KR20120080611A - Potent conjugates and hydrophilic linkers - Google Patents

Abstract

The linker that binds the agent to the cell binder is modified to a hydrophilic linker by incorporating a polyethylene glycol spacer. The efficacy or effectiveness of the cell-binding agent-drug conjugates is surprisingly improved several times in various cancer cell types, including the expression of a small number of antigens on cell surfaces or cancer cells that are resistant to treatment. There is essentially provided a method for preparing a maytansinoid containing a reactive group and a thioether moiety which allows the maytansinoid to be linked to a cell-binding agent in one step.

Description

EFFECTIVE CONJUGATES AND HYDROPHILIC LINKERS}

This application is a partial-continued application of US Non-Provisional Application No. 12 / 433,668, filed April 30, 2009, which claims priority to US Provisional Application No. 61 / 049,289, filed April 30, 2008. The entire description of prior applications 12 / 433,668 and 61 / 049,289 is considered part of the description of the attached continuing application, which is incorporated herein by reference.

The present invention relates to novel linkers that link an agent (eg a cytotoxic agent) to a cell-binding agent (eg an antibody) in such a way that the linker contributes to increasing the activity of the agent. In particular, the present invention relates to the use of a novel hydrophilic linker, wherein the linker expresses a small number of antigens on a cell surface or cancer that is resistant to treatment, in which the cell-binding agent-pharmaceutical conjugate ( Improve the efficacy or effectiveness of cell-binding agent-drug conjugates several times. The present invention also relates to a process for preparing maytansinoids containing reactive groups and thioether moieties that allow maytansinoids to be linked to cell-binding agents.

Antibody conjugates of cytotoxic agents are being developed as target-specific therapeutic agents. Antibodies to a variety of cancer cell-surface antigens include microtubules (maytansinoids, auristatins, taxanes; US Pat. Nos. 5,208,020; 5,416,064; 6.3333,410; 6,441,163; 6,340,701; 6,372,738 6,436,931; 6,596,757; 7,276.497), DNA (calikimycin, doxorubicin, CC-1065 homologue; U.S. Patents 5,475,092; 5,585,499; 5,846,545; 6,534,660; 6,756,397; 6,630,579) have been conjugated with different cytotoxic agents that inhibit various essential cellular targets. Antibody conjugates with some of these cytotoxic agents are being actively studied in the clinic for the treatment of cancer (Richart, AD, and Tolcher, AW, 2007, Nature). Clinical Practice , 4, 245-255).

Antibody-cytotoxic agent conjugates are usually prepared by initial modification of reactive moieties on the antibody, such as lysine amino groups or cysteine groups (by reduction of natural disulfide bonds or using molecular biology methods). By engineering treatment of additional non-natural cysteine residues to the antibody). Thus, antibodies may first be prepared by SPDB, SMCC and SIAB (US Pat. No. 6,913,758 and US Patent Publication No. 20050169933) to incorporate a linker with a reactive group (e.g., mixed pyridyldisulfide, maleimide or haloacetamide). Modified with a heterobifunctional linker reagent, such as described above, exemplified by. The reactive linker group incorporated into the antibody is then conjugated with a cytotoxic agent containing a reactive moiety (eg thiol group). Another conjugation route is by reaction of a thiol group on a cell-binding agent with a cytotoxic agent derivative containing a thiol-reactive group (eg haloacetamide or maleimide). Thiol groups can be obtained by reduction of natural disulfide residues ((R. Singh et al., Anal . Biochem ., 2002, 304, 147-156), or reduction of incorporated disulfide moieties (SPDP, succinimidyl 3- ( 2-pyridyldithio) propionate) followed by reduction of dithiothreitol (DG Gilliland et al., Proc . Natl . Acad . Sci . USA ., 1980, 77, 4539-4543) Or by incorporation of additional non-natural cysteine residues (JB Stimmel et al., J. Biol . Chem ., 2000, 275, 30445-30450), or 2-iminothiolane (R. Jue et al., Biochemistry , 1978, 17, 5399-5406) or cell-binding agents by incorporation of thiol groups by reaction with methyl 3-mercaptopropionimate esters (TP King et al., Biochemistry , 1978, 17, 1499-1506). For example, antibody).

Antibody-cytotoxic agent conjugates with disulfide or thioether bonds are probably intracellularly cleaved in lysosomes to deliver active cytotoxic agents into cancer cells (HK Erickson et al., 2006, Cancer Research , 66, 4626-4433). In addition to killing target cells, antibody-cytotoxic agent conjugates with reducing disulfide bonds also kill the closest antigen-negative cells in a mixed population of antigen-negative and antigen-positive cells in vitro and in vivo in a xenograft model. Thus, the role of target-cell releasing cytotoxic agents with improved efficacy against neighboring non-antigen-expressing cells in heterologous antigen-expressing tumors is suggested (YV Kovtun et al., Cancer Research , 2006, 66, 3214-3221).

Although antibody-cytotoxic drug conjugates exhibit apoptosis activity and in vivo anti-tumor activity in vitro, their efficacy is in many cases, particularly when antigen expression is low on target cancer cells or when the target cells are treated for treatment. Reduced if resistant. This is often the case in clinical settings and results in low or moderate anti-tumor activity in the patient. A potential approach to avoid resistance is to synthesize novel agents with hydrophilic or lipophobic functionality (G. Szokacs et al., Nature Reviews, 5; 219-235, 2006). However, this method is complex and some analogs have to be synthesized, and often the modification of the drug structure causes a loss of biological activity. Thus, there is a need for different approaches.

Methods described in the art for the preparation of cytotoxic conjugates of cell binders and medicaments via non-cleavable linkers require two reaction steps (US Pat. No. 5,208,020 and US Publication No. 2005/0169933). First, a cell binder (e.g. an antibody) is formed by a bifunctional crosslinker which reacts with a reactive group of the cell binder, such as an amine group on a lysine residue or a sulfhydryl group on a cysteine residue, to form a covalent chemical bond. Reform. Following modification of the cell binder, the product is purified to separate the desired modified cell binder from the unreacted crosslinker. In a second step, known as the conjugation step, a reactive drug derivative (eg thiol-containing maytansinoid) is added to the modified cell-binding agent for reaction with the modified cell-binding agent. Following this reaction, additional purification is required to remove unreacted drug groups and other byproducts from the final conjugate. These multiple reaction and purification steps produce low yield final conjugates and can be costly and complex when trying to run these steps on a large scale. An additional drawback to these methods is the heterogeneity introduced when there is an unreacted crosslinker linked to the cell-binding agent without a bound agent. The unreacted crosslinker can then undergo additional side reactions, such as hydrolysis and intramolecular or intermolecular reactions. Thus, there is a need for functionalized, reactive drug derivatives (eg maytansinoids) that can be covalently bound through non-cleavable binding to cell binding agents (eg antibodies) in essentially one reaction step. do.

The present invention solves the problem of tolerance by designing novel linkers that link the drug to the cell-binding agent in a manner that allows the linker to contribute to increasing the activity of the drug. Thus, the present invention improves the manner in which the drug is linked to the cell-binding agent such that the linker design provides conjugates that are active against a broad spectrum of tumors, particularly in low antigen expression or tolerant tumors.

The present invention relates to a hydrophilic linker by the incorporation of a conventional linker (eg SMCC, SIAB, described in US Patent Publication No. 20050169933) by incorporation of a polyethylene glycol [PEG _n , (-CH ₂ CH ₂ O) _n ) When modified, the efficacy or efficacy of cell-binding agent-drug conjugates is surprisingly based on new findings that are improved several times in various cancer cell types, including the expression of a small number of antigens on the cell surface.

In addition, these PEG-containing conjugates are unexpectedly more potent than the previously described conjugates for treatment resistant cell lines.

In addition, in the case of antibody conjugates, the incorporation of hydrophilic linkers allows for conjugation of up to 15 drug molecules per antibody molecule without high yield and aggregation or precipitation. These conjugates, with hydrophilic linkers linked with up to 15 drug molecules per antibody molecule, bind with high affinity to the target antigen (similar to that of an unmodified antibody).

The present invention is also directed to amine or thiol groups of the cell binder such that thioether-linked maytansinoid conjugates with the cell binder can be prepared in essentially one reaction step, without prior chemical modification of the cell binder. A new maytansinoid is described.

The present invention describes a process for the synthesis of novel maytansinoids comprising thioether- moieties and reactive groups. These new maytansinoids are useful for the preparation of thioether-linked conjugates in essentially one reaction step. Also described are methods of making cell binder conjugates using these novel reactive maytansinoids.

Accordingly, the present invention provides a compound of Formula 1 or a specific compound of Formula 1 ':

[Formula 1]

ZX,-(-CH ₂ -CH ₂ -O-) _n -Y _p -D

[Formula 1 ']

DY _p -(-CH ₂ -CH ₂ -O-) _n -Xi-Z

In the above formulas,

Z represents reactive functionality capable of forming an amide or thioether bond with the cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y is an aliphatic, aromatic or heterocyclic group bonded to the drug through a covalent bond selected from the group consisting of thioether bond, amide bond, carbamate bond, ether bond, amine bond, carbon-carbon bond and hydrazone bond Represent;

l is 0 or 1;

p is 0 or 1;

n is an integer from 1 to 2000.

Another aspect of the invention is a cell-binding agent conjugate of Formula 2 or a specific compound of Formula 2 '

[Formula 2]

CB- [XK-CH ₂ -CH ₂ -O-) _n -Y _p -D] _m

[Formula 2 ']

[DY _p -(-CH ₂ -CH ₂ -O-) _n -X ₁ ] _m -CB

In the above formulas,

CB represents a cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y is an aliphatic, aromatic or heterocyclic group bonded to the drug through a covalent bond selected from the group consisting of thioether bond, amide bond, carbamate bond, ether bond, amine bond, carbon-carbon bond and hydrazone bond Represent;

l is 0 or 1;

p is 0 or 1;

m is an integer from 2 to 15;

n is an integer from 1 to 2000.

Another aspect of the invention is a compound of formula 3 or a specific compound of formula 3 '

(3)

ZX,-(-CH ₂ -CH ₂ O-) _n -YD

[Formula 3 ']

DY-(-CH ₂ -CH ₂ O-) _n -X _i -Z

In the above formulas,

Z represents a reactive functional group capable of forming an amide or thioether bond with the cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y represents an aliphatic, non-aromatic heterocyclic or aromatic heterocyclic group bonded to the medicament via a disulfide bond;

l is 0 or 1;

n is an integer of 1-14.

Another aspect of the invention is a cell-binding agent conjugate of Formula 4 or a specific compound of Formula 4 '

[Formula 4]

CB- (Xi-(-CH ₂ -CH ₂ O-) _n -YD) _m

[Formula 4 ']

[DY-(-CH ₂ -CH ₂ O-) _n -X,] _m -CB

In the above formulas,

CB represents a cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y represents an aliphatic, aromatic or heterocyclic group which is bound to the medicament via a disulfide bond;

l is 0 or 1;

m is an integer from 3 to 8;

n is an integer of 1-14.

Another aspect of the invention is a method of treating cancer sensitive to treatment by the method comprising parenteral administration of an effective amount of a composition comprising a conjugate of Formula 2 or Formula 4 to a patient in need thereof.

In still another aspect of the present invention, there is provided a novel maytansinoid containing a reactive group and having a thioether moiety represented by the following formula (5):

[Chemical Formula 5]

D'-Y'-V-Q-W-Z '

In the above formulas,

D 'is a sulfhydryl-containing maytansinoid, e.g., N ^2' - having acetyl -iV ^{2 '-} (3- mercapto-1-oxopropyl) - MEI tansin (DM1) or _TV ^2' - Deacetyl-N ^{2 ′} -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4);

Y 'represents a thioether bond;

V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Q represents any aromatic or heterocyclic moiety;

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Z 'represents an amine or sulfhydryl reactive group.

Reactive maytansinoid derivatives containing thioether moieties are prepared from sulfhydryl-containing maytansinoids such as DM1 and DM4 and heterodifunctional crosslinkers. The reaction is represented by the following chemical scheme:

D '+ V-V-Q-W-Z' → D'-Y'-V-Q-W-Z '

In the above chemical scheme,

D ′ is a sulfhydryl-containing maytansinoid, for example N ^{2 ′} -deacetyl-TV ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (DM1) or N ^{2 ′} − Deacetyl-N ^{2 ′} -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4);

V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Q represents any aromatic or heterocyclic moiety;

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Z 'is an amine or sulfhydryl reactive group;

Y ″ represents a sulfhydryl-reactive moiety;

Y 'represents a thioether bond between the sulfhydryl-containing maytansinoid and the crosslinking agent.

The present invention also discloses a process for preparing cytotoxic conjugates of maytansinoids and cell binders linked via non-cleavable bonds (Formula 10), wherein the method comprises a cell binder and a chemical formula Z'-WQV-Y'- Reacting a compound of D 'to provide a cell binder conjugate of formula CB- (Z "-WQV-Y'-D') _m , wherein

Z 'represents an amine or sulfhydryl reactive group;

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Q represents any aromatic or heterocyclic moiety;

V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;

Y 'represents a thioether bond;

D ′ is a sulfhydryl-containing maytansinoid, for example N ^{2 ′} -deacetyl-iV ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (DM1) or N ^{2 ′} - Deacetyl-N ^{2 ′} -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4);

CB represents a cell-binding agent;

Z "represents an amide bond; m is an integer of 2-8.

The cell-binding maytansinoid conjugate can be further purified.

1 shows the structural formulas of representative PEG-containing thiosuccinimidyl-linked conjugates of the invention (mAb = monoclonal antibody).
2 shows the structural formulas of representative PEG-containing thioacetamidil-linked conjugates of the present invention.
3 shows the structural formulas of representative PEG-containing disulfide linked compounds of the invention.
4 shows a synthetic scheme for the PEG-containing thiosuccinimidyl-linked conjugates of the present invention.
5 shows a synthetic scheme for the PEG-containing thioacetamidil-linked conjugates of the present invention.
6 shows a synthetic scheme for an exemplary PEG-containing disulfide linked compound of the present invention: a.) Synthesis of PEG-containing disulfide linked compound for one-step conjugation to cell-binding agent; And b.) Synthesis of heterodifunctional PEG-containing disulfide linked crosslinking compounds.
Figure 7 shows the conjugation method for PEG-containing thiosuccinimidyl-linked conjugates of the invention (one-step conjugation).
8 shows the conjugation method for PEG-containing thiosuccinimidyl-linked conjugates of the invention (two-step conjugation).
9 shows a conjugation method for PEG-containing thioether-linked (thioacetamidyl-linked) conjugates of the invention (one-step conjugation).
10 shows the conjugation method for PEG-containing thioether-linked (thioacetamidyl-linked) conjugates of the present invention (two-step conjugation).
Figure 11 shows the conjugation method for PEG-containing disulfide linked conjugates of the present invention (one-step conjugation).
12 shows the conjugation method for PEG-containing disulfide linked conjugates of the invention (two-step conjugation).
FIG. 13 shows a synthetic scheme for PEG-containing sulfhydryl-reactive, thiosuccinimidyl-linked compounds of the invention.
Figure 14 shows a conjugation method for PEG-containing thiosuccinimidyl-linked conjugates of the invention (one-step conjugation).
Figure 15 shows a conjugation method for PEG-containing thiosuccinimidyl-linked conjugates of the present invention (two-step conjugation).
FIG. 16 shows a synthetic scheme for the PEG-containing sulfhydryl-reactive, thioacetamidyl-linked compounds of the present invention; FIG. a.) synthesis of PEG-containing sulfhydryl-reactive, thioacetamide linked compounds for one-step conjugation; And b.) Synthesis of heterobifunctional PEG-containing sulfhydryl-reactive crosslinking compounds for two-step conjugation.
Figure 17 shows a conjugation method for PEG-containing thioacetamidil-linked conjugates of the invention (one-step conjugation).
18 shows the conjugation method for PEG-containing thioacetamidil-linked conjugates of the invention (two-step conjugation).
Figure 19 shows a synthetic scheme for PEG-containing sulfhydryl-reactive, thioether-linked compounds of the present invention: a.) PEG-containing sulfhydryl-reactive, thioacetamide linked for one-step conjugation Synthesis of compounds; And b.) Synthesis of heterobifunctional PEG-containing sulfhydryl-reactive crosslinking compounds for two-step conjugation.
20 shows the conjugation method for PEG-containing thioacetamidil-linked conjugates of the invention (one-step conjugation).
Figure 21 shows a conjugation method for PEG-containing thioacetamidil-linked conjugates of the present invention (two-step conjugation).
FIG. 22 shows the mass spectrum (MS) of the deglycosylated HuAb-PEG ₄ Mal-DM1 conjugate (10.7 DMl / Ab, average).
Figure 23 shows size exclusion chromatography (SEC) of HuAb-PEG ₄ Mal-DM1 conjugate (10.7 DMl / Ab, average).
24 shows that FACS binding of HuAb-PEG4Mal-DM1 conjugate (10.7 maytansinoid / antibody) is similar to that of unmodified antibody.
25 shows cytotoxicity of anti-EpCAM antibody-maytansinoid conjugates against multi-drug resistant COLO205-MDR cells.
Figure 26 shows cytotoxicity of anti-CanAg antibody-maytansinoid conjugates against multi-drug resistant COLO205-MDR cells.
Figure 27 shows cytotoxicity of anti-CD56 antibody-maytansinoid conjugates against Molp-8 multiple myeloma cells.
28 shows cytotoxicity of anti-EpCAM antibody-maytansinoid conjugates on HCTl 5 cells.
29 shows cytotoxicity of anti-EpCAM antibody-maytansinoid conjugates on COLO 205 mdr cells.
30 shows in vivo anti-tumor activity of anti-EpCAM antibody-maytansinoid conjugates on HCTl 5 xenografts.
FIG. 31 shows in vivo anti-tumor activity of anti-EpCAM antibody-maytansinoid conjugates for COLO205 mdr xenografts.
32 shows in vivo anti-tumor activity of anti-EpCAM antibody-maytansinoid conjugates for COLO 205 xenografts.
33 shows in vivo anti-tumor activity of anti-CanAg antibody-maytansinoid conjugates for COLO 205 mdr xenografts.
34 shows binding of anti-CanAg antibody (huC242) -PEG24-Mal-DMl conjugates that are 17 D / A or less.
FIG. 35 shows in vitro efficacy of anti-CanAg antibody (huC242) -PEG24-Mal-DMl conjugate that is 4 to 17 D / A against COLO 205 cells.
36 shows in vitro efficacy of anti-CanAg antibody (huC242) -PEG24-Mal-DMl conjugate that is 4 to 17 D / A against multi-drug resistant (pgp +) COLO205-MDR cells.
37 shows cytotoxicity of anti-EGFR antibody-maytansinoid conjugates against UO-31 cells.
38 shows plasma pharmacokinetics of anti-PEG4-Mal-DM1.
Figure 39 shows the structural formula of thiosuccinimidyl-linked conjugates of the invention (mAb = antibody, m = 2-8).
40 shows the structural formulas of thioacetamidyl-linked conjugates of the invention (mAb = antibody, m = 2-8).
Figure 41 shows the conjugation method for thiosuccinimidyl-linked conjugates (mAb = antibody, m = 2-8) prepared by the compounds of the present invention.
42 shows conjugation methods for thioacetamidil-linked conjugates (mAb = antibody, m = 2-8) prepared by the compounds of the present invention.
Figure 43 shows a synthetic scheme for the amine-reactive thiosuccinimidyl-linked compound of the present invention.
FIG. 44 shows a synthetic scheme for the preparation of amine-reactive thiosuccinimidyl-linked compounds containing straight chain hydrocarbons between maleimide and NHS esters.
45 shows the mass spectrum (MS) of the deglycosylated mAb-SMCC-DM1 conjugate (3.42 DMl / mAb, average) prepared by the compounds of the present invention.
FIG. 46 shows a synthetic scheme for two-step preparation of amine-reactive thiosuccinimidyl-linked compounds containing a cycloalkyl group between maleimide and NHS ester.
FIG. 47 shows a synthetic scheme for two-step preparation of amine-reactive non-cleavable thiosuccinimidyl-linked compounds containing cycloalkyl groups between maleimide and NHS esters.
48 shows a schematic of the synthesis of thioacetamidyl-linked compounds of the present invention.
49 shows a synthetic scheme for two-step preparation of thioacetamidil-linked compounds of the present invention.
50 shows the structural formula of thiosuccinimidyl-linked conjugates of the invention (mAb = antibody, m = 2-8).
FIG. 51 is a conjugation method for thiosuccinimidyl-linked conjugates (mAb = antibody, m = 2-8) prepared by the compounds of the present invention.
Figure 52 is a synthetic scheme for the preparation of sulfhydryl-reactive thiosuccinimidyl-linked compounds of the present invention.
Figure 53 shows anti-CanAg (huC242) -Mal- (CH ₂ ) ₆ -Mal-DMl conjugate with 3.8 or D / A binds to antigen-positive COL205 cells.
54 shows anti-CanAg antibody (huC242) -Mal- (CH ₂ ) ₆ -Mal-DM1 conjugates with 3.8 D / A are potent against antigen-positive COLO205 cells, but not antigen-negative Namalwa cells. Indicates less.

The present invention relates to aliphatic linkers wherein conjugates of cell-binding agents (e.g. antibodies) linked to pharmaceuticals (e.g. cytotoxic agents) by polyethylene glycol or polyethylene oxide linkers ((-CH ₂ CH ₂ 0) _n ) and A novel discovery describes several times greater cytotoxicity against target cancer cells than would be expected based on comparison with conventional cell-binding drug conjugates with similar drug load. Importantly, the conjugates described herein are quite potent or effective against multidrug resistant cancer cells that are poorly sensitive to treatment with cytotoxic agents. Cancer therapies have the difficulty of overcoming the mechanism of drug resistance often encountered after several treatments with different chemotherapeutic agents. One such mechanism observed in cancer cells called multidrug resistance is caused by improved delivery of drugs by ATP-binding cassette (ABC) carriers (C. Drumond, BI Sikic, J Clin . Oncology , 1999, 17, 1061 -1070, G, Szokacs et al., Nature Reviews , 5; 219-234, 2006). Therapies that overcome these mechanisms of drug resistance, such as hindering or overcoming these outflows of drugs by cancer cells, are quite useful. Cytotoxicity of PEG-linked conjugates of cell-binding agents and cytotoxic agents is evaluated on multidrug resistant cancer cells to test whether PEG-linkers confer benefits on these resistant cells. In these assays for mdr cells, PEG-linked conjugates of cell-binding agents and cytotoxic agents unexpectedly exhibit potent cell death of mdr cells compared to conjugates with much less potency derived from conventional linkers. In addition, the conjugates of the present invention also exhibit significantly higher anti-tumor activity in animal models established by multidrug resistant tumor cells.

The use of hydrophilic polyethylene glycol or polyethylene oxide linkers (PEG or PEO; (-CH ₂ CH ₂ O) _n ) also resulted in higher protein monomer levels greater than 90% at concentrations above 1 mg / ml, which is desirable for therapeutic use. While having a relatively large number of drug incorporation per cell-binding molecule. Moreover, polyethylene glycol (PEG) -linked conjugates of cell-binding agents with a range of cytotoxic drug loads (a large number of agents, such as a small value of 2 to 15 linked per cell-binding agent) may be used to increase the increased drug loading of the conjugate. Based on the cytotoxicity of target cancer cells significantly improved than expected from the stoichiometric increase in drug delivery. Compared to conventionally prepared conjugates with similar drug loads, PEG spacers are shown by a 260 to 650-fold improvement in potency improvement (see Example; FIG. 29) in a superstoichiometric increase in cytotoxicity against target cancer cells. Conjugates of having cell-binding agents and agents are described herein.

Thus, in one aspect of the present invention, a medicament is disclosed having a polyethylene glycol spacer (-CH ₂ CH ₂ O) _n and a linker having a reactive group that can react with the cell-binding agent.

Modified compounds of formula (I) or certain compounds of formula (I ') have been particularly tried in this respect:

[ Formula 1]

ZX,-(-CH ₂ -CH ₂ -O-) _n -Y _p -D

[Formula 1 ']

DY _p -(-CH ₂ -CH ₂ -O-) _n -X _i -Z

In the above formulas,

Z represents a reactive functional group capable of forming an amide or thioether bond with the cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y is an aliphatic, aromatic or heterocyclic group bonded to the drug through a covalent bond selected from the group consisting of thioether bond, amide bond, carbamate bond, ether bond, amine bond, carbon-carbon bond and hydrazone bond Represent;

l is 0 or 1;

p is 0 or 1;

n is an integer from 1 to 2000.

Preferably, the covalent bond that binds Y to the agent is a thioether bond or an amide bond.

Preferably, n is an integer from 1 to 100. Even more preferably, n is an integer from 1 to 14. In the most preferred aspect n is an integer from 1 to 4.

In a second aspect of the invention, a novel conjugate of cell-binding agents and medicaments with polyethylene glycol linker (-CH ₂ CH ₂ O) _n is described. These conjugates are more potent on cancer cells than conjugates with conventional linkers and equivalent drug loads.

Conjugates of cell-binding agents and agents of Formula 2 or certain compounds of Formula 2 '

[Formula 2]

CB- [X,-(-CH ₂ -CH ₂ -O-) _n -Y _p -D] _m

[Formula 2 ']

(DY _p -(-CH ₂ -CH ₂ -O-) _n -X _{! m} -CB

In the above formulas,

CB represents a cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y is an aliphatic, aromatic or heterocyclic group bonded to the drug through a covalent bond selected from the group consisting of thioether bond, amide bond, carbamate bond, ether bond, amine bond, carbon-carbon bond and hydrazone bond Represent;

l is 0 or 1;

p is 0 or 1;

m is an integer from 2 to 15;

n is an integer from 1 to 2000.

Preferably, the covalent bond is a thioether bond or an amide bond.

Preferably, m is an integer from 3 to 8.

Preferably, n is an integer from 1 to 100. Even more preferably, n is an integer from 1 to 14. In the most preferred aspect n is an integer from 1 to 4.

The invention also provides an important correlation between the number of linked drugs and the length of the polyethylene glycol spacer in the improvement of the efficacy or efficacy of the immunoconjugate in the case of antibody conjugates, wherein the antibody is linked to the cytotoxic agent via disulfide bonds. Based on new findings that a relationship exists. An additional advantage of this linker design is the desired high monomer ratio and minimal aggregation of antibody-drug conjugates. Thus, in one aspect, the present invention provides that the polyethylene glycol spacer for the disulfide-linked conjugate consists of 2 to 8 ethyleneoxy groups, where the number of linked agents is 3 to 8, which means that the antibody-pharmaceutical conjugate It is based on the critical finding that it provides the best biological efficacy or efficacy and also provides the desired high monomer content.

In a preferred aspect, cells linked through disulfide groups (-SS-) having short polyethylene glycol spacers ((CH ₂ CH ₂ O) _{n = I-I4} ) with functional groups capable of reacting with cell-binding agents. Toxic agents are described.

Modified cytotoxic compounds of formula (3) or certain compounds of formula (3 ') have been particularly tried in this respect:

(3)

Z-XH-CH ₂ -CH ₂ O-) _n -YD

[Formula 3 ']

DY-(-CH ₂ -CH ₂ O-) _n -X, -Z

In the above formulas,

Z represents a reactive functional group capable of forming an amide or thioether bond with the cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y represents an aliphatic, non-aromatic heterocyclic or aromatic heterocyclic group bonded to the medicament via a disulfide bond;

l is 0 or 1;

n is an integer of 1-14.

Preferably, n is an integer from 2 to 8.

In another preferred aspect, a drug load of 3-8 is narrow, with relatively high potent biological activity against cancer cells, with minimal protein aggregation, and with the desired biochemical properties of high conjugation yield and high monomer ratio. Conjugates of cell-binding agents and medicaments linked through disulfide groups (-SS-) having a polyethylene glycol spacer ((CH ₂ CH ₂ O) _{n = I-I4} ) in the range are described.

Cell-binding agent conjugates of formula (4) or certain compounds of formula (4 ') have been particularly tried in this respect:

[Formula 4]

CB- (XK- ^ H ₂ -CH ₂ OVYD) _1n

[Formula 4 ']

[DY-(-CH ₂ -CH ₂ O-) _n -X,] _m -CB

In the above formulas,

CB represents a cell-binding agent;

D represents a medicament;

X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, amide bond, carbamate bond or ether bond;

Y represents an aliphatic, aromatic or heterocyclic group which is bound to the medicament via a disulfide bond;

l is 0 or 1;

m is an integer from 3 to 8;

n is an integer of 1-14.

Preferably, m is an integer from 3 to 6.

Also preferably, n is an integer of 2 to 8.

In the present invention, the medicament is a lipophilic molecule, which, when conjugated to a cell binding agent (eg an antibody), often causes a loss of yield due to protein aggregation or precipitation. Increasing the number of agents per cell-binding agent usually results in worse protein aggregation and precipitation, followed by poor% monomer and low yield. In contrast to conventional conjugate behavior with conventional linkers, PEG linkers provide monomer (%> 90% monomer) and yield (> 70%) of the conjugate of the drug and cell-binder at high concentrations of 1 mg / ml or more useful for therapeutic applications. ), Which leads to a desirable improvement. In addition, these conjugates are stable upon extended storage at 4 ° C.

In the present invention, novel maytansinoids having thioether moieties containing reactive groups are described such that these compounds are represented by the following general formula (5):

[Chemical Formula 5]

D'-Y'-V-Q-W-Z '

In the above formulas,

V is preferably any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms; More preferably straight chain alkyl having 1 to 5 carbon atoms, even more preferably V is one carbon alkyl group (CH ₂ );

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, more preferably 2 to 8 carbon atoms; Even more preferably W is a cyclohexyl group;

D 'represents sulfhydryl-containing maytansinoids, more preferably selected from DM1, DM3 and DM4;

Y 'represents a thioether bond;

Q represents any aromatic or heterocyclic moiety, preferably Q is absent;

Z 'is, but is not limited to, N-hydroxy succinimide ester, N-hydroxysulfosuccinimide ester, para or ortho-nitrophenyl ester, dynitrophenyl ester, pentafluorophenyl ester and sulfo Tetrafluorophenyl ester; An amine reactive group or thiol reactive group selected from maleimide or haloacetamide; More preferably, Z is N-hydroxysuccinimide, N-hydroxysulfosuccinimide ester or maleimide.

In another embodiment, reactive maytansinoid derivatives containing thioether moieties are prepared from sulfhydryl-containing maytansinoids (eg, DM1 and DM4) and heterodifunctional crosslinkers. The reaction sequence is represented by the following chemical reaction formula 6.

[Scheme 6]

D '+ γ "-V-Q-W-Z' → D'-Y'-V-Q-W-Z '

In the above chemical scheme,

V is preferably any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms; More preferably straight chain alkyl having 1 to 5 carbon atoms, even more preferably V is one carbon alkyl group (CH ₂ );

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, more preferably 2 to 8 carbon atoms; Even more preferably W is a cyclohexyl group;

Y ″ represents a thiol-reactive group selected from maleimide or haloacetamide, preferably maleimide;

D 'represents sulfhydryl-containing maytansinoids, more preferably selected from DM1, DM3 and DM4;

Y 'represents a thioether bond;

Q represents any aromatic or heterocyclic moiety, preferably Q is absent;

Z 'is, but is not limited to, N-hydroxy succinimide ester, N-hydroxysulfosuccinimide ester, para or ortho-nitrophenyl ester, dynitrophenyl ester, pentafluorophenyl ester and sulfo Tetrafluorophenyl ester; An amine reactive group or thiol reactive group selected from maleimide or haloacetamide; More preferably Z is N-hydroxysuccinimide, N-hydroxysulfosuccinimide ester or maleimide;

Y 'represents a thioether bond.

The invention also comprises reacting a cell binder with a compound of formula Z'-WQV-Y'-D 'to provide a cell binder conjugate of formula CB- (Z "-WQV-Y'-D') _m A cytotoxic conjugate of maytansinoids and cell binders linked via cleavable bonds, wherein W is any straight, branched or cyclic having 1 to 10 carbon atoms, more preferably 2 to 8 carbon atoms Alkyl, alkenyl, or alkynyl groups; even more preferably W is a cyclohexyl group; Q represents any aromatic or heterocyclic moiety, preferably Q is absent; V preferably has 1 carbon atom Any straight, branched or cyclic alkyl, alkenyl or alkynyl group having from 10 to 10, more preferably a straight chain alkyl group having from 1 to 5 carbon atoms, even more preferably V is one carbon alkyl group (CH ₂ ); Y 'is a thioether texture D 'represents a sulfhydryl-containing maytansinoid, more preferably selected from DM1, DM3 and DM4; CB is an antibody, single chain antibody, antibody fragment, peptide, growth factor , A cell binding agent selected from hormones, vitamins or ankyrin repeat protein (DARPin), preferably the cell binding agent is an antibody or antibody fragment, but Darpin; Z 'is not limited thereto, but N-hydroxy succinimide Esters, N-hydroxysulfosuccinimide esters, para or ortho-nitrophenyl esters, dynatrophenyl esters, pentafluorophenyl esters, and sulfotetrafluorophenyl esters; amines selected from maleimide or haloacetamides Reactive group or thiol reactive group; more preferably Z is N-hydroxysuccinimide, N-hydroxysulfosuccinimide De ester or maleimide; Z ″ represents a thioether bond or an amide bond.

The method optionally comprises a solution of a cell binder (e.g., an antibody) in an aqueous buffer containing up to 20% organic solvent in an organic solvent or a mixture of organic solvent and aqueous buffer or water in the formula Z'-WQV-Y'-D '. After mixing with a compound of, the reaction can be carried out by running for 5 minutes to 72 hours.

The conjugate can be further purified by chromatography, dialysis, tangential flow filtration or a combination thereof.

In all aspects, an "aliphatic group" is defined as an alkyl, alkenyl or alkynyl group. The alkyl group is preferably an aliphatic hydrocarbon group which may be straight or branched chain having 1 to 20, preferably 3 to 10 carbon atoms in the chain or cyclic. More preferable alkyl groups have 1 to 12 carbon atoms in the chain. "Branched" means that one or more lower alkyl groups (eg, methyl, ethyl or propyl) are bonded to a straight alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl and cyclohexyl.

Alkenyl groups are aliphatic hydrocarbon groups which contain carbon-carbon double bonds and may be straight or branched chains, preferably having 2 to 15 carbon atoms in the chain. More preferred alkenyl groups have 2 to 12 carbon atoms in the chain, and more preferably about 2 to 4 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, nonenyl and decenyl.

Alkynyl groups are aliphatic hydrocarbon groups which contain carbon-carbon triple bonds and may be straight or branched chains, preferably having 2 to 15 carbon atoms in the chain. More preferred alkynyl groups have 2 to 12 carbon atoms in the chain, and more preferably 2 to 4 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, octinyl and decinyl.

As used herein, the term "aromatic group" means a substituted or unsubstituted aryl group consisting of an aromatic monocyclic or multicyclic hydrocarbon ring system having 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms. do. Exemplary aryl groups include phenyl and naphthyl. Substituents include, but are not limited to, alkyl groups, halogens, nitro, amino, hydroxyl and alkoxy groups.

Halogen includes fluorine, chlorine, bromine and iodine atoms. Preference is given to fluorine and chlorine atoms.

As used herein, the term “heterocyclic group” refers to a saturated, partially unsaturated or unsaturated, non-aromatic, stable, 3 to 14 membered, preferably 5 to 10 membered mono, bi or multicyclic ring ( Wherein at least one element of the ring is a hetero atom or aromatic), preferably a 5 to 10 membered mono-, non- or multicyclic ring having at least one hetero atom. Typically, heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, selenium, and phosphorus atoms. Preferred hetero atoms are oxygen, nitrogen and sulfur.

Preferred heterocyclic groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl, dioxanyl, dioxolanyl , Piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianil, thiomorpholinyl, dihydro Pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydro-pyridyl, dihydropyridyl, tetrahydropyridinyl, dihydrothiopyranyl, azepanyl, pyrrolyl, pyridyl, pyrazolyl, thienyl , Pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, furinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadia Zolyl, isothiazolyl, Fusion system resulting from condensation with lyazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl and isoxazolyl, pyridyl-N-oxide and phenyl groups It includes.

Aliphatic, aromatic and heterocyclic groups represented by X and Y may also have charged substituents. Charged substituents may be, but are not limited to, negative charges selected from carboxylates, sulfonates and phosphates, or positive charges selected from tertiary or quaternary amino groups.

As used herein, the expression “linked to a cell-binding agent” refers to a conjugate molecule comprising at least one pharmaceutical derivative bound to the cell-binding agent via an appropriate linking group or precursor thereof. it means. Preferred linking groups are thiol or disulfide bonds, or precursors thereof.

As used herein, “precursor” of a given group means any group capable of deriving the group by deprotection, chemical modification or coupling reaction. For example, the precursor may be a suitably protected functional group where thioesters or thioethers are exemplified as thiol precursors.

As used herein, the term "reactive functionality" means amine-, thiol- or hydroxyl-reactive functional groups. In other words, reactive functional groups can react with amine, sulfhydryl (thiol), or hydroxyl groups present on the cell-binding agent. For example, amine-reactive functional group, if the functional groups are reactive carboxyl ester to provide the amide bond (iV- succinimidyl, N-sulfo-succinimidyl, N-phthalimido pyridyl, N-sulfonic Pope already de-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dynitrophenyl, 3-sulfo-4-nitrophenyl, 3-carboxy-4-nitrophenyl, tetrafluorophenyl, ester ), Reactive sulfonic acid derivatives, or reactive thioesters; For thiol-reactive functional groups, the functional groups can be maleimide, haloacetamide or vinyl sulfone to provide thioether bonds; In the case of a hydroxyl-reactive functional group, the functional group may be a reactive carboxyl ester to provide an ester bond.

A. with hydrophilic linker Reformed  Pharmaceutical and Reformed  Cell binder

Linkers are chemical moieties capable of linking agents (eg, maytansinoids) to cell-binding agents in a stable, covalent manner. The linker is susceptible or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage, under conditions where the drug or cell-binding agent remains active. Can be. 1, 2 and 3 provide exemplary structural formulas of the conjugates of the present invention.

Suitable crosslinking reagents comprising a hydrophilic PEG chain that forms a linker between the agent and the cell-binding agent are well known in the art or commercially available (eg from Quanta Biodesign, Powell, Ohio). Suitable PEG-containing crosslinkers can also be synthesized from commercial PEG itself using standard synthetic chemistry techniques known to those skilled in the art. The medicament may be reacted with a bifunctional PEG-containing crosslinker to provide a compound of Formula 1, Z-Xi-(-CH ₂ -CH ₂ -O-) _n -Y _p -D by the methods described herein. Can be. For example, thiol-containing maytansinoid agents can be reacted with bismaleimido crosslinkers with PEG spacers to provide maytansinoid agents linked via thioether bonds to PEG spacers (see, for example, 13). This modified maytansinoid with PEG spacer and terminal maleimido groups may then be reacted with a cell binder, eg, as shown in FIG. 14, to provide a cell binder-pharmaceutical conjugate of Formula 2 of the present invention. have.

In contrast, the cell binder is first reacted at one end of a bifunctional PEG containing crosslinker containing an amine reactive group (e.g., N -hydroxysuccinimide ester) to form a modified cell binder covalently linked to the linker via amide bonds. May be provided (see, eg, FIG. 15). In subsequent steps, maytansinoids are reacted with maleimido substituents at the other end of the PEG spacer to provide the cell binder-drug conjugates of the present invention.

16 and 17 illustrate the synthesis of PEG crosslinkers and their reaction with maytansinoids via thioacetamido linkages. The maleimido substituent is then incorporated into PEG to allow for reaction with the cell binder via thioether binding. Alternatively, for example, as shown in FIG. 18, the cell binder is first linked to a PEG crosslinker via thioether binding. The modified cell binder is then reacted with a maytansinoid agent to provide a conjugate. Of a homofunctional PEG crosslinker, wherein both ends of the PEG spacer contain an iodoacetamido moiety that can be linked to both the cytotoxic agent and the cell binder via a thioether bond to provide a conjugate containing a hydrophilic PEG spacer. Synthesis is shown, for example, in FIG. 19. A joining method for providing a conjugate of the present invention is shown, for example, in FIGS. 20 and 21.

Those skilled in the art will appreciate that other PEG-containing crosslinkers containing various reactive groups can be readily synthesized by the methods described herein. For example, a medicament containing a hydroxyl group, such as 19-demethylmaytansinoid (US Pat. No. 4,361,650), may be prepared by using an iodo-acetyl-PEG linker (eg, potassium carbonate) in the presence of a base (e.g., potassium carbonate). 5) and may be linked to maytansinoid through ether bonding. Similarly, amine-containing maytansinoids (synthesized as described in US Pat. No. 7,301,019) can be reacted with iodoacetyl PEG (shown in FIG. 5) in the presence of a base such as pyridine or triethylamine. Maytansinoids linked to PEG via amine linkages can be provided. For drug linkage to PEG via amide bonds, carboxy-PEG (shown in FIG. 5) is reacted with an amine-containing maytansinoid in the presence of a condensing agent (e.g. dicyclohexylcarbodiimide) May provide maytansinoids. To link the drug to the PEG spacer via carbamate linkage, the PEG first reacts with diphosgene to give PEG chloroformate and then with an amine-containing maytansinoid in the presence of a base (e.g. triethylamine). The reaction can provide a carbamate linked PEG-maytansinoid.

Examples of suitable linkers include linkers having maleimido- or haloacetyl-based moieties for reaction with the agent, as well as iV-succinimidyl ester or N -sulfosuccinimidyl ester moieties for reaction with cell-binding agents. Include. PEG spacers can be incorporated into crosslinking agents known in the art by the methods described herein. Crosslinking reagents comprising maleimido-based moieties that can be incorporated into PEG spacers include, but are not limited to, N -succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), 7V-succinimi Diyl-4- (Λ'-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidocaproate) (which is the "long chain" homologue of the SMCC) (LC-SMCC), k-maleimundodecano Acid N -succinimidyl ester (KMUA), γ-maleimidobutyric acid N -succinimidyl ester (GMBS), ε-maleimidocaproic acid N -hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl- N -hydroxysuccinimide ester (MBS), N- (α-maleimidoacetoxy) -succinimide ester (AMAS), succinimidyl-6- (β-maleimidopropionamido) hexanoate ( SMPH), N -succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB) and N- (p-maleimidophenyl) iocyanate (PMPI). Crosslinking reagents comprising haloacetyl-based moieties include N -succinimidyl-4- (iodoacetyl) aminobenzoate (SIAB), N -succinimidyl iodoacetate (SIA), N -succinimidyl bromine Moacetate (SBA) and N -succinimidyl 3- (bromoacetamido) propionate (SBAP).

Other crosslinking reagents lacking sulfur atoms can also be used in the process of the invention. The linker may be derived from a dicarboxylic acid based moiety. Suitable dicarboxylic acid based moieties include, but are not limited to, α, ω-dicarboxylic acids of the formulas shown below:

HOOC-A ' _p -E' _q- (CH ₂ CH ₂ O) _n G ' _r -COOH

Wherein A 'is any straight or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, E' is any cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, and G ' Is any substituted or unsubstituted aromatic group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the hetero atom is selected from N, O or S; p, q and r are each 0 or 1, except that p, q and r are not all zero at the same time, and n is an integer of 1-2000.

Many linkers described herein are described in detail in US Patent Publication No. 20050169933.

In another aspect of the invention, the cell-binding agent reacts with the bifunctional crosslinking reagent and the cell-binding agent, thereby creating and modifying the covalent linkage of the linker molecule to the cell-binding agent. As used herein, a "bifunctional crosslinking reagent" is a chemical moiety that covalently links a cell-binding agent to a medicament, such as a medicament described herein. In a preferred aspect of the invention, part of the linking portion is provided by a medicament. In this regard, the medicament comprises a linking moiety that is part of a larger linker molecule used to link the cell-binding agent to the medicament. For example, to form maytansinoid DM1, the side chains at the C-3 hydroxyl group of maytansine are modified to have free sulfhydryl groups (SH). This thiolated form of maytansine can be reacted with modified cell-binding agents to form conjugates. Thus, the final linker is assembled from two components, one of which is provided by the crosslinking reagent, while the other is provided by the side chain from DM1.

In another aspect of the invention, the medicament is linked to the cell-binding agent through disulfide bonds. Linker molecules include reactive chemical groups capable of reacting with cell-binding agents. Preferred reactive chemical groups for reaction with cell-binding agents are N -succinimidyl esters and N -sulfosuccinimidyl esters. The linker molecule also contains a reactive chemical group, preferably a dithiopyridyl group capable of reacting with a medicament to form a disulfide bond. Particularly preferred linker molecules include, for example, N - succinimidyl 3- (2-pyridyl die-thio) propionate (SPDP) (see, for example: Carlsson et al, Biochem J, 173: 723-737 (.. 1978)), N -succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) (see eg US Pat. No. 4,563,304), N -succinimidyl 4- (2-pyridyldithio Pentanoate (SPP) (see, eg, CAS Registry number 341498-08-6), and other reactive crosslinking agents (such as those described in US Pat. No. 6,913,748, which is incorporated by reference in its entirety herein). Include.

Alternatively, as described in US Pat. No. 6,441,163 B1, a medicament may first be modified to introduce a reactive ester suitable for reacting with the cell-binding agent. The reaction of these agents containing the activated linker moiety with the cell-binding agent provides another method of preparing cell-binding agent conjugates. For linkage of siRNAs, siRNAs can be linked to the crosslinking agents of the present invention by methods commonly used in the modification of oligonucleotides (see, eg, US Patent Publication Nos. 20050107325 and 20070213292). Thus, in its 3 'or 5'-phosphoromidite form, the siRNA reacts with one end of the crosslinking agent containing hydroxyl functional groups to provide an ester bond between the siRNA and the crosslinking agent. Similarly, the reaction of the siRNA phosphoramidite with the crosslinking agent containing terminal amino groups causes the linkage of the crosslinking agent to the siRNA via the amine.

B. Cell-Binding Agents

Cell-binding agents used in the present invention are proteins (e.g., immunoglobulins and non-immunoglobulin proteins) that specifically bind to target antigens on cancer cells. These cell-binding agents include:

Resurfaced antibodies (US Pat. No. 5,639,641)

Humanized or human antibodies (humanized or human antibodies include, but are not limited to huMy9-6, huB4, huC242, huN901, DS6, CD38, IGF-IR, CNTO 95, B-B4, trastuzumab, vivazumab (See, eg, US Pat. Nos. 5,639,641, 5,665,357, and 7,342,110; US Provisional Application No. 60 / 424,332, International Patent Application WO 02 /) 16,401, US Patent Publication No. 20060045877, US Patent Publication No. 20060127407, US Patent Publication No. 200501 18183, Pedersen et al, (1994) J MoI . Biol . 235, 959-973, Roguska et al., (1994 Proceedings of the National Academy of Sciences , VoI 9 1, 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

Epitope binding fragments of antibodies (eg sFv, Fab, Fab ', and F (ab') ₂ ) (Parham, J Immunol. 131: 2895-2902 (1983); Spring et al, J Immunol . 113: 470- 478 (1974); Nisonoff et a ', Arch . Biochem . Biophys . 89: 230-244 (I960)).

Additional cell-binding agents include, but are not limited to

Ankyrin repeat protein (DARPins; Zahnd et al., J Biol . Chem ., 281, 46, 35167-35175, (2006); Binz, HK, Amstutz, P. & Pluckthun, A. (2005) Nature Biotechnology, 23, 1257-1268) or, for example, US Patent Publication No. 20070238667; US Patent No. 7,101,675; WO / 2007/147213; And ankyrin-like repeat proteins or synthetic peptides described in WO / 2007/062466;

Interferons (eg α, β, γ);

Lymphokines (eg, IL-2, IL-3, IL-4, IL-6);

Hormones such as insulin, thyrotropin releasing hormones (TRH), melanocyte-stimulating hormone (MSH), steroid hormones (eg androgens and estrogens); And

Growth factors and colony-stimulating factors (e.g., EGF, TGF-α, IGF-I, G-CSF, M-CSF and GM-CSF) (Burgess, Immunology Today 5: 155-158 (1984)), and other cell-binding proteins and polypeptides.

If the cell-binding agent is an antibody, it binds to an antigen that is a polypeptide and may be a transmembrane molecule (eg a receptor) or a ligand (eg a growth factor). Exemplary antigens include molecules such as lenin; Growth hormones including human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipoprotein; Alpha-1-antitrypsin; Insulin A-chain; Insulin B-chain; Proinsulin; Follicle stimulating hormone; Calcitonin; Progesterone; Glucagon; Coagulation factors (eg, factor vmc, factor IX, tissue factor (TF) and von Willebrands factor); Anti-coagulation factors (eg protein C); Atrial natriuretic factor; Waste surfactants; Plasminogen activating factors such as urokinase or human urine or tissue-type plasminogen activating factor (t-PA); Bombesin; Thrombin; Hematopoietic growth factor; Tumor necrosis factor-alpha and -beta; Enkephalinase; Regulated on activation normally T-cell expressed and secreted; Human macrophage inflammatory protein (MIP-I-alpha); Serum albumin (eg, human serum albumin); Muellerian-inhibiting substance; Relaxine A-chain; Relaxine B-chain; Prolelaxacin; Mouse gonadotropin-binding peptides; Microbial proteins (eg beta-lactamase); DNase; IgE; Cytotoxic T-lymphocyte binding antigen (CTLA) (eg CTLA-4); Inhivin; Actibin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors; Protein A or D; Rheumatic factor; Neurodegenerative factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT4, NT-5 or NT-6), or nerve growth factor (Eg NGF-β)]; Platelet-derived growth factor (PDGF); Fibroblast growth factors such as aFGF and bFGF; Epidermal growth factor (EGF); Transforming growth factor (TGF) (eg, TGF-alpha and TGF-beta, including TGF-βl, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); Insulin-like growth factor-I and -II (IGF-I and IGF-II); des (l-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein, EpCAM, GD3, FLT3, PSMA, PSCA, MUCl, MUC16, STEAP, CEA, TENB2, EphA receptor, EphB receptor , Folic acid receptor, FOLRl, mesothelin, krypto, alpha beta, integrin, VEGF, VEGFR, transferrin receptor, IRTAl, IRTA2, IRTA3,

IRTA4, IRTA5; CD proteins (e.g. CD2, CD3, CD4, CD5, CD6, CD8, CDl 1, CD14, CDl9, CD20, CD21, CD22, CD23, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CDl03, CDl05, CDl34, CD137, CDl38, CDl52); Erythropoietin; Osteoinduction factors; Immunotoxins; Bone morphogenetic protein (BMP); Interferons such as interferon-alpha, -beta and -gamma; Colony stimulating factor (CSF) (eg, M-CSF, GM-CSF, and G-CSF); Interleukin (ILs) (eg, IL-I to IL-IO); Superoxide dismutase; T-cell receptor; Surface membrane proteins; Corrosion promoters; Viral antigens (eg, portions of HIV envelopes); Transport protein; Homing receptor; Addressin; Regulatory proteins; Integrins (eg, CDl Ia, CDl Ib, CDl Ic, CDl8, ICAM, VLA-4 and VCAM); Tumor binding antigens such as HER2, HER3 or HER4 receptors; And the above-presented polypeptides, the antibody mimic Adnectin ((US Patent Application No. 20070082365), or US Publication No. 20080171040 or US Publication No. 20080305044, which are incorporated by reference in their entirety herein). Fragments of any one or more of the tumor-binding antigens or antibodies that bind to cell-surface receptors described in the call.

In addition, GM-CSF that binds to bone marrow cells can be used as cell-binding agents for diseased cells from acute myeloid leukemia. IL-2 that binds to activated T-cells can be used for the prevention of graft graft rejection, for the treatment and prevention of graft-versus-host disease, and for the treatment of acute T-cell leukemia. MSH that binds to melanocytes can be used in the treatment of melanoma. Folic acid can be used to target folic acid receptors expressed in ovaries and other tumors. Epithelial growth factors can be used to target squamous cancers such as lungs and head and neck. Somatostatin can be used to target neuroblastoma and other tumor forms.

Breast and testicular cancers can be successfully targeted by estrogens (or estrogen analogs) or androgens (orrogenic analogs), respectively, as cell-binding agents.

Preferred antigens for the antibodies included in the present invention are CD proteins (e.g., CD2, CD3, CD4, CD5, CD6, CD8, CDl1, CD 14, CD18, CD19, CD20, CD 21, CD22, CD 25, CD26, CD27). , CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, and CD152); Members of an ErbB receptor group (eg, EGF receptor, HER2, HER3 or HER4 receptor); Cell adhesion molecules (eg LFA-I, Macl, pi 50.95, VLA-4, ICAM-I, VCAM, EpCAM, alpha or beta subunits thereof (eg anti-CD1 Ia, anti-CD18 or anti-CD1 Ib antibodies) Alpha4 / beta7 integrin and alphav / beta3 integrin); Growth factors (eg VEGF); Tissue factor (TF); TGF-β; Alpha interferon (alpha-IFN); Interleukin (eg, IL-8); IgE; Blood group antigen Apo2, a death receptor; flk2 / flt3 receptor; Obesity (OB) receptor; mpl receptor; CTLA-4; Protein C and the like. Most preferred targets in the present invention are IGF-IR, CanAg, EphA2, MUCl, MUCl 6, VEGF, TF, CDl9, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56, CDl38, CA6, Her2 / neu, EpCAM, CRIPTO (protein produced at increased levels in most human breast cancer cells), darpin, alphav / beta ₃ integrin, alphav / beta ₅ integrin, alpha _v / beta ₆ integrin, TGF-β, CDl Ia , CDl 8, Apo2 and C242, or antibodies that bind to one or more tumor-binding antigens or cell-surface receptors described in US Patent Publication No. 20080171040 or US Patent Publication No. 20080305044, which are incorporated by reference in their entirety.

Preferred antigens for the antibodies encompassed by the invention also include CD proteins (eg, CD3, CD4, CD8, CD19, CD20, CD27, CD34, CD36, CD37, CD38, CD46, CD56, CD70 and CD138); Members of an ErbB receptor group (eg, EGF receptor, HER2, HER3 or HER4 receptor); Cell adhesion molecules [eg LFA-I, Macl, p150.95, VLA-4, ICAM-I, VCAM, EpCAM, alpha or beta subunits thereof (eg anti-CD 1Ia, anti-CD 18 or anti-CD) Alpha 4 / beta7 integrins and alpha v / beta3 integrins); Growth factors (eg VEGF); Tissue factor (TF); TGF-β; Alpha interferon (alpha-IFN); Interleukin (eg, IL-8); IgE; Blood group antigen Apo2, a death receptor; flk2 / flt3 receptor; Obesity (OB) receptor; mpl receptor; CTLA-4; Protein C and the like. Most preferred targets in the present invention are IGF-IR, CanAg, EGF-R, EGF-RvIII, EphA2, MUCl, MUCl6, VEGF, TF, CDl9, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56 , CD70, CDl38, CA6, Her2 / neu, EpCAM, CRIPTO (protein produced at increased levels in most human breast cancer cells), alpha _v / beta ₃ integrin, alpha _v / beta ₅ integrin, TGF-β, CDl Ia , CDl 8, Apo2, EpCAM and C242.

Monoclonal antibody technology allows the preparation of specific cell-binding agents in the form of monoclonal antibodies. Antigens of interest (eg, complete target cells), antigens isolated from target cells, whole virus, attenuated whole virus, and viral proteins (eg viruses) in mice, rats, hamsters or other mammals Techniques for producing monoclonal antibodies prepared by immunization with envelope proteins) are particularly well known in the art. Sensitized human cells can also be used. Another method of producing monoclonal antibodies is the use of phage libraries of sFv (single chain variable region), in particular human sFv (see, eg, Griffiths et al, US Pat. No. 5,885,793; McCafferty et al, WO 92/01047; Liming et al, WO 99/06587).

The choice of the appropriate cell-binding agent is a matter of choice depending on the particular cell population being targeted, but in general, where appropriate, monoclonal antibodies and epitope binding fragments thereof are preferred.

For example, the monoclonal antibody My9 is a murine IgG _2a antibody specific for the CD33 antigen found in acute myeloid leukemia (AML) cells (Roy et al. Blood) . 77: 2404-2412 (1991)), which can be used to treat AML patients. Similarly, the monoclonal antibody anti-B4 is a murine IgGi that binds to a CD 19 antigen on B cells (Nadler et al, J Immunol. 131: 244-250 (1983)) and the target cell is a non-antigen (e.g., non- -Hodgkin lymphoma or chronic lymphocytic leukemia) or cells with disease. Antibody N901 is a murine monoclonal IgGi antibody that binds to CD56 found in small cell lung cancer cells and other tumor cells of neuroendocrine origin (Roy et al. J Nat . Cancer Inst . 88: 1 136-1 145 (1996)) ; huC242 is an antibody that binds to the CanAg antigen; Trastuzumab is an antibody that binds to HER2 / neu; Anti-EGF receptor antibodies bind to EGF receptors.

C. Pharmaceuticals

Agents used in the present invention are cytotoxic agents that can be linked to cell-binding agents. Examples of suitable agents include maytansinoids, DNA-binding agents (e.g. CC-1065 and analogues thereof), calicheamicin, doxorubicin and analogues thereof, vinca alkaloids, cryptophycins, dolastatin, auristatins and analogs thereof, Tubulicin, epothilones, taxoids and siRNAs.

Preferred maytansinoids are described in US Pat. No. 5,208,020; 5,416,064; 6,333.410; 6,333.410; No. 6,441,163; 6,716,821; 6,716,821; Described in RE39,151 and 7,276,497. Preferred CC-1065 analogues are described in US Pat. No. 5,475,092; 5,595,499; No. 5,846,545; 6,534,660; 6,534,660; 6,586,618; 6,586,618; 6,756,397 and 7,049,316. Preferred doxorubicin and analogs thereof are those described in US Pat. No. 6,630,579. Preferred taxoids are described in US Pat. No. 6,340,701; 6,372,738; 6,372,738; No. 6,36,931; No. 6,596,757; 6,706,708; 6,706,708; 7,008,942; 7,217,819 and 7,276,499. Calicheamicins are described in US Pat. Nos. 5,714,586 and 5739,1 16.

Vinca alkaloid compounds, dolastatin compounds and cryptophycin compounds are described in detail in WO01 / 24763. Auristatins include Auristatin E, Auristatin EB (AEB), Auristatin EEP (AEEP), Monomethyl Auristatin E (MMAE) and are described in US Pat. No. 5,635,483, Int . J. Oncol . 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); US Patent Application No. 11/134826, US Patent Publication No. 20060074008, and 2006022925. Tubulicin compounds are described in US Patent No. 20050249740. Cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021. Epothilones are described in US Pat. Nos. 6,956,036 and 6,989,450.

siRNAs are described in detail in U.S. Pat.

Analogs and Derivatives

One skilled in the art of cytotoxic agents will readily understand that the cytotoxic agents described herein can be modified in such a way that each resultant compound still retains the specificity and / or activity of the starting compound. The skilled person will also understand that many of these compounds may be used in place of the cytotoxic agents described herein. Thus, cytotoxic agents of the invention include analogs and derivatives of the compounds described herein.

Cell-binding agents can be conjugated to cytotoxic agents by the methods described above (US Pat. Nos. 6,013,748; 6,441,1631, and 6,716,821; US Patent Publications 20050169933; and WO2006 / 034488 A2). ).

D. Therapeutic Uses

The cell-binding agent conjugates (eg immunoconjugates) of the invention can also be used with chemotherapeutic agents. Such chemotherapeutic agents are described in US Pat. No. 7,303,749.

Cell-binding agent conjugates (e.g., immunoconjugates) of the invention treat patients and / or, for example, lung, blood, plasma, breast, colon, prostate, kidney, pancreas, brain, bone, ovary, testes and Cancer of the lymphoid organs; Autoimmune diseases such as systemic lupus, rheumatoid arthritis and multiple sclerosis; Graft rejection (eg, kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection); Graft versus host disease; Viral infections (eg CMV infection, HIV infection and AIDS); And in vitro, in vivo and / or ex vivo to regulate the growth of selected cell populations, including parasitic infections (eg, flagella, amoebiasis, schistosomiasis, etc.). Preferably, the immunoconjugates and chemotherapeutic agents of the present invention are for treating cancer in a patient and / or for example, blood, plasma, lung, breast, colon, prostate, kidney, pancreas, brain, bone, ovary Cancer of the testes and lymphoid organs; More preferably, it is administered in vitro, in vivo and / or ex vivo to regulate the growth of cancer cells, including lung, colon, prostate, plasma, blood or colon cancer. In the most preferred aspect, the cancer is multiple myeloma.

"Modulating the growth of a selected cell population" refers to a cell population selected from the division (eg, multiple myeloma cell population, eg, MOLP-8, for example, to produce more cells than untreated cells). Inhibition of proliferation of cells, OPM2 cells, H929 cells, etc.); Killing of selected cell populations; And / or prevention of a cell population (eg cancer cells) selected from metastasis. Growth of the selected cell population can be regulated in vitro, in vivo or ex vivo.

In the methods of the invention, cell-binding agent conjugates (eg, immunoconjugates) can be administered in vitro, in vivo or ex vivo. Cell-binding agent conjugates (eg, immunoconjugates) can be used with well known, appropriate pharmaceutically acceptable carriers, diluents and / or excipients and can be determined by one of ordinary skill in the art as a guarantee of clinical situation. Examples of suitable carriers, diluents and / or excipients are (1) Dulbecco's phosphate buffered saline, pH about 6.5, (2) 0.9% saline (containing about 1-25 mg / ml human serum albumin) 0.9% w / v NaCl) and (3) 5% (w / v) dextrose.

The compounds and compositions described herein can be administered in a suitable form, preferably parenterally, more preferably intravenously. For parenteral administration, the compound or composition may be an aqueous or non-aqueous sterile solution, suspension or emulsion. Propylene glycol, vegetable oils and injectable organic esters such as ethyl oleate may be used as the solvent or vehicle. The composition may also contain adjuvants, emulsifiers or dispersants.

The composition may also be in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium.

A “therapeutically effective amount” of a cell-binding agent conjugate (eg, an immunoconjugate) described herein refers to a dosing regimen for regulating the growth of a selected cell population and / or treating a patient's disease, including the age of the patient, Body weight, sex, diet and medical condition, severity of disease, route of administration and pharmacological considerations, for example, the activity, efficacy, pharmacokinetics and toxicological profile of the particular compound used are selected. A "therapeutically effective amount" can also be determined by reference to standard medical texts (eg, Physicians Desk Reference 2004). The patient is preferably an animal, more preferably a mammal, most preferably a human. The patient can be male or female and can be an infant, child or adult.

Examples of suitable methods of administration of cell-binding agent conjugates (eg immunoconjugates) are as follows. The conjugate may be administered daily for about 5 days as an intravenous bolus daily for about 5 days or as a continuous infusion for about 5 days.

Alternatively, the conjugate may be administered once a week for six weeks or longer. As another alternative, the conjugate may be administered once every two or three weeks. The bolus dose is provided in about 50 to about 400 ml of normal saline to which about 5 to about 10 ml of human serum albumin can be added. Continuous infusion is provided every 24 hours with about 250 to about 500 ml of normal saline to which about 25 to about 50 ml of human serum albumin can be added. The dose is about 10 pg to about 1000 mg / kg per person, intravenously (in the range of about 100 ng to about 100 mg / kg).

About 1 to about 4 weeks after treatment, the patient may receive a second course of treatment. The particular clinical method with respect to the route of administration, excipient, diluent, dose and time can be determined by the skilled person as a guarantee of clinical situation.

Compounds and conjugates (eg immunoconjugates) can also be used in the manufacture of a medicament useful for treating or lessening the severity of a disease, eg, characterized by abnormal cell growth (eg cancer).

The invention also provides a pharmaceutical kit comprising one or more containers filled with one or more components of the pharmaceutical compounds and / or compositions of the invention, including one or more immunoconjugates and one or more chemotherapeutic agents. The kit may also be, for example, written instructions in the form of other compounds and / or compositions, device (s) for administering the compounds and / or compositions, and government agencies regulating the manufacture, use of medicaments or May contain a sale or biological product.

Cancer therapies and their doses, routes of administration, and suggested uses are known in the art and have been described in documents such as the Physicians Desk Reference (PDR). PDR describes the doses of agents that have been used in the treatment of various cancers. Therapeutic regimens and dosages of these above-mentioned chemotherapeutic agents will depend on the particular cancer to be treated, the severity of the disease, and other factors familiar to those skilled in the art, and may be determined by a physician. For example, the 2006 edition of the Physicians Desk Reference shows that taxotere (see p. 2947) is an inhibitor of tubulin depolymerization; Doxorubicin (ref p 786), doxyl (ref p 3302) and oxaliplatin (ref p 2908) are DNA interaction agents, irinotecal (ref p 2602) is a topoisomerase I inhibitor, and erybitux (ref p 937) ) And tarceva (see p 2470) describe interacting with epidermal growth factor receptors. The contents of the PDR are hereby incorporated by reference in their entirety. One skilled in the art can review the PDR using one or more of the following parameters to determine the dosage regimen and dose of chemotherapeutic agents and conjugates that may be used in accordance with the teachings of the present invention. These parameters include the following:

1. Comprehensive index

a) by manufacturer

b) product (in the name of a company or branded drug)

c) category indexes (eg, "antihistamines", "DNA alkylating agents", taxanes, etc.)

d) general / chemical index (non-trade name conventional drug name)

2. Color Image of Drugs

3. Matched with FDA indication, product information,

a) chemical information

b) function / action

c) instructions & contraindications

d) research, side effects, warnings;

The entirety of each of the above references, patent applications, and patents is expressly incorporated by reference in its entirety, including, without limitation, numerical text, tables, or drawings thereof, as well as the specification, claims, and abstracts.

E. Reactive group  Containing Thioether  Having part Of maytansinoids  synthesis

A novel maytansinoid having a non-cleavable thioether moiety containing a reactive group is the compound of formula 5, D'-Y'-V-Q-W-Z '. The chemical group Z 'for reaction with cell-binding agents is, but is not limited to, N-succinimidyl ester, N-sulfosuccinimidyl ester, pentafluorophenyl ester, tetrafluorosulfophenyl and nitrophenyl Contains esters. Methods of making these compounds are described herein. The sulfhydryl-containing maytansinoids are covalently linked to heterodifunctional crosslinkers containing reactive groups via non-cleavable thioether bonds and separated prior to conjugation with cell binders.

Non-cleavable linkers are chemical moieties capable of linking cytotoxic agents to cell-binding agents in a stable covalent manner. Non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage. Examples of non-cleavable linkers include linkers having iV-succinimidyl ester or TV-sulfosuccinimidyl ester portions and maleimido- or haloacetyl-based portions for reaction with maytansinoids. Crosslinking reagents comprising maleimido-based moieties include iV-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), iV-succinimidyl-4- ( N -maleimidomethyl) -cyclohexane -1-carboxy- (6-amidocaproate) (which is the "long chain" homologue of the SMCC (LC-SMCC)), κ-maleimidodecanoic acid N -succinimidyl ester (KMUA), γ-maleimido Butyric acid iV-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-iV-hydroxysuccinimide ester (MBS), N- ( α-maleimidoacetoxy) -succinimide ester (AMAS), succinimidyl-6- (β-maleimidopropionamido) hexanoate (SMPH), N -succinimidyl 4- (p-maleimido Phenyl) butyrate (SMPB) and 7V- (p-maleimidophenyl) iocyanate (PMPI). Crosslinking reagents comprising haloacetyl-based moieties include iV-succinimidyl-4- (iodoacetyl) aminobenzoate (SIAB), 7V-succinimidyl iodoacetate (SIA), iV-succinimidyl bromine Moacetate (SBA) and iV-succinimidyl 3- (bromoacetamido) propionate (SBAP).

Maytansinoids that can be used in the present invention to prepare reactive maytansinoid derivatives containing thioether moieties that can be covalently bound to cell binding agents are well known in the art, According to known methods, or synthetically prepared according to known methods.

The synthesis of maytansinoids having thioether moieties containing reactive groups may be described with reference to FIGS. 1 to 4, wherein thioether-containing maytansinoids containing reactive groups are prepared.

Representative compounds of the invention are prepared from thiol-containing maytansinoids: DMl, DM1 called N ^{2 ′} -deacetyl- N ^{2 ′} -(3-mercapto-l-oxopropyl) -maytansine of Formula 1 and N ² of formula ^(2), - having ^{-N-acetyl-2 '-} are shown for DM4 is called a make-tansin (4-mercapto-4-methyl-1-oxo-pentyl)

Novel maytansinoids having thioether moieties containing reactive groups are prepared by the following newly described method.

The synthesis of novel maytansinoids having a thioether moiety containing a reactive group of D'-Y'-V-Q-W-Z 'of formula 5 comprising the following steps is described:

a.) Covalently linking the heterodifunctional linker of formula VVQW-Z 'to a thiol-containing maytansinoid, D', wherein Y "is a thiol-reactive group such as maleimide or haloacetamide; V Is preferably any straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and W preferably has 1 to 10 carbon atoms. And more preferably any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 5 carbon atoms, Q represents any aromatic or heterocyclic moiety; But is an amine-reactive group such as N-succinimidyl ester, 7V-sulfosuccinimidyl ester, pentafluorophenyl ester, tetrafluorosulfophenyl or nitrophenyl ester). Chemical formula 6.

[Scheme 6]

D '+ V-V-Q-W-Z' → D'-Y'-V-Q-W-Z '

b.) A stoichiometric or excess equivalent of heterodifunctional crosslinker, V-V-Q-W-Z ', can be used for thiol-containing maytansinoids D' (e.g. DM1 and DM4). The reaction can proceed to completion and can be monitored by analytical methods such as HPLC and TLC. The reaction can also be carried out using an excess thiol-containing maytansinoid, D ', for the heterodifunctional linker V-V-Q-W-Z'.

c.) The reaction can be carried out in a suitable organic solvent or a mixture of an aqueous buffer and an organic solvent to make the thiol-containing maytansinoid D 'and the heterodifunctional linker, V-V-Q-W-Z' completely soluble. Examples of suitable organic solvents are tetrahydrofuran (THF), 1,2-dimethoxyethane, Λ /, iV-dimethylformamide (DMF), iV, 7V-dimethylacetamide (DMA), methanol and ethanol It includes. When the reaction is carried out in a mixture of an organic solvent and an aqueous buffer, the pH is the reaction of sulfhydryl-containing maytansinoids with maleimide or haloacetamide, Y ″ while minimizing the hydrolysis potential of the amine reactive group Z. Silver should be adjusted to facilitate the appropriate pH range for this reaction is pH 3-10, preferred pH 5-9, and most preferred pH 6-8.

d.) Thioether formation between thiol-containing maytansinoids, D ′ and thiol-reactive groups Y ″ may also be prepared using appropriate organic bases and pure organic solvents, as described above. Bases such as N, N -diisopropylethylamine (DIPEA), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), triethylamine and 4-methylmorpholine It can be used to form the desired thioether containing maytansinoid containing the reactive group of D'-Y'-VQW-Z 'which is 5.

e.) The compound of D'-Y'-V-Q-W-Z 'of formula 5 can be separated following completion of the reaction. Suitable techniques for the purification of maytansinoids having a thioether moiety containing an amine reactive group Z '(e.g. iV-succinimidyl ester) can be prepared by silica gel chromatography, normal or reverse phase preparative high performance liquid chromatography (HPLC). ), Preparative thin layer chromatography (TLC) and crystallization.

f.) Purification and identification of the isolated product of D'-Y'-VQW-Z 'of formula 5 is carried out by analytical methods including HPLC, mass spectroscopy (MS), LC / MS, ¹ H NMR and ¹³ C NMR I can tell.

F. Thioether  Having part Maytansinoid  Preparation of Derivatives

Novel maytansinoid derivatives containing thioether moieties useful for the preparation of maytansinoids containing non-cleavable thioether moieties containing reactive groups are described herein. The preparation of these derivatives from DM1 (1) and DM4 (2) is described, which is represented by the following formula (10):

[Formula 10]

D'-Y'-V-Q-W-COOH

In the above formulas,

V is preferably any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms;

W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms;

D 'represents sulfhydryl-containing maytansinoid, DM1 or DM4;

Y 'represents a thioether bond;

Q represents any aromatic or heterocyclic moiety.

The synthesis of novel maytansinoid derivatives having a thioether moiety containing a carboxylic acid of Formula 10 is described, comprising the following steps:

a.) Covalently linking a carboxylic acid linker of formula VVQW-COOH to a thiol containing maytansinoid, D ', wherein Y "is a thiol-reactive group such as maleimide or haloacetamide; Q is any Aromatic or heterocyclic moiety) The reaction sequence is represented by the following chemical formula 11:

[Chemical Scheme 11]

D '+ Y "-V-Q-W-COOH → D'-Y'-V-Q-W-COOH

Wherein V and W are as defined above, D 'is as defined above; Y "is as defined above; Y' is a thioether between sulfhydryl containing maytansinoids, carboxylic acids, and COOH. Is a bond; Q represents any aromatic or heterocyclic moiety).

b.) A stoichiometric or excess equivalent of a carboxylic acid linker of formula Y "-VQW-COOH can be used for thiol-containing maytansinoids, D '(e.g. DM1 and DM4). And can be monitored by analytical methods such as HPLC and TLC. The reaction can also be carried out using an excess thiol-containing maytansinoid, D ′, for the carboxylic acid linker of formula Y ″ -VQW-COOH. Can be.

c.) The reaction can be carried out in a suitable organic solvent or a mixture of an aqueous buffer and an organic solvent to make the thiol-containing maytansinoid D 'and the carboxylic acid linker of formula VVQW-COOH completely soluble. Examples of suitable organic solvents include tetrahydrofuran (THF), 1,2-dimethoxyethane, NJN -dimethylformamide (DMF), Λ /, N -dimethylacetamide (DMA), methanol and ethanol do. When the reaction is carried out in a mixture of organic solvent and aqueous buffer, the pH should be adjusted to promote the reaction of sulfhydryl with maleimide or haloacetamide, Y ". Suitable pH ranges for this reaction are pH 3 to 10, preferred pH is 5-9, most preferred pH is 6-8.

d.) Thioether formation between thiol-containing maytansinoids, D ′ and thiol-reactive groups Y ″ also occurs when using suitable organic bases and pure organic solvents, as described above. N, N -diisopropylethylamine (DIPEA), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), triethylamine and 4-methylmorpholine] contain carboxylic acid It can be used to form the desired thioether containing maytansinoid.

e.) Compounds of D'-Y'-V-Q-W-COOH of Formula 10 may be separated following completion of the reaction. Suitable techniques for purification of maytansinoids having thioether moieties containing carboxylic acids include silica gel chromatography, normal or reverse phase preparative high performance liquid chromatography (HPLC), preparative thin layer chromatography (TLC) and crystallization. .

f.) Purification and identification of the isolated product of D'-Y'-VQW-COOH of formula 10 will be revealed by analytical methods including HPLC, mass spectroscopy (MS), LC / MS, ¹ H NMR and ¹³ C NMR Can be.

 Novel maytansinoid derivatives having a thioether moiety containing a terminal carboxylic acid of D'-Y'-VQW-COOH of formula 11 are prepared in preparation of the compound of D'-Y'-VQW-Z 'of formula 5 useful. Maytansinoid derivatives of D'-Y'-V-Q-W-COOH of Formula 11 may be further derivatized to provide an amine reactive group, Z. A preferred amine reactive group, Z ', is an active ester such as, but not limited to, iV-succinimidyl ester, 7V-sulfosuccinimidyl ester, pentafluorophenyl ester, tetrafluorosulfophenyl and nitrophenyl ester. . The method for preparing the compound of Formula 5 from the compound of Formula 11 follows the following reaction sequence:

D'-Y'-V-Q-W-COOH + X → D-Y'-V-Q-W-Z '

Wherein X is N-hydroxysuccinimide to provide Z ', which is an activated ester that is amine reactive.

Maytansinoids of D'-Y'-VQW-Z 'of Formula 5 are prepared from the novel maytansinoid derivatives of D'-Y'-VQW-COOH of Formula 11 by known methods described herein. It can manufacture.

a.) Maytansinoids of D'-Y'-VQW-COOH of Formula 11 are anhydrous organic solvents in the presence of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (EDCHCl). (E.g. methylene chloride, dimethylformamide, tetrahydrofuran, dioxane, diethylether) and may be reacted with a rather excess TV-hydroxysuccinimide (X). Condensing agents other than EDCHCl may be used for the reaction.

b.) Completion of the reaction can be monitored using standard chemical techniques (eg TLC or HPLC).

c.) Following completion of the reaction, maytansinoid derivatives containing reactive 7V-hydroxysuccinimidyl esters can be purified by silica gel chromatography, or by HPLC or by crystallization.

d.) Purification and identification of the isolated product of D'-Y'-VQW-Z 'of formula 5 is carried out by analytical methods including HPLC, mass spectroscopy (MS), LC / MS, ¹ H NMR and ¹³ C NMR I can tell.

G. Reactive group  Containing Thioether  Having part With maytansinoids  Preparation of Antibody Conjugates

Non-cleavable thio with cell binders using an isolated, reactive maytansinoid derivative having a thioether moiety containing a reactive group of CB- (Z "-WQV-Y'-D ') _m A process for preparing ether linked maytansinoid conjugates (FIGS. 39 and 40) is described and consists of the following steps:

a.) through the covalent amide bond formation between the amine reactive group linked to a maytansinoid having a thioether bond and the lysine residue of the cell binder, to the cell binder, CB (antibody), D'- Covalently linking purified amine-reactive maytansinoids containing the thioether portion of Y'-WQV-Z '.

b.) The conjugation reaction can be carried out at a concentration of 0.5 to 10 mg / ml of the antibody, depending on the nature of the antibody.

c.) Stock solutions of amine-reactive non-cleavable maytansinoids containing thioether moieties can be prepared in a pure organic solvent prior to conjugation with the antibody. Suitable organic solvents for preparing stock solutions include methanol, ethanol, N , iV-dimethylacetamide (DMA), Λ /, iV-dimethylformamide (DMF) and dimethylsulfoxide (DMSO).

c.) Due to the hydrophobic nature of maytansinoids, it may be necessary to perform conjugation to the antibody in a mixture of aqueous buffer and organic solvent to ensure that the maytansinoid remains in solution during conjugation. Preferred amounts of organic solvents in aqueous buffers range from 0 to 30% (v / v) depending on the solubility of the reagents and the behavior of the antibody under these conditions. Conjugation can be carried out at pH 3-10, preferred pH is 5-9, most preferred pH is 6-8. Buffers used for the conjugation reaction are buffers with pKa values around this pH range (eg phosphate and HEPES buffers).

c.) 5 to 50 fold excess of amine-reactive maytansinoid containing thioether moiety to the antibody is used in the conjugation reaction to obtain a conjugate having the desired number of maytansinoid molecules linked per antibody molecule. Can be obtained. Preferably, a range of 2 to 8 maytansinoids per antibody is preferred for the final conjugate. Conditions such as antibody concentration, solubility of the reagents and pH can affect the number of maytansinoid molecules linked per mole of antibody in the final conjugate, as well as the fold excess of required maytansinoid reagents.

d.) CB- (Z "-VQW-Y'-D ') _m by tangential flow filtration, dialysis or chromatography (gel filtration, ion exchange chromatography, hydrophobic interaction chromatography) or a combination thereof. Purification of non-cleavable conjugates.

Example

Without limitation to particular aspects, methods of synthesizing polyethylene glycol ((CH ₂ CH ₂ O) _n ) -linked agents by different reactive linkers for conjugation with cell-binding agents are described. These conjugation methods are described in the 1V of drugs and antibodies such as maytansinoids linked via polyethylene glycol ((CH ₂ CH ₂ O) _n ) linkers by reaction in a 7V-hydroxysuccinimide (NHS) reactive group. Step junctions.

Also described are methods of synthesizing disulfide group containing polyethylene glycol ((CH ₂ CH ₂ θ) _n ) -linked agents by different reactive linkers for conjugation with antibodies. These conjugation methods are combined with agents such as maytansinoids linked by disulfide groups with polyethylene glycol ((CH ₂ CH ₂ O) _n ) linkers via reactions in N-hydroxysuccinimide (NHS) reactive groups. One-step conjugation of the antibody.

The following examples are illustrative only and are not intended to limit the invention.

Example  I

Conventional aliphatic carbon Spacer  Several linked per antibody molecule by containing disulfide linker Maytansinoid  Conjugation of Molecules and Antibodies:

In a two-step process for conjugating an antibody with several molecules of maytansinoid DM4 or DM1, the humanized antibody first comprises an amine-reactive N -hydroxysuccinimide group (NHS group) and a thiol-reactive 2-pyridyldithiate. Some molecules of the linker are incorporated into the antibody molecule by modification by a commercially available heterodifunctional linker (SPDB) containing all Ogi (-SSPy groups) (WC Widdison et al., J Med . Chem ., 2006, 49, As described in 4392-4408). Following incorporation of the reactive linker into the antibody molecule, in a second step, a maytansinoid DM4 or DM1 having a reactive thiol group is added to the linker-modified antibody to conjugate the maytansinoid to the antibody by disulfide bonds. In certain embodiments, humanized antibodies at concentrations of 5-10 mg / ml are commercially available heterobifunctional linkers (e.g., having-(CH ₂ ) _n -alkyl groups in an aqueous buffer of pH 6.5-8 for 0.25-3 hours at ambient temperature. SPDB, SPP, SPDP) modified using 10-15 fold molar excess, followed by purification by gel filtration (e.g. using Sephadex G25 chromatography) to yield antibody molecules in high yield (typically 80-90% yield). An antibody modified with an average of 8 to 12 linker groups per sugar is obtained. Linked groups are based on their absorbance (ε _{343 nm} = 8080 M ^-1 cm ^-1 ) at _{343 nm} upon addition of excess 1,4-dithiothreitol (DTT) reagent to a small mother liquor of a linker-modified antibody sample. Expected by measuring the release of 2-thiopyridone. After measuring the reactive groups linked on the antibody, the linker-modified antibody at a concentration of 2.5 mg / ml is conjugated with excess maytansinoid DM4 (1.7 fold-molar excess of DM4 thiol for reactive linkers) at pH 6.5 . However, precipitation is observed during the antibody-maytansinoid conjugation reaction, and poor yield (-38 to 60% yield) of the antibody-maytansinoid conjugate is obtained upon purification of the antibody-maytansinoid conjugate by gel filtration. do. The number of linked maytansinoids per antibody molecule is determined from absorbance measurements at 252 nm and 280 nm, and using the absorption coefficients of the maytansinoid and antibody at 252 nm and 280 nm. In addition to precipitation and poor yield of antibody-maytansinoid conjugates at ˜1 to 1.5 mg / ml, the number of maytansinoids incorporated per antibody molecule is much larger average number of initial reactive linker groups incorporated per antibody molecule ( Much lower than expected based on -8-12 reactive linker groups per antibody molecule (-5.2-5.5 average maytansinoid molecules per antibody molecule), resulting in higher maytansinoid-containing antibody conjugates Suggests precipitation. In another embodiment, the humanized antibody is first modified by an SPDB heterodifunctional linker to incorporate 11 pyridyldithio groups per antibody molecule, which reacts upon a second reaction with 1.7-fold molar excess of DM4 maytansinoid thiol. Shows significant precipitation, resulting in very poor recovery of antibody-maytansinoid conjugates of <30%. Using commercially available heterobifunctional linkers with aliphatic spacers (eg SPDB or SPDP), typically 4 or per antibody at high conjugation yields for antibody-maytansinoid conjugate concentrations of 1 mg / ml or greater. It is difficult to incorporate maytansinoid molecules larger than five. This observed precipitation and low yield of antibody-maytansinoid conjugates with SPDB- or SPDP-induced linkers do not occur upon initial SPDB- or SPDP-linker modification of the antibody (prior to conjugation with maytansinoids) It is suggested that aggregation and precipitation of the maytansinoid conjugates are probably caused by the binding of hydrophobic molecules.

Example II

Hydrophilic polyethylene Oxide  Spacer PEG _n , or (- CH ₂ - CH ₂ -O) _{n = 1-14} Several linked per antibody molecule by a disulfide linker containing Maytansinoid  Conjugation of Molecules and Antibodies

Hydrophilic spacers (e.g., polyethylene oxide (PEG _n , or (-CH ₂ -CH ₂ -O) _{n = Ui4} ) antibody-maytansi, possibly with multiple maytansinoid molecules (average> 4 per antibody molecule) To investigate whether it prevents aggregation and precipitation of nodoid conjugates, several new heterozygotes that can be conjugated to the antibody by direct modification or two-step reaction, including initial derivatization of the antibody at lysine residues followed by the reaction of maytansinoids Sex and monofunctional maytansinoid derivatives are prepared (see, eg, FIGS. 3, 6, 11 and 12).

15- (2- Pyridyldithio ) -4,7,10,13- Of tetraoxapentadecanoic acid  synthesis

A solution of Aldritol-2 (1.17 g, 5.31 mmol) was prepared in 5.0 mL of 1,2-dimethoxyethane in a 10 mL round bottom flask. To the reaction flask was added a solution of 3- (2-thiotetraethyleneglycol) propionic acid ((QuantaBiodesign, 490 mg, 1.73 mmol) dissolved in 1.0 ml of 1,2-dimethoxyethane. Under 3.5 h and the product is purified by silica chromatography eluting with 5% methanol in methylene chloride The solvent is removed in vacuo to give 432 mg (64% yield) of the desired product.

PySS - PEG ₄ -NHS [15- (2- Pyridyldithio ) -4,7,10,13- Tetraoxapentadecanoic acid  Synthesis of N-hydroxysuccinimide Ester

A 10 ml round bottom flask is charged with 15- (2-pyridyldithio) -4,7,10,13-tetraoxapentadecanoic acid (431 mg, 1.10 mmol), 5.0 ml of methylene chloride and a stirring bar. 7V-hydroxysuccinimide (3.6 mg, 0.31 mmol) and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (6.8 mg, 0.036 mmol) were added to the reaction vessel The reaction was carried out for 2 hours at room temperature under stirring. The product is purified by silica chromatography eluting with 7% 1,2-dimethoxyethane in methylene chloride. The solvent is removed in vacuo to yield 206 mg (38% yield) of the desired product. MS: m / z found: 511.1 (M + Na) ⁺ , calcd: 511.2.

15- ( DM4 - Dithio ) -4,7,10,13- Of tetraoxapentadecanoic acid  synthesis

N ^{2 '-} acetyl-de-N ^2' - (4- mercapto-4-methyl-1-oxo-pentyl) mate tansin (DM4, 18.6㎎, 0.0239m㏖) and 15- (2-pyridyl die-thio) - A solution of 4,7,10,13-tetraoxapentadecanoic acid (14.0 mg, 0.0358 mmol) was prepared in 0.75 ml of 1,2-dimethoxyethane. 4-methylmorpholine (6.0 mg, 0.0597 mmol) is added to the reaction vessel and the reaction proceeds for 24 hours at room temperature under stirring. Once the reaction is complete, the crude reaction mixture is dried under vacuum and used without further purification (FIG. 6).

15- ( DM4 Dithio) -4,7,10,13- Tetraoxapentadecanoic acid -N-hydroxysuccinimide ester ( DM4 - SPEG ₄ - NHS ) Synthesis of

Crude 15- (DM4-dithio) -4,7,10,13-tetraoxapentadecanoic acid was dissolved in 2.0 ml of methylene chloride, 7V-hydroxysuccinimide (3.6 mg, 0.31 mmol) and 1 -[3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (6.8 mg, 0.036 mmol). The solution is stirred for 2.5 h and the product is purified by silica chromatography eluting with 4% methanol in methylene chloride. The solvent is removed in vacuo to yield 15.0 mg (54% yield) of the desired product. MS: m / z found: 1179.3 (M + Na) ⁺ , calcd: 1179.4 (Figure 6).

Hydrophilic polyethylene Oxide  Spacer PEG _n , or (- CH ₂ - CH ₂ -O) _{n = J-14} Containing) Disulfide  High number of molecules per antibody molecule using linker Maytansinoid  2-step conjugation of antibodies to link molecules

New observations have shown that a new heterodifunctional reagent with a hydrophilic spacer (e.g. polyethylene oxide (PEG _n , or (-CH ₂ -CH ₂ -O) _{n = 1-14} )) modifies the antibody, followed by DM4 thiols. Is made when used for bonding. Conjugation mixtures of antibody-maytansinoid conjugates with hydrophilic PEG _n spacers show no precipitation and consistently provide high conjugate yields (> 70%) with very high monomer fractions (> 90%). As an example, a humanized antibody at a concentration of 8 mg / ml is modified by PySS-PEG ₄ -NHS reagent, which is several times molar excess of antibody concentration in a buffer of pH 8 for 1 hour at 30 ° C., and then purified by gel filtration. do. Linked dithiopyridyl groups per antibody molecule were added 1.4 times molar excess of DM4 maytansinoid thiol to each dithiopyridyl-PEG _n -linker modified antibody solution for conjugation step at pH 6.5 overnight at 25 ° C. Based on the following purification of the conjugates by gel filtration (FIG. 12), an excess of dithiothritol is estimated to be -4-16 by 2-thiopyridone release analysis of the mother liquor. The final incorporated maytansinoid values per antibody for different conjugation mixtures by different initial linker incorporation ranged from 3 to 9 maytansinoids on average per antibody molecule, with no precipitation observed,> 70% yield and very high Monomer (> 90% monomer based on size-exclusion TSK-GEL G3000 HPLC with 20% isopropanol or 0.4 M sodium perchlorate). Unconjugated drug in the final conjugate was determined to be less than 0.6% by HiSep Mixed-Mode chromatography (HiSep column, Supelco), indicating that the maytansinoid is covalently linked to the antibody. In another example, a humanized antibody at a concentration of 8 mg / ml is modified with PySS-PEG ₄ -NHS reagent, which is several molar excess of antibody concentration in a buffer of pH 6.5 for 1.5 hours at 25 ° C., and then purified by gel filtration. . The dithiopyridyl-PEG _n -containing linker group per antibody sample is estimated to be 6-18 per antibody molecule, which is then reacted with 1.3-1.7-fold molar excess of DM4 maytansinoid thiol at pH 6.5 overnight at 25 ° C. Purification by gel filtration. No precipitation was observed, and the final antibody-maytansinoid conjugate sample of ˜1-2 mg / ml showed a high monomer fraction (> 90%), which showed no aggregation and the unconjugated maytansinoid was very low (<1.7% unconjugated maytansinoid estimated by HiSep chromatography), a covalently bound maytansinoid molecule with a high number of ˜3.1 to 7.1 per antibody. Conjugates with high drug load per antibody are stable upon storage at 4 ° C., even up to the longest time of analysis (1.5 months).

Hydrophilic polyethylene Oxide Spacer (PEG _n , Or (-CH ₂ -CH ₂ -O) _{n = L} i ₄ )  Containing Disulfide  High number of molecules per antibody molecule using linker Maytansinoid  1-step conjugation of antibodies to link molecules

In a one-step conjugation approach, the antibody-maytansinoid conjugate by disulfide linker containing hydrophilic polyethylene oxide spacer (PEG _n , or (-CH ₂ -CH ₂ -O) _{n = I-I4} ) was 4 mg / 10-20 fold molar excess of DM4-SPEG ₄ -NHS reagent was prepared by conjugation in a buffer of pH 8 for 2 hours at 30 ° C, followed by purification by gel filtration to purify 6.6 per molecule of antibody. The conjugated maytansinoids yield an antibody-maytansinoid conjugate at a concentration of 1.4 mg / ml (82% monomer) (FIG. 11). Thus, the two- and one-stage approaches are both linked in high numbers per antibody molecule by disulfide linkers containing hydrophilic polyethylene oxide spacers (PEG _n , or (-CH ₂ -CH ₂ -O) _{n = M4} ). Used to obtain maytansinoids.

Example HI

Hydrophilic polyethylene Oxide  Spacer PEG _n , or (- CH ₂ - CH ₂ - OU Several linked per antibody molecule by a thioether linker containing Maytansinoid  Conjugation of Molecules and Antibodies

To directly modify the lysine residues of the antibody, iV-hydroxysuccinimide esters of maytansinoids with conventional aliphatic linkers (eg, alkyl linkers derived from SPP) (WC Widdison et al., J Med . Chem) , 2006, 49, 4392-4408, are first used to conjugate antibodies in a one-step method. Attempts to conjugate the 8-fold molar excess of the DM1-SPP-NHS reagent as a 5 mg / ml test reagent with pH 5 buffer for 2 hours at 30 ° C. resulted in significant precipitation and aggregation, resulting in a final conjugate. Only 3.3% monomer with about 3.3 linked maytansinoids per antibody. In contrast, the use of DM1-MaI-PEG ₄ -NHS reagent under similar conditions yields a conjugate with 5.4 linked maytansinoid molecules per antibody at 1.1 mg / ml without precipitation in the final conjugate (FIG. 7 or 9). . Similarly, DM1-MaI-PEG ₂ -NHS reagents are used to obtain multiple conjugated maytansinoids linked per antibody molecule via thioether bonds. In another example, a murine IgG ₁ antibody was conjugated with 10-20 fold molar excess of DM1-Mal-PEG ₄ -NHS reagent at 4 mg / ml in a buffer of pH 8 for 2 hours at 30 ° C., followed by gel filtration. Antibody-maytane at a concentration of ˜1 mg / ml with covalently conjugated maytansinoid molecules of 4.1 and 7.8 (98% monomer) per antibody molecule with an uncountable level of unconjugated agent (HiSep HPLC assay) Obtain a sinoid conjugate. In another embodiment, the humanized antibody is conjugated with excess DM1-MaI-PEG ₄ -NHS reagent to yield an average of 10.7 linked maytansinoid molecules (99% monomer; 1.1 mg / ml concentration) per antibody. PEG ₄ -linked thioether conjugates are also prepared from antibodies using the two-step conjugation method shown in FIGS. 8 and 10. Thus, multiple maytansinoid molecules can be introduced per antibody molecule by the use of hydrophilic linkers (eg PEG _n , or (-CH ₂ -CH ₂ -O) _n ) (see, eg, FIGS. 1, 2). , 4, 5, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20 and 21).

DM1 - MaI - PEG ₂ - NHS Synthesis of

N ^{2 '-} having acetyl -7V ^2' - (3- mercapto-1-oxopropyl) mate tansin preparing a solution of (DM1, 13.4㎎, 0.0182m㏖) in THF 0.70㎖, and succinimidyl - [N -Maleimidopropionamido) -diethyleneglycol] ester (NHS-PEG ₂ -maleimide, Quanta Biodesign, 11.6 mg, 0.0273 mmol) was dissolved in aqueous potassium phosphate buffer (50 mM, pH 6) and THF 2: To 1.5 ml of 1 (v / v) mixture is added. The reaction was run for 1 hour under stirring at room temperature and TLC analysis indicated the reaction was complete. The crude reaction mixture is purified by silica chromatography eluting with 8% ethanol in methylene chloride and the solvent is removed in vacuo to yield 6.0 mg (28% yield) of the desired product. MS: m / z found: 1185.3 (M + Na) ⁺ , calcd .: 1184.4 (Figure 4).

DM1 - MaI - PEG ₄ - NHS Synthesis of

N ^{2 '-} de-acetyl - N ^2' - (3- mercapto-1-oxopropyl) mate tansin preparing a solution of (DM1, 28.1㎎, 0.0381m㏖) in THF 0.50㎖, and succinimidyl - [N -Maleimidopropionamido) -tetraethyleneglycol] ester (NHS-PEG ₄ -maleimide, Quanta Biodesign, 39.1 mg, 0.0762 mmol) was dissolved in aqueous potassium phosphate buffer (50 mM, pH 6) and THF 2: To 1.5 ml of 1 (v / v) mixture is added. The reaction was run for 1 hour under stirring at room temperature and TLC analysis indicated the reaction was complete. The crude reaction mixture is purified by silica chromatography eluting with 6% ethanol in methylene chloride and the solvent is removed in vacuo to give 9.6 mg (20% yield) of the desired product. MS: m / z found: 1273.5 (M + Na) ⁺ , calcd: 1273.5 (Figure 4).

Example IV

High Maytansinoid  Mass Spectrometry of Containing Antibody Groups:

To analyze a high maytansinoid containing antibody group with a hydrophilic PEG linker, a very high maytansinoid containing Ab-PEG ₄ -MaI-DM1 conjugate with an average of 10.7 DM1 per antibody is selected. The conjugate is deglycosylated and then analyzed by ESI-TOF MS (FIG. 22). Mass spectra represent various groups of antibodies labeled with different numbers of maytansinoids having a maximum of approximately 8 to 9 agents per antibody, linked in the range of 4 to 15 drugs per antibody. This distribution usually does not show selective disappearance for the high drug containing group, suggesting that it is consistent with the high solubility of the final conjugate. Size exclusion chromatography HPLC of high maytansinoid containing Ab-PEG ₄ -MaI-DM1 conjugates with an average of 10.7 DM1 per antibody shows surprisingly high> 99% amounts of monomers (FIG. 23).

Example  V

High Maytansinoid  Containing antibody groups FACS  Combined Unmodified  Similar to that of an antibody:

Binding of the high maytansinoid containing conjugates of some antibodies is compared to unmodified antibodies to different targets (eg, EpCAM, CanAg and CD56) by flow cytometry. Briefly, antigen-positive cells are incubated with conjugated or unmodified antibodies at 4 ° C., then with secondary antibody-FITC conjugates at 4 ° C., fixed with formaldehyde (1% in PBS), and then flow cytometry. Analyze No significant difference was observed between binding of the conjugate to binding of the unmodified antibody to all conjugates evaluated. One example is shown in FIG. 24, wherein the maytansinoid containing Ab-PEG ₄ -MaI-DM1 conjugate of 10.7 binds to antigen-positive cells with high affinity similar to that of an unmodified antibody.

Example VI

Hydrophilic Polyethylene oxide Spacer (PEG _n , Or (-CH ₂ -CH ₂ -O) _n )  Antibodies with Containing Thioethers and Disulfide Linkers Maytansinoid  In vitro Cytotoxicity Assessment of Conjugates:

Cytotoxic effects of antibody-maytansinoid conjugates with thioethers and disulfide linkers containing PEG _n spacers are typically WST-8 cell viability after 4-5 consecutive days of culture with cancer cells and conjugates. ) Is evaluated using the assay. Antigen-expressing cancer cells (˜1000-5000 cells / well) are incubated in 96-well plates in normal growth medium containing bovine serum at various concentrations of antibody-maytansinoid conjugates for about 5 days. Then, WST-8 reagent is added and plate absorbance is measured at 450 nm after 2-5 hours. Survival fractions are plotted against conjugate concentrations to determine the / C ₅₀ value of the conjugate (50% cell death concentration).

FIG. 25 shows improved efficacy of anti-EpCAM Ab-maytansinoid conjugates with increased drug load for PEG ₄ linked thioether conjugates (Ab-PEG ₄ -MaI-DM1), which also shows EpCAM antigen-positive It exhibits greater efficacy against thioether-linked SMCC-DM1 and disulfide-linked SPDB-DM4 at similar drug loads of about 4 maytansinoids per antibody against COLO205-multidrug resistant cells (COLO205-MDR cells). The efficacy of thioether-linked anti-EpCAM Ab-PEG ₄ -MaI-DM1 conjugates at maytansinoid loads of 4.1 and 7.8 is new and potentially very promising for therapeutic applications.

FIG. 26 shows cytotoxic activity of anti-CanAg Ab-maytansinoid conjugates on CanAg antigen-positive COLO205-MDR cells. In addition, thioether-linked Ab-PEG ₄ -MaI-DM1 and Ab-PEG ₂ -MaI-DM1 conjugates have greater efficacy compared to thioether-linked Ab-SMCC-DM1 conjugates with similar maytansinoid loads. Indicates.

27 shows the cytotoxic activity of anti-CD56 antibody-maytansinoid conjugates with PEG-containing thioether and disulfide linker on CD56-expressing Molp-8 multiple myeloma cells. Thioether-linked PEG4 conjugates with 7.7 agents per antibody (Ab-PEG ₄ -MaI-DM1) are conjugates containing 3.8 agents (/ C ₅₀ = 1.9 nM), an unexpected 100-fold increase in cytotoxic potency (/ C ₅₀ value of 0.019 nM).

FIG. 28 contains PEG ₄ linked thioether conjugates compared to conventional thioether-linked SMCC-DM1 at similar drug load, which is about 4 maytansinoids per antibody for EpCAM-positive multidrug resistant HCTl 5 cells. An improvement in the efficacy of the anti-EpCAM Ab-maytansinoid conjugate (Ab-PEG ₄ -MaI-DM1) is shown. The high efficacy of thioether-linked anti-EpCAM Ab-PEG ₄ -MaI-DM1 conjugates is a new discovery for therapeutic applications and potentially very promising.

FIG. 29 contains PEG ₄ linked thioether conjugates, as compared to conventional thioether-linked SMCC-DM1, at a similar drug load of about 4 maytansinoids per antibody for EpCAM-positive multidrug resistant COLO 205 cells. An improvement in the efficacy of the anti-EpCAM Ab-maytansinoid conjugate (Ab-PEG ₄ -MaI-DM1) is shown. The improved efficacy of thioether-linked anti-EpCAM Ab-PEG ₄ -MaI-DM1 conjugates is a new discovery for therapeutic applications and potentially very promising. FIG. 37 shows a hydrophilic thioether-linked PEG ₄ linker (Ab-PEG ₄ -MaI-) compared to a non-hydrophilic SMCC-DM1 conjugate that is 3.7 maytansinoids / Ab for EGFR-positive UO-31 human kidney cancer cells Potency improvement in cytotoxicity of anti-EGFR Ab-maytansinoid conjugates with DM1). The efficacy of PEG _4- MaI-DM1 is about 10-fold greater than that of SMCC-DM1 conjugates with conventional linkers.

Example VII

In vivo pharmacokinetics:

Plasma pharmacokinetics of humanized anti-CD56 antibody (Ab) -PEG ₄ -MaI-DM1 conjugates containing a hydrophilic PEG ₄ linker and having 6.7 D / A (maytansinoid / antibody) contain a conventional aliphatic carbon chain linker and Compared to that of Ab-SMCC-DM1 conjugate with 4D / A (FIG. 38A). CD1 mice are injected intravenously with a single bolus of 5 mg / kg conjugate (antibody-base dose; three mice per group). Plasma samples are collected at several time points up to 4 weeks. Plasma samples are analyzed for antibody concentrations and conjugate concentrations using ELISA. For antibody ELISA, plasma samples are added to coated microtiter plates containing immobilized, goat-anti-human IgG (H + L) antibodies, washed, and then horseradish peroxidase-conjugated goat-anti-human Detection is done using IgG (Fcγ) antibodies. For conjugate concentrations, plasma samples are added to microtiter plates containing coated immobilized, goat-anti-human IgG (H + L) antibodies, washed, and then biotinylated anti-maytansinoid antibodies and alkaline phosphatase. Detection using conjugated streptavidin. Antibody Concentrations and Conjugate Concentration ELISA results show that Ab-PEG ₄ -MaI-DM1 conjugates containing a hydrophilic PEG ₄ linker with both high 6.7 DM1 / Ab loads are well maintained in plasma over a four week study period.

38A shows in vivo pharmacokinetics of antibody-maytansinoid conjugates using PEG ₄ linker with high maytansinoid load (6.7 DM1 / Ab) compared to standard linker conjugate with 4 DM1 / Ab. Even at high maytansinoid loads, PEG ₄ linked thioether conjugates with 6.7 maytansinoids / Ab (Ab-PEG ₄ -MaI-DM1) have longer half-lives than standard conjugates. As another example, ³ H- plasma pharmacokinetics is from 10 to 12㎎ / ㎏ (iv) capacity with labeled DM1 humanized ^{_{C242 Ab-PEG 4 -MaI- 3 H}} -DM1 conjugate (3.3 maytansinoid / Ab) In CD-I mice, comparisons are made with Ab-SMCC- ³ H-DM1 conjugates and unconjugated antibodies containing conventional aliphatic carbon chain linkers and having similar 4.2 D / A loads (FIG. 38B). Ab-PEG ₄ -MaI- ³ H-DM1 conjugates are conventional SMCCs with similar maytansinoid loads, as measured by both antibody concentration (ELISA: FIG. 38B) and conjugate concentration ( ³ H-label count). Higher plasma concentrations over 4 weeks compared to the linker conjugate. The half-life of PEG _4- MaI linked conjugates is 16 days compared to 12.6 days for SMCC-linked conjugates, which is much improved over SMCC conjugates (FIG. 38B). Importantly, the area under the curve (AUC) (AUC = 38790 h μg / ml) of the Ab-PEG ₄ -MaI-DM1 conjugate at 3.3 mg / kg (intravenous) dose of 3.3 D / A was determined in CD-I mice, Ab-SMCC-DM1 which is similar to that of the unconjugated antibody at a similar dose of 12 mg / kg (intravenous) (AUC = 38798 h μg / ml) and 4.2 D / A at 10 mg / kg (intravenous) dose Much better than that of the conjugate (AUC = 25910 h μg / ml) (FIG. 38B).

Example VIII

Resistant colon cancer ( HCTl  5) Heterogeneous On the graft  Against EpCAM - Maytansinoid  Conjugate, muB38.1-MCC-DM1 and muB38 .One- PEG4 - mal - DM1  Comparison of In Vivo Anti-Tumor Activity of Conjugates

Anti-tumor effects of muB38.1-MCC-DM1 and muB38.1-PEG4-mal-DM1 conjugates have been shown to overexpress P-glycoprotein and are a heterologous graft of human colon cancer, HCTl 5, resistant to various drugs Evaluate in the model. HCTl 5 cells are injected subcutaneously into the area under the right shoulder of SCID mice (1 × 10 ⁷ cells / animal). When the tumor volume reaches approximately 140 mm 3 in size (9 days after tumor cell inoculation), mice are randomized by tumor volume, divided into three groups (5 animals / group), each group muB38.1-MCC- Treatment with a single intravenous bolus of DM1 (20 mg conjugate protein / kg), muB38.1-PEG4-mal-DM1 (20 mg conjugate protein / kg) or phosphate-buffered saline (control vehicle). Tumor growth is monitored by measuring tumor size twice per week. Tumor size is calculated by the formula: length × width × height × 1/2.

The average change in tumor volume is shown in FIG. 30, for example. In the PBS control, tumors reached a tumor volume of 600 mm 3 until 20 days after cell inoculation. Treatment with muB38.1-MCC-DM1 results in tumor growth delay of 15 days. Treatment with muB38.1-PEG4-mal-DM1 persisted for 44 days with 2 of 5 animals having complete tumor suppression and 3 animals with larger anti-tumor tumors with 32 days of tumor growth delay Effect.

Thus, the conjugate of the present invention, muB38.1-PEG4-mal-DM1, is significantly more effective than muB38.1-MCC-DM1 in this human colon cancer xenograft model.

Example IX

Resistant colon cancer ( COLO205 - MDR Heterogeneity of) On the graft  Against EpCAM - Maytansinoid  Junction ( muB38 .One- MCC - DM1  And muB38 .One- PEG4 - mal - DM1 Comparison of anti-tumor activity in vivo

Anti-tumor effects of muB38.1-MCC-DM1 and muB38.1-PEG4-mal-DM1 conjugates are evaluated in a xenograft model of human colon cancer, COLO205-MDR, engineered to overexpress P-glycoprotein. COLO205-MDR cells are injected subcutaneously into the area under the right shoulder of SCID mice (1 × 10 ⁷ cells / animal). When the tumor volume reached approximately 170 mm 3 in size (8 days after tumor cell inoculation), mice were randomized into three groups (6 animals / group), each group muB38.1-MCC-DM1 (20 mg conjugate protein). / Kg), muB38.1-PEG4-mal-DM1 (antibody dose 20 mg / kg) or phosphate-buffered saline (vehicle control) with a single intravenous bolus. Tumor growth is monitored by measuring tumor size twice per week. Tumor size is calculated by the formula: length x width x height x 1/4.

The average change in tumor volume is shown, for example, in FIG. 31. In the PBS control, tumors grew to about 1000 mm 3 on 38 days. Treatment with muB38.1-MCC-DM1 results in tumor growth delay of 14 days. Treatment with muB38.1-PEG4-mal-DM1 has a significant anti-tumor effect resulting in complete tumor suppression in all six animals (FIG. 31).

Similar experiments are also performed for COLO 205 xenografts. Treatment with B38.1-PEG4-mal-DM1 is more effective, resulting in complete tumor suppression, whereas standard SMCC conjugates only show moderate tumor growth delay (FIG. 32).

Similar results are obtained with conjugates of humanized anti-CanAg antibodies (FIG. 33).

Thus, the conjugate of the present invention, muB38.1-PEG4-mal-DM1, is significantly more effective than the conjugate muB38.1-MCC-DM1, prepared by the linker described above in this human colon cancer xenograft model.

Example  X

PEG  Evaluation of the length:

Several Ab-PEG _n -MaI-DM _x conjugates are prepared using PEG ₄ , PEG ₈ , PEGi ₂ , PEG ₂₄ linker and various DMxes incorporated per antibody. 34 shows Ab-PEG ₂₄ -MaI-DM1 conjugates with very high 17.1 D / A loads similar binding to antigen-expressing cancer cells as unmodified antibodies (binding measured in relative mean fluorescence RMF units by flow cytometry). It shows that. In addition, Ab-PEG ₈ -MaI-DM1 and Ab-PEG ₁₂ -MaI-DM1 conjugates with 4 to 8 D / A exhibit similar binding as antibodies unmodified by cell-bound flow cytometry. Ab-PEG _n -MaI-DM _x conjugates prepared by PEG ₄ , PEG ₈ , PEGj ₂ , PEG ₂₄ linker are potent in cytotoxicity against antigen-positive cells. 35 shows CanAg antigen-positive COLO205 cells with an anti-CanAg antibody (huC242) -PEG _n -Mal-DM1 conjugate of 4 to 17 D / A having an effective IC ₅₀ of about 0.1 to 0.5 nM when cultured for 5 days. To kill. pgp-expressing multi-drug resistant COLO205-MDR cells are killed by huC242-PEG _n -Mal-DM1 conjugate with 4 to 17 D / A in an efficient manner with an IC ₅₀ of about 0.05 to 0.5 nM (FIG. 36). . PEG ₂₄ -MaI-DM1 conjugates with high 17.1 D / A are more potent in cytotoxicity than PEG _n -Mal-DM1 conjugates with 4 D / A (FIGS. 34, 36).

Example XI

Amines against antibodies Reactive group  Non-containing Cutability Thiosuccinimide  Having part Of maytansinoids  join

N -hydroxysuccinimide esters of maytansinoids with non-cleavable thiosuccinimidyl groups are used to conjugate the antibodies using a one-step method (FIG. 41). The sulfhydryl-containing maytansinoids are modified with a heterodifunctional maleimide-containing crosslinker and separated prior to conjugation with the antibody (FIG. 43) to allow non-cleavable thisuccinimidyl-linked antibody maytansi Provide a noid conjugate (FIG. 39). The reaction is carried out using an SMCC reagent containing a hydrocarbon ring between the maleimide and the NHS ester, and a similar method is used to sulfhydryl-containing maytane to a heterodifunctional linker containing a straight chain hydrocarbon between the maleimide and the NHS ester. Can be used to react the sinoids (FIG. 44).

DM1 - SMCC Synthesis of

To a round bottom flask, N ^{2 '-} having acetyl -iV ^2' - (3- mercapto-1-oxopropyl) mate tansin (DM1, 67.9㎎, 0.092m㏖), succinimidyl -4- (7V- maleimido Methyl) cyclohexane-1-carboxylate (SMCC, 32.1 mg, 0.096 mmol, 1.05 equiv) and THF (4 mL) are charged. The solution is stirred so that the reagent is dissolved in the solvent to yield a clear colorless solution. Then phosphate buffer, pH 6 (4 mL, 100 mM potassium phosphate, 2 mM EDTA) is added. The reaction flask was equipped with septum and stir bar and the reaction proceeded under vigorous stirring at room temperature. The reaction was shown to complete within 30 minutes as shown by reversed phase HPLC. Following completion of the reaction, the reaction volume is reduced in vacuo to yield a white solid / residue. The product is separated by silica gel chromatography eluting with 3% methanol in methylene chloride. The product containing fractions were combined and concentrated to dryness in vacuo to give 63 mg (63.9% yield) of DM1-SMCC as a white solid. Mass spectral analysis of the isolated product gives the expected m + Na ⁺ (m / z 1094.4) and m + Cl ⁻ (m / z 1106.2) major molecular ions (FIG. 43).

DM1 - SMCC One-step conjugation of an antibody with an antibody

10 mM stock (w / v) of DM1-SMCC reagent is prepared in DMA (10.7 mg / ml). Stock solution is diluted with EtOH and absorbance is measured at 280 nm for reagent blanks of EtOH and DMA. The concentration of the stock DM1-SMCC reagent is calculated using an extinction coefficient of 5700M- ¹ at 280 nm, which is the extinction coefficient of DM1 at this wavelength. Since the actual extinction coefficient of DM1-SMCC has not been measured, this is only an estimate of concentration.

Antibodies are conjugated with DM1-SMCC at 5 mg / ml using 7-fold molar excess of reagent. Titration of the antibody by several-fold excess of DM1-SMCC is first performed to determine the desired DM1: Ab ratio, usually in the range of 6 to 10-fold molar excess for human antibodies. The reaction is carried out in DMA (5% v / v) and pH 7.5 buffer for 90 minutes at room temperature. The reaction mixture is then held at 4 ° C. for 12 to 36 hours. The conjugate is then purified on a NAP-5 (Sephadex G25) column equilibrated with pH 5.5 citrate buffer, filtered and dialyzed against pH 5.5 citrate buffer to remove unreacted free agent. Following dialysis, the conjugate is linked with 3.1 DM1 molecules per mole of antibody, and there is no detectable free agent in the conjugate (FIG. 39, m = 3.1). The number (average) of DM1 molecules per Ab antibody molecule in the final conjugate is determined by measuring the absorbance of the conjugate at 252 and 280 nm and using the known extinction coefficients for DM1 and the antibody at these two wavelengths.

SEC HPLC is performed on the conjugates to show 96.8% monomerity following conjugation.

Final conjugates are also analyzed by size exclusion LC / MS. The conjugate prepared via the method described in the present invention shows the desired MS spectrum of the deglycosylated conjugate containing only the expected peak distribution (FIG. 45).

DM4 - SMCC Synthesis of

To a round bottom flask, N ^{2 '-} de-acetyl - N ^2' - (4- mercapto-4-methyl-1-oxo-pentyl) mate tansin (DM4, 22.0㎎, 0.0282m㏖), succinimidyl-4- ( Charge 7V-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 24.2 mg, 0.0723 mmol, 2.50 equiv) and free distilled THF (1 mL). The solution is stirred so that the reagent is dissolved in the solvent to yield a clear colorless solution. Then phosphate buffer, pH 6 (1 mL, 100 raM potassium phosphate, 2 mM EDTA) is added. The reaction flask is equipped with a septum and stirring bar and the reaction proceeds under vigorous stirring at room temperature. The reaction was shown to be complete after 7 hours as shown by reversed phase HPLC. Following completion of the reaction, the product is extracted with ethyl acetate (3 x 25 mL), washed with brine (5 mL) and dried under vacuum. The product is separated by silica gel chromatography eluting with a mixture of 3% ethanol in methylene chloride. The product containing fractions were combined and dried under vacuum to afford 19.74 mg (62.8% yield) of DM4-SMCC. Mass spectral analysis of the isolated product provides the expected m + Na ⁺ (m / z 1136.4) and m + Cl ⁻ (m / z 1148.4) major molecular ions.

DM4 - SMCC One-step conjugation of an antibody with an antibody

A solution of humanized antibody (2.5 mg / ml) in aqueous buffer (100 mM sodium phosphate), pH 8.0 was incubated with 10-fold molar excess of DM4-SMCC in dimethyl sulfoxide (DMSO) to obtain a final DMSO concentration of 20%. do. The bonding is carried out for 1 hour at ambient temperature. The conjugate is purified by passing through a Sephadex G25 gel filtration column equilibrated with pH 5.5 buffer (10 mM histidine, 130 mM glycine, 5% (w / v) sucrose, pH 5.5). The number (average) of linked DM4 molecules per antibody molecule in the final conjugate is determined by measuring the absorbance of the conjugate at 252 and 280 nm and using the known extinction coefficients for DM4 and antibody at these two wavelengths.

Following purification, the conjugate is linked to 3.7 DM4 molecules per antibody molecule. SEC analysis is> 95% monomeric, but is performed on the final conjugate to indicate that> 5% free drug group is present in the final conjugate. Dialysis of the conjugate may reduce the presence of undesirable free drug groups.

Example XII

Amines against antibodies Reactive group  Non-containing Cutability Thioacetamidil  Having part Of maytansinoids  join

Sulhydryl containing maytansinoids DM1 are reacted with bromoacetic acid to obtain thioacetamidyl-linked carboxylic acid derivatives. Esterification with N -hydroxysuccinimide yields an amine reactive non-cleavable, thioacetamidyl-linked maytansinoid (FIG. 48). One-step conjugation of the isolated compound with the antibody (FIG. 42) yields a non-cleavable antibody maytansinoid conjugate (FIG. 40).

In contrast, sulfhydryl-containing maytansinoids DM4 are modified with heterodifunctional haloacetamide-containing crosslinkers to yield amine reactive maytansinoids containing thioacetamidyl moieties (FIG. 49). One-step conjugation of the isolated compound with the antibody (FIG. 42) yields a non-cleavable thioacetamidyl-linked antibody maytansinoid conjugate (FIG. 40).

DM1 - SBA Synthesis of

To a round bottom flask, N ^{2 '-} de-acetyl - N ^2' - (3- mercapto-1-oxo-propionic O- mate tansin (DM1, 183.4㎎, 0.248m㏖) and anhydrous Λ /, TV- dimethylformamide (DMF, 3 mL) was charged bromoacetic acid (37.9 mg, 0.273 mmol, 1.1 equiv) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU, 75.6 mg, 0.497 m) The reaction solution is stirred in succession, followed by the addition of a septum and a stirring bar, and the reaction is allowed to proceed for 1 hour under vigorous stirring at room temperature, following completion of the reaction, the reaction volume is reduced to crude oil in vacuo. The crude product is dissolved in a minimum amount of methylene chloride and the product is separated by silica gel chromatography eluting with a mixture of 5% ethanol, 0.5% acetic acid and 94.5% methylene chloride The product containing fractions are combined and the volume is vacuumed. Thioacetase which is reduced to 94.6% purity by HPLC Yidil-linked to give a carboxylic acid derivative DM1 158.8㎎ (80.4% yield).

The round bottom flask was charged with the previous reaction product (158.8 mg, 0.199 mmol) and methylene chloride (15 mL). N -hydroxysuccinimide (NHS, 25.2 mg, 0.219 mmol, 1.1 equiv) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 57.2 mg, 0.298 mmol) Add. The reaction proceeds under stirring at room temperature until completion. Following completion of the reaction (-45 min), the reaction mixture was diluted with ethyl acetate (20 mL) and transferred to a separatory funnel, followed by phosphate buffer pH 6 (15 mL, 100 mM potassium phosphate, 2 mM EDTA) and brine (7 mL). Washed with anhydrous Na ₂ SO ₄ and concentrated in vacuo to afford a crude off-white solid. The product is separated by silica gel chromatography eluting with a mixture of 10% 1,2-dimethoxyethane in ethyl acetate. The product containing fractions were combined and concentrated in vacuo to yield 34.5 mg (19.4% yield) of DM1-SBA with 96.0% purity by reverse phase HPLC. Mass spectral analysis of the isolated product provides the expected m + Na ⁺ (m / z 915.2) and m + Cl ⁻ (m / z 927.0) main molecular ions.

DM1 - SBA One-step conjugation of an antibody with an antibody

10.8 mM stocks (w / v) of DB1-SBA reagents are prepared in DMA. Antibodies are conjugated with DM1-SBA at 2.5 mg / ml using 10.2-fold molar excess of reagent. The reaction is carried out in DMA (10% v / v) and pH 7.5 buffer (100 mM potassium phosphate) for 90 minutes at room temperature. The conjugate is then purified on S300 gel filtration (Sephadex S300) column, eluting with pH 6.5 buffer (10 mM potassium phosphate, 140 mM sodium chloride). Following purification, the conjugate has 3.7 DM1 molecules and -0.18% free agent linked per mole of antibody (FIG. 40, m = 3.7).

SEC HPLC is performed on the conjugates to show 99.6% monomerity following conjugation.

N- Succinimidyl Bromoacetate  Synthetic (or Commercially Available)

A 100 mL round bottom flask was charged with 2-bromoacetic acid (2.79 g, 20.08 mmol), 7V-hydroxysuccinimide (2.54 g, 22.12 mmol) and methylene chloride (30 mL). When TV-TV-dicyclohexylcarbodiimide (DCC, 4.46 g, 22.14 mmol) is added, the solution is stirred in an ice bath. The reaction is stirred for 1 hour in an ice bath and again for 1 hour at room temperature.

The reaction mixture is filtered through a sintered glass funnel and concentrated in vacuo to yield a crude white solid. The solid is dissolved in warm methylene chloride (30 mL) and recrystallized with hexane (25 mL). The solid is collected by filtration, washed with hexane and dried under vacuum to afford 3.99 g (84% yield) of iV-succinimidyl bromoacetate as a white solid. ¹ H NMR (CDCl ₃ ) δ2.864 (s, 4H) and 4.100 (s, 2H) ppm.

DM4 - SBA Synthesis of

To a round bottom flask, N ^{2 '-} de-acetyl -N ^2' - (4- mercapto-4-methyl-1-oxo-pentyl) mate tansin (DM4, 71.9㎎, 0.092m㏖) and anhydrous N, N - dimethyl Formamide (DMF, 2.5 mL) is charged. The reaction was placed under argon atmosphere, iV-succinimidyl bromoacetate (SBA, 23.9 mg, 0.101 mmol, 1.1 equiv) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU, 14.7 Mg, 0.097 mmol, 1.05 equiv) was added sequentially. The flask is equipped with septum and stir bar and the reaction proceeds to completion under vigorous stirring at room temperature. The product is separated by preliminary cyano HPLC in a single injection, eluting with a gradient of 15-65% ethyl acetate in hexanes over 30 minutes, followed by an increase of 65-95% ethyl acetate over 10 minutes. Under these conditions, DM4-SBA elutes between 22 and 24 minutes. The product is collected and concentrated in vacuo to give 50.1 mg (55.2% yield) of the desired product DM4-SBA (95% purity). Mass spectral analysis of the isolated product provides the expected m + Na ⁺ (m / z 957.4) and m + Cl ⁻ (m / z 969.2) main molecular ions.

Example XIII

Carboxy  Non-containing part Cutability Thiosuccinimide -Connected Maytansinoid  Preparation of Derivatives

The sulfhydryl-containing maytansinoids (eg DM1 and DM4) are modified to obtain non-cleavable thiosuccinimidyl-containing carboxylic acid derivatives (FIGS. 45, 46 and 48). These derivatives are useful in the preparation of the amine-reactive non-cleavable thisuccinimidyl-linked maytansinoids described herein. 45, 46 and 48 show the formation of N -hydroxysuccinimide activated esters, but it is clear to those skilled in the art that some other activated esters may be formed, which are not limited thereto. N -sulfosuccinimidyl esters, pentafluorophenol esters, tetrafluorosulfophenol and nitrophenol esters. Sulhydryl-containing maytansinoids (eg, DM1 and DM4) are modified with homofunctional maleimide reagents to yield maytansinoids containing maleimide groups as shown in FIG. 50.

DM1 - MCC Manufacturing

A 10 ml round bottom flask was charged with DM1 (44.6 mg, 0.0571 mmol) and 1,2-dimethoxyethane (2.5 ml) and equipped with a stirring bar. A solution of N- [4- (carboxycyclohexylmethyl)] maleimide (Toronto Research Chemicals, Inc., MCC, 20.3 mg, 0.08430 mmol) in 1,2-dimethoxyethane (0.5 mL) was added to the reaction flask. Then, pH 7.5 buffer (2.5 mL, 50 mM potassium phosphate, 2 mM EDTA) is added. A few drops of saturated aqueous sodium bicarbonate solution are added to the reaction solution to maintain the reaction pH. The reaction proceeds at room temperature and is complete after 2 hours. The reaction volume is reduced to 1/2 under vacuum, acidified to pH 3 and the product is extracted with ethyl acetate (3 × 10 mL). The extracts are combined, washed with brine (5 mL) and then concentrated in vacuo to afford the crude product. The product is separated by silica gel chromatography eluting with a 93: 7 mixture of methylene chloride and ethanol. The product containing fractions were combined and concentrated to give 46.5 mg (83.5% yield) of DM1-MCC (99.3% purity) as a white solid. Mass spectral analysis of the isolated product gives the expected m + Na ⁺ major molecular ions (m / z 997.3).

DM4 - SMCC Manufacturing

DM4 (24.3 mg, 0.031 mmol), N- [A- (carboxycyclohexylmethyl)] maleimide (MCC, Toronto Research Chemicals, Inc., 8.1 mg, 0.0342 mmol) and 1,2 in 3 ml glass vials Charge dimethoxyethane (1 ml). The solution is stirred while pH 7.5 buffer (1 mL, 50 mM potassium phosphate, 2 niM EDTA) is added to the reaction. The reaction proceeds at room temperature and is complete within 2 hours. The reaction volume is reduced to 1/2 under vacuum, acidified to pH 3 and the product is extracted with ethyl acetate (3 × 10 mL). The extracts are combined, washed with brine (5 mL) and then concentrated in vacuo to afford the crude product. The product is separated by silica gel chromatography eluting with a 9: 1 mixture of methylene chloride and ethanol. The product containing fractions were combined and concentrated to give 14.8 mg (46.9% yield) of DM4-MCC (96.7% purity) as a white solid. Mass spectral analysis of the isolated product gives the expected m + Na ⁺ major molecular ions (m / z 1039.5).

Example XIV

Thiol  Non-containing reactive moieties Cutability Thiosuccinimide -Connected Maytansinoid  Preparation of Derivatives

Maleimide containing maytansinoids containing non-cleavable thiosuccinimidyl groups were conjugated to thiol containing antibodies (FIG. 51). Maleimide containing maytansinoids containing a non-cleavable thiosuccinimidyl group are prepared by coupling a thiol containing maytansinoid to a bis-maleimide crosslinker (FIG. 52). The reaction carried out in the present invention is carried out using a Ma1- (CH ₂ ) ₆ -Mal reagent, but the bis-maleimide reagent in which the sulfhydryl-containing maytansinoid contains different spacer units between maleimide moieties. It can be appreciated by those skilled in the art.

DM1 - Mal -( CJLyMal Manufacturing

A solution of bis (maleimide) hexanoate (17.9 mg, 0.0648 mmol, 3 equiv) in THF (0.75 mL) was prepared in a reaction vial. The solution is stirred while DMI (15.9 mg, 0.0216 mmol) is added to THF (0.75 mL). Then, N, N-diisopropylethylamine (3.3 mg, 0.0259 mmol, 1.2 equiv) is added and the reaction proceeds under stirring at room temperature. Following completion of the reaction, the reaction volume is reduced to obtain crude oil under vacuum. The crude product is dissolved again in a minimum volume of CH ₂ Cl ₂ , and CH ₂ Cl ₂ Purification by preparative thin layer chromatography on a 20 cm × 20 cm 1000 μ glass plate, eluting with 7% methanol in water. The product containing band was scraped off the plate, CH ₂ Cl ₂ Extracted in 20% MeOH, filtered through a sintered glass funnel and concentrated in vacuo to give 10 mg (45.9% yield) of DM1-Mal- (CH,) ₃ -Mal (95.8% purity). Mass spectral analysis of the isolated product shows the expected m + Na ⁺ (m / z 1036.4) and m + Cr (m / z 1048.3). Provide major molecular ions.

DM4 - Mal -( CH ₂ ) ₆ - Mal Manufacturing

A round bottom flask is charged with bis (maleimide) hexanoate (25.0 mg, 0.090 mmol, 3 equiv) and THF (1 mL). A solution of (4-mercapto-4-methyl-1-oxo-pentyl) mate tansin (DM4, 23.5㎎, 0.030m㏖) - When fully dissolved, THF (1㎖) of N ^{2 '-} de-acetyl -N ^2' Is added to the reaction flask. Then phosphate buffer pH 6 (2 mL, 100 mM potassium phosphate, 2 mM EDTA) is added to the reaction flask. The reaction flask is equipped with a stirring bar and septum, and the reaction proceeds under stirring at room temperature. Following completion of the reaction (~ 8 hours), the reaction volume is reduced by drying under vacuum. The crude product is again dissolved in a minimum volume of acetonitrile and the product is separated by semi-pre C18 HPLC. The product containing fractions were combined and concentrated in vacuo to give 10.6 mg (33.3% yield) of DM4-Ma1- (CH ₂ ) ₆ -MaI (99.9% purity). Mass spectral analysis of the isolated product shows the expected m + Na ⁺ (m / z 1078.4) and m + Cl ⁻ (m / z 1090.3) Provide major molecular ions.

huC242 - Mal -( CH ₂ ) ₆ - Mal - DM1 Manufacturing

HuC242 (8 mg / ml) in 150 mM HEPES buffer (pH 8.0) containing 5% dimethylacetamide was modified by SPDB 9 equivalents at 30'C for 1 hour, followed by 50 mM phosphate, 50 mM NaCl, pH 7.5 buffer Was eluted through the NAP5 sizing column. To the recovered modified antibody is added 2 [mu] l of 1M dithiothreitol at 30'C. After 10 minutes, the reaction is purified on a NAP10 column, eluting with 50 mM phosphate, 50 mM NaCl, pH 6.5 buffer. DM1-mal- (CH ₂ ) ₆ -mal (1.7 molar equivalents) in dimethylformamide is added to the fraction containing the desired product to give 10% v / v dimethylformamide / buffer. After 1 hour, the crude conjugate is purified on a NAP 25 column, eluting with 10 mM citrate, 135 mM NaCl pH 5.5 buffer. Using the binding assay described above, huC242-Mal- (CH ₂ ) ₆ -Mal-DM1 was shown to bind antigen-positive COLO205 cells to the same extent as the original huC242 antibody (FIG. 53). The huC242-Mal- (CH ₂ ) ₆ -Mal-DM1 conjugate was found to be much more cytotoxic to antigen positive COLO205 cells than antigen negative Namalwa cells (FIG. 54).

Claims (33)

A compound of Formula 1 or Formula T:
[Formula 1]
ZX,-(-CH ₂ -CH ₂ -O-) _n -Y _p -D
(V)
DY _p -(-CH ₂ -CH ₂ -O-) _n -Xi-Z
In the above formulas,
Z represents reactive functionality capable of forming an amide or thioether bond with the cell-binding agent;
D represents a medicament;
X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, an amide bond, a carbamate bond or an ether bond;
Y is an aliphatic, aromatic or heterocyclic bond to the agent via a covalent bond selected from the group consisting of thioether bonds, amide bonds, carbamate bonds, ether bonds, amine bonds, carbon-carbon bonds and hydrazone bonds Group;
l is 0 or 1;
p is 0 or 1;
n is an integer from 1 to 2000. Cell-binding agent cytotoxic drug conjugate of Formula 2 or Formula 2 ′:
(2)
CB- [X, -CH ₂ -CH ₂ -O-) _n -Y _p -D] _m
[Formula T]
[DY _p -C-CH ₂ -CH ₂ -OV-XJ _ln -CB
In the above formulas,
CB represents a cell-binding agent;
D represents a medicament;
X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, an amide bond, a carbamate bond or an ether bond;
Y is an aliphatic, aromatic or heterocyclic bond to the agent via a covalent bond selected from the group consisting of thioether bonds, amide bonds, carbamate bonds, ether bonds, amine bonds, carbon-carbon bonds and hydrazone bonds Group;
l is 0 or 1;
p is 0 or 1;
m is an integer from 2 to 15;
n is an integer from 1 to 2000. A compound of Formula 3 or Formula 3 '
(3)
Z-XK-CH ₂ -CH ₂ OVYD
[Formula 3 ']
DY-(-CH ₂ -CH ₂ O-) _n -X _i -Z
In the above formulas,
Z represents a reactive functional group capable of forming an amide or thioether bond with the cell-binding agent;
D represents a medicament;
X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, an amide bond, a carbamate bond or an ether bond;
Y represents an aliphatic, non-aromatic heterocyclic or aromatic heterocyclic group bonded to the agent via a disulfide bond;
l is 0 or 1;
n is an integer of 1-14. The cell-binding cytotoxic drug conjugate of Formula 4 or Formula 4 '
[Chemical Formula 4]
CB- (Xi-(-CH ₂ -CH ₂ O-) _n -YD) _m
[Formula 4 ']
[DY-(-CH ₂ -CH ₂ O-) _n -X,] _m -CB
In the above formulas,
CB represents a cell-binding agent;
D represents a medicament;
X represents an aliphatic, aromatic or heterocyclic group bonded to the cell-binding agent via a thioether bond, an amide bond, a carbamate bond or an ether bond;
Y represents an aliphatic, aromatic or heterocyclic group bonded to the agent via a disulfide bond;
l is 0 or 1;
m is an integer from 3 to 8;
n is an integer of 1-14. The method of claim 2 or 4, wherein the cell-binding agent is an antibody, a single chain antibody, an antibody fragment preferentially bound to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody, bispecific ) An antibody, a fragment that specifically binds to a target cell, an antibody mimic adnectin, DARPin, lymphokine, cytokines, hormones, growth factors, enzymes or nutrient-transporting molecules- Binder Cytotoxic Pharmaceutical Conjugates. The monoclonal antibody according to claim 2 or 4, wherein the cell-binding agent is a resurfaced monoclonal antibody, a resurfaced single chain monoclonal antibody, or a resurfaced monoclonal antibody preferentially bound to a target cell. A cell-binding cytotoxic drug conjugate that is a fragment. 5. The cell-binding cytotoxicity of claim 2, wherein the cell-binding agent is a humanized monoclonal antibody, a humanized single chain monoclonal antibody, or a humanized monoclonal antibody fragment preferentially bound to a target cell. Pharmaceutical conjugates. The antibody of claim 5, wherein the antibody is a chimeric antibody, chimeric antibody fragment, domain antibody. Or a domain antibody fragment thereof. The antibody of claim 5, wherein the antibody is MY9, anti-B4, EpCAM, CD2, CD3, CD4, CD5, CD6, CD11, CD19, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56 Cell-binding cells, which are CD79, CD105, CD138, EphA receptor, EphB receptor, EGFR, EGFRvIII, HER2, HER3, mesothelin, krypto, alpha _v beta ₃ , alpha _v betan ₅ , alpha _v beta ₆ integrin or C242 Toxic Pharmaceutical Conjugates. The antibody of claim 5, wherein the antibody is My9-6, B4, C242, N901, DS6, EphA2 receptor, CD38, IGF-IR, CNTO 95, B-B4, Trastuzumab, Pertuzumab, Vivatuzumab, Shiv A cell-binding cytotoxic drug conjugate, wherein the antibody is a humanized, human or resurfaced antibody selected from rotuzumab or rituximab. The method of claim 2 or 4, wherein the cell-binding agent is a tumor cell; Viral infected cells, microbial infected cells, parasitic infected cells, autoimmune cells, activated cells, bone marrow cells, activated T-cells, B-cells or melanocytes; IGF-IR, CanAg, EGFR, MUCl, MUCl6, VEGF, TF, MY9, Anti-B4, EpCAM, CD2, CD3, CD4, CD5, CD6, CDl1, CD11a, CD18, CD19, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD79, CD105, CD138, EphA receptor, EphB receptor, EGFRvIII, HER2 / neu, HER3, mesothelin, crypto, alpha _v beta ₃ integrin, alpha _v betan ₅ integrin Cells which express one or more of alpha _v beta ₆ integrin, Apo2 and C242 antigens; Or a target cell selected from cells expressing insulin growth factor receptor, epidermal growth factor receptor and folic acid receptor. The cell of claim 11, wherein the tumor cells are selected from breast cancer cells, prostate cancer cells, ovarian cancer cells, colon cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, and testicular cancer cells. Binder Cytotoxic Pharmaceutical Conjugates. A pharmaceutical composition comprising an effective amount of the drug-cell-binding conjugate of claim 2 or 4, a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier, diluent or excipient. A method of treating a disease sensitive to treatment by the method, the method comprising parenterally administering an effective amount of the conjugate of claim 2 to a patient in need thereof. The method of claim 14, wherein the disease is selected from tumors, autoimmune diseases, graft rejection, graft-to-host disease, viral infections, and parasitic infections. The method of claim 15, wherein the tumor is selected from one or more of cancers of the lung, blood, plasma, breast, colon, prostate, kidney, pancreas, brain, bone, ovary, testes, and lymphoid organs. The method of claim 15, wherein the tumor is IGF-IR, FOLRl, CanAg, EGFR, EphA2, MUCl, MUC16, VEGF, TF, MY9, anti-B4, EpCAM, CD2, CD3, CD4, CD5, CD6, CDl1, CD 1Ia, CDl8, CDl9, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD79, CDl05, CDl38, EphA, EphB, EGFRvIII, HER2 / neu, HER3, Mesothelin, Crypto , At least one of alpha _v beta ₃ integrin, alpha _v betan ₅ integrin, alpha _v beta ₆ integrin, Apo2 and C242 antigens. A maytansinoid of Formula 5 having a thioether moiety containing a reactive group:
[Chemical Formula 5]
D'-Y'-VQWZ '
In the above formulas,
D 'represents sulfhydryl-containing maytansinoids;
V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
Y 'represents a thioether bond;
Q represents any aromatic or heterocyclic moiety;
Z 'represents an amine or sulfhydryl reactive group. The method of claim 18, wherein the sulfhydryl-containing maytansinoid is N ^{2 ′} -deacetyl- N ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (DM1) or N ^{2 ′} -decane. Maytansinoid, which is acetyl-N ^{2 ′} -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4). The maytansinoid of claim 18 represented by the following structural formula:

The maytansinoid of claim 18 represented by the following structural formula:

19. The maytansinoid of claim 18 represented by the following structural formulas 7a or 7b:

A method for preparing the maytansinoid of claim 18, comprising the step of reacting a thiol-containing maytansinoid represented by Chemical Scheme 6 with a heterodifunctional crosslinking agent:
[Scheme 6]
D '+ Y ". VQWZ' → D'-Y'-VQW-Z '
In the above chemical scheme,
D 'represents sulfhydryl-containing maytansinoids,
V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
Q represents any aromatic or heterocyclic moiety;
W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
Z 'is an amine or sulfhydryl reactive group;
Y ″ represents a sulfhydryl-reactive moiety;
Y 'represents a thioether bond between the sulfhydryl-containing maytansinoid and the crosslinking agent. The method of claim 23, wherein the sulfhydryl-containing maytansinoid is N ^{2 ′} -deacetyl-iV ^{2 ′} -(3-mercapto-1-oxopropyl) -maytansine (DM1) or N ^{2 ′} − It is deacetyl-N2 ^' -(4-mercapto-4-methyl- 1-oxopentyl) maytansine (DM4), The manufacturing method of the maytansinoid. The method for producing maytansinoid according to claim 23, wherein Y ″ is maleimido or haloacetamide. A method for preparing cytotoxic conjugates of maytansinoids and cell binders linked through non-cleavable bonds, the method comprising: reacting a cell binder with a compound of formula Z'-WQV-Y'-D 'to formula CB- (Z "-WQV-Y'-D') providing a cell binder conjugate of _m , wherein
Wherein,
Z 'represents an amine or sulfhydryl reactive group;
W is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
Q represents any aromatic or heterocyclic moiety;
V is any straight, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms;
Y 'represents a thioether bond;
D 'represents sulfhydryl containing maytansinoids;
CB represents a cell-binding agent;
Z "represents a thioether bond or an amide bond;
m is an integer of 2 to 8, the method for producing a cytotoxic conjugate. The method of claim 26, wherein the sulfhydryl-containing maytansinoid N ^{2 '-} acetyl-de-N ^2' - (3- mercapto-1-oxopropyl) - MEI tansin (DM1) or N ^{2 '-} A process for producing a cytotoxic conjugate, which is deacetyl-N ^{2 '} -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4). The method of claim 26, wherein the CB is an antibody, single chain antibody or antigen-binding fragment of the antibody. 29. The method of any one of claims 26-28, wherein the cell binder conjugate of formula CB- (Z "-WQV-Y'-D ') _m is purified. 30. The cell binder according to any one of claims 26 to 29, wherein said cell-binding agent and said compound of formula Z'-WQV-Y'-D 'are optionally cell binding agents in aqueous buffer containing up to 20% organic solvent. Of a solution of the compound of formula Z'-WQV-Y'-D 'with an organic solvent or a mixture of organic solvent and aqueous buffer or water, followed by reaction for 5 minutes to 72 hours Process for the preparation of phosphorus, cytotoxic conjugates. The method of any one of claims 26-29, wherein the conjugate is purified by chromatography, dialysis, tangential flow filtration or a combination of these modes. Compound represented by the following structural formula:

Compound represented by the following structural formula:

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