will be sufficient to determine
the relative quantity of antibody or antibody conjugate in a
plurality of samples. For example, it may be sufficient to know
that sample 1 has more antibody than sample 2 which has more
antibody than sample 3, and so forth. Many methods for determining
protein quantity are known in the art and can be used herein. In
some embodiments, an absorbance assay will be used to determine
antibody concentration. In embodiments where a fluorophore is part
of the antibody conjugate, antibody concentration can be determined
using a fluorescence assay. In embodiments where fluorescence is
used for protein quantitation, a standard may be necessary to
convert the raw fluorescence values into a concentration. Methods
of using fluorescence and generating standard curves to determine
protein concentration are known in the art. In one example,
approximately 200 .mu.g of a standard antibody will be conjugated
during the conjugation step after being spiked into blank media.
After elution, the concentration of this standard will be
determined by conventional methods, e.g., a conventional A280
absorbance assay, and a standard curve prepared by a dilution
series will be assayed for fluorescence alongside the conjugate
samples. Alternatively, a liquid-handling robot can be used to
normalize plates thereby eliminating the need for serial
dilutions.
[0073] In some embodiments, the results of a cytotoxicity assay and
knowledge of the relative or actual antibody concentration in the
antibody conjugate compositions will be used to identify antibodies
with desired characteristics. The methods described herein for
making antibody conjugates allow for comparisons to be made between
a plurality of antibodies of varying concentration and, in some
embodiments, unknown quantity. The methods described herein for
making antibody conjugates allow for a selection of antibodies with
desirable characteristics when starting with, for example, a panel
of antibodies resulting from a hybridoma fusion. In some preferred
embodiments, it is the substantial uniform drug loading between
samples that allows for relevant comparisons to be made between
samples. Failure to ensure substantially uniform loading levels,
could, for example, lead to erroneous results from a screen of a
panel of antibodies for use as ADCs. This is because it would not
be known if an ADC sample exhibited greater cytotoxicity because of
the characteristics of the antibody as an ADC or because the sample
contains more drug per antibody. For example, an antibody conjugate
composion comprising antibody "A" and having an average drug
loading of 4 would typically be expected to exhibit more
cytotoxicity than an antibody conjugate composition comprising
antibody "B" and having an average drug loading of 1. This greater
cytotoxicity would not be an indicator of the relative
characteristics of antibodies A and B as ADCs, but simply an
indicator of the greater drug loading on antibody A. If both
antibody conjugate compositions had an average drug loading of
about 4, if one showed greater cytotoxicity, it could be attributed
to the antibody and not simply the drug loading. Similarly, the
ability to determine the actual or relative quantity of antibody or
antibody conjugate in the samples also allows for relevant
comparisons to be made between samples. Without knowledge of actual
or relative quantity of antibody or antibody conjugate in the
sample, it would not be known if an ADC exhibited greater
cytotoxicity because of the particular antibody or simply because
there is more antibody or ADC in the sample.
[0074] In addition to providing methods for making antibody
conjugates for use in antibody screening assays and antibody
conjugates produced by the claimed methods, the present invention
provides antibodies and/or antibody conjugates (e.g., antibody drug
conjugates) for therapeutic use wherein the antibody was selected
using the methods described herein.
[0075] As previously discussed, the drug or drug-linker used in the
present methods can be any drug or drug-linker that is effective
for use as an ADC and that is thiol reactive. The drug can be any
cytotoxic, cytostatic or immunosuppressive drug. Methods of
selecting drug and drug-linker for use as ADCs are known in the
art. See, for example, WO 2004010957, WO 2007/038658, U.S. Pat. No.
6,214,345, U.S. Pat. No.7,498,298, and U.S. Publication No.
2006/0024317, each of which is incorporated herein by reference in
its entirety and for all purposes.
[0076] Useful classes of cytotoxic or immunosuppressive agents
include, for example, antitubulin agents (e.g., auristatins,
maytansinoids, vinca alkaloids), topoisomerase inhibitors (e.g.,
camptothecins), DNA minor groove binders (e.g., calicheamicins,
duocarmycins, enediynes, lexitropsins, chloromethylbenzindolines),
DNA replication inhibitors (e.g., anthracyclines), alkylating
agents (e.g., platinum complexes such as cis-platin,
mono(platinum), bis(platinum) and tri-nuclear platinum complexes
and carboplatin), protein kinase inhibitors, cytotoxic enzymes, and
protein toxins.
[0077] In some embodiments, suitable cytotoxic agents include, for
example, antibiotics, antifolates, antimetabolites, chemotherapy
sensitizers, etoposides, fluorinated pyrimidines, ionophores,
nitrosoureas, platinols, pre-forming compounds, purine
antimetabolites, radiation sensitizers, steroids, puromycins,
doxorubicins, and cryptophysins.
[0078] Individual cytotoxic or immunosuppressive agents include,
for example, an androgen, anthramycin (AMC), asparaginase,
5-azacytidine, azathioprine, bleomycin, busulfan, buthionine
sulfoximine, gamma calicheamicin, N-acetyl gamma dimethyl hydrazide
calicheamicin, camptothecin, carboplatin, carmustine (BSNU),
CC-1065, cemadotin, chlorambucil, cisplatin, colchicine,
cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B,
dacarbazine, dactinomycin (formerly actinomycin), daunorubicin,
decarbazine, discodermolide, docetaxel, doxorubicin,
morpholino-doxorubicin, cyanomorpholino-doxorubicin, echinomycin,
eleutherobin, epothilone A and B, etoposide, estramustine, an
estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gemcitabine,
gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan,
lomustine (CCNU), maytansine, mechlorethamine, melphalan,
6-mercaptopurine, methotrexate, mithramycin, mitomycin C,
mitoxantrone, netropsin, nitroimidazole, paclitaxel, palytoxin,
plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide,
6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine,
vinorelbine, VP-16 and VM-26.
[0079] In some embodiments, the drug 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)),
and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and
vinorelbine). Other antitubulin agents include, for example,
baccatin derivatives, taxane analogs (e.g., epothilone A and B),
nocodazole, colchicine and colcimid, estramustine, cryptophysins,
cemadotin, combretastatins, discodermolide, and eleutherobin.
[0080] 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 or
DM-4 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.
52:127-131).
[0081] In some embodiments, the drug is an auristatin, another
group of anti-tubulin agents. Auristatins include, but are not
limited to, auristatin E and derivatives thereof. AFP, AEB, AEVB,
MMAF, and MMAE are examples of auristatins that can be used herein.
The synthesis and structure of auristatins are described in U.S.
Patent Application Publication Nos. 2003-0083263, 2005-0238649 and
2005-0009751; International Patent Publication No. WO 04/010957,
International Patent Publication No. WO 02/088172, and U.S. Pat.
Nos. 7,498,298, 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, each of which is
incorporated herein by reference in its entirety and for all
purposes.
[0082] The linker part of a drug-linker is a compound that can be
used to link the antibody to the drug. The linker can comprise more
than one chemical moiety. In some embodiments, the linker is
cleavable under intracellular conditions, such that cleavage of the
linker releases the drug unit from the antibody in the
intracellular environment. In some embodiments, the linker is a
peptidyl linker (e.g. a linker that comprises two or more amino
acids) that is cleaved by an intracellular peptidase or protease
enzyme, including, but not limited to, a lysosomal or endosomal
protease. Cleaving agents can include cathepsins B and D and
plasmin, all of which are known to hydrolyze dipeptide drug
derivatives resulting in the release of active drug inside target
cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics
83:67-123). In some embodiments, the peptidyl linker cleavable by
an intracellular protease comprises a Val-Cit dipeptide or a
Phe-Lys dipeptide (see, e.g., U.S. Pat. No. 7,659,241, incorporated
by reference herein in its entirety and for all purposes). In yet
other embodiments, the linker is not cleavable and the drug is
released by antibody degradation.
[0083] In some embodiments, the cleavable linker is pH-sensitive,
i.e., sensitive to hydrolysis at certain pH values and/or cleavable
under reducing conditions (e.g., a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-S-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0084] Exemplary linkers that can be used with the present methods
are described in WO 2004010957, WO 2007/038658, U.S. Pat. Nos.
6,214,345, 7,659,241, 7,498,298 and U.S. Publication No.
2006/0024317, each of which is incorporated herein by reference in
its entirety and for all purposes.
[0085] In some exemplary embodiments of the present invention, the
drug-linker is of Formula I or Formula II wherein Val-Cit refers to
the dipeptide valine-citrullline.
##STR00004##
Proteins
[0086] The methods described herein for making antibody conjugates
can also be used to make fusion proteins for use in fusion protein
screening assays. The term "fusion protein" is used herein to refer
to binding domain-Ig fusions, wherein the binding domain may be,
for example, a ligand, an extracellular domain of a receptor, a
peptide, a non-naturally occurring peptide or the like with the
proviso that the binding domain does not include a variable domain
of an antibody. Like the antibodies described herein, the Ig
portion of the fusion protein must comprise at least one reducible
disulfide bond, and a domain capable of binding to a solid phase.
In one aspect, the Ig domain will be the Fc region of an antibody.
Examples of domain-Ig fusion proteins include etanercept which is a
fusion protein of sTNFRII with the Fc region (U.S. Pat. No.
5,605,690), alefacept which is a fusion protein of LFA-3 expressed
on antigen presenting cells with the Fc region (U.S. Pat. No.
5,914,111), a fusion protein of Cytotoxic T Lymphocyte-associated
antigen-4 (CTLA-4) with the Fc region (J. Exp. Med. 181:1869
(1995)), a fusion protein of interleukin 15 with the Fc region (J.
Immunol. 160:5742 (1998)), a fusion protein of factor VII with the
Fc region (Proc. Natl. Acad. Sci. USA 98:12180 (2001)), a fusion
protein of interleukin 10 with the Fc region (J. Immunol. 154:5590
(1995)), a fusion protein of interleukin 2 with the Fc region (J.
Immunol. 146:915 (1991)), a fusion protein of CD40 with the Fc
region (Surgery 132:149 (2002)), a fusion protein of Flt-3
(fins-like tyrosine kinase) with the antibody Fc region (Acta.
Haemato. 95:218 (1996)), a fusion protein of OX40 with the antibody
Fc region (J. Leu. Biol. 72:522 (2002)), and fusion proteins with
other CD molecules (e.g., CD2, CD30 (TNFRSF8), CD95 (Fas), CD106
(VCAM-I), CD137), adhesion molecules (e.g., ALCAM (activated
leukocyte cell adhesion molecule), cadherins, ICAM (intercellular
adhesion molecule)-1, ICAM-2, ICAM-3) cytokine receptors (e.g.,
interleukin-4R, interleukin-5R, interleukin-6R, interleukin-9R,
interleukin-10R, interleukin-12R, interleukin-13Ralpha1,
interleukin-13Ralpha2, interleukin-15R, interleukin-21Ralpha),
chemokines, cell death-inducing signal molecules (e.g., B7-H1, DR6
(Death receptor 6), PD-1 (Programmed death-1), TRAIL R1),
costimulating molecules (e.g., B7-1, B7-2, B7-H2, ICOS (inducible
co-stimulator)), growth factors (e.g., ErbB2, ErbB3, ErbB4, HGFR),
differentiation-inducing factors (e.g., B7-H3), activating factors
(e.g., NKG2D), and signal transfer molecules (e.g., gpl30), BCMA,
and TACI.
[0087] All of the steps described herein can easily be adapted to
embodiments wherein the starting material is not antibody but
fusion protein. For example, in some embodiments, fusion
protein-containing samples would be provided in lieu of
antibody-containing samples. The fusion protein samples would vary
with respect to quantity and sequence. In preferred embodiments,
substantially all of the fusion protein present in a single sample
would be of the same sequence. "Substantially all of the fusion
protein present in a single sample is of the same sequence"
reflects the preference that a single sample contain one fusion
protein with the recognition that there may be a minor amount
(e.g., up to 20%, preferably less than 15%, less than 10%, less
than 5%, less than 4%, or less than 3%) of contamination with
another fusion protein.
[0088] As with the antibodies, the methods would not require a
purification step prior to fusion protein immobilization. In some
aspects, the fusion protein provided in the fusion
protein-containing sample is not purified. As with the antibodies,
in some embodiments, unpurifed cell culture supernatatant is
provided as the fusion protein-containing sample. Methods of
generating fusion proteins in cell culture are known in the art and
not discussed herein. In some embodiments, fusion protein in the
fusion protein-containing samples was grown in IgG depleted culture
medium, and, in particular, culture medium depleted of bovine IgG.
As with the antibodies, the present methods can be performed with
samples that contain very small amounts (e.g., 1 to 500 .mu.g) of
fusion protein. In some embodiments, there will be from 1 .mu.g to
100 .mu.g, from 1 .mu.g to 50 .mu.g, from 1 .mu.g to 20 .mu.g, from
5 .mu.g to 100 .mu.g, from 5 .mu.g to 50 .mu.g, from 5 .mu.g to 20
.mu.g of fusion protein present in a single sample.
[0089] The present methods comprise a step of immobilizing the
fusion protein on a solid support to provide immobilized fusion
proteins. In some embodiments, the solid support has the capacity
to bind more fusion protein than the amount present in the fusion
protein-containing sample or the amount of bound fusion protein is
less than the capacity of the solid support. In other embodiments,
the solid support will have reduced binding capacity.
[0090] Once the fusion proteins are immobilized on the solid
support, a reduction step is performed in order to fully reduce the
reducible disulfide bonds of the immobilized fusion protein and to
generate reactive thiols. Following the reduction step, the fusion
proteins are loaded with the desired chemical entity (in other
words, conjugated to the desired chemical entity). Again, the
selection of the chemical entities to be used depends in part of
the purpose of the assay. In some embodiments of the present
invention, the fusion proteins will be screened for the purpose of
selecting fusion protein for use as a fusion protein drug
conjugates. In these embodiments, it is desirable for the fusion
proteins to be conjugated to a drug. The fusion proteins can be
conjugated directly to the drug or indirectly via a linker. The
drug and drug-linker can be any drug or drug-linker described
herein. As with the antibodies, the fusion proteins can be
contacted with a reaction mixture comprising drug, capping agent
and optionally a detection agent. As with the antibodies, the
present invention encompasses embodiments wheren the fusion
proteins are screened not for the purpose of selecting a fusion
protein for use as an fusion protein drug conjugate but for the
purpose of selecting a fusion protein for use as an unconjugated
fusion protein. In these embodiments, the conjugation reaction
mixture will not include a drug or drug linker but instead a
mixture of detection agent and capping agent. As with the antibody
conjugates, in some embodiments, the methods described herein for
making fusion protein conjugates will result in a plurality of
fusion protein conjugate compositions with substantial uniform
loading between samples. Following elution of the fusion proteins,
activity assays and/or other assays can be performed in order to
characterize the fusion proteins. The results of the assays and
knowledge of the relative or actual protein concentration in the
fusion protein conjugate compositions can be used to identify
fusion proteins that have desired properties either as unconjugated
fusion proteins or as fusion protein drug conjugates.
[0091] Using the methods described herein, antibodies that perform
well as unconjugated antibodies and fusion proteins that perform
well as unconjugated fusion proteins can be identified and can be
selected for further development. In some embodiments, antibodies
or fusion proteins identified by the present methods will be
formulated for therapeutic and/or non-therapeutic applications.
Similarly, antibodies or fusion proteins identified as those with
desired activites as drug conjugates can also be selected for
further development. In some embodiments, such antibodies or fusion
proteins will be conjugated to the desired drug or drug-linker
using known methods and will be formulated for therapeutic and/or
non-therapeutic applications. In some embodiments, the antibodies,
antibody drug conjugates, fusion proteins, and fusion protein
conjugates will be formulated as pharmaceutical compositions and
will comprise a therapeutically or prophylactically effective
amount of the antibody, antibody-drug conjugate, fusion protein, or
fusion protein conjugate and one or more pharmaceutically
compatible (acceptable) ingredients. For example, a pharmaceutical
or non-pharmaceutical composition typically includes one or more
carriers (e.g., sterile liquids, such as water and oils). Water is
a more typical carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable excipients include,
for example, amino acids, 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
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E.W. Martin. Such
compositions will typically contain a therapeutically effective
amount of the protein, typically in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulations correspond to the
mode of administration.
[0092] Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. When necessary, the
pharmaceutical can also include a solubilizing agent and 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. When the pharmaceutical is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
When the pharmaceutical 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.
[0093] The invention is further described in the following
examples, which are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Reduction of Antibodies in Solution and by Solid Phase
[0094] It is well recognized that under conditions in which
antibodies retain their native folded structure, TCEP readily
reduces the interchain disulfides without reducing the intrachain
disulfides of the immunoglobulin domains, which are inaccessible to
water-soluble reagents. When an antibody is bound to protein G
affinity media, this selectivity for the interchain disulfides
remains unchanged. This is illustrated in FIG. 1. This figure shows
chromatograms made by reducing a protein G-immobilized murine
antibody with 10 mM TCEP, followed by conjugation with an excess of
mc-MMAF. These chromatograms are overlaid with chromatograms of the
same antibody reduced with TCEP by conventional solution chemistry
and reacted with mc-MMAF. The comparable results between the
standard solution method and the solid phase method indicate that
the reactivity of the antibody is not significantly changed upon
binding to protein G affinity media. This feature allows a large
panel of antibodies to all be reduced to the same number of
reactive thiols without regard to the quantity of each antibody
present, by using a quantity of TCEP that is in excess to the
number of reducible disulfides in the most abundant antibody. In
the absence of any knowledge of how much antibody may be present,
the most theoretically abundant antibody may be defined as the
capacity of the affinity resin (ug antibody per uL resin) times the
volume of the resin bed (uL).
Example 2
Tailoring the Ratio of Drug to Capping Agent in the Drug
Conjugation Reaction Mixture for a Desirable Drug Loading
[0095] FIG. 2 illustrates the degree of loading of the maleimido
drug mcMMAF when added as a mixture with N-ethyl maleimide (NEM) to
a murine IgG1 immobilized on protein G and fully reduced with
excess TCEP. The figure illustrates the slightly lower reactivity
of mcMMAF relative to NEM, such that, in this example, if a
conjugate with an average mcMMAF mole fraction of 0.4 is desired (a
drug loading of 4), the mole fraction of mcMMAF in the maleimide
mixture must be 0.53. The loading of mcMMAF on each conjugate was
determined by reversed-phase chromatography with a PLRP-S column,
which effectively separates the heavy and light chains on the basis
of their drug loading; the hydrophobicity of mcMMAF results in
later retention times for species with increasing degree of mcMMAF
conjugation (FIG. 3). A mixture of mcMMAF and NEM was prepared at
this ratio and applied to a small panel of murine antibodies to
assess the generality of this ratio across different IgG isotypes.
As shown in the table below, murine IgG1's and IgG2a's, both of
which possess 5 interchain disulfides, gave mcMMAF drug loading
levels between 3.9 and 4.2 as determined by PLRP-S chromatography.
A murine IgG2b, which possesses 6 interchain disulfides, gave a
correspondingly greater average mcMMAF loading as a result of the
greater number of reactive thiols per antibody which result from
complete reduction. This result illustrates the importance of
tailoring the maleimide mixture according to the number of
reducible antibody disulfides if a specific loading level is
desired.
TABLE-US-00001 Isotype mIgG1 mIgG2a mIgG2b Drugs/Ab 3.9 4.0 3.9 4.2
4.2 4.1 3.9 5.3
Example 3
Method for Determining Exemplary Fluorophore Loading Level and for
Preparing a Standard for Determining Fluorophore Loading in
Antibody Conjugates
[0096] Mixed conjugates can be prepared with both drug and a
fluorophore present on the conjugate in a controllable manner The
presence of a fluorophore can enable more sensitive quantitation of
the conjugates resulting from a large panel of antibodies or as a
reporter group for binding assays or other assays performed on the
panel. Alexa Fluor.RTM. 647 maleimide can be included in a mixture
of maleimides, along with mcMMAF and NEM, to create a panel of
antibody conjugates with a desired average loading for Alexa
Fluor.RTM. 647 and mcMMAF. To assess a targeted loading level of
AlexaFluor 647, a series of murine IgG1 conjugates was prepared
using a binary mixture of AlexaFluor 647 maleimide and mcMMAF. The
average loading of mcMMAF on these conjugates was determined by
PLRP-S chromatography, and the loading of Alexa Fluor.RTM. 647 was
calculated as (10-mcMMAF loading), as the total conjugation sites
on fully reduced murine IgG1 is 10. The fluorescence output of
these conjugates was then determined using a fluorescence plate
reader, and plotted as a function of Alexa Fluor.RTM. 647 loading
(FIG. 4). FIG. 4 illustrates that fluorescence rapidly increases
with increasing loading level up to a maximum value corresponding
to about 2.5 to about 3 fluorophores per antibody, then steadily
declines with further fluorophore loading. This decrease in
fluorescence output with increasing fluorophore loading is
presumably due to self-quenching which arises from the close
spatial proximity of the fluorophores when conjugated to the
reduced disulfides of an antibody. Based on this result, a
fluorophore loading of approximately 3 per antibody was selected.
At this loading level not only would the fluorescence output be
maximal (resulting in greatest sensitivity in fluorescent assays),
but also the variation in fluorescence as a function of fluorophore
loading will be minimal. This will ensure that small variations in
fluorophore loading across an antibody panel will not result in
large differences in fluorescent output, an important point if
fluorescence is to be used to quantify the conjugate
concentrations.
[0097] The ratio of the absorbance at 650 nm to 280 nm was also
determined for each of the fluorophore-drug conjugates described
above. These ratios are shown in FIG. 5, plotted against the
fluorophores per antibody data. In the region of 2.5 to 3
fluorophores per antibody, the change in the 650 nm/280 nm
absorbance ratio is linear with the change in loading level, and
the equation of this line can be used to determine the fluorophore
loading in mixed AF647-mcMMAF antibody conjugates from the measured
absorbance values.
Example 4
Exemplary Method for Tailoring the Ratio of Chemical Entities in
Order to Achieve a Desired Drug Loading Level
[0098] As described in example 3, an exemplary number of
fluorophores per antibody is about 3. Assuming that the
antibody-containing samples are of the murine IgG1 and IgG2a
isotypes and the desired loading level for flurophores is 3, a drug
loading level of 7 could be achieved by preparing the appropriate
mixture of AF647 maleimide and mcMMAF. However, a lower level of
drug loading may be achieved by including a capping reagent such as
N-ethyl maleimide (NEM). Thus, a ternary mixture of AF647, mcMMAF,
and NEM could be prepared in an appropriate ratio to achieve any
desired level of AF647 and mcMMAF loading (provided that the sum of
the two is no greater than 10 for a murine IgG1 or IgG2a). To
determine the correct mixture of these three reagents necessary to
achieve a desired loading level, their relative reactivities were
determined. This was done by preparing 1:1 mixtures of mcMMAF:NEM
and AF647:NEM and reacting these mixtures with a fully reduced
murine IgG1 immobilized on Protein G. The level of fluorophore
loading in the resulting AF647 conjugate was determined from its
650 nm/280 nm absorbance ratio by reference to FIG. 5, while the
mcMMAF loading in the resulting drug conjugate was determined by
PLRP chromatography. These data are shown in the table below; the
mole fraction on antibody is the loading of each reagent (AF647 or
mcMMAF) divided by 10, the total number of maleimides which
conjugate to the reduced murine IgG1; the NEM mole fraction is 1
minus the reagent mole fraction; and the relative reactivity is the
ratio of the reagent mole fraction to the NEM mole fraction. In
this analysis, NEM is assigned a relative reactivity value of
1.
TABLE-US-00002 NEM mole Mole fraction fraction on Relative 1:1 mix
Loading on antibody antibody Reactivity AF647:NEM 2.88 0.288 0.712
0.404 mcMMAF:NEM 3.75 0.375 0.625 0.6
[0099] To convert these relative reactivity values into an
appropriate ratio of maleimides to use in the ternary mixture, it
is first necessary to define the desired loading levels of each
reagent on the final conjugated antibody. For this example, a
target loading of 4.5 mcMMAF, 3 AF647, and 2.5 NEM will be used,
again assuming that the antibody conjugate will have 10 available
thiols when reduced. This corresponds to a conjugated mole fraction
of 0.45, 0.3, and 0.25, respectively. The necessary calculations
are then summarized in the table below.
TABLE-US-00003 Conjugated required mole Mole Fraction Relative
Reactivity Mole Fraction fraction in Reagent Target (see table
above) Relative Reactivity maleimide mix mcMMAF 0.45 0.6 0.75 0.43
AF647 0.3 0.404 0.74 0.43 NEM 0.25 1 0.25 0.14
[0100] Briefly, the target value for the conjugated mole fraction
of each reagent is divided by its relative reactivity factor which
was determined above using 1:1 mixes of the different reagents.
This value is then converted to a required mole fraction in the
maleimide mixture by dividing the value by the sum of the values
for all reagents. For example, for mcMMAF, 0.45/0.6=0.75;
0.75/(0.75+0.74+0.25)=0.43. In this manner, a mixture of mcMMAF,
AF647, and NEM in a ratio of 0.43:0.43:0.14 would be predicted to
yield antibody conjugates with an average loading 4.5, 3, and 2.5
for the three reagents, respectively. In like manner, different
ratios of the reagents could be calculated to achieve different
loading levels on the conjugate, or ratios for other reagents could
be calculated once their relative reactivities had been
determined.
Example 5
Demonstration of the Consistency of Drug Loading Across Samples
[0101] A solution of mcMMAF, AF647 maleimide, and NEM was prepared
in a ratio of 0.43:0.43:0.14 and used to conjugate a panel of
antibodies by the present methods. These antibodies had been
generated from 1.5 mL of bovine IgG-depleted hybridoma culture
media, using newly fused hybridomas resulting from a murine
immunization campaign. One 96-well plate of samples was subjected
to analysis to determine the drug and fluorophore loading
consistency of the resulting mixed conjugates. The fluorophore
loading was determined by the 650 nm/280 nm absorbance ratio of
each sample, measured in an absorbance plate reader, by reference
to the linear relationship shown in FIG. 5. The resulting data are
shown in FIG. 6, plotted against the quantity of conjugate that
each sample yielded; the data are shown only for those samples
which yielded at least 2.5 .mu.g of conjugate, as lower quantities
than this did not produce 280 nm absorbance values significantly
above the baseline. There were 65 samples on the plate which met
this 2.5 .mu.g threshold and are plotted in FIG. 6. As can be seen
in the figure, the loading is scattered between 2 and 4
fluorophores per antibody, with a calculated mean of 3.26 and a
coefficient of variation of 10.2%. The mean loading of 3.26 differs
by less than 10% from the targeted loading of 3. Importantly, the
range of observed fluorophore loading levels fell within the region
of the fluorescence vs loading curve (FIG. 4) where the
fluorescence does not change greatly, due to the self-quenching
phenomenon. In other words, the difference in observed fluorescence
between antibodies with 2, 3, or 4 fluorophores per antibody is
expected to be less than 20%. The mcMMAF loading was determined by
PLRP chromatography; a sample PLRP chromatogram of an
mcMMAF-AF647-NEM antibody conjugate is shown in FIG. 7. This figure
is an overlay of two analytical wavelengths, 280 nm to detect all
of the peaks containing protein, and 620 nm to detect those peaks
containing at least one Alexa Fluor.RTM. 647. As can be seen in
this figure, the light chain with NEM (2.2 minutes) is slightly
resolved from the light chain with Alexa Fluor.RTM. 647 (2.5
minutes), but both are well resolved from the light chain with
mcMMAF (3.8 minutes), illustrating that the PLRP column separates
species well on the basis of mcMMAF loading but not AF647 loading.
Since the light chain contains only one cysteine which is reduced
by the TCEP treatment, these are the only light chain species
present, and the NEM and mcMMAF peaks do not have absorbance at 620
nm as they contain no AF647. The heavy chain peak cluster is more
complicated due to the fact that with 4 available thiols, each peak
is not a single species. For example, the peak corresponding to
heavy chain with 2 copies of mcMMAF (7.7 minutes) is a collection
of heavy chain species which also contain 2 AF647, 2 NEM, or 1
AF647 and 1 NEM; these various species are not separated by the
PLRP column This feature of the separation permits these data to be
used to assess strictly the mcMMAF loading without being affected
by the presence of AF647 or NEM. Using this method, the mcMMAF
loading levels were determined for 34 samples from the plate of
hybridoma supernatants, and are plotted in FIG. 8 against the
quantity of conjugate that each sample yielded. As can be seen in
the figure, the loading is scattered between 4 and 6 copies of
mcMMAF per antibody, with a calculated mean of 4.51 and a
coefficient of variation of 7.75%. The mean loading of 4.51 is
exactly at the targeted level of 4.5. As can be seen in the figure,
there are 3 outliers with loading levels greater than 5; the PLRP
chromatograms from these samples contain a heavy chain species with
5 copies of mcMMAF, indicating the presence of an additional
reducible disulfide on the heavy chain of these antibodies (see
FIG. 9 as an example), suggesting that these antibodies are of the
murine IgG2b isotype. Thus, the higher loading observed in these
samples is not due to disproportionate loading of mcMMAF compared
to the other antibodies, but rather that these antibodies have 20%
more available thiols (12 rather than 10) and therefore would be
expected to be loaded with each reagent at levels 20% higher. If
these three are excluded from the analysis and only those
antibodies with 10 available thiols are considered, the mean mcMMAF
loading for the 31 antibodies is 4.42 and the coefficient of
variation falls to 3.45%. These results illustrate the consistency
of reagent loading (mcMMAF and AF647) achieved by the present
method across a panel of antibodies of variable isotype and in
variable quantities from a panel of newly fused hybridomas.
Example 6
Exemplary Method for Making Antibody Conjugates Loaded with
Drug-Linker and Capping Agent
[0102] Hybridoma supernatants were prepared as 4.5 mL solutions in
5 mL round-bottom tubes. 150 uL Protein G resin slurry (Millipore
ProSep-G) was added to each. Tubes were capped and rotated
overnight at 5.degree. C. Two control tubes were also prepared, one
with bovine IgG-depleted growth media (to serve as a blank) and one
with this same media spiked with 100 ug of a control antibody.
[0103] On the following morning, resin was transferred from tubes
to a 2 mL, 96-well filter plate with a 2.5 um polypropylene frit
(Seahorse Bioscience) using a 1250 uL Matrix pipettor. The
supernatant in the filter plate was pulled through by brief
application of vacuum. After all wells had dried (<30 seconds),
the plate was centrifuged at 500.times.g for 3 minutes to ensure
complete pulldown of all fluids and resin. After spinning, the
filter plate was replaced on the manifold and each well received
500 uL PBS. The plate was then shaken at 1200 RPM on the
Thermomixer for 30 seconds to slurry the resin. The PBS was then
pulled through by vacuum. This process was repeated twice, for a
total of 3 PBS washes. This process was then repeated with 3x PBS,
and then followed by a another wash with PBS. Following this final
wash, the plate was spun as before.
[0104] The bound antibodies were then reduced by adding 500 uL of
20 mM TCEP in 250 mM KPO.sub.4, 150 mM NaCl, pH 7, 1 mM EDTA and
shaking for 2 hours at 37.degree. C. on the Thermomixer. Following
reduction, the TCEP solution was pulled through by vacuum and then
spun as above, then washed with PBS+1 mM EDTA as described above.
This was repeated 4.times., for a total of 5 washes.
[0105] The bound antibodies were then conjugated to a mixture of
NEM and mc-MMAF in a molar ratio of 4:6. A stock of NEM+mc-MMAF at
a total maleimide concentration of 12 mM was prepared in advance.
1.1 mL of this solution was added to 55 mL of 10% DMSO, transferred
to a multichannel reservoir, and 500 uL added to each well of the
filter plate, which was then shaken for 30 minutes at 22.degree. C.
Following conjugation, the maleimide solution was pulled through by
vacuum and then spun as above. The centrifuge speed was increased
to 1500.times.g to complete the drying. Wells were then washed
twice with 500 uL of 10% DMSO in PBS, then three times with
PBS.
[0106] The bound ADCs were then eluted by adding 200 uL of 100 mM
glycine, pH 2.0 to each well and shaking for 3 minutes at 1200 RPM,
22.degree. C. on the Thermomixer. While shaking, 20 uL of
neutralization buffer (1M dibasic phosphate+0.1% Tween-20) was
added to each well of a 350 uL collection plate. When 3 minutes had
elapsed, the ADCs were eluted into the collection plate by spinning
at 1500.times.g for 6 minutes.
[0107] 200 uL of each ADC solution were transferred to a Costar UV
assay plate. A second plate was prepared with neutralized elution
buffer to serve as a blank. A280 measurements were carried out with
a Molecular Devices SpectraMax plate reader to determine protein
concentrations.
[0108] Finally, ADCs were sterile filtered. In the BSC, a sterile
0.2 um filter plate (Millipore) was fastened to a sterile 1 mL
collection plate (Matrix) using lab tape. The ADC solutions were
then added to the filter plate and spun at 500.times.g for 3
minutes. The assembly was then transferred to the BSC and
disassembled, then the collection plate capped with a sterile cap
mat (Matrix).
Example 7
Exemplary Method for Making Antibody Conjugates Loaded with
Drug-Linker, Capping Agent, and Fluorophore
[0109] Newly fused hybridomas were plated in methylcellulose media
(Genetix) containing HAT and fluorescently labeled anti-mouse IgG
(Genetix). Clonal, IgG-producing colonies were selected and
deposited into a 96 W plate containing HSFM (InVitrogen) plus
IgG-depleted cloning factor (Roche). Four-fold dilutions of
unpurified hybridoma culture supernatants were incubated with
target tumor cells in a homogenous assay containing 100 ng/ml of
Cy5-labeled anti-mouse secondary antibody (Jackson Labs). Hybridoma
binding to the tumor cells was detected using an FMAT8200 (Applied
Biosytems) and positive wells were expanded into 48 W dishes
containing 2 mls of HSFM (InVitrogen) plus IgG-depleted cloning
factor. Antibodies from 48 W extinguished supernatants were used
for solid-phase purification and conjugation.
[0110] Hybridoma supernatants (1.5 mL) were transferred to 96-well
deep well plates with a 0.45 um polypropylene frit (Seahorse
Bioscience). To enable quantitation of conjugate concentration by
fluorescence, a standard murine antibody was included in the
conjugation. 50 ug of the standard antibody was placed in 4 wells
of the plate with blank media (200 ug total). Additionally, 3 wells
contained only blank media for determination of background
fluorescence.
[0111] 100 uL of PBS was placed in each well of a 96-well deep well
filter plate fitted with a 2.5 um polypropylene filter (Seahorse
Bioscience). 20 uL Protein G resin slurry (GE Life Sciences
GammaBind Plus) was added to each well.
[0112] The filter plate containing the Protein G resin was placed
as the receiver plate in a vacuum manifold, and the manifold
assembled. The 0.45 um filter plate containing the antibody samples
and standards was placed on top of the manifold, and the
supernatants transferred to it. By application of vacuum,
supernatants were then filtered through the 0.45 um filters into
the plate containing the Protein G resin. The resin plate was then
shaken for 2 hours at room temperature at 1200 RPM using an
Eppendorf Thermomixer to effect binding to the Protein G. The
residual supernatant was then filtered into a 2 mL deep well
receiver plate by centrifugation at 500.times.g for 5 minutes.
[0113] A solution of 10 mM tricarboxyethyl phosphine (TCEP) in 100
mM potassium phosphate, pH 7.4, 150 mM NaCl, was added to the plate
(150 uL per well). The plate was then shaken as above for 30
minutes, then removed from the shaker and centrifuged for 2 minutes
at 500.times.g. The resin was washed four times with 500 uL of PBS
containing 1 mM EDTA, with vacuum filtration following each wash.
Following the final wash, another 500 uL PBS/EDTA added and removed
by centrifugation for 3 minutes at 500.times.g.
[0114] Individual stocks of drug-linker (mcMMAF), Alexa Fluor.RTM.
647, and NEM were prepared at 10 mM in DMSO. These stocks were then
blended into a single solution at the following ratio for
conjugation:
3:3:1 mcMMAF:Alexa Fluor.RTM. 647:NEM
140 uL of this solution was dissolved in 15 mL of PBS/EDTA, and 150
uL added to each well of the washed plate. The plate was then
shaken as above for 15 minutes, then removed from the shaker and
centrifuged for 2 minutes at 500.times.g. The resin was washed four
times with 500 uL of PBS, with vacuum filtration following each
wash. Following the final wash, another 500 uL of PBS was added and
removed by centrifugation for 3 minutes at 500.times.g.
[0115] 10 uL of 1M potassium phosphate pH 7.4 was added to each
well of a 350 uL 96-well clear-bottom assay plate. The resin plate
was placed atop the assay plate and 100 uL elution buffer (50 mM
glycine pH 2.5+0.08% Tween-20) added to each well. The plate was
gently agitated by manual rocking for 2 minutes, then centrifuged
for 2 minutes at 500.times.g to collect the eluted antibody
conjugates in the assay plate. The assay plate was immediately
placed in a fluorescence plate reader (Molecular Devices) and
shaken for 10 seconds using the plate reader shaker to ensure
complete mixing of the neutralization buffer into the elution
buffer. The fluorescence of each well at 675 nm was then measured
using an excitation of 635 nm with a 665 nm cutoff filter. The
solutions in the wells containing the standards were removed and
pooled into a single standard solution, and the concentration of
this standard was determined by a conventional A280 absorbance
method in a 1 cm cuvet. A dilution series of this standard was then
prepared (using neutralized elution buffer as the diluent) down to
a concentration of 1 ug/mL. 110 uL of each standard was then
transferred to a clean 350 uL clear bottom assay plate and the
fluorescence again measured on the plate reader. A second-order
polynomial curve was fit to the fluorescence values of the
standards, and the concentrations of the samples were assigned by
interpolation to this standard curve.
[0116] Finally, ADCs were sterile filtered. In the BSC, a sterile
0.2 um filter plate (Millipore) was fastened to a sterile 1 mL
collection plate (Matrix) using lab tape. The ADC solutions were
then added to the filter plate and spun at 500.times.g for 3
minutes. The assembly was then transferred to the BSC and
disassembled, then the collection plate capped with a sterile cap
mat (Matrix).
[0117] Mixed antibody conjugates containing fluorophore and drug
were tested in cell binding and cytoxicity assays. For cell-based
binding assays, the antibody panel was diluted at 1:200 and 1:1000
in PBS+2% serum and incubated on target cells for 2 hours at room
temperature in 96 W black plates. A control antibody was used on
each plate to generate a saturation binding curve for human and
cyno forms of the antigen. Plates were then analyzed in an FMAT8200
and mean fluorescence intensity values for each dilution were
plotted on the saturation binding curve to estimate test antibody
affinity on human and cyno forms of the antigen. Hybridomas that
showed equivalent binding to human and cyno antigen were advanced
for cytotoxicity studies. Cytoxicity studies were done by plating
5,000 cells per well in the appropriate growth media. Mixed
conjugates were added to a final dilution of 1:100 and 1:1000,
respectively. Tumor cells were incubated with drug/fluorophore
conjugates for 96 hours at 37.degree. C. Cell Titer Glo (Promega)
was used to measure cell viability and the potency of
drug/fluorophore conjugates was assessed based on the percent
viability relative to untreated control cells. Drug/fluorophore
conjugates that resulted in <70% viability of tumor cells at 1
nM concentrations were advanced for further testing.
Example 8
IgG Depletion
[0118] Cloning Factor is commonly used as a media component in
expanding hybridoma cell lines after fusion with murine B cells.
Cloning Factor contains important cell mediators that where
harvested from the supernatant of healthy thriving cells and these
help the new hybridoma fusions recover and begin to grow more
robustly. It is suspected that the harvested supernatant from the
healthy thriving cells that makes up the cloning factor contains
bovine serum as a media component which would include bovine
albumin, IgG and other serum proteins. It is the bovine IgG that is
of concern in this case because even a small amount of
contaminating IgG can affect the quantitative recovery of the
antibodies and quantitation of the resulting ADCs.
[0119] The method for removing bovine IgG from Cloning Factor is as
follows. 5 ml Protein G column is equilibrated with 1.times. PBS (5
Column Volumes, CV), 25 ml. Contents of the Hybridoma Cloning
factor are loaded into a 60 cc syringe. A syringe is attached to
the Protein G column and connected to a syringe pump. The pump is
set to 3 ml/min, the Cloning Factor is passed over the Protein G
column and the effluent is collected. The effluent contains the IgG
depleted Cloning factor. Bovine IgG will bind to the Protein G
column. The IgG depleted Hybridoma Cloning factor is sterile
filtered in a biosafety cabinet using a 0.22 .mu.m syringe
filter.
* * * * *