Antibody-drug conjugates for therapeutic use

Abstract

The invention relates to a conjugate of formula (I) for use as a medicament: (I) in which Ac, the attachment head, the linker arm, the spacer, M and u are as defined in the description.

Description

Title of the invention: Antibody-drug conjugates for therapeutic use Technical field

[0001] The present invention relates to antibody-drug conjugates (ADCs) for use as medicaments, in particular for use in the treatment of cancer. 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 a complement to "classical" therapies for the targeted delivery of an active ingredient, in particular a cytotoxic drug. The antibody-drug conjugate therefore makes it possible to combine the specificity of targeting by antibodies with new powerful effector functions by the agents conjugated to them.

[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 of which 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 mainly used as a vector and delivers the drug into the targeted cell.

[0005] Antibody-drug conjugates are described in the application WO 2015 / 004400. Furthermore, application PCT / FR2021 / 051345, filed on July 19, 2021, describes attachment heads which make it possible to obtain compounds which, when conjugated to proteins, in particular antibodies, make it possible to obtain a “structure” such that on average the number of attachment heads conjugated per protein (antibody) is controlled: the majority conjugate targeted carrying either 1 molecule per protein (antibody) or 2 molecules per protein (antibody). Summary of the invention

[0006] The present invention relates to a conjugate of formula (I) for use as a medicament:

[0007] [Chem.l] (I)

[0008] in which:

[0009] - Ac is an antibody or an antibody fragment;

[0010] - the attachment head, the connecting arm, the spacer, M and u are as defined above- After.

[0011] The present invention also relates to a conjugate of formula (I) for use in a method of treating cancer. Definitions

[0012] The term "antibody", also called "immunoglobulin", designates a hetero-tetramer 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 bridges. Each chain is made up, in the N-terminal position, of a variable region or domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, made up of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.

[0013] The term "antibody fragment" means any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction comprising at least two disulfide bridges, for example Fab', F(ab')2, or scFv-Fc.

[0014] The terms "Fab'", "F(ab')2" and "scFv-Fc" designate antibody fragments that retain 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 formed from two Fab' fragments linked by interchain disulfide bridges. The Fab' fragment consists of the Fab region (consisting of the variable regions and the CH1 and CL domains) and a hinge region. The scFv-Fc fragment is a fragment resulting from protein engineering and consists of the scFv fragment (single chain variable fragment) and linked to an Fc fragment. The scFv fragment consists of only the variable domains VH and VL with a structure stabilized by a short flexible peptide arm, called a linker, which is placed between the two domains.

[0015] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or EU European, or in another generally recognized pharmacopoeia, and applies to a product intended for use in animals and / or humans. A "pharmaceutical composition" means a composition comprising a pharmaceutically acceptable carrier. For example, a pharmaceutically acceptable carrier may be a diluent, an adjuvant, an excipient, or a vehicle with which the therapeutic agent is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid carriers, particularly for injectable solutions.Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol and the like. Where the pharmaceutical composition is suitable for oral administration, the tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable vehicles such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).The pharmaceutical compositions may also contain buffer salts, flavorings, colorings and sweeteners, as appropriate. The composition according to the invention is preferably a pharmaceutical composition.

[0016] The term “treat” or “treatment” encompasses any beneficial or desirable effect on a pathology or condition, and may even include a minimal reduction in one or more measurable markers of the pathology or condition. Treatment may, for example, involve either reducing or improving the symptoms of the pathology or condition, or delaying the progression of the disease or condition. The term "treatment" does not necessarily mean complete eradication or cure of the pathology or associated symptoms. Brief description of the figures

[0017] [Fig.lA] [Fig.lA] represents the evaluation of the performances of representative conjugates of the invention, compared to controls, on a HER2 positive breast cancer cell line (BT-474).

[0018] [Fig.lB] [Fig.lB] represents the evaluation of the performances of representative conjugates of the invention, compared to controls, on a HER2 negative breast cancer cell line (MCF-7).

[0019] [Fig.2A] [Fig.2A] represents the evaluation of the performances of representative conjugates of the invention, compared to controls, on a HER2 positive breast cancer cell line (BT-474).

[0020] [Fig.2B] [Fig.2B] represents the evaluation of the performances of representative conjugates of the invention, compared to controls, on a HER2 negative breast cancer cell line (MCF-7).

[0021] [Fig.3A] [Fig.3A] represents the evaluation of the performances of a representative conjugate of the invention, compared to controls, on a HER2 positive breast cancer cell line (BT-474).

[0022] [Fig.3B] [Fig.3B] represents the evaluation of the performances of a representative conjugate of the invention, compared to controls, on a HER2 negative breast cancer cell line (MCF-7). Detailed description of the invention

[0023] According to a first aspect, the invention relates to a conjugate of formula (I) for use as a medicament:

[0024] [Chem.l] (I)

[0025] in which:

[0026] a) Ac is an antibody or an antibody fragment;

[0027] b) u is such that 0.5 < u < 3.5;

[0028] c) the attachment head is a compound of formula (II) or (II'):

[0029] [Chem.2] O (he);

[0030] [Chem.3]

[0031] in which:

[0032] - West -ORa, -COR2, -CONR3R4 or -NR3COR4;

[0033] - 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 or -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5 ;

[0034] - 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-R5 or -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5 ;

[0035] - R2 est -OH, -(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-(CH2CH 2O)q-(CH2)r-R5, -O(CH2CH2O)q-(CH2)r-NHCO-(CRcRd)r-R5ou -O(CH2CH2O)q-(CH2)r -CONH-(CRcRd)r-R5 ;

[0036] - R3 is -H, -(Ci-C6)alkyl or -(CH2)V-SO3H, preferably R3 is -H or -(Ci-C6 )alkyl;

[0037] - 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, -(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;

[0038] - each R5 is -(CH2)SR6 or -(CH2)SR7;

[0039] - R6 is -COOH or -NR8R9;

[0040] - each R7 is chosen from:

[0041] [Chem.4]

[0043] - Rc is H;

[0044] - each Rd is chosen from -H, -CH2-SO3H or -SO3H;

[0045] - R8 is -H or -(Ci-C6)alkyl;

[0046] - R9 is -H or -(Ci-C6)alkyl;

[0047] - R10 is -H or -CH3;

[0048] - each q is an integer ranging from 1 to 24;

[0049] - each r is an integer ranging from 1 to 8;

[0050] - each s is an integer ranging from 0 to 6;

[0051] - each v is an integer ranging from 1 to 6;

[0052] d) the linker arm is a direct bond; a bridge -SS-; or a group of formula -Rn-(A)Z- in which:

[0053] - Ru is a direct bond, a R7-(CRcRd)r-CO- group or a R7-(CH2CH2O) group q-(CH2)s-CO-, or a group R7-(CRcRd)r-NH- where R7, Rc, Rd, q, r and s are as defined above;

[0054] - A is an amino acid residue;

[0055] - z is equal to 1, 2, 3, 4 or 5.

[0056] e) the spacer is a direct bond or is chosen from:

[0057] [Chem.5]

[0058] - G is a sulfate, a sugar, a glucuronide, or a galactoside, said sugar being a saccharide group preferably selected from beta-glucuronic acid, beta-D-galactose, beta-D-glucose, alpha-D-mannose, N-acetyl-D-glucosaminyl, N-acetyl-D-galactosaminyl, D-glucuronyl, L-iduronyl, D-glucopyranosyl, D-galactopyranosyl, D-mannopyranosyl or L-fucopyranosyl, preferably G is sulfate, beta-glucuronic acid, or beta-D-galactose;

[0059] - G can also represent a cleavage site by a sulfatase, a glucosidase, galactosidase, iduronidase, beta-glucuronidase, mannosidase, N-acetyl-D-glucosaminidase or by N-acetyl-D-galactosaminidase;

[0060] - Rn is -H or -NO2.

[0061] f) M is an active ingredient or a radionuclide chelator, preferably M is an active ingredient.

[0062] In some embodiments, the attachment head is a compound of formula (II).

[0063] In some embodiments, W is -CONR3R4 or -NR3COR4, preferably W is -CONR3R4 ;

[0064] - R3 is -H or -(Ci-C6)alkyl;

[0065] - IG is -(CH2CH2O)q-(CH2)r-R5, or -(CRcRd)r-R5;

[0066] - R5 is -(CH2)sR6 or -(CH2)sR7;

[0067] - R6 is -COOH;

[0068] - R7 is chosen from:

[0069] [Chem.6]

[0070] - Rc, Rd, R8 and R9 are as defined above;

[0071] - q is an integer ranging from 1 to 12, preferably q is an integer ranging from 1 to 8;

[0072] - r is an integer ranging from 1 to 6.

[0073] In some embodiments, the attachment head is a compound of formula (lia), (Ilb), (Ile), (ïïd), (Ile), (Ilf), (Ilg), (Ilh) or (II'a):

[0074] [Chem.7] (lia);

[0075] [Chem. 8] (Ilb);

[0076] [Chem.9] (Ild);

[0078] [Chem. 11] (iif);

[0080] [Chem. 13] (iig);

[0081] [Chem. 14] (He has).

[0083] In some embodiments, the link arm is a direct link.

[0084] In some embodiments, the linker arm is a group of formula -Rn- (A)z- as defined above, with z = 2, 3 or 4.

[0085] The formula -(A)z- represents a succession of z identical or different, natural or unnatural, amino acid residues. Said amino acids may be chosen from the group consisting of: a valine, a citrulline, a phenylalanine, a lysine, an aspartic acid, an alanine, an arginine, a glycine or a glutamic acid.

[0086] Advantageously, z is equal to 2, 3 or 4 and -(A)z- is a group of amino acids chosen from: a valine and a citrulline; a phenylalanine and a lysine; a valine and an alanine; two alanines; an aspartic acid, a valine and a citrulline; a glutamic acid, a valine and a citrulline; three glycines; two glycines, a phenylalanine and a glycine. Preferably, z is equal to 2 and -(A)z- is a group of amino acids chosen from: a valine and a citrulline or a valine and an alanine.

[0087] Also advantageously, -(A)z- may represent a cleavage site by an enzyme chosen from enzymes of the cathepsin B, cathepsin C, cathepsin D type, enzymes chosen from plasmin, a lysosomal enzyme, urokinase (also called urokinase-type plasminogen activator (uPA)), elastase, proteinase 3, cathepsin G.

[0088] -(A)z- may also represent a cleavage site by a metallo-type enzyme matrix proteinase (MMP). In this case, the matrix metalloproteinase (MMP) enzyme is preferably chosen from collagenases 1, 2 and 3 (MMP-1,

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] MMP-8, MMP-13), gelatinases A and B (MMP-2 and MMP-9), stromelysins 1 and 2 (MMP-3 and MMP-10), matrilysins 1, 2 and 3 (MMP-7, MMP-26, MMP-11), macrophage elastase (MMP-12), membrane MMPs (MMP-14, MMP-15, MMP-16 MMP-17, MMP-24 and MMP-25), enamelysin (MMP-20), CA-MMP (MMP-23), epilysin (MMP-28), MMP-19, MMP-21 and MMP-27. -(A)z- can also represent a cleavage site by an esterase, a carboxy-lesterase, proteases and cathepsins. In the context of the present invention, the term "cleavage site" means the position where the cleavage of the peptide chain takes place by an enzyme, for example the V aline-Citrulline site. In some embodiments, the spacer is a direct bond or a group of the formula: [Chem. 16] O .... OGQ h H is p VJ W y X y OO OH In a particular embodiment, the spacer is a direct bond or a group of formula: [Chem. 17] 9 / f'A°A 'X / - H In certain embodiments, M is an active ingredient. As an active ingredient that may be used in the context of the invention, mention may be made of the active ingredients of drugs already authorized and molecules currently undergoing therapeutic evaluation, in particular: - alkylating agents such as: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, no-vembichin, phenesterin, prednimustine, thiotepa, trofosfamide, uracil mustard, CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin), duocarmycin (including synthetic analogues KW-2189 and CBI-TMI), benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD) or to-maymycin dimers, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), nitroureas (carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine), alkylsulfonates (busulfan, treosulfan, improsulfan, and pi-posulfan), triazenes (dacarbazine), platinum compounds (carboplatin, cisplatin, oxaliplatin), aziridines (benzodopa, carboquone, meturedopa, and uredopa), ethyleneimines and melamines (including altretamine, triethylenemelamine, triethylenephos-phoramide, triethylenethio-phosphaoramide and trimethylolomelamine);

[0097] - plant alkaloids such as: Vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, navelbine), taxoids (paclitaxel, docetaxol) and their analogues, maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their analogues, cryptophycins (in particular cryptophycin 1 and cryptophycin 8), epothilones, eleutherobine, discodermolide, bryostatins, dolastatins, auristatins, tubulysins, cephalostatins, pancratistatins, sarcodictyin, spongistatins

[0098] - DNA topoisomerase inhibitors such as: epipodophyllin (9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), te-niposide, topotecan, 9-nitrocamptothecin (RFS 2000), mitomycins (mitomycin C), bortezomib;

[0099] - anti-metabolites such as: anti-folates (DHFR inhibitors (methotrexate, tri- metrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) and other folic acid analogues), IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR), ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine), pyrimidine analogues such as: uracil analogues (ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, di-deoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitexed), cytosine analogues (cytarabine, cytosine arabinoside, fludarabine), purine analogues (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine), folinic acid;

[0100] - hormonal agents such as: anti-estrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (goserelin, leuprolide acetate), antiandrogens (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), proteins human containing a TNF domain);

[0101] - 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;

[0102] - poly(ADP-ribose)polymerase (PARP) inhibitors such as: BGB-290, CEP 9722, E7016, 3-aminobenzamide, niraparib, olaparib, talazoparib, veliparib;

[0103] - immunomodulators such as: thalidomide, lenalidomide, pomalidomide;

[0104] - a toxin such as pseudomonas exotoxin (PE), deBouganin, Bouganin, there

[0105] In a particular embodiment, the active ingredient is selected from methotrexate, an immunomodulator, duocarmycin, combretastatin, cali-cheamicin, 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:

[0106] [Chem. 18] RMAE

[0107] In some embodiments, M is a radionuclide chelator. As radionuclide chelator that may be used in the context of the invention, mention may be made of sarcophagine, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid), NODA (1,4,7-triazacyclononane-1,4-diacetic acid), NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and MANOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) 2,2',2"-[2-(aminomethyl)-1,4,7-triazacyclononane-1,4,7-triyl]triacetic).

[0108] In some embodiments, the antibody or Fab', F(ab')2, or scFv-Fc 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, GPNMB, PSMA, PSA, PAP, PSM, Cripto, 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,Carcinoembryonal antigen (CEA), TAG-72, folate-binding protein, G250, gangliosides, collagen type 4 (collagen IV), collagen type 18 (collagen XVIII), CA19-9, p185HER2, fibroblast activation 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, CD11b-CD18 integrin heterodimer, DNA / Histone H1, proteoglycan, fibrinogen, SV40 large T antigen, SC-Ag, ES A, mucin, CCR4, MTX1, MTX2, PECAM, Tn, cathepsin D, TYRO-3, MER, or a PF4 / heparin complex.

[0109] In certain embodiments, the antibody or a Fab', F(ab')2 or scFv-Fc 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.

[0110] The antibody, the Fab' fragment or the F(ab')2 fragment, or the scFv-Fc fragment may be of mammalian origin (for example human or murine), chimeric, humanized. It is preferably a monoclonal antibody produced recombinantly by genetically modified cells according to techniques widely described in the prior art.

[0111] In some embodiments, the antibody is of the IgG type, for example IgG1, IgG2, IgG3 or IgG4.

[0112] The conjugates of formula (I) can be obtained as described in application PCT / FR2021 / 051345, for example by conjugation between an antibody, or a Fab', F(ab')2, or scFv-Fc fragment of said antibody, and a compound of formula (F):

[0113] [Chem. 19] ï Heads i___j Arms ]___। | dtaccroche [ | de liaison ] ] (D

[0114] in which the connecting arm, the spacer and M are as defined above, and the attachment head corresponds to formula (III) or (III'):

[0115] [Chem.20] (III)

[0116] [Chem.21] (III')

[0117] in which W is as defined above.

[0118] The conjugation conditions are for example those described in application PCT / FR2021 / 05134 or those specified below.

[0119] The antibody, or Fab', F(ab')2, or scFv-Fc fragment, binds to the attachment head by a substitution reaction of the Br groups present in formula (III) or (IIF).

[0120] The compounds of formula (I') can be obtained by coupling the different elements which compose the structure (attachment head / connecting arm / spacer / M). For example, and in a non-limiting manner, the compounds of formula (I') can be obtained by coupling between a compound of formula (III) or (IIF) and a compound of formula (V):

[0121] [Chem.22] Connecting arm Spacer (V).

[0122] The coupling between the attachment head and the linker arm can be done either conventionally, for example by peptide-type coupling between a carboxylic group and an amino group, or by implementing a so-called "click" reaction. A peptide-type coupling conventionally takes place between a carboxylic group carried by the attachment head, and an amine group carried by the linker arm or, where appropriate, with an amine group carried by the spacer (if the linker arm is a direct bond). Those skilled in the art will understand that the reactive group of W which will form the peptide-type bond with the linker arm (or the spacer) is, in the context of the present invention, defined as it is before the coupling reaction with the reactive group of the linker arm (or the spacer). A click reaction coupling can take place when the attachment head and the linker arm each carry a substituent R7.More specifically, the click reaction is carried out between a diene (for example an azide or a diazo) and a dienophile (for example an alkene or an alkyne), each of these functions being provided by the R7 group. Thus, the click reaction can be carried out between a diene provided by the R7 group of the attachment head and a dienophile provided by the R7 group of the linker arm, or between a dienophile provided by the R7 group of the attachment head and a diene provided by the R7 group of the linker arm. These click reactions are well known to those skilled in the art, it being understood that the two R7 groups are judiciously chosen to be compatible with each other. The coupling between the linker arm and the spacer, and between the spacer and M, is carried out in a conventional manner, for example by peptide-type coupling.

[0123] The conjugates of formula (I) can also be obtained as described in application PCT / FR2021 / 051345, by conjugation between an antibody, or a fragment Fab', F(ab')2, or scFv-Fc of said antibody, with a compound of formula (III) or (III') to form an intermediate compound of formula (IV) followed by click reaction coupling between a compound of formula (IV) and a compound of formula (V):

[0124] [Chem.23] (IV)

[0125] [Chem.22] Arm of Reason Spacer (V).

[0126] wherein the click reaction, the hook head, the connecting arm, the spacer and M are as defined above.

[0127] The conjugates of formula (I) have a ratio of molecules "attached" or "conjugated" per antibody (or per Fab', F(ab')2, or scFv-Fc fragment), designated by the letter u, in the range from about 0.50 to about 3.50. Herein, the term "molecule" means the structure of formula (F).

[0128] In some embodiments, the antibody (or Fab', F(ab')2, or scFv-Fc fragment) is conjugated on average to 1.00+0.50 (i.e., any value from 0.50 to 1.50, for example, 0.50; 0.51;.....; 1.49; 1.50) molecule(s), preferably to 1.00+0.30 molecule(s).

[0129] In some embodiments, the antibody (or F(ab')2 fragment) is conjugated to an average of 2.00+0.50 (i.e., any value from 1.50 to 2.50, for example, 1.50; 1.51;.....; 2.49; 2.50) molecules, preferably 2.00+0.30 molecules.

[0130] In some embodiments, the antibody (or F(ab')2 fragment) is conjugated to an average of 3.00+0.50 (i.e., any value from 2.50 to 3.50, e.g., 2.50; 2.51;.....; 3.49; 3.50) molecules, preferably 3.00+0.30 molecules.

[0131] The ratio "u", also called hereinafter "DAR" (for "drug to antibody ratio"), is determined for each species (LHHL, LH, L, H, HH, LHH) by HRMS (High Resolution Mass Spectrometry) analysis under denaturing conditions. Here, "drug" means the active ingredient or chelator of radionuclide M. The average DAR is obtained from the DAR per species weighted by the proportions of the species observed in analysis on SDS-PAGE gel under non-reducing denaturing conditions.

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Only the majority species LHHL and LH were considered for this calculation, the sum of the proportions of the other species (L, H, HH and LHH) being less than 18%. The sum of the proportions of the LHHL and LH species was therefore brought to 100% without taking into account the other species. The LH "half-antibody" species is observed under denaturing conditions. In solution (in native conditions) this species is not present in isolation; weak interactions hold the two LHs together. This is why the DAR of the unreconstructed LH-LH species corresponds to 2 times the DAR observed on the LH species. The average DAR was therefore calculated using the following formula: Average DAR % LHHL * LHHL * % LH * DAR LH* 2 1QS In some embodiments, the conjugate of formula (I) is present in a composition, which may be, for example, a pharmaceutical composition containing one or more pharmaceutically acceptable excipients and / or carriers. According to a second aspect, the invention relates to a conjugate of formula (I) as defined above for use in a method of treating cancer. In some embodiments, the antibody or a Fab', F(ab')2, or scFv-Fc fragment of said antibody binds to HER2 and the cancer is a HER2+ cancer. The term "HER2" refers to "Human Epidermal Growth Factor Receptor 2," which is a membrane protein in the human epidermal growth factor receptor family. "HER2" is also commonly referred to as "ErbB2." The term “HER2+ cancer” or “HER2-positive cancer” refers to a cancer involving exacerbated expression of HER2. In particular, the term “HER2+ cancer” refers to any case of cancer in which cancer cells exhibit deregulation of the HER2 gene.Preferably, in the context of the present invention, the HER2+ cancer is selected from breast cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, pancreatic cancer, urothelial cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, vulvar cancer, biliary tract cancer, endothelial cancer, colorectal cancer, colon cancer, anal cancer, salivary gland cancer, brain cancer, esophageal cancer, bowel cancer, liver cancer, lacrimal gland cancer, laryngeal cancer, peritoneal cancer, prostate cancer, testicular cancer, renal cancer, skin cancer, adenoid carcinoma cystic, angiosarcoma, gastroesophageal cancer, leukemias, thyroid cancer, lymphomas.In a preferred embodiment, the HER2+ cancer is selected from breast cancer, gastric cancer, gastroesophageal cancer, and . bladder, gallbladder cancer, extrahepatic cholangiocarcinoma, preferably breast cancer.

[0140] In a particular embodiment, the antibody is trastuzumab; in this embodiment, the structure of formula (F) may also be conjugated to a Fab', F(ab')2, or scFv-Fc of trastuzumab.

[0141] In some embodiments, the antibody or a Fab', F(ab')2, or scFv-Fc fragment of said antibody binds to CD30 and the cancer is a CD30+ cancer.

[0142] The term "CD30" refers to "Cluster of Differentiation 30", which is a membrane glycoprotein of the human tumor necrosis factor receptor superfamily (TNFRSF). "CD30" is also frequently referred to as "TNFRSF8".

[0143] The term “CD30+ cancer” or “CD30 positive cancer” refers to a cancer involving exacerbated expression of CD30. In particular, the term “CD30+ cancer” refers to any case of cancer in which cancer cells exhibit deregulation of the TNFRSF8 gene. Preferably, in the context of the present invention, the CD30+ cancer is chosen from Hodgkin's lymphoma, T-cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, hepatosplenic T-cell lymphoma, anaplastic large cell lymphoma, non-Hodgkin's lymphoma, systemic anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, enteropathy-associated T-cell lymphoma, mycosis fungoides, diffuse large B-cell lymphoma, adult T-cell leukemia, Sézary syndrome, primary mediastinal B-cell lymphoma, nodular sclerosis Hodgkin's lymphoma, angiocentric lymphoma, myeloid splenomegaly.In a preferred embodiment, the CD30+ cancer is selected from Hodgkin's lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma and mycosis fungoides.

[0144] In a particular embodiment, the antibody is brentuximab; in this embodiment, the structure of formula (!') may also be conjugated to a Fab', F(ab')2, or scFv-Fc fragment of brentuximab.

[0145] In some embodiments, the antibody or a Fab', F(ab')2, or scFv-Fc fragment of said antibody binds to CD56 and the cancer is a CD56+ cancer.

[0146] The term "CD56" refers to "Cluster of Differentiation 56", which is a membrane glycoprotein member of the immunoglobulin (Ig) superfamily containing 5 Ig-like domains and two fibronectin-like 3 domains in its extracellular portion. CD56 is also frequently referred to as "Neural-Cell Adhesion Molecule 1 (NCAM 1)".

[0147] The term “CD56+ cancer” or “CD56 positive cancer” refers to a cancer expressing CD56. In particular, the term “CD56+ cancer” refers to any case of cancer for which cancer cells exhibit CD56 expression. CD56 expression is frequently observed in neuroendocrine carcinomas, pediatric tumors, and certain blood diseases. It is also reported in certain melanomas and soft tissue sarcomas. Preferably, in the context of the present invention, the CD56+ cancer is selected from leukemias, lymphomas, nerve sheath cancer, Merkel cell carcinoma, myelodysplastic syndromes, myelomas, neuroblastoma, ovarian cancer, lung cancer, neuroendocrine cancers, rhabdomyosarcoma, Wilms tumor, glioblastoma, synovial sarcoma, pleuropulmonary blastoma, myeloid splenomegaly. In a preferred embodiment, the CD56+ cancer is selected from neuroendocrine cancers, lung cancer or Merkel cell carcinoma, preferably Merkel cell carcinoma.

[0148] In a particular embodiment, the antibody or Fab', or F(ab')2, or anti-CD56 scFv-Fc comprises:

[0149] - a variable domain of a light chain comprising a CDR1 of sequence of amino acids SEQ ID NO: 1, a CDR2 of amino acid sequence SEQ ID NO: 2, and a CDR3 of amino acid sequence SEQ ID NO: 3; and

[0150] - a variable domain of a heavy chain comprising a CDR1 of sequence of amino acids SEQ ID NO: 4, a CDR2 of amino acid sequence SEQ ID NO: 5, and a CDR3 of amino acid sequence SEQ ID NO: 6.

[0151] In this particular embodiment, the light chain variable domain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID NO: 9; and the heavy chain variable domain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID NO: 10.

[0152] Thus, the antibody or Fab', or F(ab')2, or anti-CD56 scFv-Fc may have an amino acid sequence SEQ ID NO: 9 for the variable domain of the light chain and an amino acid sequence SEQ ID NO: 10 for the variable domain of the heavy chain.

[0153] In a particular embodiment, the antibody or Fab', or F(ab')2, or anti-CD56 scFv-Fc may have an amino acid sequence SEQ ID NO: 7 for the light chain and an amino acid sequence SEQ ID NO: 8 for the heavy chain. The heavy chain of amino acid sequence SEQ ID NO: 8 may also have an additional lysine in the C terminal position.

[0154] In a particularly preferred embodiment, the antibody may be the antibody described under the reference m906 in application US 2018 / 0214568 A1. The antibody m906 is a chimeric anti-CD56 antibody of the IgG1 type with amino acid sequence SEQ ID NO: 7 for the light chain and amino acid sequence SEQ ID NO: 8 for the heavy chain.

[0155] The invention is illustrated by the following examples, given purely for illustrative purposes. In these examples, the following abbreviations are used:

[0156] DCC = dicyclohexylcarbodiimide

[0157] DIPEA = A,A-diisopropylethylamine

[0158] DMAP = 4-dimethylaminopyridine

[0159] DMF = A,A-dimethylformamide

[0160] DMSO = dimethyl sulfoxide

[0161] EDTA = ethylenediaminetetraacetic acid

[0162] HCl = hydrochloric acid

[0163] HOBt = hydroxybenzotriazole

[0164] MeCN = acetonitrile

[0165] MeOH = methanol

[0166] NaCl = sodium chloride

[0167] TA = room temperature (20°C unless otherwise stated)

[0168] TFA = trifluoroacetic acid

[0169] tR = Retention time

[0170] v / v = volume to volume ratio Analysis methods

[0171] Nuclear magnetic resonance (NMR) spectroscopy

[0172] Proton 'H nuclear magnetic resonance (NMR) spectra were performed on a Bruker Ultrashield 300 instrument (300 MHz (*H)). Analyses were performed in deuterated methanol (CD3OD). Chemical shifts (δ) are measured in parts per million (ppm) relative to the residual signal of deuterated methanol (δ'H = 3.31 ppm).

[0173] 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. In order to clarify the reading of the NMR analyses, the numbering of the atoms for the assignment of the signals has been fixed arbitrarily. High-resolution mass spectrometry (HRMS)

[0174] The exact mass of the synthesized compounds was determined by high-resolution mass spectrometry (HRMS) in positive or negative mode with the electrospray ionization technique ESI, on a Bruker maXis mass spectrometer. coupled with a Dionex Ultimate 3000 RSLC system from the ICOA / CBM “Research Federation” platform (FR2708).

[0175] Denaturing high-resolution mass spectrometry (HRMS)

[0176] Method 1: The analysis of the conjugates was carried out on a sample previously deglycosylated or not. In the case of a deglycosylated sample, it was diluted to a concentration of 1 pg / pL then A-glycosidase F (0.02 unitc / pg of sample) was added and the sample was incubated at 37 °C for at least 16 h.

[0177] The analysis was performed on a Vion IMS Qtof mass spectrometer coupled to an Acquity UPLC H-Class system from Waters (Wilmslow, UK). Before analysis, samples (800 ng) were injected onto an XBridge BEH300 C4 2.1 x 50 mm, 1.7 pm column or onto an XBridge BEH300 C4 2.1 x 30 mm, 5 pm column heated to 90 °C. A desalting step was performed with an isocratic gradient of 95% solvent A (H2O + 0.1% formic acid) and 5% solvent B (MeCN + 0.1% formic acid) for 1.5-2 min at 0.5 mL / min. Then, the sample elution was carried out with a gradient of 20% to 35% of solvent B over 7 min, of 50% to 90% of solvent B over 3 min, and an isocratic of 1 min to 90% of B, or with a gradient of 5% to 50% of solvent B over 2.9 min, of 50% to 90% of solvent B over 0.5 min, an isocratic of 0.5 min to 90% of B, with a flow rate of 0.4 mL / min. A bypass valve was programmed to allow solvent to enter the spectrometer between 3 and 7.5 min only. Mass spectrometry data were acquired in positive mode with an ESI source over a m / z range of 500 to 4000 at a scan rate of 1 Hz and analyzed using UNIFI 1.9.4 software and the MaxEnt algorithm for deconvolution. The average DAR per species (= average number of molecules conjugated to the antibody used for the bioconjugation reaction) was determined using the intensity of the observed species peaks.

[0178] Method 2: Spectrometric analysis of selected conjugates was performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, samples (5 pg) were desalted on a MassPREP desalting column (2.1x10 mm, Waters), heated to 80 °C using 0.1% aqueous formic acid solution as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 pL / min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied. MS data were acquired in positive mode with an ESI source over a 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 average DAR per species (= average number of molecules conjugated to the antibody used for the bioconjugation reaction) was determined using the intensity of the observed species peaks.

[0179] SDS-PAGE gel under denaturing, non-reducing or reducing conditions

[0180] Samples were analyzed by SDS-PAGE tris-HCl acrylamide gel. A 4% acrylamide stacking gel on a 6-7% acrylamide running gel was used. 4X Laemmli buffer (0.3 mM bromophenol blue; 2 M glycerol, 20 mM TrisBase; 0.04% sodium dodecyl sulfate) was added into the samples (1.6 μg). Under reducing conditions, samples were reduced using a 10% dithiothreitol (DTT) solution in water (10% v / v). Then, the samples were incubated at 95 °C for 10 min. A high-amplitude molecular weight marker (Invitrogen SeeBlue® Plus2 Prestained Standard) and the native antibody were used to estimate protein molecular weights. The gel was run at 100 V for 10 min then at 140 V for 35 min, in NuPAGE running buffer (50 mM MOPS; 50 mM TrisBase; 0.1% SDS (v / v); 1 mM EDTA, pH 7.3). After a water wash, the gel was stained with Coomassie Blue (Thermo Scientific Imperial TM Protein Stain). Densitometric analysis was performed using ImageJ software and a Windows Vanilla filter was applied for black and white analysis. Under non-reducing denaturing conditions, the relative optical density of the LHHL and LH species was used to determine the average DAR of the conjugate. Under reducing denaturing conditions, the relative optical density measured for the LHHL species determined the antibody reconstruction (in %). Bioconjugation reactions Preparation of solutions

[0181] Bioconjugation Buffer 1: Phosphate buffer IX at pH 8.3, with a final NaCl concentration of 180 mM and a final EDTA concentration of 1 mM.

[0182] Bioconjugation Buffer 2: Borate buffer IX at pH 8.3, with a final NaCl concentration of 25 mM and a final EDTA concentration of 1 mM.

[0183] Reducer 1: Tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HC1) solution at a concentration of 1 mM in the bioconjugation buffer.

[0184] Reducer 2: Dithiothreitol (DTT) solution at a concentration of 1 mM in the bioconjugation buffer. Bioconjugation reaction 1:

[0185] The antibody solution in the bioconjugation buffer (1.0 eq) was placed under argon. The reducing agent (1.0-12.0 eq) was then added and the reaction medium was incubated at 37°C for 2h. Then the solution of compound to be conjugated (1.0-15.0 eq, preferably 5.0-12.0 eq or 10.0-15.0 eq) was added under argon and the reaction medium was stirred at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 2.5 hours. Bioconjugation reaction 2:

[0186] The antibody solution in the bioconjugation buffer (1.0 eq) was placed under argon. The solutions of compound to be conjugated (1.0-15.0 eq, preferably 8.0-12.0 eq) then of reducer (1.0-12.0 eq) were added and the reaction medium was stirred under argon at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 2h30. Bioconjugation reaction 3:

[0187] The antibody solution in the bioconjugation buffer (1.0 eq) was placed under argon. The solutions of the compound of formula (I) (1.0-15.0 eq, preferably 8.0-12.0 eq or 10.6-12.0 eq) then of reducer (1.0-12.0 eq) were added and the reaction medium was stirred under argon at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 2h30. The solution of the compound of formula (V) (1.0-30.0 eq preferably 8.8-14.4 eq, for example 11.7 eq) was then added and the reaction medium was stirred at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 17h. Bioconjugation reaction 4:

[0188] The antibody solution in the bioconjugation buffer (1.0 eq) was placed under argon. The reducing agent (1.0-12.0 eq) was then added and the reaction medium was incubated at 37 C for 2 h. Then the solution of compound to be conjugated (1.0-15.0 eq) was added under argon and the reaction medium was stirred at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 2 h 30 min. The solution of the compound of formula (V) (1.0-30.0 eq) was then added and the reaction medium was stirred at a temperature between 4°C and 40°C, preferably 25°C or 37°C for 17 h. Examples

[0189] Example 1: 4-nitrophenyl l-[4-(6-methyl-l,2,4,5-tetrazin-3-yl)phenoxy]-3,6,9,12-tetraoxapentadecan-15-oate (1)

[0190] [Chem.24] (1)

[0191] 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 medium was placed under stirring, under argon at RT for 18h30. The mixture was purified by semi-preparative high-pressure 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 μm (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% B over 32 min then 100% B over 6 min at 17.1 mL / min) to give (1) (7.8 mg; 50%) as a pink oil.

[0192] '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).

[0193] HRMS (ESI): m / z calculated for C26H32N5O9 [M+H]+: 558.2195; observed 558.2203.

[0194] 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]

[0195] butanamido]-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl](methoxy)methyl]p ropanamido]-3-phenylpropanoic acid (2)

[0196] [Chem.25] ; » ---0 'Xk ... . - j «xi'V* * yY; GYH 0 Y 7 « X-.-v- 0 .A - A. A .N, A. .A. $.. / TX <■ V- "o- SX- -y 'yv (2)

[0197] To a solution of HOBt (2.0 mg; 0.0151 mmol; 2.1 eq), solubilized in anhydrous DMF (100 pL) in the presence of anhydrous DIPEA (2.5 pL; 0.0144 mmol; 2.0 eq) was added 4-nitrophenyl l-[4-(6-methyl-l,2,4,5-tetrazin-3-yl)phenoxy]-3,6,9,12-tetraoxapentadecan-15-oate (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 μL), was added to the reaction medium under stirring, under argon at 25 °C for 19 h.The mixture was purified by semi-preparative high-pressure 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 μm (250 mm x 19.00 mm); elution performed with 0.1% TFA (by volume) in water (solvent A), and MeCN (solvent B); gradient 20 to 90% B over 32 min then 90% B over 6 min at 17.1 mL / min) to give (2) (1.4 mg; 17%) as a purple oil.

[0198] '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).

[0199] HRMS (ESI): m / z calculated for C59H92N9O14 [M+H]+: 1150.6758; observed 1150.6754.

[0200] The commercial compounds or those from application PCT / FR2021 / 051345, used for bioconjugation reactions are summarized in Table 1 below.

[0201] [Tables 1] Number o(#) Name and structure (3) N-(2-((((2,6-bis(bromomethyl)pyridin-4-yl)carbonyl)amino)methyl)-19-( 11,1 2-didehydrodibenzo[b,f]azocin-5(6H)-yl)-3,14,19-trioxo-7,10-dioxa-4,13-dia zanonadec-l-yl)-2,6-bis(bromomethyl)pyridine-4-carboxamide [Chem26] A Br. JL À HH. q 0 H 0 A: / Br'^-YfAvSr (4) 6-Azidohexanamido-A-hexanamide-valine-citrulline-p-aminobenzoyl carbamate of MMAE [Chem27] sio Y « ? he YHG i H <0 to n.fr'Y (5) 3-(2,6-bis(bromomethyl)isonicotinamido)-2-((2,6-bis(bromomethyl)isonicoti namido)methyl)propamido-A-hexanamide-valine-citrulline-p-aminobenzoyl carbamate from MMAE [Chem28] o.T Jul x, Q 0 ' NYV □ Ap | « 0 M himsn « i » u® T j Y YjS'TYl Br O^yY'6' AY ' ^'&r (6) 2-(2-(2-(2-(4-4-(methyltetrazinylphenoxy)ethoxy)ethoxy)ethoxy)-ethyl)carbam oylpropane-1,3-diyl(2,6-bis(bromomethyl)isonicotinamide) [Chem29] . Six A 1 H O.Ym 0 UCH*-!! 6 V -8' N (7) 1 -trans-cyclooctenyl-1 -oxo-5,8,11,14-tetraoxa-2-azahetaptadecan-17-amide-valine-citrulline-p-aminobenzoyle carbamate of MMAE [Chem30] / ? 9 g And | fyAV H YUhY Vv «U h « ? m H à ' K $ A HïH 0 (8) 2-(2-(2-(tra«5-cyclooctenylcarbamoyl)ethoxy)ethoxy)ethyl)carbamoylpropan e-1,3-diyl(2,6-bis(bromomethyl)isonicotinamide) (Chem3-N15" a“1 "a9V"] Sü "a / U1" 4-methyleteatrazinylphenoxy-3,6,9,12-tetraoxapentadecane-15-amide-valine-c itrulline-p-aminobenzoyle carbamate from MMAE [Chem32] P AX / A\ notr N'A / / ) N'Xn Y' oy M «! i X "H'z's k.H is *. <K 0 ^■NH A HjH O (10) 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-oxobutanamide [Chem33] 8r^ “i - ? MX) .à . N (11) Commercial compound N3-PEG4-Val-Ala-PBD dimer obtained from Levena Biopharma [Chem34] 0 XJ 0 * à = K (12) MA'-(2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-carbamoyl)propane-l, 3-diyl)bis(2,6-bis(bromomethyl)isonicotinamide) [Chem35] K' [ O^NH 0 -Si Pi (13) (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-(carbamoylamino)pe ntanamido} phenyl 1 ) melhy N- {l-[(l-{[l-(2-{2-[(l -hydroxy-1 -phenylpropan-2-yl)carbamoyl]-l-methoxy-2-methylethyl}pyrrolidin-l-yl)-3-methoxy-5-meth yl-1 -oxoheptan-4-yl] (methyl)carbamoyl} -2-methylpropyl)carbamoyl] -2-meth ylpropyl} -N- methyl Icarbamate [Chem36] 0 $ । vj-yo Y V ?Y« î jYC° AH t Y “‘YY YYtVyY^ 0 ■ 0 b 0 1 0^ Q HH (14) bicyclo[6.1.0]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-diazacosane-20-yl)carbamate [Chem37] “1 N‘ "X Y H ? o H / $ Six Jk.Br N v (15) commercial compound N3-Cap-Val-Cit-PAB-C6-amanitine obtained from Levena Biopharma [Chem38] 0 î o aJ « \ YY H » YX ' lA 1 )0,ÎW l M Y SA / oA H j ©si \ ff 0' )C, HH“X MY A Y.HjN'OHN-yS M ) o (16) 3-(2,6-bis(bromomethyl)isonicotinamido)-2-((2,6-bis(bromomethyl)isonicoti namido)-methyl)propanamido)-3-sulfopropanamido-valine-citrulline-p-amino benzoyl carbamate from MMAE [Chem39] S! gg Y hfhi Y yY Yj i rYV^ n Y <NH” o ” o \ ‘ o । o ■'& oîJ / ~'A''er 'Br (17) A1,A5-bis(2-(2-(2-(2-azidoéthoxy)éthoxy)éthoxy)éthyl)-3-(3-(2,6-bis(bromom . ethyl)-isonicotinamido)-2-((2,6-bis(bromomethyl)isonicotinamido)methyl)pr opanamido)-pentanediamide [Chem40]

[0202] Example 3: compound (18): trastuzumab conjugate - compound (3) - compound (4) Reagents

[0203] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (3) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, compound (4) (2nd compound) (11.7 eq) at a concentration of 10 mM in DMSO. Method

[0204] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 1

[0205] The results are shown in Table 2 below.

[0206] [Tables2] LHHL LH L Intensity (%) MM (Da) > Intensity (%) MM (Da) > Intensity (%) MM (Da) DAR0 no2 14 72585 100 23439 DAR1 83 147259 86 74674 no2 DAR 2 17 149352 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.17 1.00 0.00

[0207] 1: molecular mass of the deglycosylated species

[0208] 2: not observed

[0209] The SMHR analysis determined an average DAR of 1.17 for the species LHHL and an average DAR of 1.00 for the LH species. The species LHH, HH, H and L were not observed. No mass increment corresponding to compound (3) is observed: the trastuzumab-compound (3) conjugate was fully converted.

[0210] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0211] The results are presented in Table 3 below.

[0212] [Tables3] Species DTT LHHL LHH HH LH HL Optical density (%) - 97 no1 no1 3 no1 no1 + 75 no1 no1 8 10 7

[0213] 1: not observed

[0214] SDS-PAGE gel analysis made it possible to determine a reconstruction of 75% under reducing conditions and an average DAR of 1.19 under non-reducing conditions.

[0215] Example 4: compound (19): trastuzumab conjugate - compound (3) - compound (4) Reagents

[0216] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (3) (1st compound) (10.6 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (4) (2nd compound) (11.7 eq) at a concentration of 10 mM in DMSO. Method

[0217] Bioconjugation reaction 3.

[0218] The reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4. Denaturing HRMS analysis according to method 1

[0219] The results are shown in Table 4 below.

[0220] [Tables4] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 74674 DAR 2 100 149347 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0221] 1: molecular mass of the deglycosylated species

[0222] 2: not observed

[0223] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed. No mass increment corresponding to compound (3) is observed: the trastuzumab-compound (3) conjugate was fully converted.

[0224] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0225] The results are shown in Table 5 below.

[0226] [Tables5] Species DTT LHHL LHH HH LH HL Optical density (%) - 90 no1 no1 10 no1 no1 + 97 no1 no1 3 no1 no1

[0227] 1: not observed

[0228] SDS-PAGE gel analysis made it possible to determine a reconstruction of 97% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0229] Example 5: compound (20): trastuzumab conjugate - compound (5) Reagents

[0230] Bioconjugation buffer 2, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 2 (8.0 eq), compound (5) (5.0 eq) at a concentration of 0.4 mM in a mixture of 80% DMF and 20% MeOH. Method

[0231] Bioconjugation reaction 1. Denaturing HRMS analysis according to method 1

[0232] The results are shown in Table 6 below.

[0233] [Tableauxô] LHHL LH Intensity (%) MM (Da) i Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 74184 DAR 2 100 148367 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0234] 1: molecular mass of the deglycosylated species

[0235] 2: not observed

[0236] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0237] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0238] The results are shown in Table 7 below.

[0239] [Tables7] Species DTT LHHL LHH HH LH HL Optical density (%) - 68 no1 no1 32 no1 no1 + 61 no1 5 27 6 2

[0240] 1: not observed

[0241] SDS-PAGE gel analysis made it possible to determine a reconstruction of 61% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0242] Example 6: compound (21): trastuzumab conjugate - compound (6) - compound (7) Reagents

[0243] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (6) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, compound (7) (2nd compound) (11.7 eq) at a concentration of 10 mM in DMSO. Method

[0244] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0245] The results are shown in Table 8 below.

[0246] [Tables8] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 100 147391 100 74804 DAR 2 no2 no2 DAR 3 no2 no2 DAR average 1.00 1.00

[0247] 1: molecular mass of the deglycosylated species

[0248] 2: not observed

[0249] The HRMS analysis determined an average DAR of 1.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed. The mass increment is correct.

[0250] SDS-PAGE gel analysis under denaturing, non-reducing and reducing conditions

[0251] The results are shown in Table 9 below.

[0252] [Tables9] Species DTT LHHL LHH HH LH HL Optical density (%) - 84 no1 no1 6 no1 10 + 58 no1 no1 10 17 15

[0253] 1: not observed

[0254] SDS-PAGE gel analysis made it possible to determine a reconstruction of 58% under reducing conditions and an average DAR of 1.07 under non-reducing conditions.

[0255] Example 7: compound (22): trastuzumab conjugate - compound (6) - compound (7) Reagents

[0256] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (6) (1st compound) (8.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (7) (2nd compound) (8.8 eq) at a concentration of 10 mM in DMSO. Method

[0257] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0258] The results are shown in Table 10 below.

[0259] [TableauxlO] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 74805 DAR 2 100 149610 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0260] 1: molecular mass of the deglycosylated species

[0261] 2: not observed

[0262] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0263] SDS-PAGE gel analysis under non-reducing denaturing conditions

[0264] The results are shown in Table 11 below.

[0265] [Tableauxll] Species DTT LHHL LHH HH LH HL Optical density (%) - 75 no1 no1 25 no1 no1 + 69 no1 no1 26 no1 5

[0266] 1: not observed

[0267] SDS-PAGE gel analysis made it possible to determine a reconstruction of 69% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0268] Example 8: compound (23): trastuzumab conjugate - compound (8) - compound (9) Reagents

[0269] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (8) (1st compound) (3.0 eq) at a concentration of 0.25 mM in a mixture of 80% DMF and 20% MeOH, compound (9) (2nd compound) (3.3 eq) at a concentration of 10 mM in DMSO. Method

[0270] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0271] The results are shown in Table 12 below.

[0272] [Tablesl2] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 80 150241 100 76206 DAR 2 20 152421 no2 DAR 3 no2 no2 Average DAR 1.20 1.00

[0273] 1: molecular mass of the non-deglycosylated species

[0274] 2: not observed

[0275] The HRMS analysis determined an average DAR of 1.20 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0276] SDS-PAGE gel analysis under non-reducing denaturing conditions

[0277] The results are shown in Table 13 below.

[0278] [Tablesl3] Species DTT LHHL LHH HH LH HL Optical density (%) - 81 no1 no1 14 no1 5 + 63 no1 no1 25 no1 12

[0279] 1: not observed

[0280] SDS-PAGE gel analysis made it possible to determine a reconstruction of 63% under reducing conditions and an average DAR of 1.32 under non-reducing conditions.

[0281] Example 9: compound (24): trastuzumab conjugate - compound (8) - compound (9) Reagents

[0282] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (8.0 eq), compound (8) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 30% DMF and 70% MeOH, compound (9) (2nd compound) (13.2 eq) at a concentration of 10 mM in DMSO. Method

[0283] Bioconjugation reaction 4. Denaturing HRMS analysis according to method 2

[0284] The results are shown in Table 14 below.

[0285] [Tablesl4] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 76208 DAR 2 100 152418 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0286] 1: molecular mass of the non-deglycosylated species

[0287] 2: not observed

[0288] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0289] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0290] The results are shown in Table 15 below.

[0291] [Tables 15] Species DTT LHHL LHH HH LH HL Optical density (%) - 66 no1 no1 34 no1 no1 + 62 no1 no1 33 no1 5

[0292] 1: not observed

[0293] SDS-PAGE gel analysis made it possible to determine a reconstruction of 62% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0294] Example 10: compound (25): trastuzumab conjugate - compound (10) - compound (4) Reagents

[0295] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (10) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, compound (4) (2nd compound) (11.7 eq) at a concentration of 10 mM in DMSO. Method

[0296] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0297] The results are shown in Table 16 below.

[0298] [Tables 16] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 100 147233 100 74645 DAR 2 no2 no2 DAR 3 no2 no2 DAR average 1.00 1.00

[0299] 1: molecular mass of the deglycosylated species

[0300] 2: not observed

[0301] The HRMS analysis determined an average DAR of 1.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0302] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0303] The results are shown in Table 17 below.

[0304] [Tables 17] Species DTT LHHL LHH HH LH HL Optical density (%) - 87 no1 no1 no1 7 6 + 77 no1 no1 14 1 8

[0305] 1: not observed

[0306] SDS-PAGE gel analysis made it possible to determine a reconstruction of 77% under reducing conditions and an average DAR of 1.00 under non-reducing conditions.

[0307] Example 11: compound (26): trastuzumab conjugate - compound (10) - compound (4) Reagents

[0308] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (10) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (4) (2nd compound) (13.2 eq) at a concentration of 10 mM in DMSO. Method

[0309] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0310] The results are shown in Table 18 below.

[0311] [Tables 18] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 74649 DAR 2 100 149324 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0312] 1: molecular mass of the deglycosylated species

[0313] 2: not observed

[0314] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0315] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0316] The results are shown in Table 19 below.

[0317] [Tablesl9] Species DTT LHHL LHH HH LH HL Optical density (%) - 86 no1 no1 14 no1 no1 + 89 no1 no1 11 no1 no1

[0318] 1: not observed

[0319] SDS-PAGE gel analysis made it possible to determine a reconstruction of 84% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0320] Example 12: compound (27): trastuzumab conjugate - compound (10) - commercial compound N3-PEG4-Val-Ala-PBD dimer (11) Reagents

[0321] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (10) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, commercial compound N3-PEG4-Val-Ala-PBD dimer (11), (2nd compound) (10 eq) at a concentration of 0.5 mM in DMF. Method

[0322] Bioconjugation reaction 3. In this case, the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4, the concentration of the intermediate trastuzumab-compound conjugate (10) is adjusted to 1.5 mg / mL before the addition of the commercial compound N3-PEG4-Val-Ala-PBD dimer (11) and the reaction medium was stirred for 17h40. Denaturing HRMS analysis according to method 2

[0323] The results are shown in Table 20 below.

[0324] [Tables20] LHHL LH L Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 29 148861 no2 100 23405 DAR1 41 150069 100 76018 no2 DAR 2 30 152071 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.01 1.00 0

[0325] 1: molecular mass of the non-deglycosylated species

[0326] 2: not observed

[0327] The HRMS analysis determined an average DAR of 1.01 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH and H species were not observed.

[0328] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0329] The results are shown in Table 21 below.

[0330] [Tables21] Species DTT LHHL LHH HH LH HL Optical density (%) - 95 no1 no1 5 no1 no1 + 86 no1 3 6 3 1

[0331] 1: not observed

[0332] SDS-PAGE gel analysis made it possible to determine a reconstruction of 86% under reducing conditions and an average DAR of 1.06 under non-reducing conditions.

[0333] Example 13: compound (28): trastuzumab conjugate - compound (10) - commercial compound N3-PEG4-Val-Ala-PBD dimer (11) Reagents

[0334] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (8.0 eq), compound (10) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, commercial compound N3-PEG4-Val-Ala-PBD dimer (11), (2nd compound) (20 eq) at a concentration of 1 mM in DMF. Method

[0335] Bioconjugation reaction 4. In this case, the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4, the concentration of the intermediate trastuzumab-compound conjugate (10) is adjusted to 1.5 mg / mL before the addition of the commercial compound N3-PEG4-Val-Ala-PBD dimer (11) and the reaction medium was stirred for 17h40. Denaturing HRMS analysis according to method 2

[0336] The results are shown in Table 22 below.

[0337] [Tables22] LHHL LH L Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 no2 4 74831 100 23439 DAR1 16 150883 96 76014 no2 DAR 2 84 152066 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.84 0.96 0

[0338] 1: molecular mass of the non-deglycosylated species

[0339] 2: not observed

[0340] The SMHR analysis determined an average DAR of 1.84 for the LHHL species and an average DAR of 0.96 for the LH species. The LHH, HH and H species were not observed.

[0341] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0342] The results are shown in Table 23 below.

[0343] [Tables23] Species DTT LHHL LHH HH LH HL Optical density (%) - 76 no1 no1 24 no1 no1 + 67 no1 no1 33 no1 no1

[0344] 1: not observed

[0345] SDS-PAGE gel analysis made it possible to determine a reconstruction of 67% under reducing conditions and an average DAR of 1.86 under non-reducing conditions.

[0346] Example 14: compound (29): trastuzumab conjugate - compound (12) - compound (13) Reagents

[0347] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (8.0 eq), compound (12) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (13) (2nd compound) (13.2 eq) at a concentration of 10 mM in DMSO. Method

[0348] Bioconjugation reaction 4 Denaturing HRMS analysis according to method 2

[0349] The results are shown in Table 24 below.

[0350] [Tables24] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 2 148639 no2 DAR1 17 150086 100 76049 DAR 2 81 152101 no2 DAR 3 no2 no2 Average DAR 1.79 1.00

[0351] 1: molecular mass of the non-deglycosylated species

[0352] 2: not observed

[0353] The HRMS analysis determined an average DAR of 1.81 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0354] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0355] The results are shown in Table 25 below.

[0356] [Tables25] Species DTT LHHL LHH HH LH HL Optical density (%) - 68 no1 no1 24 no1 8 + 63 no1 no1 28 no1 9

[0357] 1: not observed

[0358] SDS-PAGE gel analysis made it possible to determine a reconstruction of 63% under reducing conditions and an average DAR of 1.84 under non-reducing conditions.

[0359] Example 15: compound (30): trastuzumab conjugate - compound (14) - compound (4) Reagents

[0360] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (14) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, compound (4) (2nd compound) (11.7 eq) at a concentration of 10 mM in DMSO. Method

[0361] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0362] The results are shown in Table 26 below.

[0363] [Tables26] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 6 148841 51 74032 DAR1 78 150099 49 76067 DAR 2 16 152139 no2 DAR 3 no2 no2 Average DAR 1.08 0.49

[0364] 1: molecular mass of the non-deglycosylated species

[0365] 2: not observed

[0366] The HRMS analysis determined an average DAR of 1.16 for the LHHL species and an average DAR of 0.49 for the LH species. The LHH, HH, H and L species were not observed.

[0367] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0368] The results are shown in Table 27 below.

[0369] [Tables27] Species DTT LHHL LHH HH LH HL Optical density (%) - 85 no1 no1 7 2 6 + 70 no1 no1 8 12 10

[0370] 1: not observed

[0371] SDS-PAGE gel analysis made it possible to determine a reconstruction of 70% under reducing conditions and an average DAR of 1.07 under non-reducing conditions.

[0372] Example 16: compound (31): trastuzumab conjugate - compound (14) - compound (4) Reagents

[0373] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (8.0 eq), compound (14) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (4) (2nd compound) (13.2 eq) at a concentration of 10 mM in DMSO. Method

[0374] Bioconjugation reaction 4. Denaturing HRMS analysis according to method 2

[0375] The results are shown in Table 28 below.

[0376] [Tables28] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 76068 DAR 2 100 152138 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0377] 1: molecular mass of the non-deglycosylated species

[0378] 2: not observed

[0379] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0380] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0381] The results are shown in Table 29 below.

[0382] [Tables29] Species DTT LHHL LHH HH LH HL Optical density (%) - 73 no1 no1 22 no1 5 + 56 no1 no1 37 no1 7

[0383] 1: not observed

[0384] SDS-PAGE gel analysis made it possible to determine a reconstruction of 56% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0385] Example 17: compound (32): trastuzumab conjugate - compound (14) - commercial compound N3-Cap-Val-Cit-PAB-C6-amanitin(15) Reagents

[0386] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (14) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, commercial compound N3-Cap-Val-Cit-PAB-C6-amanitin (15), (2nd compound) (12.7 eq) at a concentration of 10 mM in DMSO. Method

[0387] Bioconjugation reaction 3. Denaturing HRMS analysis according to method 2

[0388] The results are shown in Table 30 below.

[0389] [Tables30] LHHL LH L Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 no2 5 74032 100 23439 DAR1 61 150395 95 76367 no2 DAR 2 39 152747 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.39 0.95 0

[0390] 1: molecular mass of the non-deglycosylated species

[0391] 2: not observed

[0392] The HRMS analysis determined an average DAR of 1.39 for the LHHL species and an average DAR of 0.95 for the LH species. The LHH, HH and H species were not observed.

[0393] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0394] The results are shown in Table 31 below.

[0395] [Tables31] Species DTT LHHL LHH HH LH HL Optical density (%) - 86 no1 no1 9 3 2 + 72 no1 4 14 8 2

[0396] 1: not observed

[0397] SDS-PAGE gel analysis made it possible to determine under reducing conditions a 72% reconstruction and in non-reducing conditions an average DAR of 1.44.

[0398] Example 18: compound (33): trastuzumab conjugate - compound (14) - commercial compound N3-Cap-Val-Cit-PAB-C6-amanitin(15) Reagents

[0399] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (8.0 eq), compound (14) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, commercial compound N3-Cap-Val-Cit-PAB-C6-amanitin (15) (2nd compound) (13.2 eq) at a concentration of 10 mM in DMSO. Method

[0400] Bioconjugation reaction 4. Denaturing HRMS analysis according to method 2

[0401] The results are shown in Table 32 below.

[0402] [Tables32] LHHL LH Intensity (%) MM (Da) i Intensity (%) MM (Da) i DAR0 no2 no2 DAR1 no2 100 76367 DAR 2 100 152735 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0403] 1: molecular mass of the non-deglycosylated species

[0404] 2: not observed

[0405] The HRMS analysis determined an average DAR of 2.00 for the LHHL species and an average DAR of 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0406] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0407] The results are shown in Table 33 below.

[0408] [Tables33] Species DTT LHHL LHH HH LH HL Optical density (%) - 55 no1 1 39 4 1 + 52 no1 2 38 7 1

[0409] 1: not observed

[0410] SDS-PAGE gel analysis made it possible to determine a reconstruction of 52% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0411] Example 19: compound (34): trastuzumab conjugate - compound (16) Reagents

[0412] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (16) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH. Method

[0413] Bioconjugation reaction 2. Denaturing HRMS analysis according to method 2

[0414] The results are shown in Table 34 below.

[0415] [Tables34] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 39 74029 DAR1 100 149703 61 75664 DAR 2 no2 no2 DAR 3 no2 no2 DAR average 1.00 0.61

[0416] 1: molecular mass of the non-deglycosylated species

[0417] 2: not observed

[0418] The HRMS analysis determined an average DAR of 1.00 for the LHHL species and 0.61 for the LH species. The LHH, HH, H and L species were not observed.

[0419] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0420] The results are shown in Table 35 below.

[0421] [Tables35] Species DTT LHHL LHH HH LH HL Optical density (%) - 85 no1 no1 7 no1 8 + 60 no1 no1 15 15 10

[0422] 1: not observed

[0423] SDS-PAGE gel analysis made it possible to determine under reducing conditions a 60% reconstruction and in non-reducing conditions an average DAR of 1.02.

[0424] Example 20: compound (35): trastuzumab conjugate - compound (16) Reagents

[0425] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 1 (7.0 eq), compound (16) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH. Method

[0426] Bioconjugation reaction 2. Denaturing HRMS analysis according to method 2

[0427] The results are shown in Table 36 below.

[0428] [Tables36] LHHL LH Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 DAR0 no2 no2 DAR1 no2 100 75666 DAR 2 100 151332 no2 DAR 3 no2 no2 DAR average 2.00 1.00

[0429] 1: molecular mass of the non-deglycosylated species

[0430] 2: not observed

[0431] The SMHR analysis determined an average DAR of 2.00 for the LHHL species and 1.00 for the LH species. The LHH, HH, H and L species were not observed.

[0432] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0433] The results are shown in Table 37 below.

[0434] [Tables37] Species DTT LHHL LHH HH LH HL Optical density (%) - 56 no1 no1 34 no1 10 + 54 no1 no1 37 no1 9

[0435] 1: not observed

[0436] SDS-PAGE gel analysis made it possible to determine a reconstruction of 54% under reducing conditions and an average DAR of 2.00 under non-reducing conditions.

[0437] Example 21: compound (36): trastuzumab conjugate - compound (17) - compound (13) Reagents

[0438] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reductant 1 (7.0 eq), compound (17) (1st compound) (10.6 eq) at a concentration of 1 mM in a mixture of 80% DMF and 20% MeOH, compound (13) (2nd compound) (15.0 eq) at a concentration of 1 mM in DMSO. Method

[0439] Bioconjugation reaction 3. In this case, the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4, the concentration of the intermediate trastuzumab-compound (17) conjugate is adjusted to 1.5 mg / mL before the addition of compound (13) and the reaction medium was stirred for 22h. Denaturing HRMS analysis according to method 2

[0440] The results are shown in Table 38 below.

[0441] [Tables38] LHHL LH L Intensity (%) MM (Da)> Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 2 148057 15 74026 80 23405 ND3 10 150747 5 76712 no2 DAR1 73 151852 80 77817 20 27223 DAR 2 15 155650 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.02 0.80 0.20

[0442] 1: molecular mass of the non-deglycosylated species

[0443] 2: not observed

[0444] 3: ND: impurity of undetermined structure

[0445] The HRMS analysis determined an average DAR of 1.02 for the LHHL species and an average DAR of 0.80 for the LH species. The LHH, HH, and H species were not observed.

[0446] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0447] The results are shown in Table 39 below.

[0448] [Tables39] Species DTT LHHL LHH HH LH HL Optical density (%) - 91 no1 no1 9 no1 no1 + 63 no1 6 15 12 4

[0449] 1: not observed

[0450] SDS-PAGE gel analysis made it possible to determine a reconstruction of 63% under reducing conditions and an average DAR of 1.07 under non-reducing conditions.

[0451] Example 22: compound (37): trastuzumab conjugate - compound (17) - compound (13) Reagents

[0452] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reducer 2 (8.0 eq), compound (17) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 20% DMF and 80% MeOH, compound (13) (2nd compound) (30.0 eq) at a concentration of 1 mM in DMSO. Method

[0453] Bioconjugation reaction 4. In this case, the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4, the concentration of the intermediate trastuzumab-compound (17) conjugate is adjusted to 1.4 mg / mL before the addition of compound (13) and the reaction medium was stirred for 22h. Denaturing HRMS analysis according to method 2

[0454] The results are shown in Table 40 below.

[0455] [Tables40] LHHL LH L Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 no2 no2 100 23405 DAR1 14 151858 90 77817 no2 ND3 8 154529 10 76712 no2 DAR 2 78 155640 no2 no2 DAR 3 no2 no2 no2 Average DAR 1.70 0.90 0

[0456] 1: molecular mass of the non-deglycosylated species

[0457] 2: not observed

[0458] 3: ND: impurity of undetermined structure

[0459] The SMHR analysis determined an average DAR of 1.70 for the LHHL species and an average DAR of 0.90 for the LH species. The LHH, HH, and H species were not observed.

[0460] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0461] The results are shown in Table 41 below.

[0462] [Tables41] Species DTT LHHL LHH HH LH HL Optical density (%) - 61 no1 no1 39 no1 no1 + 56 no1 3 36 5 no1

[0463] 1: not observed

[0464] SDS-PAGE gel analysis made it possible to determine a reconstruction of 56% under reducing conditions and an average DAR of 1.74 under non-reducing conditions.

[0465] Example 23: compound (38): trastuzumab conjugate - compound (8) - compound (2) Reagents

[0466] Bioconjugation buffer 1, trastuzumab at 5 mg / mL in bioconjugation buffer, reductant 1 (8.0 eq), compound (8) (1st compound) (12.0 eq) at a concentration of 3 mM in a mixture of 30% DMF and 70% MeOH, compound (9) (2nd compound) (30 eq) at a concentration of 2 mM in DMSO. Method

[0467] Bioconjugation reaction 4. In this case, the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco® PBS buffer pH 7.4, the concentration of the intermediate trastuzumab-compound (8) conjugate is adjusted to 1.4 mg / mL before the addition of compound (2) and the reaction medium was stirred for 17 h. Denaturing HRMS analysis according to method 2

[0468] The results are shown in Table 42 below.

[0469] [Tables42] LHHL LH L Intensity (%) MM (Da)1 Intensity (%) MM (Da)1 Intensity (%) MM (Da) DAR0 no2 12 74693 100 23468 DAR1 no2 88 75812 no2 DAR 2 62 151625 no2 no2 DAR 3 38 153443 no2 no2 Average DAR 2.38 0.88 0

[0470] 1: molecular mass of the non-deglycosylated species

[0471] 2: not observed

[0472] The HRMS analysis determined an average DAR of 2.38 for the LHHL species and an average DAR of 0.88 for the LH species. The LHH, HH and H species were not observed.

[0473] SDS-PAGE gel analysis under non-reducing and reducing denaturing conditions

[0474] The results are shown in Table 43 below.

[0475] [Tables43] Species DTT LHHL LHH HH LH HL Optical density (%) - 63 no1 1 29 4 4 + 57 no1 4 29 5 5

[0476] 1: not observed

[0477] SDS-PAGE gel analysis made it possible to determine a reconstruction of 57% under reducing conditions and an average DAR of 2.18 under non-reducing conditions.

[0478] Example 24: In vitro evaluation of the cytotoxicity of the conjugates by XTT test on a positive line (BT-474) and a negative line (MCF-7) for the HER2 receptor

[0479] Material and method

[0480] Cells were obtained from ATCC (BT-474 and MCF-7). An aliquot of frozen BT-474 cells or MCF-7 cells was rapidly thawed in a 37°C water bath and the cells were washed twice with culture medium respectively with F12 / DMEM supplemented with 8% S VF, 100 pg / mL L-glutamine, 100 pg / mL penicillin G sodium, 100 pg / mL streptomycin sulfate for BT-474 cells or with DMEM GlutaMAX™ supplemented with 10% FCS, 1% penicillin G sodium, 1% streptomycin sulfate for MCF-7 cells. Then the cells were deposited in a 75 cm2 cell culture flask at a density of at least 10,000 cells / cm2. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 for at least one week.

[0481] Then, MCF-7 and BT-474 cells were plated in 96-well plates at densities of 2,500 and 50,000 cells per well respectively for cytotoxicity assays. The cells were incubated for 24 h at 37°C before the addition of the conjugates according to the invention or the tested controls and the vehicle (medium alone). The percentages of DMSO never exceeded 0.5%. The tested conjugates were added at the following final concentrations: 200,000 at 0.2 pM; and incubated for 96 h.

[0482] After 4 days of exposure to the conjugates, 25 μL of 1 mg / mL XTT reagent with 25 mM activator (N-methyl dibenzopyrazine methyl sulfate) was added per well and the absorbance was measured at 450 nm after 4 h of incubation at 37°C. The absorbance at 620 nm was used as a reference. Cell viability is expressed as the mean (+ / -SEM) of the percentage obtained.

[0483] Each compound concentration was performed in triplicate and two or three “N” independent experiments were conducted.

[0484] Results

[0485] The evaluation of the cytotoxicity on the HER2 positive breast cancer cell line BT-474 showed that compounds (24), (25), (26), (34), and (35) are as cytotoxic as MMAE (toxin alone) and as the control ADC (Trastuzumab - MMAE conjugate from application PCT / FR2020 / 050833). Trastuzumab (TTZ) alone (i.e. not coupled to MMAE), has no cytotoxic effect on this same line at the lowest concentrations tested, demonstrating the absence of intrinsic toxicity of the antibody ([Fig.lA]). On the HER2-negative breast cancer cell line MCF-7, compounds (24), (25), (26), (34), (35), and the control ADC have no cytotoxic effects at the lowest effective concentrations tested, demonstrating the absence of intrinsic toxicity of these constructs ([Fig.lB]). Thus, compounds (24), (25), (26), (34), and (35) are effective on cells expressing the target on their surface only.

[0486] SEQUENCE LISTING

[0487] <110> MCSAF

[0488] <120> Antibody-drug conjugates for use

[0489] therapeutic

[0490] <130> 1H317490 008 FR / BN

[0491] <160> 10

[0492] <170> Patentln version 3.5

[0493] <210> 1

[0494] <211> 10

[0495] <212> PRT

[0496] <213> Artificial Sequence

[0497] <220>

[0498] <223> CDR1 of the light chain of the anti-CD56 antibody

[0499] <400> 1

[0500] Gin Ser Leu Leu His Ser Asn Gly Tyr Asn

[0501] 15 10

[0502] <210> 2

[0503] <211> 3

[0504] <212> PRT

[0505] <213> Artificial Sequence

[0506] <220>

[0507] <223> CDR2 of the light chain of the anti-CD56 antibody

[0508] <400> 2

[0509] Tyr Leu Gly

[0510] 1

[0511] <210> 3

[0512] <211> 10

[0513] <212> PRT

[0514] <213> Artificial Sequence

[0515] <220>

[0516] <223> CDR3 of the light chain of the anti-CD56 antibody

[0517] <400> 3

[0518] Cys Met Gin Ser Leu Gin Thr Pro Trp Thr

[0519] 15 10

[0520] <210> 4

[0521] <211> 11

[0522] <212> PRT

[0523] <213> Artificial Sequence

[0524] <220>

[0525] <223> CDR1 of the heavy chain of the anti-CD56 antibody

[0526] <400> 4

[0527] Gly Gly Thr Phe Thr Gly Tyr Tyr Met His Trp

[0528] 15 10

[0529] <210> 5

[0530] <211> 9

[0531] <212> PRT

[0532] <213> Artificial Sequence

[0533] <220>

[0534] <223> CDR2 of the heavy chain of the anti-CD56 antibody

[0535] <400> 5

[0536] Asn Ser Gly Gly Thr Asn Tyr Ala Gin

[0537] 15

[0538] <210> 6

[0539] <211> 15

[0540] <212> PRT

[0541] <213> Artificial Sequence

[0542] <220>

[0543] <223> CDR3 of the heavy chain of the anti-CD56 antibody

[0544] <400> 6

[0545] Leu Ser Ser Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly Gin Gly

[0546] 15 10 15

[0547] <210> 7

[0548] <211> 219

[0549] <212> PRT

[0550] <213> Artificial Sequence

[0551] <220>

[0552] <223> Anti-CD56 antibody light chain

[0553] <400> 7

[0554] Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

[0555] 15 10 15

[0556] Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser

[0557] 20 25 30

[0558] Asn Gly Tyr Asn Phe Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser

[0559] 35 40 45

[0560] Pro Gin Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

[0561] 50 55 60

[0562] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[0563] 65 70 75 80

[0564] Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gin Ser

[0565] 85 90 95

[0566] Leu Gin Thr Pro Trp Thr Phe Gly His Gly Thr Lys Val Glu Ile Lys

[0567] 100 105 110

[0568] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[0569] 115 120 125

[0570] Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[0571] 130 135 140

[0572] Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin

[0573] 145 150 155 160

[0574] Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser

[0575] 165 170 175

[0576] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[0577] 180 185 190

[0578] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser

[0579] 195 200 205

[0580] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[0581] 210 215

[0582] <210> 8

[0583] <211>451

[0584] <212> PRT

[0585] <213> Artificial Sequence

[0586] <220>

[0587] <223> Chaîne lourde de l'anticorps anti-CD56

[0588] <400> 8

[0589] Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

[0590] 15 10 15

[0591] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Gly Tyr

[0592] 20 25 30

[0593] Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met

[0594] 35 40 45

[0595] Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gin Lys Phe

[0596] 50 55 60

[0597] Gin Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[0598] 65 70 75 80

[0599] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[0600] 85 90 95

[0601] Ala Arg Asp Leu Ser Ser Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly

[0602] 100 105 110

[0603] Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

[0604] 115 120 125

[0605] Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

[0606] 130 135 140

[0607] Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

[0608] 145 150 155 160

[0609] Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

[0610] 165 170 175

[0611] Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

[0612] 180 185 190

[0613] Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His

[0614] 195 200 205

[0615] Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

[0616] 210 215 220

[0617] Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

[0618] 225 230 235 240

[0619] Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

[0620] 245 250 255

[0621] Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

[0622] 260 265 270

[0623] Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

[0624] 275 280 285

[0625] His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr

[0626] 290 295 300

[0627] Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly

[0628] 305 310 315 320

[0629] Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

[0630] 325 330 335

[0631] Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val

[0632] 340 345 350

[0633] Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

[0634] 355 360 365

[0635] Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

[0636] 370 375 380

[0637] Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

[0638] 385 390 395 400

[0639] Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

[0640] 405 410 415

[0641] Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

[0642] 420 425 430

[0643] His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

[0644] 435 440 445

[0645] Pro Gly Lys

[0646] 450

[0647] <210> 9

[0648] <211> 113

[0649] <212> PRT

[0650] <213> Artificial Sequence

[0651] <220>

[0652] <223> Variable domain of the light chain of the anti-CD56 antibody

[0653] <400> 9

[0654] Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

[0655] 15 10 15

[0656] Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser

[0657] 20 25 30

[0658] Asn Gly Tyr Asn Phe Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser

[0659] 35 40 45

[0660] Pro Gin Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

[0661] 50 55 60

[0662] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[0663] 65 70 75 80

[0664] Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gin Ser

[0665] 85 90 95

[0666] Leu Gin Thr Pro Trp Thr Phe Gly His Gly Thr Lys Val Glu Ile Lys

[0667] 100 105 110

[0668] Arg

[0669] <210> 10

[0670] <211> 120

[0671] <212> PRT

[0672] <213> Artificial Sequence

[0673] <220>

[0674] <223> Domaine variable de la chaîne lourde de l'anticorps anti-CD56

[0675] <400> 10

[0676] Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

[0677] 15 10 15

[0678] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Gly Tyr

[0679] 20 25 30

[0680] Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met

[0681] 35 40 45

[0682] Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gin Lys Phe

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691] 50 55 60 Gin Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Ser Ser Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly 100 105 110 Gin Gly Thr Leu Val Thr Val Ser 115 120

Claims

Claims

1. Conjugate of formula (I) for use as a medicament: [Cheml] Head "hangs on" 8 tiatecn ras Spacer f in which: a) Ac is an antibody or antibody fragment; b) u is such that 0.5 < u < 3.5; (c) the attachment head is a compound of formula (II) or formula (II'): [Chem. 2] (II) [Chem. 3] (he') in which: - W is -ORa, -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-R5 or -(CH2CH2O)q-(CH2)r-CONH-(CRcRd)r-R5; - Rb is -(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-R5ou -(CH2CH2O)q-(CH2)r -CONH-(CRcRd)r-R5 ; - R2 est -OH, -(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-R5 ou -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 -(CrC6)alkyle ; - 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, -(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, ou -CH-[(CRcRd )r-NHCO-(CRcRd)r-R5]2, de préférence R4 est -(CH2CH2O)qR5, -(CRcRd)r R5, -(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; - each R5 is -(CH2)SR6 or -(CH2)SR7; - R6 is -COOH or -NR8R9; - each R7 is chosen from:

2.

3. - Rc is H; - each Rd is chosen from -H, -CH2-SO3H or -SO3H; - R8 is -H or -(Ci-C6)alkyl; - R9 is -H or -(Ci-C6)alkyl; - Rio 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; (d) the linker arm is a direct bond; a sugar; a glucuronide; a bridge -SS-; or a group of formula -Rn-(A)Z- in which: - Rn is a direct bond, a group R7-(CRcRd)r-CO- or a group R7 -(CH2CH2O)q-(CH2)s-CO-, or a group R7-(CRcRd)r-NH-, where R7, 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; e) the spacer is a direct bond or a group of formula: [Chem. 5] 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; - R[2 is -H or -NO2; f) M is an active ingredient or a radionuclide chelator, preferably M is an active ingredient. Conjugate for use according to claim 1, wherein the attachment head is a compound of formula (II). A conjugate for use according to claim 1 or 2, wherein 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; - R5 is -(CH2)sR6 or -(CH2)sR7; - R6 is -COOH; - R7 is chosen from: [Chem. 6] - Rc, Rd, R8 and R9 are as defined in claim 1; - q is an integer ranging from 1 to 12, preferably q is an integer ranging from 1 to 8; - r is an integer ranging from 1 to 6.

4. A conjugate for use according to any preceding claim, wherein the attachment head is a compound of formula (IIa), (IIb), (IIe), (IId), (IIe), (IIf), (IIg), (IIh) or (IIa): [Chem. 7] (Ha); [Chem. 8] (Ilb); [Chem.9] (Island) ; (üd); [Chem. 11] (Island) ; [Chem. 12] (iif); [Chem. 13] (Hg); [Chem. 14] (Ilh); [Chem. 15]

5.

6. (Ha). A conjugate for use according to any preceding claim, wherein the linker arm is a group of formula -Rn-(A)z- in which: - Rn and A are as defined in claim 1; - z is equal to 2, 3 or 4. A conjugate for use according to any preceding claim, wherein the spacer arm is a direct bond or a group of formula: [Chem. 16]

7. A conjugate for use according to any preceding claim, wherein the spacer arm is a direct bond or a group of formula: [Chem. 17]

8.

9.

10. A conjugate for use 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 pyrro-lopyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, ricin. A conjugate for use according to claim 8, wherein the active ingredient is amanitin, a pyrrolobenzodiazepine dimer, MMAF or MMAE. A conjugate for use according to any preceding claim, wherein the antibody or an antibody fragment of said antibody binds to a cancer-specific antigen, e.g., 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, CD 194, 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, GPNMB, PSMA, PSA, PAP, PSM, Cripto, 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,carcinoembryonal antigen (CEA), TAG-72, folate-binding protein, G250, gan-gliosides, collagen type 4 (collagen IV), collagen type 18 (collagen XVIII), CA19-9, pl85HER2, fibroblast activation 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-1, CD11, IFAR, CADM, CADM BCAM, CEACAM6, JAM-A, PTGFRN (CD9P-1), MCAM, MCP, EMMPRIN, TfR, ClqR, hTERT, Survivine, MDM2, CYP1B1, MART-1, MART-2, melanosomal proteins, gplOO, CDC27, MAGE1, WT, MUM-2, 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, EBV-1 protein, taxa, HTLVNA, HTLV-1 HPV16-E7, HLA-A2 mutated, HAÏ, SART3, CEACAM5, ESAT-6, RANK, fibrin, TF, PRAME, CA19-9, CA50, CA195, CAM17.1 / WGA, beta-MG, DU-PAN2, HE4, transferrin, transthyrin, Apol-A2, TROP, TROP GITR, PD-1, PD-L1, c-KIT, CDllb-CD18 integrin heterodimer, DNA / Histone Hl, proteoglycan, fibrinogen, large T SV40 antigen, SC-Ag, ESA, mucin, CCR4, MTX1, MTX2, PECAM, TER, TER, D-3, T or a PF4 / heparin complex.

11. A conjugate for use according to any preceding claim, wherein the antibody or a Fab', F(ab')2, or scFv-Fc fragment of said antibody binds to HER2, CD30 or CD56.

12. A conjugate for use according to any preceding claim, wherein the antibody is an anti-HER2 antibody, an anti-CD30 antibody or an anti-CD56 antibody.

13. A conjugate for use according to claim 12, wherein the antibody is an anti-CD56 antibody.

14. A conjugate for use according to any preceding claim, said conjugate being present in a composition, for example a pharmaceutical composition containing one or more pharmaceutically acceptable excipients and / or carriers.

15. A conjugate of formula (I) as defined in any one of claims 1 to 14 for use in a method of treating cancer.

16. A conjugate for use according to claim 15, wherein: - the antibody or a Fab', F(ab')2, or scFv-Fc fragment thereof binds to HER2 and the cancer is a HER2+ cancer, or - the antibody or a Fab', F(ab')2, or scFv-Fc fragment thereof binds to CD30 and the cancer is a CD30+ cancer, or - the antibody or a Fab', F(ab')2, or scFv-Fc fragment thereof binds to CD56 and cancer is CD56+ cancer.

17. A conjugate for use according to claim 15, wherein: - the antibody is an anti-HER2 antibody and the cancer is selected from breast cancer, gastric cancer, gastroesophageal cancer, bladder cancer, gallbladder cancer, extrahepatic cholangiocarcinoma; or - the antibody is an anti-CD30 antibody and the cancer is selected from Hodgkin's lymphoma, anaplastic large cell lymphoma and peripheral T-cell lymphoma, mycosis fungoides; or - the antibody is an anti-CD56 antibody and the cancer is chosen from neuroendocrine cancers, lung cancer or Merkel cell carcinoma.

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