AU2019275619B2 - Conjugation methods - Google Patents

Abstract

CONJUGATION METHODS Abstract This invention describes a method of conjugating a cell binding agent such as an antibody with an effector group (e.g., a cytotoxic agent) or a reporter group (e.g., a radionuclide), whereby the reporter or effector group is first reacted with a bi-functional linker and the mixture is then used without purification for the conjugation reaction with the cell binding agent. The method described in this invention is advantageous for preparation of stably-linked conjugates of cell binding agents, such as antibodies with effector or reporter groups. This conjugation method provides in high yields conjugates of high purity and homogeneity that are without inter-chain cross-linking and inactivated linker residues.

Description

22081847.1:DCC-08 11/2021

CONJUGATION METHODS

This application claims priority of United States provisional application no. 61/183,774, filed June 3, 2009.

FIELD OF THE INVENTION

[01] This invention relates to a novel method of conjugating an effector group (e.g., a cytotoxic agent) or a reporter group (e.g., a radiolabel) to a cell binding agent, such as an antibody or a fragment thereof, via a bifunctional linker. More specifically, this invention relates to a novel method of conjugating an effector group (e.g., maytansinoids) or a reporter group (e.g., a radiolabel) to a cell binding agent (e.g., an antibody or a fragment thereof) via a bifunctional linker such that the process eliminates the steps that result in formation of undesired hydrolyzed species or undesired cross-linked species formed due to intra-molecular or inter-molecular reactions.

BACKGROUND OF THE INVENTION

[02] Conjugates of cell binding agents, such as antibodies, with effector groups, such as small cytotoxic agents or cytotoxic proteins, are of immense interest for the development of anti-cancer therapeutics (Richart, A. D., and Tolcher, A. W., 2007, Nature Clinical Practice, 4, 245-25). These conjugates are tumor-specific due to the high specificity of the selected antibodies toward antigens expressed on the cell surface of tumor cells. Upon specific binding to the tumor cell, the antibody-cytotoxic agent conjugate is internalized and degraded inside the target cancer cell thereby releasing the

I active cytotoxic agent that inhibits essential cellular functions such as microtubule dynamics or DNA replication resulting in the killing of the cancer cell. Various linkers have been employed to link the antibodies with cytotoxic agents with the goal of enhancing the delivery of the agent inside the cell upon internalization and processing of the conjugate, while maintaining the desired stability of the conjugate in plasma. These linkers include disulfide linkers designed with different degrees of steric hindrance to influence their reduction kinetics with intracellular thiol, cleavable peptide linkers such as valine-citrulline linkage, and non-cleavable linkers such as thioether linkage (Widdison,

W., et al., J. Med. Chem., 2006, 49,4392-4408; Erickson, H., et al., Cancer Res., 2006,

66, 4426-4433).

[03] Conjugates of cell binding agents such as antibodies with labels or reporter groups

are useful for tumor-imaging applications in cancer patients, immunoassay applications

for diagnosis of various diseases, cancer therapy using radioactive nuclide-ligand

conjugates, and affinity chromatography applications for purification of bioactive agents

such as proteins, peptides, and oligonucleides. The labels or reporter groups that are

conjugated with cell-binding agents include fluorophores, and affinity labels such as

biotin.

[04] The conventional method of conjugation of the cell-binding agent such as an

antibody (Ab) with an effector group (e.g., a cytotoxic agent) or a reporter group (e.g., a

radiolabel) linked via a non-reducible linkage (such as thioether linkage) employs two

distinct reaction steps with the antibody and necessitates the use of purification steps. In

the first reaction step, the antibody is reacted with a heterobifunctional linker bearing two

different reactive groups (e.g., X and Y). For example, in one approach, the reaction of an antibody's reactive residues (such as lysine amino residues) with the X reactive group

(such as N-hydroxysuccinimide ester) of the heterobifunctional reagent results in the

incorporation of the linker with Y reactive group at one or more reactive residues in the

antibody (such as lysine amino residues). The initially modified antibody product must

be purified from the excess linker or hydrolyzed linker reagent before the next step can

occur. In the second reaction step, the linker-modified antibody containing the Y reactive

group (such as maleimide or haloacetamide) is reacted with the effector such as an

effector group (C) (e.g., a cytotoxic agent) containing a reactive group such as thiol to

generate the antibody-effector conjugate, which is again purified in an additional

purification step (see, e.g., U.S. patents 5,208,020, 5,416,064, or 5,024,834). Thus, in

the above process, at least two purification steps are needed.

[05] Another approach that involves two reaction and purification steps to conjugate

antibody with an effector or reporter group uses the reaction of thiol residues in antibody

(generated via modification of antibody with thiol-generating reagents such as 2

iminothiolane, or via mutagenesis to incorporate non-native cysteine residues, or via

reduction of native disulfide bonds) with a homobifunctional linker Y-L-Y containing Y

reactive groups (such as maleimide or haloacetamide).

[06] Major drawbacks of incorporating a reactive group Y such as maleimide (or

haloacetamide) in an antibody or peptide are the propensity of the reactive maleimide (or

haloacetamide) groups to undergo intra- or inter-molecular reaction with the native

histidine, lysine, tyrosine, or cysteine residues in antibody or peptide (Papini, A. et al.,

Int. J. Pept. Protein Res., 1992, 39, 348-355; Ueda, T. et al., Biochemistry, 1985, 24,

6316-6322), and aqueous inactivation of the Y maleimide group. The undesired intra molecular or inter-molecular reaction of maleimide (or haloacetamide) groups Y incorporated in antibody with the native histidine, lysine, or cysteine residues in antibody, and aqueous inactivation of the Y maleimide group before the second reaction with the effector or reporter group C give rise to cross-linked proteins or heterogeneous conjugates and lower the efficiency of the second reaction with the effector or reporter group C. The heterogeneous conjugate product-cross-linked protein or peptide generated from the undesired reaction of the initially incorporated group Y (such as maleimide group) with native groups in the antibody or peptides (such as histidine, lysine, tyrosine, or cysteine), or with inactive maleimide residues generated by aqueous inactivation-may have inferior activity and stability than the desired homogeneous conjugate product.

[07] Processes for conjugating antibodies to thiol-containing cytotoxic agents via

disulfide linkages have been described previously (see, e.g., U.S. patents 5,208,020,

,416,064, 6,441,163, U.S. Patent Publication No. 2007/0048314 Al). These processes

involve the initial reaction of antibody with a heterobifunctional reagent, followed by a

second reaction with a thiol-containing cytotoxic agent. An alternative process has been

described in U.S. patent 6,441,163 BI in which the disulfide-linked reactive ester of the

cytotoxic agent is first purified and then reacted with the antibody, but which involves an

additional reaction and purification step starting from the thiol group-containing

cytotoxic agent before the reaction step with the antibody.

[08] A further drawback of the current process to make conjugates of cell binding

agents is the need for two purification steps, which lowers the overall yield and also

makes the process cumbersome and uneconomical for scale-up.

22081847. DCC -08/11/2021

[09] In view of the foregoing, there is a need in the art to develop improved methods of preparing cell-binding agent-drug conjugate compositions that are of substantially high purity and can be prepared avoiding cumbersome steps and by reducing time and cost to the user. Embodiments of the present invention provide such a method. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

SUMMARY OF THE INVENTION

[10] The present invention describes a conjugation method for preparing non-reducible, thioether-linked conjugates of the formula C-L-CBA, wherein C represents an effector or reporter molecule (e.g., a cytotoxic agent or a radiolabel), L is a linker and CBA is a cell binding agent (e.g., an antibody or a fragment thereof), by utilizing a direct reaction of the thiol-containing cytotoxic agent (e.g., maytansinoids) with a hetero-or a homo-bifunctional reagent, (e.g., cleavable or a non-cleavable linker) followed by mixing of the unpurified reaction mixture with a cell binding agent (e.g., an antibody or a fragment thereof), thereby generating the non-reducible, thioether-linked conjugate by a process that is more efficient, has a high yield, and is amenable for scale up. Another important advantage is that such conjugation method yields thioether-linked non-reducible conjugates with no inter-chain protein cross-linking or inactivated residues(e.g., maleimide or haloacetamide residues). The novel methods disclosed in this application can be applied to the preparation of any conjugate represented by the above formula.

[10a] According to a first aspect, the present disclosure provides a process for preparing a purified conjugate in a solution, wherein the conjugate comprises an effector or a reporter molecule linked to a cell binding agent through a disulfide bond, the process comprising the steps of: (a) contacting an effector or a reporter molecule comprising a thiol group with a bifunctional linker reagent represented by the following formula:

22081847.:DCC -08/112021

S O-N SO 3 H 0 to covalently attach the linker to the effector or the reporter molecule and thereby prepare an unpurified first mixture of step (a) comprising the effector or the reporter molecule having linkers bound thereto, (b) conjugating a cell binding agent to the effector or the reporter molecule having linkers bound thereto by reacting the unpurified first mixture with the cell binding agent in a solution having a pH to prepare a second mixture, and (c) subjecting the second mixture to tangential flow filtration, dialysis, gel filtration, adsorptive chromatography, selective precipitation or a combination thereof to thereby prepare the purified conjugate.

[10b] According to a second aspect, the present disclosure provides a process for preparing a purified conjugate in a solution, wherein the conjugate comprises an effector or a reporter molecule comprising a thiol group linked to a cell binding agent, the process comprising the steps of: (a) contacting the effector or the reporter molecule comprising a thiol group with a bifunctional linker reagent represented by one of the following formulas

5A

22081847.1:DCC -08/11/2021

ON

S 0 0

S O-N: N0 0 SOM

-- Nh

S0311 > 0

O3H 0 S0 311

00

0 0N1

H0 :0. N- o o 4or o 0 0 0

to covalently attach the linker to the effector or the reporter molecule and thereby prepare an unpurified first mixture comprising the effector or the reporter molecule having linkers bound thereto, (b) mixing the unpurified first mixture comprising the effector or the reporter molecule having linkers bound thereto with a cell binding agent at a pH of about 5, to form an unpurified second mixture, (c) conjugating the cell binding agent to the effector or the reporter molecule having linkers bound thereto by increasing the pH of the unpurified second mixture to about 6.5 to about 8.5 to prepare a third mixture, and (d) subjecting the third mixture to tangential flow filtration, dialysis, gel filtration, adsorptive chromatography, selective precipitation or a combination thereof to thereby prepare the purified conjugate.

[10c] According to a third aspect, the present disclosure provides a purified conjugate in a solution obtained by the process according to the first or second aspect.

5B

BRIEF DESCRIPTION OF THE DRAWINGS

[11] Figure 1 shows conjugation of antibody with a reaction mixture of the

maytansinoid DM1 (or DM4) and Maleimide-PEG,-NHS linker

[12] Figure 2 shows reducing SDS-PAGE of Ab-(PEG 4 -Mal)-DM4 conjugates

prepared using the method described in this invention versus conjugates prepared using

the traditional 2-step method. Each sample lane contained 10 pg protein; the gel was

stained with Coomassie Blue. Lanes 1 and 2 contained molecular weight markers. Lane

3 contained conjugate prepared by the traditional two-step method with 6.1 DM4 per Ab.

Lane 4 contained conjugate prepared by the method described in this invention and

contained 6.2 DM4 per Ab.

[13] Figure 3 shows Protein LabChip electrophoresis of Ab-(PEG 4-Mal)-DM4

conjugates prepared using the method described in this invention versus conjugates

prepared using the traditional 2-step method. A. Protein LabChip electrophoresis under

reducing condition (Agilent 2160 Bioanalyzer/Agilent Protein 230 kit) of Ab-(PEG 4

Mal)-DM4 conjugates. Lane 1: molecular weight markers; lane 2: Ab-PEG 4 -Mal-DM4,

6.2 D/Ab, synthesized using the method described in this invention; lane 3: Ab-PEG 4

Mal-DM4, 6.1 D/Ab, synthesized using the 2 step conjugation method; lane 4:

unconjugated Ab (0.24 microgram total protein in each lane). The upper marker, system

peak and lower marker bands represent external markers added from kit. B. Quantitation

of protein bands from Protein LabChip electrophoresis.

[14] Figure 4 shows MS of Ab-(PEG 4 -Mal)-DM4 conjugates prepared using the

method described in this invention versus conjugates prepared using the traditional 2-step

method. A. MS of conjugate prepared by the traditional two-step method with 6.1 DM4 per Ab. Due to significant heterogeneity of the conjugate the MS peaks could not be resolved well. B. MS of conjugate prepared by the method described in this invention and contained 6.2 DM4 per Ab. Due to homogeneity of the conjugate, the MS peaks were well resolved.

[15] Figure 5 shows binding of an anti-CanAg antibody-PEG 4-Mal-DM1 conjugate

with 6.7 DM1 per antibody (prepared using the method described in this invention)

versus binding of unmodified antibody toward CanAg antigen-expressing COL0205

cells. The binding was measured in fluoresence units.

[16] Figure 6 shows in vitro cytotoxicity of an anti-CanAg Antibody-PEG 4 -Mal-DM1

conjugate with 6.7 DM1 per antibody (prepared using the method described in this

invention) toward CanAg antigen-expressing COL0205 cells. The conjugate was added

to COL0205 cells, and after 5 days of continuous incubation with the conjugate, the

viability of the cells was measured using WST-8 assay. A control experiment to

demonstrate the specificity of the conjugate was carried out using an excess of

unconjugated anti-CanAg antibody to block the binding and cytotoxicity of the conjugate

toward target cancer cells.

[17] Figure 7 shows conjugation of antibody with a reaction mixture of DM1 (or

DM4) and Maleimide-Sulfo-NHS linker.

[18] Figure 8 shows reducing SDS-PAGE ofAb-(Sufo-Mal)-DM1 conjugates

prepared using the method described in this invention versus conjugates prepared using

the traditional 2-step method. Each sample lane contained 10 pg protein; the gel was

stained with Coomassie Blue. Lane 1 contained molecular weight markers. Lanes 3 and

contained conjugates prepared by the method described in this invention and contained

3.6 and 5.6 DM1 per Ab, respectively. Lanes 2 and 4 contained conjugates prepared by

the traditional two-step method and contained 4.0 and 5.7 DM1 per Ab, respectively.

[19] Figure 9 shows Protein LabChip electrophoresis of Ab-(Sulfo-Mal)-DM1

conjugate prepared using the method described in this invention versus conjugate

prepared using the traditional 2-step method. A. Protein LabChip electrophoresis under

reducing condition (Agilent 2100 Bioanalyzer/Agilent Protein 230 kit) of Ab-(Sulfo

Mal)-DM1 conjugates. Lane 1: molecular weight markers; lane 2: unconjugated Ab; lane

3: Ab-Sulfo-Mal-DM1, 5.7 D/Ab, synthesized using the 2 step conjugation method; lane

4: Ab-Sulfo-Mal-DM1, 5.6 D/Ab, synthesized using the method described in this

invention; 0.22 microgram total protein loaded per well. The upper marker, system peak

and lower marker bands represent external markers added from kit (0.24 microgram total

protein per well). B. Quantitation of protein bands from Protein LabChip

electrophoresis.

[20] Figure 10 shows LC-MS comparison of Antibody-(Sulfo-Mal)-DM1 conjugate

prepared by the method described in this invention versus by the traditional two-step

conjugation method. A. MS of conjugate with 3.6 DMl/Ab prepared using the method

described in this invention shows a homogeneous conjugate with 1-6 DM1-bearing

discrete conjugate peaks. B. MS of conjugate with 4.0 DMl/Ab prepared by the

traditional two-step conjugation method. The MS for the conjugate prepared by the

traditional two-step method shows peaks corresponding to conjugates, and conjugates

with hydrolyzed or cross-linked linkers (such as conjugate with 2 DM1, plus one L, 2L,

and 3 L), indicating a heterogeneous product.

[21] Figure 11 shows binding of an anti-CanAg antibody-Sulfo-Mal-DM1 conjugate

with 5.6 DM4 per antibody (prepared using the method described in this invention)

versus binding of unmodified antibody toward CanAg antigen-expressing COL0205

cells. The binding was measured in fluoresence units.

[22] Figure 12 shows in vitro cytotoxicity of an anti-CanAg Antibody-Sulfo-Mal-DM1

conjugate with 5.6 DM4 per antibody (prepared using the method described in this

invention) toward CanAg antigen-expressing COL0205 cells. The conjugate was added

to COL0205 cells and after 5 days of continuous incubation with the conjugate, the

viability of the cells was measured using WST-8 assay. A control experiment to

demonstrate the specificity of the conjugate was carried out using an excess of

unconjugated anti-CanAg antibody to block the binding and cytotoxicity of the conjugate

toward target cancer cells.

[23] Figure 13 shows conjugation of antibody with a reaction mixture of DM1 (or

DM4) and Sulfo-NHS SMCC linker.

[24] Figure 14 shows reducing SDS-PAGE of Ab-(SMCC)-DM1 conjugate prepared

using the method described in this invention versus conjugate prepared using the

traditional 2-step method. Each sample lane contained 10 microgram total protein; the

gel was stained with Coomassie Blue. Lane 1 contains molecular weight markers, Lane 2

contains unconjugated Ab, Lane 3 contains conjugate prepared by the traditional two-step

method with 3.1 DM1 per Ab and Lane 4 contains conjugate prepared by the method

described in this invention with 3.1 DM1 per Ab.

[25] Figure 15 shows Protein LabChip electrophoresis of Ab-(SMCC)-DM1 conjugate

prepared using the method described in this invention versus conjugate prepared using the traditional 2-step method. A. Protein LabChip electrophoresis under reducing condition (Agilent 2100 Bioanalyzer/Agilent Protein 230 kit) of Ab-SMCC-DM1 conjugates. Lane 1: molecular weight markers; lane 2: Ab-SMCC-DM1, 3.1 D/Ab, synthesized using the method described in this patent; lane 3: unconjugated Ab; lane 4:

Ab-SMCC-DM1, 3.1 D/Ab, synthesized using the 2 step conjugation method; (0.24

microgram total protein in each lane). The upper marker, system peak and lower marker

bands represent external markers added from kit. B. Quantitation of protein bands from

Protein LabChip electrophoresis.

[26] Figure 16 shows LC-MS comparison of Antibody-(SMCC)-DM1 conjugate

prepared by the method described in this invention versus conjugate prepared by the

traditional two-step conjugation method. A. MS of conjugate prepared by the sequential

two-step method with 3.1 DM1 per Ab. Each major conjugate peak has associated side

peaks due to the presence of hydrolyzed and cross-linked linker fragments. B. MS of

conjugate prepared by the method described in this invention with 3.1 DM1 per Ab. Due

to homogeneity of the conjugate, the MS peaks were well resolved.

[27] Figure 17 shows proposed mechanisms for inter-chain cross-linking and

maleimide inactivation during conjugation by the traditional 2-step method.

[28] Figure 18 shows non-reducing SDS PAGE of Ab-(Sufo-Mal)-DM4 conjugate

prepared using the method described in this invention and quenching of free DM4 thiol

(after the initial DM4 + NHS-Sulfo-Mal heterobifunctional reagent coupling reaction)

using 4-maleimidobuytric acid prior to the antibody conjugation reaction. Each sample

contained 10 g protein; the gel was stained with Coomassie Blue. Lanes 1 and 5

contained molecular weight markers. Lane 2 contained Ab alone. Lane 3 contained conjugate prepared by the method described in this invention without addition of 4 maleimidobuytric acid. Lane 4 contained conjugate prepared by the method described in this invention with addition of 4-maleimidobutyric acid after the initial DM4 + NHS

Sulfo-Mal heterobifunctional reagent (prior to the antibody conjugation step).

[29] Figure 19 shows preparation of disulfide-linked conjugate of antibody using a

reaction mixture of DM1 (or DM4) and SPDB linker.

[30] Figure 20 shows preparation of antibody-maytansinoid conjugate with both

disulfide- and non-cleavable PEG4-Mal linkers via antibody conjugation with an

unpurified reaction mixture of DM1 (or DM4) and both SPDB and NHS-PEG 4 -Mal

linkers.

[31] Figure 21 shows MS of antibody-maytansinoid conjugate with both disulfide- and

non-cleavable PEG4 -Mal linkers (prepared by conjugation of antibody with an unpurified

reaction mixture of DM1, or DM4, and both SPDB and NHS-PEG 4 -Mal linkers).

[32] Figure 22 shows the conjugation of antibody with a reaction mixture of DM1 (or

DM4) and SMCC linker.

[33] Figure 23 shows the MS of antibody-SMCC-DM1 conjugate prepared using

SMCC by the method described in this invention, containing average 3.1 DM1 per

antibody.

[34] Figure 24 shows the preparation of disulfide-linked conjugate of antibody using a

reaction mixture of DM1 (or DM4) and SSNPB linker.

[35] Figure 25 shows the conjugation of antibody with a reaction mixture of DM1 (or

DM4) and heterobifunctional linker with aliphatic linear carbon chain.

DETAILED DESCRIPTION OF THE INVENTION

[36] Reference will now be made in detail to certain embodiments of the invention,

examples of which are illustrated in the accompanying structures and formulas. While the

invention will be described in conjunction with the enumerated embodiments, it will be

understood that they are not intended to limit the invention to those embodiments. On the

contrary, the invention is intended to cover all alternatives, modifications, and

equivalents which may be included within the scope of the present invention as defined

by the claims. One skilled in the art will recognize many methods and materials similar or

equivalent to those described herein, which could be used in the practice of the present

invention.

[37] This invention describes a novel method of conjugating a thiol-containing effector

(e.g., a cytotoxic agent) or a reporter group (e.g., a radiolabel) with a cell binding agent

(e.g., an antibody), in which the thiol-group containing effector or reporter is first reacted

with a bifunctional linker reagent in organic, aqueous, or mixed organic/aqueous solvent,

followed by reaction of the unpurified reaction mixture with the cell binding agent in

organic, aqueous or mixed organic/aqueous solvents.

legend abbreviations

[38] Abbreviations which have been used in the descriptions of the Schemes and the

Examples that follow are:

[39] C = Effector or a reporter group (e.g., a cytotoxic agent or a radiolabel)

[40] L = Linker (e.g., cleavable or a non-cleavable linker)

[41] X = amine-reactive group (e.g., N-hydroxysuccinimide ester (NHS ester), sulfo

NHS ester, p-nitrophenol ester, tetrafluorosulfonate phenyl ester, 1-hydroxy-2-nitro

benzene-4-sulfonic acid ester)

[42] Y = Maleimide, or haloacetamide (iodoacetamide, bromoacetamide)

[43] Yb is a reactive mixed disulfide group (e.g., 2-pyridyldithio, 4-pyridyldithio, 2

nitro-pyridyldithio, 5-nitro-pyridyldithio, 2-carboxy-5-nitro-pyridyldithio)

[44] X'= amide linkage

[45] Y'= thioether (R-S-R') or selenoether (R-Se-R') linkage

[46] Y'= disulfide (R-S-S-R') linkage

[47] In one embodiment of this invention, a process for the preparation of a thioether

linked conjugate of a cell-binding agent with an effector or a reporter molecule is

described, the process comprising the following steps: a) contacting a heterobifunctional

linker of formula X-L-Y with a thiol-containing effector or reporter molecule C (e.g., a

maytansinoid or a radionuclide) in aqueous solvent, organic solvent, or mixed

organic/aqueous reaction mixtures which yields an intermediate product of formula X-L

Y'-C; b) mixing of the reaction mixture without purification with a cell-binding agent

such as an antibody (Ab) to produce a conjugate of formula Ab-(X'-L-Y'-C)m, wherein,

L is a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, or alkynyl

group bearing from 1-10 carbon atoms, a simple or substituted aryl unit (substituents

selected from alkyl, alkoxy, halogen, nitro, fluoro, carboxy, sulfonate, phosphate, amino,

carbonyl, piperidino) or a polyethylene glycol containing unit (preferably 1-500 PEG

spacer, or more preferably 1-24 PEG spacer, or still more preferably 2-8 PEG spacer); X and Y are amine or thiol-reactive group such as N-hydroxysuccinimide ester and maleimide or haloacetamide; Ab is an antibody; m is an integer from 1-20; X' is modified X site (e.g., an amide linkage) upon reaction with antibody; Y' is modified Y site (e.g., thioether linkage) upon reaction with, for example, a cytotoxic agent or a radiolabel of the effector or reporter group; and c) purification of the conjugate by tangential flow filtration, dialysis, or chromatography (e.g., gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography) or a combination thereof.

Preferably, Y is a thiol-reactive group selected from maleimide or haloacetamide.

Preferably, L is a linear or branched alkyl group with 1-6 carbons or 2-8 PEG spacer.

Preferably, C is a cytotoxic agent selected from a maytansinoid, a CC-1065 analog, a

taxane, a DNA-binding agent, and more preferably it is a maytansinoid.

[48] This reaction sequence represented in formulae 1 and 2:

X-L-Y + C --+ X-L-Y'-C (1)

Ab + X-L-Y'-C (unpurified from reaction 1)-+ Ab-(X'-L-Y'-C)m (2)

does not involve any purification of the intermediate product X-L-Y'-C, and therefore

provides the advantage of directly mixing it with the antibody (the unpurified

intermediate product is added to the antibody or, the antibody is added to the unpurified

intermediate product) thereby making the method advantageous for conjugation because

it eliminates the need of a cumbersome purification step. Importantly, this method yields

homogeneous conjugate with no inter-chain protein cross-linking or inactivated '

maleimide residues, in contrast to the inter-chain protein cross-linking and inactivated

maleimide residues observed in conjugates prepared by the traditional two step reaction

and purification sequence.

[49] The reaction 1 can be carried out at high concentrations of the heterobifunctional

linker, X-L-Y, and the effector or reporter group C in aqueous solvent, organic solvent, or

organic/aqueous reaction mixtures, resulting in faster reaction rates than at lower

concentrations in aqueous solutions for conjugates prepared by the traditional two step

reaction and purification sequence.

[50] The intermediate product X-L-Y'-C generated in reaction 1 can be stored

unpurified in a frozen state, at low temperatures in aqueous solvent at appropriate low pH

(e.g., pH -4-6), in organic solvents, or in mixed organic/aqueous mixtures, or in

lyophilized state, for prolonged periods and can be mixed later with the antibody solution

for the final conjugation reaction at a higher pH value of about 4-9, therefore adding to

the convenience of this reaction sequence. The intermediate product can be diluted as

required with organic solvent or with aqueous buffer, or a mixture of organic solvent and

aqueous buffer prior to mixing with the cell binding agent. The term "about" as used

herein in connection with a numerical should be understood to refer to all such numbers,

including all numbers and small variations therefrom. The reaction of the intermediate

product X-L-Y'-C with antibody can be carried out at pH values of about 4 to about pH 9,

preferably in the pH range of about 5 to 8.7, more preferably, in the pH range of about

6.5 to about 8.5, such as, pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,

7.9, 8.0, 8.1, 8.2, 8.3, 8.4, and 8.5, a pH range therein or small variations therefrom. The

buffers used for the reaction of the antibody with the intermediate product X-L-Y'-C in

the preferential pH range of about 6.5 to 8.5 are buffers with pKa values around this pH

range, such as phosphate and HEPES buffer. These preferred buffers should not have primary or secondary amino groups, or other reactive groups, that can react with linker X

(such as N-hydroxysuccinimide ester).

[51] A stoichiometric or a slight excess of C over the heterobifunctional linker X-L-Y

is used in the first reaction to ensure that all Y group (such as maleimide) is reacted

before the unpurified mixture is added to the antibody. An optional additional treatment

with a quenching reagent (such as 4-maleimidobutyric acid, 3-maleimidopropionic acid,

or N-ethylmaleimide, or iodoacetamide, or iodoacetamidopropionic acid) can be done to

ensure that any unreacted C is quenched before mixing with the antibody to minimize any

unwanted thiol-disulfide interchange reaction with the native antibody disulfide groups.

Upon quenching with polar, charged thiol-quenching reagents (such as 4

maleimidobutyric acid or 3-maleimidopropionic acid), the excess, unreacted C is

converted into a polar, charged adduct that can be easily separated from the. covalently

linked conjugate. Optionally, the final reaction mixture 2, before purification, is treated

with nucleophiles, such as amino group containing nucleophiles (e.g., lysine, taurine,

hydroxylamine) to quench any unreacted linker (X-L-Y'-C).

[52] An alternative method for the reaction of antibody with the unpurified initial

reaction mixture of maytansinoids (DMx) and heterobifunctional linker involves mixing

the initial reaction mixture of DMx and heterobifunctional linker (upon completion of the

DMx-linker reaction) with antibody at low pH (pH ~5) followed by addition of buffer or

base to increase the pH to about 6.5-8.5 for the conjugation reaction.

[53] This new method is applied to the preparation of an antibody conjugate with the

cytotoxic maytansinoid drug. The antibody-maytansinoid conjugates prepared using this

method outlined in the reaction sequence 1-2 unexpectedly were much superior in homogeneity compared to the conjugates prepared by the traditional two step reaction and purification sequence, based on characterization of the conjugates by reducing SDS

PAGE, protein LabChip electrophoresis, and mass spectrometry. The conjugation

method described in this invention that involves the reaction sequence 1-2 also does not

require any intermediate purification step and is therefore significantly more convenient

than the traditional two-step method.

[54] In a second embodiment of the invention, a process for the preparation of a

thioether-linked conjugate of a cell-binding agent with an effector or reporter molecule is

described comprising the following steps: a) contacting a homobifunctional linker of

formula Y-L-Y with a thiol- or amine-containing effector or reporter group C (such as a

cytotoxic agent) in aqueous solvent, organic solvent, or mixed aqueous/organic reaction

mixtures to yield Y-L-Y'-C, b) mixing of the reaction mixture without purification with

an antibody in aqueous solution or aqueous/organic mixture to produce a conjugate of

formula Ab-(Y'-L-Y'-C)m, wherein, L is as defined above; Y is a thiol- or amine-reactive

group such as a maleimide or haloacetamide, or N-hydroxysuccinimide or sulfo N

hydroxysuccinimide; Ab is an antibody; mis an integer from 1 to 20; Y' is the modified

Y site (such as a thioether or amide linkage) upon reaction with antibody or a modified Y

site (such as a thioether or amide linkage) upon reaction with the cytotoxic agent or

effector or reporter group, and c) purification of the conjugate by tangential flow

filtration, dialysis, or chromatography (gel filtration, ion-exchange chromatography,

hydrophobic interaction chromatography) or. a combination thereof. The reaction

sequence represented in formulae 3 and 4:

Y-L-Y + C -+ Y-L-Y'-C (3)

Ab + Y-L-Y'-C (unpurified from reaction 3)-+ Ab-(Y'-L-Y'-C)m (4)

does not involve any purification of the intermediate product Y-L-Y'-C, and therefore is

an advantageous method for conjugation.

[55] In a third embodiment, a process for the preparation of a disulfide-linked

conjugate of a cell binding agent with an effector or reporter molecule is described that

comprises of the following steps: a) contacting a heterobifunctional linker of formula X

L-Yb with the effector or reporter group C (such as a cytotoxic agent) in aqueous solvent,

organic solvent or mixed organic/aqueous reaction mixtures to yield intermediate product

X-L-Y'-C; b) mixing of the reaction mixture without purification with the antibody in an

aqueous solution or aqueous/organic mixture to produce a conjugate of formula Ab-(X'

L-Yb'-C)m, wherein, L is as described above; Yb is a reactive disulfide such as a pyridyl

disulfide or a nitro-pyridyl disulfide; X is an amine-reactive group such as N

hydroxysuccinimide ester or sulfo N-hydroxysuccinimide ester; Ab is an antibody; m is

an integer from 1 to 20; X'is modified X site (such as amide linkage) upon reaction with

antibody; Yb is modified Yb site (disulfide) upon reaction with the cytotoxic agent or

effector or reporter group; and c) purification of the conjugate by tangential flow

filtration, dialysis, or chromatography (gel filtration, in-exchange chromatography,

hydrophobic interaction chromatography) or a combination thereof. The reaction

sequence is represented in formulae 5 and 6:

X-L-Yb + C -+ X-L-Yb'-C (5)

Ab + X-L-Yb'-C (unpurified from reaction 5) - Ab-(X'-L-Yb'-C)m (6)

[56] In a fourth embodiment, a process for the preparation of conjugates of antibody

with effector or reporter groups with two types of linkers -non-cleavable (thioether linkage) and cleavable (disulfide linkage)--comprising the following steps is described: a) contacting X-L-Y and X-L-Yb linkers with the cytotoxic agent C to generate intermediate compounds of formulae X-L-Y'-C and X-L-Y'-C, b) mixing of the reaction mixtures without purification with the antibody either in a sequence or simultaneously as indicated in reaction formulae 7-9:

X-L-Y + C -> X-L-Y'-C (7)

X-L-Yb + C -+ X-L-Yb'-C (8)

Ab + X-L-Y'-C + X-L-Yb'-C (unpurified from reactions 7-8)

-+ Ab-(X'-L-Y'-C)m(X'-L-Yb'-C)m' (9)

to provide a conjugate Ab-(X'-L-Y'-C)m(X'-L-Y'-C)m, wherein, the definitions of X, L,

Y', C, Yb', and m are as given above, and m' is an integer from 1 to 20; and c) purification

of the conjugate by tangential flow filtration, dialysis, or chromatography (gel filtration,

ion-exchange chromatography, hydrophobic interaction chromatography) or a

combination thereof. These two linker effector intermediates (X-L-Y'-C and X-L-Yb'-C)

are mixed without purification with the antibody in different sequences (first X-L-Y'-C

then X-L-Y'-C, or first X-L-Yb'-C then X-L-Y'-C or simultaneously) in various ratio.

[57] The reactions 1, 3, 5, and 7-8, can be carried out at high concentrations of the

bifunctional linker (X-L-Y, X-L-Yb, or Y-L-Y) and the effector or reporter group C in

aqueous solvent, organic solvent, or organic/aqueous reaction mixtures, resulting in faster

reaction rates than at lower concentrations in aqueous solutions for conjugates prepared

by the traditional two step reaction and purification sequence where the solubility of

reagents is limiting.

[58] The intermediate products X-L-Y'-C, or Y-L-Y'-C, or X-L-Yb'-C generated in

reactions 1, 3, 5, and 7-8 can be stored unpurified in a frozen state, at low temperatures in

aqueous solvent at appropriate pH, in organic solvents, or in mixed organic/aqueous

mixtures, or in lyophilized state, for prolonged periods and can be added later to the

antibody solution for the final conjugation reaction, therefore adding to the convenience

of this reaction sequence.

[59] A stoichiometric or a slight excess of C over the heterobifunctional linker X-L-Y,

or Y-L-Y, or X-L-Yb is used in the first reaction to ensure that all Y group (such as

maleimide) is reacted before the unpurified mixture is added to the antibody. An optional

additional treatment with a quenching reagent (such as 4-maleimidobutyric acid, or 3

maleimidopropionic acid, or N-ethylmaleimide, or iodoacetamide, or iodoacetic acid) can

be done to ensure that any unreacted group (such as thiol) in C is quenched before the

addition to the antibody to minimize any unwanted thiol-disulfide interchange reaction

with the native antibody disulfide groups. The quenching of the excess C using a

charged, polar thiol-quenching reagent, after the initial reaction of C with the bifunctional

linker, converts excess C into a highly polar, water-soluble adduct that is easily separated

from the covalently-linked conjugate by gel filtration, dialysis, or TFF. The final

conjugate product does not contain any non-covalently associated C. Optionally, the

final reaction mixtures 2, 4, 6, and 9, before purification, are treated with nucleophiles,

such as, amino group containing nucleophiles (e.g., lysine, taurine, hydroxylamine) to

quench any unreacted linkers (X-L-Y'-C, Y-L-Y'-C, or X-L-Yb'-C).

[60] An alternate method of the reaction of antibody with the unpurified initial reaction

mixture of DMx and bifunctional linker involves mixing the initial reaction mixture of

DMx and bifunctional linker (upon completion of the DMx-linker reaction) with antibody

at low pH (pH -5) followed by addition of buffer or base to increase the pH to about 6.5

8.5 for the conjugation reaction.

[61] Multiple copies of more than one type of effector can be conjugated to the

antibody by adding two or more linker-effector intermediates derived from two or more

different effectors, without purification, to the antibody either in a sequence or

simultaneously.

EFFECTOR GROUP(S)

[62] The terms Effector group or Effector molecule are used interchangeably and the

term "Effector group(s)" or "Effector molecule(s)", as used herein, is meant to include

cytotoxic agents. In certain respects, it may be desirable that the effector groups or

molecules are attached by spacer arms of various lengths to reduce potential steric

hindrance. Multiple copies of more than one type of effector can be conjugated to the

antibody by adding two or more linker-effector intermediates derived from two or more

different effectors, without purification, to the antibody either in a sequence or

simultaneously.

[63] Cytotoxic agents that can be used in the present invention include

chemotherapeutic agents or structural analogues of chemotherapeutic agents.

"Chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.

Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and

cyclophosphamide (CYTOXAN TM); alkyl sulfonates such as busulfan, improsulfan and

piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW

2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen

mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine,

ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such

as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics,

such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin

.gammaland calicheamicin theta I, see, e.g., Angew Chem Intl. Ed. Engl. 33:183-186

(1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin

chromophore and related chromoprotein enediyne antiobiotic chromomophores),

aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin,

carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin,

detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino

doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and

deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins,

mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,

quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin,

zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimnidine analogs such as, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2 ethylhydrazide; procarbazine; PSK*; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T

2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;

mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");

cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL*, Bristol-Myers Squibb

Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE*, Rhone-Poulenc Rorer, Antony,

France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;

platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP

16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;

novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;

topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;

capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; siRNA and pharmaceutically acceptable salts, acids or derivatives of any of the above. Other chemotherapeutic agents that can be used with the present invention are disclosed in US

Publication No. 20080171040 or US Publication No. 20080305044 and are incorporated

in their entirety by reference.

[64] In a preferred embodiment, chemotherapeutic cytotoxic agents are essentially

small molecule cytotoxic agents. A "small molecule drug" is broadly used herein to refer

to an organic, inorganic, or organometallic compound that may have a molecular weight

of for example 100 to 1500, more suitably from 120 to 1200, favorably from 200 to 1000,

and typically having a molecular weight of less than about 1000. Small molecule

cytotoxic agents of the invention encompass oligopeptides and other biomolecules having

a molecular weight of less than about 1000. Small molecule cytotoxic agents are well

characterized in the art, such as in W005058367A2, European Patent Application Nos.

85901495 and 8590319, and in U.S. Patent No. 4,956,303, among others and are

incorporated in their entirety by reference.

[65] Preferable small molecule cytotoxic agents are those that allow for linkage to the

cell-binding agent. The invention includes known cytotoxic agents as well as those that

may become known. Especially preferred small molecule cytotoxic agents include

cytotoxic agents.

[66] The cytotoxic agent may be any compound that results in the death of a cell, or

induces cell death, or in some manner decreases cell viability, wherein each cytotoxic

agent comprises a thiol moiety.

[67] Preferred cytotoxic agents are maytansinoid compounds, taxane compounds, CC

1065 compounds, daunorubicin compounds and doxorubicin compounds,

pyrrolobenzodiazepine dimers, calicheamicins, auristatins and analogues and derivatives

thereof, some of which are described below.

[68] Other cytotoxic agents, which are not necessarily small molecules, such as

siRNA, are also encompassed within the scope of the instant invention. For example,

siRNAs can be linked to the crosslinkers of the present invention by methods commonly

used for the modification of oligonucleotides (see, for example, US Patent Publications

20050107325 and 20070213292). Thus the siRNA in its 3' or 5'-phosphoromidite form

is reacted with one end of the crosslinker bearing a hydroxyl functionality to give an ester

bond between the siRNA and the crosslinker. Similarly reaction of the siRNA

phosphoramidite with a crosslinker bearing a terminal amino group results in linkage of

the crosslinker to the siRNA through an amine. siRNA are described in detail in U.S.

Patent Publication Numbers: 20070275465, 20070213292, 20070185050, 20070161595,

20070054279,20060287260,20060035254,20060008822,20050288244,20050176667,

which are incorporated herein in their entirety by reference.

Maytansinoids

[69] Maytansinoids that can be used in the present invention are well known in the art

and can be isolated from natural sources according to known methods or prepared

synthetically according to known methods.

[70] Examples of suitable maytansinoids include maytansinol and maytansinol

analogues. Examples of suitable maytansinol analogues include those having a modified

aromatic ring and those having modifications at other positions.

[71] Specific examples of suitable analogues of maytansinol having a modified

aromatic ring include:

[72] (1) C-19-dechloro (U.S. Patent No. 4,256,746) (prepared by LAH reduction of

ansamitocin P2);

[73] (2) C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (U.S. Patent Nos.

4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces

or dechlorination using LAH); and

[74] (3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Patent No.

4,294,757) (prepared by acylation using acyl chlorides).

[75] Specific examples of suitable analogues of maytansinol having modifications of

other positions include:

[76] (1) C-9-SH (U.S. Patent No. 4,424,219) (prepared by the reaction of maytansinol

with H2S or P2S5);

[77] (2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Patent No. 4,331,598);

[78] (3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH20Ac) (U.S. Patent

No. 4,450,254) (prepared from Nocardia);

[79] (4) C-15-hydroxy/acyloxy (U.S. Patent No. 4,364,866) (prepared by the

conversion of maytansinol by Streptomyces);

[80] (5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (isolated from

Trewia nudiflora);

[81] (6) C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared by

the demethylation of maytansinol by Streptomyces); and

[82] (7) 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared by the titanium

trichloride/LAH reduction of maytansinol).

[83] The synthesis of thiol-containing maytansinoids useful in the present invention is

fully disclosed in U.S. Patent Nos. 5,208,020, 5,416,064, and U. S. Patent Application

No.20040235840.

[84] Maytansinoids with a thiol moiety at the C-3 position, the C-14 position, the C-15

position or the C-20 position are all expected to be useful. The C-3 position is preferred

and the C-3 position of maytansinol is especially preferred. Also preferred are an N

methyl-alanine-containing C-3 thiol moiety maytansinoid, and an N-methyl-cysteine

containing C-3 thiol moiety maytansinoid, and analogues of each.

[85] Specific examples of N-methyl-alanine-containing C-3 thiol moiety maytansinoid

derivatives useful in the present invention are represented by the formulae M1, M2, M3,

M6 and M7.

OH 3 0

N (CH 2)1SH 0) HN 0 OH 3

May

Ml

wherein:

/ is an integer of from 1 to 10; and

May is a maytansinoid.

CH 3 0 R, R2

N) CH-CH-(CH 2 )mSH 0 CH 3

May M2

wherein:

R 1 and R2 are H, CH3 or CH 2CH 3, and may be the same or different;

m is 0, 1, 2 or 3; and

May is a maytansinoid.

N (CHa 2) SH 01 hay M3

wherein:

n is an integer of from 3 to 8; and

May is a maytansinoid.

0 0 N ,(CH2)/SH

Yo \ O X30 N 00 O

N HL-O MeO M6

wherein:

lis1,2or3;

Yo is Cl or H; and

X 3 is H or CH3 .

CH 3 0 0J I I N CH-CH-(CR 3R 4)mSH

) OH 3 May

M7

wherein:

R 1, R2 , R3, R4 are H, CH3 or CH2 CH 3, and may be the same or different;

m is 0, 1, 2 or 3; and

May is a maytansinoid.

[86] Specific examples of N-methyl-cysteine-containing C-3 thiol moiety

maytansinoid derivatives useful in the present invention are represented by the formulae

M4 and M5.

SH (CH 2 )0 0

N (CH 2)pCH 3 0 may M4

wherein:

ois1,2or3;

p is an integer of 0 to 10; and

May is a maytansinoid.

SH (OH2)o O

N (CH 2)qCH 3

x30 N' O

YO O

N OH MeO M5

wherein:

ois1,2or3;

q is an integer of from 0 to 10;

Yo is Cl or H; and

X3 is H or CH3 .

[87] Preferred maytansinoids are those described in U.S. Patent Nos. 5,208,020;

,416,064; 6,333.410; 6,441,163; 6,716,821; RE39,151 and 7,276,497.

Taxanes

[88] The cytotoxic agent according to the present invention may also be a taxane.

[89] Taxanes that can be used in the present invention have been modified to contain a

thiol moiety. Some taxanes useful in the present invention have the formula T1 shown

below:

0 ~ R2 0 0 OR5

RNH - ~ 1 10 09 876 GRc 4 N O R515 5

4 H 0s 3 OAc OR6 RI"

[90] Preferred taxoids are those described in U.S. Patent Nos. 6,340,701; 6,372,738;

6.436,931; 6,596,757; 6,706,708; 7,008,942; 7,217,819 and 7,276,499.

[91] CC-1065 analogues

[92] The cytotoxic agent according to the present invention may also be a CC-1065

analogue.

[93] According to the present invention, the CC-1065 analogues contain an A subunit

and a B or a B-C subunit. Preferred CC-1065 analogs are those described in U.S. Patent

Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316.

Daunorubicin/DoxorubicinAnalogues

[94] The cytotoxic agent according to the present invention may also be a

daunorubicin analogue or a doxorubicin analogue.

[95] The daunorubicin and doxorubicin analogues of the present invention can be

modified to comprise a thiol moiety. The modified doxorubicin/daunorubicin analogues

of the present invention, which have a thiol moiety, are described in WO 01/38318. The

modified doxorubicin/daunorubicin analogues can be synthesized according to known

methods (see, e.g., U.S. Patent No. 5,146,064).

[96] Auristatin include auristatin E, auristatin EB (AEB), auristatin EFP (AEFP),

monomethyl auristatin E (MMAE) and are described in U.S. Patent No. 5,635,483, Int. J.

Oncol. 15:367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-932

(2004); U.S. Application Number 11/134826. U.S. Patent Publication Nos.

20060074008,2006022925.

[97] The cytotoxic agents according to the present invention include

pyrrolobenzodiazepine dimers that are known in the art (US Patent Nos 7,049,311;

7,067,511; 6,951,853; 7,189,710; 6,884,799; 6,660,856).

Analogues and derivatives

[98] One skilled in the art of cytotoxic agents will readily understand that each of the

cytotoxic agents described herein can be modified in such a manner that the resulting

compound still retains the specificity and/or activity of the starting compound. The

skilled artisan will also understand that many of these compounds can be used in place of

the cytotoxic agents described herein. Thus, the cytotoxic agents of the present invention

include analogues and derivatives of the compounds described herein.

REPORTER GROUP(S)

[99] The terms Reporter group or Reporter molecule are used interchangeably and the

term "Reporter group(s)" or "Reporter molecule(s)", as used herein, refers to a substance

which is delivered to the specific substance or cells by the specific affinity portion of the

reagent, for a diagnostic or therapeutic purpose; examples are radioisotopes,

paramagnetic contrast agents, and anti-cancer agents. Various labels or reporter groups

are useful for tumor-imaging applications in cancer patients, immunoassay applications for diagnosis of various diseases, cancer therapy using radioactive nuclide-ligand conjugates, and affinity chromatography applications for purification of bioactive agents such as proteins, peptides, and oligonucleides. The labels or reporter groups that are conjugated with cell-binding agents include fluorophores, and affinity labels such as biotin. Such reporter group references can be found in US publication number

2007/0092940. Reporter groups including, for example, biotin or fluorescein can also be

attached to a PEG conjugate moiety. A number of suitable reporter groups are known in

the art, e.g., U.S. Pat. No. 4,152,411 and Hirschfeld U.S. Pat. No. 4,166,105, U.S. Pat.

No. 5,223,242, U.S. Pat. No. 5,501,952, US publication 20090136940 and are

incorporated in their entirety by reference.

LINKERS

[100] The conjugates may be prepared by in vitro methods. In order to link a drug to

the cell-binding agent, a linking group is used. Suitable linking groups are well known in

the art and include non-cleavable or cleavable linkers. A non-cleavable linker is any

chemical moiety that is capable of linking a cytotoxic agent to a cell-binding agent in a

stable, covalent manner. Non-cleavable linkers are substantially resistant to acid-induced

cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage,

and disulfide bond cleavage. Examples of non-cleavable linkers include linkers having

an N-succinimidyl ester, N-sulfosuccinimidyl ester moiety, maleimido- or haloacetyl

based moiety for reaction with the drug, the reporter group or the cell binding agent.

Crosslinking reagents comprising a maleimido-based moiety include N-succinimidyl 4

(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), x-maleimidoundecanoic acid N-succinimidyl ester

(KMUA), y-maleimidobutyric acid N-succinimidyl ester (GMBS), e-maleimidocaproic

acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide

ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(p

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-4-(iodoacetyl)

aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate

(SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[101] Other crosslinking reagents lacking a sulfur atom that form non-cleavable linkers

can also be used in the inventive method. Such linkers can be derived from dicarboxylic

acid based moieties. Suitable dicarboxylic acid based moieties include, but are not

limited to, a,o-dicarboxylic acids of the general formula shown below:

HOOC-Xi-Yn-Zm-COOH

[102] 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, 0 or S, and wherein 1, m, and n are each 0 or 1, provided that 1, m, and n are all not

zero at the same time.

[103] Many of the non-cleavable linkers disclosed herein are described in detail in U.S.

Patent publication number 20050169933.

[104] Cleavable linkers are linkers that can be cleaved under mild conditions, i.e.

conditions under which the activity of the cytotoxic agent is not affected. Many known

linkers fall in this category and are described below.

[105] 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

), and provide conditions suitable to cleave acid-labile linkers.

[106] Linkers that are photo-labile are useful at the body surface and in many body

cavities that are accessible to light. Furthermore, infrared light can penetrate tissue.

[107] Some linkers can be cleaved by peptidases. Only certain peptides are readily

cleaved inside or outside cells, see e.g. Trouet et al., 79 Proc. Natl. Acad. Sci. USA, 626

629 (1982), Umemoto et al. 43 Int. J. Cancer, 677-684 (1989), and lysosomal-hydrolase

cleavable valine-citrulline linkage (US Patent 6,214,345 B1). Furthermore, peptides are

composed of .alpha.-amino acids and peptidic bonds, which chemically are amide bonds

between the carboxylate of one amino acid and the .alpha.-amino group of a second

amino acid. Other amide bonds, such as the bond between a carboxylate and the.epsilon.

amino group of lysine, are understood not to be peptidic bonds and are considered non

cleavable.

[108] Some linkers can be cleaved by esterases. Again only certain esters can be

Cleaved by esterases present inside or outside cells. Esters are formed by the condensation

of a carboxylic acid and an alcohol. Simple esters are esters produced with simple

alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols. For

example, the present inventors found no esterase that cleaved the ester at C-3 of maytansine, since the alcohol component of the ester, maytansinol, is very large and complex.

[109] Preferred cleavable linker molecules 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 341498-08-6), and other reactive cross-linkers, such as those described in U.S.

Patent 6,913,748, which is incorporated herein in its entirety by reference.

[110] Other linkers which can be used in the present invention include charged linkers

or hydrophilic linkers and are described in U.S. Patent Application Nos., 12/433,604 and

12/433,668, respectively, which are incorporated herein in its entirety by reference.

CELL BINDING AGENTS

[111] The cell-binding agents used in this invention are proteins (e.g., immunoglobulin

and non-immunoglobulin proteins) which bind specifically to target antigens on cancer

cells. These cell-binding agents include:

-antibodies including:

-resurfaced antibodies (U.S. patent no. 5,639,641);

-humanized or fully human antibodies (Humanized or fully human antibodies are

selected from, but not limited to, huMy9-6, huB4, huC242, huN901, DS6, CD38, IGF-IR,

CNTO 95, B-B4, trastuzumab, bivatuzumab, sibrotuzumab, and rituximab (see, e.g., U.S.

Patent Nos. 5,639,641, 5,665,357, and 7,342,110, International Patent Application WO

02/16,401, U.S. publication number 20060045877, U.S. publication number

20060127407, U.S. publication number 20050118183, Pedersen et al., (1994) J. Mol.

Biol. 235, 959-973, Roguska et al., (1994) Proceedingsof the NationalAcademy of

Sciences, Vol 91, 969-973, 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)); and

-fragments of antibodies such as sFv, Fab, Fab', and F(ab') 2 that preferentially

bind to a target cell (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));

[112] Additional cell-binding agents include other cell binding proteins and

polypeptides exemplified by, but not limited to:

-Ankyrin repeat proteins (DARPins; Zahnd et al., J. Biol. Chem., 281, 46, 35167

35175, (2006); Binz, H.K., Amstutz, P. & Pluckthun, A. (2005) Nature Biotechnology,

23, 1257-1268) or ankyrin-like repeats proteins or synthetic peptides described, for

example, in U.S. publication number 20070238667; U.S. Patent No. 7,101,675;

WO/2007/147213; WO/2007/062466);

-interferons (e.g. a, P, y);

-lymphokines such as IL-2, IL-3, IL-4, IL-6;

-hormones such as insulin, TRH (thyrotropin releasing hormones), MSH

(melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens;

and

-growth factors and colony-stimulating factors such as EGF, TGF-a, IGF-1,

G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158 (1984)).

[113] 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; alpha

1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone;

calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vmc, factor IX,

tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C;

atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or

human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin;

hemopoietic growth factor; tumor necrosis factor-a and -P; enkephalinase; RANTES

(regulated on activation normally T-cell expressed and secreted); human macrophage

inflammatory protein (MIP-1-alpha); a serum albumin such as human serum albumin;

Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse

gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase;

IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;

activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth

factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived

neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6),

or a nerve growth factor such as NGF-p.; 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 betal, TGF-p2, TGF- P3, TGF-P4, or TGF- P5; 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;

CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD40; 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, alpha-V subunit of a

heterodimeric human integrin receptor; a tumor associated antigen such as HER2, HER3

or HER4 receptor; and fragments of any of the above-listed polypeptides.

[114] Preferred antigens for antibodies encompassed by the present invention include

CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD46; members of the

ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell

adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM,

alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits

thereof (e.g. anti-CDl la, anti-CD18 or anti-CD11b antibodies); growth factors such as

VEGF; tissue factor (TF); TGF-p.; alpha interferon (alpha-IFN); an interleukin, such as

IL-8; IgE; blood group antigens Apo2, death receptor; fik2/flt3 receptor; obesity (OB)

receptor; mpl receptor; CTLA-4; protein C etc. The most preferred targets herein are

IGF-IR, CanAg, EGF-R, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22,

CD33, CD37, CD38, CD40, CD44, CD56, CD138, CA6, Her2/neu, CRIPTO (a protein

produced at elevated levels in a majority of human breast cancer cells), alpha v/beta3

integrin, alpha v/beta5 integrin, TGF- P, CD11a, CD18, Apo2 and C24.

[115] Monoclonal antibody techniques allow for the production of specific cell-binding

agents in the form of monoclonal antibodies. Particularly well known in the art are

techniques for creating monoclonal antibodies produced by immunizing mice, rats,

hamsters or any other mammal with the antigen of interest such as the intact target cell,

antigens isolated from the target cell, whole virus, attenuated whole virus, and viral

proteins such as viral coat proteins. Sensitized human cells can also be used. Another

method of creating monoclonal antibodies is the use of phage libraries of sFv (single

chain variable region), specifically human sFv (see, e.g., Griffiths et al, U.S. patent no.

,885,793; McCafferty et al, WO 92/01047; Liming et al, WO 99/06587.)

[116] Selection of the appropriate cell-binding agent is a matter of choice that depends

upon the particular cell population that is to be targeted, but in general monoclonal

antibodies and fragments thereof that preferentially bind to a target cell are preferred, if

an appropriate one is available.

[117] For example, the monoclonal antibody My9 is a murine IgG 2 a antibody that is

specific for the CD33 antigen found on Acute Myeloid Leukemia (AML) cells (Roy et al.

Blood 77:2404-2412 (1991)) and can be used to treat AML patients. Similarly,the

monoclonal antibody anti-B4 is a murine IgG 1, that binds to the CD19 antigen on B cells

(Nadler et al, J. Immunol. 131:244-250 (1983)) and can be used if the target cells are B

cells or diseased cells that express this antigen such as in non-Hodgkin's lymphoma or

chronic lymphoblastic leukemia. Similarly, the antibody N901 is a murine monoclonal

IgGi antibody that binds to CD56 found on small cell lung carcinoma cells and on cells of

other tumors of neuroendocrine origin (Roy et al. J. Nat. CancerInst. 88:1136-1145

(1996)), huC242 antibody that binds to the CanAg antigen, Trastuzumab that binds to

HER2/neu, and anti-EGF receptor antibody that binds to EGF receptor.

PURIFICATION METHODS

[118] The conjugate, i.e., the finalized product, of the present invention is purified to

remove any unreacted or unconjugated effector or reporter molecule, or unreacted linker

or unconjugated, hydrolyzed linker. The purification method can be a tangential flow

filtration (TFF, also known as cross flow filtration, ultrafiltration, or diafiltration), gel

filtration, adsorptive chromatography, selective precipitation, or combinations thereof.

The adsorptive chromatography methods include ion-exchange chromatography,

hydroxyapatite chromatography, hydrophobic interaction chromatography (HIC),

hydrophobic charge induction chromatography (HCIC), mixed mode ion exchange

chromatography, immobilized metal affinity chromatography (IMAC), dye ligand

chromatography, affinity chromatography, and reversed phase chromatography. For

example, the conjugate Ab-(X'-L-Y'-C)m described in formula 2 is purified from

unreacted C or unreacted/hydrolyzed linker X-L-Y or X-L-Y'-C. Similarly, the

conjugates described in formulae 4, 6, and 9 are purified. Such methods of purification

are known to one of skill in the art and can be found, for example, in US Publication No.

2007/0048314.

UNDESIRED HYDROLYZED LINKER OR PROTEIN CROSS-LINKING IN CONJUGATES

[119] Traditional conjugation methods employing the initial reaction of a protein with a

heterobifunctional linker with reactive maleimide or haloacetamide residue suffer from

two major drawbacks: (i) the conjugate product may consist of hydrolyzed linker, due to

aqueous inactivation of the incorporated linker in the antibody before reaction with the

effector or reporter molecule; and (ii) inter-or intrachain cross-linking of conjugate, due

to reaction of maleimide (or haloacetamide) group with the native histidine, lysine,

tyrosine, or cysteine residues in protein or peptide (A. Papini et al., Int. J. Pept. Protein

Res., 1992, 39, 348-355; T. Ueda et al., Biochemistry, 1985, 24, 6316-6322). Such

interchain cross-linking in antibody would result in various non-reducible covalent

linkages between the heavy and light chains, or between two heavy chains, which would

be apparent in reducing SDS-PAGE analysis as bands of higher molecular weights than

the expected heavy and light chain bands. Such interchain or intrachain cross-linking in

antibody would also be apparent by MS as peaks of aberrant masses different than the

expected masses of antibody plus linked reporter or effector groups. Unlike traditional

conjugation methods, the method described in this application results in conjugates with

high homogeneity with no substantial interchain cross-linking or hydrolyzed linker.

[120] All references cited herein and in the examples that follow are expressly

incorporated by reference in their entireties.

EXAMPLES

[121] Thefollowing examples, which are illustrative only, are not intended to limit the

present invention.

Example 1. Conjugation of antibody with cytotoxic agent DM1/DM4 using

heterobifunctional linker Maleimide-PEG 1 -NHS by this method (Figure 1) versus

traditional two-step method.

[122] Stock solutions of DM1 [N2 -deacetyl-N2 -(3-mercapto-1-oxopropyl)-maytansine],

or DM4 [N2-deacetyl-N -(4-mercapto-4-methyl-1-oxopentyl)maytansine] (DMx) thiol

and the Maleimide-PEG,-NHS bifunctional linker were made up in N,N

dimethylacetamide (DMA) at concentrations of 30-60 mM. The linker and DMx thiol

were mixed together in DMA containing up to 50% v/v of 200 mM succinate buffer, 2

mM EDTA, pH 5.0 to give a molar ratio of DMx to linker of 1.6:1 and a final

concentration of DMx equal to 15 mM. After mixing, the reaction mixture was left for 1

4 h and then an aliquot of the reaction mixture was diluted 10 fold and its absorbance

measured at 302-320 nm to determine the presence of any remaining unreacted

maleimide using the extinction coefficient (s) of maleimide at 302 nm= 620 M-1 cm-1,

and 320 nm 450 M~1 cm-1 . (Additional reverse phase HPLC analysis of a frozen

aliquot of the reaction mixture was carried out later with absorbance monitoring at 302

nm and 252 nm to verify complete disappearance of linker maleimide and formation of

the desired linker-DMx reagent at the time of addition of the reaction mixture to

antibody). When no further maleimide was present by UV, an aliquot of the reaction

mixture was added without purification to a solution of antibody in phosphate buffer (pH

7.5) under final conjugation conditions of 4 mg/ml Ab, 90% phosphate buffer/10%

DMA, pH 7.5. The conjugation reaction was allowed to proceed at ambient temperature

for 2 h. Ab-DMx conjugate was purified from the excess small-molecule DMx and linker

reactants using a G25 gel filtration column equilibrated in pH 7.5 phosphate buffer, or

using tangential flow filtration (TFF). The conjugation mixture was further kept at 4 °C

for 2 days in pH 7.5 buffer to allow the dissociation of any DMx species attached to

antibody non-covalently or via labile linkage. The conjugate was then dialyzed overnight

into pH 5.5 histidine/glycine buffer and then filtered through a 0.22 m filter for final

storage. The number of DMx molecule per Ab molecule (average) in the final conjugate

was measured by determining absorbance of the conjugate at 252 and 280 nm and using

known extinction coefficients for DMx and antibody at these two wavelengths.

[123] Several different reaction conditions were used for the initial reaction of DMx

thiol with the heterobifunctional maleimide-PEG 4-NHS reagent: 50% DMA/50%

aqueous 200 mM succinate buffer pH 5.0,2 mM EDTA (v/v); or 60% DMA/40% 200

mM succinate buffer pH 5.0, 2 mM EDTA (v/v); or 100% DMA with 1.5 molar

equivalents of an organic base (for example N,N'-diisopropyl ethylamine, DIPEA, or 4

methylmorpholine) per mol DM4 thiol.

[124] In one series of experiments, the molar equivalent of DMx to maleimide-PEG 4

NHS linker (purchased from Pierce Endogen) was varied from 1.2 - 2.4, and the reaction

time was 30 min. The number of DMx/Ab measured on purified conjugates were

measured as a function of added equivalents of DMx per linker. Conditions of 1.2 - 2.0

equivalents of DM1/Linker gave conjugates with similar DMx/Ab loads, indicating that

the undesired reaction of the DMx thiol at the NHS ester side of the linker is not a significant problem. The amount of cross-linking present in the final conjugates was also analyzed by reducing SDS PAGE showing that the presence of cross-linked contaminants decreases significantly with increasing DM1/linker ratio.

[125] One optional quenching step using maleimide or haloacetamide reagents (such as

maleimidobutyric acid, or maleimidopropionic acid, or N-ethylmaleimide, or

iodoacetamide, or iodoacetic acid) was introduced after the completion of the initial DMx

and heterobifunctional linker reaction (before the addition of the reaction mixture to the

antibody) to quench the excess DMx thiol group in order to prevent any unwanted

reaction of DMx thiol with the antibody.

[126] An alternate method of the reaction of antibody with the unpurified initial reaction

mixture of DMx and heterobifunctional linker involved mixing the initial reaction

mixture of DMx and heterobifunctional linker (upon completion of the DMx-linker

reaction) with antibody at low pH (pH -5) followed by the addition of buffer or base to

increase the pH to 6.5-8.5 for the conjugation reaction.

[127] An antibody-PEG 4-Mal-DM1 or DM4 conjugate was made by the traditional two

step conjugation method for comparison with the conjugation method described in this

invention. The humanized antibody at a concentration of 8 mg/ml in pH 7.5 phosphate

buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA, pH 7.5) and

% DMA was modified with excess heterobifunctional maleimide-PEG 4-NHS linker

reagent (purchased from Pierce Endogen). After 2 h at 25 °C, the modified antibody was

gel purified by G25 chromatography to remove excess unreacted, unincorporated linker.

The recovery of purified Ab was determined by UV absorbance at 280 nm. The number

of linked maleimide groups in the modified Ab was determined using a small aliquot of modified antibody by addition of a known amount of thiol (such as 2-mercaptoethanol), added in excess over the maleimide, to react with the maleimide residues in the modified antibody and then assaying the remaining thiol by Ellman's assay using DTNB reagent

(extinction coefficient of TNB thiolate at 412 nm= 14150 M- cm-; Riddles, P. W. et al.,

Methods Enzymol., 1983, 91, 49-60; Singh, R., Bioconjugate Chem., 1994, 5, 348-351).

The conjugation of modified Ab with DM1 or DM4 thiol was carried out at an Ab

concentration of 2.5 mg/ml in a reaction mixture consisting of 95% phosphate buffer pH

7.5 (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA, pH 7.5) and

% DMA. An excess of 1.7 molar equivalents of DM1 or DM4 thiol was added per mol

of linked maleimide on the Ab. After reacting overnight at 25 °C, the conjugate was

sterile filtered using a 0.22 pm filter and gel purified from excess unreacted DMl or

DM4 by a G25 column equilibrated in phosphate buffer pH 7.5 (50 mM potassium

phosphate, 50 mM sodium chloride, 2 mM EDTA, pH 7.5). The purified conjugate was

held at 4 °C for 2 days in phosphate buffer pH 7.5 (50 mM potassium phosphate, 50 mM

sodium chloride, 2 mM EDTA, pH 7.5) to allow for the dissociation of any DM1 or DM4

species attached to antibody non-covalently or via a labile linkage. The conjugate was

subsequently dialyzed for 2 days in histidine/glycine buffer pH 5.5 (130 mM glycine/10

mM histidine, pH 5.5) and sterile filtered using a 0.22 pm filter. The number of DM1 or

DM4 molecules per Ab molecule in the final conjugate was measured by determining

absorbance of the conjugate at 252 and 280 nm using known extinction coefficients for

DM1/DM4 and Ab at these two wavelengths.

[128] Reducing SDS PAGE was carried out on conjugate and antibody samples using

the NuPage electrophoresis system with a 4 -12% Bis Tris Gel (Invitrogen). Heat denatured and reduced samples were loaded at 10 tg/lane. The reducing SDS-PAGE of the conjugates prepared using the method described in this invention showed only the expected heavy and light chain bands (50 kDa and 25 kDa respectively) as the major bands (Figure 2). In contrast, the conjugates prepared by the traditional two-step conjugation method showed undesired cross-linked bands with molecular weights of 75,

100, 125, and 150 kDa, presumably corresponding to inter-chain cross-linked species HL,

H2 , H 2L, and H2 L 2 respectively (Figure 2).

[129] Protein LabChip electrophoresis analysis (under reducing condition) of the

antibody-PEG 4 -Mal-DM4 conjugate prepared by the method described in this invention

showed the expected heavy and light chain bands with percentages of 58 and 30% (of

total protein), which are similar to those for unconjugated antibody of 65 and 30%

respectively (Figure 3). In contrast, the conjugate prepared using the traditional two-step

conjugation method showed heavy and light bands of only 16 and 8% respectively, and

major bands of higher molecular weights ranging from 94-169 kDa presumably due to

inter-chain cross-linking. Based on the quantitative Protein LabChip analysis, the

conjugate prepared by the method described in this application is highly superior to that

prepared using the conventional two-step process (Figure 3).

[130] The MS analysis of the conjugates prepared by the method described in this

invention showed discrete DMx-antibody conjugate peaks for antibody bearing

increasing numbers of maytansinoid molecules per antibody molecule (Figure 4). In

contrast, the MS of the conjugate obtained using the traditional 2-step method was nearly

unresolved suggesting inhomogeneity of the conjugate preparation presumably due to

cross-linking or inactivated maleimide linker. Based on MS, therefore, the conjugate prepared using the method described in this invention is superior to that synthesized by the traditional two-step method.

[131] The binding of an anti-CanAg Ab-PEG 4-Mal-DM1 conjugate prepared by the

method described in this invention was measured by flow cytometry using the antigen

expressing COL0205 cells, and was found to be similar to that of unconjugated antibody

suggesting that the conjugation had no detrimental effect on the binding of the antibody

(Figure 5). The cytotoxic activity of the anti-CanAg Ab-PEG 4-Mal-DM1 conjugate

prepared by the method described in this invention was measured in vitro using

COL0205 colon cancer cells expressing the CanAg antigen (Figure 6). The antigen

expressing cancer cells were plated at around 1000 cells/well in a 96 well plate in cell

culture media containing fetal bovine serum and exposed to varying concentrations of

Ab-DMx conjugate. After a 5 day exposure to the conjugate, the viable cells remaining in

each well were measured using the WST-8 assay (Dojindo Molecular Technologies). As

shown in Figure 6, the anti-CanAg Ab-PEG 4-Mal-DM1 conjugate prepared using this

method was highly potent at low concentrations toward CanAg antigen-expressing

COL0205 colon cancer cells. The cytotoxicity of the anti-CanAg Ab-PEG 4 -Mal-DM1

conjugate prepared by the method described in this invention was specific to COL0205

cells as it could be blocked by the addition of excess, unconjugated antibody.

Example 2. Conjugation of antibody with DM1/DM4 using maleimide-Sulfo-NHS

linker by this method (Figure 7) versus traditional sequential two-step method.

[132] Stock solutions of DMx thiol and the maleimide-Sulfo-NHS heterobifunctional

linker were made up in N,N-dimethylacetamide (DMA) at concentrations of 30-60 mM.

The linker and DMx thiol were mixed together in DMA containing up to 40 % v/v of 200

mM succinate buffer, 2 mM EDTA, pH 5.0 to give a ratio of DMx to linker of 1.6 and a

final concentration of DMx equal to 15 mM. After mixing, the reaction was left for 1-4 h

and then an aliquot of the reaction mixture was diluted 10 fold to measure the absorbance

at 302-320 nm for assessing the completion of reaction and the absence of maleimide.

(Additional reverse phase HPLC analysis of a frozen aliquot of the reaction mixture was

carried out later with absorbance monitoring at 302 nm and 252 nm to verify complete

disappearance of linker maleimide and formation of the desired linker-DMx reagent at

the time of addition of the reaction mixture to antibody). When no further maleimide was

present by UV, an aliquot of the reaction mixture was added to a mixture of antibody in

phosphate buffer (pH 7.5) under final conjugation conditions of 4 mg/ml Ab, 90%

phosphate buffer/10% DMA, pH 7.5. The conjugation reaction was allowed to proceed

at ambient temperature for 2 h. The Ab-DMx conjugate was purified from excess

unreacted DMx and unconjugated linker products using a G25 gel filtration column

equilibrated in pH 7.5 phosphate buffer or by tangential flow filtration. The conjugate

was kept at 4 °C for 2 days in pH 7.5 buffer to allow the dissociation of any DMx species

attached to antibody non-covalently or via labile linkage. The conjugate was then

dialyzed overnight into pH 5.5 histidine/glycine buffer and then filtered through a 0.22

m filter for final storage. The number of DMx molecules per Ab antibody molecule

(average) in the final conjugate was measured by determining absorbance of the

conjugate at 252 and 280 nm and using known extinction coefficients for DMx and

antibody at these two wavelengths.

[133] For comparison, the Ab-Sulfo-Mal-DMx conjugates were prepared using the

traditional 2-step conjugation method. The antibody (Ab) at a concentration of 8 mg/ml

in pH 7.5 phosphate buffer/5% DMA buffer was modified with excess bifunctional

maleimide-Sulfo-NHS linker. The reaction was allowed to proceed at 20 °C for 2 h and

then the modified Ab was purified away from excess unreacted linker using G25

chromatography. The recovery of purified Ab was determined by UV absorbance at 280

rm. The number of linked maleimide groups in the modified Ab was determined using a

small aliquot of modified antibody by addition of a known amount of thiol (such as 2

mercaptoethanol), added in excess over the maleimide, to react with the maleimide

residues in the modified antibody and then assaying the remaining thiol by Ellman's assay

using DTNB reagent (extinction coefficient of TNB thiolate at 412 nm= 14150 M-1 cm';

Riddles, P. W. et al., Methods Enzymol., 1983, 91, 49-60; Singh, R., Bioconjugate

Chem., 1994, 5, 348-351). The conjugation of modified Ab with DMx was carried out at

an antibody concentration of 2.5 mg/ml in 95% pH 7.5 phosphate buffer/5% DMA (v/v),

with 1.7 molar equivalents of DMx thiol added per mol of linked maleimide in the Ab.

The reaction was left for 8-24 h at 18 °C and the conjugate was separated from excess,

unreacted DMx via G25 size-exclusion chromatography. After purification the conjugate

was kept at 4 °C for 2 days in pH 7.5 buffer to allow the dissociation of any DMx species

attached to antibody non-covalently or via labile linkage. The conjugate was then

dialyzed overnight into pH 5.5 histidine/glycine buffer and then filtered through a 0.22

pm filter for final storage. The number of DMx molecule per Ab molecule in the final

conjugate was measured by determining absorbance of the conjugate at 252 and 280 nm

and using known extinction coefficients for DMx and antibody at these two wavelengths.

[134] Reducing SDS PAGE was carried out using conjugate and antibody samples (10

pg/lane) using the NuPage electrophoresis system (Invitrogen) with a NuPage 4 -12% Bis

Tris Mini Gel and NuPAGE MOPS SDS running buffer (Figure 8). Bands on the gel

with molecular weights of 75, 125, and 150 kDa were indicative of inter-chain cross

linked species (HL, H 2L and H 2 L 2 respectively). A comparison of Ab-Sulfo-Mal-DM1

conjugates with -4 DM1/Ab (lane 3, by this method, and lane 2, by traditional 2-step

conjugation method, respectively) and -6 DMl/Ab (lane 5, by this method, and lane 4,

by traditional 2-step conjugation method, respectively) clearly shows that conjugates

made via the method described in this invention (lanes 3 and 5) have much smaller

proportion of high molecular weight cross-linked species than conjugates made by the

traditional 2-step method (lanes 2 and 4).

[135] Protein LabChip electrophoresis analysis (under reducing condition) of the

antibody-Sulfo-Mal-DM1 conjugate prepared by the method described in this invention

showed the heavy and light chain major bands with percentages of 70 and 28% (of total

protein), which are similar to those for unconjugated antibody of 70 and 30% respectively

(Figure 9). In contrast, the conjugate prepared using the traditional two-step method

showed heavy and light bands of only 53 and 23% respectively, and major bands of

higher molecular weights ranging from 99-152 kDa presumably due to inter-chain cross

linking. Based on the quantitative Protein LabChip analysis, the conjugate prepared by

the method described in this application is much superior in terms of lack of inter-chain

cross-linking compared to that prepared using the conventional two-step process (Figure

9).

[136] The Ab-Sulfo-mal-DMlconjugates with similar drug loads made via the method

described in this invention and by the traditional two step method were compared by size

exclusion LC/MS analysis (Figure 10). The conjugates made via the method described in

this invention show the desired MS spectrum containing only the expected distribution of

peaks with mass equal to Ab-(linker-DMx)n. In the case of conjugates made using the

traditional two-step method, the major peaks in the spectra all contain one or more

hydrolyzed or cross-linked linker fragments in addition to the desired Ab-(inker-DMx)n

moieties. The putative mechanism of the inter-chain cross-linking or aqueous inactivation

of maleimide in the traditional 2-step reaction sequence is shown in Figure 17, whereby

the incorporated maleimide (or haloacetamide) residue from the initial reaction of

antibody with the heterobifunctional linker can react with intramolecular (or

intermolecular) histidine, lysine, tyrosine, or cysteine residues resulting in inter-chain

cross-linking, or the initially incorporated maleimide (or haloacetamide) residue can get

inactivated (such as by hydrolytic maleimide ring cleavage or by aqueous addition to

maleimide) and therefore become unavailable for the rapid reaction with thiol-bearing

effector or reporter group. Thus the LC-MS analysis clearly shows that the method

described in this invention has the advantage of producing homogeneous conjugate with

little or no hydrolyzed or cross-linked linker fragments attached to antibody.

[137] The binding of an anti-CanAgAb-Sulfo-Mal-DM1 conjugate with 5.6

maytansinoid load per antibody molecule (average) prepared by the method described in

this invention was measured by flow cytometry using the antigen-expressing COL0205

cells, and was found to be similar to that of unconjugated antibody suggesting that the

conjugation did not affect the binding of the antibody to target antigen (Figure 11). The cytotoxic activity of the anti-CanAg Ab-Sulfo-Mal-DM1 conjugate prepared by the method described in this invention was measured in vitro using COL0205 colon cancer cells expressing the CanAg antigen (Figure 12). The antigen-expressing cancer cells were plated at around 1000 cells/well in a 96 well plate in cell culture media containing fetal bovine serum and exposed to varying concentrations of Ab-DMx conjugate. After a day exposure to the conjugate, the viable cells remaining were measured using the

WST-8 assay (Dojindo Molecular Technologies). As shown in Figure 12, the anti

CanAg Ab-Sulfo-Mal-DM1 conjugate prepared using this method was highly potent at

low concentrations toward CanAg antigen-expressing COL0205 colon cancer cells. The

cytotoxicity of this conjugate was specific as it could be blocked by competition with

excess, unconjugated antibody.

[138] An alternative method of conjugation using the method described in this invention

involved a quenching step using maleimide or haloacetamide reagents (such as 4

maleimidobutyric acid, or 3-maleimidopropionic acid, or N-ethylmaleimide, or

iodoacetamide, or iodoacetic acid) after the completion of the initial DMx and

heterobifunctional linker reaction (before the addition of the reaction mixture to the

antibody) to quench the excess DMx thiol group in order to prevent any unwanted

reaction of DMx thiol with the antibody. In a specific example, following completion of

the initial DMx and heterobifunctional linker reaction (before the addition of the reaction

mixture to the antibody), 4-maleimidobutyric acid was added to quench the excess DMx

thiol group in order to prevent any unwanted reaction of DMx thiol with the antibody

during the conjugation reaction. To a reaction mixture of DM4 and Sulfo-Mal-NHS

heterobifunctional reagent that contained an excess of DM4 (3 mM), upon completion of the desired DM4 thiol coupling to the maleimide group of the heterobifunctional reagent, a two-fold molar excess of 4-maleimidobutyric acid (6 mM) was added to the reaction mixture at ambient temperature for 20 minutes to quench the remaining DM4 from the initial coupling reaction. Without purification of the reaction mixture, an aliquot was mixed with a solution of antibody in phosphate buffer (pH 7.5) under final conjugation conditions of 4 mg/mi Ab, 90% aqueous phosphate buffer/10%DMA, pH 7.5. The conjugation reaction was allowed to proceed at ambient temperature for 2 h. The antibody-DM4 conjugate was purified from the excess small-molecule DM4 and linker reactants using a G25 gel filtration column equilibrated in pH 7.5 phosphate buffer. The conjugation mixture was further kept at 4 °C for 2 days in pH 7.5 buffer to allow the dissociation of any DMx species attached to antibody non-covalently or via labile linkage. The conjugate was then dialyzed overnight into pH 5.5 histidine/glycine buffer and filtered through a 0.22 pm filter for final storage. The average number of DM4 molecules per Ab molecule in the final conjugate was measured by determining absorbance of the conjugate at 252 and 280 nm and using known extinction coefficients for DM4 and antibody at these two wavelengths. The conjugate samples were analyzed by non-reducing SDS PAGE using the NuPage electrophoresis system with a 4-12% Bis

Tris Gel (Invitrogen). The heat-denatured samples were loaded at 10 tg/lane. The non

reducing SDS-PAGE of the conjugate prepared using the method described in this

invention (without quenching) showed evidence of a light chain band (~25 kDa) and half

antibody band (heavy-light chain; -75 kDa) (Figure 18). On the other hand, the

conjugate prepared using the method described in this invention which was treated with

4-maleimidobutyric acid (to cap excess DMx thiol) had significantly lower amounts of these undesirable bands (at levels comaparable to unmodifed antibody sample). Another advantage of the quenching of the initial DMx and heterobifunctional reaction mixture

(before conjugation with the antibody) by thiol-quenching reagents such as 4

maleimidobutyric acid is that during the antibody conjugation reaction there is no "free"

DMx (DM1 or DM4) species and therefore the final conjugate after purification does not

contain "free" or unconjugated DMx species. The DMx-adduct with 4-maleimidobutyric

acid (or other polar thiol-quencing reagents) is more water soluble than DMx and

therefore can be more easily separated from the covalently linked antibody-DMx

conjugate.

Example 3. Conjugation of antibody with maytansinoid (DM1/DM4) using sulfo

NHS-SMCC linker (Figure 13).

[139] Stock solutions of DM1 or DM4 thiol (DMx) and the sulfo-SMCC

heterobifunctional linker with sulfo-NHS group (purchased from Pierce Endogen; Figure

13) were prepared in DMA at concentrations of 30-60 mM. Linker and DM1 or DM4

thiol were mixed together in DMA containing up to 40% v/v of aqueous 200 mM

succinate buffer, 2 mM EDTA, pH 5.0 to give a ratio of DM1 or DM4 (DMx) to linker of

1.6:1 and a final concentration of DMx of 6 mM. After mixing, the reaction was left for

1-4 h at ambient temperature and then an aliquot of the reaction mixture was diluted 10

fold to measure absorbance at 302-320 nm to assess whether all of the maleimide had

reacted. (Additional reverse phase HPLC analysis of a frozen aliquot of the reaction

mixture was carried out later with monitoring at 302 nm and 252 nm to verify complete

disappearance of linker maleimide and formation of the desired sulfo-NHS-linker-Mal

DMx reagent at the time of addition of the reaction mixture to antibody). When no

further maleimide was present by UV an aliquot of the reaction was added to an aqueous

solution of an antibody in phosphate buffer (pH 7.5) under final conjugation conditions of

4 mg/ml Ab, 90% phosphate buffer (aqueous)/10% DMA (v/v), pH 7.5. The conjugation

reaction was allowed to proceed at ambient temperature for 2 h. Ab-DMx conjugate was

purified from excess unreacted reagent and excess DMx using a G25 gel filtration

column equilibrated in pH 7.5 phosphate buffer (aqueous). Conjugate was kept at 4 °C for

2 days in pH 7.5 buffer to allow the dissociation of any DMx species attached to Ab non

covalently or via labile linkage. The conjugate was then dialyzed overnight into pH 5.5

histidine/glycine buffer and then filtered through a 0.22 m filter for final storage. The

number of DMx molecule per Ab molecule in the final conjugate was measured by

determining absorbance of the conjugate at 252 and 280 nm and using known extinction

coefficients for DMx and antibody at these two wavelengths.

[140] For comparison, the Ab-SMCC-DMx conjugates were prepared using the

traditional 2-step conjugation method. The antibody (Ab) at a concentration of 8 mg/ml

in 95% pH 6.5 phosphate buffer/5% DMA buffer was modified with excess bifunctional

sulfo-SMCC linker with sulfo-NHS group (purchased from Pierce Endogen). The

reaction was allowed to proceed at 25 °C for 2 h and then the modified Ab was purified

away from excess unreacted linker using G25 chromatography. The recovery of purified

Ab was determined by UV absorbance at 280 nm. The number of linked maleimide

groups in the modified Ab was determined using a small aliquot of modified antibody by

addition of a known amount of thiol (such as 2-mercaptoethanol), added in excess over

the maleimide, to react with the maleimide residues in the modified antibody and then assaying the remaining thiol by Ellman's assay using DTNB reagent (extinction coefficient of TNB thiolate at 412 nm= 14150 M' cm'; Riddles, P. W. et al., Methods

Enzymol., 1983, 91, 49-60; Singh, R., Bioconjugate Chem., 1994, 5, 348-351). The

conjugation of modified Ab with DM1 or DM4 was carried out at an antibody

concentration of 2.5 mg/ml in 95% pH 6.5 phosphate buffer/5% DMA (v/v), with 1.7

molar equivalents of DM1 or DM4 thiol added per mol of linked maleimide in the Ab.

The reaction was left for 8-24 h at 18 °C and the conjugate was separated from excess,

unreacted DM1 (or DM4) via G25 chromatography. After purification the conjugate was

kept at 4 °C for 2 days in pH 6.5 buffer to allow the hydrolysis of any weakly linked

DM1/DM4 species. The conjugate was then dialyzed overnight into pH 5.5

histidine/glycine buffer and then filtered through a 0.22 pm filter for final storage. The

number of DM1/DM4 molecules per Ab molecule in the final conjugate was measured by

determining absorbance of the conjugate at 252 and 280 nm and using known extinction

coefficients for DM1/DM4 and antibody at these two wavelengths.

[141] Reducing SDS PAGE was carried out on conjugate and antibody samples (10

[tg/lane) using the NuPage electrophoresis system (Invitrogen) with a NuPage 4 -12% Bis

Tris Mini Gel and NuPAGE MOPS SDS running buffer (Figure 14). Bands on the gel

with molecular weights of 75, 125, and 150 kDa were indicative of inter-chain cross

linked species (HL, H 2L and H 2L 2 respectively). A comparison of Ab-SMCC-DMI

conjugates with 3.1 D/Ab (lane 4, by this method, and lane 3, by the traditional 2-step

method, respectively) clearly shows that conjugate made via the method described in this

invention (lane 4) has much fewer high molecular weight cross-linked species than

conjugates made by the traditional 2 step method (lane 3).

[142] Protein LabChip electrophoresis analysis (under reducing condition) of the

antibody-SMCC-DM1 conjugate prepared by the method described in this invention

showed the heavy and light chain major bands with percentages of 67 and 30% (of total

protein), which are similar to those for unconjugated antibody of 68 and 30% respectively

(Figure 15). In contrast, the conjugate prepared using the traditional two-step method

showed heavy and light bands of only 54 and 24% respectively, and major bands of

higher molecular weights ranging from 96-148 kDa presumably due to inter-chain cross

linking. Based on the quantitative Protein LabChip analysis, the conjugate prepared by

the method described in this application is much superior in terms of lack of inter-chain

cross-linking compared to that prepared using the conventional two-step process (Figure

).

[143] The Ab-SMCC-DM1 conjugates with similar drug loads made via the method

described in this invention and by the traditional two step method were compared by size

exclusion LC/MS analysis (Figure 16). The conjugate made via the method described in

this invention shows the desired MS spectrum containing only the expected distribution

of peaks with mass equal to Ab-(linker-DMx)n. In the case of conjugate made using the

traditional two-step method the spectrum shows a heterogeneous mixture of species

which includes the desired Ab-(inker-DMx)n species plus additional species containing

inactivated maleimide and cross-linked linker fragments. The putative mechanisms of

the inter-chain cross-linking and maleimide inactivation in the traditional 2-step reaction

sequence are shown in Figure 17 whereby the incorporated maleimide (or haloacetamide

residue) from the initial reaction of antibody with the heterobifunctional linker can react

with intramolecular (or intermolecular) histidine, lysine, tyrosine, or cysteine residues resulting in inter-chain cross-linking, or the initially incorporated maleimide (or haloacetamide) residue can get inactivated by hydrolysis or hydration of the maleimide residue before the reaction step with the thiol-bearing DM1 or DM4 (DMx) agent. Thus the LC-MS analysis clearly shows that the method described in this invention has the advantage of producing homogeneous conjugate with little or no inactivated maleimide or cross-linked linker fragments attached to antibody.

Example 4. Conjugation of antibody with DM/DM4 (DMx) with cleavable,

disulfide linkers by this method (Figure 19).

[144] Stock solutions containing DM1 or DM4 thiol (DMx) and heterobifunctional

linker 4-(2-pyridyldithio)butanoic acid-N-hydroxysuccinimide ester (SPDB) were

prepared in DMA at concentrations of 30-60 mM. Linker and DMx thiol were mixed

together in DMA containing up to 40% v/v of aqueous 200 mM succinate buffer, 2 mM

EDTA, pH 5.0 to give a ratio of DM1 or DM4 (DMx) to linker of 1.6:1 and a final

concentration of DMx of 8 mM. After mixing, the reaction was left for 1 h at ambient

temperature and then an aliquot of the reaction was added to an aqueous solution of

antibody in phosphate buffer (pH 7.5) under final conjugation conditions of 4 mg/ml Ab,

% phosphate buffer (aqueous)/10% DMA (v/v), pH 7.5. The conjugation reaction was

allowed to proceed at ambient temperature for 2 h. The Ab-DMx conjugate was purified

from excess unreacted reagent and excess DMx using a G25 gel filtration column

equilibrated in pH 7.5 phosphate buffer (aqueous). Conjugate was kept at 4°C for 2 days

in pH 7.5 buffer to allow for the dissociation of any DMx species attached to Ab non

covalently or via labile linkage. The conjugate was then dialyzed overnight into pH 5.5 histidine/glycine buffer and then filtered through a 0.22 tm filter for final storage. The number of DMx molecules per Ab molecule on the final conjugate was measured by determining absorbance of the conjugate at 252 and 280 nm using known extinction coefficients for DMx and antibody at these two wavelengths.

Example 5. Preparation of antibody-DM/DM4 (Ab-DMx) conjugate with both

disulfide- and non-cleavable linkers using this method (Figure 20).

[145] Stock solutions of DM1 or DM4 thiol (DMx) and the NHS-PEGn-Maleimide

heterobifunctional linker were prepared in N,N-dimethylacetamide (DMA) at

concentrations of 30-80 mM. The NHS-PEG 4-Maleimide linker and DMx thiol were

mixed together in DMA containing up to 40% v/v of 200 mM succinate buffer, 2 mM

EDTA, pH 5.0 to give a molar ratio of DMx to linker of 1.6:1 and a final concentration of

DMx equal to 8.0 mM. The reaction mixture was left to react for 2 h at ambient

temperature. In a separate parallel reaction, SPDB linker and DMx thiol were mixed

together and reacted in a similar fashion to the conditions used for NHS-PEG 4-maleimide

reaction except for a reaction time of 1 h. After the completion of both reactions and

without purification, equal volumes of PEG 4 -Mal-DM4 mixture and SPDB-DM4 mixture

were combined. An aliquot of the combined reaction mixtures was added without

purification to a solution of antibody in phosphate buffer (pH 7.5) under final conjugation

conditions of 4 mg/ml Ab, 90% phosphate buffer (aqueous)/10% DMA (v/v), pH 7.5.

The conjugation reaction was allowed to proceed at ambient temperature for 2 h. Ab

DMx conjugate was purified from excess unreacted reagents and excess DMx using a

G25 gel filtration column equilibrated in pH 7.5 phosphate buffer (aqueous). The conjugate was kept at 4°C for 2 days in pH 7.5 buffer to allow for the dissociation of

DMx species attached to Ab non-covalently or via labile linkage. The conjugate was then

dialyzed overnight into pH 5.5 histidine/glycine buffer and then filtered through a 0.22

im filter for final storage. The number of DMx molecules per Ab molecule on the final

conjugate was measured by determining absorbance of the conjugate at 252 and 280 nm

using known extinction coefficients for DMx and antibody at these two wavelengths.

[146] The Ab-(mixed SPDB and PEG4 -Mal linker)-DMx conjugate made via the

method described in this invention was tested to determine the percent of incorporation of

cleavable versus non-cleavable linker on the Ab by comparing DMx per antibody (D/A)

ratio before and after DTT (dithiothreitol) treatment of the conjugate to reduce the

disulfide linkage. In order to maintain reaction pH at 7.5 during DTT reduction, the

conjugate was first dialyzed into 250 mM HEPES buffer pH 7.5. The conjugate was then

reduced by reacting with 25 mM DTT for 20 min at 37C. After the DTT reaction, the

released DMx and DTT were separated from the reaction mixture using a G25 gel

filtration column equilibrated in 250 mM HEPES buffer pH 7.5. The average number of

DMx molecules per Ab molecule in the purified product was measured by determining

the absorbance of the conjugate at 252 and 280 nm and using known extinction

coefficients for DMx and antibody at these two wavelengths. The ratio between D/A of

DTT-treated conjugate and D/A of non-DTT treated conjugate was used to calculate the

percent of DMx attached to Ab via non-cleavable linkage. Two additional samples, Ab

SPDB-DM4 and Ab-PEG 4 -Mal-DM4 conjugates, were treated with DTT as positive and

negative controls, respectively. By comparing D/A ratio before and after DTT treatment,

the control non-cleavable Ab-PEG 4 -Mal-DM4 conjugate showed that approximately all linkers bound were found to be non-cleavable (93%) as expected. The Ab-(mixed SPDB and PEG 4Mal linker)-DMx conjugate containing both non-cleavable and disulfide linkers made via the method described in this invention had 41% less DMx cleaved by DTT treatment relative to the amount of DMx loss from the Ab-SPDB-DMx conjugate that consists entirely of cleavable linker. This demonstrated that the Ab-(mixed SPDB and

PEG 4-Mal)-DMx conjugate made via the method described in this invention was

composed of approximately 40% non-cleavable and 60% cleavable linkers. By changing

the initial ratio of the non-cleavable and cleavable linker reagents, conjugates of antibody

with maytansinoid or other effector can be prepared with different ratio of non-cleavable

and cleavable linkers. Figure 21 shows the mass spectrum of deglycosylated conjugate

described above, which comprises of antibody with an average of 3.5 maytansinoid

molecules per antibody molecule linked via both disulfide linkers (SPDB) and non

cleavable linkers (PEG). The MS shows discrete conjugate species bearing both

cleavable and non-cleavable linkers (Figure 21). For example, the conjugate peak

designated D2-PEG-SPDB bears one disulfide-linked and one non-cleavable thioether

linked maytansinoid molecule; the conjugate peak designated D3-PEG-2SPDB bears two

disulfide-linked and one non-cleavable thioether-linked maytansinoid molecules; and the

conjugate peak designated D3-2PEG-SPDB bears one disulfide-linked and two non

cleavable thioether-linked maytansinoid molecules.

Example 6. Conjugation of antibody with maytansinoid using SMCC linker (Figure

22).

[147] Stock solutions of DM1 thiol and SMCC heterobifunctional linker (Pierce) were

prepared in DMA at concentrations of 30-60 mM. Linker and DM1 thiol were mixed

together in DMA containing up to 50% v/v of aqueous 200 mM succinate buffer, 2 mM

EDTA, pH 5.0 to give a ratio of DM1 to linker of 1.4:1 mole equivalent and a final

concentration of DM1of 1 to 6 mM. After mixing, the reaction was left for up to 4 h at

ambient temperature and then an aliquot of the reaction mixture was diluted 10-fold to

measure absorbance at 302-320 nm to assess whether all of the maleimide had reacted

with thiol. When no further maleimide was present by UV, an aliquot of the reaction was

added to an aqueous solution of an antibody in phosphate buffer (pH 7.5-8.5) under final

conjugation conditions of 2.5 mg/ml Ab, 70-80% phosphate buffer (aqueous)/30-20%

DMA (v/v). The conjugation reaction was allowed to proceed at ambient temperature for

3 h. Ab-DMl conjugate was purified from excess unreacted or hydrolyzed reagent and

excess DM1 using a G25 gel filtration column equilibrated in pH 7.4 phosphate buffer

(aqueous). The conjugate was then dialyzed overnight into pH 7.4 phosphate buffer

(aqueous) and then filtered through a 0.22 pm filter for final storage. The number of

DM1 molecule per Ab molecule in the final conjugate was measured by determining

absorbance of the conjugate at 252 and 280 nm and using known extinction coefficients

for DM1 and antibody at these two wavelengths. Similarly, conjugates of antibody with

DM4 thiol and SMCC can be prepared. These conjugates of antibody with DM1 or DM4

using SMCC linker contain thioether non-cleavable linker.

[148] The Ab-SMCC-DMl conjugate made via the method described in this invention

was characterized by MS analysis of deglycosylated conjugate (Figure 23). The conjugate

made via the method described in this invention shows the desired MS spectrum

containing the expected distribution of peaks with mass equal to Ab-(linker-DM1).

Example 7. Conjugation of antibody with maytansinoid using heterobifunctional

disulfide-containing linkers (SSNPB, SPP).

[149] Disulfide containing heterobifunctional linkers SSNPB (N-sulfosuccinimidyl-4

(5-nitro-2-pyridyldithio)butyrate) and SPP (N-succinimidyl-3-(2

pyridyldithio)propionate) can be used to prepare disulfide-linked antibody- maytansinoid

conjugates by the method similar to that described for SPDB linker

in Example 4. The structure of the disulfide-linked conjugate prepared using SPDB

(Figure 19) is identical to that of the conjugat'e prepared with SSNPB (Figure 24). The

MS of a disulfide-linked conjugate prepared using SPDB showed discrete peaks with

mass values corresponding to different numbers of maytansinoid molecules attached to

antibody.

Example 8. Conjugation of antibody with maytansinoid containing non-cleavable

linkers with linear alkyl carbon chain.

[150] Conjugates containing non-cleavable linker with linear alkyl carbon chain were

prepared using reaction mixture of maytansinoid and heterobifunctional linkers with

linear alkyl carbon chain, similar to the method described for SMCC linker in example 6.

For example, conjugates of a humanized antibody with DM1 were prepared using BMPS

(N-[p-maleimidopropyloxy]succinimide ester) or GMBS ((N-[y

maleimidobutyryloxy]succinimide ester) linker as shown in Figure 26. The initial

reaction mixture containing BMPS or GMBS (8 mM) and DM1 thiol (10.4 mM) in 60%

DMA/40% (v/v) 200 mM succinate buffer, pH 5, showed complete reaction of maleimide

moiety (based on decay of maleimide absorbance at 302-320 nm) when checked at 15

min. This reaction mixture was added, in two portions 30 min apart, to a humanized

antibody solution at 2.5 mg/ml in 80% aqueous EPPS buffer, pH 8.1, containing 20%

DMA (v/v) with the total linker added at 8 molar equivalents to antibody. The conjugate

mixture was gel purified after 4 h and subjected to 2 rounds of dialysis. Conjugates with

DM/antibody ratio of 3.8 and 5.1 were prepared with 71-75% recovery, and high

monomer % (96.2-97.6%). These conjugates prepared with GMBS or BMPS showed no

unconjugated free drug by HISEP HPLC analysis. Similar conjugates containing non

cleavable linkers with linear alkyl chains can be prepared using AMAS (N-[P

maleimidoacetoxy]succinimide ester) or EMCS (N-[p-maleimidocaproyloxy]succinimide

ester) or the sulfo-N-hydroxysuccinimide esters (sulfo-GMBS, sulfo-EMCS) as shown in

Figure 25. Table 1 shows the monomer % for select conjugates prepared by the method

described in this invention, which all showed high monomer % by size-exclusion

chromatography analysis. For comparison, monomer % are also shown for conjugates

prepared by the traditional two-step conjugation method (by the initial reaction of

antibody with heterobifunctional linker followed by reaction with mayansinoid thiol).

Table 1. Monomer % for select conjugates made by the method described in this

application versus by traditional two-step conjugation methods

22081847.1:DCC -08/11/2021

Conjugate D/A Conjugation method % Monomer

Ab-PEG4 -Mal-DM1 6.6 this invention 99.0

Ab-PEG 4 -Mal-DM1 6.8 two-step 98.0

Ab-SulfoMaI-DM1 3.6 this invention 99.0

Ab-Sulfo-Mal-DM1 4.0 two-step 96.7

Ab-SMCC-DMI 4.0 this invention 98.6

Ab-SMCC-DMI 3.8 two-step 97.0

Ab-PEG 4-MaI-DM4 6.2 Xhis invention 6.9

Ab-PEG 4 -Ma1-DM4 6.1 tvo-step 84.5

Ab-SPDB-DM4 4.1 this invention 99.4

b-SPDB-DM4 3.9 two-step, one-pot 53

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (29)

C:\Interwo n\NRPortbl\DCC\MDT\19686498_l.docx-20/12/2019 The claims defining the invention are as follows:

1. A process for preparing a purified conjugate in a solution, wherein the conjugate comprises an effector or a reporter molecule linked to a cell binding agent through a disulfide bond, the process comprising the steps of: (a) contacting an effector or a reporter molecule comprising a thiol group with a bifunctional linker reagent represented by the following formula:

S O-N SO 3 H 0 to covalently attach the linker to the effector or the reporter molecule and thereby prepare an unpurified first mixture of step (a) comprising the effector or the reporter molecule having linkers bound thereto, (b) conjugating a cell binding agent to the effector or the reporter molecule having linkers bound thereto by reacting the unpurified first mixture with the cell binding agent in a solution having a pH to prepare a second mixture, and (c) subjecting the second mixture to tangential flow filtration, dialysis, gel filtration, adsorptive chromatography, selective precipitation or a combination thereof to thereby prepare the purified conjugate.

2. The process of claim 1, wherein the pH of the solution in step (b) is from about 4 to about 9.

3. The process of claim 1, wherein the pH of the solution in step (b) is from about 5 to about 8.7.

4. The process of claim 1, wherein the pH of the solution in step (b) is from about 6.5 to about 8.5.

5. The process of any one of claims 1-4, wherein the second mixture in step (b) is substantially free of undesired cross-linked, hydrolyzed species formed due to intra molecular or inter-molecular reactions.

C:\Interwoven\NRPortbl\DCC\MDT\19686498_1.dox-20/12/2019

6. The process of any one of claims 1-5, wherein the process further comprises the step of quenching the excess maytansinoid in the unpurified first mixture with a quenching reagent between steps (a) and (b).

7. The process of claim 6, wherein the quenching reagent is selected from 4 maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid.

8. A process for preparing a purified conjugate in a solution, wherein the conjugate comprises an effector or a reporter molecule comprising a thiol group linked to a cell binding agent, the process comprising the steps of: (a) contacting the effector or the reporter molecule comprising a thiol group with a bifunctional linker reagent represented by one of the following formulas 02N

O SN

OE

O3

N 0 00

to covalently attach the linker to the effector or the reporter molecule and thereby prepare an unpurified first mixture comprising the effector or the reporter molecule having linkers bound thereto,

C:\Interwo n\NRPortbl\DCC\MDT\19686498_l.docx-20/12/2019

(b) mixing the unpurified first mixture comprising the effector or the reporter molecule having linkers bound thereto with a cell binding agent at a pH of about 5, to form an unpurified second mixture, (c) conjugating the cell binding agent to the effector or the reporter molecule having linkers bound thereto by increasing the pH of the unpurified second mixture to about 6.5 to about 8.5 to prepare a third mixture, and (d) subjecting the third mixture to tangential flow filtration, dialysis, gel filtration, adsorptive chromatography, selective precipitation or a combination thereof to thereby prepare the purified conjugate.

9. The process of claim 8, wherein in step (c) the pH is increased to about 6.5.

10. The process of claim 8, wherein in step (c) the pH is increased to about 7.0.

11. The process of claim 8, wherein in step (c) the pH is increased to about 7.5.

12. The process of claim 8, wherein in step (c) the pH is increased to about 8.0.

13. The process of any one of claims 8-12, wherein the third mixture in step (c) is substantially free of undesired cross-linked, hydrolyzed species formed due to intra molecular or inter-molecular reactions.

14. The process of any one of claims 8-13, wherein the process further comprises the step of quenching the excess maytansinoid in the unpurified first or second mixture with a quenching reagent between steps (a) and (b) or between steps (b) and (c).

15. The process of claim 14, wherein the quenching reagent is selected from 4 maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid.

16. The process of any one of claims 1-15, wherein the effector or the reporter molecule is a cytotoxic agent.

17. The process of claim 16, wherein the cytotoxic agent is selected from maytansinoids, taxanes, CC1065, or analogs thereof.

18. The process of claim 17, wherein the cytotoxic agent is a maytansinoid.

22081847. DCC -08/11/2021

19. The process of claim 18, wherein the maytansinoid is DM.

20. The process of claim 18, wherein the maytansinoid is DM4.

21. The process of any one of claims 1-20, wherein the cell binding agent is selected from interferons, interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, EGF, TGF-a, FGF, G-CSF, VEGF, MCSF, GM-CSF, transferrin, and antibodies.

22. The process of claim 21, wherein the cell binding agent is an antibody.

23. The process of claim 22, wherein the antibody is a monoclonal antibody.

24. The process of claim 23, wherein the antibody is a human or a humanized monoclonal antibody.

25. The process of claim 22, wherein the antibody is MY9, anti-B4, C242, or an antibody that binds to an antigen selected from EpCAM, CD2, CD3, CD4, CD5, CD6, CD1l, CD19, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD79, CD105, CD138, EphA receptors, EphB receptors, EGFR, EGFRvIII, HER2, HER3, mesothelin, cripto, alphavbeta3, alphavbeta5, and alphavbeta6 integrin.

26. The process of claim 24, wherein the human or the humanized antibody is huMy9 6, huB4, huC242, huN901, DS6, CNTO 95,B-B4, trastuzumab, pertuzumab, bivatuzumab, sibrotuzumab, rituximab, or a human or humanized antibody that binds to an antigen selected from EphA2 receptor, CD38, and IGF-IR.

27. The process of any one of claims 1-26, wherein the reporter molecule is a radioisotope.

28. The process of any one of claims 1-27, wherein an excess of maytansinoid relative to the bifunctional linker reagent is used.

29. A purified conjugate in a solution obtained by the process according to any one of claims 1 to 28.