Pegylated linkers and uses thereof

Abstract

The present invention concerns a compound having the following formula (I) wherein: - m represents an integer comprised from 1 to 3, - L1 represents a bioconjugation unit, - L2 and L3 represent a spacer group, - L4 represents a release system, - L5 represents an anticancer agent, and - L6 represents a PEG moiety.

Description

[0001] PEGYLATED LINKERS AND USES THEREOF

[0002] The present invention concerns new pegylated linkers, in particular for antibodydrug conjugates (ADC), as well as uses thereof, in particular for the preparation of conjugates, especially for treating cancer.

[0003] In the past few years, since the advent of Glivec®, cancer treatment has been revolutionized by the arrival of several targeted therapies that have greatly increased the efficacy and tolerance of treatments. Along with tyrosine kinase inhibitors, antibody-drug conjugates (ADCs) are among the personalized targeted therapies that have been widely developed in recent years, since the approval of Mylotarg® by the Food and Drug Administration (FDA) in 2000. Today, the FDA approved 13 ADCs. ADCs are targeted biotherapies against one antigen, vectorized for the transport of cytotoxic molecules and injectable via intravenous route. An important characteristic of ADCs is the drug-to-antibody ratio (DAR) defined by the average number of cytotoxic drugs conjugated onto the antibody. The general structure of this molecular architecture is thus characterized by an immunoglobulin, a bioconjugation head, a linker (cleavable or not) and a vectorized drug.

[0004] In oncology, the first ADC approved by the FDA in 2013 to treat a solid tumor was trastuzumab emtansine (T-DM1 , Kadcyla®), used as a monotherapy against HER2-positive metastatic (or locally advanced unresectable) breast cancer. Its indication has now been extended to the treatment of patients with HER2-positive early breast cancer (after neoadjuvant therapy). Unfortunately, T-DM1 is a second- generation ADC, associated with several limitations. The stochastic conjugation of the hydrophobic maytansine derivative DM1 , through a hydrophobic non-cleavable linker onto the side chains of accessible lysines, lead to a very heterogeneous distribution of immunoconjugates characterized by an average DAR of only 3.5. The release of its active metabolite (lysine-linker-DM1 ), charged at physiological pH, prevents it to exhibit a bystander killing effect and limits its activity. Moreover, Kadcyla® is already associated with emerging resistances in patients, urging the need for improved design in ADCs.

[0005] In December 2018, the FDA approved the trastuzumab deruxtecan (T-Dxd, Enhertu®) as a treatment for patients with unresectable or metastatic HER2-positive or HER2-low breast cancer (IHC score 1 + or IHC 2+ / HIS-). T-Dxd is a third generation ADC, characterized by a homogeneous DAR of 8, where trastuzumab is conjugated to a membrane-permeable payload Dxd (exhibiting a bystander killing effect to kill HER2-low cancer cells), through an innovative enzymatically cleavable linker designed to control hydrophobicity. The innovative linker (with a tetrapeptidyl-spacer MC-GlyGlyPheGly) and the more hydrophilic aminomethylene self-immolative spacer and drug DXd allowed the development of a stable ADC with a high homogeneous DAR of 8, as tolerable as a second-generation ADC with an average DAR of 4. These two ADCs are derived from an lgG1 , so they can bind to FcRn, which is ubiquitous and responsible for recycling immunoglobulins in the body, and to FcyR, during plasma circulation and biodistribution in the body. These nonspecific bindings therefore results in unwanted off-target off-site toxicity, which are associated to strong side effects (thrombocytopenia and hepatic cytolysis). In addition, the use of high molecular weight proteins such as IgG (150 kDa), could lead to a suboptimal tumor penetration.

[0006] To overcome the limitations associated with the IgG format to develop immunoconjugates, more and more studies are focusing on conjugated smaller antibody formats or fragments. They have the advantage of being much smaller than IgG-based ADCs, have no Fc part (responsible for non-specific binding and antigenindependent internalization) and potentially diffuse better in the tumor microenvironment. It has been shown in the literature that the larger the macromolecule is, the more rapidly it is eliminated from the extravascular tumor environment. Indeed, Zhe Li's study shows that the tissue penetration of entities increases as the size of the molecules decreases. Deonarain et al. also argued that small antibody conjugate sizes may have better tumor penetration properties. The minibody format was first described in preclinical studies in 1996 and in clinical first- in-human phase I trial in 2004 as an anti-CEA radiotracer in colon cancer patients. This type of conjugated antibody format showed a rapid distribution in the tumor associated with a fast clearance of the body. To date, interestingly the use of minibody fragments has been scarcely reported in a only some preclinical studies and few clinical trials as a radiotracer (completed: NCT02092948, NCT05279027, in progress: NCT04874818, NCT03542669; “Clinical Trials”). Although, a very recent study proved promising results about the efficacy of a minibody conjugated to 2 molecules of monomethyl auristatin E (MMAE) (DAR 2) against internalizing GD2 overexpressed at the surface of cancer cells nevertheless, the authors pointed out that the low DAR 2 was associated with limited efficacy, hence higher DAR is needed to reach clinical translation. Currently, the FDA approved four ADCs (Adcetris®, Polivy®, Padcev®, Tivdak®) on the market with the drug MMAE, with an average DAR of only 4. In fact, MMAE and its associated release trigger (valine-citrulline-para-aminobenzylcarbamate or VC-PABC) are very hydrophobic, which leads to physicochemical stability issues and a propension for aggregation of the corresponding ADCs. This also limits the possibility to efficiently conjugate a high number of MMAE payloads onto antibody smaller formats (e.g. usually no more than 1 MMAE onto a scFv or 2 MMAE onto a Mb). On the other hand, MMAE remains a very interesting molecule with a cytotoxic activity in the subnanomolar range and with the potential to exert a bystander effect on neighboring tumor cells. It is therefore necessary and essential to control this hydrophobicity as it limits the number of MMAE that can be vectorized either onto an IgG or onto a smaller antibody fragment (e.g. Mb).

[0007] An aim of the present invention is thus to provide new linkers for the preparation of antibody-drug conjugates, for improving the bioconjugation on the antibodies and the pharmacokinetic properties of the corresponding bioconjugated compounds, in particular in terms of serum stability and internalization in targeted cells.

[0008] Another aim of the present invention is to provide new linkers for the preparation of antibody-drug conjugated, allowing doubling the DAR on each disulfide bridge in comparison with the current conjugates.

[0009] Another aim of the present invention is to provide new linkers for the preparation of antibody-drug conjugated, with high DAR, preferably of 8 or 12, with satisfying yields.

[0010] Thus, the present invention relates to a compound having the following formula (I): wherein:

[0011] - m represents an integer comprised from 1 to 3, m being preferably equal to 1 , - Li represents a bioconjugation unit capable of binding to at least one antibody or antibody fragment by reacting with an amino, hydroxyl, amide or thiol function,

[0012] - L2represents a spacer group of formula -Ai-C(=O)-, Ai representing a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,

[0013] - L3represents a spacer group of formula -A2-C(=O)-, A2representing a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,

[0014] - L4represents a release system selected from the group consisting of:

[0015] * the group having the following formula (II):

[0016] -[NH-CH(Ai)-C(=O)]i-[A9]j- (II) wherein:

[0017] . i is 0 or is an integer from 1 to 8,

[0018] . j is 0 or 1 , provided that i is not 0 when j=0, and when i=0 then j=1 ,

[0019] . each Ai represents, independently from each other, the side chain of an amino acid residue, in particular a linear or branched (Ci-Ce)alkyl chain, and

[0020] . A9represents a -NH-CH2- group or a group having the following formula (H-1 ):

[0021] X representing

[0022] * the group having the following formula (III): wherein:

[0023] . X’ is H or NO2, . L7is a group of formula H-[NH-CH(Ai)-C(=O)]i-, i and A being as defined above in formula (II), or l_7 is selected from the group consisting of: beta-glucuronic acid, beta- D-galactose, beta-D-glucose, alpha-D-mannose, N-acetyl-D- glucosaminyle, N-acetyl-D-galactosaminyle, D-glucuronyle, L-iduronyle, D-glucopyranosyle, D-galactopyranosyle, D-mannopyranosyle, and L- fucopyranosyle,

[0024] . L8represents a group -CH2-CH2-C(=O)-NH-, a bond or a linear or branched alkylene radical comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,

[0025] * the group having the following formula (IV): wherein:

[0026] . X” is H or NO2, and

[0027] . L7and L8are as defined in formula (III);

[0028] - L5represents an anticancer agent, and

[0029] - L6represents a PEG moiety selected from the group consisting of:

[0030] * the group of formula -(CH2-CH2-O)n-CH3, n representing an integer from 1 to 24, and

[0031] * the group of the following formula (V):

[0032] -(CH2)3-C(=O)-NH-(CH2-CH2-O)4-(CH2)2-C(=O)-NH-C(R)3(V) wherein R is a group of the following formula (V-1 ): -CH2-O-(CH2)2-C(=O)-NH-(CH2)2-(O-CH2-CH2)7-OCH3.

[0033] The compounds according to the invention thus include a PEG moiety (L6) that plays the role of a hydrophobicity masker, and thus allows the control of hydrophobicity of the linker. The compounds according to the invention are also advantegous in that they can form conjugates with antibodies, but also with antibody fragments, while obtaining high DAR.

[0034] In some embodiments of the invention, the compounds of the invention can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereoisomeric mixtures. All such isomeric forms of these compounds are included in the present invention, unless expressly provided otherwise.

[0035] According to the present application, the term “alkyl”’ means a linear or branched, saturated, hydrocarbon-based aliphatic group comprising, unless otherwise mentioned, from 1 to 12 carbon atoms. The preferred alkyl groups according to the invention comprise 1 to 6 carbon atoms in the chain. “Branched” means that one or several inferior alkyl groups such as methyl, ethyl or propyl are linked to a linear alkyl chain. “Inferior alkyl” corresponds to an alkyl group comprising 1 to 4 carbon atoms in the chain, said chain being linear or branched.

[0036] The term "alkylene" as used herein refers to a divalent radical comprising, unless otherwise specified, from 1 to 6 carbon atoms. An alkylene radical corresponds to an alkyl radical with one less hydrogen atom. The said radical, when it is linear, can be represented in particular by the formula (CH2)nin which n is an integer varying from 1 to 6.

[0037] Within the present invention, a “bioconjugation unit capable of binding to at least one antibody or antibody fragment by reacting with an amino, hydroxyl or thiol function” refers to a unit, generally a hydrocarbon radical, that has a chemical function or chemical moiety, that is able to react with a secondary amino, hydroxyl or thiol function, and thus forms a covalent bond between the compound of formula (I) and a distinct chemical unit carrying this function and being compatible with the formation of this covalent function.

[0038] According to an embodiment, in formula (I), Li comprises or is a maleimide group, a NHS ester group, an amine, an azide (N3) or an isothiocyanate group.

[0039] According to an embodiment, in formula (I), Li comprises or is a maleimide group, a NHS ester group, or an isothiocyanate group.

[0040] Preferably, in formula (I), Li is a maleimide group or an amine (NH2). According to an embodiment, the compounds of the invention have the following formula (1-1 ):

[0041] L2, l_3, l_4, L5, and L6being as defined above in formula (I).

[0042] According to an embodiment, in formula (I) or (1-1 ), L2and L3, independently from each other, represent a spacer group of formula -(CH2)P-C(=O)-, p being an integer comprised from 2 to 20, or of formula -(CH2-CH2-O)m-(CH2)r-C(=O)-, m being an integer comprised from 1 to 10 and r being 1 or 2.

[0043] According to an embodiment, the compounds of the invention have the following formula (I-2): wherein: - L4, L5, and L6are as defined above in formula (I), and

[0044] - p and p’, independently from each other, are integers comprised from 1 to 20. According to an embodiment, in formula (I) or in formulae (1-1 ) or (I-2), L4 is selected from the group consisting of:

[0045] - the group having the following formula (VI):

[0046] - the group having the following formula (VII):

[0047] - and the group having the following formula (VIII):

[0048] In one embodiment, in the above formula (I), or in one of the formulae (1-1 ) or (I-2) as defined above, L5is selected from the group consisting of: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exatecan, duocarmycin and analogues thereof, dolastatins, combretastatin, calicheamicin, N-acetyl-y- calicheamycin (CMC), calicheamycin derivatives, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), a tubulysin (for example tubulysin A), AZ13599185, disorazole, epothilones, paclitaxel, le docetaxel, echinomycin, estramustin, cemadotine, eleutherobin, methopterin, actinomycin, mitomycin A, camptothecin, camptothecin derivatives, SN38, Dxd, exatecan, maytansin, maytansinoid derivatives, DM1 , DM4, TK1 , amanitin (such as alpha, beta or HDP 30.21 15), a pyrrolobenzodiazepine, a dimer of pyrrolobenzodiazepine, SGN-3199, an inhibitor of histone deacetylase, an inhibitor of tyrosine kinases, an inhibitor of serine-threonine kinase, a vinca-alcaloid (for example vincristine), mitomycin C, a retinoid (for example retinoic acid), an anti-metabolic agent (for example methotrexate), inhibitors of PARP such as niraparib or rucaparib, panobinostat, a duocarmazin, seco-DUBA, exotoxin of Pseudomonas (PE), of deBouganin, of Bouganin, Diphtheria toxin (DT), and ricine. As inhibitor of tyrosine kinases, one may for example cite the antiangiogenic compounds such as for example nintedanib, axitinib or sunitinib, or also lapatinib, gefitinib, dasatinib, PD153035, ceritinib, crizotinib, lorlatinib, ibrutinib, and acalabrutinib.

[0049] As inhibitor of serine-threonine kinase, one may cite for example dabrafenib, binimetinib, or an inhibitor of CDK such as palbociclib or ribociclib, bortezomib, and anthracyclines as for example daunorubicin, daunorubicine analogues or PNU- 159682.

[0050] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_5 is an anticancer agent selected from the group consisting of: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exatecan, and derivatives thereof.

[0051] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_5 is an anticancer agent selected from the group consisting of: monomethyl auristatin E (MMAE), and derivatives thereof.

[0052] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_5 is an anticancer agent selected from the group consisting of: exatecan, and derivatives thereof.

[0053] Preferably, L5is represented by the following formula (A-1 ):

[0054] In one embodiment, the compounds of the invention have the above formula (I), or one of the above-mentioned formulae (1-1 ) or (I-2), wherein:

[0055] - L4has the formula (VI) as defined above and L5is monomethyl auristatin E; or

[0056] - L4has the formula (VIII) as defined above and L5is exatecan.

[0057] In one embodiment, in formula (I), or in one of the above-mentioned formulae (1-1 ) or (I-2), l_6 is a group having the formula -(CH2-CH2-O)n-CH3, n being 12 or 24. In one embodiment, the compounds of the invention have the following formula

[0058] (I-3): wherein:

[0059] - L4and L5are as defined above in formula (I),

[0060] - p and p’, independently from each other, are integers from 1 to 20, and

[0061] - n is an integer comprised from 1 to 24, and preferably equal to 12 or 24.

[0062] Preferably, p is comprised from 4 to 10, more preferably from 4 to 8, still more preferably from 4 to 6, notably p is equal to 4.

[0063] Preferably, p’ is comprised from 4 to 10, more preferably from 4 to 8, still more preferably from 4 to 6, notably p’ is equal to 5.

[0064] A preferred compound according to the invention has the following formula:

[0065] The present invention also relates to a conjugate comprising at least one compound of formula (I) as defined above, said compound being covalently bound to at least one cell binding agent, in particular selected from the antibodies and the antibody fragments.

[0066] The term “cell binding agent”, refers to a molecule with affinity for a biological target. The function of the binding agent is to direct the biologically active compound towards the biological target. The cell binding agent may be, for example, a ligand, a protein, an antibody, more particularly a monoclonal antibody, a protein or antibody fragment, a peptide, an oligonucleotide or an oligosaccharide. Preferably, the present invention relates to a conjugate comprising at least one compound of formula (I) as defined above, said compound being covalently bound to at least one cell binding agent selected from the antibodies and the antibody fragments. Such conjugate will also be named ADC or antibody-drug conjugate.

[0067] By “antibody”, also commonly called “immunoglobulin”, is meant a heterotetramer constituted by 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), joined together by intra- and interchain disulphide bridges. Each chain is constituted, in the N-terminal position, by a variable region or domain, called VL for the light chain, VH for the heavy chain and, in the C-terminal position, by a constant region, constituted by a single domain called CL for the light chain and of three or four domains called CH1 , CH2, CH3, CH4, for the heavy chain.

[0068] By “antibody fragment” is meant any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction.

[0069] The antibody according to the present invention can be a chimeric monoclonal antibody, humanized or human, monospecific or bispecific.

[0070] By “chimeric antibody” is meant an antibody in which the sequences of the variable regions of the light chains and of the heavy chains belong to a species different from that of the sequences of constant regions of the light chains and of the heavy chains. For the purposes of the invention, the sequences of the variable regions of the heavy and light chains are preferably of murine origin whereas the sequences of the constant regions of the heavy and light chains belong to a non-murine species. In this respect, for the constant regions, all the species of non-murine mammals can be used, and in particular human, monkey, Old-World swine, bovines, equines, felids, canines or birds, this list not being exhaustive.

[0071] Preferably, the chimeric antibodies according to the invention contain sequences of constant regions of heavy and light chains of human origin and the sequences of variable regions of heavy and light chains of murine origin.

[0072] By “humanized antibody” is meant an antibody for which some or all of the sequences of the regions involved in antigen recognition (the hypervariable regions or CDR: Complementarity Determining Region) and sometimes certain amino acids of the FR regions (FR: Framework Regions)) are of non-human origin whereas the sequences of the constant regions and variable regions not involved in antigen recognition are of human origin. By “human antibody” is meant an antibody containing only human sequences, both for the variable and constant regions of the light chains and for the variable and constant regions of the heavy chains.

[0073] In another particular embodiment of the invention, the antibody fragment is selected from the group constituted by: Fab, F(ab)'2, Fc, F'c, pFc', ScFv, Fv, Fd, Fabc, diabody, minibody, ScFv-Fc or ScFv-Fv.

[0074] Enzymatic digestion of the immunoglobulins by papain generates two identical fragments, which are called the Fab (Fragment antigen binding) fragments, and one Fc (Fragment crystallizable) fragment.

[0075] Enzymatic digestion of the immunoglobulins by pepsin generates a F(ab')2 fragment and an Fc fragment split into several peptides.

[0076] F(ab')2 is formed by two Fab' fragments linked by interchain disulphide bridges. The Fab parts are constituted by the variable regions and the CH1 and CL domains, whereas the Fc region is constituted by the two globular domains CH2 and CH3 (and CH4 when it is present).

[0077] The Fab' fragment is constituted by the Fab region and a hinge region. Fab'-SH refers to a Fab' fragment in which the cysteine residue of the hinge region bears a free thiol group (Carter et al., Nature BioTechnology 10: 163-167 (1992)). The Fv fragment is composed of the domains VH and VL linked by disulphide bridges. It is the smallest fragment retaining antigen binding activity.

[0078] The Fd fragment is formed by the VH and CH1 domains.

[0079] The scFv (single chain Fragment variable) is a fragment originating from protein engineering which is constituted only by the variable domains VH and VL. The structure is stabilized by a short flexible peptide arm, called a linker, which is placed between the two domains. The ScFv fragment can be bound to an Fc fragment in order in order to produce an ScFv-Fc or to an Fv fragment in order in order to produce an ScFv-Fv.

[0080] If the size of the binding peptide is reduced, new steric constraints appear between the scFvs; the VH and VL domains can no longer combine into a functional structure and multimeric structures are obtained (dimeric: “diabody”, trimeric: “triabody” and tetrameric: “tetrabody”).

[0081] In the context of the present invention, diabodies can be used. They can have multiple valences and specificities. The term “valent” denotes the presence of a defined number of antigen binding sites in an antibody molecule.

[0082] The term “specific” refers to the different types of antigens that can bind to the same antibody.

[0083] Thus, in the present invention, by “diabody” is meant an scFv dimer, said diabody being divalent, mono- or bispecific according to whether it binds two identical or different antigens.

[0084] The Fab, Fv and scFv fragments are monovalent and monospecific.

[0085] By “minibody” is meant a fragment composed of a light chain, of a heavy chain bound to a CH3 group.

[0086] In the context of the present invention, the antibody or antibody fragment is directed against a tumour antigen or inflammation antigen.

[0087] In a particular embodiment of the invention, the antibody or antibody fragment is directed against one of the antigens of the cluster of differentiation (CD), the identification number of which varies between CD1 a and CD363; in this list, the following CDs are preferred: CD1 a, CD363, CD3, CD4, CD13, CD19, CD20, CD21 , CD22, CD25, CD30, CD31 , CD33, CD34, CD37, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD79b, CD80, CD86, CD1 17, CD138, CD194, CD205, CD227 or CD248. The antibody or antibody fragment can also be directed against one of the antigens selected from the list formed by the following: CA125, G250, GD2, HLA-DRp, MUC1 , VEGF, VEGFA, VEGF-R1 / 2, TRAIL-R2 (DR5), EpCAM, GPIIb, GPIIIa, TNF alpha, TNFR, TNT, Lewis Y, EGFR, HER-2, HER-3, HER-3 MM-11 1 , HER-4, homodimer or heterodimer between members of the erbbn family (n between 1 and 4), AXL, Protein F, IgE-Fc, VEGF-A, integrin a4, integrin a4p7, integrin aV, C5, IL-6R, IL-6Ra, IL12, IL15, IL18, IL23, IL- 1 P, IL-1 , TPO-R, GPNMB, PSMA, PSA, PAP, PSM, integrin av, Cripto, TACSTD2 (TROP2 or EGP1 ), CEA, Folate receptor 1 , Mucin 16, Endothelin Receptor ETB, STEAP1 , SLC44A4 (AGS-5), Nectin 4, AGS-16, Guanylyl cyclase C, Mucin 1 , EGFRvlll, Mesothelin, IL2R, A33, Can, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGFR-1 , VEGFR-2, VEGFR-3, TGFP, TGFPR, FGF, FGF8b, FGFR, PDGF, PDGFR, PDGFRa, PDGFRp, Ang-1 , Ang-2, integrin, avp3, avp5, a3pi , a6p4, a2pi , anti-integrin a4, RANK-L, BLyS, c-MET, DR, DR10, TCRap, ICOS, CTLA-4, CAIX (MN), EphA2, CA6, ovarian CA6, cervical CA6, breast CA6, angiopoietin-2, Cripto, ENPP3, Mesothelin, FOLR1 , Nectin-4, TIM-1 , Muc-16, Tissue Factor, LIV-1 , GM2, a5 integrin, TLR-7, PD-1 , AFP, CA125 (MUC16), Sialyl LewisY, CAMPATH-1 , HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), TAG-72, Folate-binding protein, A33, G250, gangliosides (including GD2, GD3, GM2), LeY, collagen 4 (collagen IV), collagen 18 (collagen XVIII), SC6, CA-125, CA19-9, p185HER2, de2-7 EGFR, Fibroblast activation protein (FAP), Tenascin, metalloproteinases, Endosialin, Carbonic anhydrase, Galectin 9, Aldolase A, elFy4, Tyrosinase, Galectin 4, HERKV- K10, p53, NY-LU-12, Restin, NY-CO-38, MAGE-1 , MAGE-4a, SSX2, NY-ESO-1 , SCP-1 , HGFR, PTK 7, CCK-4, PDGFR, PTP-LAR, CDCP1 , CADM1 , IGSF4, Lu, BCAM, CEACAM6, JAM-A, PTGFRN (CD9P-1 ), MCAM, MUC18, MCP, EMMPRIN, TfR, TRAILR2, ClqR, hTERT, Survivin, MDM2, CYP1 B1 , Melan-A, MART-1 , MART- 2, Melanosomal proteins, gp1 OO, neo-PAP, CDC27, MAGEs, WT1 , MUM-1 , MUM-2, MUM-3, BRAF, TPI, fibronectin, K-ras, p-catenin, CDK4, caspase-8, p14ARF, p16INK4a, TGF RII, bcr-abl, SYT-SSX, TRP-1 , TRP-2, GnT-V, tyrosinase, FGF5, TEL-AML1 , Proteinase 3, HER2 / neu, AFP, MUC-1 , EBV-EBNA, HTLV-1 tax, HPV16- E7, mutated HLA-A2, HAL SART3, GnT-V, CEACAM5, AGS-16, GPNMB, ESAT-6, RANK, CanAg, fibrin, TF, PRAME, CA19-9, CA50, CA125, CA195, CAM17.1 / WGA, AFP, P2-MG, DU-PAN2, HE4, b-2 microglobulin, transferrin, transthyretin, ApoA1 , TROP-2, CTLA-4, GITR, PD-1 , PD-L1 , c-KIT, CD1 1 b-CD18 integrin heterodimer, DNA / Histon H1 , Folate, EpCAM, Tenascin-c, ECM (proteoglycan or fibronectin), fibrinogen, SV40 large T antigen, SC6-Ag, SC-Ag, DR4 (death receptor 4), DR5, ESA, mucin, hPAM4, hRS7, HLA-DR, CCR4, MTX1 , MTX2, PECAM, thrombomodulin Tn, cathepsin D, TYRO-3, MER.

[0088] The present invention also relates to the compound according to the invention, having the formula (I) as defined above or the conjugate as mentioned above, for use as drug.

[0089] The present invention also relates to a medicament comprising a compound of formula (I) as defined above or the conjugate as defined above, or a pharmaceutically acceptable salt thereof.

[0090] The present invention also relates to a pharmaceutical composition, comprising a compound of formula (I) as defined above or the conjugate as defined above, or a pharmaceutically acceptable salt thereof, and also at least one pharmaceutically acceptable excipient.

[0091] The present invention relates to a compound of formula (I) as defined above, or the above conjugate, for use as a medicine.

[0092] The present invention relates to a compound of formula (I) as defined above, or the above conjugate, for use as a drug. Although it is possible to administer the compounds of the invention of formula (I) or the conjugates alone, it is preferable to present them in the form of pharmaceutical compositions. Pharmaceutical compositions, both for veterinary and human use, useful according to the present invention comprise at least one compound of formula (I) as defined above or a conjugate, together with one or more pharmaceutically acceptable excipients or vehicles and optionally other therapeutic ingredients.

[0093] In some preferred embodiments, the active ingredients required for the combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.

[0094] As used herein, the term "pharmaceutically acceptable" and its grammatical variations, when referring to compositions, carriers, diluents and reagents, are used interchangeably and mean that the materials are capable of being administered to or on a mammal without producing adverse physiological effects, such as nausea, dizziness, gastric disturbances, etc.

[0095] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited on the basis of formulation. Typically, these compositions are prepared as injectable products either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in a liquid prior to use can also be prepared. The preparation may also be emulsified. In particular, the pharmaceutical compositions may be formulated in solid dosage form, e.g., capsules, tablets, pills, powders, dragees or granules.

[0096] The choice of vehicle and the content of active substance in the vehicle are generally determined by the solubility and chemical properties of the active compound, the particular mode of administration, and the requirements of pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates together with lubricants such as magnesium stearate, sodium lauryl sulfate and talc can be used for the preparation of tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used, they may contain emulsifying agents or agents that facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used. The compounds of formula (I) or conjugates, or the pharmaceutical compositions according to the present invention can be administered orally, parenterally (subcutaneous, intravenous or intramuscular) or locally by topical application to the skin and mucous membranes.

[0097] The conjugates, compounds or pharmaceutical compositions in accordance with the present invention may in particular be administered alone or in combination with chemotherapy or radiotherapy or in combination, for example, with other therapeutic agents, in particular anticancer and antimitotic agents, but also in combination with anti-inflammatory agents.

[0098] An appropriate dosage for the invention can be determined according to a routine approach normally used in the field of the invention. The adjustment of said dosage is clearly within the general competence of the man of the art.

[0099] It is indeed dependent, in particular, on the weight, age and sex of the individual to be treated, and on the state of evolution of the disease to be treated.

[0100] The present invention also relates to a compound having the formula (I) or to a conjugate as defined above, for its use for treating cancer.

[0101] The present invention also relates to a method for treating cancer, that comprises the administration of an effective dose of a compound of formula (I), a conjugate or a pharmaceutical composition according to the invention, to a patient in need thereof.

[0102] The present invention also relates to a method for treating cancer comprising the administration of a compound of formula (I), a conjugate as defined above, or a pharmaceutical composition according to the invention, in combination with another treatment selected from the group consisting of: a chemotherapy, a radiotherapy, a treatment with at least one anti-inflammatory agent, and combinations thereof.

[0103] A compound having the above formula (I), a conjugate according to the invention, or a pharmaceutical composition according to the invention, may be implemented for its use for the prevention and / or the treatment of a solid cancer (or solid tumor), preferably selected from the group consisting of: neuroblastoma, glioblastoma, osteosarcoma, retinoblastoma, soft tissue sarcoma, central nervous system cancer, nephroblastoma, lung cancer, breast cancer, prostate cancer, colorectal cancer, thyroid cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer liver cancer, brain cancer, testicular cancer, pancreatic cancer, bone cancer, skin cancer, small bowel cancer, stomach cancer, pleural cancer, esophageal cancer, laryngeal cancer, and bladder cancer. In one embodiment, the solid cancer is selected from the group consisting of: pancreatic cancer, lung cancer, and breast cancer, and more particularly is breast cancer.

[0104] FIGURES

[0105] In the figures and in the description, MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2- OMe, MC-Lys-PEGi2-OMe-(Cap-VC-PABC-MMAE) and m-(b-PEG12)-VC-MMAE are equivalent notations.

[0106] In the figures and in the description, MC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe, MC-Lys-PEGi2-OMe-(Cap-AA-PABC-EXA) and m-(b-PEG12)-AA-EXA are equivalent notations.

[0107] Figure 1 : HER2 binding affinities of lgG(8)-m-(b-PEG12)-VC-MMAE (A: square curve) and Mb(4)-m-(b-PEG12)-VC-MMAE (B: square curve) - obtained by bioconjugation of compound 16 (MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe) according to example 2.18 - in comparison to trastuzumab (lgG(8)) (A: circle black curve) or non-conjugated minibody (Mb(4)) (B: circle black curve)

[0108] Figure 2: HER2 binding affinities of anti-HER2 immunoconjugates lgG(8)-MC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (square curve) - obtained by bioconjugation of compound 24 (MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe) according to example 2.10 and 2.19 - in comparison to trastuzumab (lgG(8)) (diamond black curve), determined by indirect ELISA (MC = maleimidocaproic).

[0109] Figure 3: Internalization of (from left to right) T-DM1 , lgG(8)-m-(b-PEG12)-VC- MMAE, Mb(4)-m-(b-PEG12)-VC-MMAE and ppL-PE alone after complexation with ppL-PE in SK-BR-3 cell lines, as analyzed by flow cytometry (exposure time = 4 h at 37 °C (A) and 24 h at 37°C (B)). ANOVA test (**p<0.01 and ™p<0.0001).

[0110] Figure 4: Internalization of (from left to right) ppL-PE alone, irrelevant immunoconjugate lgGirr(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.11) (lgG(8)IRR-EXA), anti-HER2 immunoconjugate lgG(8)-MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe (according to examples 2.10 and 2.19) and trastuzumab deruxtecan (T-DXd) after complexation with ppL-PE in SK-BR-3 cell lines (figure 4B) and in HER2-negative MDA-MB-468 cells (figure 4A), as analyzed by flow cytometry (exposure time = 24 h at 37°C). ANOVA test (ns: non significative, and ™p<0.0001). Figure 5: In vitro cytotoxic activity of lgG(8)-m-(b-PEG12)-VC-MMAE (example 2.18.2) and Mb(4)-m-(b-PEG12)-VC-MMAE (example 2.18.1 .1 ) in comparison to their respective native entity (empty squares) and free MMAE (black curve) on SK-BR-3 (A) and MDA-MB-231 (B) cancer cell lines after five days of incubation at 37°C - CellTiter Gio assay (Promega®).

[0111] Figure 6: In vitro cytotoxic activity of anti-HER2 immunoconjugates lgG(8)-MC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (up triangle curve - examples 2.10 and 2.19) in comparison to irrelevant immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)- PEGi2-OMe (lgG(8)IRR-EXA (example 2.11 ), down triangle curve), trastuzumab deruxtecan (T-DXd, square curve) and free exatecan (cross curve) on SK-BR-3 (figure 6A) and HER2-negative MDA-MB-468 (figure 6B) cancer cell lines after five days of incubation at 37°C - CellTiter Gio assay (Promega®).

[0112] Figure 7: Schematic schedule and tumoural size evolution in BALB-c nude mice after (from top to bottom) one injection of PBS, one injection (3 mg / kg) of T-DM1 , one injection (3 mg / kg) of lgG(8)-m-(b-PEG12)-VC-MMAE (example 2.18.2) and five injections (3 mg / kg) of Mb(4)-m-(b-PEG12)-VC-MMAE (example 2.18.1.1 ). (A): Schematic representation of administration schedule of lgG(8)-m-(b-PEG12)-VC- MMAE and T-DM1 (B, b) and Mb(4)-m-(b-PEG12)-VC-MMAE. (B): Mice were injected at Day 0 with a mean tumoral volume of 160 mm3. The arrow corresponds to injections and the crossed out circle corresponds to mice sacrifice ANOVA test (**p<0.01 and »»»»p<0.0001 ).

[0113] Figure 8: Weight of the tumors at day 64 post-treatment (A), from left to right, T-DM1 , lgG(8)-m-(b-PEG12)-VC-MMAE (example 2.18.2), Mb(4)-m-(b-PEG12)-VC- MMAE (example 2.18.1.1 ) and placebo. Comparison of the size of isolated tumors after animal sacrifice in the different groups treated (B) by placebo (PBS), T-DM1 , lgG(8)-m-(b-PEG12)-VC-MMAE or Mb(4)-m-(b-PEG12)-VC-MMAE. ANOVA test (**p<0.01 , ™p<0.001 and ™ p<0.0001 ).

[0114] Figure 9: Deconvoluted mass spectra for anti-HER2 immunoconjugates lgG(8)- MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (examples 2.10 and 2.19), Mb(8)-MC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.12), Mb(4)-MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe (example 2.9), Db(4)-MC-Lys-(Cap-AA-PABC-EXA)- PEGi2-OMe (example 2.8) and irrelevant immunoconjugate lgG(8)IRR-MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe (example 2.1 1 ). Linker-cytotoxic 187 is MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe (compound 24).

[0115] Figure 10: Stability profile overtime in PBS at 4°C over 120 days for anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (examples 2.10 and 2.19), Mb(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.12) and irrelevant immunoconjugate lrr(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.1 1 ).

[0116] Figure 11 : Purity profile by size exclusion chromatography (SEC), indicating the percentage of monomer, for anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe (examples 2.10 and 2.19), Mb(8)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe (example 2.12), Mb(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.9), Db(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.8) and irrelevant immunoconjugate lgG(8)IRR-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.1 1 ).

[0117] Figure 12: HER2 binding affinities for anti-HER2 immunoconjugates lgG(8)- MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (examples 2.10 and 2.19), Mb(8)-MC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, Mb(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe and Db(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe determined by indirect ELISA.

[0118] Figure 13: Tumor / blood (T / B) and tumor / muscle (T / M) ratios overtime for [125l]- anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEG12-OMe (figure 13A) and [125l]-trastuzumab deruxtecan (figure 13B).

[0119] Figure 14: Tumoral volume evolution (in mm3) in Balb- / c nude mice after one injection (0.07 pmol / kg equivalent to 10.0 mg / kg of trastuzumab deruxtecan (T-DXd)), of anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgG(8)-mal-EXA, black square solid curve) in comparison to one injection of trastuzumab deruxtecan (T-DXd, gray circle solid curve), and two injections (0.07 pmol / kg equivalent to 10.0 mg / kg of T-DXd) of anti-HER2 immunoconjugates lgG(8)- MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (black diamond dotted curve) in comparison to two injections of trastuzumab deruxtecan (T-DXd, gray square dotted curve), or with PBS injection (black triangle curve), figure 14 being a zoomed view of a portion of figure 14A.

[0120] EXAMPLES

[0121] MATERIAL AND METHODS

[0122] All chemicals were obtained from commercial suppliers and were used without additional purification. All solvents were anhydrous reagents from commercial sources. TFA salt of VC-PABC-MMAE was purchased from Levena Biopharma (#T1004).

[0123] Trastuzumab (Ontruzant®, Samsung Bioepis) was kindly provided by the Hospital Pharmacy of the Tours Teaching Hospital. Thin layer chromatography (TLC) was performed using commercial pre-coated aluminum sheets silica gel (60 A, F254; Merk) and revealed by irradiation with light at 254 nm. Column chromatography was carried out on an ISCO purification unit, Combi Flash RF 75 PSI, with Redisep flash silica gel columns (60 A, 230-400 mesh, grade 9385). Purity of final compounds was determined by high performance liquid chromatography (HPLC). HPLC analyses were carried out with a LaChrom Elite system [Hitachi L-2130 (pump), L-2200 (autosampler) and L-2400 (UV-detector)], with a UV detection at 254 nm at 25 °C, and a XBridge C-18 column (250 x 10 mm, 4 pm, 135 A); elution was performed with (gradient 1 ) 0.1% trifluoroacetic acid (TFA) in water (solvent A), and 0.1% TFA in acetonitrile (MeCN) (solvent B), with a gradient from 20 to 100% of B over 35 minutes with a flow rate of 1 mL.min-1; injection was realized at 1 mg / mL in DMSO (10 pL). Final compounds were obtained in a purity > 95%. Semi-preparative HPLC was carried out on a Gilson PLC 2050 system [ARMEN V2 (pump), ECOM TOYDAD600 (UV-detector)], with a UV detection at 254 nm at 25 °C, and a Waters XBridge™ C- 18 column (250 mm x 19 mm, 5 pm); elution was performed with (gradient A) 0.1% TFA in water (solvent A), and 0.1% TFA in MeCN (solvent B); with a gradient from 20 to 100% of B over 32 min and then 100% of B for 6 min at 17.1 mL / min. NMR spectra were recorded at 300 MHz (1H) or 75 MHz (13C) on a Broker Avance (300 MHz) spectrometer, and at 400 MHz (1H) or 100 MHz (13C) on a Broker Avance Neo (400 MHz) spectrometer. The chemical shifts are reported in parts per million (ppm, 5) relative to residual deuterated solvent peaks. The abbreviations s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet and bs = broad signal were used throughout. For small-molecule mass analysis, high-resolution accurate mass spectrometry measurements (HRMS) were performed using an Acquity UPLC H- Class system hyphenated to a Vion IMS QTof mass spectrometer (Waters). Before MS analysis, 1 ng of sample was injected onto a BEH C18 column (2.1 x 50 mm, 1 .7 pm) heated to 50 °C. A 6 min gradient from 5% to 90% solvent B was applied with a 0.5 mL / min flow rate to elute the sample (solvent A: H2O + 0.1 % formic acid; solvent B: MeCN + 0.1% formic acid). MS data were acquired in positive mode with an ESI source over a 50-1400 m / z window with 0.2 Hz scan rate and collision energy ramp from 20 to 40 eV. Data were processed using IINIFI software version 1 .9.4.

[0124] Denaturing High-Resolution Mass Spectrometry (HRMS) of conjugates

[0125] Denaturing high-resolution mass spectrometric analyses of ADCs were performed on a Broker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, samples (ca. 5pg) were desalted on a MassPREP desalting cartridge (2.1 x10 mm, Waters) heated at 80°C using 0.1 % formic acid as solvent A and 0.1 %formic acid in MeCN as solvent B at 500 pL / min. After 1 min, a linear gradient from 5 to 90% B in 1 .5 min was applied; the first 1 .5 min were diverted to waste. HRMS data were acquired in positive mode with ESI source over the m / z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 software (Broker) and the MaxEnt algorithm for spectral deconvolution.

[0126] Size-Exclusion Chromatography (SEC) analysis

[0127] ADCs were diluted to 1 mg / mL with PBS pH 7.4 and filtered on a 0.22 pm PVDF membrane. A sample of size 40 pg was injected on an AdvanceBio SEC (7.8 x 300 mm, 2.7 pm) from Agilent Technologies, connected to a Waters Alliance (e2695) apparatus equipped with a photo-diode array detector (2998) set for detection at 280 nm. Samples were run with an isocratic gradient (1 mM potassium phosphate monobasic, 155 mM sodium chloride, 3mM sodium phosphate dibasic, 3 mM sodium azide, pH 7.0) over 24 min at a flow rate of 1 mL / min. The column oven temperature was maintained at 25°C.

[0128] General protocol of bioconjugation

[0129] BBS buffer

[0130] The conjugation buffer Borate Buffered Saline: BBS 1 X was made at pH 8.0 with 25 mM NaCI, 1 mM EDTA and was titrated with NaOH 1 M and purified through 0.2 pm membrane filtration. Before use, the pH of the solutions is checked with a pH meter and adjusted if necessary, with 0.1 M hydrochloric acid solution. The PBS buffer (Phosphate-Buffered Saline) 1X at pH 7.2 was used for the purification step of the ADC. The antibody trastuzumab was provided by le Centre Hospitalier Regional Universitaire (CHRU) de Tours.

[0131] Purification and buffer exchange of antibodies

[0132] Desalting and Buffer Exchange Procedure is proceed by selecting the appropriate MWCO (50 kDa for IgG before bioconjugation and 10 kDa for IgG- conjugates and FDCs (antibody Fragment-Drug Conjugates)) of centrifugal concentrator Vivaspin® SARTORIUS STEDIM biotech Polyethersulfone membrane - REF VS0102) 500 pL for the sample. When the sample is smaller than the maximum device volume, it can be diluted up to the maximum volume before the first centrifugation step. Centrifuge at 4°C for the recommended amount of time at an appropriate spin speed : 3 cycles of 3 min for FDCs at 10 000 rpm. 5-8-10 min for ADCs at 5xG.

[0133] TCEP reduction and linker-cytotoxic bioconjugation

[0134] The antibody solution at the desired concentration in borate buffer was added in HPLC vial of 1 .5 mL when the solution is up to 200 pL and in insert of 200 pL when the experimentation is in small scale. This insert is put into a 1 .5 mL HPLC vial fill with 750 pL of bioconjugation buffer and argonized. Disulfide bonds of proteins were reduced using a solution of TCEP ((tris(2-carboxyethyl)phosphine)) in BBS conjugation buffer at 1 mM : VTECP= neq * (CmAb*VmAb) / (MWmAb * [TCEP]). (VTCEP = volume of TCEP solution in pL; VmAb = volume of mAb solution in mL, mAb; MWmAb = Molecular Weight of the mAb in g / mol ; neq= number of equivalent relative to the mAb ; CmAb = concentration of mAb solution in mg / mL (or g / L); [TCEP] = concentration of TCEP solution in mmol / L; e.g. TTZ reduction with 6 eq of TCEP solution ([TCEP] = 1 mM) with CmAb = 4.5 mg / mL and VmAb = 0,2 mL: VTCEP = 6 * (4.5*0.2) / (148068*0,001 ) = 36 pL)

[0135] 5.9 mg of TCEP were solubilized in 2 mL of BBS buffer of bioconjugation to get a 10X solution with a concentration of 10 mmol / L (10 mM). The 10X solution was flushed under argon and a 10-fold dilution afforded the TCEP 1X solution (1 mM), which was kept under argon for storage. The TCEP volume desired was injected to the antibody solution under argon to prevent reoxidation of disulfide bridges. The solution mixture is incubated at 37°C for 1 h15 with agitation (550 rpm). Next, the desired linker-cytotoxic was prepared in dry DMSO to reach a concentration of 1 mM and added at the desired number of equivalents to the reduced antibodies (The percentage of organic solvent (DMSO) depends on the antibodies format, but in the majority of cases the percentage reach a maximum of 20% to prevent a possible effects on proteins). The reaction was incubated at 4°C (or 25°C or 37°C in some cases) under external mild agitation for 16h (or the time needed). Afterwards, the mixture was immediately purified.

[0136] Conjugates purification and final buffer exchange

[0137] Vivaspin® protocol for FDCs purification

[0138] Prepare a container filled with ice, immerse the vials FDCs solutions in it and set the centrifuge to 4 °C. Place the antibody solution in a Vivaspin® 500 and dilute by 2 the concentrate with a new buffer PBS, pH 7.4 - Because of the tendency of the small fragment antibody to precipitate at higher concentrations. Place it open in the centrifuge and perform 1 cycle of fast filtration “diafiltration steps in sequence” at 10 000 rpm (3min). While proteins are retained by ultrafiltration membrane, salts, TCEP and linker in excess can pass freely through. Remove the pellet and make up the solution in the Vivaspin® with the desired buffer to regain the starting volume. Homogenize the entire Vivaspin® solution and repeat the centrifugation cycle. Repeat until a minimum of 3 cycles is reached (2min + 2min).

[0139] Vivaspin® protocol for ADCs purification

[0140] The same procedure is done for ADCs except for the time of centrifugation and the speed of rotation: 3 cycles at 5xG of 5, 8 and 10 min were performed.

[0141] Superdex® purification

[0142] Size-exclusion chromatography (SEC) was performed on a Superdex® 200 10 / 300 GL, column (1 .0 x 300 mm, 13 pM, molecular mass range 10, 000 - 600,000) from Cytiva (GE Healthcare Life Sciences, 17-5174-01 ), connected to an Akta purifier (Cytiva). The column was used at a rate of 0.8 mL / min with a UV detector at 280 nm. After loading the desired conjugate, the column was eluted with PBS, pH 7.4 for 0.2 CV, then with 0.3 CV of H2O / ACN (7 : 3) + 0.2% TFA and finally with PBS pH 7.4 (1 CV).

[0143] Determination of conjugates concentrations and alternative way to DAR determination

[0144] Antibody trastuzumab and ADCs concentrations were determined by UV absorbance using NanoDrop-spectrophotometer (Thermo Fisher Scientific). SDCs and FDCs concentrations were determined by UV absorbance using UV biophotometer. When exatecan was used the method of determination of conjugates concentrations change.

[0145] The basis for quantitative analysis in the UV / Vis spectrophotometric experimentation is the Beer-Lambert law:

[0146] A = sCl where A is the absorbance, E is the extinction coefficient (L / mol / cm), / is the path length through the cell containing the analyte (usually 1 cm), and c is the concentration (mol / L).

[0147] We could also apply the Beer-Lambert law to a multicomponent system. In this case, the linker and the protein have different absorption spectra and we approximate no interactions among these components. The total absorbance of the solution at a given wavelength (A) is the sum of the individual absorbance for each species:

[0148] Atot =AmAb +Adrug (1)

[0149] The first step is the determination of the extinction coefficient (E) of the linker at two wavelengths, the first one is at 280 nm were both linker and mAb absorbed. The second one is at 400 nm which is the wavelength were the linker absorbance is the highest. To determine this coefficient we used a calibration line in our concentrations range.

[0150] Then we determine the concentration of the drug:

[0151] The concentration of conjugate is directly linked to the concentration of mAb which is determined with the parallel equation (3) of equation (1):

[0152] The average drug to antibody ratio is expressed as moles of drugs to moles of antibody calculated with (4):

[0153] Sample preparation for biological evaluation and storage

[0154] The protein sample can be concentrated by Vivaspin® or diluted in PBS buffer to reach the desired concentration. The samples were stored at 4 °C. EXAMPLE 1 : PREPARATION OF COMPOUNDS ACCORDING TO THE INVENTION

[0155] Example 1.1. Synthesis of MC-Lys-PEGi2OMe(Cap-VA-PABC-EXA)

[0156] 6-Maleimidohexanoic acid (1)

[0157] Chemical Formula: C10H13NO4Molecular Weight: 211,22

[0158] Maleic anhydride (2.00 g, 20.40 mmol) and 6-aminocaproic acid (2.68 g, 20.40 mmol) were stirred under argon in 20 mL of acetic acid. The reaction medium is heated to reflux and stirred overnight. On next day, the mixture turned orange. The acetic acid was co-evaporated three times with toluene to give a crud product as an orange viscous oil. The crude product was taken up in DCM and washed with H2O. The aqueous phase was re-extracted two times with DCM and the combined organic phases were dried over MgSO4 and evaporated under vacuum then purified through combiflash (sorbent 80 g) with DCM / MeOH (0 to 4%) to give the desired product 1as white solid (1 .92 g, 45%). HPLC purity = 99.07%, tR = 8.53 min.

[0159] 1H NMR (300 MHz, CDCI3) 66.69 (s, 2H), 3.52 (t, 2H, J= 7.2 Hz), 2.34 (t, 2H, J = 7.4 Hz), 1 .63 (m, 4H), 1 .42-1 .27 (m, 2H).

[0160] 13C NMR (75 MHz, CDCI3) 5 179.4, 171.4, 134.6, 38.2, 34.2, 28.7, 26.7, 24.7.

[0161] (S)-6-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxy- carbonyl)amino)hexanamido)hexanoic acid (3)

[0162] Chemical Formula: C32H43N3O7

[0163] Molecular Weight: 581 ,71 To 2 mL of DMF were added aminocaproic acid (53.3 mg, 0.407 mmol, 1 eq) and Fmoc-Lys(Boc)-OH (200.1 mg, 0.427 mmol, 1.05 eq). HOBt (60.6 mg, 0.448 mmol, 1 .1 eq) and DIPEA (7.0 pL, 0.41 mmol, 0.1 eq) were diluated into the solution and EDC.HCI (86.0 mg, 0.448 mmol, 1.1 eq) was added. The solution was stirred overnight at room temperature. Once the reaction was over, 50 mL of water were added to the solution which was subsequently extracted 3 times with 50 mL of DCM.

[0164] Organic layers were dried over MgSO4, concentrated under vacuum and the resulting oil was purified by flash chromatography (DCM / MeOH - 1 to 15%) to afford a yellow oil (264.1 mg, 0.454 mmol, quant.)

[0165] 1H NMR (400 MHz, DMSO-cfe) 5 11 .63 (s, 1 H), 7.90 (d, J = 7.5 Hz, 2H), 7.83 (t, J = 5.6 Hz, 1 H), 7.77-7.57 (m, 2H), 7.47-7.36 (m, 3H), 7.33 (t, J = 7.5 Hz, 2H), 6.76

[0166] (t, J = 5.3 Hz, 1 H), 4.31—4.16 (m, 3H), 3.89 (dd, J = 13.8, 8.4 Hz, 1 H), 3.11-2.96 (m, 2H), 2.95-2.83 (m, 2H), 2.18 (t, J = 7.3 Hz, 2H), 1.63-1.16 (m, 21 H).

[0167] HRMS (ESI+) calcd for C32H44N3O7 (M+H+) 582.3179, found 582.3223.

[0168] (S)-6-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-amino- hexanamido)hexanoic acid (4)

[0169] Chemical Formula: C2? H35N3O5 Molecular Weight: 481,59

[0170] To a solution of 3 (780.0 mg, 1.31 mmol, 1eq) in DCM (15 mL) were added 3.0 mL (20%v) of TFA. The mixture was stirred 3h at room temperature. Volatiles were removed and the crude product was dried to afford an orange oil (TFA salt) (288.5 mg, 0.484 mmol, quant.).

[0171] 1H NMR (400 MHz, DMSO-cfe) 57.90 (d, J= 7.5 Hz, 2H), 7.85 (t, J= 5.4 Hz, 1H), 7.73 (t, J = 6.9 Hz, 2H), 7.65 (s, 3H), 7.43 (t, J = 7.3 Hz, 3H), 7.33 (t, J = 7.4 Hz, 2H), 4.30-4.19 (m, 3H), 3.91 (td, J = 8.6, 5.3 Hz, 2H), 3.11-2.96 (m, 2H), 2.81-2.71 (m, 2H), 2.18 (t, J= 7.3 Hz, 2H), 1.66-1.24 (m, 12H).

[0172] 13C NMR (101 MHz, DMSO-cfe) 5174.9 (1C), 172.0 (1C), 159.3 (1C), 158.9 (1C), 158.5 (1C), 158.2 (1C), 156.4 (1C), 144.3 (1C), 144.2 (1C), 141.2 (1C), 128.1 (1C), 127.5 (1C), 125.7 (2C), 120.6 (1C), 66.0 (1C), 54.9 (1C), 47.1 (1C), 39.1 (1C), 38.7 (1C), 34.0 (1C), 31.9 (1C), 29.2 (1C), 27.1 (1C), 26.3 (1C), 24.6 (1C), 22.9 (1C).

[0173] 19F NMR (376 MHz, DMSO-c / 6) 5 -74.14.

[0174] HRMS (ESI+) calcd for C27H36N3O5 (M+H+) 482.2781 , found 482.2646. (S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38,45-dioxo-

[0175] 2, 5, 8,11 ,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazadopentacontan-52-oic acid (5)

[0176] Chemical Formula: C53H85N3O 18

[0177] Molecular Weight: 1052,27

[0178] To 500 pL of DMF were added 4 (21 .7 mg, 0.0365 mmol, 1 eq) and NHS-PEG12- OMe (25.0 mg, 0.0365 mmol, 1 eq). DIPEA (12.5 pL, 0.073 mmol, 2 eq) was added and the solution was stirred overnight at room temperature. The reaction was monitorred by analytic-HPLC. The solution was then purified by semipreparative HPLC (gradient A) to afford a clear oil (22.9 mg, 0.022 mmol, 60%).

[0179] 1H NMR (400 MHz, DMSO-cfe) 5 7.89 (d, = 7.5 Hz, 2H), 7.86-7.77 (m, 2H), 7.73 (dd, J= 7.3, 3.8 Hz, 2H), 7.40 (dd, J= 16.5, 8.7 Hz, 3H), 7.32 (t, J= 7.1 Hz, 2H), 4.34-4.15 (m, 3H), 3.94-3.84 (m, 1 H), 3.63-3.47 (m, 46H), 3.23 (s, 3H), 3.10-2.96

[0180] (m, 4H), 2.28 (t, J = 6.5 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.62-1.16 (m, 12H).

[0181] HRMS (ESI+) calcd for C53H86N3O18 (M+H+) 1052.5906, found 1052.5908

[0182] (9H-fluoren-9-yl)methyl((S)-3-methyl-1 -(((S)-1 -((4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1 - oxobutan-2-yl)carbamate (6)

[0183] Chemical Formula: C37H36N4O9Molecular Weight: 680,71

[0184] To oven-dried glassware Fmoc-ValAla-PAB (100.0 mg, 0.194 mmol, 1 eq) was dissolved in pyridine (6 mL) and subsequently bi-PNP carbonate (354.1 mg, 1.164 mmol, 6 eq) was added. The mixture was stirred at room temperature overnight and the solvent was removed under reduced pressure to afford a brown oil. The oil was purified by chromatofash (DCM / MeOH -1 to 2%) to afford a yellow powder (60.0 mg,

[0185] 0.088 mmol, 45%).

[0186] 1H NMR (400 MHz, DMSO-cfe) 5 10.08 (s, 1 H), 8.34-8.28 (m, 2H), 8.20 (d, J = 6.9 Hz, 1 H), 7.89 (d, J= 7.5 Hz, 2H), 7.74 (t, J= 7.2 Hz, 2H), 7.63 (d, J= 8.5 Hz, 2H), 7.59-7.53 (m, 2H), 7.46-7.36 (m, 6H), 7.32 (t, J = 7.4 Hz, 1 H), 5.24 (s, 2H), 4.47- 4.38 (m, 1 H), 4.34-4.18 (m, 3H), 3.95-3.87 (m, 1 H), 2.04-1.93 (m, 1 H), 1.31 (d, J =

[0187] 7.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H).

[0188] 1H NMR in accordance with literature. (Hochdorffer K. & Al, J. Med. Chem. 2012, 55, 17, 7502-7515)

[0189] (S)-1 -(((S)-1 -((4-(((((1 S,9S)-9-ethyl-5-f luoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1 ,2-b]quinolin-1 -yl)carbamoyl)oxy)methyl)phenyl)amino)-1 - oxopropan-2-yl)amino)-3-methyl-1 -oxobutan-2-aminium (7)

[0190] Chemical Formula: C4oH43FN608Molecular Weight: 754,82

[0191] 880 pL of DMF / pyridine (8 / 2) were used to dissolve 6 (53.7 mg, 0.079 mmol, 1 .2 eq). To the solution Exatecan mesylate (35.1 mg, 0.066 mmol, 1 .0 eq), HOBt (8.9 mg, 0.066 mmol, 1.0 eq), DIPEA (1 1.5 pL, 0.066 mmol, 1.0 eq) were dissolved and the mixture was stirred 2 hours at 40 °C. Piperidine (20%v) was added and the solution was stirred 3 hours at room temperature. The mixture was purified by preparative HPLC (gradient A) to afford the product as a yellow paste (TFA salt) (49.0 mg, 0.057 mmol, 86%).

[0192] 1H NMR (400 MHz, DMSO-cfe) 5 10.16 (s, 1 H), 8.71 (d, = 7.0 Hz, 1 H), 8.07 (d, J = 5.0 Hz, 4H), 7.78 (d, J = 10.9 Hz, 1 H), 7.59 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (s, 1 H), 5.45 (s, 2H), 5.36-5.20 (m, 3H), 5.14-5.04 (m, 2H), 4.51 (p, J = 6.9 Hz, 1 H), 3.30-3.05 (m, 4H), 2.38 (s, 3H), 2.26-2.04 (m, 3H), 1 .95-1 .80 (m, 2H), 1.36 (d, J = 7.1 Hz, 3H), 0.96 (dd, J = 6.8, 4.2 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0193] 19F NMR (377 MHz, DMSO-cfe) 5 -74.51 , -1 11 .32.

[0194] HRMS (ESI+) calcd for C4oH44FN608 (M+H+) 755.3199, found 755.3201 . 4-((44S,54S,57S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-54- isopropyl-57-methyl-38,45,52,55-tetraoxo-2,5,8,11 ,14,17,20,23,26,29,32,35- dodecaoxa-39,46,53,56-tetraazaoctapentacontan-58-amido)benzyl((1 S,9S)-9- ethyl-5-f luoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 11-1,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)carbamate (8’)

[0195] Chemical Formula: CggH^eFNgC^g Molecular Weight: 1789,07

[0196] To 900 pL of DMF were dissolved 7 (13.9 mg, 0.016 mmol, 1 eq) before the addition of 5 (22.1 mg, 0.021 mmol, 1 .3 eq). HATLI (8.0 mg, 0.021 mmol, 1 .3 eq) was dissolved and finally, 2,6-Lutidine (2.43 pL, 0.021 mmol, 1.3 eq) was added to the mixture. The solution was stirred 72h at room temperature. The solution was purified by semi-preparative HPLC (80 / 20 gradient method) to afford a green paste (13.0 mg, 0.0073 mmol, 46%). 1H NMR (400 MHz, DMSO-cfe) 59.93 (s, 1 H), 8.15 (d, J = 6.9 Hz, 1 H), 8.05 (d,

[0197] J= 8.8 Hz, 1 H), 7.87 (d, J= 7.5 Hz, 2H), 7.84-7.74 (m, 4H), 7.72 (dd, J= 7.3, 3.4 Hz, 2H), 7.59 (d, J= 8.5 Hz, 2H), 7.46-7.25 (m, 8H), 5.44 (s, 2H), 5.34-5.22 (m, 3H), 5.07 (s, 2H), 4.38 (p, J = 7.0 Hz, 1 H), 4.29-4.13 (m, 4H), 3.94-3.83 (m, 1 H), 3.57 (t, J = 6.5 Hz, 2H), 3.53-3.44 (m, 44H), 3.23 (s, 3H), 3.13-2.92 (m, 6H), 2.37 (s, 3H), 2.28 (t, J = 6.5 Hz, 2H), 2.23-2.06 (m, 4H), 2.05-1.76 (m, 4H), 1.63-1.43 (m, 4H), 1.42- 1.16 (m, 1 1 H), 0.91-0.80 (m, 9H).

[0198] 19F NMR (377 MHz, DMSO-cfe) 5 -74.53, -1 11 .36 (d, J= 10.4 Hz).

[0199] HRMS (ESI+) calcd for C93H127FN9O25 (M+H+) 1788.8922, found 1788.8908

[0200] According to the procedure described previously we obtained approximatively 75% of drug release for the enzymatic reaction with Cathepsin B over 16 hours.

[0201] (44S,54S,57S)-58-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1 ,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-54- isopropyl-57-methyl-38,45,52,55,58-pentaoxo-2,5,8,11 ,14,17,20,23,26,29,32,35- dodecaoxa-39,46,53,56-tetraazaoctapentacontan-44-aminium (9’)

[0202] Chemical Formula: C78H116FNgO23 Molecular Weight: 1566,82

[0203] 8’ (10.2 mg, 0.0057 mmol) was dissolved in 500 pL of DMF. Piperidine (100 pL, 20%v) was added to the mixture which was stirred during 3 hours. The solution was purified by semi-preparative HPLC (gradient A) to afford a yellow paste (TFA salt) (7.4 mg, 0.0044 mmol, 77%)

[0204] 1H NMR (400 MHz, DMSO-cfe) 5 9.95 (s, 1 H), 8.35 (t, J = 5.5 Hz, 1 H), 8.16 (d, J = 6.9 Hz, 1 H), 8.04 (s, 4H), 7.85-7.73 (m, 3H), 7.59 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.31 (s, 1 H), 5.44 (s, 2H), 5.28 (s, 3H), 5.08 (s, 2H), 4.43-4.33 (m, 1 H), 4.19 (dd, J = 8.6, 6.9 Hz, 1 H), 3.60-3.38 (m, 46H), 3.23 (s, 3H), 3.17-2.95 (m, 5H), 2.37 (s, 3H), 2.29 (t, J = 6.5 Hz, 2H), 2.25-2.06 (m, 4H), 2.01-1.81 (m, 3H), 1.69- 1.64 (m, 2H), 1.56-1.20 (m, 15H), 0.92-0.78 (m, 9H).

[0205] 19F NMR (377 MHz, DMSO-cfe) 5 -74.51 , -1 11 .32 (d, J= 10.4 Hz). 4-((44S,54S,57S)-44-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido) -54-isopropyl-57-methyl-38,45,52,55-tetraoxo-2,5,8,11 ,14,17,20,23,26,29,32,35- dodecaoxa-39,46,53,56-tetraazaoctapentacontan-58-amido)benzyl((1 S,9S)-9- ethyl-5-f luoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 11-1,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)carbamate (10’) Linker MC-Lys-PEGi2OMe(Cap-VA-PABC-EXA)

[0206] Chemical Formula: CggH-^FNioC^B Molecular Weight: 1760,03

[0207] To 800 pL of DMF were dissolved 9’ (7.4 mg, 0.0044 mmol, 1 eq) before the addition of 1 (1.4 mg, 0.0066 mmol, 1.5 eq). HATLI (5 mg, 0.0132 mmol, 3 eq) was dissolved and finally, 2,6-Lutidine (2.5 pL, 0.022 mmol, 5 eq) was added to the mixture. Few milligrams of HOBt were also added. The solution was stirred overnight at room temperature. The solution was purified by semi-preparative HPLC (gradient A) to afford a yellow green paste (3.0 mg, 0.0017 mmol, 39%).

[0208] 1H NMR (400 MHz, DMSO-cfe) 59.93 (s, 1 H), 8.15 (d, J = 6.8 Hz, 1 H), 8.05 (d, J = 8.6 Hz, 1 H), 7.85-7.68 (m, 5H), 7.59 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.31 (s, 1 H), 7.00 (d, J = 3.7 Hz, 2H), 6.51 (s, 1 H), 5.45 (s, 2H), 5.29 (s, 3H), 5.07 (s, 2H), 4.44-4.31 (m, 1 H), 4.21^.11 (m, 2H), 3.60-3.38 (m, 46H), 3.23 (s, 3H), 3.03- 2.95 (m, 4H), 2.38 (s, 3H), 2.28 (t, J = 6.5 Hz, 2H), 2.22-2.06 (m, 7H), 2.04-1 .80 (m, 4H), 1 .57-1 .1 1 (m, 23H), 0.92-0.78 (m, 9H).

[0209] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .36 (d, J = 11 .2 Hz).

[0210] HRMS (ESI+) calcd for C88Hi28FNi0O26 (M+H+) 1759.8980, found 1759.8974 Example 1.2. Synthesis of linker MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) tert-butyl (((9 H-f luoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (16’)

[0211] Chemical Formula: C25H3QN2O5 Molecular Weight: 438,52

[0212] To 4 mL of DMF was dissolved FmocAla-OH (250.1 mg, 0.803 mmol, 1.3 eq) before the addition of Ala-OtBu.HCI (112.0 mg, 0.618 mmol, 1 eq). HATLI (305.3 mg, 0.803 mmol, 1 .3 eq) was dissolved and finally, 2,6-Lutidine (93.0 pL, 0.803 mmol, 1 .3 eq) was added to the mixture. The solution was stirred over weekend at room temperature.

[0213] Once the reaction was over, DMF was removed under reduced pressure and the crude product was purified by flash chromatography (DCM / MeOH - 1 to 5%) to afford a translucent paste (130.6 mg, 0.298 mmol, 48%)

[0214] 1H NMR (400 MHz, DMSO-cfe) 5 8.16 (d, J = 6.9 Hz, 1 H), 7.89 (d, J = 7.5 Hz, 2H), 7.78 - 7.69 (m, 2H), 7.49 (d, J = 7.9 Hz, 1 H), 7.42 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.31 - 4.02 (m, 5H), 1 .38 (s, 9H), 1 .28-1 .20 (m, 6H).

[0215] 13C NMR (101 MHz, DMSO-cfe) 5 172.4 (s, 1 C), 171.7 (s, 1 C), 155.6 (s, 1 C), 143.9 (s, 1 C), 143.8 (s, 1 C), 140.7 (s, 2C), 127.6 (s, 2C), 127.1 (s, 2C), 125.3 (s, 2C), 120.1 (s, 2C), 80.3 (s, 1 C), 65.6 (s, 1 C), 49.6 (s, 1 C), 48.6 (s, 1 C), 46.6 (s, 1 C), 27.6 (s, 3C), 18.2 (s, 1 C), 16.9 (s, 1 C).

[0216] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (17)

[0217] Chemical Formula: C21H22N2O5 Molecular Weight: 382,42 To a solution of 16’ (125.4 mg, 0.286 mmol, 1 eq) in DCM (5 mL) were added 1 mL of TFA. The mixture was stirred 3h at room temperature. Volatiles were removed and the crude product was dried to afford a yallowy paste (175.3 mg, 0.35 mmol, quant.)

[0218] 1H NMR (400 MHz, DMSO-cfe) 5 12.51 (s, 1 H), 8.12 (d, J= 7.3 Hz, 1 H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 7.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 1 H), 7.42 (t, J = 7.2 Hz, 2H), 7.33 (td, J = nA, 0.9 Hz, 2H), 4.30-4.14 (m, 4H), 4.08 (p, J = 7.1 Hz, 1 H), 1.27 (d, J = 7.3 Hz, 3H), 1 .22 (d, J= 7.1 Hz, 3H).

[0219] 1H NMR in accordance with literature (Bukya H. & Al, Eur. J. Org. Chem., 2020, 5358-5362).

[0220] 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (18)

[0221] Chemical Formula: C13H23NOSi Molecular Weight: 237,42

[0222] To a solution of 4-aminophenyl)methanol (300.1 mg, 2.44 mmol, 1 eq) in DMF (8.5 mL) was added DMAP (98.3 mg, 0.804 mmol, 0.3 eq) and triethylamine (0.41 mL, 2.92 mmol, 1 .2 eq). Tertbutylchlorodimethylsilane (403.2 mg, 2.68 mmol, 1.1 eq) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered to remove the salt. The filtrate was concentrated and dissolved in EtOAc (20 mL), washed with water (3x 10 mL) and organic layers were dried over MgSC . Filtration and concentration afforded our product as a yellow oil (301 .2 mg, 1.27 mmol, 52%)

[0223] 1H NMR (400 MHz, DMSO-cfe) 5 6.94 (d, J = 8.3 Hz, 2H), 6.51 (d, J = 8.3 Hz, 2H), 4.98 (s, 2H), 4.49 (s, 2H), 0.87 (s, 9H), 0.03 (s, 6H).

[0224] 1H NMR in accordance with literature (W02009011850). (9H-f luoren-9-yl)methyl((S)-1 -(((S)- 1 -((4-(hydroxymethyl)phenyl)amino)-1 - oxopropan-2-yl)amino)-1 -oxopropan-2-yl)carbamate (19)

[0225] Chemical Formula: C28H29N3O5 Molecular Weight: 487,56

[0226] A mixture of compound 17 (119.5 mg, 0.31 mmol, 1 eq), EEDQ (153.3 mg, 0.62 mmol, 2 eq), PAB-OH (176.7 mg, 1.43 mmol, 4.6 eq) diluted in DCM / MeOH 2:1 was stirred at r.t. overnight. Volatiles were removed under reduced pressure and the crude product was sonicated and triturated in Et20. The product was collected by filtration and washed with Et20. A white powder was afforded (135.2 mg, 0.277 mmol, 89%).

[0227] 1H NMR (400 MHz, DMSO-cfe) 59.88 (s, 1 H), 8.10 (d, J = 7.2 Hz, 1 H), 7.90 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 8.0 Hz, 2H), 7.60 - 7.49 (m, 3H), 7.42 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 5.10 (t, J = 5.7 Hz, 1 H), 4.47 - 4.35 (m, 3H), 4.32 - 4.18 (m, 3H), 4.10 (p, J = 7.1 Hz, 1 H), 1.31 (d, J = 7.1 Hz, 3H), 1.24 (d, J = 7.1 Hz, 3H).

[0228] 1H NMR in accordance with literature (Widdison W. C., Bioconjugate Chem., 2015, 26, 11 , 2261-2278).

[0229] HRMS (ESI+) calcd for C28H30N3O5 (M+H+) 488.2180, found 488.2180

[0230] (9H-f luoren-9-yl)methyl ((S)-1 -(((S)-1 -((4-((((4-nitrophenoxy)carbonyl)oxy) methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-1 -oxopropan-2-yl)carbamate (20)

[0231] Chemical Formula: C35H32N4O9 Molecular Weight: 652,66 To oven-dried glassware was added 19 (100.2 mg, 0.205 mmol, 1 eq), pyridine (6 mL) and subsequently bi-PNP carbonate (374.2 mg, 1 .23 mmol, 6 eq). The mixture was stirred at room temperature overnight and the solvent was removed under reduced pressure to afford a brown oil.

[0232] The oil was purified by chromatoflash (DCM / 1 -2%MeOH) to afford a yellow powder as our product (118.8 mg, 0.182 mmol, 89%).

[0233] 1H NMR (400 MHz, DMSO-cfe) 5 10.05 (s, 1 H), 8.58 (d, J = 4.2 Hz, 2H), 8.36 - 8.28 (m, 2H), 8.15 (d, J = 7.1 Hz, 1 H), 7.89 (d, J = 7.4 Hz, 2H), 7.85 - 7.53 (m, 6H), 7.44 - 7.31 (m, 5H), 5.25 (s, 2H), 4.44 - 4.35 (m, 1 H), 4.28 - 4.22 (m, 3H), 4.15 - 4.05 (m, 1 H), 1.32 (d, J= 7.1 Hz, 3H), 1.24 (d, = 6.8 Hz, 3H).

[0234] HRMS (ESI+) calcd for C35H33N4O9 (M+H+) 653.2242, found 653.2242.

[0235] 4-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl((1S,9S)-9-ethyl-5- f luoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)carbamate (21)

[0236] Chemical Formula: C38HsgFNeOs Molecular Weight: 726,76

[0237] 880 pL of DMF / pyridine (8 / 2) were used to dissolve 20 (52.2 mg, 0.079 mmol, 1 .2 eq). To the solution Exatecan mesylate (35.1 mg, 0.066 mmol, 1 .0 eq), HOBt (9.0 mg, 0.066 mmol, 1.0 eq) and DIPEA (8.5 mg, 0.066 mmol, 1.0 eq) were added and the mixture was stirred 2 hours at 40 °C.

[0238] To the previous solution 20%v of piperidin were added and the solution was stirred 20 minutes at RT.

[0239] The mixture was purified by semi-preparative HPLC (gradient A) to afford a flashy yellow paste (TFA salt) (40.1 mg, 0.048 mmol, 73%)1H NMR (400 MHz, DMSO-c / 6) <5 10.15 (s, 1 H), 8.67 (d, J = 7.2 Hz, 1 H), 8.08- 8.01 (m, 4H), 7.79 (d, J = 10.9 Hz, 1 H), 7.60 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (s, 1 H), 5.45 (s, 2H), 5.35-5.28 (m, 3H), 5.13-5.05 (m, 2H), 4.49 (m, 1 H), 3.92-3.82 (m, 1 H), 3.15-3.00 (m, 1 H), 2.39 (s, 3H), 2.25-2.13 (m, 4H), 1 .92-1 .83 (m, 2H), 1 .36 (m, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0240] 19F NMR (377 MHz, DMSO-cfe) 5 -73.93, -111 .32 (d, J= 10.9 Hz).

[0241] HRMS (ESI+) calcd for CssH^FNeOs (M+H+) 727.2897, found 727.2886.

[0242] 4-((44S,54S,57S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-54,57- dimethyl-38,45,52,55-tetraoxo-2,5,8,11 ,14,17,20,23,26,29,32,35-dodecaoxa- 39,46,53,56-tetraazaoctapentacontan-58-amido)benzyl((1S,9S)-9-ethyl-5-fluoro- 9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1 -yl)carbamate (22)

[0243] Chemical Formula: Cgf H-^FNgC^s Molecular Weight: 1761 ,01

[0244] To 900 pL of DMF were dissolved 21 (13.5 mg, 0.016 mmol, 1 eq) before the addition of 5 (22.0 mg, 0.021 mmol, 1 .3 eq). HATLI (8.0 mg, 0.021 mmol, 1 .3 eq) and HOBt (4.3 mg, 0.032 mmol, 2 eq) were dissolved and finally, 2,6-Lutidine (2.43 pL, 0.021 mmol, 1 .3 eq) was added to the mixture. The solution was stirred 72h at room temperature. The solution was purified by semi-preparative HPLC (80 / 20 gradient method) to afford a yellow paste (20.0 mg, 0.0114 mmol, 71%).

[0245] 1H NMR (400 MHz, DMSO-d6) 5 9.90 (s, 1 H), 8.08-8.05 (m, 3H), 7.99 (d, J = 7.2 Hz, 1 H), 7.87 (d, J = 7.6 Hz, 2H), 7.83-7.76 (m, 3H), 7.72 (dd, J = 7.6, 3.6 Hz, 2H), 7.62 (d, J= 8.4 Hz, 2H), 7.42-7.27 (m, 8H), 5.45 (s, 2H), 5.35-5.22 (m, 3H), 5.08 (s, 2H), 4.37 (m, 1 H), 4.31-4.16 (m, 4H), 3.89 (m, 1 H), 3.60-3.40 (m, 46H), 3.23 (s, 3H), 3.15-2.95 (m, 5H), 2.38 (s, 3H), 2.32-2.15 (m, 8H), 1.95-1.80 (m, 2H), 1.65- 1.29 (m, 15H), 1.20 (d, J= 7.2 Hz, 3H), 0.88 (t, J= 7.2 Hz, 3H).

[0246] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .32 (d, J = 10.5 Hz).

[0247] HRMS (ESI+) calcd for C91H123FN9O25 (M+H+) 1760.8609, found 1760.8592

[0248] According to the procedure described previously (Gikanga, B.; Adeniji, N. S.; Patapoff, T. W.; Chih, H.-W.; Yi, L. Cathepsin B Cleavage of VcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific. Bioconjug. Chem. 2016, 27 (4), 1040-1049. https: / / doi.org / 10.1021 / acs.bioconjchem.6b00055.), we obtained approximatively 100% of drug release for the enzymatic reaction with Cathepsin B over 16 hours.

[0249] (44S,54S,57S)-58-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-

[0250] 10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1 ,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-54,57- dimethyl-38,45,52,55,58-pentaoxo-2,5,8,11 ,14,17,20,23,26,29,32,35-dodecaoxa-

[0251] 39,46,53,56-tetraazaoctapentacontan-44-aminium (23)

[0252] Chemical Formula: CygHi-^FNgC^a Molecular Weight: 1538,77

[0253] 22 (10.2 mg, 0.0056 mmol) was dissolved in 500 pL of DMF. Piperidine (100 pL, 20%v) was added to the mixture which was stirred during 3 hours. The solution was purified by semi-preparative hplc (gradient A) to afford a yellow paste (TFA salt) (7.3 mg, 0.0044 mmol, 78%).

[0254] 1H NMR (400 MHz, DMSO-c / 6) 5 9.93 (s, 1 H), 8.34 (t, = 5.6 Hz, 1 H), 8.11- 7.96 (m, 6H), 7.82-7.76 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.31 (s, 1 H), 5.45 (s, 2H), 5.35-5.20 (m, 3H), 5.08 (s, 2H), 4.38 (m, 1 H), 4.28 (m, 1 H), 3.63 (m, 1 H), 3.60-3.40 (m, 46H), 3.23 (s, 3H), 3.17-2.97 (m, 5H), 2.38 (s, 3H), 2.29 (t, J = 6.4 Hz, 2H), 2.22-2.10 (m, 4H), 1.94-1.81 (m, 2H), 1.69-1.64 (m, 2H), 1.53-

[0255] 1.25 (m, 15H), 1.20 (d, J= 7.2 Hz, 3H), 0.88 (t, J= 7.2 Hz, 3H).

[0256] 19F NMR (377 MHz, DMSO-cfe) 5 -73.68, -1 11 .33 (d, J= 10.2 Hz).

[0257] HRMS (ESI+) calcd for C76H113FN9O23 (M+H+) 1538.7928 found 1538.791 1.

[0258] MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA)

[0259] 4-((44S,54S,57S)-44-(6-(2,5-dioxocyclopent-3-en-1-yl)hexanamido)-54,57- dimethyl-38,45,52,55-tetraoxo-2,5,8,11 ,14,17,20,23,26,29,32,35-dodecaoxa- 39,46,53,56-tetraazaoctapentacontan-58-amido)benzyl((1 S,9S)-9-ethyl-5-fluoro- 9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1 -yl)carbamate (24)

[0260] Chemical Formula: C86H123FN10O26

[0261] Molecular Weight: 1731 ,97

[0262] To 800 pL of DMF were dissolved 23 (15.0 mg, 0.009 mmol, 1 eq) before the addition of 1 (2.9 mg, 0.0135 mmol, 1 .5 eq). HATLI (10.3 mg, 0.027 mmol, 3 eq) was dissolved and finally, 2,6-Lutidine (5.2 pL, 0.045 mmol, 5 eq) was added to the mixture. A bit of HOBt (2 eq) was also added. The solution was stirred overnight at room temperature. The solution was purified by semi-preparative HPLC (gradient A) to afford a yellow green paste (14.0 mg, 0.0081 mmol, 90%)

[0263] 1H NMR (400 MHz, DMSO-c / 6) 5 9.90 (s, 1 H), 8.08-8.04 (m, 2H), 7.98 (d, J = 6.8 Hz, 1 H), 7.84-7.74 (m, 4H), 7.61 (d, J= 8.4 Hz, 2H), 7.36 (d, J= 8.4 Hz, 2H), 7.31 (s, 1 H), 6.99 (s, 2H), 6.51 (s, 1 H), 5.45 (s, 2H), 5.35-5.20 (m, 3H), 5.07 (s, 2H), 4.37 (m, 1 H), 4.26 (m, 1 H), 4.13 (m, 1 H), 3.60-3.40 (m, 46H), 3.23 (s, 3H), 3.15-2.90 (m, 7H), 2.38 (s, 3H), 2.27 (t, J= 6.4 Hz, 2H), 2.22-2.03 (m, 8H), 1 .93-1 .80 (m, 2H), 1 .53- 1.17 (m, 16H), 1.21-1.10 (m, 8H), 0.87 (t, J = 7.2 Hz, 3H).

[0264] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .32 (d, J = 10.5 Hz). HRMS (ESI+) calcd for C86HI24FNIO026 (M+H+) 1731 .8672, found 1731 .8651

[0265] Example 1.3. Synthesis of MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe (16)

[0266] Scheme: Reagents and conditions: (a) TFA, rt, 3 h, 98%; (b) MeO-PEGi2-NHS (1.2 eq), DIPEA (1.2 eq), rt, 2 h, 73%; (c) NaHCO3(2 eq), Fmoc-CI (1.2 eq), H20 / Dioxane, 69% (d) TFA.NH2-VC-PABC-MMAE (0.8 eq), HATU (1.3 eq), 2,6- lutidine (2.3 eq), DMF, rt, overnight; (e) Piperidine (20%), DMF, rt, 2 h, 96% over two steps; (f) Intermediate 9 (1.4 eq), EEDQ (2 eq), ACN, rt, 30 min and then DIPEA (2 eq), DMF, rt, overnight ; (g) Piperidine (20%), DMF, rt, 1 h30, 96% over two steps; (h) Intermediate 15 (3 eq), HATU (2 eq), 2,6-lutidine (3.5 eq), DMF, rt, 6 h, 64%. (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-lysine (8)

[0267] Chemical Formula: C21H24N2O4 Molecular Weight: 368,43

[0268] A -Fmoc-AA-Boc-L-Lysine (250 mg, 0.53 mmol) was dissolved in 2.0 mL of DCM and stirred 5 min at room temperature. TFA (2.0 mL) was then added to the solution and the resulting mixture was stirred 3 h at room temperature. The solvent was then removed under reduced pressure and TFA was co-evaporated three times with DCM. The crude compound was dried under reduced pressure, triturated in diethyl ether and finally concentrated under reduced pressure to afford compound 8 as a white powder (252 mg, 98%); tR= 13.42 min; purity (HPLC) 97.94%.

[0269] 1H NMR (300 MHz, DMSO-cfe) 5 7.89 (d, 2H, J = 7.5 Hz), 7.75 - 7.63 (m, 5H), 7.42 (t, 2H, J = 7.2 Hz), 7.34 (td, 2H, J = 10.7, 4.1 Hz), 4.36 - 4.17 (m, 3H), 3.92 (td, 1 H, J= 9.3, 4.7 Hz), 2.81 - 2.70 (m, 2H), 1.78 - 1 .45 (m, 4H), 1 .41 - 1 .30 (m, 2H).

[0270] 13C NMR (101 MHz, DMSO-oy 3 173.9, 156.2, 143.9, 143.8, 140.8, 127.7, 127.1 , 125.3, 120.2, 65.6, 53.6, 46.7, 38.6, 30.2, 26.5, 22.6.

[0271] HRMS (ESI) m / z: [M+H]+calcd for C21H25N2O4 369,1736; found, 369.1803.

[0272] Fmoc-Lys-PEGi2-OMe (9)

[0273] MeO-PEGi2-NHS (60 mg, 0.09 mmol,) was dissolved in 2 mL of anhydrous DMF and added to Fmoc-Lys-OH (35.2 mg, 0.07 mmol). DIPEA (13 pL, 0.08 mmol) was added and the reaction mixture was stirred for 2 h at room temperature. The crude compound was purified by semi-preparative HPLC. Fractions were concentrated under reduced pressure to afford the desired compound 9 as a transparent oil (49.9 mg, 73%); tR= 16.17 min; purity (HPLC) 100%.

[0274] 1H NMR (300 MHz, DMSO-cfe) 5 12.54 (br, 1 H), 7.89 (d, 2H, J = 7.4 Hz), 7.82 (br, 1 H), 7.73 (d, 2H, J = 7.4 Hz), 7.62 (br, 1 H), 7.42 (t, 2H, J = 7.1 Hz), 7.33 (td, 2H, J = nA, 0.9 Hz), 4.29 - 4.21 (m, 3H), 3.99 - 3.85 (m, 1 H), 3.77 - 3.68 (m, 1 H), 3.61 - 3.43 (m, 44H, under water of DMSO), 3.23 (s, 3H), 3.07 - 2.97 (m, 2H), 2.28 (t, 2H, J= 6.4 Hz), 1 .77 - 1 .53 (m, 3H), 1 .30 - 1 .21 (m, 1 H), 1 .09 - 0.94 (m, 1 H), 0.91 - 0.66 (m, 2H).

[0275] 13C NMR (75 MHz, DMSO-cfe) 5174.4, 170.3, 156.6, 144.3, 144.3, 141.2, 128.1 , 127.5, 125.7, 120.6, 71.7, 70.2, 70.1 , 70.0, 70.0, 67.3, 66.1 , 58.5, 54.2, 47.1 , 38.7, 36.7, 30.9, 29.1 , 23.5.

[0276] HRMS (ESI) m / z: [M+Na]+calcd for C47H74N2NaOi7, 961 .4880; found, 961 .4840.

[0277] AZ-Fmoc-6-Aminohexanoic acid (10)

[0278] FmocHN

[0279] Chemical Formula: C21H23NO4 Molecular Weight: 353,42

[0280] 6-Aminohexanoic acid (1.00 g, 7.62 mmol) and NaHCOs (s) (1.28 g, 15.25 mmol) were dissolved in a solution of H20 / dioxane (18 mL - 27 mL), then Fmoc-CI (2.17 g, 8.39 mmol) was added, and the reaction mixture was stirred over night at room temperature. The solution was acidified by HCI (1 M), solution became cloudy, then dioxane was evaporated under reduced pressure. The crude compound was extracted three times with EtOAc, dried over MgSO4, filtrated, concentrated under reduced pressure, and purified via silica gel chromatography (combiflash RS 80 SiOH, 40-30 pm), eluting with DCM-MeOH: 95 / 05 to yield Fmoc-6-aminohexanoic acid (1 .87 g, 69%) as a white solid. t« = 13.42 min, purity (HPLC) = 97.94%.

[0281] 1H NMR (300 MHz, CDCI3) 67.76 (d, 2H, = 7.5 Hz), 7.59 (d, 2H, = 7.5 Hz), 7.40 (t, 2H, J = 7.2 Hz), 7.31 (td, 2H, J = 7.4, 1.2 Hz), 4.79 (br, 1 H), 4.41 (d, 2H, J = 6.8 Hz), 4.21 (t, 1 H, J = 6.7 Hz), 3.19 (dd, 2H, J = 12.9, 6.5 Hz), 2.36 (t, 2H, J = 7.3 Hz), 1.65 (dt, 2H, J = 14.8, 7.3 Hz), 1.53 (dt, 2H, J = 14.0, 7.0 Hz), 1.38 (dd, 2H, J = 14.9, 8.3 Hz).

[0282] 13C NMR (101 MHz, DMSO-cfe) 5 174.5, 156.1 , 144.0, 140.8, 127.6, 127.1 , 125.2, 120.1 , 65.2, 46.8, 40.1 , 33.6, 29.1 , 25.8, 24.2.

[0283] HRMS (ESI) m / z: [M+Na]+calcd for C2iH23NO4Na, 376.1519; found, 376.1510. TFA.NH2-Cap-VC-PABC-MMAE (12)

[0284] A solution of compound 10 (21 mg 0.06 mmol) in anhydrous DMF (500 pL) was added to a solution of TFA.NH2-VC-PABC-MMAE (44 mg 0.04 mmol) in anhydrous DMF (500 pL). HATLI (29 mg, 0.08 mmol) and then 2,6-lutidine (15 mg, 0.14 mmol, 16 pL) were added under argon and the medium was left overnight with stirring at room temperature to give intermediate 11 in the crude mixture. [HRMS (ESI) m / z: [M+Na]+calcd for CygHnsNuNaOis 1480.8574 ; found. 1480.8457],

[0285] 20% of piperidine were added to remove the protective group Fmoc for 2 h. The crude was diluted with DMSO and purified by semi-preparative HPLC (ACN / H2O + 0.1 % TFA) to give the TFA salt of compound 12 as a white solid (51 mg, 96% on the two steps). tR = 15.47 min, purity (HPLC) = 100%.

[0286] 1H NMR (300 MHz, DMSO) <510.01 (s, 1 H), 8.11 (d, J= 7.8 Hz, 1 H), 7.98 - 7.87 (m, 1 H), 7.83 (d, J = 8.6 Hz, 1 H), 7.70 - 7.53 (m, 4H), 7.38 - 7.21 (m, 5H), 7.20 - 7.1 1 (m, 1 H), 6.03 (s, 1 H), 5.15 - 4.92 (m, 3H), 4.87 - 4.10 (m, 25H, under water of DMSO), 4.05 - 3.88 (m, 3H), 3.78 (d, 1 H, = 9.4 Hz), 3.65 - 3.62 (m, 1 H), 3.34 - 3.09 (m, 8H), 3.07 - 2.68 (m, 1 1 H), 2.32 - 1 .88 (m, 6H), 1.87 - 1 .62 (m, 4H), 1 .61 - 1 .42 (m, 6H), 1 .40 - 1 .18 (m, 4H), 1 .03 - 0.94 (m, 6H), 0.92 - 0.68 (m, 22H).

[0287] HRMS (ESI) m / z: [M+H]2+calcd for C64H106N11O13, 618.9019; found, 618.9021 .

[0288] TFA.NH2-Lys-PEGi2OMe-Cap-VC-PABC-MMAE (14) To a solution of compound 9 (49.90 mg, 0.05 mmol) in ACN (1 mL) was added EEDQ (18.79 mg, 0.08 mmol) under argon and the mixture was stirred for 30 min at room temperature under protection from the light. Then, compound 12 (51 mg, 0.04 mmol) was solubilized in dry DMF (1 .2 mL) and added to the mixture before adding DIPEA (9.82 mg, 0.08 mmol, 13 pL).

[0289] The reaction was stirred overnight and after complete conversion shown by HPLC monitoring, deprotection of Fmoc was occurred by adding 20% (440 pL) of piperidine in DMF. The reaction mixture was stirred for 1 h 30 then the resulting solution was purified by semi-preparative HPLC (0.1% TFA, H2O / ACN 80 / 20) to give the desired compound 14 (51 .4 mg 96% on the two steps) as a white solid.

[0290] 1H NMR (300 MHz, DMSO-cfe) 5 10.00 (s, 1 H), 8.50 - 8.23 (m, 2H), 8.17 - 7.99 (m, 4H), 7.96 - 7.77 (m, 3H), 7.72 - 7.47 (m, 3H), 7.42 - 7.10 (m, 7H), 6.05 (s 1 H), 5.29 - 4.10 (m, 6H, under water of DMSO), 4.06 - 3.89 (m, 3H), 3.81 - 3.68 (m, 1 H), 3.68 - 3.39 (m, 47H), 3.27 — 2.81 (m, 23H), 2.34 - 2.24 (m, 3H), 2.24 - 1 .89 (m, 7H), 1 .86 - 1 .15 (m, 25H), 1 .09 - 0.92 (m, 7H), 0.92 - 0.68 (m, 24H).

[0291] 6-Maleimidohexanoic acid (15)

[0292] Chemical Formula: C10HI3NO4Molecular Weight: 211,22

[0293] Maleic anhydride (2.00 g, 20.40 mmol) and 6-aminocaproic acid (2.68 g, 20.40 mmol) were stirred under argon in 20 mL of acetic acid. The reaction medium is heated to reflux and stirred overnight. On next day, the mixture turned orange. The acetic acid was co-evaporated three times with toluene to give a crud product as an orange viscous oil. The crude product was taken up in DCM and washed with H2O. The aqueous phase was re-extracted two times with DCM and the combined organic phases were dried over MgSO4 and evaporated under vacuum then purified through combiflash (sorbent 80 g) with DCM / MeOH (0 to 4%) to give the desired product 15 as white solid (1 .92 g, 45%). HPLC purity = 99.07%, t« = 8.53 min.

[0294] 1H NMR (300 MHz, CDCI3) 66.69 (s, 2H), 3.52 (t, 2H, J= 7.2 Hz), 2.34 (t, 2H, J = 7.4 Hz), 1 .63 (m, 4H), 1 .42-1 .27 (m, 2H).

[0295] 13C NMR (75 MHz, CDCI3) 5 179.4, 171.4, 134.6, 38.2, 34.2, 28.7, 26.7, 24.7. MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe (16)

[0296] Compound 14 (51.4 mg, 0.03 mmol), compound 15 (16 mg, 0.08 mmol) and HATLI (19 mg, 0.05 mmol) were solubilized in dry DMF (2.5 mL) under argon. Then, 2,6-lutidine (10 mg, 10.25 pL, 0.088 mmol) was added and the mixture was stirred 6 h at room temperature. The solution was diluted by 2 with DMSO and purified by semipreparative HPLC (tR = 16.83 min) to give the desire compound 16 (34.3 mg, 64%) as a white solid. Purity (HPLC) = 99.55%.

[0297] 1H NMR (300 MHz, DMSO-cfe) 5 10.00 (s, 1 H) , 8.19 - 8.01 (m, 1 H), 7.95 - 7.71 (m, 5H), 7.70 - 7.52 (m, 3H), 7.46 - 7.09 (m, 6H), 7.21 - 7.09 (m, 1 H), 7.07 (s, 2H),

[0298] 5.98 (s, 1 H), 5.16 - 4.93 (m, 2H), 4.53 - 3.86 (m, 9H), 3.86 - 3.30 (m, 44H, under water of DMSO), 3.28 - 2.80 (m, 23H), 2.34 - 1.9 (m, 10H), 1.87 - 1.09 (m, 35H), 1.08 - 0.94 (m, 8H), 0.92 - 0.67 (m, 27H). HRMS (ESI) m / z: [M+2Na]2+calcd for Cio6Hi78Ni4Na203o, 1086.6278; found, 1086.6309.

[0299] NH2-c-Lys(PEGi2)-Cap-AA-PABC-EXA (TFA salt) (17)

[0300] Chemical Formula: C^H^FN^C^

[0301] To a solution of compound 14 (16.0 mg, 0.01 mmol) in dry DMF (800 pL), under an argon atmosphere, were added 10 (5.3 mg, 0.015 mmol), HATU (11.4 mg, 0.03 mmol), HOBt (2.0 mg, 0.015 mmol), and 2,6-lutidine (5.8 pL, 0.05 mmol). The resulting mixture was stirred overnight at room temperature.

[0302] To the previous solution, was added piperidine (160 pL). The resulting mixture was stirred 3 h at room temperature, and purified by semi-preparative HPLC (0.1 % TFA, H2O / ACN 80 / 20 method) to afford drug-linker 17 (3.7 mg, 20%) as a yellow green paste (TFA salt).

[0303] 1H NMR (400 MHz, DMSO-c / 6) 5 9.90 (s, 1 H), 8.08-8.04 (m, 2H), 7.98 (d, J = 6.8 Hz, 1 H), 7.84-7.74 (m, 7H), 7.61 (d, J= 8.4 Hz, 2H), 7.36 (d, J= 8.4 Hz, 2H), 7.31 (s, 1 H), 5.45 (s, 2H), 5.35-5.20 (m, 3H), 5.07 (s, 2H), 4.37 (m, 1 H), 4.26 (m, 1 H), 4.13 (m, 1 H), 3.60-3.40 (m, 46H), 3.23 (s, 3H), 3.15-2.90 (m, 7H), 2.38 (s, 3H), 2.27 (t, J = 6.4 Hz, 2H), 2.22-2.03 (m, 8H), 1.93-1.80 (m, 2H), 1.53-1.17 (m, 16H), 1.21-1.10 (m, 8H), 0.87 (t, J= 7.2 Hz, 3H).

[0304] 19F NMR (377 MHz, DMSO-cfe) 5 -73.68, -1 11 .33 (d, J= 10.2 Hz).

[0305] HRMS (ESI+) calcd for C82H125FN10O24 (M+H)2+826.4419, found 826.4421. EXAMPLE 2 - BIOCONJUGATION

[0306] Anti-HER 2 antibodies:

[0307] Example 2.1. Bioconjugation of MC-Lys-PEGi2OMe(Cap-VA-PABC-MMAE) in solution in DMSO (1 mM), Mb(12)- MMAE (11): Mb(12) = minibody DAR 12.

[0308] To 500 pL of antibody solution at 835.2 pg / mL in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37 °C, 1 h 15 and 16 eq of X, 4°C, 16 h.

[0309] Example 2.2. Bioconjugation of MC-Lys-PEGi2OMe(Cap-VA-PABC-MMAE) in solution in DMSO (1 mM), Mb(4)- MMAE (12): Mb(4) = minibody DAR 4

[0310] To 500 pL of antibody solution at 859.2 pg / mL in BBS, the general procedure for bioconjugation has been applied: 6 eq of TCEP, 37°C, 1 h 15 and 16 eq of X, 4°C, 16 h.

[0311] Example 2.3. Bioconjugation of MC-Lys-PEGi2OMe(Cap-VA-PABC-MMAE) in solution in DMSO (1 mM), Db(4)- MMAE (13): Db(4) = diabody DAR 4

[0312] To 500 pL of antibody solution at 708.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 4 eq of TCEP, 37°C, 1 h 15 and 8 eq of X, 4°C, 16 h.

[0313] Example 2.4. Bioconjugation of MC-Lys-PEGi2OMe(Cap-VA-PABC-EXA) in solution in DMSO (1 mM), Mb(4)- EXA (14): Mb(4) = minibody DAR 4

[0314] To 500 pL of antibody solution at 276.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of X, 4°C, 16 h.

[0315] Example 2.5. Bioconjugation of MC-Lys-PEGi2OMe(Cap-VA-PABC-EXA) in solution in DMSO (1 mM), TTZ-EXA (ou equivalemment lgG(8)- MC-Lys-(Cap- VA-PABC-EXA)-PEGi2-OMe) (15): TTZ = trastuzumab

[0316] To 500 pL of antibody solution at 968.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 16 eq of TCEP, 37°C, 1 h 15 and 20 eq of X, 4°C, 16 h. Example 2.6. Bioconjugation of MC-Lys-PEGi20Me(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), Mb(12)-EXA (25): Mb(12) = minibody DAR 12

[0317] To 500 pL of antibody solution at 835.2 pg / mL in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0318] Example 2.7. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), Mb(4)-EXA (26): Mb(4) = minibody DAR 4

[0319] To 500 pL of antibody solution at 859.2 pg / mL in BBS, the general procedure for bioconjugation has been applied: 6 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0320] Example 2.8. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), Db(4)-EXA (27): Db(4) = diabody DAR 4

[0321] To 500 pL of antibody solution at 708.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 4 eq of TCEP, 37°C, 1 h 15 and 8 eq of Y, 4°C, 16 h.

[0322] Example 2.9. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-MMAE) in solution in DMSO (1 mM), Mb(8)- MMAE (28): Mb(8) = minibody DAR 8

[0323] To 500 pL of antibody solution at 276.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0324] Example 2.10. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), TTZ-EXA (ou equivalemment lgG(8)- MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe) (29): TTZ = trastuzumab

[0325] To 500 pL of antibody solution at 968.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 16 eq of TCEP, 37°C, 1 h 15 and 20 eq of Y, 4°C, 16 h.

[0326] Example 2.11. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), IRR-EXA (ou equivalemment lgG(8)IRR- MC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe ) (30): IRR = irrelevant antibody (negative control) To 500 pL of antibody solution at 730.1 pg / mL in BBS, the general procedure for bioconjugation has been applied: 16 eq of TCEP, 37°C, 1 h 15 and 20 eq of Y, 4°C, 16 h.

[0327] Anti-TROP2 antibodies:

[0328] Example 2.12. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), Mb(8)-EXA (31): Mb(8) = minibody DAR 8

[0329] To 500 pL of antibody solution at 1 .28 mg / mL in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0330] Example 2.13. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), SCZ-EXA (32): SCZ = sacituzumab

[0331] To 500 pL of antibody solution at 708.0 pg / mL in BBS, the general procedure for bioconjugation has been applied: 16 eq of TCEP, 37°C, 1 h 15 and 20 eq of Y, 4°C, 16 h.

[0332] Anti-HER2 antibodies:

[0333] Example 2.14. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), scFv(2)-EXA (33): scFv(2) = minibody DAR 2

[0334] To 500 pL of antibody solution at 265.0 pg / mL (or 794.0 pg / mL) in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0335] Example 2.15. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), scFv(4)-EXA (34): scFv(4) = minibody DAR 4

[0336] To 500 pL of antibody solution at 270.0 pg / mL (or 809.0 pg / mL) in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 24 eq of Y, 4°C, 16 h.

[0337] Example 2.16. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), scFv(6)-EXA (35): scFv(6) = minibody DAR 6 To 500 pL of antibody solution at 275.0 pg / mL (or 826.0 pg / mL) in BBS, the general procedure for bioconjugation has been applied: 8 eq of TCEP, 37°C, 1 h 15 and 16 eq of Y, 4°C, 16 h.

[0338] Example 2.17. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), scFv(8)-EXA (36): scFv(8) = minibody DAR 8

[0339] To 500 pL of antibody solution at 281.1 pg / mL (or 842.1 pg / mL) in BBS, the general procedure for bioconjugation has been applied : 8 eq of TCEP, 37 °C, 1 h 15 and 16 eq of Y, 4 °C, 16 h.

[0340] Example 2.18. Bioconjugation of MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2- OMe (16)

[0341] 1 . FDCs conjugation

[0342] 1 .1 . Mb4-MC-Lys-PEGi2OMe(Cap-VC-PABC-MMAE)

[0343] Into 4 vials (500 pL of protein for each), were added Mb4 (minibody DAR 4) (1 .7 mg / mL, 0.85 mg, 21.4 pM, 500 pL) and TCEP (85 pL, 8 eq, 1 mM in BBS buffer of bioconjugation) and the reaction was incubated for 1.25 h at 37 °C under agitation (300 rpm). Linker 16 (278 pL, 26 eq, 1 mM in DMSO) was then added, and the reaction was incubated for 16 h at 4 °C under agitation (600 rpm). The mixtures of 4 vials were finally purified on a Superdex® 200 10 / 300 GL column to give the desired conjugate (0.9 mg / mL, 1.989 mg, 10.26 pM, 12800 pL, 78%). For the quantity’s calculation, the molecular weight of ADC with the average DAR was used: 87962.02 Da (Mb4 conjugated).

[0344] Into 2 vials (500 pL of protein for each), were added Mb4 (1 .5 mg / mL, 0.72 pg, 18 pM, 500 pL) and TCEP (72 pL, 8 eq, 1 mM in BBS buffer of bioconjugation) and the reaction was incubated for 1.25 h at 37 °C under agitation (300 rpm). Linker 16 (235 pL, 26 eq, 1 mM in DMSO) was then added, and the reaction was incubated for 16 h at 4 °C under agitation (600 rpm). The mixture of these 2 last vials were purified by dialysis as described for TTZ-16 to afford the desired conjugate Mb4-16 (1 .4 mg, 11.8 pM, 1400 pL, 100%). The conjugates concentration was quantified using the extinction coefficient of Mb4 (1 .732), the measured absorbance values at 280 nm and the molecular weight of Mb4 79450.02 Da. For the quantity’s calculation, the molecular weight of average DAR was used: 87962.02 Da (Mb4 conjugated).

[0345] 1.2. Mb12-MC-Lys-PEGi2OMe(Cap-VC-PABC-MMAE) Into 7 vials (500 pL of protein for each), were added Mb12 (minibody DAR 12) (20.35 pM, 1.7 mg / mL, 838 pg, 500 pL) and TCEP (92 pL, 9 eq, 1 mM in BBS buffer of bioconjugation) and the reaction was incubated for 1 .25 h at 37 °C under agitation (300 rpm). Linker 16 (265 pL, 26 eq, 1 mM in DMSO) was then added, and the reaction was incubated for 16 h at 4 °C under agitation (600 rpm). The mixture was finally purified on a Superdex® 200 10 / 300 GL column to give the desired FDC Mb12-16 (0.5 mg / mL, 3.1 mg, 4.68 pM, 41.036 mL, 52%). The conjugates concentration was quantified using the extinction coefficient of Mb12 (1 .677), the measured absorbance values at 280 nm and the molecular weight of Mb12. For the quantity’s calculation, the molecular weight of average DAR was used: 107885.48 Da (Mb12 conjugated).

[0346] 2. ADCs conjugation

[0347] TTZ-MC-Lys-PEGi2OMe(Cap-VC-PABC-MMAE) i.e. lgG(8)-MC-Lys- PEGi2OMe(Cap-VC-PABC-MMAE)

[0348] Commercial solution of Ontruzant® (trastuzumab) was first purified by Vivaspin® with three cycles of centrifugation in BBS bioconjugation buffer of pH 8.0 and then diluted to give the antibody with a concentration of 5.9 mg / mL. Into 3 vials, were added TTZ (5.9 mg / mL, 2.97 mg, 40.8 pM, 500 pL) and TCEP (361 pL, 18 eq, 1 mM in BBS buffer of bioconjugation) and the reaction was incubated for 1.25 h at 37 °C under stirring (300 rpm). Linker 16 (401 pL, 20 eq, 1 mM in DMSO) was then added, and the reaction was incubated for 16 h at 4 °C under stirring (600 rpm). The two first vials were finally purified on a Superdex® 75 column to give the desired ADC TTZ-16 (2.1 mg / mL, 4.18 mg, 12.58 pM, 15600 pL, 70%).

[0349] The third crude mixture (third vial) was purified by dialyzing three times during 3 h for the two firsts cycles and overnight for the last cycle over a 10 kDa membrane against a PBS buffer (1 L) at pH 7.4 to afford the desired ADC TTZ-16 (2.28 mg / mL, 2.51 mg, 13.8 pM, 1100 pL, 84%). The conjugates concentration was quantified using the extinction coefficient of TTZ (1 .481 ), the measured absorbance values at 280 nm and the molecular weight of TTZ 145387.5 Da. For the quantity’s calculation, the molecular weight of average DAR was used: 165092 Da (TTZ conjugated).

[0350] Example 2.19. Bioconjugation of MC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe (24) onto trastuzumab

[0351] TTZ-MC-Lys-PEGi2OMe(Cap-VC-PABC-EXA) i.e. lgG(8)-MC-Lys- PEGi2OMe(Cap-VC-PABC-EXA) General procedure: into a vial, were added lgG(8) (968.0 pg / mL, 500 pL, 6.7 pM in BBS buffer (pH 8)) and TCEP (53.24 pL, 16 eq, 1 mM in BBS buffer) and the reaction was incubated for 1 .25 h at 37 °C under agitation (300 rpm). Drug-linker MC- Lys-PEGi2OMe(Cap-VC-PABC-EXA) (24) (66.57 pL, 20 eq, 1 mM in DMSO) was then added, and the reaction was incubated for 16 h at 4 °C under agitation (600 rpm). The mixture was finally purified by size-exclusion chromatography (SEC) on a Superdex 200 10 / 300 GL column (1.0 x 300 mm, 13 pM, molecular mass range 10, 000 - 600,000 Da) from Cytiva (GE Healthcare Life Sciences, 17-5174-01 ), connected to an Akta purifier (Cytiva). The column was used at a rate of 0.8 mL / min with a UV detector at 280 nm. After the loading of immunoconjugate, the column was eluted with PBS for 0.2 column volume (cv), then with 0.3 cv of H2O / ACN (7 / 3) + 0.2% TFA and finally with PBS (1 cv) to give lgG(8)-mc-(b-PEG12)-AA-PABC-EXA (445.3 pg, 92%), with a purity of >95%, confirmed by size exclusion chromatography.

[0352] EXAMPLE 3 - EVALUATION OF IMMUNOCONJUGATES WITH LINKER MC- Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe AND WITH LINKER MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe

[0353] ELISA assays

[0354] HER2 recombinant protein (Sino Biologicals, Beijing, P. R. China) was coated in a 96-well plates at 1 pg.mL-1in PBS (Phosphate-Buffered Saline) and incubated overnight at 4 °C. The wells were then saturated with 3% FBS (Foetal Bovine Serum)- PBS for 1 h at 37 °C, and washed with PBS prior to incubation with PBS (negative control), ADCs, unconjugated minibodies or trastuzumab (Ontruzant®, MSD laboratory, France) from 0.001 to 1000 nM during 1 h at 37 °C. Wells were then washed with PBS-Tween 20 (0.05%) and incubated with 100 pL of protein-L- peroxydase (ThermoScientific, Pierce®, Massachusetts, USA) at 1.25 pg.mL-1for 1 h at 37 °C, added to 100 pL of 3,3',5,5'-tetramethylbenzidine substrate (TMB) (Sigma, St Louis, USA). Enzymatic reactions were stopped by the addition of 50 pL of 1 M H2SO4 and the absorbance was measured at 450 nm, using an absorbance microplate reader (Bio-Tek® instruments, Inc., Vermont, USA).

[0355] Flow Cytometry Studies

[0356] Protein L coupled with phycoerythrin (ppL-PE, Sinobiological, Beijing, China) was used as an intracellular tracer. Protein L was obtained from Peptostreptococcus magnus. This protein possesses a high affinity for kappa light chain of anti-HER2-like antibodies. Briefly, 50,000 SK-BR-3, MDA-MB-468 and MDA-MB-231 cells were seeded 24 h on 24-well plates. ADCs and FDCs were then incubated 30 minutes with ppL-PE (1 :2 ratio) at 37 °C. Cells were incubated with 10 nmol / L of ADC-ppL-PE or FDC-ppL-PE complexes for 4 h at 37 °C or 4 °C. Cells were then washed three times with cold PBS and were transferred on 96-round bottom well plates in a PBS, BSA 1% and EDTA 2 mM solution. Finally, cells were analyzed by a MACSQuant 10 flow cytometer (Miltenyi Biotec®, Bergisch Gladbach, Germany) with an excitation laser at 488 nm (filter 565-605 nm) to detect PE fluorescence. Data were analyzed with MACSQuantify Software®.

[0357] Microscopy studies

[0358] 50,000 SK-BR-3 cells were incubated for 48 h on poly-D-lysine-coated coverslips in p24 plate wells. Entities were pre-incubated as in the flow cytometry experiment and then incubated on cells for 24 h after removing cell culture medium. PpL-PE alone is also incubated on cells during 24h as a negative control. After rinsing the cells, the coverslips are mounted on a superfrost slide with DAPI-free Fluoromount (Invitrogen®, Waltham, Massachusetts, USA). Fluorescence detection at 550nm is done at x20 objective with the IXplore Standard microscope (Olympus®, Tokyo, Japan).

[0359] Cell Cultures and Reagents

[0360] SK-BR-3 and BT-474 cell lines were obtained from Cell Lines Service (CLS Eppelheim, Germany). MDA-MB-231 and MDA-MB-468 cell lines were respectively obtained from ECACC (92020424) and ATCC. SK-BR-3 are epithelial adenocarcinoma of breast cells and were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution (Gibco®), in humidified atmosphere at 37°C with 5% CO2. MDA-MB-231 human triple-negative breast carcinoma cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco®), with 10% foetal bovine serum (FBS, Gibco®, Life Technologies), glucose, L-glutamine, 1% penicillin-streptomycin solution (10,000 U / mL, Gibco®) and 1% of non-essential amino acid (NEAA) sol 100X at 37°C and 5% CO2. MDA-MB-468 are epithelial cells from breast adenocarcinoma characterised by an absence of expression of HER2, estrogen receptor and progesterone receptor. They were cultured in MEM-Glutamax with 10% FBS, 1 % sodium pyruvate (Gibco®), 1% non-essential amino acid solution (Gibco®), 1 % 100X vitamins (Gibco®), 0.08% gentamicin (Gibco®) and 1 % penicillin- streptomycin solution (Gibco®), in humidified atmosphere at 37°C with 5% CO2. BT- 474 are epithelial ductal carcinoma of breast cells and are characterised by membrane HER2 overexpression and estrogen receptor expression. They are cultured in DMEM / HAM F12 (Gibco®) medium supplemented with 5% FBS, 1 % penicillin / streptomycin (Gibco®), and with 0.5% insulin transferrin selenium (Gibco®). Incubation studies with radiolabelled antibodies or antibody fragments were performed under the same conditions.

[0361] Cell Proliferation Assays

[0362] Cell viability and proliferation were studied, using a luminescent test based on quantification of ATP, using the CellTiter-Glo cell proliferation assay (Promega®, Wisconsin, USA). Briefly, 6,000 cells of SK-BR-3, 6,000 cells of MDA-MB-468 or 3,000 cells of MDA-MB-231 were incubated in 100 pL of medium in 96-well plates for 24 h and then treated with concentrations ranging from 0.0001 nM to 100 nM of tested compounds. Cells were incubated with 100 pL of each derivative at 37°C with 5% CO2 for 5 days. A 20 mM H2O2 (Sigma-Aldrich®, Missouri, USA) solution was used as positive control; culture medium alone was tested as negative control. MMAE (100 nM, stock solution at 2 pM in PBS), exatecan (100 nM, stock solution at 5 mM in DMSO), trastuzumab (100 nM, Ontruzant® stock solution at 26 pM in PBS), trastuzumab emtansine T-DM1 (100 nM, Kadcyla® stock solution at 2 pM in PBS (primary concentration at 20 mg / mL) and trastuzumab deruxtecan T-DXd (primary concentration at 20 mg / mL) were used as reference. Cells were incubated with 100 pL of each solution at 37°C with 5% CO2 for 5 days. Cell viability was then determined using CellTiter-Glo reagent (Promega, Wisconsin, USA). Briefly, 100 pL of medium were removed and 100 pL of CellTiter-Glo reagent were added to each well. The plates were shaken for 2 min and then incubated at room temperature for 10 min. The luminescence values were measured with a gain at 135 with an acquisition at 0.5 s, using a microplate reader (Bio-Tek® instruments, Inc., Vermont, USA). When a dosedependent activity was observed, 50% inhibitory concentration (IC50) were calculated using GraphPad® PRISM 7 software (San Diego, USA) (n = 4 in quadruplicate).

[0363] Animal model

[0364] Ten weeks old BALB / c nude female mice (Janvier Labs®, Le Genest-Saint-lsle, France) were used in this study as an orthotopic graft model of breast cancer development. Mice were housed in standard conditions (4 to 6 animals per cage on ventilated racks, temperature 21-24 °C, 60% humidity, 12 / 12 light / dark cycle) with free access to standard food and water). After gaseous anesthesia (Isoflurane, Iso- Vet® 1000 mg / g) animals were tumorised in the mammary fat pad (sub-cutaneous) at eleven weeks with 5 million of BT-474 cells in Matrigel matrix high concentration (Corning Life Science, New York, USA) (50:50 with cells culture media). Mice were observed and weighted to evaluate general clinical state, in order to detect end points. L*l2

[0365] Formula — was used to calculate tumoural volume, where L is the largest and I is the largest perpendicular dimension. Tumours were measured with a caliper every week or two weeks.

[0366] All experiments were conducted in accordance with the European directive 2010 / 63 / EU after approval by the local animal ethical committee C2E2A (authorization #24881 -202003311 1188075 v4. + authorization

[0367] #2019011814134419.v2).

[0368] Biodistribution study

[0369] 1 / 125l-radiolabelling of antibodies and antibody fragments

[0370] General: [125l]Nal (3.60-3.63 GBq / mL, 643.8 MBq / mg) was purchased from PerkinElmer Life and Analytical Sciences (331 Treble Cove Road, Billerica, MA 01862, US) as a no-carrier-added solution in reductant free 1.0 x 10-5M aqueous sodium hydroxide solution (pH 8-11 ). Pierce iodination tubes were purchased from Thermo Fischer Scientific (28601 ). NAP-5 columns (NAP-5 columns Sephadex G-25 DNA grade) were purchased from GE Healthcare. Radio-instant thin-layer chromatography (radio-ITLC) analyses were measured on a miniGITA Dual radio- TLC system (Elysia-Raytest) using silica gel-impregnated chromatography paper (Varian inc.) eluted with PBS. Size exclusion high-performance liquid chromatography (SEC-HPLC) analyses were performed on a Superose 12 column (10 / 300 GL, 1 1 pm, GE Healthcare) using a system consisting of a HP1100 (Hewlett Packard, Les Ulis, France) and a Flo-One A-500 Radiomatic detector (Packard, Canberra, Australia). Isocratic elution with DMSO in PBS (5 / 95, v / v) was applied at a flow rate of 0.5 mL.min-1( = 220 / 254 / 280 nm). The anti-HER2 antibody, Trazimera® (Pfizer laboratory, New York, US) biosimilar of trastuzumab (also called trastuzumab-qyyp, PF-05280014) was provided by the Cancer Center Jean Perrin, Clermont-Ferrand, France. All the glass vessels used for radiolabelling (except Pierce tubes) were pre-treated overnight with a solution of bovine serum albumin (BSA) in PBS (10 mg / mL) and rinsed twice with PBS. All radiolabelled compounds were compared by analytical radio / UV-SEC- HPLC to the authentic non-radioactive material. Radiolabelling with iodine-125: Antibodies and antibody fragments were radiolabelled via direct electrophilic radioiodination on tyrosine residues using Pierce tubes pre-coated with iodogen, a mild oxidative reagent, according to a well- established protocol developed for the radiosynthesis of [125l]trastuzumab (Aurelie Maisonial-Besset et al. Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates. Molecules. 2023 Jan; 28(1 ): 75). Typically, a solution of antibody or antibody fragment (quantity adapted to reach molar activities (Am) of 12.6-15.3 GBq / pmol) in PBS (200- 250 |_iL) was introduced in the Pierce tube before addition of carrier-free [125l]Nal (4- 18 pL, 8-38 MBq). The Pierce tube was sealed and maintained under gentle agitation at room temperature. After 20 min, the reaction media was transferred to a glass vial. The final radiochemical purity (RCP) of all radiotracers was determined using radio- SEC-HPLC and ITLC analyses.

[0371] [125l]Trastuzumab: RCY: 68±7%; RCP: 96.3±2.8%; Am: 13.5±1 .2 GBq / pmol (n=3).

[0372] [125l]Mb(4)-MC-Lys-PEGi2OMe(Cap-VC-PABC-MMAE): RCY: 59±3%; RCP: 98.8±0.4%; Am: 14.2±0.1 GBq / pmol (n=3).

[0373] [125l]lgG(8)-MC-Lys-PEGi2OMe(Cap-VC-PABC-MMAE): RCY: 48±8%; RCP: 97.0±0.8%; Am: 14.3±0.4 GBq / pmol (n=3).

[0374] [125l]lgG(8)-MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA): RCY: 59±3%; RCP: 97.8±0.9%; Am: 16.0±0.4 GBq / pmol (n=3).

[0375] 2 / Immunoreactivity of radiolabelled entities to BT-474 cells

[0376] All experiments were performed with low retention tubes pre-treated (Mb(4)-m- (b-PEG12)-VC-MMAE, lgG(8)-m-(b-PEG12)-VC-MMAE) or not (trastuzumab) with BSA (10 mg / mL) for 24 h at 4 °C before rinsing twice with PBS. BT474 cells (1 .106, 2.106, 5.106and 10.106) were incubated in 0.5 mL of binding media (25 mM HEPES pH = 7, containing 0.2% of BSA, completed with Dulbecco’s Modified Eagle’s Medium (DMEM) F12 (1 / 1 , v / v) glutamax) with 0.65 pmol of [125l]trastuzumab (31 GBq / pmol) or 0.46 pmol of [125l]lgG(8)-m-(b-PEG12)-VC-MMAE (15 GBq / pmol) or 0.60 pmol of [125l]lgG(8)-m-(b-PEG12)-AA-EXA (7.38 GBq / pmol) or 0.60 pmol of Enhertu® (12.08 GBq / pmol) or 0.57 pmol of [125l]Mb(4)-m-(b-PEG12)-VC-MMAE (14 GBq / pmol) in a final volume of 1 mL. After 30 min under gentle shaking, samples were centrifuged at 460 g for 8 min at 4 °C. The supernatants were removed, the cell pellets were washed with PBS containing 0.2% of BSA and centrifuged again at 460 g for 8 min. The collected supernatants and pellets were separately recovered for radioactivity counting using a y-counter (Wallac 1480 Wizard® 3“, Perkin Elmer). Based on the Lindmo method (Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA. Determination of the immunoreactive fraction of radiolabelled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods. 1984; 72: 77- 89), the immunoreactive fraction (IRF) was determined as the inverse of the y- intercept value of the linear regression line obtained by plotting (B+S) / B as a function of 1 / [cell concentration] where B and S are the radioactivity counted in pellets and supernatants, respectively.

[0377] 3 / Injection schedule

[0378] Biodistribution studies were carried out 10-11 weeks post tumour cell inoculation, corresponding to a mean tumour volume of 185 ± 49.6 mm3. Mice were randomly divided in different groups containing three to five mice per time points considered for the biodistribution studies. To block radioiodide cell uptake by sodium / iodide symporter, the animals were treated with a solution of lugol dissolved in water, starting 4 days prior to study. For ex vivo biodistribution, intravenous (i.v.) injections of the radiolabelled entities were performed in the lateral tail vein of vigil mice. Precisely, mice were injected with (i) 1 ,45±0.03 or 16.5±0.18 MBq (T = 72 h) of [125l]trastuzumab (n=3 / time point) and sacrificed 4, 24, 48 and 72 h post injection (p.i.), (ii) 0.99±0.08 or 9.31 ±0.18 MBq (T = 120 h) of [125l]lgG(8)-m-(b-PEG12)-VC- MMAE (n=3 / time point) and sacrificed 3, 6, 24, 48, 72 and 120 h p.i.; (iii) 1.27±0.11 , or 6.77±0.33 (T = 36 h), or 10.1 ±0.66 MBq (T = 72 h) of [125l]Mb(4)-m-(b-PEG12)-VC- MMAE (n=3 / time point, except for T = 6 h (n=5) and T = 24 h (n=4)) and sacrificed 1 , 3, 6, 24, 36, 48, and 72 h p.i. For each experiment, the mice of the last time point were also used for SPECT / CT imaging follow-up studies. For all groups, mice were sacrificed after gaseous anesthesia followed by cervical dislocation and tumour, blood and major organs were excised, weighted and counted with a calibrated y-counter (Wallac 1480 Wizard® 3“, Perkin Elmer). After radioactive decay correction, results were expressed as a percentage of injected dose per gram of tissue (%ID / g) and / or per organ.

[0379] 4 / SPECT / CT imaging studies

[0380] All imaging experiments were performed at IVIA facility (Clermont-Ferrand, France; https: / / www.ibisa.net / plateformes / in-vivo-imaging-auvergne-ivia-495.html). SPECT / CT images were acquired using a hybrid camera (NanoScan Mediso, Ltd, Budapest, Hungary) equipped with 4 multi pinholes collimators on anesthetized mice (Isoflurane, Iso-Vet®, 1.5%, 1 L / min, air / 02, 70 / 30, v / v). All animals were secured on a dedicated bed with temperature control. SPECT-CT acquisitions were performed at different time (n = 3 animals) ranging from 15 min to 120 h post-injection depending on radiolabeled entities. Precisely, mice were injected (i.v.) with (i) 16.5±0.18 MBq of [125l]trastuzumab; (ii) 9.31 ±0.18 MBq of [125l]lgG(8)-m-(b-PEG12)-VC-MMAE; (iii) 10.1 ±0.66 MBq of [125l]Mb(4)-m-(b-PEG12)-VC-MMAE., (iv) 4.23±0.20 MBq of [125l]lgG(8)-m-(b-PEG12)-AA-EXA and (v) 3.45±0.08 MBq of [125l]trastuzumab deruxtecan. Anatomic CT acquisitions of helical scan were carried-out with 480 projections, 300 ms per projection, 50 kV and 590 pA. SPECT acquisitions consisted in 128 projections, 20 s per projection, and an energy window set at 140 keV ± 20%. After image reconstruction using the 3D OSEM algorithm, analyses were performed with InterView FUSION (v3.03.074, Mediso Ltd, Budapest, Hungary).

[0381] Efficacy in vivo studies

[0382] For MMAE-based ADC study:

[0383] Mice were randomly divided to form five groups with homogenous tumoural volume (mean tumoural volume was 160 mm3). 200 pL of treatment were injected in the mouse intraperitoneal cavity (under safety cabinet) five weeks after tumorization. The reference treatment T-DM1 (Kadcyla®, Roche, Bale, Switzerland) was injected at 3 mg / kg in a single dose. Treated groups received lgG(8)-m-(b-PEG12)-VC-MMAE at 6 mg / kg in a single dose, or Mb(4)-m-(b-PEG12)-VC-MMAE at 3 mg / kg five times every two days. Untreated group received phosphate-buffered saline (PBS) in a single dose (Figure 7B). At day 64, the animals were sacrificed by cervical dislocation, tumors were removed, measured, and weighed.

[0384] For EXA-based ADC study:

[0385] Mice were randomly divided to form five groups with homogenous tumoural volume (mean tumoural volume was 160 mm3). 200 pL of treatment were injected in the mouse intraperitoneal cavity (under safety cabinet) at 25 days (1 dose) after tumorization, or at 25 and 29 days (2 doses) after tumorization. The reference treatment T-DXd (Enhertu®, Daiichi Sankyo, Japan) and lgG(8)-m-(b-PEG12)-AA- EXA were injected at 10 mg / kg per dose. Untreated group received phosphate- buffered saline (PBS) in a single dose (Figure 14).

[0386] Statistical analysis Statistical tests were performed with Graphpad software (PRISM®, San Diego, USA). ANOVA tests were used in the biological cited studies. Stars on graphics indicates significant differences related to adjusted p-value (*p<0.05, **p<0.01 , ™p<0.001 , ™p<0.0001 as graphpad PRISM® convention).

[0387] EXAMPLE 4 - RESULTS OF EVALUATION OF IMMUNOCONJUGATES WITH LINKER MC-Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe

[0388] Grafting of linker m-(b-PEG12)-VC-MMAE onto lgG(8) (IgG DAR 8) or Mb(4) (minibody DAR 4) does not modify binding of corresponding conjugates lgG(8)-m-(b- PEG12)-VC-MMAE and Mb(4)-m-(b-PEG12)-VC-MMAE to HER2 in comparison to native respective counterparts (Figure 1 ).

[0389] Grafting of pegylated linker m-(b-PEG12)-VC-MMAE onto lgG(8) or Mb(4) improves internalization of corresponding conjugates lgG(8)-m-(b-PEG12)-VC- MMAE and Mb(4)-m-(b-PEG12)-VC-MMAE into cancer cell lines in comparison to T- DM1 (standard of care Kadcyla®) (Figure 3).

[0390] Table 1 IC50 (in nM) cell-based assays of tested entities on two breast cancer cell lines

[0391] SK-BR-3 (HER2+++) and MDA-MB-231 (HER2 low)

[0392] Grafting of linker m-(b-PEG12)-VC-MMAE onto lgG(8) or Mb(4) confers to corresponding conjugates lgG(8)-m-(b-PEG12)-VC-MMAE and Mb(4)-m-(b-PEG12)- VC-MMAE the ability to kill selectively HER2-positive cancer cell lines with IC50 lower than 30 pM (Figure 5 and Table 1 ).

[0393] Grafting of linker m-(b-PEG12)-VC-MMAE onto lgG(8) or Mb(4) confers to corresponding conjugates lgG(8)-m-(b-PEG12)-VC-MMAE and Mb(4)-m-(b-PEG12)- VC-MMAE better ability to reduce tumor size in mouse in an in vivo BT-474 murine model than T-DM1 (standard of care Kadcyla®) (Figure 7 and 8). EXAMPLE 5 - RESULTS OF EVALUATION OF IMMUNOCONJUGATES WITH LINKER MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe

[0394] HER2 binding:

[0395] ADC lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgG(8)-mal-EXAwas tested for antigen recognition to assess the immunoconjugate ability to recognize the HER2 protein.

[0396] Binding to HER2 was assessed by enzyme-linked immunosorbent assay (ELISA). lgG(8)-mal-EXA was compared to native trastuzumab lgG(8) .

[0397] As visible in figure 2, lgG(8)-mal-EXA has the same binding profile compared to native lgG(8) , even though the absorbance is a bit smaller for the conjugated entity.

[0398] Without wishing to be bound by a theory, the inventors are of the opinion that the slight shift (<log10) could be related to the large number of drugs coupled to the lgG(8)-mal-EXA. The conjugation onto the cysteine side chains of the CH1 and CL domains might disturb their conformation, and indirectly the conformation of the VH and V domains, thus inducing a slight loss of affinity.

[0399] Nevertheless, the trend of the curves showed that the recognition of HER2 is still maintained after the bioconjugation.

[0400] Under these conditions, the tested entities can be compared in cell-based assays without affinity bias.

[0401] It is worth noting that a slight decreased affinity may theoretically increase the homogeneous distribution of the entity in the tumor.

[0402] Moreover, figure 12 shows that grafting of linker MC-Lys-(Cap-AA-PABC-EXA)- PEGi2-OMe onto anti-HER2 lgG(8) (IgG DAR 8), Mb(8) (minibody DAR 8), Mb(4) (minibody DAR 4), Db(4) (diabody DAR 8) does not modify binding to HER2 of corresponding conjugates immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)- PEGi2-OMe, Mb(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, Mb(4)-MC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe, Db(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe in comparison to native respective counterparts.

[0403] To sum up, grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti-HER 2 lgG(8) (IgG DAR 8) does not modify binding of corresponding conjugates lgG(8)-MC-Lys-(Cap-AA-PABC-MMAE)-PEGi2-OMe to HER2 in comparison to trastuzumab. HRMS:

[0404] Grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti-HER2 lgG(8) (IgG DAR 8), Mb(8) (minibody DAR 8), Mb(4) (minibody DAR 4), Db(4) (diabody DAR 8) and IRR lgG(8) (irrelevant antibody IgG) afforded respectively homogeneous and site-specific anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe, Mb(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, Mb(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, Db(4)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe and irrelevant immunoconjugate lgG(8)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe (Figure 9).

[0405] Grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, with hydrophobicity control, onto antibodies, leads to immunoconjugates mainly as monomers, according to size exclusion chromatography (SEC) (Figure 11 ). SEC profiles of native and conjugated proteins are comparable.

[0406] Internalization:

[0407] As shown in figure 4, after 24 h of exposure at 37°C on cells, lgG(8)-mal-EXA and T-DXd were internalized compared to the negative control ppL-PE alone and to lgGirr(8)-EXA (figure 4B). lgGirr(8)-EXA is considered as a second negative control in so far as this irrelevant IgG of DAR 8 does not recognize HER2. The two negative controls were not internalized on HER2 positive cells SK-BR-3 (figure 4B) or negative cells MDA-MB-468 (figure 4A) (MFI < 5), whereas lgG(8)-mal-EXA and T-DXd are internalized only on the positive HER2 positive cells SK-BR-3 and not on the negative cells MDA-MB-468, showing their specificity against HER2.

[0408] Further, the MFI measured for the cells incubated with lgG(8)-mal-EXA was significantly increased and was more than doubled compared to the reference compound T-DXd ADC (MFI of 120 versus 55 respectively).

[0409] In these studies, the MFI values represents mainly the intracellular signal of the labelled immunoconjugates and a minority of membrane label, since a fluorescence merge of the images was performed after incubation of lgG(8)-mal-EXA / ppL-PE and T-DXd / ppL-PE for 24 h at 37°C. The recorded signal is mainly cytoplasmic and very little at the membrane level for both entities.

[0410] These qualitative results are correlated with semi-quantitative results of flow cytometry. Accordingly, lgG(8)-mal-EXA and T-DXd are well internalized in HER2 positive cells with a better internalization for lgG(8)-mal-EXA. As each condition is standardized with the same cells, MFI is not subject to variations in the number of HER2 receptors at the cell surface.

[0411] No signal of internalization was recorded on flow cytometry nor on microscopy images when the same acquisition parameters as SK-BR-3 were applied.

[0412] To sum up, grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti-HER2 lgG(8) (IgG DAR 8) improves internalization of corresponding conjugate lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe into cancer cell lines in comparison to trastuzumab deruxtecan (Figure 4).

[0413] Cytotoxicity:

[0414] As shown in figure 6 and in Table 2 hereafter, the cytotoxicity of lgG(8)-mal- EXA was tested on breast cancer cell lines and compared to their irrelevant lgGirr(8)- EXA counterpart and to free exatecan (EXA).

[0415] Firstly, on SK-BR-3 cells overexpressing HER2 (Figure 6A), as expected, no activity of the irrelevant lgGirr(8)-EXA was observed. For the lgG(8)-mal-EXA conjugate, a high cytotoxicity with an IC50 value of 0.42 nM was highlighted.

[0416] Secondly, on MDA-MB-468 cells having no HER2 gene amplification (Figure 6B), only a slight cytotoxicity was observed with concentrations of the conjugates above 100 nM (no IC50 values below 30 nM was measured with any of the tested compounds).

[0417] These results shows that the immunoconjugates according to the invention are very specific of HER2 as targeted therapies, and that they don’t release drug in the cell media during incubation.

[0418] To sum up, grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti HER-2 lgG(8) confers to corresponding conjugates lgG(8)-MC-Lys-(Cap-AA- PABC-EXA)-PEG12-OMe the ability to kill selectively HER2-positive cancer cell lines with IC50 lower than 1 nM (Figure 6 and Table 2).

[0419] Table 2: IC50 (in nM) cell-based assays of tested entities on two breast cancer cell lines

[0420] SK-BR-3 (HER2+++) and MDA-MB-468 (HER2 negative) Biodistribution:

[0421] Grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-0Me onto anti HER-2 lgG(8) allows to obtain an analogous, and even slightly improved, biodistribution of corresponding conjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe over time as with reference compound trastuzumab deruxtecan (T-Dxd), as shown by figure 13A representing the evolution over time of the ratio of percentage of injected dose in tumor over percentage of injected dose in blood (T / S) and of the ratio of percentage of injected dose in tumor over percentage of injected dose in muscle (T / M) for lgG(8-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, in comparison to figure 13B obtained with trastuzumab deruxtecan. Hence, grafting of linker MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe onto anti HER-2 lgG(8) allows satisfactory tumor recognition by the corresponding conjugates.

[0422] Serum Stability:

[0423] Grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEG12-OMe onto anti-HER2 and irrelevant antibodies afforded homogeneous and site-specific anti-HER2 immunoconjugates that are stable in PBS at 4°C for 120 days (Figure 10).

[0424] In addition, measurements of DAR variation over time of immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe and trastuzumab deruxtecan (T- DXd) in human serum or mouse serum showed 0.3 DAR reduction at 192h in both human and mouse serum for lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, while trastuzumab deruxtecan (T-DXd) showed significant DAR reduction in human (- 1.1 DAR) and mouse (-0.9 DAR) serum (Table 3 hereafter).

[0425] Table 3 : ratio of the DAR and of the initial DAR (in %) as a function of time after injection

[0426] Hence, grafting of linker MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-0Me onto anti HER-2 lgG(8) allows to obtain improved stability over time of the corresponding conjugates, with no or very low DAR loss, together with improved internalization as mentioned above.

[0427] In vivo efficacy:

[0428] This is confirmed by figure 14, showing tumoral volume evolution over time after either one or two injections of anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe compared to reference compound trastuzumab deruxtecan (T-DXd)).

[0429] As visible in figure 14, the tumor growth analyses showed a significantly increased anti-tumor activity of lgG(8)-mal-EXA and T-DXd compared to the control group (PBS as a vehicle) was quickly highlighted around day 35, just few days after the first injection at day 25.

[0430] At day 60, both lgG(8)-mal-EXA and T-DXd achieved complete and lasting remission after two injections (up to day 75 corresponding to the last monitoring day).

[0431] In parallel, from day 35 to day 60, lgG(8)-mal-EXA and T-DXd achieved a reduction in tumor growth after a single injection, but after day 65, tumor growth is slighlty beginning to start again. In this case, lgG(8)-mal-EXA shows no significant difference with T-DXd.

[0432] It should be noted that lgG(8)-mal-EXA and T-DXd treatments were very well tolerated by the mice, even with two injections of each immunoconjugates.

Claims

CLAIMS1. A compound having the following formula (I):wherein:- m represents an integer comprised from 1 to 3,- Li represents a bioconjugation unit capable of binding to at least one antibody or antibody fragment by reacting with an amino, hydroxyl, amide or thiol function,- L2represents a spacer group of formula -Ai-C(=O)-, Ai representing a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,- L3represents a spacer group of formula -A2-C(=O)-, A2representing a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,- L4represents a release system selected from the group consisting of:* the group having the following formula (II):-[NH-CH(Ai)-C(=O)]i-[A9]j- (II) wherein:. i is 0 or is an integer from 1 to 8,. j is 0 or 1 , provided that i is not 0 when j=0, and when i=0 then j=1 ,. each Ai represents, independently from each other, the side chain of an amino acid residue, in particular a linear or branched (Ci-Ce)alkyl chain, and. A9represents a -NH-CH2- group or a group having the following formula* the group having the following formula (III):wherein:. X’ is H or NO2,. L7is a group of formula H-[NH-CH(Ai)-C(=O)]i-, i and A being as defined above in formula (II), or l_7 is selected from the group consisting of: beta-glucuronic acid, beta- D-galactose, beta-D-glucose, alpha-D-mannose, N-acetyl-D- glucosaminyle, N-acetyl-D-galactosaminyle, D-glucuronyle, L-iduronyle, D-glucopyranosyle, D-galactopyranosyle, D-mannopyranosyle, and L- fucopyranosyle,. L8represents a group -CH2-CH2-C(=O)-NH-, a bond or a linear or branched alkylene radical comprising from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom,* the group having the following formula (IV):wherein:. X” is H or NO2, and. L7and L8are as defined in formula (III);- L5represents an anticancer agent, and- L6represents a PEG moiety selected from the group consisting of:* the group of formula -(CH2-CH2-O)n-CH3, n representing an integer from 1 to 24, and* the group of the following formula (V):-(CH2)3-C(=O)-NH-(CH2-CH2-O)4-(CH2)2-C(=O)-NH-C(R)3(V) wherein R is a group of the following formula (V-1 ):-CH2-O-(CH2)2-C(=O)-NH-(CH2)2-(O-CH2-CH2)7-OCH3.

2. The compound of formula (I) according to claim 1 , wherein L2 and L3, independently from each other, represent a spacer group of formula -(CH2)P-C(=O)-, p being an integer comprised from 2 to 20, or a spacer group of formula -(CH2-CH2- O)m-(CH2)r-C(=O)-, m being an integer comprised from 1 to 10 and r being 1 or 2.

3. The compound of formula (I) according to claim 1 or 2, wherein Li comprises or is a maleimide group, a NHS ester group or an isothiocyanate group.

4. The compound of formula (I) according to any one of the preceding claims, wherein L4 is selected from the group consisting of:- the group having the following formula (VI):- the group having the following formula (VII):- and the group having the following formula (VIII):

5. The compound of formula (I) according to any one of the preceding claims, wherein:- L4has the formula (VI) as defined in claim 5 and L5is monomethyl auristatin E; or- L4has the formula (VIII) as defined in claim 5 and L5is exatecan.

6. The compound of formula (I) according to any one of the preceding claims, wherein L6is a group having the formula -(CH2-CH2-O)n-CH3, n being 12 or 24.

7. A conjugate comprising at least one compound according to any one of the preceding claims, said compound being covalently bound to at least one cell binding agent, in particular selected from the antibodies and the antibody fragments.

8. The compound according to any one of claims 1 to 6 or a conjugate according to claim 7, for its use as drug.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 or a conjugate according to claim 7, and also at least one pharmaceutically acceptable excipient.

10. The compound according to any one of claims 1 to 6 or a conjugate according to claim 7, for its use for treating cancer.

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