NZ712414B2 - Preparation of antibody maytansinoid conjugates - Google Patents
Preparation of antibody maytansinoid conjugates Download PDFInfo
- Publication number
- NZ712414B2 NZ712414B2 NZ712414A NZ71241412A NZ712414B2 NZ 712414 B2 NZ712414 B2 NZ 712414B2 NZ 712414 A NZ712414 A NZ 712414A NZ 71241412 A NZ71241412 A NZ 71241412A NZ 712414 B2 NZ712414 B2 NZ 712414B2
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- NZ
- New Zealand
- Prior art keywords
- antibody
- mixture
- maytansinoid
- cell
- hours
- Prior art date
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Abstract
Disclosed is a process for preparing an antibody maytansinoid conjugate comprising the steps of: (a) contacting an antibody with a maytansinoid to form a first mixture comprising the antibody and the maytansinoid, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9 to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products; and b) contacting the second mixture with the maytansinoid to form a third mixture; and then contacting the third mixture with the bifunctional crosslinking reagent at a pH of about 4 to about 9 to provide a fourth mixture a linker, in a solution having a pH of about 4 to about 9 to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products; and b) contacting the second mixture with the maytansinoid to form a third mixture; and then contacting the third mixture with the bifunctional crosslinking reagent at a pH of about 4 to about 9 to provide a fourth mixture
Description
PREPARATION OF ANTIBODY MAYTANSINOID CONJUGATES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of New Zealand patent application 616509, which
is the national phase entry in New Zealand of PCT international application
(published as ), and claims the benefit of U.S.
Provisional Patent Application No. 61/468,997, filed March 29, 2011, all of which are
incorporated herein by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0001a] Incorporated by reference in its entirety herein is a computer-readable
nucleotide/amino acid sequence listing identified as follows: One 9,233 Byte ASCII (Text)
file named “710088SequenceListing.TXT,” created on April 3, 2013.
BACKGROUND OF THE INVENTION
Antibody-Drug-Conjugates (ADC’s) which are useful for the treatment of cancer
and other diseases are commonly composed of three distinct elements: a cell-binding agent; a
linker; and a cytotoxic agent. The conventional method of conjugating a cell-binding agent,
such as an antibody, to a cytotoxic agent, employs two distinct reaction steps with the
antibody. In the first reaction step (the modification step), the antibody is reacted with a
heterobifunctional linker to produce a linker-modified antibody. The modified antibody
product is then optionally purified from the excess linker or hydrolyzed linker reagent. In the
second reaction step (the conjugation step), the linker-modified antibody is reacted with the
cytotoxic agent containing a reactive group, such as thiol, to generate the antibody-cytotoxic
agent conjugate, which is again purified in an additional purification step.
The processes that have been previously described for manufacture of the
antibody-cytotoxic agent conjugates are complex because they are encumbered with steps
that are cumbersome to perform or produce immunoconjugates that are less pure or less
stable than optimally desired. Thus, it would be desirable to modify or eliminate one or more
manufacturing steps while improving the product quality, such as purity and/or stability.
In view of the foregoing, there is a need in the art to develop improved processes
for preparing cell-binding agent-cytotoxic agent conjugates that are of substantially high
purity and can be prepared avoiding cumbersome steps and by reducing time and cost to the
user. The invention provides such a process and/or at least provides the public with a useful
choice. These and other advantages of the invention, as well as additional inventive features,
will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0004a] In one aspect the invention relates to a process for preparing an antibody
maytansinoid conjugate comprising the steps of:
(a) contacting an antibody with a maytansinoid to form a first mixture comprising the
antibody and the maytansinoid, then contacting the first mixture with a bifunctional
crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9 to
provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the
antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid,
and (iii) reaction by-products; and
(b) contacting the second mixture with the maytansinoid to form a third mixture; and
then contacting the third mixture with the bifunctional crosslinking reagent at a pH of about 4
to about 9 to provide a fourth mixture.
[0004b] Certain statements that appear below are broader than what appears in the
statements of the invention above. These statements are provided in the interests of
providing the reader with a better understanding of the invention and its practice. The reader
is directed to the accompanying claim set which defines the scope of the invention.
Also described herein is a process for preparing a cell-binding agent cytotoxic
agent conjugate comprising the step of contacting a cell-binding agent with a cytotoxic agent
to form a first mixture comprising the cell-binding agent and the cytotoxic agent, and then
contacting the first mixture comprising the cell-binding agent and the cytotoxic agent with a
bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to
about 9 to provide a mixture comprising (i) the cell-binding agent cytotoxic agent conjugate,
wherein the cell-binding agent is chemically coupled through the linker to the cytotoxic
agent, (ii) free cytotoxic agent, and (iii) reaction by-products. The process can further
comprise the step of purifying the mixture to provide a purified cell-binding agent cytotoxic
agent conjugate.
The processes of the present invention provides cell-binding agent cytotoxic agent
conjugate with high purity and/or stability. To achieve the high purity and/or stability of the
conjugate, it is essential that the cytotoxic agent is contacted with the cell-binding agent first
to form a mixture comprising the cell-binding agent and the cytotoxic agent before the
mixture is contacted with a bifunctional crosslinking reagent.
Also described herein is a cell-binding agent cytotoxic agent conjugate prepared
according to the processes described herein.
DETAILED DESCRIPTION OF THE INVENTION
Described herein is a one-step process for preparing a cell-binding agent cytotoxic
agent conjugate. The process comprises contacting a cell-binding agent (e.g., an antibody)
with a cytotoxic agent to form a first mixture comprising the cell-binding agent and the
cytotoxic agent, and then contacting the first mixture comprising the cell-binding agent and
the cytotoxic agent with a bifunctional crosslinking reagent comprising a linker, in a solution
having a pH of about 4 to about 9 to provide a second mixture comprising the cell-binding
agent cytotoxic agent conjugate, free cytotoxic agent, and reaction by-products, wherein the
cell-binding agent is chemically coupled through the linker to the cytotoxic agent. The
second mixture is then subjected to purification to provide a purified cell-binding agent
cytotoxic agent conjugate.
In one embodiment, the contacting is effected by providing the cell-binding agent,
then contacting the cell-binding agent with the cytotoxic agent to form a first mixture
comprising the cell-binding agent and the cytotoxic agent, and then contacting the first
mixture comprising the cell-binding agent and the cytotoxic agent with the bifunctional
crosslinking reagent. For example, in one embodiment, the cell-binding agent is provided in
a reaction vessel, the cytotoxic agent is added to the reaction vessel (thereby contacting the
cell-binding agent), and then the bifunctional crosslinking reagent is added to the mixture
comprising the cell-binding agent and the cytotoxic agent (thereby contacting the mixture
comprising the cell-binding agent and the cytotoxic agent). In one embodiment, the cell-
binding agent is provided in a reaction vessel, and the cytotoxic agent is added to the reaction
vessel immediately following providing the cell-binding agent to the vessel. In another
embodiment, the cell-binding agent is provided in a reaction vessel, and the cytotoxic agent is
added to the reaction vessel after a time interval following providing the cell-binding agent to
the vessel (e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes,
about 40 minutes, about 50 minutes, about 1 hour, about 1 day or longer after providing the
cell-binding agent to the space). The cytotoxic agent can be added quickly (i.e., within a
short time interval, such as about 5 minutes, about 10 minutes) or slowly (such as by using a
pump).
The mixture comprising the cell-binding agent and the cytotoxic agent can be then
contacted with the bifunctional crosslinking reagent either immediately after contacting the
cell-binding agent with the cytotoxic agent or at some later point (e.g., about 5 minutes to
about 8 hours or longer) after contacting the cell-binding agent with the cytotoxic agent. For
example, in one embodiment, the bifunctional crosslinking reagent is added to the mixture
comprising the cell-binding agent and the cytotoxic agent immediately after the addition of
the cytotoxic agent to the reaction vessel comprising the cell-binding agent. Alternatively,
the mixture comprising the cell-binding agent and the cytotoxic agent can be contacted with
the bifunctional crosslinking reagent at about 5 minutes, about 10 minutes, about 20 minutes,
about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, or longer after contacting the cell-binding agent
with the cytotoxic agent.
In another embodiment, the cytotoxic agent and the bifunctional agent are added
through multiple cycles (e.g., 1, 2, 3, 4, 5 or more cycles). For example, described herein is a
process comprising the steps of: a) contacting a cell-binding agent with a cytotoxic agent to
form a first mixture comprising the cell-binding agent and the cytotoxic agent; and then
contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a
solution having a pH of about 4 to about 9 to provide a second mixture comprising the cell-
binding agent cytotoxic agent conjugate, free cytotoxic agent, and reaction by-products,
wherein the cell-binding agent is chemically coupled through the linker to the cytotoxic
agent; b) contacting the second mixture with the cytotoxic agent to form a third mixture; and
then contacting the third mixture with the bifunctional crosslinking reagent at a pH of about 4
to about 9 to provide a fourth mixture; and c) purifying the fourth mixture to provide the
purified cell-binding agent cytotoxic agent conjugate. In one embodiment, step b) is carried
out after a time interval (e.g., about 1 hour, about 2 hours, about 3 hours or longer) following
step a). In another embodiment, step b) can be repeated several times (e.g., 1, 2, 3, 4 or more
times) before step c) is carried out. The additional step b) can be carried out after a time
interval (e.g., about 1 hour, about 2 hours, about 3 hours or longer) following the initial step
In another embodiment, the bifunctional crosslinking reagent is added before the
complete addition of the cytotoxic agent. For example, in one embodiment, the cytotoxic
agent is added to the cell-binding agent continuously over a time interval (e.g., over about 5
minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, or
longer) to form a mixture comprising the cell-binding agent and the cytotoxic agent. Before
the addition of the cytotoxic agent is complete, the bifunctional crosslinking reagent is added
to the mixture comprising the cell-binding agent and the cytotoxic agent, provided that at any
time, the cytotoxic agent is in molar excess of the bifunctional crosslinking reagent. In one
embodiment, the bifunctional crosslinking reagent is added continuously over a time interval
(e.g., over about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours,
about 3 hours, or longer).
After the mixture comprising the cell-binding agent and the cytotoxic agent is
contacted with the bifunctional crosslinking reagent, the reaction is allowed to proceed for
about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about
7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about
13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,
about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, or longer (e.g., about 30 hours, about 35 hours, about 40 hours, about 45 hours, or
about 48 hrs).
Contacting the cell-binding agent with the cytotoxic agent and then the
bifunctional crosslinking reagent (i.e., the reaction step) occurs in a solution having a pH of
about 4 to about 9 (e.g., about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7,
about 7.5, about 8, about 8.5, or about 9). In one embodiment, the reaction step occurs in a
solution having a pH of about 6 or less (e.g., about 4 to about 6, about 4 to about 5.5, or about
4.5 to about 5.5).
In another embodiment, the inventive process comprises contacting a cell-binding
agent with a cytotoxic agent and then a bifunctional crosslinking reagent in a solution having
a pH of about 6 or greater (e.g., about 6 to about 9, about 6 to about 7, about 7 to about 9,
about 7 to about 8.5, about 7.5 to about 8.5, about 7.5 to about 8.0, about 8.0 to about 9.0, or
about 8.5 to about 9.0). For example, the inventive process comprises contacting a cell-
binding agent with a cytotoxic agent and a bifunctional crosslinking reagent in a solution
having a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6,
about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about
7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3,
about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In a specific
embodiment, the inventive process comprises contacting a cell-binding agent with a cytotoxic
agent and a bifunctional crosslinking reagent in a solution having a pH of about 7.8 (e.g., a
pH of 7.6 to 8.0 or a pH of 7.7 to 7.9).
The inventive process comprises performing the one-step reaction (i.e., contacting
a cell-binding agent with a cytotoxic agent and then a bifunctional crosslinking reagent) at
any suitable temperature known in the art. For example, the one-step reaction can occur at
about 20 ºC or less (e.g., about -10º C (provided that the solution is prevented from freezing,
e.g., by the presence of organic solvent used to dissolve the cytotoxic agent and the
bifunctional crosslinking reagent) to about 20 ºC, about 0 ºC to about 18 ºC, about 4 ºC to
about 16 ºC), at room temperature (e.g., about 20 ºC to about 30 ºC or about 20 ºC to about
ºC), or at an elevated temperature (e.g., about 30 ºC to about 37 ºC). In one embodiment,
contacting a cell-binding agent with a cytotoxic agent and a bifunctional crosslinking reagent
occurs at a temperature of about 16 °C to about 24 °C (e.g., about 16 ºC, about 17 ºC, about
18 ºC, about 19 ºC, about 20 ºC, about 21 ºC, about 22 ºC, about 23 ºC, about 24 ºC, or about
ºC).
In another embodiment, contacting a cell-binding agent with a cytotoxic agent and
then a bifunctional crosslinking reagent occurs at a temperature of about 15 °C or less (e.g.,
about -10 ºC to about 15 ºC, or about 0 °C to about 15 °C). In this respect, the inventive
process comprises contacting a cell-binding agent with a cytotoxic agent and then a
bifunctional crosslinking reagent at a temperature of about 15 °C, about 14 °C, about 13 °C,
about 12 °C, about 11 °C, about 10 °C, about 9 °C, about 8 °C, about 7 °C, about 6 °C, about
°C, about 4 °C, about 3 °C, about 2 °C, about 1 °C, about 0 °C, about -1 °C, about -2 °C,
about -3 °C, about -4 °C, about -5 °C, about -6 °C, about -7 °C, about -8 °C, about -9 °C, or
about -10 °C, provided that the solution is prevented from freezing, e.g., by the presence of
organic solvent(s) used to dissolve the bifunctional crosslinking reagent. In one embodiment,
the inventive process comprises contacting a cell-binding agent with a cytotoxic agent and
then a bifunctional crosslinking reagent at a temperature of about -10° C to about 15° C,
about 0 °C to about 15 °C, about 0 °C to about 10 °C, about 0 °C to about 5 °C, about 5 °C to
about 15 °C, about 10 °C to about 15 °C, or about 5 °C to about 10 °C. In another
embodiment, the inventive process comprises contacting a cell-binding agent with a cytotoxic
agent and then a bifunctional crosslinking reagent at a temperature of about 10 °C (e.g., a
temperature of 8 ºC to 12 ºC or a temperature of 9 ºC to 11 ºC).
In one embodiment, the inventive process comprises contacting a cell-binding
agent with a cytotoxic agent and then a bifunctional crosslinking reagent in a solution having
a high pH (e.g., about 7 or greater) at a low temperature (e.g., about 15 ºC or less). For
example, in one embodiment, the inventive process comprises contacting a cell-binding agent
with a cytotoxic agent and then a bifunctional crosslinking reagent in a solution having a pH
of about 7.5 at a temperature of about 15 ºC, in a solution having a pH of about 7.8 at a
temperature of about 10 ºC, in a solution having a pH about 8.2 at a temperature of about
0° C, or in a solution having a pH about 8.5 at a temperature of about 0° C. In another
embodiment, the inventive process comprises contacting a cell-binding agent with a cytotoxic
agent and then a bifunctional crosslinking reagent in a solution having a pH of 7.0 to 8.5
(e.g., a pH of 7.5 to 8.0) at a temperature of 5 °C to 15 °C.
In one embodiment, the inventive process further comprises a quenching step to
quench any unreacted cytotoxic agent and/or unreacted bifunctional crosslinking reagent.
The quenching step is performed prior to purification of the cell-binding agent cytotoxic
agent. For example, the inventive process comprises (a) contacting a cell-binding agent with
a cytotoxic agent to form a mixture comprising the cell-binding agent and the cytotoxic agent
and then contacting the mixture comprising the cell-binding agent and the cytotoxic agent
with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of
about 4 to about 9 to provide a mixture comprising (i) the cell-binding agent cytotoxic agent
conjugate, wherein the cell-binding agent is chemically coupled through the linker to the
cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, (b) quenching the
mixture prepared in step (a) to quench any unreacted cytotoxic agent and/or unreacted
bifunctional crosslinking reagent, and (c) purifying the mixture to provide a purified cell-
binding agent cytotoxic agent conjugate.
In one embodiment, the mixture is quenched by contacting the mixture with a
quenching reagent. As used herein, the “quenching reagent” refers to a reagent that reacts
with the free cytotoxic agent and/or the bifunctional crosslinking reagent.
In one embodiment, maleimide or haloacetamide quenching reagents, such as 4-
maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, or
iodoacetamidopropionic acid, can be used to ensure that any unreacted group (such as thiol)
in the cytotoxic agent is quenched. The quenching step can help prevent the dimerization of
the cytotoxic agent, particular the cytotoxic agent having an unreacted thiol group (such as
DM1). The dimerized cytotoxic agent can be difficult to remove. The quenching step may
also minimize any unwanted thiol-disulfide interchange reaction with the native antibody
disulfide groups. Upon quenching with polar, charged thiol-quenching reagents (such as 4-
maleimidobutyric acid or 3-maleimidopropionic acid), the excess, unreacted cytotoxic agent
is converted into a polar, charged, water-soluble adduct that can be easily separated from the
covalently- linked conjugate during the purification step. Quenching with non-polar and
neutral thiol-quenching reagents can also be used.
In one embodiment, the mixture is quenched by contacting the mixture with a
quenching reagent that reacts with the unreacted bifunctional crosslinking reagent. For
example, nucleophiles can be added to the mixture in order to quench any unreacted
bifunctional crosslinking reagent. The nucleophile preferably is an amino group containing
nucleophile, such as lysine, taurine and hydroxylamine.
In a preferred embodiment, the reaction (i.e., contacting a cell-binding agent with
a cytotoxic agent and then a bifunctional crosslinking reagent) is allowed to proceed to
completion prior to contacting the mixture with a quenching reagent. In this regard, the
quenching reagent is added to the mixture about 1 hour to about 48 hours (e.g., about 1 hour,
about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,
about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or
about 25 hours to about 48 hours) after the mixture comprising the cell-binding agent and the
cytotoxic agent is contacted with the bifunctional crosslinking reagent.
The inventive process may optionally include the addition of sucrose to the
reaction step (i.e., contacting a cell-binding agent with a cytotoxic agent and a bifunctional
crosslinking reagent) to increase solubility and recovery of the cell-binding agent-cytotoxic
agent conjugates. Desirably, sucrose is added at a concentration of about 0.1% (w/v) to about
% (w/v) (e.g., about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v), 15% (w/v), or 20%
(w/v)). Preferably, sucrose is added at a concentration of about 1% (w/v) to about 10% (w/v)
(e.g., about 0.5% (w/v), about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 3% (w/v),
about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9%
(w/v), about 10% (w/v), or about 11% (w/v)). In addition, the reaction step also can comprise
the addition of a buffering agent. Any suitable buffering agent known in the art can be used.
Suitable buffering agents include, for example, a citrate buffer, an acetate buffer, a succinate
buffer, and a phosphate buffer. In one embodiment, the buffering agent is selected from the
group consisting of HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic
acid)), POPSO (piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-
(2-hydroxyethyl)piperazineethanesulfonic acid), HEPPS (EPPS) (4-(2-
hydroxyethyl)piperazinepropanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]
aminoethanesulfonic acid), and a combination thereof.
Following the reaction step, the cell-binding agent cytotoxic agent conjugate is
subjected to a purification step. In this regard, the cell-binding agent cytotoxic agent
conjugate can be purified from the other components of the mixture (e.g., free cytotoxic agent
and reaction by-products) using tangential flow filtration (TFF), which is a membrane-based
tangential flow filtration process, non-adsorptive chromatography, adsorptive
chromatography, adsorptive filtration, selective precipitation, or any other suitable
purification process, as well as combinations thereof. In one embodiment, the cell-binding
agent cytotoxic agent conjugate is purified using a single purification step (e.g., TFF).
Preferably, the conjugate is purified and exchanged into the appropriate formulation using a
single purification step (e.g., TFF). In another embodiment, the cell-binding agent cytotoxic
agent conjugate is purified using two sequential purification steps. For example, the
conjugate can be first purified by selective precipitation, adsorptive filtration, absorptive
chromatography or non-absorptive chromatography, followed by purification with TFF. One
of ordinary skill in the art will appreciate that purification of the cell-binding agent cytotoxic
agent conjugate enables the isolation of a stable conjugate comprising the cell-binding agent
chemically coupled to the cytotoxic agent.
Any suitable TFF systems may be utilized for purification, including a Pellicon
type system (Millipore, Billerica, MA), a Sartocon Cassette system (Sartorius AG,
Edgewood, NY), and a Centrasette type system (Pall Corp., East Hills, NY).
Any suitable adsorptive chromatography resin may be utilized for purification.
Preferred adsorptive chromatography resins include hydroxyapatite chromatography,
hydrophobic charge induction chromatography (HCIC), hydrophobic interaction
chromatography (HIC), ion exchange chromatography, mixed mode ion exchange
chromatography, immobilized metal affinity chromatography (IMAC), dye ligand
chromatography, affinity chromatography, reversed phase chromatography, and combinations
thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT
Type I and Type II, Bio-Rad Laboratories, Hercules, CA), HA Ultrogel hydroxyapatite (Pall
Corp., East Hills, NY), and ceramic fluoroapatite (CFT Type I and Type II, Bio-Rad
Laboratories, Hercules, CA). An example of a suitable HCIC resin is MEP Hypercel resin
(Pall Corp., East Hills, NY). Examples of suitable HIC resins include Butyl-Sepharose,
Hexyl-Sepaharose, Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare,
Piscataway, NJ), as well as Macro-prep Methyl and Macro-Prep t-Butyl resins (Biorad
Laboratories, Hercules, CA). Examples of suitable ion exchange resins include SP-
Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway,
NJ), and Unosphere S resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable
mixed mode ion exchangers include Bakerbond ABx resin (JT Baker, Phillipsburg NJ).
Examples of suitable IMAC resins include Chelating Sepharose resin (GE Healthcare,
Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, CA). Examples
of suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, NJ)
and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable affinity
resins include Protein A Sepharose resin (e.g., MabSelect, GE Healthcare, Piscataway, NJ),
where the cell-binding agent is an antibody, and lectin affinity resins, e.g. Lentil Lectin
Sepharose resin (GE Healthcare, Piscataway, NJ), where the cell-binding agent bears
appropriate lectin binding sites. Alternatively an antibody specific to the cell-binding agent
may be used. Such an antibody can be immobilized to, for instance, Sepharose 4 Fast Flow
resin (GE Healthcare, Piscataway, NJ). Examples of suitable reversed phase resins include
C4, C8, and C18 resins (Grace Vydac, Hesperia, CA).
Any suitable non-adsorptive chromatography resin may be utilized for
purification. Examples of suitable non-adsorptive chromatography resins include, but are not
limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g., S-200 and S-
300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL®
resins (e.g., P-6, P-10, P-30, P-60, and P-100), and others known to those of ordinary skill in
the art.
In one embodiment, the inventive process further comprises a holding step to
release the unstably bound linkers from the cell-binding agent. The holding step comprises
holding the mixture prior to purification of the cell-binding agent-cytotoxic agent conjugate
(e.g., after the reaction step, between the reaction step and the quenching step, or after the
quenching step). For example, the inventive process comprises (a) contacting a cell-binding
agent with a cytotoxic agent to form a mixture comprising the cell-binding agent and the
cytotoxic agent; and then contacting the mixture comprising the cell-binding agent and the
cytotoxic agent with a bifunctional crosslinking reagent, which provides a linker, in a
solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the cell-
binding agent cytotoxic agent conjugate, wherein the cell-binding agent is chemically
coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction
by-products, (b) holding the mixture prepared in step (a) to release the unstably bound linkers
from the cell-binding agent, and (c) purifying the mixture to provide a purified cell-binding
agent cytotoxic agent conjugate.
In another embodiment, the inventive process comprises (a) contacting a cell-
binding agent with a cytotoxic agent to form a mixture comprising the cell-binding agent and
the cytotoxic agent; and then contacting the mixture comprising the cell-binding agent and
the cytotoxic agent with a bifunctional crosslinking reagent, which provides a linker, in a
solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the cell-
binding agent cytotoxic agent conjugate, wherein the cell-binding agent is chemically
coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction
by-products, (b) quenching the mixture prepared in step (a) to quench any unreacted
cytotoxic agent and/or unreacted bifunctional crosslinking reagent, (c) holding the mixture
prepared in step (b) to release the unstably bound linkers from the cell-binding agent, and (d)
purifying the mixture to provide a purified cell-binding agent cytotoxic agent conjugate.
Alternatively, the holding step can be performed after purification of the cell-
binding agent-cytotoxic agent conjugate, followed by an additional purification step.
In a preferred embodiment, the reaction (i.e., contacting a cell-binding agent with
a cytotoxic agent and then a bifunctional crosslinking reagent) is allowed to proceed to
completion prior to the holding step. In this regard, the holding step can be performed about
1 hour to about 48 hours (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,
about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about
22 hours, about 23 hours, about 24 hours, or about 24 hours to about 48 hours) after the
mixture comprising the cell-binding agent and the cytotoxic agent is contacted with the
bifunctional crosslinking reagent.
The holding step comprises maintaining the solution at a suitable temperature
(e.g., about 0º C to about 37º C) for a suitable period of time (e.g., about 1 hour to about 1
week, about 1 hour to about 24 hours, about 1 hour to about 8 hours, or about 1 hour to about
4 hours) to release the unstably bound linkers from the cell-binding agent while not
substantially releasing the stably bound linkers from the cell-binding agent. In one
embodiment, the holding step comprises maintaining the solution at about 20 ºC or less (e.g.,
about 0 ºC to about 18 ºC, about 4 ºC to about 16 ºC), at room temperature (e.g., about 20 ºC
to about 30 ºC or about 20 ºC to about 25 ºC), or at an elevated temperature (e.g., about 30 ºC
to about 37 ºC). In one embodiment, the holding step comprises maintaining the solution at a
temperature of about 16 °C to about 24 °C (e.g., about 15 ºC, about 16 ºC, about 17 ºC, about
18 ºC, about 19 ºC, about 20 ºC, about 21 ºC, about 22 ºC, about 23 ºC, about 24 ºC, or about
ºC). In another embodiment, the holding step comprises maintaining the solution at a
temperature of about 2 °C to about 8 °C (e.g., about 0 ºC, about 1 ºC, about 2 ºC, about 3 ºC,
about 4 ºC, about 5 ºC, about 6 ºC, about 7 ºC, about 8 ºC, about 9 ºC, or about 10 ºC). In
another embodiment, the holding step comprises maintaining the solution at a temperature of
about 37 °C (e.g., about 34 ºC, about 35 ºC, about 36 ºC, about 37 ºC, about 38 ºC, about 39
ºC, or about 40 ºC).
The duration of the holding step depends on the temperature and the pH at which
the holding step is performed. For example, the duration of the holding step can be
substantially reduced by performing the holding step at elevated temperature, with the
maximum temperature limited by the stability of the cell-binding agent-cytotoxic agent
conjugate. The holding step can comprise maintaining the solution for about 1 hour to about
1 day (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6
hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14
hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours),
about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 14 hours to about
24 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours
to about 24 hours, about 5 hours to about 1 week, about 20 hours to about 1 week, about 12
hours to about 1 week (e.g., about 12 hours, about 16 hours, about 20 hours, about 24 hours,
about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days), or
about 1 day to about 1 week.
In one embodiment, the holding step comprises maintaining the solution at a
temperature of about 2 ºC to about 8 ºC for a period of at least about 12 hours for up to a
week. In another embodiment, the holding step comprises maintaining the solution at a
temperature of about 2 ºC to about 8 ºC overnight (e.g., about 12 to about 24 hours,
preferably about 20 hours).
The pH value for the holding step preferably is about 4 to about 10. In one
embodiment, the pH value for the holding step is about 4 or more, but less than about 6 (e.g.,
4 to 5.9) or about 5 or more, but less than about 6 (e.g., 5 to 5.9). In another embodiment, the
pH values for the holding step range from about 6 to about 10 (e.g., about 6.5 to about 9,
about 6 to about 8). For example, pH values for the holding step can be about 6, about 6.5,
about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.
In specific embodiments, the holding step can comprise incubating the mixture at
°C at a pH of about 6-7.5 for about 12 hours to about 1 week, incubating the mixture at 4
°C at a pH of about 4.5-5.9 for about 5 hours to about 5 days, or incubating the mixture at 25
°C at a pH of about 4.5-5.9 for about 5 hours to about 1 day.
Also described herein is a process for preparing compositions of stable conjugates
comprising a cell-binding agent chemically coupled to a cytotoxic agent, wherein the
compositions are substantially free of unstable conjugates. In this respect, described herein is
a process for preparing cell-binding agent-cytotoxic agent conjugate of substantially high
purity and stability. Such compositions can be used for treating diseases because of the high
purity and stability of the conjugates. Compositions comprising a cell-binding agent, such as
an antibody, chemically coupled to a cytotoxic agent, such as a maytansinoid, are described
in, for example, U.S. Patent 7,374,762, the entire teaching of which is incorporated herein by
reference in its entirety. As described herein, a cell-binding agent-cytotoxic agent conjugate
of substantially high purity has one or more of the following features: (a) greater than about
90% (e.g., greater than or equal to about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%), preferably greater than about 95%, of conjugate species are monomeric, (b)
unconjugated linker level in the conjugate preparation is less than about 10% (e.g., less than
or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%) (relative to total linker), (c)
less than 10% of conjugate species are crosslinked (e.g., less than or equal to about 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%), (d) free cytotoxic agent level in the conjugate
preparation is less than about 2% (e.g., less than or equal to about 1.5%, 1.4%, 1.3%, 1.2%,
1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) (mol/mol
relative to total cytotoxic agent) and/or (e) no substantial increase in the level of free
cytotoxic agent upon storage (e.g., after about 1 week, about 2 weeks, about 3 weeks, about 1
month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months,
about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years). “Substantial
increase” in the level of free cytotoxic agent means that after certain storage time (e.g., about
1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years), the increase in the level of free cytotoxic agent is less than
about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about
0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about
1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.2%, about
2.5%, about 2.7%, about 3.0%, about 3.2%, about 3.5%, about 3.7%, or about 4.0%.
As used herein, the term “unconjugated linker” refers to the cell-binding agent
that is covalently linked with the bifunctional crosslinking reagent, wherein the cell-binding
agent is not covalently coupled to the cytotoxic agent through the linker of the bifunctional
crosslinking reagent (i.e., the “unconjugated linker” can be represented by CBA-L, wherein
CBA represents the cell-binding agent and L represents the bifunctional crosslinking reagent.
In contrast, the cell-binding agent cytotoxic agent conjugate can be represented by CBA-L-D,
wherein D represents the cytotoxic agent).
In one embodiment, the average molar ratio of the cytotoxic agent to the cell-
binding agent in the cell-binding agent cytotoxic agent conjugate is about 1 to about 10, about
2 to about 7, about 3 to about 5, about 2.5 to about 4.5 (e.g., about 2.5, about 2.6, about 2.7,
about 2.8, about 2.9, about 3.0, about 3.1, about 3.3, about 3.4, about 3.5, about 3.6, about
3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5),
about 3.0 to about 4.0, about 3.2 to about 4.2, or about 4.5 to 5.5 (e.g., about 4.5, about 4.6,
about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about
.5).
The present invention provides a more efficient process for preparing
compositions of stable conjugates comprising a cell-binding agent chemically coupled to a
cytotoxic agent. In one embodiment, as compared to the traditional processes for preparing
conjugates of a cell-binding agent and a cytotoxic agent, less amount of the cytotoxic agent is
required to achieve the same average molar ratio of the cytotoxic agent to the cell-binding
agent for the conjugates.
The cell-binding agent can be any suitable agent that binds to a cell, typically and
preferably an animal cell (e.g., a human cell). The cell-binding agent preferably is a peptide
or a polypeptide. Suitable cell-binding agents include, for example, antibodies (e.g.,
monoclonal antibodies and fragments thereof), interferons (e.g. alpha., beta., gamma.),
lymphokines (e.g., IL-2, IL-3, IL-4, IL-6), hormones (e.g., insulin, TRH (thyrotropin
releasing hormone), MSH (melanocyte-stimulating hormone), steroid hormones, such as
androgens and estrogens), growth factors and colony-stimulating factors such as EGF, TGF-
alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158
(1984)), nutrient-transport molecules (e.g., transferrin), vitamins (e.g., folate) and any other
agent or molecule that specifically binds a target molecule on the surface of a cell.
Where the cell-binding agent is an antibody, it binds to an antigen that is a
polypeptide or a glycotope and may be a transmembrane molecule (e.g., receptor) or a ligand
such as a growth factor. Exemplary antigens include molecules such as renin; a growth
hormone, including human growth hormone and bovine growth hormone; growth hormone
releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha
antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone;
calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vmc, factor IX,
tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial
natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine
or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor;
tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation
normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP
alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance;
relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated
antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor
(VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5,
or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF- β; platelet-derived
growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth
factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including
TGF- β1, TGF-β2, TGF- β3, TGF-β4, or TGF- β5; insulin-like growth factor-I and -II (IGF-I
and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins;
EpCAM; GD3; FLT3; PSMA; PSCA; MUC1; MUC16; STEAP; CEA; TENB2; EphA
receptors; EphB receptors; folate receptor; FOLR1; mesothelin; crypto; alpha beta ;
integrins; VEGF, VEGFR; EGFR; transferrin receptor; IRTA1; IRTA2; IRTA3; IRTA4;
IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20,
CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52,
CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138,
CD152 or an antibody which binds to one or more tumor-associated antigens or cell-surface
receptors disclosed in U.S. Patent Application Publication No. 2008/0171040 or U.S. Patent
Application Publication No. 2008/0305044 and are incorporated in their entirety by
reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic
protein (BMP); an interferon, such as interferon-alpha, -beta, and -gamma; colony stimulating
factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;
superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating
factor; viral antigen such as, for example, a portion of the HIV envelope; transport proteins;
homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c,
CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3, or
HER4 receptor; endoglin; c-Met; IGF1R; prostate antigens such as PCA3, PSA, PSGR,
NGEP, PSMA, PSCA, TMEFF2, and STEAP1; LGR5; B7H4; and fragments of any of the
above-listed polypeptides.
Additionally, GM-CSF, which binds to myeloid cells can be used as a cell-binding
agent to diseased cells from acute myelogenous leukemia. IL-2 which binds to activated T-
cells can be used for prevention of transplant graft rejection, for therapy and prevention of
graft-versus-host disease, and for treatment of acute T-cell leukemia. MSH, which binds to
melanocytes, can be used for the treatment of melanoma, as can antibodies directed towards
melanomas. Folic acid can be used to target the folate receptor expressed on ovarian and
other tumors. Epidermal growth factor can be used to target squamous cancers such as lung
and head and neck. Somatostatin can be used to target neuroblastomas and other tumor
types.
Cancers of the breast and testes can be successfully targeted with estrogen (or
estrogen analogues) or androgen (or androgen analogues) respectively as cell-binding agents
The term “antibody,” as used herein, refers to any immunoglobulin, any
immunoglobulin fragment, such as Fab, Fab’, F(ab’) , dsFv, sFv, minibodies, diabodies,
tribodies, tetrabodies (Parham, J. Immunol., 131: 2895-2902 (1983); Spring et al. J.
Immunol., 113: 470-478 (1974); Nisonoff et al. Arch. Biochem. Biophys., 89: 230-244 (1960),
Kim et al., Mol. Cancer Ther., 7: 2486-2497 (2008), Carter, Nature Revs., 6: 343-357
(2006)), or immunoglobulin chimera, which can bind to an antigen on the surface of a cell
(e.g., which contains a complementarity determining region (CDR)). Any suitable antibody
can be used as the cell-binding agent. One of ordinary skill in the art will appreciate that the
selection of an appropriate antibody will depend upon the cell population to be targeted. In
this regard, the type and number of cell surface molecules (i.e., antigens) that are selectively
expressed in a particular cell population (typically and preferably a diseased cell population)
will govern the selection of an appropriate antibody for use in the inventive composition.
Cell surface expression profiles are known for a wide variety of cell types, including tumor
cell types, or, if unknown, can be determined using routine molecular biology and
histochemistry techniques.
The antibody can be polyclonal or monoclonal, but is most preferably a
monoclonal antibody. As used herein, “polyclonal” antibodies refer to heterogeneous
populations of antibody molecules, typically contained in the sera of immunized animals.
“Monoclonal” antibodies refer to homogenous populations of antibody molecules that are
specific to a particular antigen. Monoclonal antibodies are typically produced by a single
clone of B lymphocytes (“B cells”). Monoclonal antibodies may be obtained using a variety
of techniques known to those skilled in the art, including standard hybridoma technology
(see, e.g., Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976), Harlow and Lane (eds.),
Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.),
Immunobiology, 5 Ed., Garland Publishing, New York, NY (2001)). In brief, the hybridoma
method of producing monoclonal antibodies typically involves injecting any suitable animal,
typically and preferably a mouse, with an antigen (i.e., an “immunogen”). The animal is
subsequently sacrificed, and B cells isolated from its spleen are fused with human myeloma
cells. A hybrid cell is produced (i.e., a “hybridoma”), which proliferates indefinitely and
continuously secretes high titers of an antibody with the desired specificity in vitro. Any
appropriate method known in the art can be used to identify hybridoma cells that produce an
antibody with the desired specificity. Such methods include, for example, enzyme-linked
immunosorbent assay (ELISA), Western blot analysis, and radioimmunoassay. The
population of hybridomas is screened to isolate individual clones, each of which secretes a
single antibody species to the antigen. Because each hybridoma is a clone derived from
fusion with a single B cell, all the antibody molecules it produces are identical in structure,
including their antigen binding site and isotype. Monoclonal antibodies also may be
generated using other suitable techniques including EBV-hybridoma technology (see, e.g.,
Haskard and Archer, J. Immunol. Methods, 74(2): 361-67 (1984), and Roder et al., Methods
Enzymol., 121: 140-67 (1986)), bacteriophage vector expression systems (see, e.g., Huse et
al., Science, 246: 1275-81 (1989)), or phage display libraries comprising antibody fragments,
such as Fab and scFv (single chain variable region) (see, e.g., U.S. Patents 5,885,793 and
,969,108, and International Patent Application Publications WO 92/01047 and WO
99/06587).
The monoclonal antibody can be isolated from or produced in any suitable animal,
but is preferably produced in a mammal, more preferably a mouse or human, and most
preferably a human. Methods for producing an antibody in mice are well known to those
skilled in the art and are described herein. With respect to human antibodies, one of ordinary
skill in the art will appreciate that polyclonal antibodies can be isolated from the sera of
human subjects vaccinated or immunized with an appropriate antigen. Alternatively, human
antibodies can be generated by adapting known techniques for producing human antibodies in
non-human animals such as mice (see, e.g., U.S. Patents 5,545,806, 5,569,825, and
,714,352, and U.S. Patent Application Publication No. 2002/0197266 A1).
While being the ideal choice for therapeutic applications in humans, human
antibodies, particularly human monoclonal antibodies, typically are more difficult to generate
than mouse monoclonal antibodies. Mouse monoclonal antibodies, however, induce a rapid
host antibody response when administered to humans, which can reduce the therapeutic or
diagnostic potential of the antibody-cytotoxic agent conjugate. To circumvent these
complications, a monoclonal antibody preferably is not recognized as “foreign” by the human
immune system.
To this end, phage display can be used to generate the antibody. In this regard,
phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated
using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et
al. (eds.), Molecular Cloning, A Laboratory Manual, 3 Edition, Cold Spring Harbor
Laboratory Press, New York (2001)). Phage encoding a variable region with the desired
specificity are selected for specific binding to the desired antigen, and a complete human
antibody is reconstituted comprising the selected variable domain. Nucleic acid sequences
encoding the reconstituted antibody are introduced into a suitable cell line, such as a
myeloma cell used for hybridoma production, such that human antibodies having the
characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al.,
supra, Huse et al., supra, and U.S. Patent 6,265,150). Alternatively, monoclonal antibodies
can be generated from mice that are transgenic for specific human heavy and light chain
immunoglobulin genes. Such methods are known in the art and described in, for example,
U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., supra.
Most preferably the antibody is a humanized antibody. As used herein, a
“humanized” antibody is one in which the complementarity-determining regions (CDR) of a
mouse monoclonal antibody, which form the antigen binding loops of the antibody, are
grafted onto the framework of a human antibody molecule. Owing to the similarity of the
frameworks of mouse and human antibodies, it is generally accepted in the art that this
approach produces a monoclonal antibody that is antigenically identical to a human antibody
but binds the same antigen as the mouse monoclonal antibody from which the CDR
sequences were derived. Methods for generating humanized antibodies are well known in the
art and are described in detail in, for example, Janeway et al., supra, U.S. Patents 5,225,539,
,585,089 and 5,693,761, European Patent No. 0239400 B1, and United Kingdom Patent No.
2188638. Humanized antibodies can also be generated using the antibody resurfacing
technology described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235: 959-
973 (1994). While the antibody employed in the conjugate of the inventive composition most
preferably is a humanized monoclonal antibody, a human monoclonal antibody and a mouse
monoclonal antibody, as described above, are also contemplated herein.
Antibody fragments that have at least one antigen binding site, and thus recognize
and bind to at least one antigen or receptor present on the surface of a target cell, also are
contemplated herein. In this respect, proteolytic cleavage of an intact antibody molecule can
produce a variety of antibody fragments that retain the ability to recognize and bind antigens.
For example, limited digestion of an antibody molecule with the protease papain typically
produces three fragments, two of which are identical and are referred to as the Fab fragments,
as they retain the antigen binding activity of the parent antibody molecule. Cleavage of an
antibody molecule with the enzyme pepsin normally produces two antibody fragments, one
of which retains both antigen-binding arms of the antibody molecule, and is thus referred to
as the F(ab’) fragment. Reduction of a F(ab’) fragment with dithiothreitol or
mercaptoethylamine produces a fragment referred to as a Fab’ fragment. A single-chain
variable region fragment (sFv) antibody fragment, which consists of a truncated Fab fragment
comprising the variable (V) domain of an antibody heavy chain linked to a V domain of a
light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA
technology techniques (see, e.g., Janeway et al., supra). Similarly, disulfide-stabilized
variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g.,
Reiter et al., Protein Engineering, 7: 697-704 (1994)). Antibody fragments as described
herein, however, are not limited to these exemplary types of antibody fragments. Any
suitable antibody fragment that recognizes and binds to a desired cell surface receptor or
antigen can be employed. Antibody fragments are further described in, for example, Parham,
J. Immunol., 131: 2895-2902 (1983), Spring et al., J. Immunol., 113: 470-478 (1974), and
Nisonoff et al., Arch. Biochem. Biophys., 89: 230-244 (1960). Antibody-antigen binding can
be assayed using any suitable method known in the art, such as, for example,
radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive
inhibition assays (see, e.g., Janeway et al., supra, and U.S. Patent Application Publication
No. 2002/0197266 A1).
In addition, the antibody can be a chimeric antibody or an antigen binding
fragment thereof. By “chimeric” it is meant that the antibody comprises at least two
immunoglobulins, or fragments thereof, obtained or derived from at least two different
species (e.g., two different immunoglobulins, such as a human immunoglobulin constant
region combined with a murine immunoglobulin variable region). The antibody also can be a
domain antibody (dAb) or an antigen binding fragment thereof, such as, for example, a
camelid antibody (see, e.g., Desmyter et al., Nature Struct. Biol., 3: 752, (1996)), or a shark
antibody, such as, for example, a new antigen receptor (IgNAR) (see, e.g., Greenberg et al.,
Nature, 374: 168 (1995), and Stanfield et al., Science, 305: 1770-1773 (2004)).
Any suitable antibody can be used in the context of the invention. For example,
the monoclonal antibody J5 is a murine IgG2a antibody that is specific for Common Acute
Lymphoblastic Leukemia Antigen (CALLA) (Ritz et al., Nature, 283: 583-585 (1980)), and
can be used to target cells that express CALLA (e.g., acute lymphoblastic leukemia cells).
The monoclonal antibody MY9 is a murine IgG1 antibody that binds specifically to the CD33
antigen (Griffin et al., Leukemia Res., 8: 521 (1984)), and can be used to target cells that
express CD33 (e.g., acute myelogenous leukemia (AML) cells).
Similarly, the monoclonal antibody anti-B4 (also referred to as B4) is a murine
IgG1 antibody that binds to the CD19 antigen on B cells (Nadler et al., J. Immunol., 131:
244-250 (1983)), and can be used to target B cells or diseased cells that express CD19 (e.g.,
non-Hodgkin’s lymphoma cells and chronic lymphoblastic leukemia cells). N901 is a murine
monoclonal antibody that binds to the CD56 (neural cell adhesion molecule) antigen found
on cells of neuroendocrine origin, including small cell lung tumor, which can be used in the
conjugate to target drugs to cells of neuroendocrine origin. The J5, MY9, and B4 antibodies
preferably are resurfaced or humanized prior to their use as part of the conjugate.
Resurfacing or humanization of antibodies is described in, for example, Roguska et al., Proc.
Natl. Acad. Sci. USA, 91: 969-73 (1994).
In addition, the monoclonal antibody C242 binds to the CanAg antigen (see, e.g.,
U.S. Patent 5,552,293), and can be used to target the conjugate to CanAg expressing tumors,
such as colorectal, pancreatic, non-small cell lung, and gastric cancers. HuC242 is a
humanized form of the monoclonal antibody C242 (see, e.g., U.S. Patent 5,552,293). The
hybridoma from which HuC242 is produced is deposited with ECACC identification Number
90012601. HuC242 can be prepared using CDR-grafting methodology (see, e.g., U.S.
Patents 5,585,089, 5,693,761, and 5,693,762) or resurfacing technology (see, e.g., U.S. Patent
,639,641). HuC242 can be used to target the conjugate to tumor cells expressing the CanAg
antigen, such as, for example, colorectal, pancreatic, non-small cell lung, and gastric cancer
cells.
To target ovarian cancer and prostate cancer cells, an anti-MUC1 antibody can be
used as the cell-binding agent in the conjugate. Anti-MUC1 antibodies include, for example,
anti-HMFG-2 (see, e.g., Taylor-Papadimitriou et al., Int. J. Cancer, 28: 17-21 (1981)),
hCTM01 (see, e.g., van Hof et al., Cancer Res., 56: 5179-5185 (1996)), and DS6. Prostate
cancer cells also can be targeted with the conjugate by using an anti-prostate-specific
membrane antigen (PSMA) as the cell-binding agent, such as J591 (see, e.g., Liu et al.,
Cancer Res., 57: 3629-3634 (1997)). Moreover, cancer cells that express the Her2 antigen,
such as breast, prostate, and ovarian cancers, can be targeted with the conjugate by using anti-
Her2 antibodies, e.g., trastuzumab, as the cell-binding agent. Cells that express epidermal
growth factor receptor (EGFR) and variants thereof, such as the type III deletion mutant,
EGFRvIII, can be targeted with the conjugate by using anti-EGFR antibodies. Anti-EGFR
antibodies are described in International Patent Application Nos. PCT/US11/058385 and
PCT/US11/058378. Anti-EGFRvIII antibodies are described in U.S. Patents 7,736,644 and
7,628,986 and U.S. Application Publications 2010/0111979, 2009/0240038, 2009/0175887,
2009/0156790, and 2009/0155282. Anti-IGF-IR antibodies that bind to insulin-like growth
factor receptor, such as those described in U.S. Patent 7,982,024, also can be used in the
conjugate. Antibodies that bind to CD27L, Cripto, CD138, CD38, EphA2, integrins, CD37,
folate, CD20, PSGR, NGEP, PSCA, TMEFF2, STEAP1, endoglin, and Her3 also can be used
in the conjugate.
In one embodiment, the antibody is selected from the group consisting of huN901,
huMy9-6, huB4, huC242, an anti-HER2 antibody (e.g., trastuzumab), bivatuzumab,
sibrotuzumab, rituximab, huDS6, anti-mesothelin antibodies described in International Patent
Application Publication (such as MF-T), anti-cripto antibodies described in
U.S. Patent Application Publication 2010/0093980 (such as huB3F6), anti-CD138 antibodies
described in U.S. Patent Application Publication 2007/0183971 (such as huB-B4), anti-EGFR
antibodies described in International Patent Application Nos. PCT/US11/058385 and
PCT/US11/058378 (such as EGFR-7), anti-EGFRvIII antibodies described U.S. Patents
7,736,644 and 7,628,986 and U.S. Patent Application Publications 2010/0111979,
2009/0240038, 2009/0175887, 2009/0156790 and 2009/0155282, humanized EphA2
antibodies described in International Patent Application Publications and
(such as 2H11R35R74); anti-CD38 antibodies described in International
Patent Application Publication (such as hu38SB19), anti-folate antibodies
described in International Patent Application Publication , and U.S. Patent
Application Publication 2012/0009181 (e.g., huMov19); anti-IGF1R antibodies described in
U.S. Patents 5,958,872, 6,596,743, and 7,982,024; anti-CD37 antibodies described in U.S.
Patent Application Publication 2011/0256153(e.g., huCD37-3); anti-integrin α β antibodies
described in U.S. Application Publication 2006/0127407 (e.g., CNTO95); and anti-Her3
antibodies described in International Patent Application Publication .
Particularly preferred antibodies are humanized monoclonal antibodies described
herein. Examples include, but are not limited to, huN901, huMy9-6, huB4, huC242, a
humanized monoclonal anti-Her2 antibody (e.g., trastuzumab), bivatuzumab, sibrotuzumab,
CNTO95, huDS6, and rituximab (see, e.g., U.S. Patents 5,639,641 and 5,665,357, U.S.
Provisional Patent Application No. 60/424,332 (which is related to U.S. Patent 7,557,189),
International (PCT) Patent Application Publication WO 02/16401, Pedersen et al., supra,
Roguska et al., supra, Liu et al., supra, Nadler et al., supra, Colomer et al., Cancer Invest.,
19: 49-56 (2001), Heider et al., Eur. J. Cancer, 31A: 2385-2391 (1995), Welt et al., J. Clin.
Oncol., 12: 1193-1203 (1994), and Maloney et al., Blood, 90: 2188-2195 (1997)). Other
humanized monoclonal antibodies are known in the art and can be used in connection with
the invention.
In one embodiment, the cell-binding agent is an humanized anti-folate antibody or
antigen binding fragment thereof that specifically binds a human folate receptor 1, wherein
the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO: 1); a
heavy chain CDR2 comprising RIHPYDGDTFYNQXaa FXaa Xaa (SEQ ID NO: 2) ; and a
1 2 3
heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 3); and (b) a light chain CDR1
comprising KASQSVSFAGTSLMH (SEQ ID NO: 4); a light chain CDR2 comprising
RASNLEA (SEQ ID NO: 5); and a light chain CDR3 comprising QQSREYPYT (SEQ ID
NO: 6); wherein Xaa is selected from K, Q, H, and R; Xaa is selected from Q, H, N, and R;
and Xaa is selected from G, E, T, S, A, and V. Preferably, the heavy chain CDR2 sequence
comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 7).
In another embodiment, the anti-folate antibody is a humanized antibody or
antigen binding fragment thereof that specifically binds the human folate receptor 1
comprising the heavy chain having the amino acid sequence of
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDG
DTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQG
TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ
ID NO: 8).
In another embodiment, the anti-folate antibody is a humanized antibody or
antigen binding fragment thereof encoded by the plasmid DNA deposited with the ATCC on
April 7, 2010 and having ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.
In another embodiment, the anti-folate antibody is a humanized antibody or
antigen binding fragment thereof comprising a heavy chain variable domain at least about
90%, 95%, 99% or 100% identical to
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDG
DTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQG
TTVTVSS (SEQ ID NO: 9), and a light chain variable domain at least about 90%, 95%, 99%
or 100% identical to
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR (SEQ
ID NO: 10); or
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNL
EAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR (SEQ
ID NO: 11).
While the cell-binding agent preferably is an antibody, the cell-binding agent also
can be a non-antibody molecule. Suitable non-antibody molecules include, for example,
interferons (e.g., alpha, beta, or gamma interferon), lymphokines (e.g., interleukin 2 (IL-2),
IL-3, IL-4, or IL-6), hormones (e.g., insulin), growth factors (e.g., EGF, TGF-alpha, FGF,
and VEGF), colony-stimulating factors (e.g., G-CSF, M-CSF, and GM-CSF (see, e.g.,
Burgess, Immunology Today, 5: 155-158 (1984)), somatostatin, and transferrin (see, e.g.,
O'Keefe et al., J. Biol. Chem., 260: 932-937 (1985)). For example, GM-CSF, which binds to
myeloid cells, can be used as a cell-binding agent to target acute myelogenous leukemia cells.
In addition, IL-2, which binds to activated T-cells, can be used for prevention of transplant
graft rejection, for therapy and prevention of graft-versus-host disease, and for treatment of
acute T-cell leukemia. Epidermal growth factor (EGF) can be used to target squamous
cancers such as lung cancer and head and neck cancer. Somatostatin can be used to target
neuroblastoma cells and other tumor cell types.
The conjugate can comprise any suitable cytotoxic agents. A “cytotoxic agent,”
as used herein, refers to any compound that results in the death of a cell, induces cell death,
or decreases cell viability. Suitable cytotoxic agents include, for example, maytansinoids and
conjugatable ansamitocins (see, for example, International Patent Application No.
PCT/US11/59131, filed November 3, 2011), taxoids, CC-1065 and CC-1065 analogs, and
dolastatin and dolastatin analogs. In a preferred embodiment, the cytotoxic agent is a
maytansinoid, including maytansinol and maytansinol analogs. Maytansinoids are
compounds that inhibit microtubule formation and are highly toxic to mammalian cells.
Examples of suitable maytansinol analogues include those having a modified aromatic ring
and those having modifications at other positions. Such maytansinoids are described in, for
example, U.S. Patents 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,322,348,
4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,424,219, 4,371,533, 4,450,254, 5,475,092,
,585,499, 5,846,545, and 6,333,410.
Examples of maytansinol analogs having a modified aromatic ring include:
(1) Cdechloro (U.S. Patent 4,256,746) (prepared by LAH reduction of ansamytocin P2),
(2) Chydroxy (or Cdemethyl) +/-Cdechloro (U.S. Patents 4,361,650 and
4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination
using LAH), and (3) Cdemethoxy, Cacyloxy (-OCOR), +/-dechloro (U.S. Patent
4,294,757) (prepared by acylation using acyl chlorides).
Examples of maytansinol analogs having modifications of positions other than an
aromatic ring include: (1) CSH (U.S. Patent 4,424,219) (prepared by the reaction of
maytansinol with H S or P S ), (2) Calkoxymethyl (demethoxy/CH OR) (U.S. Patent
2 2 5 2
4,331,598), (3) Chydroxymethyl or acyloxymethyl (CH OH or CH OAc) (U.S. Patent
4,450,254) (prepared from Nocardia), (4) Chydroxy/acyloxy (U.S. Patent 4,364,866)
(prepared by the conversion of maytansinol by Streptomyces), (5) Cmethoxy (U.S.
Patents 4,313,946 and 4,315,929) (isolated from Trewia nudiflora), (6) CN-demethyl
(U.S. Patents 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by
Streptomyces), and (7) 4,5-deoxy (U.S. Patent 4,371,533) (prepared by the titanium
trichloride/LAH reduction of maytansinol).
In a preferred embodiment, the conjugate utilizes the thiol-containing
2’ 2’
maytansinoid DM1, also known as N -deacetyl-N -(3-mercaptooxopropyl)-maytansine,
as the cytotoxic agent. The structure of DM1 is represented by formula (I):
N SH
NH O
In another preferred embodiment, the conjugate utilizes the thiol-containing
2’ 2’
maytansinoid DM4, also known as N -deacetyl-N -(4-methylmercaptooxopentyl)-
maytansine, as the cytotoxic agent. The structure of DM4 is represented by formula (II):
N SH
NH O
(II)
Other maytansinoids may be used in the context of the invention, including, for
example, thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution
on the carbon atom bearing the sulfur atom. Particularly preferred is a maytansinoid having
at the C-3 position (a) C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl functionality,
and (b) an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl
group, wherein the carbon atom of the acyl group bearing the thiol functionality has one or
two substituents, said substituents being CH , C H , linear or branched alkyl or alkenyl
3 2 5
having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and further
wherein one of the substituents can be H, and wherein the acyl group has a linear chain length
of at least three carbon atoms between the carbonyl functionality and the sulfur atom.
Additional maytansinoids for use in the context of the invention include
compounds represented by formula (III):
NH O
(III),
wherein Y’ represents
(CR R ) (CR =CR ) (C ≡C) A (CR R ) D (CR =CR ) (C ≡C) B (CR R ) CR R SZ,
7 8 l 9 10 p q o 5 6 m u 11 12 r s t 3 4 n 1 2
wherein R and R are each independently CH , C H , linear alkyl or alkenyl having from 1
1 2 3 2 5
to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and wherein
R also can be H,
wherein A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or
substituted aryl, or heterocyclic aromatic, or heterocycloalkyl radical,
wherein R , R , R , R , R , R , R , R , R , and R are each independently H, CH , C H ,
3 4 5 6 7 8 9 10 11 12 3 2 5
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic, or
heterocycloalkyl radical,
wherein l, m, n, o, p, q, r, s, and t are each independently zero or an integer from 1 to 5,
provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any one time, and
wherein Z is H, SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic, or heterocycloalkyl radical.
Preferred embodiments of formula (III) include compounds of formula (III)
wherein (a) R is H, R is methyl and Z is H, (b) R and R are methyl and Z is H, (c) R is H,
1 2 1 2 1
R is methyl, and Z is –SCH , and (d) R and R are methyl, and Z is –SCH .
2 3 1 2 3
Such additional maytansinoids also include compounds represented by formula
(IV-L), (IV-D), or (IV-D,L):
H C 3
(IV-L) (IV-D) (IV-D,L)
wherein Y represents (CR R ) (CR R ) (CR R ) CR R SZ,
7 8 l 5 6 m 3 4 n 1 2
wherein R and R are each independently CH , C H , linear alkyl, or alkenyl having from 1
1 2 3 2 5
to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and wherein
R also can be H,
wherein R , R , R , R , R , and R are each independently H, CH , C H , linear alkyl or
3 4 5 6 7 8 3 2 5
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl
radical,
wherein l, m, and n are each independently an integer of from 1 to 5, and in addition n can be
zero,
wherein Z is H, SR, or COR wherein R is linear or branched alkyl or alkenyl having from 1
to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical, and
wherein May represents a maytansinoid which bears the side chain at C-3, C-14
hydroxymethyl, C-15 hydroxy, or C-20 desmethyl.
Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) include
compounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein (a) R is H, R is methyl, R ,
1 2 5
R , R , and R are each H, l and m are each 1, n is 0, and Z is H, (b) R and R are methyl,
6 7 8 1 2
R , R , R , R are each H, l and m are 1, n is 0, and Z is H, (c) R is H, R is methyl, R , R ,
6 7 8 1 2 5 6
R , and R are each H, l and m are each 1, n is 0, and Z is –SCH , or (d) R and R are
7 8 3 1 2
methyl, R , R , R , R are each H, l and m are 1, n is 0, and Z is –SCH .
6 7 8 3
Preferably the cytotoxic agent is represented by formula (IV-L).
Additional preferred maytansinoids also include compounds represented by
formula (V):
NH O
wherein Y represents (CR R ) (CR R ) (CR R ) CR R SZ,
7 8 l 5 6 m 3 4 n 1 2
wherein R1 and R2 are each independently CH3, C2H5, linear alkyl, or alkenyl having from 1
to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and wherein
R also can be H,
wherein R , R , R , R , R , and R are each independently H, CH , C H , linear alkyl or
3 4 5 6 7 8 3 2 5
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl
radical,
wherein l, m, and n are each independently an integer of from 1 to 5, and in addition n can be
zero, and
wherein Z is H, SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.
Preferred embodiments of formula (V) include compounds of formula (V)
wherein (a) R is H, R is methyl, R , R , R , and R are each H; l and m are each 1; n is 0;
1 2 5 6 7 8
and Z is H, (b) R and R are methyl; R , R , R , R are each H, l and m are 1; n is 0; and Z is
1 2 5 6 7 8
H, (c) R is H, R is methyl, R , R , R , and R are each H, l and m are each 1, n is 0, and Z is
1 2 5 6 7 8
–SCH , or (d) R and R are methyl, R , R , R , R are each H, l and m are 1, n is 0, and Z is –
3 1 2 5 6 7 8
SCH .
Still further preferred maytansinoids include compounds represented by formula
(VI-L), (VI-D), or (VI-D,L):
H H C
H C 3 3
NY 2
2 May
(VI-L) (VI-D) (VI-D, L),
wherein Y2 represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ2,
wherein R and R are each independently CH , C H , linear alkyl or alkenyl having from 1
1 2 3 2 5
to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and wherein
R also can be H,
wherein R , R , R , R , R , and R are each independently H, CH , C H , linear cyclic alkyl
3 4 5 6 7 8 3 2 5
or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from
3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl
radical,
wherein l, m, and n are each independently an integer of from 1 to 5, and in addition n can be
zero,
wherein Z is SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical, and
wherein May is the macrocyclic ring structure of the maytansinoid.
Additional preferred maytansinoids include compounds represented by formula
(VII):
NY '
NH O
(VII),
wherein Y represents
(CR R ) (CR =CR ) (C ≡C) A (CR R ) D (CR =CR ) (C ≡C) B (CR R ) CR R SZ ,
7 8 l 9 10 p q o 5 6 m u 11 12 r s t 3 4 n 1 2 2
wherein R and R are each independently CH , C H , linear branched or alkyl or alkenyl
1 2 3 2 5
having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,
phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in
addition R can be H,
wherein A, B, and D each independently is cycloalkyl or cycloalkenyl having 3 to 10 carbon
atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical,
wherein R3, R4, R5, R6, R7, R8, R9, R10, R11, and R12 are each independently H, CH3, C2H5,
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl radical,
wherein l, m, n, o, p, q, r, s, and t are each independently zero or an integer of from 1 to 5,
provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any one time, and
wherein Z is SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.
Preferred embodiments of formula (VII) include compounds of formula (VII),
wherein R is H and R is methyl.
In addition to maytansinoids, the cytotoxic agent used in the conjugate can be a
taxane or derivative thereof. Taxanes are a family of compounds that includes paclitaxel
(Taxol®), a cytotoxic natural product, and docetaxel (Taxotere®), a semi-synthetic
derivative, which are both widely used in the treatment of cancer. Taxanes are mitotic
spindle poisons that inhibit the depolymerization of tubulin, resulting in cell death. While
docetaxel and paclitaxel are useful agents in the treatment of cancer, their antitumor activity
is limited because of their non-specific toxicity towards normal cells. Further, compounds
like paclitaxel and docetaxel themselves are not sufficiently potent to be used in conjugates of
cell-binding agents.
A preferred taxane for use in the preparation of a cytotoxic conjugate is the taxane
of formula (VIII):
NH O
MeO OMe
(VIII)
Methods for synthesizing taxanes that can be used in the context of the invention,
along with methods for conjugating taxanes to cell-binding agents such as antibodies, are
described in detail in U.S. Patents 5,416,064, 5,475,092, 6,340,701, 6,372,738, 6,436,931,
6,596,757, 6,706,708, 6,716,821, and 7,390,898.
The cytotoxic also can be CC-1065 or a derivative thereof. CC-1065 is a potent
anti-tumor antibiotic isolated from the culture broth of Streptomyces zelensis. CC-1065 is
about 1000-fold more potent in vitro than commonly used anti-cancer drugs, such as
doxorubicin, methotrexate, and vincristine (Bhuyan et al., Cancer Res., 42: 3532-3537
(1982)). CC-1065 and its analogs are disclosed in U.S. Patents 5,585,499, 5,846,545,
6,340,701, and 6,372,738. The cytotoxic potency of CC-1065 has been correlated with its
alkylating activity and its DNA-binding or DNA-intercalating activity. These two activities
reside in separate parts of the molecule. In this respect, the alkylating activity is contained in
the cyclopropapyrroloindole (CPI) subunit and the DNA-binding activity resides in the two
pyrroloindole subunits of CC-1065.
Several CC-1065 analogs are known in the art and also can be used as the
cytotoxic agent in the conjugate (see, e.g., Warpehoski et al., J. Med. Chem., 31: 590-603
(1988)). A series of CC-1065 analogs has been developed in which the CPI moiety is
replaced by a cyclopropabenzindole (CBI) moiety (Boger et al., J. Org. Chem., 55: 5823-
5833 (1990), and Boger et al., Bioorg. Med. Chem. Lett., 1: 115-120 (1991)). These CC-1065
analogs maintain the high in vitro potency of the parental drug, without causing delayed
toxicity in mice. Like CC-1065, these compounds are alkylating agents that covalently bind
to the minor groove of DNA to cause cell death.
The therapeutic efficacy of CC-1065 analogs can be greatly improved by
changing the in vivo distribution through targeted delivery to a tumor site, resulting in lower
toxicity to non-targeted tissues, and thus, lower systemic toxicity. To this end, conjugates of
analogs and derivatives of CC-1065 with cell-binding agents that specifically target tumor
cells have been generated (see, e.g., U.S. Patents 5,475,092, 5,585,499, and 5,846,545).
These conjugates typically display high target-specific cytotoxicity in vitro, and anti-tumor
activity in human tumor xenograft models in mice (see, e.g., Chari et al., Cancer Res., 55:
4079-4084 (1995)).
Methods for synthesizing CC-1065 analogs are described in detail in U.S. Patents
,475,092, 5,585,499, 5,846,545, 6,534,660, 6,586,618, 6,756,397, and 7,329,760.
Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine,
melphalan, mitomycin C, chlorambucil, calicheamicin, tubulysin and tubulysin analogs,
duocarmycin and duocarmycin analogs, dolastatin and dolastatin analogs also can be used as
the cytotoxic agent as described herein. Doxarubicin and daunorubicin compounds (see, e.g.,
U.S. Patent 6,630,579) can also be used as the cytotoxic agent.
The cell-binding agent cytotoxic agent conjugates may be prepared by in vitro
methods. In order to link a cytotoxic agent to the antibody, a linking group is used. Suitable
linking groups are well known in the art and include disulfide groups, acid labile groups,
photolabile groups, peptidase labile groups, and esterase labile groups, as well as
noncleavable linking groups. For example, the cell binding agent can be chemically coupled
to the cytotoxic agent via chemical bonds selected from the group consisting of disulfide
bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, thioether bonds, and
esterase labile bonds.
As described herein, the cell-binding agent is linked with the cytotoxic agent via a
bifunctional crosslinking reagent. As used herein, a “bifunctional crosslinking reagent”
refers to a reagent that possesses two reactive groups; one of which is capable of reacting
with a cell-binding agent, while the other one is capable of reacting with the cytotoxic agent
to link the cell-binding agent with the cytotoxic agent, thereby forming a conjugate.
Any suitable bifunctional crosslinking reagent can be used as described herein, so
long as the linker reagent provides for retention of the therapeutic, e.g., cytotoxicity, and
targeting characteristics of the cytotoxic agent and the cell-binding agent, respectively,
without undue toxicity. Preferably, the linker molecule joins the cytotoxic agent to the cell-
binding agent through chemical bonds (as described above), such that the cytotoxic agent and
the cell-binding agent are chemically coupled (e.g., covalently bonded) to each other.
In one embodiment, the bifunctional crosslinking reagent comprises non-cleavable
linkers. A non-cleavable linker is any chemical moiety that is capable of linking a cytotoxic
agent, such as a maytansinoid, a taxane, or a CC-1065 analog, to a cell-binding agent in a
stable, covalent manner. Thus, non-cleavable linkers are substantially resistant to acid-
induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced
cleavage, and disulfide bond cleavage, at conditions under which the cytotoxic agent or the
cell-binding agent remains active.
Suitable crosslinking reagents that form non-cleavable linkers between a cytotoxic
agent and the cell-binding agent are well known in the art. In one embodiment, the cytotoxic
agent is linked to the cell-binding agent through a thioether bond. Examples of non-cleavable
linkers include linkers having a maleimido- or haloacetyl-based moiety for reaction with the
cytotoxic agent. Such bifunctional crosslinking agents are well known in the art (see U.S.
Patent Application Publication Nos. 2010/0129314, 2009/0274713, 2008/0050310,
20050169933, 2009/0274713, 2010/0129314, and those available from Pierce Biotechnology
Inc. P.O. Box 117, Rockland, IL 61105, USA) and include, but not limited to, N-succinimidyl
4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl(N-
maleimidomethyl)-cyclohexanecarboxy-(6-amidocaproate), which is a “long chain”
analog of SMCC (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA),
γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-
hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl(β-
maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-
butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI). Cross-linking reagents
comprising a haloacetyl-based moiety include N-succinimidyl(iodoacetyl)-aminobenzoate
(SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-
succinimidyl 3-(bromoacetamido)propionate (SBAP), bis-maleimidopolyethyleneglycol
(BMPEO), BM(PEO) , BM(PEO) , N-(β-maleimidopropyloxy)succinimide ester (BMPS), 5-
maleimidovaleric acid NHS, HBVS, 4-(4-N-maleimidophenyl)-butyric acid hydrazide•HCl
(MPBH), Succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane
(DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane
(BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-
(N-maleimido-methyl)cyclohexanecarboxylate (sulfo-SMCC), sulfosuccinimidyl(4-iodo-
acetyl)aminobenzoate (sulfo-SIAB), m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester
(sulfo-MBS), N-(γ-maleimidobutryloxy)sulfosuccinimde ester (sulfo-GMBS), N-(ε-
maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS), N-(κ-
maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), sulfosuccinimidyl 4-(p-
maleimidophenyl)butyrate (sulfo-SMPB), CX1-1, sulfo-Mal and PEG -Mal. Preferably, the
bifunctional crosslinking reagent is SMCC.
(CX1-1);
(sulfo-Mal);
n= 2to20 (e.g., 2,4, 6,8)
(PEG -Mal)
In one embodiment, the linking reagent is a cleavable linker. Examples of
suitable cleavable linkers include disulfide linkers, acid labile linkers, photolabile linkers,
peptidase labile linkers, and esterase labile linkers. Disulfide containing linkers are linkers
cleavable through disulfide exchange, which can occur under physiological conditions. Acid
labile linkers are linkers cleavable at acid pH. For example, certain intracellular
compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide
conditions suitable to cleave acid labile linkers. Photo labile linkers are useful at the body
surface and in many body cavities that are accessible to light. Furthermore, infrared light can
penetrate tissue. Peptidase labile linkers can be used to cleave certain peptides inside or
outside cells (see e.g., Trouet et al., Proc. Natl. Acad. Sci. USA, 79: 626-629 (1982), and
Umemoto et al., Int. J. Cancer, 43: 677-684 (1989)). In one embodiment, the cleavable
linker is cleaved under mild conditions, i.e., conditions within a cell under which the activity
of the cytotoxic agent is not affected.
In another embodiment, the cytotoxic agent is linked to a cell-binding agent
through a disulfide bond. The linker molecule comprises a reactive chemical group that can
react with the cell-binding agent. Preferred reactive chemical groups for reaction with the
cell-binding agent are N-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally the
linker molecule comprises a reactive chemical group, preferably a dithiopyridyl group, that
can react with the cytotoxic agent to form a disulfide bond. Bifunctional crosslinking
reagents that enable the linkage of the cell-binding agent with the cytotoxic agent via
disulfide bonds are known in the art and include, for example, N-succinimidyl 3-(2-
pyridyldithio)propionate (SPDP) (see, e.g., Carlsson et al., Biochem. J., 173: 723-737
(1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Patent
4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see, e.g., CAS Registry
number 3414986), and N-succinimidyl(2-pyridyldithio)2-sulfo butanoate (sulfo-
SPDB) (see, e.g., U.S. Application Publication 2009/0274713). Other bifunctional
crosslinking reagents that can be used to introduce disulfide groups are known in the art and
are described in U.S. Patent 6,913,748, 6,716,821 and U.S. Patent Application Publications
2009/0274713 and 2010/0129314, all of which are incorporated herein in its entirety by
reference.
Other crosslinking reagents lacking a sulfur atom that form non-cleavable linkers
can also be used in the inventive method. Such linkers can be derived from dicarboxylic acid
based moieties. Suitable dicarboxylic acid based moieties include, but are not limited to, α, ω-
dicarboxylic acids of the general formula (IX):
HOOC-X -Y -Z -COOH
l n m
(IX),
wherein X is a linear or branched alkyl, alkenyl, or alkynyl group having 2 to 20 carbon
atoms, Y is a cycloalkyl or cycloalkenyl group bearing 3 to 10 carbon atoms, Z is a
substituted or unsubstituted aromatic group bearing 6 to 10 carbon atoms, or a substituted or
unsubstituted heterocyclic group wherein the hetero atom is selected from N, O or S, and
wherein l, m, and n are each 0 or 1, provided that l, m, and n are all not zero at the same time.
Many of the non-cleavable linkers disclosed herein are described in detail in U.S.
Patent Application Publication No. 2005/0169933 A1.
The following examples further illustrate the invention but, of course, should not
be construed as in any way limiting its scope.
Example 1
Humanized CD37-3 antibody was reacted with the heterobifunctional crosslinking
reagent SMCC and the maytansinoid DM1 using a previously described process (e.g. U.S.
Patent 5,208,020), as well as the one-step process that is the subject of the present
application.
For the previously described process, huCD37-3 (15 mg/mL) first was reacted
with SMCC (6.5-fold molar excess relative to the amount of antibody) to form the modified
antibody. The modification reaction was performed at 16º C in 50 mM sodium phosphate
buffer (pH 6.9) containing 2 mM EDTA and 10% DMA for 90 minutes. The reaction was
quenched with 1 M acetate to adjust the pH to 4.5 and the modified antibody was purified
using a column of Sephadex G-25F resin equilibrated and eluted in 20 mM sodium acetate
(pH 4.5) containing 2 mM EDTA. After purification, the modified antibody (5 mg/mL) was
reacted with the maytansinoid DM1 (6.8-fold molar excess relative to the amount of
antibody; 1.3-fold excess relative to the measured amount of linker on the antibody) to form
the conjugated antibody. The conjugation reaction was performed at 20º C in 20 mM sodium
acetate buffer (pH 5.0) containing 2 mM EDTA and 5% DMA for approximately 20 hours.
The reaction mixture was then purified using a column of Sephadex G-25F resin equilibrated
and eluted in 10 mM sodium succinate (pH 5.0).
For the inventive process, huCD37-3 (2.5 mg/mL) was mixed with DM1 (6.2-fold
molar excess relative to the amount of antibody) and then with SMCC (5.2-fold excess
relative to the amount of antibody). The reaction was performed at 20º C in 50 mM EPPS [4-
(2-Hydroxyethyl)piperazinepropanesulfonic acid] buffer (pH 8.1) containing 2 mM EDTA
and 10% DMA for approximately 4 hours. The reaction was quenched by adding 1 M acetate
to adjust the pH to 5.0. The reaction mixture was then held at 2 – 8ºC for approximately 20
hours. After holding, the reaction mixture was filtered through a 0.2 µm PVDF filter and
purified and diafiltered into 10 mM sodium succinate (pH 5.0) using Tangential Flow
Filtration (TFF).
Conjugate derived from the two processes was analyzed by: UV spectroscopy for
concentration and cytotoxic agent loading (Maytansinoid to Antibody Ratio, MAR); Mass
Spectrometry for determination of unconjugated linker level; reduced SDS PAGE
electrophoresis for determination of level of non-reducible species; SEC-HPLC for
determination of conjugate monomer; and stability on storage with respect to conjugate
monomer and free maytansinoid release.
Concentration and Maytansinoid to Antibody Ratio (MAR) were determined by
measuring the absorbance of the conjugate at 252 and 280 nm in a UV-VIS
spectrophotometer and using the molar extinction coefficients of DM1 and antibody at the
two wavelengths to calculate the molar concentrations of antibody and DM1.
The un-conjugated linker level of the conjugates was analyzed by mass
spectrometry: peak areas of individual conjugate species (including conjugates with or
without un-conjugated linkers) were measured; the un-conjugated linker level was calculated
by the ratio of the sum of areas containing un-conjugated linkers (weighted by the number of
linkers) to the sum of areas of all conjugate species (also weighted by the number of linkers).
The non-reducible species level of the conjugates was analyzed by reduced SDS
gel electrophoresis: peak areas of individual reduced conjugate species (including reduced
light chain, reduced heavy chain, cross-linked light-light chains, cross-linked light-heavy
chains, etc.) were measured; the non-reducible species level was calculated by the ratio of the
sum of areas of non-reducible species to the sum of areas of all species.
The monomer level of the conjugates was analyzed by size exclusion HPLC: peak
areas of monomer, dimer, aggregates and low molecular weight species were measured using
an absorbance detector set to a wavelength of 252 nm or 280 nm; the monomer level was
calculated by the ratio of the monomer area to the total area.
The amount of free maytansinoid present in the conjugate was analyzed by dual
column (HiSep and C18 columns) HPLC: peak areas of total free maytansinoid species
(eluted in the gradient and identified by comparison of elution time with known standards)
were measured using an absorbance detector set to a wavelength of 252 nm; the amount of
free maytansinoid was calculated using a standard curve generated by the peak areas of
known amount of standards.
As shown in Table 1 below, conjugate manufactured using the inventive process
was superior to that manufactured using the previously described process with respect to
unconjugated linker, non-reducible species, and conjugate monomer. In addition, the
stability of conjugate made by the inventive process was significantly superior with respect to
free maytansinoid release after storage for five months at 4° C. Monomer levels of
conjugates made by both processes were stable.
Table 1. Comparison of key properties of CD37-3 conjugate manufactured by the
inventive process compared to previous process
Previous Process Inventive Process
Conjugate concentration 3.9 8.1
Maytansinoid to Antibody 4.1 3.4
ratio
Unconjugated Linker (%) 12 < 1
Nonreducible species (%) 9.4 0.9
Conjugate monomer, (%) 97.8 98.9
(t=0)
Conjugate monomer, (%) 97.8 98.4
(after 5 months at 4°C)
Free Maytansinoid, (%) 0.4 0.2
(t=0)
Free Maytansinoid, (%) 3.3 0.5
(after 5 months at 4°C)
The results of the experiments reflected in this example demonstrate an improved
processes for preparing cell-binding agent-cytotoxic agent conjugates that are of substantially
high purity. In addition to the improvements in conjugate purity and stability from using the
inventive process, there are also improvements in processing time and convenience, due to
elimination of two processing steps (modification reaction and purification of the modified
antibody).
Example 2
Humanized Folate Receptor antibody huMov19 (see U.S. Application Publication
2012/0009181) was reacted with the heterobifunctional crosslinking reagent sulfo-SPDB and
the maytansinoid DM4 using two previously described processes, as well as the improved
process that is the subject of the present application.
For the previously described Process A (two-step process, e.g. Chari et al., US
,208,020), huMov19 antibody (20 mg/mL) first was reacted with sulfo-SPDB (5.7-fold
molar excess relative to the amount of antibody, dissolved in DMA, dimethylacetamide) to
form the modified antibody. The modification reaction was performed at 20º C in 50 mM
EPPS (4-(2-hydroxyethyl)piperazinepropanesulfonic acid) buffer (pH 8.1) containing 5%
DMA for 180 minutes. The modified antibody was purified using a column of Sephadex G-
25F resin equilibrated and eluted in 50 mM EPPS (pH 8.1) with 2 mM EDTA
(Ethylenediaminetetraacetic acid). After purification, the modified antibody (5.0 mg/mL)
was reacted with the maytansinoid DM4 (Dissolved in DMA; 9.7-fold molar excess relative
to the amount of antibody; 1.7-fold excess relative to the measured amount of linker on the
antibody) to form the conjugated antibody. The conjugation reaction was performed at room
temperature in 50 mM EPPS (pH 8.1) containing 2 mM EDTA and 5% DMA for
approximately 18 hours. The reaction mixture was then purified using a column of Sephadex
G-25F resin equilibrated and eluted in 10 mM sodium succinate (pH 5.0).
For the previously described Process B (one-pot process, Dai et al., U.S. Patent
7,811,572), huMov19 antibody (10 mg/mL) first was reacted with sulfo-SPDB (4.9-fold
molar excess relative to the amount of antibody, dissolved in DMA) to form the modified
antibody. The modification reaction was performed at 20º C in 50 mM EPPS buffer (pH 7.5)
containing 2mM EDTA and 10% DMA for 60 minutes. The modified antibody was not
purified before the conjugation reaction. Instead, the un-purified modified antibody was
reacted at 10 mg/mL with the maytansinoid DM4 (8.3-fold molar excess relative to the
amount of antibody, dissolved in DMA) to form the conjugated antibody. The conjugation
reaction was performed at room temperature in 50mM EPPS buffer (pH 7.5) containing 2
mM EDTA and 10% DMA for approximately 18 hours. The reaction mixture was then
purified using a column of Sephadex G-25F resin equilibrated and eluted in 10 mM sodium
succinate (pH 5.0).
For the inventive process in which Sephadex G-25 was used (Process C, one-step
process) to purify the conjugate, huMov19 antibody (6.0 mg/mL) was mixed with DM4 (9.7-
fold molar excess relative to the amount of antibody, dissolved in DMA) and then sulfo-
SPDB (5.7-fold excess relative to the amount of antibody, dissolved in DMA) was added.
The reaction was performed at 20º C in 50 mM EPPS buffer (pH 8.1) containing 2 mM
EDTA and 10% DMA for approximately 20 hours. The reaction mixture was then purified
using a column of Sephadex G-25F resin equilibrated and eluted in 10 mM sodium succinate
(pH 5.0).
For the inventive process in which tangential flow filtration (TFF) (Process D,
one-step process) was used to purify the conjugate, huMov19 antibody (5.0 mg/mL) was
mixed with DM4 (10.2-fold molar excess relative to the amount of antibody, dissolved in
DMA) and then with sulfo-SPDB (6.0-fold excess relative to the amount of antibody,
dissolved in DMA). The reaction was performed at 20º C in 50 mM EPPS buffer (pH 8.5)
containing 2 mM EDTA and 10% DMA for approximately 20 hours. The reaction mixture
was then purified and diafiltered using a TFF into 10 mM sodium succinate (pH 5.0).
Conjugate derived from the different processes was analyzed by: UV spectroscopy
(for concentration and maytansinoid to antibody ratio, MAR); reversed phase HPLC for
determination of Free Maytansinoid; Mass Spectrometry for determination of unconjugated
linker level and mass distribution profile; reduced SDS PAGE electrophoresis for
determination of level of non-reducible species; non-reduced SDS PAGE electrophoresis for
determination of level of fragmentation; SEC-HPLC for determination of conjugate
monomer. Stability on storage was assessed with respect to conjugate monomer and free
maytansinoid release. Additional details on the analytical methodologies are provided in
Example 1.
As shown in Table 2 below, conjugate manufactured using the inventive process
was superior to that manufactured using the previously described processes with respect to
monomer. Conjugate manufactured using the one-pot and one-step processes in which the
final purification of conjugate was performed using Sephadex G-25, Processes B and C,
respectively, had a higher level of free maytansinoid than conjugate manufactured using the
two step process, Process A. However, when a different final purification process, TFF, was
used (Process D), the level of free maytansinoid was very low and comparable to that seen
with the two-step process, both after initial purification and after storage at 4º C for six
weeks. With respect to other important conjugate attributes (e.g. fragmentation, non-
reducible species, mass distribution profile and unconjugated linker), conjugate manufactured
using the inventive process was equivalent to that manufactured by the previously described
processes.
Table 2. Comparison of key properties of huMov19 conjugate manufactured by the
inventive process compared to previous processes
Process Process A* Process B* Process C* Process D**
Number of Unit Operations 5 4 3 3
Concentration (mg/mL) 1.0 1.0 1.0 4.2
MAR (UV) 4 3.8 3.7 3.6
Conjugate Monomer, (%) at t = 0 94.8 97.4 98.9 98.6
Conjugate Monomer, (%) at t = 6 weeks, 94.4 97.5 98.8 98.1
Free Maytansinoid, (%) at t = 0 0.6 6.3 3.1 0.1
.4 2.7 0.4
Free Maytansinoid, (%) at t = 6 weeks, 4ºC 0.6
Fragmentation By non-reducing gel-chip
14 14 12
Non-reducible species By reducing gel-chip
0.7 0.8 0.7 0.5
Mass Distribution Profile (MDP) Comparable
Unconjugated Linker (MDP) Non-Detectible
* Purified with Sephadex G-25
** Purified using TFF
The results of the experiments reflected in this example demonstrate an improved
processes for preparing cell-binding agent-cytotoxic agent conjugates that are of substantially
high purity. In addition to the improvements in conjugate purity and stability from using the
inventive process, there are also improvements in processing time and convenience, due to
elimination of two processing steps (modification reaction and purification of the modified
antibody).
Example 3
This example demonstrates that the one-step process described herein can be used
to make conjugates starting with a variety of linkers and maytansinoid cytotoxic agents.
Humanized huN901 antibody was mixed with Maytansinoid (DM1or DM4) and
then with Linker (Sulfo-SMCC, SMCC, SPDB, or SPP). The reaction was performed at
20º C in 50 mM phosphate buffer (pH 7.5) containing 2 mM EDTA and 10% DMA for
approximately 20-24 hours. The reaction mixture was then purified using a column of
Sephadex G25F resin equilibrated and eluted in 10 mM sodium succinate (pH 5.0).
As shown in Table 3 below, the one-step reaction can be performed on different
linker and maytansinoid combinations and yield conjugate with good MAR and monomer
levels.
Table 3. Making conjugates by using different linker and maytansinoid combinations
Linker DMx Conjugate MAR Conjugate Monomer (%)
Two-step SPP DM1 3.5 96.8
SPP DM1 3.4 97.5
SPDB DM1 4.6 97.7
One-step
SMCC DM4 4.5 95.1
(Inventive)
S-SMCC DM1 3.5 98.0
SPDB DM4 3.8 97.3
All references, including publications, patent applications, and patents, cited
herein are hereby incorporated by reference to the same extent as if each reference were
individually and specifically indicated to be incorporated by reference and were set forth in
its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of
describing the invention (especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms “comprising,” “having,” “including,” and
“containing” are to be construed as open-ended terms (i.e., meaning “including, but not
limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually to each separate value
falling within the range, unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all examples, or exemplary
language (e.g., “such as”) provided herein, is intended merely to better illuminate the
invention and does not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best
mode known to the inventors for carrying out the invention. Variations of those preferred
embodiments may become apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to employ such variations as
appropriate, and the inventors intend for the invention to be practiced otherwise than as
specifically described herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
Claims (34)
1. A process for preparing an antibody maytansinoid conjugate comprising the steps of: (a) contacting an antibody with a maytansinoid to form a first mixture comprising the antibody and the maytansinoid, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9 to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products; and (b) contacting the second mixture with the maytansinoid to form a third mixture; and then contacting the third mixture with the bifunctional crosslinking reagent at a pH of about 4 to about 9 to provide a fourth mixture.
2. The process of claim 1, wherein the process further comprises the step of: (c) purifying the fourth mixture to provide a purified antibody maytansinoid conjugate.
3. The process of claim 2, wherein step (b) is repeated one or more times before step (c) is carried out.
4. The process of claim 2 or 3, wherein step (b) is repeated one more times before step (c) is carried out.
5. The process of claim 3 or 4, wherein each repeated step (b) is carried out after about one or more hours following initial step (b).
6. The process of any one of claims 1–5, wherein the fourth mixture is purified by subjecting the mixture to tangential flow filtration, selective precipitation, adsorptive filtration, adsorptive chromatography, non-absorptive chromatography, or a combination thereof, to purify the antibody maytansinoid conjugate from the free maytansinoid and reaction by-products.
7. The process of claim 6, wherein the fourth mixture is purified by subjecting the mixture to non-adsorptive chromatography.
8. The process of claim 6, wherein the fourth mixture is purified by subjecting the mixture to tangential flow filtration.
9. The process of any one of claims 1–8, wherein the contacting in step (a) is effected by providing the antibody in a reaction vessel, adding the maytansinoid to the reaction vessel to form the first mixture comprising the antibody and the maytansinoid, and then adding the bifunctional crosslinking reagent to the first mixture.
10. The process of any one of claims 2–9, further comprising holding the fourth mixture between steps (a) – (c) to release the unstably bound linkers from the antibody.
11. The process of claim 10, wherein the fourth mixture is held for 20 hours at a temperature of 2º C to 8º C.
12. The process of any one of claims 2–11, further comprising quenching the fourth mixture between steps (a) – (c) to quench any unreacted maytansinoid and/or unreacted bifunctional crosslinking reagent.
13. The process of claim 12, wherein the mixture is quenched by contacting the fourth mixture with a quenching reagent that reacts with the free maytansinoid.
14. The process of claim 13, wherein the quenching reagent is selected from the group consisting of 4-maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid.
15. The process of any one of claims 1–14, wherein the contacting in step (a) occurs in a solution having a pH of 7 to 9.
16. The process of any one of claims 1–15, wherein the contacting in step (a) occurs at a temperature of 16° C to 24° C.
17. The process of any one of claims 1–15, wherein the contacting in step (a) occurs at a temperature of 0° C to 15° C.
18. The process of any one of claims 1–17, wherein the antibody is a monoclonal antibody.
19. The process of any one of claims 1–17, wherein the antibody is a humanized monoclonal antibody.
20. The process of any one of claims 1–17, wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, rituximab, an antibody that binds to Her2, an antibody that binds to epidermal growth factor receptor (EGFR), an antibody that binds to CD27L, an antibody that binds to EGFRvIII, an antibody that binds to Cripto, an antibody that binds to CD138, an antibody that binds to EphA2, an integrin targeting antibody, an antibody that binds to CD37, an antibody that binds to folate, an antibody that binds to Her3, and an antibody that binds to insulin-like growth factor 1 receptor (IGF1R).
21. The process of any one of claims 1–17, wherein the antibody is huCD37-3 antibody.
22. The process of any one of claims 1–17, wherein the antibody is trastuzumab.
23. The process of any one of claims 1–22, wherein the maytansinoid comprises a thiol group.
24. The process of any one of claims 1–23, wherein the maytansinoid is N - deacetyl-N -(3-mercaptooxopropyl)-maytansine (DM1).
25. The process of any one of claims 1–23, wherein the maytansinoid is N - deacetyl-N -(4-methylmercaptooxopentyl)-maytansine (DM4).
26. The process of any one of claims 1–25, wherein the antibody is chemically coupled to the maytansinoid via chemical bonds selected from the group consisting of disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, thioether bonds, and esterase labile bonds.
27. The process of any one of claims 1–26, wherein the bifunctional crosslinking reagent comprises an N-succinimidyl ester moiety, an N-sulfosuccinimidyl ester moiety, a maleimido-based moiety, or a haloacetyl-based moiety.
28. The process of claim 27, wherein the bifunctional crosslinking reagent is selected from the group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2- pyridyldithio)butanoate (SPDB), N-succinimidyl(2-pyridyldithio)2-sulfo butanoate (sulfo- SPDB), N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), PEG-mal, sulfo-Mal, and CX1-1.
29. The process of any one of claims 1–28, wherein the solution in step (a) comprises sucrose.
30. The process of any one of claims 1–29, therein the solution in step (a) comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
31. The process of any one of claims 1–30, wherein the solution in step (a) comprises a buffering agent selected from the group consisting of HEPPSO (N-(2- hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis- (2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-(2-hydroxyethyl)piperazine ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazinepropanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]aminoethanesulfonic acid), and a combination thereof.
32. The process of any one of claims 1–17, wherein the maytansinoid is DM1, the bifunctional crosslinking reagent is SMCC, and the antibody is huCD37-3 antibody.
33. The process of any one of claims 1–17, wherein the maytansinoid is DM1, the bifunctional crosslinking reagent is SMCC, and the antibody is trastuzumab.
34. A process as defined in anyone of claims 1 to 33 substantially as herein described with reference to any example thereof. 5801587_1
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NZ726386A NZ726386A (en) | 2011-03-29 | 2012-03-29 | Preparation of antibody maytansinoid conjugates |
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US201161468997P | 2011-03-29 | 2011-03-29 | |
US61/468,997 | 2011-03-29 | ||
NZ616509A NZ616509B2 (en) | 2011-03-29 | 2012-03-29 | Preparation of maytansinoid antibody conjugates by a one-step process |
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