Bioconjugation systems and uses thereof

Abstract

The present invention relates to a compound having the following formula (I) wherein: - A represents an aryl or heteroaryl group, - n is 1 or 3, - L1 represents a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms, - L2 represents a release system, and - L3 represents an anticancer agent.

Description

[0001] BIOCONJUGATION SYSTEMS AND USES THEREOF

[0002] The present invention concerns new bioconjugation systems, in particular for antibody-drug 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.

[0006] In parallel, the FDA approved four ADCs (Adcetris®, Polivy®, Padcev®, Tivdak®) on the market with vedotin, comprising the drug monomethyl auristatin E (MMAE), with an average DAR of 4. On one hand, the maleimide included in this linker is associated with partial deconjugation during plasma circulation, through a retro- Michael reaction, thus releasing vedotin, which is then conjugated to any sulfur component of the plasma (glutathione, albumin, etc), which mediates payload- mediated toxicities leading to side effects in patients. 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. Therefore, enhancing plasma stability, in particular at the conjugation site onto antibodies by replacing classical first generation maleimide (e.g. maleimidocaproic unit), is of particular interest, to reach better-designed ADC with enhanced stability in plasma, leading to more favorable pharmacokinetic properties for the resulted immunoconjugates, in particular to improved internalization.

[0007] Careful linker design is thus essential to reach better tolerance and efficacy of ADCs, and to this end, stability control during plasma circulation appears to be a key parameter. Indeed, the linker has a major role in the stability of immunoconjugates during plasma circulation and in the controlled release of the drug into the tumor.

[0008] An aim of the present invention is thus to provide new bioconjugation systems 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.

[0009] Another aim of the present invention is to provide new bioconjugation systems for the preparation of antibody-drug conjugated, allowing doubling the DAR on each disulfide bridge in comparison with the current conjugates.

[0010] Another aim of the present invention is to provide new bioconjugation systems for the preparation of antibody-drug conjugated, with high DAR, preferably of 8 or 12, with satisfying yields. Thus, the present invention relates to a compound having the following formula wherein:

[0011] - A represents an aryl or heteroaryl group, said aryl or heteroaryl group being optionally subsituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups,

[0012] - n is 1 or 3,

[0013] - Li represents a linear or branched alkylene radical, comprising from 1 to 20, preferably from 2 to 20 carbon atoms, optionally interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group, at least one of the carbon atoms of said radical being optionally substituted with at least one alkylene side chain comprising 2 to 80 carbon atoms, optionally interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group;

[0014] - L2represents 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 * the group having the following formula (III): wherein:

[0021] . X’ is H or NO2,

[0022] . L4is a group of formula H-[NH-CH(Ai)-C(=O)]i-, i and A being as defined above in formula (II), or l_4 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,

[0023] . L5represents 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,

[0024] * the group having the following formula (IV): wherein:

[0025] . X” is H or NO2, and

[0026] . L4and L5are as defined in formula (III);

[0027] - L3represents an anticancer agent.

[0028] 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. 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.

[0029] 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.

[0030] 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.

[0031] The term "alkoxy group" means: an -O-alkyl radical where the alkyl group is as previously defined. By way of examples, mention may be made of -O-(Ci-C4)alkyl groups, and in particular the -O-methyl group, the -O-ethyl group as -O-Csalkyl group, the -O-propyl group, the -O-isopropyl group, and as -O-C4alkyl group, the -O-butyl, - O-isobutyl or -O-tert-butyl group.

[0032] The term "aryl group" means: a cyclic aromatic group comprising between 6 and 10 carbon atoms. By way of examples of aryl groups, mention may be made of phenyl or naphthyl groups.

[0033] The term "heteroaryl group" means: a 5- to 10-membered aromatic monocyclic or bicyclic group containing from 1 to 4 heteroatoms selected from O, S or N. By way of examples, mention may be made of imidazolyl, thiazolyl, oxazolyl, furanyl, thiophenyl, pyrazolyl, oxadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, benzofuranyl, benzothiophenyl, benzoxazolyl, benzimidazolyl, indazolyl, benzothiazolyl, isobenzothiazolyl, benzotriazolyl, quinolinyl and isoquinolinyl groups. By way of a heteroaryl comprising 5 to 6 atoms, including 1 to 4 nitrogen atoms, mention may in particular be made of the following representative groups: pyrrolyl, pyrazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, tetrazolyl, and 1 ,2,3-triazinyl.

[0034] Mention may also be made, by way of heteroaryl, of thiophenyl, oxazolyl, furazanyl, 1 ,2,4-thiadiazolyl, naphthyridinyl, quinoxalinyl, phthalazinyl, imidazo[1 ,2- a]pyridine, imidazo[2,1 -b]thiazolyl, cinnolinyl, benzofurazanyl, azaindolyl, benzimidazolyl, benzothiophenyl, thienopyridyl, thienopyrimidinyl, pyrrolopyridyl, imidazopyridyl, benzoazaindole, 1 ,2,4-triazinyl, indolizinyl, isoxazolyl, isoquinolinyl, isothiazolyl, purinyl, quinazolinyl, quinolinyl, isoquinolyl, 1 ,3,4-thiadiazolyl, thiazolyl, isothiazolyl, carbazolyl, and also the corresponding groups resulting from their fusion or from fusion with the phenyl nucleus.

[0035] The term "halogen" means: a fluorine, a chlorine, a bromine or an iodine.

[0036] In one embodiment, in the formula (I) as defined above, n is 1 .

[0037] In one embodiment, in the formula (I) as defined above, A represents an aryl group comprising 6 to 10 carbon atoms, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

[0038] In one embodiment, in the formula (I) as defined above, A represents a phenyl group, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

[0039] In one embodiment, the compounds of the invention have the following formula (1-1 ):

[0040] Li, l_2, and L3being as defined above in formula (I).

[0041] In one embodiment, in the formula (I) as defined above, A represents an aromatic monocyclic group comprising 5 or 6 atoms and including at least one heteroatom selected from N, S, or O, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

[0042] In one embodiment, in the formula (I) as defined above, A represents an aromatic monocyclic group comprising 5 or 6 atoms and including at least one nitrogen atom, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (C1- Ce)alkoxy groups.

[0043] In one embodiment, in the formula (I) as defined above, A represents a pyridinyl group, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

[0044] In one embodiment, the compounds of the invention have the following formula (1-2):

[0045] Li, l_2, and L3are as defined above in formula (I).

[0046] In one embodiment, in the above formula (I), A has one of the following formulae:

[0047] (4) (5) (6) the symbols showing the bonds with the groups -NH-C(=O)-CH=CH2, and -NH-C(=O)-LI-C(=O)-L2-L3. A preferred group of compounds according to the invention consists of compounds having the formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, wherein Li represents a linear or branched alkylene radical, comprising from 2 to 20 carbon atoms.

[0048] A preferred group of compounds according to the invention consists of compounds having the formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, wherein Li represents a linear or branched alkylene radical, comprising from 1 to 20, preferably from 2 to 20, carbon atoms, and being interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group.

[0049] A preferred group of compounds according to the invention consists of compounds having the formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, wherein Li represents a linear or branched alkylene radical, comprising from 1 to 20, preferably from 2 to 20 carbon atoms, wherein at least one of the carbon atoms of said radical is substituted with at least one alkylene side chain comprising 2 to 80 carbon atoms, optionally interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group.

[0050] In a particularly preferred embodiment, Li represents a spacer group of formula or -(CH2)m-C(=O)-NH-CR’-C(=O)-NH-(CH2)m’ -, each of m and m’ being an integer comprised from 1 to 20, preferably from 2 to 20, more preferably from 3 to 6, for example equal to 4, and R’ being said alkylene side chain. In a preferred embodiment R’ is of formula - (CH2)4-NHCO-I_6, Le representing a PEG of formula -(CH2-CH2-O)q- CH3, q representing an integer from 1 to 24, for example 12.

[0051] A preferred group of compounds according to the invention consists of compounds having the formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, wherein Li represents a spacer group of formula -(CH2)P-, p being an integer comprised from 1 to 20, preferably from 2 to 20.

[0052] In one embodiment, in formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, Li represents a spacer group of formula -(CH2)P-, p being an integer comprised 1 to 20, preferably from from 2 to 20.

[0053] In one embodiment, in formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, Li represents 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.

[0054] In one embodiment, in formula (I) or one of the formulae (1-1 ) or (I-2) as defined above, Li is as defined above and includes at least one side chain -(CH2)4-NHCO-L6, L6representing a PEG moiety selected from the group consisting of: * the group of formula -(CH2-CH2-O)q-CH3, q representing an integer from 1 to 24, for example 12, and

[0055] * the group of the following formula (IX):

[0056] -(CH2)3-C(=O)-NH-(CH2-CH2-O)4-(CH2)2-C(=O)-NH-C(R)3(IX) wherein R is a group of the following formula (X-1 ):

[0057] -CH2-O-(CH2)2-C(=O)-NH-(CH2)2-(O-CH2-CH2)7-OCH3.

[0058] This group of compounds according to the invention thus includes compounds comprising a PEG moiety (L6) that plays the role of a hydrophobicity masker, and thus allows the control of hydrophobicity of the linker.

[0059] According to an embodiment, in formula (I) or in formulae (1-1 ) or (I-2), L2 is selected from the group consisting of:

[0060] - the group having the following formula (V):

[0061] - the group having the following formula (VI):

[0062] - and the group having the following formula (VII):

[0063] In one embodiment, in the above formula (I), or in one of the formulae (1-1 ) or (I-2) as defined above, L3is 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, 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, a 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.

[0064] 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.

[0065] 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.

[0066] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_3 is an anticancer agent selected from the group consisting of: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exatecan, and derivatives thereof.

[0067] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_3 is an anticancer agent selected from the group consisting of: monomethyl auristatin E (MMAE), and derivatives thereof.

[0068] In one embodiment, in the above formula (I), or in one of the above formulae (1-1 ) or (I-2), l_3 is an anticancer agent selected from the group consisting of: exatecan, and derivatives thereof.

[0069] Preferably, L3is represented by the following formula (A-1 ):

[0070] 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:

[0071] - L2has the formula (V) as defined above and L3is monomethyl auristatin E; or - L2has the formula (VII) as defined above and L3is exatecan.

[0072] Examples of preferred compounds according to the invention are as follows:

[0073] (Compound B)

[0074]

[0075] (Compound D)

[0076] 5

[0077]

[0078] (Compound 24)

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] By “antibody fragment” is meant any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction.

[0084] The antibody according to the present invention can be a chimeric monoclonal antibody, humanized or human, monospecific or bispecific.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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. Enzymatic digestion of the immunoglobulins by pepsin generates a F(ab')2 fragment and an Fc fragment split into several peptides.

[0091] 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).

[0092] 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.

[0093] The Fd fragment is formed by the VH and CH1 domains.

[0094] 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.

[0095] 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”).

[0096] In the context of the present invention, diabodies can be used. They can have multiple valences and specificities.

[0097] The term “valent” denotes the presence of a defined number of antigen binding sites in an antibody molecule.

[0098] The term “specific” refers to the different types of antigens that can bind to the same antibody.

[0099] 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.

[0100] The Fab, Fv and scFv fragments are monovalent and monospecific.

[0101] By “minibody” is meant a fragment composed of a light chain, of a heavy chain bound to a CH3 group. In the context of the present invention, the antibody or antibody fragment is directed against a tumour antigen or inflammation antigen.

[0102] 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, gp100, 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] The present invention relates to a compound of formula (I) as defined above, or the above conjugate, for use as a medicine.

[0107] The present invention relates to a compound of formula (I) as defined above, or the above conjugate, for use as a drug.

[0108] 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.

[0109] In some preferred embodiments, the active ingredients required for the combination therapy may be combined in a single pharmaceutical composition for simultaneous administration. 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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. 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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. FIGURES

[0120] In the figures and in the description, PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe, PyC-Lys-PEGi2-OMe-(Cap-AA-PABC-EXA) and py-EXA are equivalent notations.

[0121] In the figures and in the description, MC-Lys-(Cap-AA-PABC-EXA)-PEGi2- OMe, MC-Lys-PEGi2-OMe-(Cap-AA-PABC-EXA) and mal-EXA are equivalent notations.

[0122] In the figures and in the description, lgGiRR(8)-MC-Lys-(Cap-AA-PABC-EXA)- PEGi2-OMe and lgGiRR(8)-EXA are equivalent notations.

[0123] Figure 1 : Stability comparison in PBS at 4°C for 327 days of two anti-HER2 immunoconjugates Db(4)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe and Db(4)- PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe. MC = MaleimidoCaproic; PyC = N-(Q- acryloylpyridin-2-yl)-6-oxohexanamide; Db(4) = diabody DAR 4.

[0124] Figure 2: HER2 binding affinities of anti-HER2 immunoconjugates lgG(8)-PyC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.5) (square curve) in comparison to anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.3) (circle curve) and trastuzumab (lgG(8)) (diamond curve), determined by indirect ELISA.

[0125] Figure 3: Internalization of (from left to right) ppL-PE alone, irrelevant immunoconjugate lgGiRR(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (IgGiRR(S)- EXA) (example 2.4), anti-HER2 immunoconjugate lgG(8)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe (example 2.3), anti-HER2 immunoconjugate lgG(8)-PyC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.5) and trastuzumab deruxtecan (T- DXd) after complexation with ppL-PE in SK-BR-3 cell lines (figure 3A) and in HER2- negative MDA-MB-468 cells (figure 3B), as analyzed by flow cytometry (exposure time = 24 h at 37°C). ANOVA test (ns: non significative, and •** *p<0.0001 ).

[0126] Figure 4: In vitro cytotoxic activity of anti-HER2 immunoconjugates lgG(8)-PyC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.5) (up triangle curve), in comparison with anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe (up triangle curve) (example 2.3), irrelevant immunoconjugates lgGiRR(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgGiRR(8)-EXA, example 2.4) (down triangle curve), trastuzumab deruxtecan (T-DXd, square curve) and free exatecan (cross curve) on SK-BR-3 (figure 6A) and HER2-low MDA-MB-468 (figure 6B) cancer cell lines after five days of incubation at 37°C - CellTiter Gio assay (Promega®).

[0127] Figure 5: Tumor / blood (T / B) and tumor / muscle (T / M) ratios over time for [125l]- anti-HER2 immunoconjugates lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (example 2.5) (figure 5A) and [125l]-trastuzumab deruxtecan (figure 5B).

[0128] Figure 6: 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)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (gray square curve) (example 2.5, lgG(8)-Py-EXA (1 )) in comparison to one injection of trastuzumab deruxtecan (T-DXd (1 ), light gray circle alternate dotted curve), and two injections (0.07 pmol / kg equivalent to 10.0 mg / kg of T-DXd) of anti-HER2 immunoconjugates lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (black circle double alternate dotted curve) (example 2.5, lgG(8)-Py-EXA (2)) in comparison to two injections of trastuzumab deruxtecan (T-DXd (2), black bordered square black solid curve), or with PBS injection (black triangle curve), figure 6B being a zoomed view of a portion of figure 6A.

[0129] EXAMPLES

[0130] MATERIAL AND METHODS

[0131] 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).

[0132] 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 aluminium 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.

[0133] Denaturing High-Resolution Mass Spectrometry (HRMS) of conjugates

[0134] 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.

[0135] Size-Exclusion Chromatography (SEC) analysis

[0136] 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.

[0137] General protocol of bioconjugation

[0138] BBS buffer

[0139] 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. Purification and buffer exchange of antibodies

[0140] Desalting and Buffer Exchange Procedure is proceed by selecting the appropriate MWCO (50 kDa for IgG before bioconjugation and 10 kDa for IgG- conjufgates and FDCs) 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.

[0141] TCEP reduction and linker-cytotoxic bioconjugation

[0142] 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)

[0143] 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. Conjugates purification and final buffer exchange

[0144] Vivaspin® protocol for FDCs purification

[0145] 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).

[0146] Vivaspin® protocol for ADCs purification

[0147] 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.

[0148] Superdex® purification

[0149] 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).

[0150] Determination of conjugates concentrations and alternative way to DAR determination

[0151] Antibody trastuzumab and ADCs concentrations were determined by UV absorbance using NanoDrop-spectrophotometer (Thermo Fisher Scientific). SDCs and FDCs (antibody Fragment-Drug Conjugate) concentrations were determined by UV absorbance using UV biophotometer.

[0152] When exatecan was used the method of determination of conjugates concentrations change.

[0153] The basis for quantitative analysis in the UV / Vis spectrophotometric experimentation is the Beer-Lambert law: 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).

[0154] 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:

[0155] Atot =AmAb +Adrug (1)

[0156] 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.

[0157] Then we determine the concentration of the drug:

[0158] The concentration of conjugate is directly linked to the concentration of mAb which is determined with the parallel equation (3) of equation (1):

[0159] The average drug to antibody ratio is expressed as moles of drugs to moles of antibody calculated with (4):

[0160] Sample preparation for biological evaluation and storage

[0161] 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.

[0162] EXAMPLE 1 : PREPARATION OF COMPOUNDS ACCORDING TO THE INVENTION

[0163] Example 1.1. Synthesis of compound (A) Methyl 6-((3-nitrophenyl)amino)-6-oxohexanoate (A-1)

[0164] Chemical Formula: C13H16N2O5Molecular Weight: 280,28

[0165] To 1.5 mL of THF were added m- nitroanilin (100.0 mg, 0.72 mmol, 1 eq) and EtsN (1 16.1 pL, 0.86 mmol, 1.2 eq). Methyl adipoyl chloride (112.1 pL, 0.72 mmol, 1 eq) was added dropwise. The mixture was stirred overnight at room temperature. Volatiles were removed and the product was recristalized in methanol to afford a pale yellow powder (125.3 mg, 0.447 mmol, 62%)

[0166] 1H NMR (300 MHz, DMSO-cfe) 6 10.39 (s, 1 H), 8.64 (t, J= 2.1 Hz, 1 H), 7.89 (dd, J = 8.4, 2.2 Hz, 2H), 7.59 (t, J = 8.2 Hz, 1 H), 3.58 (s, 3H), 2.35 (q, J = 6.8 Hz, 4H), 1.66-1.52 (m, 4H).

[0167] 13C NMR (101 MHz, DMSO-cfe) 5 173.3 (1 C), 171.8 (1 C), 148.0 (1 C), 140.4 (1 C), 130.2 (1 C), 125.0 (1 C), 1 17.6 (1 C), 1 13.1 (1 C), 51.3 (1 C), 36.0 (1 C), 33.0 (1 C), 24.3 (1 C), 24.0 (1 C).

[0168] HRMS (ESI+) calcd for C13H17N2O5 (M+H+) 281 .1127, found 281 .1132

[0169] Methyl 6-((3-aminophenyl)amino)-6-oxohexanoate (A-2)

[0170] Chemical Formula: C13HI8N2O3Molecular Weight: 250,30

[0171] To 10 mL of ethyl acetate were added A-1 (120.1 mg, 0.43 mmol) and Pd / C (10% mol). The solution was stirred under H2overnight at room temperature. Once the reaction was over the solution was filterred off on a celite pad and volatiles were removed to afford a browny powder (107.1 mg, 0.428 mmol, quant.).1H NMR (300 MHz, DMSO-cfe) 59.54 (s, 1H), 7.92-6.85 (m, 2H), 6.65 (d, J = 7.9 Hz, 1H), 6.22 (dd, J = 7.9, 1.3 Hz, 1H), 5.02 (s, 2H), 3.58 (s, 3H), 2.35-2.23 (m, 4H), 1.59-1.52 (m, 4H).

[0172] 13C NMR (101 MHz, DMSO-cfe) 5173.3 (1C), 170.6 (1C), 148.9 (1C), 139.9 (1C), 128.8 (1C), 109.1 (1C), 107.1 (1C), 104.8 (1C), 51.2 (1C), 36.0 (1C), 33.1 (1C), 24.7 (1C), 24.1 (1C).

[0173] HRMS (ESI+) calcd for C13H19N2O3 (M+H+) 251.1385, found 251.1390

[0174] Methyl 6-((3-acrylamidophenyl)amino)-6-oxohexanoate (A-3)

[0175] Chemical Formula: C16H20N2O4 Molecular Weight: 304,35

[0176] To 1 mL of THF with 61.3 pL of EtsN, were added A-2 (100.5 mg, 0.40 mmol, 1 eq) and acryloyl chloride (32.3 pL, 0.40 mmol, 1 eq). The mixture was stirred overnight at room temperature. Once the reaction was over, the product was purified by flash chromatography (CH / EtOAc - 70 / 30 to 20 / 80) to afford a white powder (64.9 mg, 0.213 mmol, 53%).

[0177] 1H NMR (300 MHz, DMSO-cfe) 510.13 (s, 1H), 9.91 (s, 1H), 8.00 (s, 1H), 7.37 (d, =7.8 Hz, 1H), 7.29-7.18 (m, 2H), 6.45 (dd, J = 17.0, 10.0 Hz, 1H), 6.24 (dd, J = 17.0, 2.2 Hz, 1H), 5.74 (dd, J = 10.0, 2.2 Hz, 1H), 3.58 (s, 3H), 2.38-2.25 (m, 4H), 1.67-1.51 (m, 4H).

[0178] 13C NMR (101 MHz, DMSO-cfe) 5173.3 (1C), 171.0 (1C), 163.1 (1C), 139.6 (1C), 139.3 (1C), 131.9 (1C), 128.9 (1C), 126.8 (1C), 114.4 (1C), 114.2 (1C), 110.2 (1C), 51.2 (1C), 36.0 (1C), 33.1 (1C), 24.6 (1C), 24.1 (1C).

[0179] HRMS (ESI+) calcd for C16H21N2O4 (M+H+) 305.1491 , found 305.1496

[0180] 6-((3-acrylamidophenyl)amino)-6-oxohexanoic acid (A-4)

[0181] Chemical Formula: C15H18N2O4 Molecular Weight: 290,32

[0182] To 1 mL of THF, were added A-3 (60.0 mg, 0.197 mmol, 1 eq) and 1 mL (2.5 eq) of LiOH 0.5 N. The mixture was stirred 3h. Once the reaction was over, HCL 1N was added to quench the solution. 10 mL of H2O were added and aqueous layer was extracted with ethyl acetate. Organic layers were dried over MgSO4 and evaporated under vacuum to afford a white powder (50.2 mg, 0.173 mmol, 88%).

[0183] 1HNMR(400 MHz, DMSO-cfe) 512.00 (s, 1H), 10.12 (s, 1H), 9.90 (s, 1H), 8.00 (s, 1 H), 7.38 (d, J= 8.0 Hz, 1 H), 7.27 (d, J= 8.4 Hz, 1 H), 7.21 (t, J= 8.0 Hz, 1 H), 6.44

[0184] (dd, J=17.0, 10.1 Hz, 1H), 6.24 (dd, J= 17.0, 2.0 Hz, 1H),5.73 (dd, J= 10.1 , 2.0 Hz, 1 H), 2.30 (t, J = 7.1 Hz, 2H), 2.24 (t, J = 7.1 Hz, 2H), 1.65-1.48 (m, 4H).

[0185] 13C NMR (101 MHz, DMSO-cfe) 5174.4 (1C), 171.1 (1C), 163.1 (1C), 139.6 (1C), 139.3 (1C), 131.9 (1C), 128.9 (1C), 126.8 (1C), 114.4 (1C), 114.2 (1C), 110.2 (1C), 36.1 (1C), 33.4 (1C), 24.7 (1C), 24.1 (1C).

[0186] HRMS (ESI+) calcd for C15H19N2O4 (M+H+) 291.1342, found 291.1339

[0187] 4-((S)-2-((S)-2-(6-((3-acrylamidophenyl)amino)-6-oxohexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1 -(((S)-1 -(((3R,4S,5S)-1 - ((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2- methyl-3-oxopropyl)pyrrolidin-1 -yl)-3-methoxy-5-methyl-1 -oxoheptan-4- yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)(methyl)carbamate (A)

[0188] Chemical Formula: C73H110N12O15Molecular Weight: 1395,75 To 600 pL of DMF was dissolved TFA.ValCitPABMMAE (9.0 mg, 0.008 mmol,

[0189] 1 eq) before the addition of A-4 (2.9 mg, 0.010 mmol, 1.3 eq). HATU (3.8 mg, 0.010 mmol, 1.3 eq) was dissolved and finally, 2,6-Lutidine (1.16 pL, 0.010 mmol, 1.3 eq) was added to the mixture. The solution was stirred overnight at room temperature.

[0190] The reaction was monitored by analytic-HPLC. The solution was purified by semipreparative HPLC (gradient A) to afford a white powder (7.0 mg, 0.005 mmol,

[0191] 63%)

[0192] HRMS (ESI+) calcd for C73H111N12O15 (M+H+) 1395.8292, found 1395.8286 Example 1.2. Synthesis of compound (B)

[0193] Tert-butyl (2-aminophenyl)carbamate (B-1)

[0194] Chemical Formula: CII H16N2O2 Molecular Weight: 208,26

[0195] To 2 mL of THF were added o- phenylenediamine (100.0 mg, 0.92 mmol, 1 eq) and B0C2O (201.0 mg, 0.92 mmol, 1 eq). K2CO3 (0.46 mmol, 0.5 eq) was added and the mixture was stirred overnight at room temperature. The solution was diluted with 20 mL of EtOAc and washed 3 times with 50 mL of water. Organic layers were dried over MgSO4, and concentrated under vacuum to afford a white powder (152.1 mg, 0.73 mmol, 79%)

[0196] 1H NMR (400 MHz, DMSO-cfe) 58.26 (s, 1 H), 7.17 (d, J= 7.6 Hz, 1 H), 6.83 (td, J= 7.9, 1 .5 Hz, 1 H), 6.67 (dd, J= 7.9, 1 .4 Hz, 1 H), 6.51 (td, J= 7.7, 1 .4 Hz, 1 H), 4.80 (s, 2H), 1.45 (s, 9H).

[0197] 1H NMR in accordance with literature (Du F. & Al, New J. Chem., 2019,43, 6549- 6554).

[0198] Methyl 6-((2-((tert-butoxycarbonyl)amino)phenyl)amino)-6-oxohexanoate

[0199] (B-2)

[0200] Chemical Formula: C18H26N2O5Molecular Weight: 350,42

[0201] To 3 mL of THF with EtsN (128.2 pL, 0.95 mmol, 1.1 eq) were added B-1 (179.4 mg, 0.86 mmol, 1 eq) and methyl adipoyl chloride (133.8 pL, 0.86 mmol, 1 eq). The mixture was stirred overnight at room temperature. Once the reaction was over, the solution was dissolved in methanol and concentrated under reduced pressure. The product was purified by flash chromatography (DCM / MeOH - 1 to 15%) to afford a yellow oil (190.5 mg, 0.54 mmol, 63%).

[0202] 1H NMR (400 MHz, DMSO-cfe) 59.46 (s, 1 H), 8.30 (s, 1 H), 7.53 (d, J = 7.5 Hz, 1 H), 7.40 (dd, = 7.8, 1.1 Hz, 1 H), 7.13 (td, J= 7.8, 1.5 Hz, 1 H), 7.07 (td, J= 7.6, 1.5 Hz, 1 H), 3.59 (s, 3H), 2.38-2,32 (bs, 4H), 1 .64-1 .53 (m, 4H), 1 .45 (s, 9H).

[0203] 13C NMR (101 MHz, DMSO-cfe) 5173.22 (1 H), 171 .55 (1 H), 153.06 (1 H), 131.13 (1 H), 129.64 (1 H), 125.08 (1 H), 124.85 (1 H), 123.90 (1 H), 123.71 (1 H), 79.34 (1 H), 51 .23 (1 H), 35.55 (1 H), 33.05 (1 H), 28.04 (3H), 24.64 (1 H), 23.93 (1 H).

[0204] HRMS (ESI+) calcd for C18H27N2O5 (M+H+) 351.1909, found 351.1915.

[0205] Methyl 6-((2-acrylamidophenyl)amino)-6-oxohexanoate (B-3)

[0206] Chemical Formula: C18H26N2O5 Chemical Formula: C16H20N2O4Molecular Weight: 350,42 Molecular Weight: 304,35

[0207] To 3 mL of DCM were added B-2 (190.3 mg, 0.54 mmol, 1 eq) and 20% of TFA (0.6 mL). The mixture was stirred 1 h at room temperature. Once the reaction was over the solution was dried under reduced pressure to afford an orange oil.

[0208] To the obtained oil were added 3 mL of THF and EtsN (157 pL, 1.16 mmol, 2 eq). Acryloyl chloride (47.7 pL, 0.59 mmol, 1.1 eq) was added dropwise. The mixture was stirred over week-end at room temperature.

[0209] Once the reaction was over the product was diluted in MeOH and purified by flash chromatography (DCM / MeOH - 2 to 12%) to afford an orange oil (114.6 mg, 0.38 mmol, 70%).

[0210] 1H NMR (400 MHz, DMSO-cfe) 5 9.52 (s, 1 H), 9.38 (s, 1 H), 7.63-7.61 (m, 1 H), 7.51-7.49 (m, 1 H), 7.18-7.13 (m, 2H), 6.46 (dd, J= 17.0, 10.2 Hz, 1 H), 6.24 (dd, J = 17.0, 1.9 Hz, 1 H), 5.77 (dd, J = 10.2, 1.9 Hz, 1 H), 3.58 (s, 3H), 2.38-2.29 (m, 4H), 1.63-1.53 (m, 4H).

[0211] 13C NMR (101 MHz, DMSO-cfe) 5 173.2 (1 C), 171.4 (1 C), 163.4 (1 C), 131.9 (1 C), 130.6 (1 C), 130.4 (1 C), 126.8 (1 C), 125.1 (1 C), 125.0 (1 C), 124.9 (1 C), 124.7 (1 C), 51.2 (1 C), 35.6 (1 C), 33.1 (1 C), 24.5 (1 C), 24.0 (1 C). HRMS (ESI+) calcd for C16H21N2O4 (M+H+) 305.1491 , found 305.1496.

[0212] 6-((2-acrylamidophenyl)amino)-6-oxohexanoic acid (B-4)

[0213] Chemical Formula: C-|5H18N2O4 Molecular Weight: 290,32 To 1 mL of THF, were added B-3 (65.3 mg, 0.21 mmol, 1 eq) and 1 .2 mL (2.5 eq) of LiOH 0.5 N. The mixture was stirred 3h at room temperature. Once the reaction was over, HCI 1 N was added to neutralize LiOH. 10 mL of H2O were added and aqueous layer was extracted with ethyl acetate. Organic layers were dried over MgSO4 and evaporate under vacuum to afford a yellow paste (38.5 mg, 0.13 mmol, 62%).

[0214] 1H NMR (400 MHz, DMSO-cfe) 5 12.01 (s, 1 H), 9.51 (s, 1 H), 9.38 (s, 1 H), 7.64- 7.48 (m, 1 H), 7.54-7.47 (m, 1 H), 7.15 (dd, J = 5.5, 4.1 Hz, 2H), 6.46 (dd, J = 17.0, 10.2 Hz, 1 H), 6.24 (dd, J = 17.0, 1 .9 Hz, 1 H), 5.76 (dd, J = 10.2, 1 .8 Hz, 1 H), 2.34 (t, J = 6.9 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 1 .66-1 .48 (m, 4H). 13C NMR (101 MHz, DMSO-cfe) 5 174.4 (1 C), 171.5 (1 C), 163.5 (1 C), 131.9

[0215] (1 C), 130.6 (1 C), 130.5 (1 C), 126.9 (1 C), 125.1 (1 C), 125.0 (1 C), 124.9 (1 C), 124.7 (1 C), 35.8 (1 C), 33.5 (1 C), 24.6 (1 C), 24.1 (1 C).

[0216] HRMS (ESI+) calcd for C15H19N2O4 (M+H+) 291 .1342, found 291 .1339.

[0217] 4-((S)-2-((S)-2-(6-((2-acrylamidophenyl)amino)-6-oxohexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1 -(((S)-1 -(((3R,4S,5S)-1 - ((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2- methyl-3-oxopropyl)pyrrolidin-1 -yl)-3-methoxy-5-methyl-1 -oxoheptan-4- yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)(methyl)carbamate (B)

[0218] Chemical Formula: C73H11ON120I5Molecular Weight: 1395,75 To 600 pL of DMF was dissolved TFA.ValCitPABMMAE (9.0 mg, 0.008 mmol,

[0219] 1 eq) before the addition of B-4 (2.9 mg, 0.010 mmol, 1.3 eq). HATU (3.8 mg, 0.010 mmol, 1.3 eq) was dissolved and finally, 2,6-Lutidine (1.16 pL, 0.010 mmol, 1.3 eq) was added to the mixture. The solution was stirred overnight at room temperature.

[0220] The reaction was monitored by analytic-HPLC. The solution was purified by semipreparative HPLC (gradient A) to afford a white powder (8.2 mg, 0.006 mmol,

[0221] 75%).

[0222] HRMS (ESI+) calcd for C73H111N12O15 (M+H+) 1395.8292, found 1395.8286.

[0223] Example 1.3. Synthesis of compound (C)

[0224] Tert-butyl (4-aminophenyl)carbamate (C-1)

[0225] Molecular Weight: 208,26

[0226] To 2 mL of THF were added p- phenylenediamine (100.0 mg, 0.92 mmol, 1 eq) and B0C2O (201.0 mg, 0.92 mmol, 1 eq). K2CO3 (0.46 mmol, 0.5 eq) was added and the mixture was stirred overnight at room temperature. The solution was diluted with 20 mL of EtOAc and washed 3 times with 50 mL of water. Organic layers were dried over MgSO4, and concentrated under vacuum to afford a orange oil (1 12.6 mg, 0.54 mmol, 59%)

[0227] 1H NMR (300 MHz, DMSO-cfe) <58.77 (s, 1 H), 7.05 (d, J = 7.8 Hz, 2H), 6.44 (d, J = 8.7 Hz, 2H), 4.72 (s, 2H), 1 .43 (s, 9H).

[0228] 1H NMR in accordance with literature (Zhang J. & Al, J. Med. Chem. 2019, 62, 14, 6765-6784).

[0229] Methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)amino)-6-oxohexanoate (C-2)

[0230] Chemical Formula: CI8H26N2O5Molecular Weight: 350,42

[0231] To 3 mL of THF with EtsN (79.6 pL, 0.59 mmol, 1.1 eq) were added C-1 (112.6 mg, 0.54 mmol, 1 eq) and methyl adipoyl chloride (84.0 pL, 0.54 mmol, 1 eq). The mixture was stirred overnight at room temperature. Once the reaction was over the solution was dissolved in methanol and concentrated under reduced pressure. The product was then purified by flash chromatography (DCM / MeOH - 1 to 13%) to afford a white fluffy powder (133.2 mg, 0.38 mmol, 70%).

[0232] 1H NMR (300 MHz, DMSO-cfe) 59.74 (s, 1 H), 9.22 (s, 1 H), 7.44 (d, = 9.0 Hz, 2H), 7.33 (d, J= 8.9 Hz, 2H), 3.58 (s, 3H), 2.33 (t, J = 6.9 Hz, 2H), 2.26 (t, J = 6.3 Hz, 2H), 1.60 - 1 .50 (m, 4H), 1 .46 (s, 9H).

[0233] 13C NMR (101 MHz, DMSO-cfe) 5 173.2 (1 C), 170.5 (1 C), 152.8 (1 C), 134.7 (1 C), 133.8 (1 C), 1 19.5 (2C), 118.4 (2C), 78.8 (1 C), 51.2 (1 C), 35.9 (1 C), 33.0 (1 C), 28.2 (3C), 24.6 (1 C), 24.1 (1 C).

[0234] HRMS (ESI+) calcd for C18H27N2O5 (M+H+) 351.1909, found 351.1915.

[0235] Methyl 6-((4-acrylamidophenyl)amino)-6-oxohexanoate (C-3)

[0236] Chemical Formula: C18H26N2O5Chemical Formula: Ci6H20N2O4 Molecular Weight: 350,42 Molecular Weight: 304,35

[0237] To 3 mL of DCM were added C-2 (125.3 mg, 0.36 mmol, 1 eq) and 20% of TFA (0.6 mL). The mixture was stirred 1 h at room temperature. Once the reaction was over the solution was dried under reduced pressure to afford an orange oil.

[0238] To the obtained oil, were added 3 mL of THF and EtsN (97.2 pL, 0.72 mmol, 2 eq). Acryloyl chloride (32.4 pL, 0.40 mmol, 1.1 eq) was added dropwise. The mixture was stirred over weekend at room temperature.

[0239] Once the reaction was over, the solution was diluted in MeOH and purified by flash chromatography (DCM / MeOH - 2 to 12%) to afford a white fluffy powder (89.8 mg, 0.30 mmol, 83%)

[0240] 1H NMR (300 MHz, DMSO-cfe) 5 10.07 (s, 1 H), 9.85 (s, 1 H), 7.59-7.50 (m, 4H), 6.41 (dd, J = 17.0, 10.0 Hz, 1 H), 6.22 (dd, J = 17.0, 2.2 Hz, 1 H), 5.72 (dd, J = 10.0, 2.2 Hz, 1 H), 3.58 (s, 3H), 2.36-2.26 (m, 4H), 1 .58-1 .56 (m, 4H).

[0241] 13C NMR (101 MHz, DMSO-cfe) 5 173.1 (1 C), 170.5 (1 C), 162.6 (1 C), 134.8 (1 C), 134.0 (1 C), 131.8 (1 C), 126.3 (1 C), 120.1 (1 C or 2C), 119.5 (1 C), 119.2 (1 C), 51.1 (1 C), 35.7 (1 C), 32.9 (1 C), 24.4 (1 C), 23.9 (1 C). HRMS (ESI+) calcd for C16H21N2O4 (M+H+) 305.14901 , found 305.1496.

[0242] 6-((4-acrylamidophenyl)amino)-6-oxohexanoic acid (C-4)

[0243] Chemical Formula: C15H 18N2O4 Molecular Weight: 290,32

[0244] To 1 mL of THF, were added C-3 (60.0 mg, 0.197 mmol, 1 eq) and 1.0 mL (2.5 eq) of LiOH 0.5 N. The mixture was stirred 3h at room temperature. Once the reaction was over, HCL 1 N was added to neutralize LiOH. 10 mL of water were added and aqueous layer was extracted with ethyl acetate. Organic layers were dried over MgSO4 and evaporate under vacuum to afford a white powder (44.1 mg, 0.152 mmol,

[0245] 77%).

[0246] 1H NMR (300 MHz, DMSO-cfe) 5 11 .99 (s, 1 H), 10.06 (s, 1 H), 9.83 (s, 1 H), 7.61 - 7.49 (m, 4H), 6.41 (dd, J = 17.0, 10.0 Hz, 1 H), 6.23 (dd, J = 17.0, 2.2 Hz, 1 H), 5.72 (dd, J= 10.0, 2.2 Hz, 1 H), 2.26 (dt, J= 17.3, 6.9 Hz, 4H), 1.63-1.48 (m, 4H). 13C NMR (101 MHz, DMSO-cfe) 5 174.4 (1 C), 170.8 (1 C), 162.8 (1 C), 135.1

[0247] (1 C), 134.2 (1 C), 131.9 (1 C), 126.5 (10), 119.7 (20), 1 19.4 (2C), 36.0 (1 C), 33.5 (1 C), 24.7 (1 C), 24.2 (1 C).

[0248] HRMS (ESI+) calcd for C15H19N2O4 (M+H+) 291 .1345, found 291 .1350.

[0249] 4-((S)-2-((S)-2-(6-((4-acrylamidophenyl)amino)-6-oxohexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl((S)-1 -(((S)-1 -(((3R,4S,5S)-1 - ((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2- methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4- yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)(methyl)carbamate (C)

[0250] Chemical Formula: C73H110N12O15

[0251] Molecular Weight: 1395,75 To 600 pL of DMF was dissolved TFA.ValCitPABMMAE (18.0 mg, 0.016 mmol,

[0252] 1 eq) before the addition of C-4 (6.1 mg, 0.021 mmol, 1.3 eq). HATLI (12.2 mg, 0.032 mmol, 2 eq) was dissolved and finally, 2,6-Lutidine (3.7 pL, 0.032 mmol, 2 eq) was added to the mixture. The solution was stirred overnight at room temperature.

[0253] The reaction was monitored by analytic-HPLC. The solution was purified by semipreparative HPLC (gradient A) to afford a white solid (10.9 mg, 0.008 mmol, 50%) HRMS (ESI+) calcd for C73H111N12O15 (M+H+) 1395.8292, found 1395.8286. Example 1.4. Synthesis of compound (D)

[0254] Methyl 6-((6-aminopyridin-2-yl)amino)-6-oxohexanoate (D-1 )

[0255] Chemical Formula: CI2H17N3O3Molecular Weight: 251 ,29

[0256] To 1 .5 mL of THF were added 2,6-diaminopyridine (100.0 mg, 0.92 mmol, 2 eq) and Et3N (124.2 pL, 0.92 mmol, 2 eq). Methyl adipoyl chloride (71 .5 pL, 0.46 mmol, 1 eq) was added dropwise. The mixture was stirred over weekend at room temperature. Once the reaction was over, volatiles were removed and the product was purified by flash chromatography (EtOAc / Cyclohexane - 60 / 40 to 80 / 20) to afford a yellow oil (80.6 mg, 0.321 mmol, 70%)

[0257] 1H NMR (400 MHz, DMSO-cfe) 5 9.78 (s, 1 H), 7.31 (t, J = 7.9 Hz, 1 H), 7.21 (d, J = 7.8 Hz, 1 H), 6.15 (dd, J = 7.9, 0.8 Hz, 1 H), 5.69 (s, 2H), 3.58 (s, 3H), 2.32 (t, J = 6.1 Hz, 4H), 1.56-1.51 (m, 4H).

[0258] 13C NMR (101 MHz, DMSO-cfe) 5 173.3 (1 C), 171.4 (1 C), 158.4 (1 C), 150.5 (1 C), 138.8 (1 C), 103.2 (1 C), 100.8 (1 C), 51.2 (1 C), 35.7 (1 C), 33.0 (1 C), 24.5 (1 C), 24.0 (1 C).

[0259] HRMS (ESI+) calcd for C12H18N3O3 (M+H+) 252.1348, found 252.1343.

[0260] Methyl 6-((6-acrylamidopyridin-2-yl)amino)-6-oxohexanoate (D-2)

[0261] Chemical Formula: CI5H19N3O4 Molecular Weight: 305,33

[0262] To 2 mL of THF with 112.6 pL (1 .5 eq) of Et3N were added D-1 (139.7 mg, 0.556 mmol, 1 eq) and acryloyl chloride (49.5 pL, 0.612 mmol, 1.1 eq). The mixture was stirred overnight at room temperature. Once the reaction was over, volatiles were removed and the product was purified by flash chromatography (EtOAc / CH - 70 / 30) to afford a fluffy white powder (140.0 mg, 0.46 mmol, 83%).

[0263] 1H NMR (400 MHz, DMSO-cfe) 510.29 (s, 1H), 10.02 (s, 1 H), 7.84-7.81 (m, 1H), 7.75 (d, J = 5.1 Hz, 2H), 6.65 (dd, J = 17.0, 10.2 Hz, 1H), 6.30 (dd, J = 17.0, 1.9 Hz, 1H), 5.78 (dd, J = 10.2, 1.9 Hz, 1H), 3.58 (s, 3H), 2.41 (t, J = 6.9 Hz, 2H), 2.33 (t, J = 7.0 Hz, 2H), 1.60-1.53 (m, 4H).

[0264] 13C NMR (101 MHz, DMSO-cfe) 5173.2 (1C), 171.9 (1C), 163.7 (1C), 150.4 (1C), 150.2 (1C), 140.0(1C), 131.6(1C), 127.7(10), 109.5(10), 109.3 (1C), 51.2 (s), 35.7 (1C), 33.0 (1C), 24.4 (1C), 24.0 (1C).

[0265] HRMS (ESI+) calcd for C15H20N3O4 (M+H+) 306.1454, found 306.1448.

[0266] 6-((6-acrylamidopyridin-2-yl)amino)-6-oxohexanoic acid (D-3)

[0267] Chemical Formula: C14H 17N3O4 Molecular Weight: 291,31

[0268] To 1 mL of THF, were added D-2 (50.0 mg, 0.164 mmol, 1 eq) and 820 pL (2.5 eq) of LiOH 0.5 N. The mixture was stirred 3h. Once the reaction was over, HCI 1N was added to quench the solution. 10 mL of H2O were added and aqueous layer was extracted with ethyl acetate. Organic layers were dried over MgSO4 and evaporated under vacuum to afford a white powder (28.0 mg, 0.096 mmol, 59%)

[0269] 1H NMR (400 MHz, DMSO-cfe) 512.00 (s, 1H), 10.29 (s, 1H), 10.02(s, 1H),7.85 -7.73 (m, 3H), 6.64 (dd, J = 17.0, 10.2 Hz, 1H), 6.30 (dd, J= 17.0, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 2.40 (t, J =7.0 Hz, 2H), 2.23 (t, J=7.1 Hz, 2H), 1.62-1.47 (m, 4H).

[0270] 13C NMR (101 MHz, DMSO-cfe) 5174.4 (1C), 172.0 (1C), 163.7 (1C), 150.4 (1C), 150.2 (1C), 140.0 (1C), 131.6 (1C), 127.7 (1C), 109.5 (1C), 109.3 (1C), 35.8 (1C), 33.4 (1C), 24.5 (1C), 24.1 (1C).

[0271] HRMS (ESI+) calcd for C14H18N3O4 (M+H+) 292.1296, found 292.1292. 4-((S)-2-((S)-2-(6-((6-acrylamidopyridin-2-yl)amino)-6-oxohexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1 -(((S)-1 -(((3R,4S,5S)-1 - ((S)-2-((1 R,2R)-3-(((1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2- methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4- yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)(methyl)carbamate (D)

[0272] Chemical Formula: C72H109N13O15Molecular Weight: 1396,74

[0273] To 600 pL of DMF was dissolved TFA.ValCitPABMMAE (18.0 mg, 0.016 mmol, 1 eq) before the addition of D-3 (6.1 mg, 0.021 mmol, 1.3 eq). HATU (8.0 mg, 0.021 mmol, 1.3 eq) was dissolved and finally, 2,6-Lutidine (2.44 pL, 0.021 mmol, 1.3 eq) was added to the mixture. The solution was stirred overnight at room temperature.

[0274] The reaction was monitored by analytic-HPLC. The solution was purified by semipreparative HPLC (gradient A) to afford a white powder (16.5 mg, 0.012 mmol, 75%)

[0275] HRMS (ESI+) calcd for C72H110N13O15 (M+H+) 1396.8244, found 1396.8239.

[0276] Example 1.5. Synthesis of PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-0Me (compound 24) tert-butyl (((9 H-f luoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (16)

[0277] Chemical Formula: C25H30N2O5 Molecular Weight: 438,52

[0278] 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 overweek-end at room temperature.

[0279] 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%)

[0280] 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).

[0281] 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).

[0282] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (17)

[0283] 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 yellowy paste (175.3 mg, 0.35 mmol, quant.)

[0284] 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).

[0285] 1H NMR in accordance with literature. (Bukya H. & Al, Eur. J. Org. Chem., 2020, 5358-5362).

[0286] 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (18)

[0287] Chemical Formula: C13H23NOSi Molecular Weight: 237,42

[0288] 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%)

[0289] 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).

[0290] 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)

[0291] Chemical Formula: C28H29N3O5 Molecular Weight: 487,56

[0292] 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%).

[0293] 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).

[0294] 1H NMR in accordance with literature (Widdison W. C., Bioconjugate Chem., 2015, 26, 11 , 2261-2278).

[0295] HRMS (ESI+) calcd for C28H30N3O5 (M+H+) 488.2180, found 488.2180

[0296] (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)

[0297] 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.

[0298] 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%).

[0299] 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).

[0300] HRMS (ESI+) calcd for C35H33N4O9 (M+H+) 653.2242, found 653.2242.

[0301] 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)

[0302] Chemical Formula: C38H39FN6O8 Molecular Weight: 726,76

[0303] 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.

[0304] To the previous solution 20%v of piperidine were added and the solution was stirred 20 minutes at RT.

[0305] 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).

[0306] 19F NMR (377 MHz, DMSO-cfe) 5 -73.93, -111 .32 (d, J= 10.9 Hz).

[0307] HRMS (ESI+) calcd for CssH^FNeOs (M+H+) 727.2897, found 727.2886.

[0308] 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)

[0309] Chemical Formula: CgiH122FNgO25

[0310] Molecular Weight: 1761 ,01

[0311] 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%).

[0312] 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).

[0313] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .32 (d, J = 10.5 Hz).

[0314] HRMS (ESI+) calcd for C91H123FN9O25 (M+H+) 1760.8609, found 1760.8592 According to the procedure described previously, we obtained approximatively 100% of drug release for the enzymatic reaction with Cathepsin B over 16 hours.

[0315] (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)-54,57-dimethyl-38,45,52,55,58- pentaoxo-2,5,8,11 ,14,17,20,23,26,29,32,35-dodecaoxa-39,46,53,56- tetraazaoctapentaconta n-44-am i ni u m (23)

[0316] Chemical Formula: C76Hi12FNgO23 Molecular Weight: 1538,77

[0317] 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%).

[0318] 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- 1.25 (m, 15H), 1.20 (d, J= 7.2 Hz, 3H), 0.88 (t, J= 7.2 Hz, 3H).19F NMR (377 MHz, DMSO-cfe) 5 -73.68, -1 11 .33 (d, J = 10.2 Hz).

[0319] HRMS (ESI+) calcd for C76H113FN9O23 (M+H+) 1538.7928 found 1538.791 1.

[0320] 4-((44S,54S,57S)-44-(6-((6-acrylamidopyridin-2-yl)amino)-6- oxohexanamido)-54,57-dimethyl-38,45,52,55-tetraoxo-2,5,8,11 ,14,17,20,23,

[0321] 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-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1- yl)carbamate (24, “PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe”)

[0322] Chemical Formula: C90H127FN12O26 Molecular Weight: 1812,06

[0323] To 800 pL of DMF were dissolved 23 (15.0 mg, 0.009 mmol, 1 eq) before the addition of D-3 (7.9 mg, 0.027 mmol, 1 .5 eq). HATLI (20.5 mg, 0.054 mmol, 6 eq) was dissolved and finally, 2,6-Lutidine (20.8 pL, 0.09 mmol, 10 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 (7.7 mg, 0.0045 mmol, 50%)

[0324] 1H NMR (400 MHz, DMSO-cfe) 510.30 (s, 1 H), 10.02 (s, 1 H), 9.91 -9.90 (m, 1 H), 8.10-7.92 (m, 3H), 7.85-7.71 (m, 7H), 7.61 (dd, J= 8.5, 3.6 Hz, 2H), 7.36 (d, J= 8.5 Hz, 2H), 7.31 (s, 1 H), 6.64 (dd, J= 17.0, 10.2 Hz, 2H), 6.29 (dd, J= 17.0, 1.9 Hz, 1 H), 5.77 (dd, J = 10.2, 1.9 Hz, 1 H), 5.44 (s, 2H), 5.34-5.20 (m, 3H), 5.08 (s, 2H), 4.43- 4.33 (m, 1 H), 4.27 (p, J = 7.1 Hz, 1 H), 4.21^.1 1 (m, 1 H), 3.56 (td, J = 6.5, 2.3 Hz, 2H), 3.52-3.38 (m, 48H), 3.23 (s, 3H), 3.04 - 2.92 (m, 4H), 2.42-2.35 (m, 5H), 2.28 (t, J = 6.5 Hz, 2H), 2.20-2.08 (m, 5H), 1 .94-1 .80 (m, 2H), 1 .58-1 .42 (m, 6H), 1 .40-

[0325] 1.16 (m, 16H), 0.87 (t, J = 7.3 Hz, 3H).

[0326] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .36 (d, J = 11 .2 Hz).

[0327] HRMS (ESI+) calcd for C90H128FN12O26 (M+H+) 1811 .9041 , found 1811 .9019. Example 1.6. Synthesis of comparative linker MC-Lys-PEGi2OMe(Cap-AA-

[0328] PABC-EXA) 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 (25)

[0329] Chemical Formula: C86H123FN10O26 Molecular Weight: 1731 ,97

[0330] Step 1 : synthesis of 6-maleimidohexanoic acid (26)

[0331] Chemical Formula: C10H13NO4Molecular Weight: 211 ,22

[0332] 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 26 as white solid (1 .92 g, 45%). HPLC purity = 99.07%, t« = 8.53 min.

[0333] 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).

[0334] 13C NMR (75 MHz, CDCI3) 5 179.4, 171.4, 134.6, 38.2, 34.2, 28.7, 26.7, 24.7.

[0335] Step 2 :

[0336] To 800 pL of DMF were dissolved 23 (15.0 mg, 0.009 mmol, 1 eq) before the addition of 26 (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%)

[0337] 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).

[0338] 19F NMR (377 MHz, DMSO-cfe) 5 -11 1 .32 (d, J = 10.5 Hz).

[0339] HRMS (ESI+) calculated for C86H124FN10O26 (M+H+) 1731.8672, found 1731.8651 EXAMPLE 2 - BIOCONJUGATION

[0340] Example 2.1. Bioconjugation of PyC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), Db(4)-PyC-EXA: diabody-exatecan conjugate of DAR 4

[0341] To 500 pL of antibody solution at 353.9 pg / mL in BBS, the general procedure for bioconjugation has been applied: 4 eq of TCEP, 40 eq of EDTA (10 mM in BBS buffer), 37°C, 1 h 15 and 8 eq of PyC-Lys-PEGi2OMe(Cap-AA-PABC-EXA), 4°C, 16 h.

[0342] Example 2.2. Bioconjugation of PyC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), lgG(8)-PyC-EXA: ADC (antibody-exatecan conjugate) of DAR 8

[0343] To 500 pL of antibody solution at 4.6 mg / mL in BBS, the general procedure for bioconjugation has been applied with two additions: 16 eq of TCEP, 160 eq of EDTA and NaCI (10 mM in BBS buffer), 37°C, 1 h 15 and 32 eq of PyC-Lys- PEG12OMe(Cap-AA-PABC-EXA), 20°C, 16 h, then 16 eq of TCEP, 160 eq of EDTA and NaCI (10 mM in BBS buffer), 37°C, 1 h 15 and 32 eq of PyC-Lys- PEG12OMe(Cap-AA-PABC-EXA), 20°C, 16 h.

[0344] Example 2.3. Bioconjugation of MC-Lys-PEGi2OMe(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), lgG(8)-mal-EXA: ADC (antibody-exatecan conjugate) of DAR 8

[0345] 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-(Cap-AA-PABC- EXA)-PEGi2-OMe (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-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (445.3 pg, 92%), with a purity of >95%, confirmed by size exclusion chromatography.

[0346] Example 2.4. Bioconjugation of MC-Lys-PEGi20Me(Cap-AA-PABC-EXA) in solution in DMSO (1 mM), lgGiRR(8)-EXA (ou equivalemment lgG(8)IRR- MC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe ) (30): IgG IRR (8) = irrelevant antibody DAR 8 (negative control)

[0347] 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.

[0348] Example 2.5. Bioconjugation of PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (16)

[0349] To 400 pL of antibody solution at 3.9 mg / mL in BBS, the general procedure (below for example 2.6) for bioconjugation has been applied with two additions : 16 eq of TCEP, 160 eq of EDTA and NaCI (10 mM in BBS buffer), and 32 eq of PyC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe, then 16 eq of TCEP, 160 eq of EDTA and NaCI (10 mM in BBS buffer), and 32 eq of PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe to afford lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (1353.0 pg, 89%), with a purity of >95%, confirmed by size exclusion chromatography.

[0350] Example 2.6. Bioconjugation of MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (24)

[0351] General procedure for bioconjugation: 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-(Cap-AA-PABC-EXA)-PEGi2-OMe (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-Lys-(Cap-AA-PABC-EXA)-PEGi2-0Me (445.3 pg, 92%), with a purity of >95%, confirmed by size exclusion chromatography.

[0352] EXAMPLE 3 - Example of enhanced stability of conjugates comprising PyC (A / -(6-acryloylpyridin-2-yl)-6-oxohexanamide) in comparison to MC (maleimidocaproic)

[0353] The inventors compared the stability, after incubation in PBS at 4°C for 327 days, of two anti-HER2 diabody-exatecan conjugates of DAR 4: Db(4)-MC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe obtained with comparative compound 25 and Db(4)- PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe obtained with compound 24. It is clear that Db(4)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe was more stable, without any linker-payload loss over time. While the counterpart Db(4)-MC-Lys-(Cap-AA-PABC- EXA)-PEGi2-OMe, equipped with classical first generation maleimide, was associated with linker-payload loss over time (Figure 1 ).

[0354] The use of / V-(6-acryloylpyridin-2-yl)-6-oxohexanamide to generate immunoconjugates, is associated with more stable immunoconjugates, in comparison to the use of first generation maleimide.

[0355] EXAMPLE 4 - EVALUATION OF IMMUNOCONJUGATES WITH PyC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe (compound 24)

[0356] ELISA assays

[0357] 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).

[0358] Flow Cytometry Studies

[0359] Protein L coupled with phycoerythrin (ppL-PE, Sinobiological, Beijing, China) was used as an intracellular tracer, in particular to measure the amount of anti-HER2 entities internalized into the cells. 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 pre-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 24 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®.

[0360] Microscopy studies

[0361] 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).

[0362] Cell Cultures and Reagents

[0363] 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% penicillinstreptomycin 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.

[0364] Cell Proliferation Assays

[0365] 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 dose- dependent activity was observed, 50% inhibitory concentration (IC50) were calculated using GraphPad® PRISM 7 software (San Diego, USA) (n = 4 in quadruplicate).

[0366] Animal model

[0367] 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. 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.

[0368] 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

[0369] #2019011814134419.v2).

[0370] Biodistribution study

[0371] 1 / 125l-radiolabelling of antibodies and antibody fragments

[0372] 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 HP1 100 (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.

[0373] 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.

[0374] Accordingly, the radioimmunoconjugates [125l]-lgG(8)- PyC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe ([125l]-lgG(8)-py-EXA) and [125l]-trastuzumab deruxtecan were obtained in short reaction time, with good radiochemical yields, high radiochemical purities (RCP, 91.3-97.8%) and with molar activities (Am) around 17 GBq / pmol (16.0-19.5 GBq / pmol).

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

[0376] All experiments were performed with low retention tubes pre-treated 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 0.60 pmol of [125l]lgG(8)-MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe (16.0 GBq / pmol) or 0.60 pmol of [125l]lgG(8)-PyC-Lys- (Cap-AA-PABC-EXA)-PEGi2-OMe (16.3 GBq / pmol) or 0.60 pmol of [125l]trastuzumab deruxtecan (19.5 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) 3 or 10 MBq (T = 120 h) of [125l]lgG(8)- MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (n=3 / time point) and sacrificed 3, 6, 24, 48, 72 and 120 h p.i.; (ii) 3 or 10 MBq (T = 120 h) of [125l]lgG(8)-PyC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe (n=3 / time point) and sacrificed 3, 6, 24, 48, 72 and 120 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) 10.32±0.01 MBq of [125l]lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe; (ii) 12.08±0.04 MBq of [125l]lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe. 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] In vivo efficacy study

[0382] A HER2 xenografted mouse model (on BALB / c nude mice) was established and the antitumour efficacy of lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgG(8)-PyC-EXA) was analyzed by comparison to FDA-approved gold standard ADC T-DXd. Tumour volumes were assessed weekly during 60 days after injections of our anti-HER2 conjugates. Tumoural volume evolution in Balb- / c nude mice were monitored after one injection (at day 25, 0.07 pmol / kg equivalent to 10.0 mg / kg of T- DXd) of lgG(8)-py-EXA in comparison to T-DXd , and two injections (at days 25 and 29, 0.07 pmol / kg equivalent to 10.0 mg / kg of T-DXd) of lgG(8)-py-EXA in comparison to T-DXd.

[0383] Statistical analysis

[0384] 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).

[0385] EXAMPLE 5 - RESULTS OF EVALUATION OF IMMUNOCONJUGATES WITH LINKER PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe

[0386] HER2 binding: ADC lgG(8)-pyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (IgG(S)-py-EXA) was tested for antigen recognition to assess the immunoconjugate ability to recognize the HER2 protein.

[0387] Binding to HER2 was assessed by enzyme-linked immunosorbent assay (ELISA). lgG(8)-py-EXA was compared to native trastuzumab lgG(8).

[0388] As visible in figure 2, lgG(8)-py-EXA has the same binding profile compared to native I gG(8), even though the absorbance is a bit smaller for the conjugated entity.

[0389] 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)-py-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.

[0390] Nevertheless, the trend of the curves showed that the recognition of HER2 is still maintained after the bioconjugation.

[0391] Under these conditions, the tested entities can be compared in cell-based assays without affinity bias.

[0392] It is worth noting that a slight decreased affinity may theoretically increase the homogeneous distribution of the entity in the tumour.

[0393] To sum up, grafting of linker PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (compound 24) onto anti-HER2 lgG(8) (IgG DAR 8) does not modify binding of corresponding conjugates lgG(8)-PyC-Lys-(Cap-VC-PABC-MMAE)-PEGi2-OMe to HER2 in comparison to trastuzumab and in comparison to anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe having a classical maleimide head and obtained with comparative linker MC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe.

[0394] Internalization:

[0395] As shown in figure 3, after 24 h of exposure at 37°C on cells, lgG(8)-Py-EXA and T-DXd were internalized compared to the negative control ppL-PE alone and to lgGirr(8)-EXA (figure 3A). 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 3A) or negative cells MDA-MB-468 (figure 3B) (MFI < 5), whereas lgG(8)-py-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. Further, the MFI measured for the cells incubated with lgG(8)-py-EXA was significantly increased and was more than doubled compared to reference compound T-DXd ADC (MFI of 130 versus 55 respectively) and increase to comparative compound lg(8)-mal-EXA (MFI of 130 versus MFI of 120).

[0396] 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)-py-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.

[0397] These qualitative results are correlated with semi-quantitative results of flow cytometry. Accordingly, lgG(8)-py-EXA and T-DXd are well internalized in HER2 positive cells with a better internalization for lgG(8)-py-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.

[0398] No signal of internalization was recorded on flow cytometry nor on microscopy images when the same acquisition parameters as SK-BR-3 were applied.

[0399] To sum up, grafting of linker PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti-HER2 lgG(8) improves internalization of corresponding conjugate lgG(8)-PyC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe into cancer cell lines in comparison to trastuzumab deruxtecan and in comparison to comparative anti-HER2 immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe having a classical maleimide head (Figure 3).

[0400] Cytotoxicity:

[0401] As shown in figure 4 and in Table 1 hereafter, the cytotoxicity of lgG(8)-py-EXA was tested on breast cancer cell lines and compared to their irrelevant lgGirr(8)-EXA counterpart and to free exatecan (EXA).

[0402] Firstly, on SK-BR-3 cells overexpressing HER2 (Figure 4A), as expected, no activity of the irrelevant lgGirr(8)-EXA was observed. For the lgG(8)-py-EXA conjugate, a high cytotoxicity with an IC50 values of 0.69 nM was highlighted.

[0403] Secondly, on MDA-MB-468 cells having no HER2 gene amplification (Figure 4B), 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). 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.

[0404] To sum up, grafting of linker PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti-HER 2 lgG(8) onto anti-HER2 lgG(8) confers to corresponding conjugates lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEG12-OMe the ability to kill selectively HER2-positive cancer cell lines, with very low IC50, in particular lower than 1 nM, corresponding to a high cytotoxicity (Figure 4 and Table 1 ). -468

[0405] Table 1 : IC50 (in nM) cell-based assays of tested entities on two breast cancer cell lines SK-BR-3 (HER2+++) and MDA-MB-468 (HER2 negative)

[0406] Biodistribution:

[0407] Grafting of linker PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe onto anti HER2 lgG(8) allows to obtain an analogous, and even slightly improved, biodistribution of corresponding conjugates lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEG12-OMe over time as with reference compound trastuzumab deruxtecan (T-Dxd), as shown by figure 5A 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 5B obtained with trastuzumab deruxtecan. Hence, grafting of linker PyC-Lys-(Cap-AA- PABC-EXA)-PEGi2-OMe onto anti HER2 lgG(8) allows satisfactory tumor recognition by the corresponding conjugates.

[0408] Serum Stability:

[0409] In addition, measurements of DAR variation over time of immunoconjugates lgG(8)-PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgG(8)-py-Exa), comparative immunoconjugates lgG(8)-MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe (lgG(8)-mal- Exa), and trastuzumab deruxtecan (T-DXd) in human serum or mouse serum showed almost no DAR reduction at 192h in both human and mouse serum for lgG(8)-PyC- Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe, significantly lower than the 0.3 DAR reduction at 192h in both human and mouse serum obtained with comparative conjugate 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 2 hereafter).

[0410] Table 2 : ratio of the DAR and of the initial DAR (in %) as a function of time after injection

[0411] Hence, grafting of linker PyC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe 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, in comparison to linker equipped with classical maleimide head and in comparison to reference compound trastuzumab deruxtecan.

[0412] In vivo efficacy:

[0413] This is confirmed by figure 6, showing tumoral volume evolution over time after either one or two injections of anti-HER2 immunoconjugates lgG(8)-PyC-Lys-(Cap- AA-PABC-EXA)-PEGi2-OMe compared to comparative immunoconjugates lgG(8)- MC-Lys-(Cap-AA-PABC-EXA)-PEGi2-OMe and to reference compound trastuzumab deruxtecan (T-DXd)). As visible in figure 6, the tumor growth analyses showed a significantly increased anti-tumor activity of lgG(8)-py-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. At day 60, both lgG(8)-py-EXA and T-DXd achieved complete and lasting remission after two injections (up to day 75 corresponding to the last monitoring day).

[0414] In parallel, from day 35 to day 60, lgG(8)-py-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 addition, on these tumor volume follow-up curves, lgG(8)-py-EXA shows a slightly improved efficiency in comparison to T-DXd.

[0415] It should be noted that lgG(8)-py-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:- A represents an aryl or heteroaryl group, said aryl or heteroaryl group being optionally subsituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups,- n is 1 or 3,- Li represents a linear or branched alkylene radical, comprising from 1 to 20 carbon atoms, optionally interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group, at least one of the carbon atoms of said radical being optionally substituted with at least one alkylene side chain comprising 2 to 80 carbon atoms, optionally interrupted with at least one oxygen atom and / or at least one -C(=O)-NH- or -NH-C(=O)- group;- L2represents 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 (H-1 ):X representing H or NO2;* the group having the following formula (III):wherein:. X’ is H or NO2,. L4is a group of formula H-[NH-CH(Ai)-C(=O)]i-, i and Ai being as defined above in formula (II), or l_4 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,. L5represents 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, anda. L4and L5are as defined in formula (III);- L3represents an anticancer agent.

2. The compound of formula (I) according to claim 1 , wherein A represents an aryl group comprising 6 to 10 carbon atoms, optionally substituted with at least onesubstituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

3. The compound of formula (I) according to claim 1 , wherein A represents an aromatic monocyclic group comprising 5 or 6 atoms and including at least one heteroatom selected from N, S, or O, optionally substituted with at least one substituent selected from the group consisting of: halogen atoms, (Ci-Ce)alkyl groups, OH, CN, NO2, and (Ci-Ce)alkoxy groups.

4. The compound of formula (I) according to any one of the preceding claims, wherein A has one of the following formulae:the symbols showing the bonds with the groups -NH-C(=O)-CH=CH2 and -NH-C(=O)-LI-C(=O)-L2-L3.

5. The compound of formula (I) according to any one of the preceding claims, wherein Li represents 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.

6. The compound of formula (I) according to any one of the preceding claims, wherein L2is selected from the group consisting of:- the group having the following formula (V):- the group having the following formula (VI):- and the group having the following formula (VII):

7. The compound of formula (I) according to any one of the preceding claims, wherein:- L2has the formula (V) as defined in claim 5 and L3is monomethyl auristatin E; or- L2has the formula (VII) as defined in claim 5 and L3is exatecan.

8. A conjugate comprising at least one compound of formula (I) as defined in 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.

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

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a conjugate according to claim 8, and also at least one pharmaceutically acceptable excipient.

11. The compound according to any one of claims 1 to 7 or a conjugate according to claim 8, for its use for treating cancer.

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