NZ616516B2 - Process for manufacturing conjugates of improved homogeneity - Google Patents
Process for manufacturing conjugates of improved homogeneity Download PDFInfo
- Publication number
- NZ616516B2 NZ616516B2 NZ616516A NZ61651612A NZ616516B2 NZ 616516 B2 NZ616516 B2 NZ 616516B2 NZ 616516 A NZ616516 A NZ 616516A NZ 61651612 A NZ61651612 A NZ 61651612A NZ 616516 B2 NZ616516 B2 NZ 616516B2
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- New Zealand
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- cell
- maytansinoid
- mixture
- antibody
- binding agent
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Abstract
Discloses a process for preparing a conjugate comprising a cell-binding agent chemically coupled to a maytansinoid, which process comprises: (a) contacting a cell-binding agent with a bifunctional crosslinking reagent in a solution having a pH of about 7.5 to about 9 at a temperature of about -10°C to about 15°C to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto; (b) conjugating a maytansinoid to the cell-binding agents having linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto with a maytansinoid in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products; and (c) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell binding agents chemically coupled through the linkers to the maytansinoid from the other components of the second mixture and thereby prepare a purified second mixture of cell binding agents chemically coupled through the linkers to the maytansinoid. to about 15°C to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto; (b) conjugating a maytansinoid to the cell-binding agents having linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto with a maytansinoid in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products; and (c) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell binding agents chemically coupled through the linkers to the maytansinoid from the other components of the second mixture and thereby prepare a purified second mixture of cell binding agents chemically coupled through the linkers to the maytansinoid.
Description
PROCESS FOR MANUFACTURING CONJUGATES OF IMPROVED HOMOGENEITY
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Patent Application No.
61/468,981, filed March 29, 2011, which is incorporated by reference in its entirety herein.
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 9,212 Byte ASCII (Text) file named
"SequenceListing-Rule13ter.TXT," created on June 8, 2012.
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. Commonly used manufacturing processes comprise a modification step, in which
the cell-binding agent is reacted with a bifunctional linker at room temperature (about 20º C) or above
to form a cell-binding agent covalently attached to a linker having a reactive group, and a conjugation
step, in which the modified cell-binding agent is reacted with a cytotoxic agent to form a covalent
chemical bond from the linker (using the reactive group) to the cytotoxic agent.
Optimizing the modification step (reaction of the cell-binding agent with the linker)
requires maximizing the reaction of the linker with the cell-binding agent and minimizing side
reactions of the reactive group on the linker, with, for example, water and reactive groups on the cell-
binding agent. These side reactions are especially problematic where the reactive group on the linker
is a very reactive functional group, such as a maleimide. The side reactions can lead to undesirable
reaction products, such as cell-binding agents crosslinked to themselves, as well as cell-binding agents
having linkers that are unable to react with the cytotoxic agent.
In view of the foregoing, there is a need in the art to develop an improved process for
preparing cell-binding agents having a linker bound thereto that results in a high yield of the desired
species of cell-binding agents having a linker bound thereto and that is compatible with large scale
manufacturing processes. The invention provides such a process and/or to at least provide 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 a conjugate
comprising a cell-binding agent chemically coupled to a maytansinoid, which process
comprises:
(a) contacting a cell-binding agent with a bifunctional crosslinking reagent in a
solution having a pH of about 7.5 to about 9 at a temperature of about -10 C to about 15 C to
covalently attach a linker to the cell-binding agent and thereby prepare a first mixture
comprising cell-binding agents having linkers bound thereto,
(b) conjugating a maytansinoid to the cell-binding agents having linkers bound
thereto in the purified first mixture by reacting the cell-binding agents having linkers bound
thereto with a maytansinoid in a solution having a pH of about 4 to about 9 to prepare a
second mixture comprising (i) cell-binding agent chemically coupled through the linker to the
maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products, and
(c) subjecting the second mixture to tangential flow filtration, selective
precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive
chromatography, or a combination thereof to purify the cell-binding agents chemically
coupled through the linkers to the maytansinoid from the other components of the second
mixture and thereby prepare a purified second mixture of cell-binding agents chemically
coupled through the linkers to the maytansinoid.
[0004b] The invention provides a process for preparing a cell-binding agent having a
linker bound thereto, which process comprises contacting a cell-binding agent with a
bifunctional crosslinking reagent at a temperature of about 15° C or less to covalently attach a
linker to the cell-binding agent and thereby prepare a mixture comprising the cell-binding
agents having linkers bound thereto.
[0004c] 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.
In one embodiment, described herein is a process for preparing a conjugate
comprising a cell-binding agent chemically coupled to a cytotoxic agent, which process
comprises (a) contacting a cell-binding agent with a bifunctional crosslinking reagent at a
temperature of about 15° C or less to covalently attach a linker to the cell-binding agent and
thereby prepare a first mixture comprising the cell-binding agents having linkers bound
thereto, (b) subjecting the first mixture to tangential flow filtration, selective precipitation,
non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a
combination thereof and thereby prepare a purified first mixture of cell-binding agents having
linkers bound thereto, (c) conjugating a cytotoxic agent to the cell-binding agents having
linkers bound thereto in the purified first mixture by reacting the cell-binding agents having
linkers bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to
prepare a second mixture comprising (i) cell-binding agent chemically coupled through the
linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and (d)
subjecting the second mixture to tangential flow filtration, selective precipitation, non-
adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a
combination thereof to purify the cell-binding agents chemically coupled through the linkers
to the cytotoxic agent from the other components of the second mixture and thereby prepare a
purified second mixture of cell-binding agents chemically coupled through the linkers to the
cytotoxic agent.
Another embodiment described herein provides a process for preparing a
conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent, which
process comprises (a) contacting a cell-binding agent with a bifunctional crosslinking reagent
at a temperature of about 15° C or less to covalently attach a linker to the cell-binding agent
and thereby prepare a first mixture comprising cell-binding agents having linkers bound
thereto, (b) conjugating a cytotoxic agent to the cell-binding agents having linkers bound
thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto
with a cytotoxic agent in a solution having a pH of about 4 to about 9 to prepare a second
mixture comprising (i) cell-binding agent chemically coupled through the linker to the
cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and (c) subjecting the
second mixture to tangential flow filtration, selective precipitation, non-adsorptive
chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof,
to purify the cell binding agents chemically coupled through the linkers to the cytotoxic agent
from the other components of the second mixture and thereby prepare a purified second
mixture of cell binding agents chemically coupled through the linkers to the cytotoxic agent.
Also described herein is a conjugate comprising a cell-binding agent chemically
coupled to a cytotoxic agent prepared according to the processes described herein.
DESCRIPTION OF THE INVENTION
One of ordinary skill in the art will appreciate that conjugates comprising a cell-
binding agent, such as an antibody, chemically coupled to a cytotoxic agent (“antibody-
cytotoxic agent conjugates”) typically are prepared by modifying an antibody with a
bifunctional crosslinking reagent at room temperature (i.e., about 20º C or above), purifying
the antibody having linkers bound thereto, conjugating a cytotoxic agent to the antibody
having linkers bound thereto, and purifying the antibody- cytotoxic agent conjugate. The
invention improves upon such methods by optimizing the modification step in order to
maximize reaction of the linker with the cell-binding agent and minimize undesirable side
reactions. In particular, it was surprisingly discovered that performing the modification
reaction (reaction of the cell-binding agent with the linker) at a lower temperature (e.g., about
15º C or less), extends the interval during which the level of desirable species of cell-binding
agents having a linker bound thereto is maximized and before significant levels of
undesirable reaction products are formed, thereby making the process suitable for large scale
manufacturing. Accordingly, the invention provides processes for manufacturing cell-
binding agent-cytotoxic agent conjugates of improved homogeneity comprising performing
the modification reaction at a lower temperature.
Described herein is a process for preparing a cell-binding agent having a linker
bound thereto, which process comprises contacting a cell-binding agent with a bifunctional
crosslinking reagent at a temperature of about 15° C or less to covalently attach a linker to the
cell-binding agent and thereby prepare a mixture comprising cell-binding agents having
linkers bound thereto. For example, the process described herein comprises contacting a cell-
binding agent with 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 5° C, about 4° C, about 3° C, about 2° C, about 1° C, or 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 process described herein comprises contacting
a cell-binding agent with 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
° 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 process described herein comprises contacting a cell-binding agent
with a bifunctional crosslinking reagent at a temperature of about 10° C (e.g., a temperature
of 8º C to 12º C or a temperature of 9º C to 11º C).
In one embodiment, the process described herein comprises contacting a cell-
binding agent with a bifunctional crosslinking reagent in a solution having a pH of about 7.5
or greater. For example, the process described herein comprises contacting a cell-binding
agent with a bifunctional crosslinking reagent in a solution having a pH of 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 one embodiment, the
process described herein comprises contacting a cell-binding agent with a bifunctional
crosslinking reagent in a solution having a pH of about 7.5 to about 9.0, 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. In another
embodiment, the process described herein comprises contacting a cell-binding agent with 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). Any suitable buffering agent can be used. Suitable buffering
agents include, for example, a citrate buffer, an acetate buffer, a succinate buffer, and a
phosphate buffer. In a preferred 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.
In one embodiment, the process described herein comprises contacting a cell-
binding agent with a bifunctional crosslinking reagent in a solution having a high pH (e.g.,
about 7.5 or greater) at a low temperature (e.g., about 15º C or less). In a preferred
embodiment, the process described herein comprises contacting a cell-binding agent with a
bifunctional crosslinking reagent in a solution having a pH about 7.8 at a temperature of
about 10° C. In another preferred embodiment, the process described herein comprises
contacting a cell-binding agent with a bifunctional crosslinking reagent in a solution having a
pH about 8.5 at a temperature of about 0° C.
In accordance with the method described herein, contacting a cell-binding agent
with a bifunctional crosslinking reagent produces a first mixture comprising the cell-binding
agent having linkers bound thereto, as well as reactants and other by-products. In some
embodiments, the first mixture comprises the cell-binding agent having linkers stably and
unstably bound thereto, as well as reactants and other by-products. A linker is “stably”
bound to the cell-binding agent when the covalent bond between the linker and the cell-
binding agent is not substantially weakened or severed under normal storage conditions over
a period of time, which could range from a few months to a few years. In contrast, a linker is
“unstably” bound to the cell-binding agent when the covalent bond between the linker and the
cell-binding agent is substantially weakened or severed under normal storage conditions over
a period of time, which could range from a few months to a few years.
In one embodiment described herein, purification of the modified cell-binding
agent from reactants and by-products is carried out by subjecting the first mixture to a
purification process. In this regard, the first mixture can be purified using tangential flow
filtration (TFF), e.g., a membrane-based tangential flow filtration process, non-adsorptive
chromatography, adsorptive chromatography, adsorptive filtration, or selective precipitation,
or any other suitable purification process, as well as combinations thereof. This first
purification step provides a purified first mixture, i.e., an increased concentration of the cell-
binding agents having linkers bound thereto and a decreased amount of unbound bifunctional
crosslinking reagent, as compared to the first mixture prior to purification as contemplated
herein. Preferably, the first mixture is purified using tangential flow filtration.
After purification of the first mixture to obtain a purified first mixture of cell-
binding agents having linkers bound thereto, a cytotoxic agent is conjugated to the cell-
binding agents having linkers bound thereto in the first purified mixture by reacting the cell-
binding agents having linkers bound thereto with a cytotoxic agent in a solution having a pH
from about 4 to about 9, wherein a second mixture comprising (i) the cell-binding agent
chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and
(iii) reaction by-products is produced.
Optionally, purification of the modified cell-binding agent may be omitted. Thus,
in one embodiment described herein, the first mixture comprising the cell-binding agent
having linkers bound thereto, as well as reactants and other by-products, is not subjected to a
purification process. In such a situation, the cytotoxic agent may be added simultaneously
with the crosslinking reagent or at some later point, e.g., 1, 2, 3, or more hours after addition
of the crosslinking reagent to the cell-binding agent. The modified cell-binding agent is
conjugated to a cytotoxic agent (e.g., a maytansinoid) by reacting the modified cell-binding
agent with the cytotoxic agent in a solution having a pH from about 4 to about 9, wherein the
conjugation step results in formation of a mixture of stable cell-binding agent-cytotoxic agent
conjugates, non-stable cell-binding agent-cytotoxic agent conjugates, non-conjugated
cytotoxic agent (i.e., “free” cytotoxic agent), reactants, and by-products.
The conjugation reaction preferably is performed at a pH of about 4 to about pH 9
(e.g., a pH of about 4.5 to about 8.5, about 5 to about 8, about 5.5 to about 7.5, or about 6.0 to
about 7). In some embodiments, the conjugation reaction is performed at a pH of about 6 to
about 6.5 (e.g., a pH of 5.5 to 7, a pH of 5.7 to 6.8, a pH of 5.8 to 6.7, a pH of 5.9 to 6.6, or a
pH of 6 to 6.5), a pH of about 6 or below (e.g., a pH of about 4 to 6, about 4 to about 5.5,
about 5 to 6) or at a pH of about 6.5 or greater (e.g., a pH of 6.5 to about 9, about 7 to about
9, about 7.5 to about 9, or 6.5 to about 8). In one embodiment, the conjugation reaction is
performed at a pH of about 4 to a pH less than 6 or at a pH of greater than 6.5 to 9. When the
conjugation step is performed at a pH of about 6.5 or greater, some sulfhydryl-containing
cytotoxic agents may be prone to dimerize by disulfide-bond formation. In one embodiment,
removal of trace metals and/or oxygen from the reaction mixture, as well as optional addition
of antioxidants or the use of linkers with more reactive leaving groups, or addition of
cytotoxic agent in more than one aliquot, may be required to allow for efficient reaction in
such a situation.
The inventive process may optionally include the addition of sucrose to the
conjugation step used in the inventive process 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 20% (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
conjugation reaction 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 a preferred
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)piperazine
ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazinepropanesulfonic acid),
TES (N-[tris(hydroxymethyl)methyl]aminoethanesulfonic acid), and a combination
thereof.
Following the conjugation step, the conjugate is subjected to a purification step.
In this regard, the conjugation mixture can be purified using tangential flow filtration (TFF),
e.g., a membrane-based tangential flow filtration process, non-adsorptive chromatography,
adsorptive chromatography, adsorptive filtration, or selective precipitation, or any other
suitable purification process, as well as combinations thereof. One of ordinary skill in the art
will appreciate that purification after the conjugation step enables the isolation of a stable
conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent.
In one embodiment, described herein is a process for preparing a conjugate
comprising a cell-binding agent chemically coupled to a cytotoxic agent, which process
comprises a first purification step after the modification step and a second purification step
after the conjugation step. For example, described herein is a process for preparing a
conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent, which
process comprises (a) contacting a cell-binding agent with a bifunctional crosslinking reagent
at a temperature of about 15° C or less to covalently attach a linker to the cell-binding agent
and thereby prepare a first mixture comprising cell-binding agents having linkers bound
thereto, (b) subjecting the first mixture to tangential flow filtration, selective precipitation,
non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a
combination thereof and thereby prepare a purified first mixture of cell-binding agents having
linkers bound thereto, (c) conjugating a cytotoxic agent to the cell-binding agents having
linkers bound thereto in the purified first mixture by reacting the cell-binding agents having
linkers bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to
prepare a second mixture comprising (i) cell-binding agent chemically coupled through the
linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and (d)
subjecting the second mixture to tangential flow filtration, selective precipitation, non-
adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a
combination thereof to purify the cell-binding agents chemically coupled through the linkers
to the cytotoxic agent from the other components of the second mixture and thereby prepare a
purified second mixture of cell-binding agents chemically coupled through the linkers to the
cytotoxic agent.
In one embodiment described herein, tangential flow filtration (TFF, also known
as cross flow filtration, ultrafiltration and diafiltration) and/or adsorptive chromatography
resins are utilized in the purification steps. For example, the inventive process can comprise
a first purification step using TFF after the modification step and a second purification step
using TFF after the conjugation step. Alternatively, the inventive process can comprise a first
purification step using adsorptive chromatography after the modification step and a second
purification step using adsorptive chromatography after the conjugation step. The inventive
process also can comprise a first purification step using adsorptive chromatography after the
modification step and a second purification step using TFF after the conjugation step or a first
purification step using TFF after the modification step and a second purification step using
adsorptive chromatography after the conjugation step.
In one embodiment described herein, non-adsorptive chromatography is utilized
as the purification step. For example, the inventive process can comprise a first purification
step using non-adsorptive chromatography after the modification step and a second
purification step using non-adsorptive chromatography after the conjugation step.
In another embodiment, described herein is a process for preparing a conjugate
comprising a cell-binding agent chemically coupled to a cytotoxic agent, which process
comprises a single purification step after the conjugation step. For example, the inventive
process can comprise a process for preparing a conjugate wherein the mixture is not subjected
to purification following the modification step. In this respect, described herein is a process
for preparing a conjugate comprising a cell-binding agent chemically coupled to a cytotoxic
agent, which process comprises (a) contacting a cell-binding agent with a bifunctional
crosslinking reagent at a temperature of about 15° C or less to covalently attach a linker to the
cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having
linkers bound thereto, (b) conjugating a cytotoxic agent to the cell-binding agents having
linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers
bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to
prepare a second mixture comprising (i) cell-binding agent chemically coupled through the
linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and (c)
subjecting the second mixture to tangential flow filtration, selective precipitation, non-
adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a
combination thereof, to purify the cell binding agents chemically coupled through the linkers
to the cytotoxic agent from the other components of the second mixture and thereby prepare a
purified second mixture of cell binding agents chemically coupled through the linkers to the
cytotoxic agent.
In one embodiment described herein, the process as described herein comprises
two separate purification steps following the conjugation step.
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 process described herein further comprises a holding step
to release the unstably bound linkers from the cell-binding agent. The holding step comprises
holding the mixture after modification of the cell-binding agent with a bifunctional
crosslinking reagent, after conjugation of a cytotoxic agent to the cell-binding agents having
linkers bound thereto, and/or after a purification step.
The holding step comprises maintaining the solution at a suitable temperature
(e.g., about 2º C to about 37º C) for a suitable period of time (e.g., about 1 hour to about 1
week) 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 a low temperature (e.g.,
about 2º C to about 10º C or about 4º C), at room temperature (e.g., about 20º C to about
30º C or about 20º C to about 25º C), or at an elevated temperature (e.g., about 30º C to about
37º C).
The duration of the holding step depends on the temperature 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 5 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), for 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 1 day.
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.
The holding step can be performed before or after the cell-binding agent is
conjugated to the cytotoxic agent. In one embodiment, the holding step is performed directly
after the modification of the cell-binding agent with the bifunctional crosslinking reagent.
For example, the process described herein comprises a holding step after modification of the
cell-binding agent with a bifunctional crosslinking reagent and before conjugation. After
modification of the cell-binding agent, a purification step may be performed before the hold
step and/or after the hold step, but prior to the conjugation step. In another embodiment, the
holding step is performed directly after conjugation of the cytotoxic agent to the cell-binding
agent having linkers bound thereto and prior to purification step. In another embodiment, the
holding step is performed after the conjugation and purification steps and followed by an
additional purification step.
In specific embodiments, the holding step can comprise incubating the mixture at
4º C at a pH of about 6-7.5 for about 12 hours to about 1 week, incubating the mixture at 25°
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. 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%)
(relative to total cytotoxic agent), and/or (e) no substantial increase in free cytotoxic agent
level 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, or about 5 years). “Substantial increase” in free cytotoxic agent level
means that after certain storage time, 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, 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, about
.5).
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 or a glycotope. 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
: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 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; alphaantitrypsin;
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 (MIPalpha); 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, cripto, 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 composition as described herein. 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 composition described
herein 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 in the context of
the invention, 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. Patent 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. Patent 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 No. 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 heavy
1 2 3
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
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTF
YNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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,
: 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 agent. 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, PCT/US11/059131 filed November 3, 2011),
taxoids, CC-1065 and CC-1065 analogs, and dolastatin and dolastatin analogs. In a preferred
embodiment described herein, 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, 5,585,499, 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 described herein, the conjugate utilizes the thiol-
2’ 2’
containing 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 described herein, the conjugate utilizes the thiol-
2’ 2’
containing maytansinoid DM4, also known as N -deacetyl-N -(4-methylmercapto
oxopentyl)-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):
N Y'
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 3
May May
(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 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, 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 C 3
N Y 2
2 May
(VI-L) (VI-D) (VI-D, L),
wherein Y represents (CR R ) (CR R ) (CR R ) CR R SZ ,
2 7 8 l 5 6 m 3 4 n 1 2 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 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):
N Y '
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 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 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
OH OAc
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 agents 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.
In accordance with the invention, the cell-binding agent is modified by reacting a
bifunctional crosslinking reagent with the cell-binding agent, thereby resulting in the covalent
attachment of a linker molecule to the cell-binding agent. 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 in connection with the
invention, 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, while providing an acceptable toxicity profile. 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,
2005/0169933, and 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) and 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 one 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. Patent
Application Publication No. 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.
Patents 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 method as described herein. 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
This example demonstrates a processes for manufacturing cell-binding agent-
cytotoxic agent conjugates of improved homogeneity comprising performing the
modification reaction at a lower temperature.
Humanized CD37-3 antibody (huCD37-3) was reacted with the heterobifunctional
crosslinking reagent SMCC (N-succinimidyl(maleimidomethyl)cyclohexanecarboxylate)
and the maytansinoid DM1 using a previously described process, as well as the improved
process that is the subject of the present application.
For the previously described process, Process A (see, e.g., Chari et al., U.S.
,208,020), huCD37-3 (15 mg/mL) first was reacted with SMCC (6.0-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 sodium
phosphate buffer (pH 6.7) containing 2 mM EDTA (ethylenediaminetetraacetic acid) and
% DMA for 180 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 2mM EDTA. After
purification, the modified antibody (at 5 mg/mL) was adjusted to pH 5.0 with potassium
phosphate tribasic buffer and was reacted with the maytansinoid DM1 (7.2-fold molar excess
relative to the amount of antibody, dissolved in DMA) 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 Process B (involving performing the modification step at high pH and room
temperature), huCD37-3 (15 mg/mL) first was reacted with SMCC (6.0-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 sodium phosphate buffer (pH 7.5)
containing 2 mM EDTA and 10% DMA for 50 minutes. The reaction was quenched with 1
M acetic acid 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 (at 5 mg/mL) was
adjusted to pH 5.0 with potassium phosphate tribasic buffer and was reacted with the
maytansinoid DM1 (7.2-fold molar excess relative to the amount of antibody, dissolved in
DMA) to form the conjugated antibody. The conjugation reaction was performed at 20º C in
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 process described herein, Process C (involving performing the
modification step at high pH and low temperature), huCD37-3 (15 mg/mL) first was reacted
with SMCC (6.0-fold molar excess relative to the amount of antibody, dissolved in DMA) to
form the modified antibody. The modification reaction was performed at 10º C in 50 mM
sodium phosphate buffer (pH 7.5) containing 2 mM EDTA and 10% DMA for 50 minutes.
The reaction was quenched with 1 M acetic acid 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
20mM sodium acetate (pH 4.5) containing 2mM EDTA. After purification, the modified
antibody (at 5 mg/mL) was adjusted to pH 5.0 with potassium phosphate tribasic buffer and
was reacted with the maytansinoid DM1 (7.2-fold molar excess relative to the amount of
antibody, dissolved in DMA) 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
% 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
.0).
Conjugate derived from the three processes was analyzed by: UV spectroscopy
for conjugate concentration and Maytansinoid to Antibody Ratio (MAR); Free Maytansinoid
by Dual Column reversed phase chromatography; Mass Spectrometry for determination of
unconjugated linker level; reduced SDS PAGE electrophoresis for determination of level of
non-reducible species; non-reduced SDS PAGE electrophoresis for determination of level of
fragments; and SEC-HPLC for determination of conjugate monomer.
Concentration and Maytansinoid to Antibody Ratio 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
(Process C) was superior to that manufactured using the previously described process,
Process A, with respect to unconjugated linker, non-reducible species and monomer, as well
as the Process B, involving performing the modification step at high pH and room
temperature.
Table 1. Comparison of key properties of the conjugate manufactured by the inventive
process compared to other processes
Process A Process B Process C
Modification at Modification at Modification at
pH 6.7, 20º C pH 7.5, 20º C pH 7.5, 10º C
Concentration (mg/mL) 3.2 3.1 3.2
MAR 3.7 3.8 3.6
Monomer (SEC HPLC) 95.2% 94.8% 97.8%
Non-reducible species
(Reduced Gel Chip) 11.4% 10.9% 4.4%
Un-conjugated linker (MDP) 14% 16% 7%
Free Maytansinoid 0.5% 0.4% 0.4%
Fragmentation
(Non-reduced Gel Chip) 3.6% 3.0% 3.6%
The results of the experiments described in this example demonstrate that
performing the modification step at a low temperature (e.g., 10º C) produces a conjugate that
is superior to conjugate manufactured using the previously described process. In addition,
the results of the experiments described in this example demonstrate that performing the
modification step at a high pH (e.g., 7.5) produces a conjugate of superior quality only when
the modification step is performed at a low temperature (e.g., 10º C).
Example 2
This example demonstrates a processes for manufacturing cell-binding agent-
cytotoxic agent conjugates of improved homogeneity comprising performing the
modification reaction at a lower temperature and a higher pH.
A humanized antibody was reacted with the heterobifunctional crosslinking
reagent SMCC and the maytansinoid DM1 to make a conjugate with a MAR (maytansinoid
to antibody ratio, also known as drug to antibody ratio) of approximately 3.5.
The reaction was performed using a previously described process (see, e.g., U.S.
Patent Application Publications 2011/0166319 and 2006/0182750), as well as the inventive
process comprising performing the modification reaction at a higher pH and a lower
temperature.
Using the previously described process, the humanized antibody (15 mg/mL) first
was reacted with SMCC (7.5-fold molar excess relative to the amount of antibody) to form
the modified antibody. The modification reaction was performed at 21º C in 50 mM sodium
phosphate buffer (pH 6.7) containing 2 mM EDTA and 5% DMA for 120 minutes. The
reaction was quenched with 0.5 M citrate to adjust the pH to5.0, and the modified antibody
was purified using a column of Sephadex G25F. After purification, the modified antibody (at
mg/mL) was reacted with the maytansinoid DM1 (5.4-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
ambient temperature in 20 mM citrate buffer (pH 5.0) containing 2 mM EDTA and 5% DMA
for approximately 17 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).
In the inventive process, the humanized antibody (3 mg/mL) first was reacted with
SMCC (6.0-fold molar excess relative to the amount of antibody) to form the modified
antibody. The modification reaction was performed at 0º C in 50 mM sodium phosphate
buffer (pH 8.2) containing 2 mM EDTA and 5% DMA for 117 minutes. The reaction was
quenched with 0.5 M citrate to adjust the pH to 5.0, and the modified antibody was purified
using a column of Sephadex G25F. After purification, the modified antibody (2.5 mg/mL)
was reacted with the maytansinoid DM1 (5.2-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 ambient temperature in
mM citrate 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 G25F resin
equilibrated and eluted in 10 mM sodium succinate (pH 5.0).
Conjugate derived from the two processes was analyzed by: Mass Spectrometry
for determination of unconjugated linker level; reduced SDS PAGE electrophoresis for
determination of level of non-reducible species; and SEC-HPLC for determination of
conjugate monomer.
As shown in Table 2 below, conjugate manufactured using the inventive process
was superior to conjugate manufactured using the previously described process with respect
to unconjugated linker and non-reducible species.
Table 2. Comparison of key properties of conjugate manufactured by the inventive
process compared to previous process
Previous Process Inventive Process
Modification at pH 6.7, Modification at
Room Temperature pH 8.2, 0º C
MAR 3.6 3.1
Monomer% (SEC HPLC) 96.8% 97.5%
Non-reducible species
(Reduced Gel Chip) 12.9% 6.8%
Un-conjugated linker% (MDP) 12.3% 7.6%
Total Free Maytansinoid % 0.2% 0.1%
The results of the experiments described in this example demonstrate that
performing the modification step at a low temperature (e.g., 0º C) and high pH (e.g., pH 8.2)
produces a conjugate that is superior to conjugate manufactured using the previously
described process, wherein the modification step is performed at room temperature and a
lower pH (e.g., pH 6.7).
Example 3
This example illustrates a large-scale process for manufacturing cell-binding
agent-cytotoxic agent conjugates of improved homogeneity comprising performing the
modification reaction at a lower temperature and a higher pH.
A humanized antibody is reacted with the heterobifunctional crosslinking reagent
SMCC and the maytansinoid DM1 to prepare a stable humanized antibody-SMCC-DM1
conjugate.
In particular, using the inventive process described herein, a humanized antibody
is reacted with SMCC to form the modified antibody. The modification reaction is
performed for 40 minutes using a molar excess of SMCC over antibody of 5.7 at about 10º C
in a buffer having a pH of about 7.8 in 50 mM sodium phosphate, 2 mM EDTA, with 7%
(v/v) DMA. After modification, the pH of the reaction mixture is adjusted to 4.5 with 1 M
acetic acid, and the modified antibody is purified using TFF. After purification, the modified
antibody is reacted with the maytansinoid DM1 (about 1.2 fold molar excess over bound
linker) to form the conjugated antibody. The conjugation reaction is performed for 16 hours
at ambient temperature at a pH of about 5.0 in 20 mM sodium acetate, 2.0 mM EDTA, with
.0% (v/v) DMA. The reaction mixture is then purified using TFF.
Analysis of the conjugate can be conducted by: Mass Spectrometry for
determination of unconjugated linker level; reduced SDS PAGE electrophoresis for
determination of level of non-reducible species; and SEC-HPLC for determination of
conjugate monomer. The results of the analysis demonstrate that conjugate prepared by the
inventive process is superior to conjugate manufactured using previously described processes
(see, e.g., U.S. Patent Application Publications 2011/0166319 and 2006/0182750).
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 (52)
1. A process for preparing a conjugate comprising a cell-binding agent chemically coupled to a maytansinoid, which process comprises: (a) contacting a cell-binding agent with a bifunctional crosslinking reagent in a solution having a pH of about 7.5 to about 9 at a temperature of about -10 C to about 15 C to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto, (b) conjugating a maytansinoid to the cell-binding agents having linkers bound thereto in the purified first mixture by reacting the cell-binding agents having linkers bound thereto with a maytansinoid in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products, and (c) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof to purify the cell-binding agents chemically coupled through the linkers to the maytansinoid from the other components of the second mixture and thereby prepare a purified second mixture of cell-binding agents chemically coupled through the linkers to the maytansinoid.
2. The process of claim 1, wherein the solution comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
3. The process of claim 1, wherein the solution 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)piperazineethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazinepropanesulfonic acid), TES (N- [tris(hydroxymethyl)methyl]aminoethanesulfonic acid), and a combination thereof.
4. The process of claim 1, wherein the pH is about 7.8.
5. The process of claim 1, wherein the temperature is about 10° C.
6. The process of any one of claims 1-5, wherein the adsorptive chromatography is selected from the group consisting of 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.
7. The process of any one of claims 1-6, wherein the cell-binding agent is selected from the group consisting of antibodies, interferons, interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, and transferrin.
8. The process of claim 7, wherein the cell-binding agent is an antibody.
9. The process of claim 8, wherein the antibody is a monoclonal antibody.
10. The process of claim 9, wherein the antibody is a humanized monoclonal antibody.
11. The process of any one of claims 8-10, wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, rituximab, anti-CD27L, anti-Her2, anti-EGFR, anti- EGFRvIII, Cripto, anti-CD138, anti-CD38, anti-EphA2, integrin targeting antibody, anti- CD37, anti-folate, anti-Her3 and anti-IGFIR.
12. The process of claim any one of claims 1-11, wherein the maytansinoid comprises a thiol group.
13. The process of claim 12, wherein the maytansinoid is DM1.
14. The process of claim 12, wherein the maytansinoid is DM4.
15. The process of any one of claims 1-14, wherein the cell-binding agent 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.
16. The process of any one of claims 1-15, 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.
17. The process of claim 16, wherein the bifunctional crosslinking reagent comprises a maleimido-based moiety.
18. The process of claim 17, wherein the bifunctional crosslinking reagent is selected from the group consisting of N-succinimidyl 4- (maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl(N- maleimidomethyl)-cyclohexanecarboxy-(6-amidocaproate) (LC-SMCC), κ- maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N- succinimidyl ester (GMBS), β-maleimidopropyloxy-succinimidyl ester (BMPS), 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), N-(p-maleimidophenyl)isocyanate (PMPI), sulfo-Mal, PEG -Mal and CX1-1.
19. The process of claim 18, wherein the bifunctional crosslinking reagent is N- succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).
20. The process of any one of claims 1-19, wherein the solution in step (c) comprises sucrose.
21. The process of any one of claims 1-20, wherein the solution in step (c) comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
22. The process of any one of claims 1-20, wherein the solution in step (c) 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.
23. The process of any one of claims 1-22, further comprising (e) holding the mixture between at least one of steps a-b, and steps b-c to release the unstably bound linkers from the cell-binding agent.
24. The process of claim 1, which process comprises: (a) contacting a cell-binding agent with a bifunctional crosslinking reagent in a solution having a pH of about 7.5 to about 9 at a temperature of about -10 C to about 15°C to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto, (b) subjecting the first mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto, (c) conjugating a maytansinoid to the cell-binding agents having linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto with a maytansinoid in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell-binding agent chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products, and (d) subjecting the second mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to purify the cell binding agents chemically coupled through the linkers to the maytansinoid from the other components of the second mixture and thereby prepare a purified second mixture of cell binding agents chemically coupled through the linkers to the maytansinoid.
25. The process of claim 24, wherein the solution comprises a buffering agent selected from a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
26. The process of claim 24, wherein the solution 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)piperazineethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazinepropanesulfonic acid), TES (N- [tris(hydroxymethyl)methyl]aminoethanesulfonic acid), and a combination thereof.
27. The process of any one of claims 24-26, wherein the pH is about 7.8.
28. The process of any one of claims 24-27, wherein the temperature is about 10° C.
29. The process of any one of claims 24-28, wherein the adsorptive chromatography is selected from the group consisting of 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.
30. The process of any one of claims 24–29, wherein tangential flow filtration is utilized in steps (b) and (d).
31. The process of any one of claims 24–29, wherein adsorptive chromatography is utilized in steps (b) and (d).
32. The process of any one of claims 24–29, wherein non-adsorptive chromatography is utilized in steps (b) and (d).
33. The process of any one of claims 24–29, wherein tangential flow filtration is utilized in step (b) and adsorptive chromatography is utilized in step (d).
34. The process of any one of claims 24–29, wherein adsorptive chromatography is utilized in step (b) and tangential flow filtration is utilized in step (d).
35. The process of any one of claims 24-34, wherein the cell binding agent is selected from the group consisting of antibodies, interferons, interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, and transferrin.
36. The process of claim 35, wherein the cell binding agent is an antibody.
37. The process of claim 36, wherein the antibody is a monoclonal antibody.
38. The process of claim 37, wherein the antibody is a humanized monoclonal antibody.
39. The process of any one of claims 36-38, wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, rituximab, anti-CD27L, anti-Her2, anti-EGFR, anti- EGFRvIII, Cripto, anti-CD138, anti-CD38, anti-EphA2, integrin targeting antibody, anti- CD37, anti-folate, anti-Her3, and anti-IGFIR.
40. The process of any one of claims 24-39, wherein the maytansinoid comprises a thiol group.
41. The process of claim 40, wherein the maytansinoid is DM1.
42. The process of claim 40, wherein the maytansinoid is DM4.
43. The process of any one of claims 24-42, wherein the cell binding agent 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.
44. The process of any one of claims 24-43, 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.
45. The process of claim 44, wherein the bifunctional crosslinking reagent comprises a maleimido-based moiety.
46. The process of claim 45, wherein the bifunctional crosslinking reagent is selected from the group consisting of N-succinimidyl 4- (maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl(N- maleimidomethyl)-cyclohexanecarboxy-(6-amidocaproate) (LC-SMCC), κ- maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N- succinimidyl ester (GMBS), β-maleimidopropyloxy-succinimidyl ester (BMPS), 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), sulfo- Mal, PEG -Mal and CX1-1.
47. The process of claim 46, wherein the bifunctional crosslinking reagent is N- succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).
48. The process of any one of claims 24-47, wherein the solution in step (b) comprises sucrose.
49. The process of any one of claims 24-48, wherein the solution in step (b) comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
50. The process of any one of claims 24-48, wherein the solution in step (b) 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.
51. The process of any one of claims 24-50, further comprising (e) holding the mixture between at least one of steps a-b, steps b-c and steps c-d to release the unstably bound linkers from the cell-binding agent.
52. A process as claimed in any one of claims 1 to 51 substantially as herein described with reference to any example thereof. 5807633_1.txt
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201161468981P | 2011-03-29 | 2011-03-29 | |
US61/468,981 | 2011-03-29 | ||
PCT/US2012/031253 WO2012135522A2 (en) | 2011-03-29 | 2012-03-29 | Process for manufacturing conjugates of improved homogeneity |
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NZ616516A NZ616516A (en) | 2016-01-29 |
NZ616516B2 true NZ616516B2 (en) | 2016-05-03 |
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