Method for preparing antibody-drug conjugates

Abstract

The invention relates to a method for preparing a mixture of conjugates of formula (I): (I) in which the hook head, the link arm, the spacer, M and u are as defined in the description, a method which includes a reaction step between an antibody or an antibody fragment and a compound of formula (IIa), (IIb), (IIc), (IId), (III) or (IV) as defined in the description.

Description

Title of the invention: Method for preparing antibody-drug conjugates technical field

[0001] The present invention relates to a method for preparing a mixture of antibody-drug conjugates (in English "Antibody Drug Conjugale" or "ADC") and the conjugates that can be obtained by this method. State of the art

[0002] The early 2000s saw an intensification of research on antibody-drug conjugates (ADCs), these conjugates potentially representing an alternative or complement to "conventional" therapies for the targeted delivery of an active ingredient, particularly a cytotoxic drug. The antibody-drug conjugate thus makes it possible to combine the specificity of antibody targeting with powerful new effector functions provided by the conjugated agents.

[0003] The structure of an antibody-drug conjugate typically consists of an antibody linked to the drug by a molecule, one part of which will bind the antibody and another part will couple to the drug, generally via a spacer arm (or linker) of variable length and nature.

[0004] After binding to its target antigen, the antibody is most often internalized into the cell by receptor-mediated endocytosis. The vesicles fuse with lysosomes where the drug is released from the antibody via various mechanisms. The active drug then acts directly on the cell, inducing its death, and sometimes on neighboring cancer cells by transport or diffusion into the environment. The antibody is therefore primarily used as a vector, delivering the drug into the target cell.

[0005] Antibody-drug conjugates are described in the application WO 2015 / 004400. Furthermore, PCT / FR2021 / 051345, filed on July 19, 2021, describes binding heads that allow the production of compounds which, when conjugated to proteins, particularly antibodies, provide a "structure" such that, on average, the number of binding heads conjugated per protein (antibody) is controlled: the intended major conjugate carrying either 1 molecule per protein (antibody) or 2 molecules per protein (antibody). Summary of the invention

[0006] The present invention relates to a method for preparing a mixture of conjugates of formula (I):

[0007] [Chem.l] Acroehe heads Spacer (I)

[0008] in which the hook head, the connecting arm, the spacer, M and u are as defined below.

[0009] The present invention also relates to a mixture of conjugates that can be obtained by the aforementioned process.

[0010] The present invention also relates to a composition containing a mixture of conjugates of formula (I), and the use of this composition as a medicinal product, in particular for the treatment of cancer. Definitions

[0011] The term “antibody” refers to a heterotetramer consisting of two heavy chains of approximately 50-70 kDa each (called the H chains for Heavy) and two light chains of approximately 25 kDa each (called the L chains for Light), linked together by interchain disulfide bonds. Each chain consists, at the N-terminus, of a variable region or domain, called VL for the light chain, VH for the heavy chain, and at the C-terminus, of a constant region, consisting of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.

[0012] The term "F(ab')2" designates an antibody fragment that retains the ability of said antibody to bind an antigen. The F(ab')2 fragment is obtained by enzymatic digestion of immunoglobulins with pepsin or IdeS. F(ab')2 is composed of two Fab' fragments linked by interchain disulfide bridges. The Fab' fragment consists of the Fab region (comprising the variable regions and the CH1 and CL domains) and a hinge region. Brief description of the figures

[0013] Figures IA, IB, and IC represent the structure of conjugates obtained by the process according to the invention. In these figures, the gray square represents the hook head, the oval containing the letter M represents the active ingredient, and the gray oval between M and the hook head represents the link arm-spacer motif. Detailed description of the invention

[0014] According to a first aspect, the invention relates to a method for preparing a mixture of antibody-drug conjugates of formula (I):

[0015] [Chem.l] / AnUewys y- 5 ea I \ ? Hsiso's arm |—] Spacer (I)

[0016] in which:

[0017] i) the hook head is a compound of formula (lia), (Ilb), (Ile) or (Ild):

[0018] [Chem.2] (lia)

[0019] [Chem.3] (Ilb)

[0020] [Chem.4] (Island)

[0021] [Chem.5] (Ild)

[0022] in which:

[0023] - T represents -(CH2)y- , y being an integer from 0 to 6;

[0024] - W is -ORa, -COR2, -CONR3R4 or -NR3COR4;

[0025] - Ra is -(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5, -CORb, -(CRcRd)r-NHCO-(CH2CH2O)q -(CH2)r-R5, -(CRcRd)r-CONH-(CH2CH2O)q-(CH2)r-R5, -(CH2CH2O)q-(CH2)r - NHCO-(CRcRd)r-R5 ou -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5 ;

[0026] - Rb est -(CH2CH2O)q-(CH2)r-R5, -O(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5, -O(CRcRd)r-R5 -(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-CONH-(CH2CH2 O)q-(CH2)r-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5ou -(CH2CH2O)q-(CH2)r -CONH-(CRcRd)r-R5 ;

[0027] - R2 est -(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5, -O(CH2CH2O)q-(CH2)r-R5, -O(CRcRd)r - r5, -O(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R5, -O(CRcRd)r-CONH-(CH2CH2O)q-(CH2)r -r5, -O(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5ou -O(CH2CH2O)q-(CH2)r-CONH-(CRcRd) r-R5;

[0028] - R3 est -H, -(Ci-C6)alkyle ou -(CH2)V-SO3H, de préférence R3 est -H ou -(Ci-C6 )alkyle ;

[0029] - R4 est -(CH2CH2O)qR5, -(CRcRd)rR5, -(CRcRd)r-NHCO-(CH2CH2O)q-R5, -(CRcRd)r-CONH-(CH2CH2O)q-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5, -(CH2 CH2O)q-(CH2)r-CONH-(CRcRd)r-R5, -CH-[(CRcRd)r-CONH-(CRcRd)r-(OCH2CH2)q-R5]2, -CH-[(CRcRd)r-NHCO-(CRcRd)r-(OCH2CH2)q-R5]2, -CH-[(CRcRd)r-CONH-(CRcRd)r-R5] 2, or -CH-[(CRcRd)r-NHCO-(CRcRd)r-R5]2, preferably R4 is -(CH2CH2O)qR5, -(CRcRd)rR5, -(CRcRd)r-NHCO-(CH2CH2O)q-R5, -(CRcRd)r-CONH-(CH2CH2O)q-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5, or -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5;

[0030] - each R5 is -(CH2)SR6;

[0031] - each R6 is chosen from:

[0032] [Chem.6]

[0034] - Rc is H;

[0035] - each Rd is chosen from -H, -CH2-SO3H or -SO3H;

[0036] - R7 is -H or -CH3;

[0037] - each q is an integer from 1 to 24;

[0038] - each r is an integer from 1 to 8;

[0039] - each s is an integer ranging from 0 to 6;

[0040] - each v is an integer from 1 to 6;

[0041] ii) the connecting arm is a direct connection; an -SS- bridge; or a group of formula -(A)z-

[0042] - A is an amino acid residue;

[0043] - z is equal to 1, 2, 3, 4 or 5;

[0044] iii) the spacer is a direct bond or a formula group:

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] - G is a sulfate, a sugar, a glucuronide, or a galactoside, said sugar being a saccharide group preferably selected from a beta-glucuronic acid, a beta-D-galactose, a beta-D-glucose, an alpha-D-mannose, an N-acetyl-D-glucosaminyl, an N-acetyl-D-galactosaminyl, a D-glucuronyl, an L-iduronyl, a D-glucopyranosyl, a D-galactopyranosyl, a D-mannopyranosyl or an L-fucopyranosyl, preferably G is a sulfate, a beta-glucuronic acid, or a beta-D-galactose; - Rn is -H or -NO2; (iv) M is a molecule of interest; and v) u, which represents the ratio between M and the antibody (or F(ab')2), is in the range of 2 to 5; provided that the mixture contains at least 50% (by mass) of a conjugate of formula (I) in which u = 2, 3 or 5; said preparation process comprising: al) the reaction of an antibody (or an F(ab')2 fragment) with a compound of formula (lia) or (Ilb); bl) the reaction of the compound obtained in step a1) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined above; it being understood that: - if a compound (lia) is used in step a1) and a compound (lia) is used in step b1), the two compounds (lia) have a different structure; - if a compound (Ilb) is used in step a1) and a compound (Ilb) is used in step b1), the two compounds (Ilb) have a different structure; cl) the reaction of the compound obtained in step bl) with two identical or different compounds, each corresponding to formula (III): [Chem. 8] >■—I Spacer ?--1 MS » of tfatsen s: § (III) in which:

[0060] - R8 is R6-(CRcRd)r-CO-, R6-(CH2CH2O)q-(CH2)s-CO-, R6-(CRcRd)r-NH-, where R6, Rc, Rd, q, r and s are as defined above;

[0061] - the connecting arm, the spacer and M are as defined above, M being identical or not in each compound (III);

[0062] or

[0063] al) the reaction of an antibody (or an F(ab')2 fragment) with a compound of formula (lia) or (Ilb);

[0064] b2) the reaction of the compound obtained in step a1) with a compound of formula (IV):

[0065] [Chem.9] (IV)

[0066] in which:

[0067] i) the hook head is a compound of formula (II'a), (II'b), (II'c), or (II'd):

[0068] [Chem. 10] (II'a)

[0069] [Chem. 11]

[0070] [Chem. 12] (H'c)

[0071] [Chem. 13] (He'd)

[0072] in which:

[0073] - Test tel que defini ci-dessus;

[0074] - Y est -OR'a, -COR'2, -CONR'3R'4 or -NR'3COR'4;

[0075] - R'a est -(CH2CH2O)q-(CH2)r-R'5, -(CRcRd)r-R'5, -COR'b, -(CRcRd)r-NHCO-(CH2CH2 O)q-(CH2)r-R'5, -(CRcRd)r-CONH-(CH2CH2O)q-(CH2)r-R'5, -(CH2CH2O)q-(CH2)r - NHCO-(CRcRd)r-R'5ou -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R'5 ;

[0076] - R'b est -(CH2CH2O)q-(CH2)r-R'5, -O(CH2CH2O)q-(CH2)r-R'5, -(CRcRd)r-R'5, -O(CRcRd)r-R'5 -(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R'5, -(CRcRd)r-CONH-(CH2 CH2O)q-(CH2)r-R'5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R'5or -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R'5 ;

[0077] - R'2 est -OH, -(CH2CH2O)q-(CH2)r-R'5, -(CRcRd)r-R'5, -O(CH2CH2O)q-(CH2)r-R'5, - O(CRcRd)r-R'5, -O(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R'5, -O(CRcRd)r-CONH-(CH2 CH2O)q-(CH2)r-R'5, -O(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R'5ou -O(CH2CH2O)q-(CH2)r-CONH-(CRcRd )r-R'5 ;

[0078] - R'3 est -H, -(Ci-C6)alkyle or -(CH2)V-SO3H, de preference R'3 est -H or -(Ci-C6 )alkyl ;

[0079] - R'4 est -(CH2CH2O)qR'5, -(CRcRd)rR'5, -(CRcRd)r-NHCO-(CH2CH2O)q-R'5, -(CRcRd)r-CONH-(CH2CH2O)q-R'5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R'5, -(CH2 CH2O)q-(CH2)r-CONH-(CRcRd)r-R'5, -CH-[(CRcRd)r-CONH-(CRcRd)r-(OCH2CH2)q-R'5] 2, -CH-[(CRcRd)r-NHCO-(CRcRd)r-(OCH2CH2)q-R'5]2, -CH-[(CRcRd)r-CONH-(CRcRd)r -R'5]2, ou -CH-[(CRcRd)r-NHCO-(CRcRd)r-R'5]2, preferably R'4 est -(CH2CH2O)qR'5, -(CRcRd)rR'5, -(CRcRd)r-NHCO-(CH2CH2O)q-R'5, -(CRcRd)r-CONH-(CH2CH2O)q-R'5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R'5, ou -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r - R'5;

[0080] - chaque R'5 est -(CH2)SR'6 ou -(CH2)SR'7 ;

[0081] - R'6 est choisi parmi :

[0082] [Chem. 14]

[0084] - R'7 is -COOH or -NR'8R'9;

[0085] - R'8 is-H,-(Ci-C6)alkyl;

[0086] - R'9 is -H, -(CrC6)alkyl;

[0087] - R'i0 is-H or-CH3;

[0088] where Rc, Rd, q, r and s are as defined above; and

[0089] ii) the connecting arm is a direct connection; a -SS- bridge; or a group of formula -R'n-(A)Z-;

[0090] - R'h is a direct bond, a group R'6-(CRcRd)r-CO-, R'6-(CH2CH2O)q-(CH2)s - CO-, R'6-(CRcRd)r-NH-, where R'6, Rc, Rd, q, r and s are as defined above;

[0091] - A is an amino acid residue;

[0092] - z is equal to 1, 2, 3, 4 or 5; and

[0093] iii) the spacer and M are such as defined in step cl);

[0094] c2) the reaction of the compound obtained in step b2) with a compound of formula (III) such as defined in step cl), the molecule of interest M of compound (III) being identical to or different from the molecule of interest M of compound (IV);

[0095] or

[0096] a) the reaction of an antibody (or an F(ab')2 fragment) with a compound of formula (lia) or (Ilb);

[0097] b3) the reaction of the compound obtained in step a1) with a compound of formula (III) such as defined above;

[0098] c3) the reaction of the compound obtained in step b3) with a compound of formula (IV) such as defined above, the molecule of interest M of compound (IV) being identical to or different from the molecule of interest M of compound (III);

[0099] or

[0100] al) the reaction of an antibody (or an F(ab')2 fragment) with a compound of formula (lia) or (Ilb);

[0101] b3) the reaction of the compound obtained in step a1) with a compound of formula (III) such as defined above;

[0102] c4) the reaction of the compound obtained in step b3) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined above;

[0103] dl) the reaction of the compound obtained in step c4) with a compound of formula (III) as defined above, the molecule of interest M being identical or not in each compound (III).

[0104] Antibody-drug conjugates of formula (I) can also be prepared by a process comprising:

[0105] a2) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (IV) as defined above, in which the hook head is solely a compound of formula (Il'a) or (Il'b);

[0106] b4) the reaction of the compound obtained in step a2) with a compound of formula (IV) such as defined above, M being identical or not in each compound (IV), it being understood that:

[0107] - if a compound (Il'a) is used in step a2) and a compound (Il'a) is used in step b4), the two compounds (Il'a) have a different structure;

[0108] - if a compound (Il'b) is used in step a2) and a compound (Il'b) is used in step b4), the two compounds (Il'b) have a different structure;

[0109] or

[0110] a2) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (IV) as defined above, in which the hook head is solely a compound of formula (Il'a) or (Il'b);

[0111] b5) the reaction of the compound obtained in step a2) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined above;

[0112] c5) the reaction of the compound obtained in step b5) with a compound of formula (III) such as defined above, the molecule of interest M of compound (III) being identical to or different from the molecule of interest M of compound (IV).

[0113] Step a1) of the process of the invention consists of reacting an antibody (or the fragment F(ab')2 of said antibody) with a hook head of formula (lia) or (Ilb). The antibody, or fragment F(ab')2, binds to the hook head by a substitution reaction of the Br groups present in formula (lia) or (Ilb).

[0114] In some embodiments, the antibody (or the F(ab')2 fragment) is reacted with the hook head in the presence of a reducing agent. In some embodiments, the hook head and the reducing agent are present in excess relative to the antibody (or the F(ab')2 fragment). "Excess" (and this is applicable to the description and the claims) means an amount (expressed as an equivalent) at least twice as much, for example, at least five times, ten times, or fifteen times as much. In some embodiments, the hook head is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0115] At the end of step a1) we obtain a "compound" of structure:

[0116] [Chem. 15] S— S'

[0117] Step bl) of the process of the invention consists of reacting the compound obtained in step a1) with a second binding head, distinct from that used in step a1), of formula (Ha), (Ilb), (Ile) or (Ild). Similar to what is shown for step a1), the antibody, or the F(ab')2 fragment, binds to the second binding head by a substitution reaction of the Br groups present in the formula (lia), (Ilb), (Ile) or (Ild).

[0118] In some embodiments, the compound obtained in step a1) is reacted with the second attachment head in the presence of a reducing agent. In some embodiments, an excess of the second attachment head and reducing agent is used, relative to the compound obtained in step a1). In some embodiments, the compound obtained in step a1) is first reacted with the reducing agent, and then the second attachment head is added. In some embodiments, the second attachment head is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0119] At the end of step bl) we obtain a "compound" with the following structure:

[0120] [Chem. 16]

[0121] Step cl) of the process of the invention consists of reacting the compound obtained at the end of step bl) with two identical or different compounds of formula (III).

[0122] In some embodiments, an excess of each of the compounds of formula (III) is used, relative to the compound obtained in step 1b). In some embodiments, the compounds of formula (III) are each in solution in a water-miscible solvent or a mixture of water-miscible solvents. In some embodiments, the two compounds of formula (III) are added simultaneously. In some embodiments, the two compounds of formula (III) are added sequentially.

[0123] The reaction between the compound obtained in step 1b) and the two compounds of formula (III) is carried out by click chemistry. This reaction takes place between an R6 group on each of the parent groups and an R6 group on each of the compounds of formula (III). More precisely, the click reaction occurs between a diene (for example, an azide or a diazo) and a dienophile (for example, an alkene or an alkyne), each of these functional groups being provided by an R6 group. Thus, the click reaction can take place between a diene provided by the R6 group on each of the parent groups and a dienophile provided by the R6 group of compound (III), or between a dienophile provided by the R6 group on each of the parent groups and a diene provided by the R6 group of compound (III). These click reactions are well known to those skilled in the art, provided that the R6 groups are judiciously chosen to be compatible with each other.When the two compounds of formula (III) are added simultaneously, the click reaction is carried out in a single step. When the two compounds of formula (III) are added sequentially, two successive click reactions are implemented, the order of addition of the compounds of formula (III) being a function of the compatibility of the click functions (R6) used.

[0124] In some embodiments, the compound obtained in step a1) and / or the compound obtained in step b1) may be purified before being used in the next step. Similarly, when the click reaction is carried out in a single step, the mixture of conjugates obtained at the end of step c1) may also be purified. When two successive click reactions are implemented in step cl), purification can occur after one or both click reactions. The purification is performed using techniques well known to those skilled in the art.

[0125] At the end of step cl) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB, or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3, or 5).

[0126] Step b2) of the process of the invention consists of reacting the compound obtained in step al) with a compound of formula (IV).

[0127] In some embodiments, the compound obtained in step a1) is reacted with the compound of formula (IV) in the presence of a reducing agent. In some embodiments, an excess of compound of formula (IV) and reducing agent is used, relative to the compound obtained in step a1). In some embodiments, the compound of formula (IV) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0128] Step b2) yields a "compound" having one of the following structures:

[0129] [Chem. 17]

[0130] Step c2) of the process of the invention consists of reacting the compound obtained in step b2) with a compound of formula (III). This step is carried out by click reaction according to a method similar to that described for step c1).

[0131] In some embodiments, an excess of compound of formula (III) is used, relative to the compound obtained in step b2). In some embodiments, the compound of formula (III) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0132] In certain embodiments, the compound obtained in step a1) and / or the compound obtained in step b2) may be purified before being used in the next step. Similarly, the mixture of conjugates obtained at the end of step c2) may also be purified. The purification is carried out using techniques well known to those skilled in the art.

[0133] At the end of step c2) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3 or 5).

[0134] Step b3) of the process of the invention consists of reacting the compound obtained in step al) with a compound of formula (III).

[0135] This step is carried out by click reaction according to a method similar to that described for step cl).

[0136] In some embodiments, an excess of compound of formula (III) is used, relative to the compound obtained in step a1). In some embodiments, the compound of formula (III) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0137] At the end of step b3) we obtain a "compound" with the following structure:

[0138] [Chem. 18]

[0139] Step c3) of the process of the invention consists of reacting the compound obtained in step b3) with a compound of formula (IV).

[0140] In some embodiments, the compound obtained in step b3) is reacted with the compound of formula (IV) in the presence of a reducing agent. In some embodiments, an excess of compound of formula (IV) and reducing agent is used, relative to the compound obtained in step b3). In some embodiments, the compound of formula (IV) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0141] In certain embodiments, the compound obtained in step a1) and / or the compound obtained in step b3) may be purified before being used in the next step. Similarly, the mixture of conjugates obtained at the end of step c3) may also be purified. The purification is carried out using techniques well known to those skilled in the art.

[0142] At the end of step c3) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3 or 5).

[0143] Step c4) of the process of the invention consists of reacting the compound obtained in step b3) with a second hook head, of formula (lia), (Ilb), (Ile) or (Ild).

[0144] In some embodiments, the compound obtained in step b3) is reacted with the second attachment head in the presence of a reducing agent. In some embodiments, an excess of the second attachment head and reducing agent is used, relative to the compound obtained in step b3). In some embodiments, the compound obtained in step b3) is first reacted with the reducing agent, and then the second attachment head is added. In some embodiments, the second attachment head is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0145] Step c4) yields a "compound" having one of the following structures:

[0146] [Chem. 19]

[0147] Step dl) of the process of the invention consists of reacting the compound obtained in step c4) with a compound of formula (III).

[0148] This step is carried out by click reaction according to a method similar to that described for step cl).

[0149] In some embodiments, an excess of compound of formula (III) is used, relative to the compound obtained in step c4). In some embodiments, the compound of formula (III) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0150] In certain embodiments, the compound obtained in step a1) and / or the compound obtained in step b3) and / or the compound obtained in step c4) may be purified before being used in the next step. Similarly, the mixture of conjugates obtained at the end of step d1) may also be purified. The purification is carried out using techniques well known to those skilled in the art.

[0151] At the end of step dl) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3 or 5).

[0152] Step a2) of the process of the invention consists of reacting an antibody (or fragment F(ab')2) with a compound of formula (IV) as defined above, in which the binding head responds only to formula (II'a) or (II'b). The antibody, or fragment F(ab')2, binds to the binding head of compound (IV) by a substitution reaction of the Br groups present in formula (IFa) or (II'b).

[0153] In some embodiments, the antibody (or the F(ab')2 fragment) is reacted with the compound of formula (IV) in the presence of a reducing agent. In some embodiments, an excess of compound of formula (IV) and reducing agent is used, relative to the antibody. In some embodiments, the compound of formula (IV) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0154] At the end of step a2) we obtain a "compound" with the following structure:

[0155] [Chem. 18]

[0156] Step b4) consists of reacting the compound obtained in step a2) with a second compound of formula (IV).

[0157] In some embodiments, the compound obtained in step a2) is reacted with the second compound of formula (IV) in the presence of a reducing agent. In some embodiments, an excess of the second compound of formula (IV) and of the reducing agent is used, relative to the compound obtained in step a2). In some embodiments, the second compound of formula (IV) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0158] In some embodiments, the compound obtained in step a2) can be purified before being used in the next step. Similarly, the mixture of conjugates obtained at the end of step b4) can also be purified. The purification is carried out using techniques well known to those skilled in the art.

[0159] At the end of step b4) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3 or 5).

[0160] Step b5) of the process of the invention consists of reacting the compound obtained with step a2) with a formula hook head (lia), (Ilb), (Ile) or (Ild).

[0161] In some embodiments, the compound obtained in step a2) is reacted with the hook head in the presence of a reducing agent. In some embodiments, an excess of hook head and reducing agent is used, relative to the compound obtained in step a2). In some embodiments, the compound obtained in step a2) is first reacted with the reducing agent, and then the hook head is added. In some embodiments, the hook head is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0162] Step b5) yields a "compound" having one of the following structures:

[0163] [Chem. 19] Step c5) of the process of the invention consists of reacting the compound obtained in step b5) with a compound of formula (III).

[0164] This step is carried out by click reaction according to a method similar to that described for step cl).

[0165] In some embodiments, an excess of compound of formula (III) is used, relative to the compound obtained in step b5). In some embodiments, the compound of formula (III) is in solution in a water-miscible solvent or a mixture of water-miscible solvents.

[0166] In certain embodiments, the compound obtained in step a2) and / or the compound obtained in step b5) may be purified before being used in the next step. Similarly, the mixture of conjugates obtained at the end of step c5) may also be purified. The purification is carried out using techniques well known to those skilled in the art.

[0167] At the end of step c5) a mixture of conjugates is obtained, the major conjugate of which is as shown in [Fig.1A], IB or IC. This major conjugate has 2, 3 or 5 molecules of interest M carried by the antibody (u = 2, 3 or 5).

[0168] Some of the embodiments described above involve a reducing agent. A person skilled in the art will be able to choose such an agent judiciously from among the compounds

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] used conventionally in bioconjugation reactions, for example we can mention: dithiothreitol, beta-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride or tris(hydroxypropryl)phosphine. In some embodiments, the process according to the invention comprises steps a1), b1) and c1). In some embodiments, the hook head used in step a1) and step b1) conforms to one of the formulas (lia), (ilb), (ile) or (ild) in which: - W is -CONR3R4 or -NR3COR4, preferably W is -CONR3R4; - R3 is -H or -(Ci-C6)alkyl; - R4 is -(CH2CH2O)q-(CH2)r-R5, or -(CRcRd)r-R5; - R6 is chosen from: N' t H* i—N

[0176]

[0177]

[0178]

[0179] - Rc, Rd are as defined above; - q is an integer from 1 to 12, preferably q is an integer from 1 to 8; - r is an integer ranging from 1 to 6. In some embodiments, the hook head is a compound of formula (liai), (IIa2), (IIa3), (IIa4), (IIa5), or (IIa6):

[0180] [Chem.21] (liai);

[0181] [Chem.22] (IIa2);

[0182] [Chem.23] (IIa3); (IIa4);

[0184] [Chem.25] (IIa5);

[0185] [Chem.26] N' IS. (IIa6).

[0186] In certain embodiments, the process according to the invention comprises steps a1), b2) and c2).

[0187] In some embodiments, the hook head used in step b2) conforms to one of the formulas (II'a), (II'b), (II'c) or (II'd) in which:

[0188] - Y is -CONR'3R'4 or -NR'3COR'4, preferably Y is -CONR'3R'4;

[0189] - R'3 is -H or -(Ci-C6)alkyl;

[0190] - R'4 is -(CH2CH2O)q-(CH2)r-R'5, or -(CRcRd)r-R'5;

[0191] - R'5 is-(CH2)SR'6 ;

[0192] - R'6 is chosen from:

[0193] [Chem.27]

[0194] - Rc, Rd are as defined above;

[0195] - q is an integer from 1 to 12, preferably q is an integer from 1 to 8;

[0196] - r is an integer from 1 to 6.

[0197] In some embodiments, the hook head is a compound of formula (II'al), (II'a2), (II'a3), (II'a4), (II'a5), (II'a6), (II'a7), (II'a8) or (II'bl):

[0198] [Chem.28] (11'al);

[0199] [Chem.29] (II'a2);

[0200] [Chem.30] (II'a3);

[0201] [Chem.31]

[0202] (II'a4); [Chem. 32] Br Br Br Br

[0203] (II'a5); [Chem.33]

[0204] (II'a6); [Chem. 34] (II'a7);

[0205] [Chem.35] (Il'bl).

[0207] In some embodiments, the connecting arm is a direct connection.

[0208] In some embodiments, the connecting arm is a group of formula -Rn- (A)z- in which Rn and A are such as defined above, and z is equal to 2, 3 or 4.

[0209] A is an amino acid residue that may be selected from the group consisting of: a valine, a citrulline, a phenylalanine, a lysine, an aspartic acid, a methionine, an asparagine, a proline, an isoleucine, alanine, an arginine, a glycine, or a glutamic acid. Advantageously, z is equal to 2 or 3, preferably 2, and -(A)z- is an amino acid residue selected from the residue of: valine-citrulline; phenylalanine-lysine; valine-alanine; valine-aspartic acid; lysine-methionine; lysine-asparagine; proline-isoleucine; proline-lysine; valine-lysine; alanine-lysine; phenylalanine-phenylalanine-lysine; phenylalanine-phenylalanine-alanine; arginine-arginine; lysine-arginine; lysine-glutamic acid; glycine-arginine; or glycine-glycine-arginine (the order of amino acids is given here as an example, for example a citrulline-valine residue is also included in the definition of -(A)z-).

[0210] In some embodiments, the spacer arm is a direct link or a formula group:

[0211] [Chem.37] "OH , preferably a direct bond or a group of formula:

[0212] [Chem.38]

[0213] In some embodiments, M is an active ingredient. Examples of active ingredients that may be used in the context of the invention include the active ingredients of already authorized medicinal products and molecules currently undergoing therapeutic evaluation, in particular:

[0214] - alkylating agents such as: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, no-vembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard, CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin), duocarmycin (including the synthetic analogues KW-2189 and CBI-TMI), benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD) or to-maymycin dimers, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines), nitroureas (carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine), alkylsulfonates (busulfan, treosulfan, improsulfan and pi-posulfan), triazenes (dacarbazine), platinum-based compounds (carboplatin, cisplatin, oxaliplatin), aziridines (benzodopa, carboquone, meturedopa, and uredopa), ethyleneimines and melamines (including altretamine, triethylenemelamine,triethylenephos-phoramide, triethylenethio-phosphaoramide and trimethylolomelamine); ,

[0215] - plant alkaloids such as: Vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, navelbine), taxoids (paclitaxel, docetaxol) and their analogues, maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocin) and their analogues, cryptophycins (in particular cryptophycin 1 and cryptophycin 8), epothilones, eleutherobine, discodermolide, bryostatins, dolastatins, auristatins, tubulysins, cephalostatins, pancratistatins, sarcodictyin, spongistatins

[0216] - DNA topoisomerase inhibitors such as: epipodophyllin (9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000), mitomycins (mitomycin C), bortezomib;

[0217] - antimetabolites such as: antifolates (DHFR inhibitors (methotrexate, tri- metrexate, denopterin, pteropterin, aminopterin (4-aminopteric acid) and other folic acid analogues), IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurine, ribavirin, EICAR), ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine), pyrimidine analogues such as: uracil analogues (ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed), cytosine analogues (cytarabine, cytosine arabinoside, fludarabine), purine analogues (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine), folinic acid;

[0218] - hormonal agents such as: anti-estrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (goserelin, leuprolide acetate), anti-androgens (bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane), vitamin D3 analogues (CB 1093, EB 1089, KH 1060, cholecalciferol, ergocalciferol), photodynamic therapies (verteporfin, phthalocyanine, photosensitizer Pc4), cytokines (interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), human proteins containing a TNF domain);

[0219] - kinase inhibitors such as: BIBW 2992, CYT387, E7080, axitinib, bafetinib, bosutinib, cabozantinib, dasatinib, erlotinib, gefitinib, imatinib, iniparib, ispinesib, lapatinib, masitinib, mubritinib, nilotinib, pazopanib, pegaptanib, ponatinib, ruxolitinib, sorafenib, sunitinib, tivozanib, vandetanib, vismodegib;

[0220] - poly(ADP-ribose) polymerase (PARP) inhibitors such as: BGB-290, CEP 9722, E7016, 3-aminobenzamide, niraparib, olaparib, talazoparib, veliparib;

[0221] - immunomodulators such as: thalidomide, lenalidomide, pomalidomide;

[0222] - a toxin such as Pseudomonas exotoxin (PE), deBouganin, Bouganin, there

[0223] In a particular embodiment, the active ingredient is selected from methotrexate, an immunomodulator, duocarmycin, combretastatin, calicheamicin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, SN38, amanitin and its analogues, pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, pyrrolopyridodiazepine, a pyrrolo-pyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, and ricin, preferably the active ingredient is amanitin, a pyrrolobenzodiazepine dimer, MMAF or MMAE, the latter two being represented by the following formulas:

[0224] [Chem.39] MMAF MMAE

[0225] In some embodiments, M is a radionuclide chelator. Examples of radionuclide chelators that may be used in the context of the invention include sarcophagin, DOTA (1,4,7,10-tetraazacyclododecane-A,A',A",A "'-tetraacetic acid), DOTAGA (tetraazacyclododecane triacetic acid glutaric acid), NODA (1,4,7-triazacyclononane-1,4-diacetic acid), NODAGA (triazacyclononane diacetic acid glutaric acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and MANOTA (methyl amino triazacyclononane triacetic acid).

[0226] In some embodiments, the antibody or the F(ab')2 fragment of said antibody binds to a cancer-specific antigen. The cancer-specific antigen may be, for example, CD1A, CD3, CD4, CD13, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD80, CD86, CD117, CD138, CD194, CD205, CD227, VEGF, EpCAM, GPIIb, GPIIIa, TNF alpha, TNFR, TNT, Lewis Y, EGFR, HER-2, HER-3, HER-4, AXL, Protein F, IgE-Fc, C5, IL-6R, IL12, IL15, IL18, IL23, IL-1, TPO-R,

[0227]

[0228]

[0229]

[0230]

[0231] GPNMB, PSMA, PSA, PAP, PSM, Crypto, Folate Receptor 1, Endothelin Receptors ETB, STEAP1, SLC44A4 (AGS-5), AGS-16, Guanylyl cyclase C, EGFRvIII, Mesothelin, IL2R, A33, Can, VEGFR-1, VEGFR-2, VEGFR-3, TGFbeta, TGFbetaR, FGF, FGFR, PDGF, PDGFR, Ang-1, Ang-2, Integrin, RANK-L, BLyS, c-MET, DR, TCRalpha, beta, ICOS, EphA2, CA6, ENPP3, FOLR1, Nectin-4, TIM-1, Tissue Factor, LIV-1, TLR-7, AFP, HLA-DR, Carcinoembryonic Antigen (CEA), TAG-72, Binding Protein folates, G250, gangliosides, type 4 collagen (collagen IV), type 18 collagen (collagen XVIII), CA19-9, pl85HER2, fibroblast-activating protein (FAP), tenascin, metalloproteinases, endosialin, carbonic anhydrase, Galectin 9, Aldolase A, eIFgamma4, Galectin 4, HERKV-K10, p53, NY-LU-12, Restin, NY-CO-38, SSX2, NY-ESO-1, SCP-1, HGFR, PTK 7, CCK-4, PTP-LAR, CDCP1, CADM1, IGSF4, BCAM, CEACAM6, JAM-A, PTGFRN (CD9P-1), MCAM, MCP, EMMPRIN, TfR, ClqR, hTERT, Survivin, MDM2, CYP1B1, MART-1,MART-2, melanosomal proteins, gplOO, CDC27, MAGEs, WT1, MUM-1, MUM-2, MUM-3, BRAF, TPI, fibronectin, K-ras, beta-catenin, CDK4, caspase-8, pl4ARF, pl6INK4a, bcr-abl, SYT-SSX, TRP-1, TRP-2, GnT-V, tyrosinase, TEL-AML1, proteinase 3, EBV-EBNA, HTLV-1 tax, HPV16-E7, mutated HLA-A2, HAÏ, SART3, CEACAM5, ESAT-6, RANK, fibrin, TF, PRAME, CA19-9, CA50, CA195, CAM17.1 / WGA, beta-MG, DU-PAN2, HE4, transferrin, transthyretin, ApoAl, TROP-2, CTLA-4, GITR, PD-1, PD-L1, c-KIT, CD1b-CD18 integrin heterodimer, DNA / Histone H1, proteoglycan, fibrinogen, large T antigen SV40, SC-Ag, ES A, mucin, CCR4, MTX1, MTX2, PECAM, Tn, cathepsin D, TYRO-3, MER, or a PF4 / heparin complex. In some embodiments, the antibody or the F(ab')2 fragment of said antibody binds to HER2, CD30 or CD56. It may therefore, for example, be an anti-HER2 antibody, an anti-CD30 antibody or an anti-CD56 antibody. The antibody or F(ab')2 fragment can be of mammalian (e.g., human or mouse), murine, chimeric, humanized, or human origin. Preferably, it is a monoclonal antibody produced recombinantly by genetically modified cells using techniques widely described in the literature. In some embodiments, the antibody is of the IgG type, for example IgG1, IgG2, IgG3 or IgG4. Compounds of formula (III): [Chem. 8] (III)

[0232] can be obtained in a conventional manner, for example by peptide-type coupling between on the one hand the linking arm (carrying the R8 group) and the spacer, and on the other hand between the spacer and M. Such coupling is carried out in a classical manner between a carboxylic group carried by the linking arm (respectively, the spacer), and an amine group carried by the spacer (respectively M).

[0233] Compounds of formula (IV):

[0234] [Chem.9]

[0235] can be purchased or obtained, for example, as described in PCT / FR2021 / 051345 or in PCT / FR2020 / 050833.

[0236] The preparation process of the invention makes it possible to obtain a mixture of antibody-drug conjugates of formula (I). Those skilled in the art will understand that, by way of simplicity, the structure of the binding head (Ha), (Ilb), (Ile), or (Ild) as represented for the conjugates of formula (I) actually corresponds to the structure before its binding to the antibody (or the F(ab')2 fragment), a binding that occurs by substitution of the Br leaving groups of the binding head. This mixture of conjugates is characterized in that it contains predominantly (i.e., at least 50% (by mass)) an antibody-drug conjugate comprising 2, 3, or 5 molecules of interest M bound to the antibody (or the F(ab')2 fragment). The number of molecules bound to the antibody (or the F(ab')2 fragment) corresponds to the number "u" in formula (I). The proportion of the expected species (u = 2, 3 or 5) is determined by HRMS (High Resolution Mass Spectrometry) analysis under denaturing conditions.When the formula (I) conjugates comprise an entire antibody (rather than an F(ab')2 fragment), the analysis is performed after digestion of the conjugates by IdeS, on the F(ab')2 fragment. The mixture of formula (I) conjugates obtained by the process of the invention may be present in a composition, which may be, for example, a pharmaceutical composition containing one or more pharmaceutically acceptable excipients and / or vehicles.

[0237] Thus, according to another aspect, the invention relates to a composition comprising a mixture of antibody-drug conjugates of formula (I) obtainable by the process described above. As indicated above, this mixture predominantly contains an antibody-drug conjugate comprising 2, 3, or 5 molecules of interest M attached to the antibody (or the fragment F(ab')2).

[0238] In certain embodiments, when the mixture predominantly contains an antibody-drug conjugate comprising 2 molecules of interest M attached to the antibody (or the fragment F(ab')2), these 2 molecules M are different from each other.

[0239] According to another aspect, the invention relates to a composition as defined above for use as a medicinal product.

[0240] The invention is illustrated by the following examples, given for illustrative purposes only. In these examples, the following abbreviations are used:

[0241] DCC = dicyclohexylcarbodiimide

[0242] DIPEA = A,A-diisopropylethylamine

[0243] DMAP = 4-dimethylaminopyridine

[0244] DMF = A,A-dimethylformamide

[0245] DMSO = dimethyl sulfoxide

[0246] EDTA = ethylenediaminetetraacetic acid

[0247] HCl = hydrochloric acid

[0248] HOBt = hydroxybenzotriazole

[0249] MeCN = acetonitrile

[0250] MeOH = methanol

[0251] NaCl = sodium chloride

[0252] TA = ambient temperature (20 °C unless otherwise specified)

[0253] TFA = trifluoroacetic acid

[0254] tR = retention time

[0255] v / v = volume-to-volume ratio Analysis methods

[0256] Nuclear magnetic resonance (NMR) spectroscopy

[0257] Proton 'H' nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Ultrashield 300 instrument (300 MHz (*H)). The analyses were performed in deuterated methanol (CD3OD). The chemical shifts (δ) are measured in parts per million (ppm) relative to the residual signal of deuterated methanol (δ'H = 3.31 ppm).

[0258] The coupling constants (J) are expressed in Hertz (Hz) and the multiplicity is described as follows: d = doublet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet, p = pentuplet, s = singlet, t = triplet. High-resolution mass spectrometry (HRMS)

[0259] The exact mass of the synthesized compounds was determined by high-resolution mass spectrometry (HRMS) in positive or negative mode with the electron spray ionization technique ESI, on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system of the ICOA / CBM “Research Federation” platform (FR2708)).

[0260] Denaturing high-resolution mass spectrometry (HRMS)

[0261] In order to eliminate sugar-related heterogeneity and increase the mass response, The Fc regions of the antibodies to be analyzed were cleaved using the IdeS protease. Conjugate analysis was therefore performed on a sample previously cleaved with the IdeS protease: 1 pL of IdeS at a concentration of 0.8 g / L was added to 10 pL of the sample, and the sample was then incubated at 37°C for at least 16 h. Spectrometric analysis of the conjugates was performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, the samples (5 pg) were desalted on a MassPREP desalting column (2.1 x 10 mm, Waters), heated to 80°C using a 0.1% aqueous formic acid solution as solvent A and a 0.1% formic acid in acetonitrile solution as solvent B at 500 pL / min. After 1 min, a linear gradient from 5% to 90% of B was applied over 1.5 min.MS data were acquired in positive mode with an ESI source over an m / z range of 900 to 5000 at 1 Hz and analyzed using DataAnalysis 4.4 software (Bruker) and the MaxEnt algorithm for deconvolution. The proportion of species of interest obtained was determined using the intensity of the species peaks observed on fragment F(ab')2. Bioconjugation reactions; Preparation of solutions

[0262] Buffer 1: Phosphate buffer IX (1 mM KH2PO4, 180 mM NaCl and 3 mM Na2 HPO4.7H2O) at a pH of 8.3, and a final EDTA concentration of 1 mM.

[0263] Buffer 2: Phosphate buffer IX (1 mM KH2PO4, 180 mM NaCl and 3 mM Na2 HPO4.7H2O) at a pH of 7.4.

[0264] Reducer: Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP.HC1) at a concentration of 1 mM in buffer 1. Bioconjugation reaction 1

[0265] The antibody solution in buffer 1 (1.0 eq) was placed under argon. The solution of the compound of formula (Ha), (Ilb), (II'a) or (Il'b) (1.0-15.0 eq, preferably 3.0-12.0 eq) then the reducing solution (1.0-12.0 eq, preferably 3.0-8.0 eq) were added and the reaction medium was stirred under argon at 37 °C for 2.5 h. Purification 1

[0266] If necessary, the compound obtained from bioconjugation reaction 1 was purified using Sephadex® with buffer 2. The fractions containing the purified compound (as determined by NanoDrop) were collected and then concentrated using Vivaspin® (10 kDa). A buffer exchange was performed on the concentrated solution using Vivaspin® (10 kDa) to obtain the compound that was used as buffer 1 in the subsequent step. Bioconjugation reaction 2

[0267] The possibly purified compound obtained at the end of bioconjugation reaction 1 (1.0 eq) was placed under argon. The reducing agent (7.0–30.0 eq, preferably 8.0–15.0 eq) was then added and the reaction medium was incubated at 37°C for 2h. Then the solution of compound (lia), (Ilb), (Ile), (Ild), or (IV) (5.0-30.0 eq, preferably 12.0-15.0 eq) was added under argon and the reaction medium was stirred at 37°C for 2h30. Purification 2

[0268] If necessary, the compound obtained from bioconjugation reaction 2 or from the one-step click reaction (see below) was purified with Sephadex® using buffer 2. The fractions containing the purified compound (NanoDrop® assay) were collected for use in the next step. Click reaction

[0269] In one step: addition of a single compound (III) or simultaneous addition of two compounds (III)

[0270] To the possibly purified compound obtained at the end of bioconjugation reaction 2 (1.0 eq) was added the solution(s) of compound(s) (III) (5.0-30.0 eq, preferably 15.0-30.0 eq) and the reaction medium was stirred at 37°C for at least 16h.

[0271] In two steps: sequential addition of two compounds (III)

[0272] To the possibly purified compound obtained at the end of bioconjugation reaction 2 (1.0 eq), a solution of the first compound (III) (5.0–30.0 eq, preferably 15.0–30.0 eq) was added, and the reaction mixture was stirred at 37°C or 25°C for at least 16 h. The intermediate compound obtained was optionally purified (see below). Then, a solution of the second compound (III) was added (5.0–30.0 eq, preferably 15.0–30.0 eq), and the reaction mixture was stirred at 37°C or 25°C for at least 16 h. Purification 3

[0273] When two click reactions were carried out sequentially, the compound obtained at the end of the first click reaction was advantageously purified by Sephadex® with buffer 2. The fractions containing the purified compound (assay by NanoDrop®) were collected and then concentrated by Vivaspin® (10 kDa) for use in the second click reaction. Examples

[0274] Example 1: 4-nitrophenyl l-[4-(6-methyl-l,2,4,5-tetrazin-3-yl)phenoxy]-3,6,9,12-tetraoxapentadecan-15-oate (1)

[0275] [Chem.40] r <4 . G ... o x g O.....' (1)

[0276] 4-Methylteatrazinylphenoxy-3,6,9,12-tetraoxapentadecan-15-oic acid (12.2 mg; 0.028 mmol; 1.0 eq) was dissolved in anhydrous DMF (250 qL), and then 4-nitrophenol (5.1 mg; 0.036 mmol; 1.3 eq), DCC (7.5 mg; 0.036 mmol; 1.3 eq), and DMAP (1.0 mg; 0.008 mmol; 0.3 eq) were added. The reaction mixture was stirred under argon at room temperature for 18.5 h. The mixture was purified by semi-preparative high-performance liquid chromatography (tR = 26.01 min; on the Gilson PLC 2050 system [ARMEN V2 (pump) and ECOM TOYDAD600 (UV detector)] UV detection at 254 nm at 25 °C; Waters XBridge™ C-18 column; 5 qm (250 mm x 19.00 mm); elution carried out with 0.1% TFA (by volume) in water (solvent A), and MeCN (solvent B); gradient 20 to 100% of B over 32 min then 100% of B over 6 min at 17.1 mL / min) to give (1) (7.8 mg; 50%) as a pink oil.

[0277] 'H NMR (300 MHz, CD3OD) δ 8.53 - 8.42 (m; 2H); 8.32 - 8.21 (m; 2H); 7.42 -7.31 (m; 2H); 7.21 - 7.11 (m; 2H); 4.29 - 4.20 (m; 2H); 3.93 - 3.80 (m; 4H); 3.75 - 3.66 (m; 4H); 3.66 - 3.63 (m; 8H); 3.00 (s; 3H); 2.87 (t; J= 6.0 Hz; 2H).

[0278] SMHR (ESI): m / z calculated for C26H32N5O9 [M+H]+: 558.2195; observed 558.2203.

[0279] Example 2: acid (2R)-2-[(2S)-2-[(S)-[(2R)-l-[(3S,4R,5R)-4-[(2R)-N,3-dimethyl-2-[(2R)-3-methyl-2-{N-methyl-l-[4-(6-methyl-l,2,4,5-tetrazin-3-yl)phenoxy]-3,6,9,12-tetraoxapentad ecan-15-amido}butanamido]

[0280] butanamido]-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl](methoxy)methyl]propanamido]-3-phenylpropanoic (2)

[0281] [Chem.41] (2)

[0282] To a solution of HOBt (2.0 mg; 0.0151 mmol; 2.1 eq), solubilized in anhydrous DMF (100 qL) in the presence of anhydrous DIPEA (2.5 qL; 0.0144 mmol; 2.0 eq) was added 1-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]-3,6,9,12-tetraoxapentadecan-15-oate of 4-nitrophenyl (1) (7.1 mg; 0.0128 mmol; 1.8 eq). Then, a solution of MMAF trifluoroacetic acid salt (6.1 mg; 0.0072 mmol; 1.0 eq), solubilized in anhydrous DMF (100 qL), was added to the reaction mixture, which was stirred under argon at 25 °C for 19 h. The mixture was purified by semi-preparative high-performance liquid chromatography (tR = 27.44 min; on the Gilson PLC 2050 system [ARMEN]). V2 (pump) and ECOM TOYDAD600 (UV detector)] UV detection at 254 nm at 25 °C; Waters XBridge™ C-18 column; 5 pm (250 mm x 19.00 mm); elution carried out with 0.1% TFA (by volume) in water (solvent A), and MeCN (solvent B); gradient 20 to 90% of B over 32 min then 90% of B over 6 min at 17.1 mL / min) to give (2) (1.4 mg; 17%) as a purple oil.

[0283] 'H NMR (300 MHz, CD3OD) δ 8.57 - 8.45 (m; 2H); 8.41 - 8.08 (m; 1H); 8.08 -7.79 (m; 1H); 7.37 - 7.06 (m; 8H); 4.78 - 4.59 (m; 1H); 4.32 - 4.22 (m; 2H); 4.14 - 3.96 (m; 2H); 3.96 - 3.85 (m; 2H); 3.84 - 3.52 (m; 13H); 3.48 - 3.35 (m; 5H); 3.21 - 3.15 (m; 2H); 3.14 - 3.03 (m; 4H); 3.00 (s; 3H); 2.97 - 2.89 (m; 1H); 2.83 - 2.63 (m; 1H); 2.61 - 2.40 (m; 2H); 2.41 - 2.15 (m; 2H); 2.15 - 1.67 (m; 8H); 1.50 - 1.12 (m; 3H); 1.12 - 0.76 (m; 28H).

[0284] SMHR (ESI): m / z calculated for C59H92N9O14 [M+H]+: 1150.6758; observed 1150.6754.

[0285] The commercial compounds, from PCT / FR2021 / 051345 or PCT / FR2020 / 050833, used for bioconjugation reactions are summarized in Table 1 below.

[0286] [Tables 1] Number(#) Name and structure (3) bicyclo[6T.0]non-4-yn-9-ylmethyl(4-((2,6-bis(bromomethyl)isonicotinamido)methyl)-1 -(2,6-bis(bromomethyl)pyridin-4-yl)-1,5-dioxo-9,12,15,18-tetraoxa -2,6-diazaicosan-20-yl)carbamate [Chem42] ex O | H « tj (4) 2-(2-(2-(tra«5-cyclooctenylcarbamoyl)ethoxy)ethoxy)ethyl)carbamoylpropan e-1,3-diyl(2,6-bis(bromomethyl)isonicotinamide) [Chem43] O \ S $ 5 (5) 4- {2-azatricyclo [ 10.4.0.04'9]hexadeca-1(12) ,4(9) ,5,7,13,15 -hexaen-10-yn-2-yl}-N-(2-{2-[2-(3-{[2,6-bis(bromomethyl)pyridin-4-yl]formamido}-2-({[2,6-bis(bromomethyl)pyridin-4-yl]formamido}methyl)-propanamido)ethoxy]ethox y}ethyl)-4-oxobutanametamide [Chem44] By N'X't 3 H g ï I r ï ' 0 ^11 vxJ N (6) 2-(2-(2-(2-(4-(methyltetrazinylphenoxy)ethoxy)ethoxy)ethoxy)-ethyl)carbamoylpropane-1,3-diyl(2,6-bis(bromomethyl)isonicotinamide) [Chem45] AA 1 H o^Jh ù M'An AOK Bfx^AjYv^.Br (7) MA'-(2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-carbamoyl)propane-1, 3-diyl)bis(2,6-bis(bromomethyl)isonicotinamide) [Chem46] Bf^ 1 ..jcv | H aA o X Bk Jk (8) 6-azidohexanamido-1V-hexanamide-valine-citrulline-p-aminobenzoyl carbamate of MMAE [Chem47] o 1 x .j XXvO H 0 k ” ' 0 Yx ! O SjM (9) (4-{ 2-[2-(6-{ 2-azatricyclo[10.4.0.04'9]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-6-oxohexanamido)-3-methylbutanamido]-5-(carbamoylammo)pe ntanamido}phenyl)methylUV- {l-[(l-{[l-(2-{2-[(l -hydroxy-1 -phenylpropan-2-yl)carbamoyl]-l-methoxy-2-methylethyl}pyrrolidin-l-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl] (methyl)carbamoyl} -2-methylpropyl)carbamoyl] -2-methylpropyl} -A-methylcarbamate [Chem48] o  >• A 0Xh XjxXX axx WTHT fi Y r « Y1 0 © Y ! 0 1 0^ 0 A 'O (10) 1 -trans-cyclooctenyl-1 -oxo-5,8,11,14-tetraoxa-2-azahetaptadecan-17-amide-valine-citrulline-p-aminobenzoyl carbamate from MMAE [Chem49] o î j / M g Y hs fY'0 * « g YMMSLHUA ' ko (11) MMAE 6-(2,6-bis(bromomethyl)pyridin-4-yl)amido-A-hexanamide-valine-citrulline-p-aminobenzoyl carbamate [Chem50] Bt, JH g YB g 0 H 0 AH ' O' tW jYvO H oyy”\ ho YV'rVrV'7 3 G ZAX 1 O (12) commercial compound Mc-Val-Cit-PAB-MMAE (CAS: 646502-53-6) [Chem51] çAjOÇsy—a OH o 1 HO NH yvtYyv^ (13) 3-(2,6-bis(bromomethyl)isonicotinamido)-2-((2,6-bis(bromomethyl)isonicotinamido)-methyl)propanamido)-3-sulfopropanamido-valine-citrulline-p-amino benzoyl carbamate de MMAE [Chem52] Example 3 Step al) Reagents

[0287] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (6) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0288] Bioconjugation reaction 1. Step bl) Reagents

[0289] Compound from al) at 3.2 mg / mL in buffer 1, reducing agent (8.0 eq) and compound (7) (15.0 eq) at a concentration of 10 mM in MeOH. Method

[0290] Bioconjugation reaction 2. Step cl) Reagents

[0291] Compound from bl) at 2.6 mg / mL in buffer 1, compound (9) (16.5 eq) and compound (10) (11.6 eq) at a concentration of 10 mM in DMSO. Method

[0292] Click reaction at 25°C for 17h. In this case, compounds (9) and (10) were added concomitantly. A mixture of conjugates was obtained, the HRMS analysis of which below determined the major presence of conjugate (14), whose structure is as shown in [Fig. 1A]. Denaturing SMHR analysis

[0293] The results are presented in Table 2 below.

[0294] [Tables2] Expected MM (Da) Observed MM (Da) Proportion (%) (14) 101870 101871 56 Example 4 Step al) Reagents

[0295] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (3) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0296] Bioconjugation reaction 1 then purification 1. Step b2) Reagents

[0297] Compound from al) at 5.0 mg / mL in buffer 1, reducer (15.0 eq) and compound (11) (15.0 eq) at a concentration of 2 mM in a mixture of 80% DMF and 20% MeOH. Method

[0298] Bioconjugation reaction 2 then purification 2. Step c2) Reagents

[0299] Compound from b2) at 1.3 mg / mL in buffer 2 and compound (2) (15.0 eq) at a concentration of 1 mM in DMSO. Method

[0300] Click reaction at 25 °C for 23 h followed by purification 2. A mixture of was obtained conjugates whose SMHR analysis below determined the predominant presence of conjugate (15), whose structure is as represented in [Fig. 1B]. Denaturing SMHR analysis

[0301] The results are presented in Table 3 below.

[0302] [Tables3] Expected MM (Da) Observed MM (Da) Proportion (%) (15) 102263 102263 64 Example 5 Step a1) Reagents

[0303] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (3) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0304] Bioconjugation reaction 1 then purification 1. Step b2) Reagents

[0305] Compound derived from a1) at 5.0 mg / mL in buffer 1, reducing agent (15.0 eq) and compound (12) (15.0 eq) at a concentration of 2 mM in DMSO. Method

[0306] Bioconjugation reaction 2 then purification 2. Step c2) Reagents

[0307] Compound from b2) at 0.7 mg / mL in buffer 2 and compound (2) (15.0 eq) at a concentration of 1 mM in DMSO. Method

[0308] Click reaction at 25°C for 23 h followed by purification. 2. A mixture of conjugates was obtained, and the HRMS analysis below determined the major presence of conjugate (16), whose structure is as shown in [Fig. 1C]. Denaturing HRMS analysis

[0309] The results are presented in Table 4 below.

[0310] [Tables4] Expected MM (Da) Observed MM (Da) Proportion (%) (16) 104799 104799 88 Example 6 Step a1) Reagents

[0311] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (3) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0312] Bioconjugation reaction 1 then purification 1. Step b2) Reagents

[0313] Compound from a1) at 5.0 mg / mL in buffer 1, reducing agent (15.0 eq) and compound (13) (15.0 eq) at a concentration of 2 mM in DMF. Method

[0314] Bioconjugation reaction 2 then purification 2. Step c2) Reagents

[0315] Compound from b2) at 0.9 mg / mL in buffer 2 and compound (2) (15.0 eq) at a concentration of 1 mM in DMSO. Method

[0316] Click reaction at 25°C for 23h followed by purification 2. A mixture of was obtained conjugates whose SMHR analysis below made it possible to determine the majority presence of the conjugate (17) whose structure is such as represented in [Fig.1A]. Denaturing SMHR analysis

[0317] The results are presented in Table 5 below.

[0318] [Tables5] Expected MM (Da) Observed MM (Da) Proportion (%) (17) 101164 101165 63 Example 7 Step a1) Reagents

[0319] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (3) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0320] Bioconjugation reaction 1 then purification 1. Step bl) Reagents

[0321] Compound from a1) at 5.0 mg / mL in buffer 1, reducing agent (15.0 eq) and compound (4) (15.0 eq) at a concentration of 2 mM in DMF. Method

[0322] Bioconjugation reaction 2 then purification 2. Step cl) Reagents

[0323] Compound from bl) at 0.6 mg / mL in buffer 2, compound (8) (15.0 eq) at a concentration of 1 mM in DMSO (“click 1”), then compound from “click 1” at 1.8 mg / mL in buffer 2, compound (2) (15.0 eq) at a concentration of 2 mM in DMSO (“click 2”). Method

[0324] Sequential click.

[0325] Click 1: click reaction at 37°C for 23h then purification 3.

[0326] Click 2: click reaction at 37°C for 18h. A mixture of conjugates was obtained. the SMHR analysis below made it possible to determine the majority presence of the conjugate (18) whose structure is such as represented in [Fig.1A]. Denaturing SMHR analysis

[0327] The results are presented in Table 6 below.

[0328] [Tableauxô] Expected MM (Da) Observed MM (Da) Proportion (%) (18) 101453 101451 53 Example 8 Step a1) Reagents

[0329] Trastuzumab at 5.0 mg / mL in buffer 1, reducing agent (7.0 eq), compound (3) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0330] Bioconjugation reaction 1 then purification 1. Step bl) Reagents

[0331] Compound from a1) at 5.0 mg / mL in buffer 1, reducing agent (15.0 eq) and compound (5) (15.0 eq) at a concentration of 2 mM in DMF. Method

[0332] Bioconjugation reaction 2 then purification 2. Step cl) Reagents

[0333] Compound from bl) at 1.0 mg / mL in buffer 2 and compound (2) (15.0 eq) at a concentration of 1 mM in DMSO (“click 1”), then compound from “click 1” at 1.2 mg / mL in buffer 2 and compound (8) (15.0 eq) at a concentration of 1 mM in DMSO (“click 2”). Method

[0334] Sequential click.

[0335] Click 1: click reaction at 25°C for 23h then purification 3.

[0336] Click 2: click reaction at 37°C for 18h. A mixture of conjugates was obtained. the SMHR analysis below made it possible to determine the majority presence of the conjugate (19) whose structure is such as represented in [Fig.1A]. Denaturing SMHR analysis

[0337] The results are presented in Table 7 below.

[0338] [Tables7] Expected MM (Da) Observed MM (Da) Proportion (%) (19) 101588 101588 55

Claims

Demands

1. Method for preparing a mixture of antibody-drug conjugates of formula (I): [Chem.l] <k< (I) in which: i) the anchoring head is a compound of formula (Ha), (Ilb), (Ile) or (Ild): [Chem. 2] (Ha) [Chem. 3] (Ilb) [Chem. 4] (Island) [Chem. 5] (üd) in which: - T represents -(CH2)y- , y being an integer from 0 to 6; - W is -0Ra, -COR2, -CONR3R4 or -NR3COR4; - Ra is -(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5, -CORb, -(CRcRd)r -NHCO-(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-CONH-(CH2CH2O)q-(CH2)rR 5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5or -(CH2CH2O)q-(CH2)r -CONH-(CRcRd)r-R5; - Rb est -(CH2CH2O)q-(CH2)r-R5, -O(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5 -O(CRcRd)r-R5 -(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r -CONH-(CH2CH2O)q-(CH2)r-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R 5 ou -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5 ; - R2 est -(CH2CH2O)q-(CH2)r-R5, -(CRcRd)r-R5, -O(CH2CH2O)q-(CH2)r-R5 , -O(CRcRd)r-R5, -O(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R5, -O(CRcRd)r -CONH-(CH2CH2O)q-(CH2)r-R5, -O(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r -R5ou -O(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5 ; - R3 est -H, -(Ci-C6)alkyle ou -(CH2)V-SO3H, de préférence R3 est -H ou -(Ci-C6)alkyle ; - R4 is -(CH2CH2O)qR5, -(CRcRd)rR5, -(CRcRd)r-NHCO-(CH2CH2O)q-R5, -(CRcRd)r-CONH-(CH2CH2O)q-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd) r-R5, -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5, -CH-[(CRcRd)r -CONH-(CRcRd)r-(OCH2CH2)q-R5]2, -CH-[(CRcRd)r-NHCO-(CRcRd)r -(OCH2CH2)q-R5]2, -CH-[(CRcRd)r-CONH-(CRcRd)r-R5]2, or -CH-[(CRcRd)r-NHCO-(CRcRd)r-R5]2, preferably R4 is -(CH2CH2O)qR5, -(CRcRd)rR5, -(CRcRd)r-NHCO-(CH2CH2O)q-R5, -(CRcRd)r-CONH-(CH2 CH2O)q-R5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5, or -(CH2CH2O)q -(CH2)r-CONH-(CRcRd)r-R5; - each R5 is -(CH2)SR6; - Each R6 is chosen from: - Rc is H; - each Rd is chosen from -H, -CH2-SO3H or -SO3H; - R7 is -H or -CH3; - each q is an integer ranging from 1 to 24; - each r is an integer ranging from 1 to 8; - each s is an integer ranging from 0 to 6; - each v is an integer ranging from 1 to 6; ii) the connecting arm is a direct connection; a -SS- bridge; or a group of formula -(A)z- - A is an amino acid residue; - z is equal to 1, 2, 3, 4 or 5; iii) The spacer is a direct link or a formula group: [Chem. 7] G - G is a sulfate, a sugar, a glucuronide, or a galactoside, said sugar being a saccharide group preferably selected from a beta-glucuronic acid, a beta-D-galactose, a beta-D-glucose, an alpha-D-mannose, an N-acetyl-D-glucosaminyl, an N-acetyl-D-galactosaminyl, a D-glucuronyl, an L-iduronyl, a D-glucopyranosyl, a D-galactopyranosyl, a D-mannopyranosyl or an L-fucopyranosyl, preferably G is a sulfate, a beta-glucuronic acid, or a beta-D-galactose; - R12 is -H or -NO2; (iv) M is a molecule of interest; and v) u, which represents the ratio between M and the antibody (or F(ab')2), is in the range of 2 to 5; provided that the mixture contains at least 50% (by mass) of a conjugate of formula (I) in which u = 2, 3 or 5; said process comprising: al) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (lia) or (Ilb): bl) the reaction of the compound obtained in step a1) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined above; it being understood that: - if a compound (lia) is used in step a1) and a compound (lia) is used in step bl), the two compounds (lia) have a different structure; - if a compound (Ilb) is used in step a1) and a compound (Ilb) is used in step b1), the two compounds (Ilb) have a different structure; cl) the reaction of the compound obtained in step bl) with two identical or different compounds, each corresponding to formula (III): [Chem. 8] | Brss '"l of connection —] Spacer i- (HD in which: - R8 is R6-(CRcRd)r-CO-, R6-(CH2CH2O)q-(CH2)s-CO-, R6-(CRcRd)r -NH-, where R6, Rc, Rd, q, r and s are as defined above; - the connecting arm, the spacer and M are as defined above, M being identical or not in each compound (III); or al) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (lia) or (Ilb); b2) the reaction of the compound obtained in step a1) with a compound of formula (IV): [Chem.9] (IV) in which: i) the hook head is a compound of formula (Il'a), (Il'b), (II'c), or (n'd): [Chem. 10] (ira) (H’b) [Chem. 12] (n’c) [Chem. 13] G X y m-t (H’d) dans lesquelles : - T est tel que défini ci-dessus ; - Y est -OR’a, -COR’2, -CONR’3R’4 ou -NR’3COR’4 ; - R’a est -(CH2CH2O)q-(CH2)r-R’5, -(CRcRd)r-R’5, -COR’b,-(CRcRd)r -NHCO-(CH2CH2O)q-(CH2)r-R’5, -(CRcRd)r-CONH-(CH2CH2O)q-(CH2)r -R’5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R’5ou -(CH2CH2O)q-(CH2)r -CONH-(CRcRd)r-R’5 ; - R’b est -(CH2CH2O)q-(CH2)r-R’5, -O(CH2CH2O)q-(CH2)r-R’5, -(CRcRd)r -R’5, -O(CRcRd)r-R’5 -(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R’5, -(CRcRd)r -CONH-(CH2CH2O)q-(CH2)r-R’5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r -R’5ou -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R’5 ; - R'2 est -OH, -(CH2CH2O)q-(CH2)r-R'5, -(CRcRd)r-R'5, -O(CH2CH2O)q -(CH2)r-R'5, -O(CRcRd)r-R'5, -O(CRcRd)r-NHCO-(CH2CH2O)q-(CH2)r-R'5, -O(CRcRd)r-CONH-(CH2CH2O)q-(CH2)r-R'5, -O(CH2CH2O)q-(CH2)r -NHCO-(CRcRd)r-R'5ou -O(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R'5 ; - R'3 est -H, -(Ci-C6)alkyle or -(CH2)V-SO3H, preferably R'3 est -H or -(Ci-C6)alkyle ; - R'4 est -(CH2CH2O)qR'5, -(CRcRd)rR'5, -(CRcRd)r-NHCO-(CH2CH2O)q - -(CRcRd)r-CONH-(CH2CH2O)q-R'5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd )r-R'5, -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R'5, -CH-[(CRcRd)r -CONH-(CRcRd)r-(OCH2CH2)q-R'5]2, -CH-[(CRcRd)r-NHCO-(CRcRd)r -(OCH2CH2)q-R'5]2, -CH-[(CRcRd)r-CONH-(CRcRd)r-R'5]2, or -CH-[(CRc Rd)r-NHCO-(CRcRd)r-R'5]2, preferably R'4 is -(CH2CH2O)qR'5, -(CRcRd)rR'5, -(CRcRd)r-NHCO-(CH2CH2O)q-R'5, -(CRcRd)r-CONH-(CH 2CH2O)q-R'5, -(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R'5, or -(CH2CH2O) q-(CH2)r-CONH-(CRcRd)r-R'5 ; - each R'5 is -(CH2)SR'6 or -(CH2)SR'7; - R'6 is chosen from: [Chem. 14] - R'7 is -COOH or -NR'sR'ç; - R'8 is -H, -(Ci-C6)alkyl; - R'ç is -H, -(Ci-C6)alkyl; - R'10 is -H or -CH3; where Rc, Rd, q, r and s are as defined above; and ii) the connecting arm is a direct connection; a -SS- bridge; or a group of formula -R'n-(A)Z-; - R'n is a direct bond, a group R'6-(CRcRd)r-CO-, R'6-(CH2CH2 O)q-(CH2)s-CO-, R'6-(CRcRd)r-NH-, where R'6, Rc, Rd, q, r and s are as defined above; - A is an amino acid residue; - z is equal to 1, 2, 3, 4 or 5; and iii) the spacer and M are as defined in step cl); c2) the reaction of the compound obtained in step b2) with a compound of formula (III) as defined in step cl), the molecule of interest M of compound (III) being identical to or different from the molecule of interest M of compound (IV); or al) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (lia) or (Ilb); b3) the reaction of the compound obtained in step a1) with a compound of formula (III) as defined above; c3) the reaction of the compound obtained in step b3) with a compound of formula (IV) as defined above, the molecule of interest M of compound (IV) being identical to or different from the molecule of interest M of compound (III); or al) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (lia) or (Ilb); b3) the reaction of the compound obtained in step a1) with a compound of formula (III) as defined above; c4) the reaction of the compound obtained in step b3) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined above; dl) the reaction of the compound obtained in step c4) with a compound of formula (III) as defined above, the molecule of interest M being identical or not in each compound (III).

2. A preparation method according to claim 1, comprising steps a1), b1) and c1).

3. A preparation method according to claim 2, wherein the hook head used in step a1) and step b1) conforms to one of the formulas (Ha), (Ilb), (Ile) or (Ild) in which: - W is -CONR3R4 or -NR3COR4, preferably W is -CONR3R4; - R3 is -H or -(Ci-C6)alkyl; - R4 is -(CH2CH2O)q-(CH2)r-R5, or -(CRcRd)r-R5; - each R5 is -(CH2)SR6; - R6est is chosen from: [Chem. 20]

4. - Rc, Rd are as defined above; - q is an integer from 1 to 12, preferably q is an integer from 1 to 8; - r is an integer ranging from 1 to 6. Preparation method according to claim 3, wherein the hook head is a compound of formula (liai), (IIa2), (IIa3), (IIa4), (IIa5), or (IIa6): [Chem.21] Br Br Br Br (Hai); [Chem. 22] (Üa2); [Chem.23] (Üa3); [Chem. 24] (IIa4); [Chem. 25] (Ha5); [Chem.26]

5. A preparation method according to claim 1, comprising steps a1), b2) and c2).

6. A preparation method according to claim 5, wherein the hook head used in step b2) conforms to one of the formulas (II'a), (II'b), (II'c) or (II'd) in which: - Y is -CONR'3R'4 or -NR'3COR'4, preferably Y is -CONR'3R'4; - R'3 is -H or -(Ci-C6)alkyl; - R'4 is -(CH2CH2O)q-(CH2)r-R'5, or -(CRcRd)r-R'5; - R'5 is -(CH2)sR'6; - R'6est is chosen from: [Chem.27] - Rc, Rd are as defined above; - q is an integer from 1 to 12, preferably q is an integer from 1 to 8; - r is an integer ranging from 1 to 6.

7. A preparation method according to claim 6, wherein the hook head is a compound of formula (II'al), (II'a2), (II'a3), (II'a4), (II'a5), (II'a6), (II'a7), (II'a8) or (II'bl): [Chem.28] (I will go); [Chem.29] (II'a2); [Chem. 30] (II'a3); [Chem.31] (Ü'a4); [Chem.32] (II'a5); [Chem.33] (n'a6); [Chem.34] (H'a7); [Chem. 35] (H'a8); (n'bi).

8. A method for preparing a mixture of antibody-drug conjugates of formula (I) as defined in claim 1, comprising: a2) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (IV) as defined in claim 1, wherein the hook head is solely a compound of formula (II'a) or (II'b); b4) the reaction of the compound obtained in step a2) with a compound of formula (IV) as defined in claim 1, M being identical or not in each compound (IV), it being understood that: - if a compound (II'a) is used in step a2) and a compound (II'a) is used in step b4), the two compounds (II'a) have a different structure; - if a compound (Il'b) is used in step a2) and a compound (Il'b) is used in step b4), the two compounds (Il'b) have a different structure; or a2) the reaction of an antibody (or fragment F(ab')2) with a compound of formula (IV) as defined in claim 1, wherein the hook head is solely a compound of formula (II'a) or (II'b); b5) the reaction of the compound obtained in step a2) with a compound of formula (lia), (Ilb), (Ile) or (Ild) as defined in claim 1;

9.

10. c5) the reaction of the compound obtained in step b5) with a compound of formula (III) as defined in claim 1, the molecule of interest M of compound (III) being identical to or different from the molecule of interest M of compound (IV). A preparation method according to any one of the preceding claims, wherein the linking arm is a direct link or a formula group -Rn-(A)Z- in which Rn and A are as defined in claim 1, and z is equal to 2, 3 or 4. A preparation method according to any one of the preceding claims, wherein the spacer is a direct bond or a formula group: [Chem.37] ; preferably a formula group: [Chem. 3 8]

11.

12. A preparation method according to any one of the preceding claims, wherein M is an active ingredient selected from: methotrexate, an immunomodulator, duocarmycin, combretastatin, calicheamicin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansine, DM1, DM4, SN38, amanitin and its analogues, pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, pyrrolopyridodiazepine, a pyrrolopyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, ricin; preferably M is amanitin, a pyrrolobenzodiazepine dimer, MMAF or MMAE. a mixture of antibody-drug conjugates of formula (I) obtainable by the process according to any one of the preceding claims, said mixture comprising predominantly a conjugate in which u = 2, 3 or 5, it being understood that when the mixture mainly contains a conjugate comprising two molecules of interest M fixed on the antibody or on the fragment F(ab')2 (u = 2), these 2 molecules of interest M are different from each other.

13. Composition comprising a mixture of antibody-drug conjugates according to claim 12.

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