Preparation of protein conjugates via one-pot multicomponent reaction

Abstract

The invention relates to the preparation of conjugates comprising a molecule of interest, such as a drug or a drug analogue, and a protein, such as an antibody, via the multicomponent Ugi reaction. It also relates to the obtained conjugates, and to the use of the obtained conjugates in the treatment of proliferative diseases.

Description

[0001] PREPARATION OF PROTEIN CONJUGATES VIA ONE-POT

[0002] MULTICOMPONENT REACTION

[0003] TECHNICAL FIELD

[0004] The invention relates to the preparation of conjugates comprising a molecule of interest, such as a drug or a drug analogue, and a protein, such as an antibody. It also relates to the obtained conjugates, and to the use of the obtained conjugates in the treatment of proliferative diseases.

[0005] TECHNICAL BACKGROUND

[0006] Protein-drug conjugates are by far the fastest growing class of highly potent active pharmaceutical ingredients. Protein-drug conjugate constructs generally involve a protein, such as an antibody, covalently attached to one end of a linker group, on the other end of which is a cytotoxin, i.e. a highly potent cell killing toxin. The protein component of the biomolecule provides target specificity. Once the conjugate enters the cell, the toxin is released, for instance by action of cellular enzymes. Most protein-drug conjugates are indeed directed to cancer treatment. Besides the 14 marketed antibody-drug-conjugates, including trastuzumab emtansine (referred to as T-DM1 or Kadcyla®) and the brentuximab vedotin (Adcetris®), a number of protein-drug conjugates are currently undergoing clinical trials for a variety of cancer indications.

[0007] One of the main prerequisites for the activity of such conjugates is the efficient release of the cytotoxic drug upon internalisation by cancer cells, while the linker between the protein and drug should be stable in blood circulation before the conjugate reaches its biological target.

[0008] Another key parameter of the activity of such conjugates is the site of conjugation of the molecule of interest on the protein backbone. In order for the conjugate to have the desired activity, conjugation should not alter the biological activity of the protein. When the protein is an antibody, conjugation should not affect its affinity to its targets.

[0009] Due to their large size - 150 kDa on average - antibodies, in particular monoclonal antibodies (mAbs), are inherently more complicated to conjugate in a site-selective manner than their smaller fragments via a chemical approach.

[0010] Multicomponent reactions can be defined as cascade processes where three or more reagents combined in one-pot fashion, lead to the formation of one more complex product presenting significant portions of all starting materials. The Ugi reaction, reported by Ivar Karl Ugi in 1959, is probably the most famous member of this family of reactions and consists in the combination of a carbonyl, a carboxylic acid, an amine and an isocyanide to generate a- aminoacylamide derivatives. From a mechanistic point of view, the reaction consists first in the condensation between the carbonyl compound and the amine to form an imine, which is then protonated by the acid leading to an iminium ion. This gets then attacked by the isocyanide forming an isonitrilium ion, which reacts with the carboxylate generating an intermediate. At this point, the irreversible Mumm rearrangement takes place forming the bis-amide final product.

[0011] Patent applications WO2022 / 245299, WO2022 / 169415 and WO2022 / 169416 disclose the synthesis of linkers for antibody-drug conjugates by Ugi reaction.

[0012] Ziegler et al. (Angew. Chem. Int. Ed. 2000, 39, 12, p.2109-2112) discloses the use of Ugi reaction to conjugate proteins to other compounds. A single functional group of the protein is involved in the Ugi reaction. While the work from Ziegler and coworkers showed the potential of the Ugi reaction for protein conjugation, its limitation to single-residue modification (either aspartate / glutamate or lysine, depending on the reagents used) hampered the development of a site-selective approach.

[0013] Sornay etal. (Chem. Eur. J. 2020, 2, p.13797-13805) discloses the site-selective conjugation of an antibody to a compound comprising an azide moiety, allowing further incorporating of different payloads to obtain antibody-drug conjugates. At least three reaction steps are necessary to obtain the antibody-drug conjugate: the Ugi reaction to insert the azide moiety onto the antibody (1), the modification of the drug with a strained alkyne moiety (2), and the azide / alkyne coupling to obtain the conjugate (3). The obtained conjugate presents the classical structure of antibody-drug conjugates, with a linker between the antibody and the drug moiety. Steps (2) and (3) involve a cytotoxic moiety.

[0014] Minimising the number of steps of chemical synthesis processes is a constant concern in the field, in order to increase the productivity and limit the exposition to potentially toxic reagents. This is particularly important when high-molecular weight compounds, such as proteins or antibodies, are involved in the process because reacting and purifying the obtained modified protein or antibody is generally laborious. In addition, the use of toxic reagents, such as cytotoxic reagents, is to be as much as possible avoided.

[0015] It would thus be desirable to develop new synthetic processes for the synthesis of protein conjugates with molecules of interest, such as drugs, which would involve as few steps as possible, and which would not imply toxic reagents. Advantageously, especially in the case of antibody-drug conjugates involving cytotoxic agents, the obtained conjugate should be toxic, ie the toxicity should be generated simultaneously to the formation of the conjugate.

[0016] SUMMARY OF THE INVENTION

[0017] In this respect, the Inventors have evidenced that multicomponent Ugi reaction could be successfully implemented in a single step for site-selective conjugation of desired fragments to proteins, thus affording a new and highly promising way for synthesising new potent conjugates.

[0018] The process of the invention allows coupling a protein, such as an antibody, to a molecule of interest, such as a cytotoxic drug, in a site-specific way, in a single step, without the need of a linker, and by involving nontoxic reagents.

[0019] The moiety corresponding to the molecule of interest is created upon implementation of the Ugi coupling, thus even if the molecule of interest is a toxic molecule, the conjugate can be accessed by using only nontoxic reagents.

[0020] In addition, the process according to the invention involves two residues concomitantly or two positions in the same residue, thus reducing the number of potential conjugation sites on the protein backbone, and thus increasing the site- selectivity.

[0021] A first object of the present invention is a process for preparing a conjugate of a protein and a

[0022] (I) molecule of interest of formula the process comprising the step of reacting a protein with an isocyanide compound of formula

[0023] (la) R-i- NC and a carbonyl compound of formula , wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein the isocyanide compound of formulaRl NCis an isocyanide derivative of a molecule of interest or a fragment thereof. In some embodiments, the step of reacting the protein with the isocyanide compound and the carbonyl compound comprises contacting the protein with the isocyanide compound and the carbonyl compound in conditions suitable for an Ugi multicomponent reaction.

[0024] In some embodiments, the molecule of interest is a drug, a drug analogue or a fragment thereof.

[0025] In some embodiments, the drug is a chemotherapeutic drug and / or a cytotoxic drug.

[0026] In some embodiments, the drug is selected from the group consisting of auristatins, dolastatins, tubulysins, camptothecins, mertansines, non-ribosomal peptides, antibiotics, nitrogen mustards, taxanes, analogues thereof and derivatives thereof.

[0027] In some embodiments, the drug is an auristatin, preferably selected from the group consisting of monomethyl dolastatin 10 (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and monomethyl soblidotin (MMAPE).

[0028] In some embodiments, the drug is a camptothecin, preferably selected from the group consisting of irinotecan, SN-38, topotecan; deruxtecan, belotecan, 9-aminocamptothecin and exatecan, and more preferably, the drug is exatecan.

[0029] In some embodiments, the primary amine group of the protein and the carboxylic acid group of the protein are separated by a moiety comprising from 2 to 20 atoms.

[0030] In some embodiments, the primary amine group of the protein is part of a lysine side-chain or a N-terminal residue, preferably a N-terminal aspartic acid or glutamic acid, and / or the carboxylic acid group of the protein is part of an aspartate or a glutamate amino acid, preferably a N-terminal aspartate or glutamate amino acid, or a C-terminal amino acid, preferably an aspartate or glutamate amino acid side chain.

[0031] In some embodiments, the primary amine group of the protein and the carboxylic acid group of the protein are part of a single amino acid or of two different amino acids.

[0032] In some embodiments, the protein is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof, in particular trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab, sacituzumab, gemtuzumab, cetuximab, a variant thereof or a fragment thereof.

[0033] In some embodiments, the isocyanide compound is a compound of formula diastereoisomer thereof, wherein R’ is a

[0034] C1-C7 alkyl group or arylalkyl group, wherein the alkyl and arylalkyl groups may be terminated and / or interrupted by at least one heteroatom, wherein R4 is a terminal group which is bonded to the remainder of the molecule through a hydroxyl or a primary amine group.

[0035] R4 may be as defined in the compounds disclosed in Maderna et al. Mol. Pharmaceutics 2015, 12, 1798.

[0036] In some embodiments, R4 is a group of formula O-R4’ or NH-R4’, wherein R4’ is a hydrocarbon chain, preferably a C1-C5 alkyl group, or a heteroaryl group, the alkyl group and / or the heteroaryl group being optionally substituted with at least one of a carboxylic acid COOH group, and amide group, a hydroxyl group, an alkyl group, an aryl group, a heteroaryl group and an alkylphosphonate group.

[0037] In some embodiments, R4 is selected from the group consisting of (+)-norephedrine, (S)- dolaphenine, L-phenylalanine and phenethylamine and R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L- phenylalanine or phenethylamine.

[0038] In some embodiments, one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkylaryl group, preferably selected from the group consisting of a methyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group.

[0039] In some embodiments, R4 is (+)-norephedrine.

[0040] In some embodiments, R’ is an ethyl or an isopropyl group.

[0041] In some embodiments, the step of reacting the protein with the isocyanide compound and the carbonyl compound is the only step of the process for preparing the conjugate.

[0042] In some embodiments, the toxicity of the conjugate of formula (I) is higher than a first threshold, and the toxicity of the isocyanide compound and of the carbonyl compound is lower than a second threshold, the second threshold being lower than the first threshold. Another object of the invention is a conjugate of a protein and a molecule of interest of formula wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein Ri is a molecule of interest or a fragment thereof.

[0043] In some embodiments:

[0044] - one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is selected from the group consisting of a methyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group, and wherein R’ is an alkyl group, wherein represents the position of the bond to the nitrogen atom of the remainder of the protein conjugate of formula (I), and wherein R4 is a terminal group which is bonded to the remainder of the molecule through a hydroxyl or a primary amine group.

[0045] R4 may be as defined in the compounds disclosed in Maderna et al. Mol. Pharmaceutics 2015, 12, 1798.

[0046] In some embodiments, R4 is a group of formula O-R4’ or NH-R4’, wherein R4’ is a hydrocarbon chain, preferably a C1-C5 alkyl group, or a heteroaryl group, the alkyl group and / or the heteroaryl group being optionally substituted with at least one of a carboxylic acid COOH group, and amide group, a hydroxyl group, an alkyl group, an aryl group, a heteroaryl group and an alkylphosphonate group.

[0047] In some embodiments, R4 is selected from the group consisting of (+)-norephedrine, (S)- dolaphenine, L-phenylalanine and phenethylamine and R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L- phenylalanine or phenethylamine.

[0048] In some embodiments, R4 is (+)-norephedrine and / or R’ is an ethyl or an isopropyl group.

[0049] In some embodiments, the protein is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof, in particular trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab, sacituzumab, gemtuzumab, cetuximab, a variant thereof or a fragment thereof.

[0050] In some embodiments, the molecule of interest : protein ratio is comprised between 1 and 20, preferably between 1 and 10.

[0051] Another object of the invention is a conjugate obtained with a process according to the invention, or a conjugate according to the invention, for use as a medicament.

[0052] Another object of the invention is a conjugate obtained with a process according to the invention, or a conjugate according to the invention, for use in the treatment of a proliferative disease such as a cancer.

[0053] Another object of the invention is an isocyanide compound of formula diastereoisomer thereof, wherein R’ is an alkyl group, wherein R4 is a terminal group which is bonded to the remainder of the molecule through a hydroxyl or a primary amine group.

[0054] R4 may be as defined in the compounds disclosed in Maderna et al. Mol. Pharmaceutics 2015, 12, 1798.

[0055] In some embodiments, R4 is a group of formula O-R4’ or NH-R4’, wherein R4’ is a hydrocarbon chain, preferably a C1-C5 alkyl group, or a heteroaryl group, the alkyl group and / or the heteroaryl group being optionally substituted with at least one of a carboxylic acid COOH group, and amide group, a hydroxyl group, an alkyl group, an aryl group, a heteroaryl group and an alkylphosphonate group.

[0056] In some embodiments, R4 is selected from the group consisting of (+)-norephedrine, (S)- dolaphenine, L-phenylalanine and phenethylamine and R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L- phenylalanine or phenethylamine.

[0057] Another object of the invention is a process for preparing an antibody-drug conjugate by a one- step four-centre three-component Ugi reaction, wherein the active pharmaceutical ingredient of the drug is an isocyanide derivative thereof or an isocyanide fragment thereof.

[0058] FIGURES

[0059] Figure 1 is the synthesis scheme of compound 19 of example 1.

[0060] Figure 2 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 10 mg / mL trastuzumab, 45 equivalents of isonitrile and aldehyde, and diastereoisomer A of compound 19.

[0061] Figure 3 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 10 mg / mL trastuzumab, 45 equivalents of isonitrile and aldehyde, and diastereoisomer B of compound 19.

[0062] Figure 4 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 15 mg / mL trastuzumab, 45 equivalents of isonitrile and aldehyde, and diastereoisomer A of compound 19.

[0063] Figure 5 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 15 mg / mL trastuzumab, 45 equivalents of isonitrile and aldehyde, and diastereoisomer B of compound 19.

[0064] Figure 6 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 10 mg / mL trastuzumab, 90 equivalents of isonitrile and aldehyde, and diastereoisomer A of compound 19.

[0065] Figure 7 presents the mass spectrum and the histogram of the DAR repartition for the conjugate Cl obtained with 10 mg / mL trastuzumab, 90 equivalents of isonitrile and aldehyde, and diastereoisomer B of compound 19. Figure 8 presents the mass spectrum and the histogram of the DAR repartition for the conjugate C5 obtained with 10 mg / mL sacituzumab, 90 equivalents of isonitrile and aldehyde, and 90 equivalents of compound 19.

[0066] Figure 9 presents the mass spectrum and the histogram of the DAR repartition for the conjugate C6 obtained with 10 mg / mL rituximab, 90 equivalents of isonitrile and aldehyde, and 90 equivalents of compound 19.

[0067] Figure 10 presents the mass spectrum and the histogram of the DAR repartition for the conjugate C7 obtained with 10 mg / mL ramucirumab, 90 equivalents of isonitrile and aldehyde, and 90 equivalents of compound 19.

[0068] Figure 11 presents the mass spectrum for the conjugate C8 obtained with 10 mg / mL cetuximab, 90 equivalents of isonitrile and aldehyde, and 90 equivalents of compound 19.

[0069] Figure 12 presents the mass spectrum and the histogram of the DAR repartition for the conjugate C9 obtained with 10 mg / mL gemtuzumab, 90 equivalents of isonitrile and aldehyde, and 90 equivalents of compound 19.

[0070] Figure 13 presents the results of in vitro cytotoxicity assays on SKBR3 and MDA-MB-231 of both diastereoisomers of conjugate Cl with different DAR.

[0071] Figure 14 presents the results of in vitro cytotoxicity assays on SKBR3 and MDA-MB-231 of both diastereoisomers of compound 19.

[0072] Figure 15 presents the results of in vivo toxicity studies with diastereoisomer B of conjugate C3

[0073] DETAILED DESCRIPTION OF THE INVENTION

[0074] Definitions

[0075] Unless otherwise indicated, when a range is expressed using the expression “between”, the limit values are included within the range described.

[0076] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical value, it modifies that numerical value by extending it 10% above and 10% below the numerical value, preferably 5% above and 5% below the numerical value, more preferably 1% above and 1% below the numerical value. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries 10% above and 10% below the numerical values set forth, preferably 5% above and 5% below the numerical values, more preferably 1% above and 1% below the numerical values.

[0077] The term “alkyl” means a saturated, linear or branched aliphatic hydrocarbon-based group. A “C1-C12 alkyl” is an alkyl containing from 1 to 12 carbon atoms. Examples of alkyl (or C1-C12 alkyl) are notably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl. Preferably, the C1-C12 alkyl is a Ci-Ce alkyl, for example: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl or hexyl.

[0078] The term “alkenyl” means a linear or branched, unsaturated aliphatic hydrocarbon-based group comprising at least one carbon-carbon double bond. A “C2-C12 alkenyl” is an alkenyl containing from 2 to 12 carbon atoms. Examples of alkenyl (or C2-C12 alkenyl) are notably ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl or dodecenyl. Preferably, the C2-C12 alkenyl is a C2-C6 alkenyl, for example: ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, or hexenyl.

[0079] The term “alkynyl” means a linear or branched, unsaturated aliphatic hydrocarbon-based group comprising at least one carbon-carbon triple bond. A “C2-C12 alkynyl” is an alkynyl containing from 2 to 12 carbon atoms. Examples of alkynyl (or C2-C12 alkynyl) are notably ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl or dodecynyl. Preferably, the C2-C12 alkynyl is a C2-C6 alkynyl, for example: ethynyl, propynyl, butynyl, pentynyl or hexynyl.

[0080] An “alkoxy group” is an O-alkyl group.

[0081] An “amino acid" refers to an alpha amino carboxylic acid, that is to say a molecule comprising a carboxylic acid functional group and an amine functional group in the alpha position of the carboxylic acid group, for example a proteinogenic amino acid or a non-proteinogenic amino acid such as 2-aminoisobutyric acid. A “proteinogenic amino acid" relates to an amino acid that is incorporated into proteins during the translation of messenger RNA by ribosomes in living beings, i.e. Alanine, Arginine, Asparagine, Aspartate (Aspartic Acid), Cysteine, Glutamate (Glutamic Acid), Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Pyrrolysine, Selenocysteine, Serine, Threonine, Tryptophan, Tyrosine or Valine.

[0082] An “amine” or “amino group” is a group of formula -NR5R6, wherein Rs and Re are independently a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group. A “ primary amine" or a “ primary amino group" refers to the case where Rs and Re are both hydrogen atoms.

[0083] An “ analogue" refers broadly to the modification or substitution of one or more chemical moieties on a parent compound and may include functional derivatives, isoelectronic groups, inferior homologues, superior homologues, positional isomers, tautomers, zwitterions, enantiomers, diastereomers, racemates, isosteres or stereochemical mixtures thereof. The modified part of the analogue is preferably structurally similar to that of the parent compound.

[0084] The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antigen-binding antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In particular, the antibody according to the invention may correspond to a monoclonal antibody (e.g. a chimeric, humanised or human antibody), or a fragment of monoclonal antibody. The term antibody refers to classical antibodies as well as to heavy-chain antibodies and fragments and derivatives thereof such as (VHH)2 fragments and single domain antibodies.

[0085] Antibody fragments that recognise specific epitopes can be generated by known techniques. The antibody fragments are antigen binding portions of an antibody, such as F(ab’)2, Fab, Fv, scFv and the like. Other antibody fragments include, but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab' fragments, which can be generated by reducing disulfide bridges of the F(ab')2 fragments. Alternatively, Fab' expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.

[0086] The term “antibody", when used in relation with a specific sequence, encompasses the exact sequence, and variants thereof. Preferably, the variants present at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% homology with the sequence.

[0087] The term “aryl" means a monocyclic or polycyclic aromatic carbocyclic group, preferably containing from 6 to 20 ring members. Examples of aryl groups are phenyl, biphenyl and naphthyl, preferably phenyl. The aryl group, in particular phenyl, is optionally substituted.

[0088] “Chemotherapeutic drugs" or agents, also referred to as antineoplastic agents, are agents used to directly or indirectly inhibit the uncontrolled growth and proliferation of cancer cells during a standardised chemotherapy regimen. “Cytotoxic drugs’" refers to a group of medicines which are toxic to cells, preventing their replication or growth, and so are used to treat cancer.

[0089] A “derivative” refers broadly to the modification or substitution of one or more chemical moieties on a parent compound. A derivative of a compound may be modified for instance with at least one peptide tag(s), and / or by addition of a thioester moiety allowing further reaction with proteins.

[0090] An “isocyanide derivative of a molecule of interest” refers broadly to a molecule of interest that has been modified to include at least one isocyanide moiety, preferably exactly one isocyanide moiety. More particularly, an ’isocyanide derivative of a molecule of interest” refers to the modification or substitution of at least one nitrogen atom-containing group, preferably exactly one nitrogen atom-containing group, on a molecule of interest by an isocyanide moiety, preferably by exactly one isocyanide moiety.

[0091] An “isocyanide fragment of a molecule of interest” refers broadly to a fragment (or a portion, or a segment) of a molecule of interest that contains at least one isocyanide moieties.

[0092] The term “halogen” means chlorine, fluorine, bromine or iodine. Preferably, a halogen is bromine, chlorine or fluorine, better still chlorine.

[0093] A “heterocyclic group” or a “heterocycle” is a cyclic alkyl, alkenyl, alkynyl or aryl group, preferably comprising from 5 to 20 carbon atoms, and which is interrupted by at least one heteroatom. Examples of heterocyclic groups include furan, thiophene, pyrrole, pyridine, pyran, oxazine, thiazine, pyrimidine, piperazine and thiine.

[0094] The term “heteroary means an aromatic, mono- or polycyclic group preferably containing 5 to 20 carbon atoms and also comprising at least one heteroatom such as N, O, P, Se or S. Examples of heteroaryl are notably: pyridinyl, thiazolyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthal enyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, triazinyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, P- carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, indolinyl, isoindolinyl, oxazolidinyl, benzotri azolyl, benzoisoxazolyl, oxindolyl, benzoxazolinyl, benzothienyl, benzothiazolyl, isatinyl, dihydropyridyl, pyrimidinyl, s-triazinyl, oxazolyl, arylphosphine, indole, indoline, phosphindoline or thiofuranyl.

[0095] Unless otherwise specified, all chemical groups, especially the alkyl, alkenyl, alkynyl, aryl, heterocyclic and / or heteroaryl groups are optionally substituted. The term “optionally substituted" means unsubstituted or substituted with one or more (for example, one, two, three or four, preferably one or two, better still only one) substituents.

[0096] Examples of substituents are notably nitro (-NO2), cyano (-CN), -SO3H, -OH, -SH, -NH2, - COOH, halogen, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce heteroalkyl, C3-C6 cycloalkyl, C2-C6 heterocycloalkyl, aryl, heteroaryl, -S(O)2-R and -C(O)-R, in which R is independently chosen from Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce heteroalkyl, C3- Ce cycloalkyl, C2-C6 heterocycloalkyl, aryl, and heteroaryl.

[0097] A “pharmaceutically acceptable excipient"" is an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA

[0098] A “protein"" refers to a compound comprising amino acid residues covalently linked by peptide bonds. A protein contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that can be comprised in a protein sequence. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Proteins"" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogues, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0099] A “salt"" of a compound includes the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Non-limiting examples include the acetate, trifluoroacetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, tetrafluoroborate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinafoate salts. Suitable base salts are formed from bases which form nontoxic salts. Non-limiting examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, 2-(diethylamino)ethanol, ethanolamine, morpholine, 4-(2- hydroxyethyl)morpholine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

[0100] A “therapeutic index" (TI) of a drug is the ratio of the dose that exerts toxicity in 50% of the population (TD50) to the dose that exerts a therapeutic or effective response (ED50) in 50% of the population (TI=TD50 / ED50).

[0101] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and / or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and / or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0102] The compounds according to the present invention and / or the compounds used in the present invention may comprise one or several stereogenic centre(s). Unless otherwise specified, mention of such compound encompasses all isomers, diastereoisomers, and enantiomers of the compound. Process for preparing a conjugate

[0103] A first object of the present invention is a process for preparing a conjugate of a protein and a molecule of interest of formula

[0104] The process comprising the step of reacting a protein with an isocyanide compound of formula . wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein the isocyanide compound of formula Ri—NCs aniSOcyanide derivative of a molecule of interest or a fragment thereof.

[0105] O

[0106] In an embodiment, the molecule of formula wherein R is a substituted nitrogen atom, is the molecule of interest.

[0107] It is understood that formula (Ic) represents a fragment of formula (I) that results when the bond between the N moiety and the protein, as well as the bond between the N and C=O moieties, are cleaved.

[0108] More particularly, when the isocyanide compound is an isocyanide derivative of a molecule of interest, the formula (Ic) preferably represents an analogue of a molecule of interest. In other words, in this case, Ri represents the molecule of interest and the formula (Ic) - which comprises Ri - represents an analogue of the molecule of interest.

[0109] When the isocyanide compound is an isocyanide fragment of a molecule of interest, the formula (Ic) preferably represents a molecule of interest. In other words, in this case, Ri represents a fragment of a molecule of interest and the formula (Ic) represents the molecule of interest. In this case, the part of the conjugate corresponding to the molecule of interest of formula (Ic) is advantageously created upon implementation of the reaction between the protein, the isocyanide and carbonyl compounds.

[0110] The step of reacting the protein, the isocyanide compound and the carbonyl compound may be implemented in any conditions suitable for forming the conjugate of formula (I).

[0111] In an embodiment, said reacting step is implemented by contacting the protein with the isocyanide compound and the carbonyl compound in conditions suitable for an Ugi multicomponent reaction. Preferably, the Ugi reaction is a three-component, four-centre Ugi reaction.

[0112] Since the Ugi reaction can be conducted under mild conditions - e.g., room temperature, under air and in aqueous environment -, it has been explored for multiple purposes in the field of chemical biology.

[0113] In some embodiments, the step of reacting the protein, the isocyanide compound and the carbonyl compound is implemented at room temperature, ie at a temperature comprised between 15°C and 30°C, preferably at a temperature of about 25°C.

[0114] In some embodiments, the step of reacting the protein, the isocyanide compound and the carbonyl compound is implemented for a duration comprised between 30 minutes and 7 days, preferably between 4h and 48h, especially for a duration of about 16h.

[0115] In some embodiments, the number of equivalents of carbonyl compound and / or of isocyanide compound to the protein is independently strictly higher than 1, preferably higher than 5, more preferably higher than 25, in particular comprised between 40 and 100 equivalents. In specific embodiments, the number of equivalents of carbonyl compound and / or of isocyanide compound to the protein is about 45 or about 90.

[0116] In a preferred embodiment, the molar ratio of the carbonyl compound to the isocyanide compound is comprised between 0.5 and 1.5, preferably between 0.8 and 1.2, more preferably it is about 1.

[0117] One skilled in the art is able to adapt the conditions such as duration and / or temperature and the number of equivalents to be implemented depending on the structure of the protein, of the carbonyl compound, of the isocyanide compound and the desired ratio between the molecule of interest and the protein in the conjugate, such as the desired DAR (drug-antibody ratio).

[0118] In some embodiments, the step of reacting the protein, the isocyanide compound and the carbonyl compound is implemented in aqueous medium. In some embodiments, the step of reacting the protein, the isocyanide compound and the carbonyl compound is implemented in phosphate-buffered saline (PBS) buffer at a pH comprised between 6 and 9, preferably at a pH about 7.5.

[0119] The presence of alternative buffers, especially amine buffers or carboxylate buffers, is preferably avoided as these may compete with the protein for the Ugi reaction.

[0120] In some preferred embodiments, the Ugi multicomponent reaction is the last synthesis step in the process for preparing the conjugate according to the invention. In some embodiments, purification steps, such as steric exclusion chromatography, gel permeation chromatography or membrane filtration, may be implemented after the Ugi reaction.

[0121] The invention also pertains to a process for preparing a conjugate of a protein and a molecule of interest of formula , the process comprising the step of reacting a protein with an isocyanide compound of formulala) R-i- NOan(ja carbonyl compound of formula , wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and

[0122] O wherein , wherein R is a substituted nitrogen atom, is a molecule of interest.

[0123] Isocyanide compound

[0124] The isocyanide compound implied in the process according to the invention is a compound of formula Ri—NC ,wh erein Ri is an organic moiety different from a hydrogen atom.

[0125] In some embodiments, the isocyanide compound is derived from the amine group of an amino acid or an analogue thereof. In some embodiments, the isocyanide compound is an isocyanide derivative of a molecule of interest. In that case Ri is advantageously the molecule of interest.

[0126] In some embodiments, the isocyanide compound is derived from a primary amine group of a molecule of interest.

[0127] In some embodiments, the isocyanide compound is an isocyanide fragment of a molecule of interest. In that case Ri is advantageously a fragment of a molecule of interest.

[0128] In some embodiments, Ri is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heterocyclic and heteroaryl groups.

[0129] Each of said groups may independently be terminated and / or interrupted by heteroatoms, such as O, S, N, or P atoms.

[0130] Each of said groups may independently be substituted by at least one substituent, said substituent being for instance selected from the group consisting of halogen atoms, hydroxy groups, amino groups, alkoxy groups, and alkyl, alkenyl, alkynyl, aryl, heterocyclic and heteroaryl groups.

[0131] One skilled in the art is able to design the suitable structure for Ri in order for the desired molecule of interest to be formed by the coupling reaction.

[0132] In some embodiments, Ri does not comprise any carboxylic acid COOH moiety. In some embodiments, Ri does not comprise any primary amine NH2 moiety.

[0133] In some embodiments where the molecule of interest comprises a terminal N-methylvaline moiety, the isocyanide compound may consist in the entire molecule of interest but its first N- methyl valine residue, the latter being formed upon conjugation to the protein.

[0134] In some embodiments, Ri is a drug or a drug analogue.

[0135] In some embodiments, Ri is a fragment of a drug or of a drug analogue.

[0136] In some embodiments, diastereoisomer thereof, wherein R’ is a C1-C7 alkyl group or aralkyl group, wherein the alkyl and aralkyl groups may be terminated and / or interrupted by at least one heteroatom, wherein represents the position of the bond to the nitrogen atom of the remainder of the protein conjugate of formula (I), and wherein R4 is a terminal group which is bonded to the remainder of the molecule through a hydroxyl or a primary amine group.

[0137] R4 may be as defined in the compounds disclosed in Maderna et al. Mol. Pharmaceutics 2015, 12, 1798.

[0138] In some embodiments, R4 is a group of formula O-R4’ or NH-R4’, wherein R4’ is a hydrocarbon chain, preferably a C1-C5 alkyl group, or a heteroaryl group, the alkyl group and / or the heteroaryl group being optionally substituted with at least one of a carboxylic acid COOH group, and amide group, a hydroxyl group, an alkyl group, an aryl group, a heteroaryl group and an alkylphosphonate group.

[0139] In some embodiments, R4 is selected from the group consisting of (+)-norephedrine, (S)- dolaphenine, L-phenylalanine and phenethylamine and R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L- phenylalanine or phenethylamine.

[0140] In some embodiments, R’ is a Ci-Ce alkyl group. In some embodiments, R’ is selected from the group consisting of a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n- butyl group, an isobutyl group, a tert-butyl group, a n-pentyl group, a ramified pentyl group, a n-hexyl group, a ramified hexyl group and a cyclohexyl group. In some embodiments, R’ is an ethyl group, an isopropyl group or an isobutyl group, preferably an ethyl group or an isopropyl group, more preferably an isopropyl group.

[0141] In some embodiments, R4 is (+)-norephedrine and the conjugate is a protein-MMAE conjugate.

[0142] In some embodiments, R4 is (L)-phenylalanine and the conjugate is a protein-MMAF conjugate.

[0143] In some embodiments, R4 is (S)-dolaphenine and the conjugate is a protein-dolastatin 10 conjugate.

[0144] In some embodiments, R4 is phenethylamine and the conjugate is a protein- monom ethyl soblidotin conjugate. It is understood that when defined above, the isocyanide compound is preferably a fragment of a molecule of interest.

[0145] Carbonyl compound

[0146] The carbonyl compound implied in the process according to the invention is a compound of O formula , wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom.

[0147] In some embodiments, R2 and R3 are not simultaneously hydrogen atoms.

[0148] In some embodiments, the carbonyl compound has a molecular weight lower than 1000 Da.

[0149] In some embodiments, one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is not a hydrogen atom.

[0150] In some embodiments, each of R2 and R3 is independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group and an optionally substituted alkylaryl group. In some embodiments, each of R2 and R3 is independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group.

[0151] In some embodiments, one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is selected from the group consisting of an optionally substituted alkyl group, an optionally substituted aryl group and an optionally substituted alkylaryl group. Preferably, one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is selected from the group consisting of a methyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group.

[0152] In some embodiments, each of R2 and R3 is independently selected from the group consisting of an optionally substituted alkyl group, an optionally substituted aryl group and an optionally substituted alkylaryl group.

[0153] Molecule of interest

[0154] The molecule of interest that is conjugated to the protein thanks to the process according to the invention may be: - Ri of the isocyanide compound, in particular when the isocyanide compound is an isocyanide derivative of a molecule of interest, and / or

[0155] O a compound of formula , wherein Rl, R2 and R3 are as defined above, and wherein R is a substituted nitrogen atom, in particular when the isocyanide compound is an isocyanide fragment of a molecule of interest.

[0156] In some embodiments, especially when the molecule of interest is a compound of formula (Ic), R is a monosubstituted nitrogen atom. In other embodiments, R is a disubstituted nitrogen atom. The substituents of the nitrogen atom may each independently be selected in the list of examples of substituents provided above for the substitution of alkyl groups. In some embodiments, R is a NH(CH3) group.

[0157] In some embodiments, the molecule of interest is a pharmaceutical interest unit. A pharmaceutical interest unit is a unit that can have or contribute to a direct or indirect pharmaceutical effect when administered in suitable conditions.

[0158] In some embodiments, the molecule of interest is a drug, a drug analogue or a fragment thereof. In some embodiments, the drug is a chemotherapeutic drug and / or a cytotoxic drug.

[0159] In some embodiments, the drug is selected from the group consisting of auristatins, dolastatins, tubulysins, camptothecins, mertansines, non-ribosomal peptides, antibiotics, nitrogen mustards, analogues thereof and derivatives thereof.

[0160] In some embodiments, the drug is selected from the group consisting of auristatins, dolastatins, tubulysins, camptothecins, mertansines, non-ribosomal peptides, antibiotics, nitrogen mustards, taxanes, analogues thereof and derivatives thereof.

[0161] Dolastatins and auristatins are analogues of dolastatin 10, and the latter is a biologically active polypeptide isolated from the marine mollusc, dolastatin. Dolastatin 10 inhibits tubulin polymerisation by binding to tubulin (the same binding region as vincristine). Dolastatin 10, auristatin peptide PE, and auristatin peptide E are all linear polypeptides, containing four amino acids (three of which are unique to donotoxin compounds) and a C-terminal amide group. Two representative auristatin compounds, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), are the preferred drug parts of antibody-drug conjugates.

[0162] Examples of auristatins include monomethyl auristatin D (MMAD), also known as monomethyl dolastatin 10, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and monomethyl auristatin phenylethyl (MMAPE), also known as monomethyl soblidotin, preferably MMAE.

[0163] Examples of auristatin analogues include monomethyl auristatin D (MMAD) analogues, monomethyl auristatin E (MMAE) analogues, monomethyl auristatin F (MMAF) analogues and monomethyl auristatin phenylethyl (MMAPE) analogues, preferably MMAE analogues.

[0164] Auristatin analogues are cytotoxic drugs which structure differs from that of a known auristatin in one or several positions. For instance, auristatin analogues may be obtained by varying the group R’ as defined above.

[0165] Examples of dolastatins include dolastatin 10, dolastatin 14 and dolastatin 15. Dolastatins are oligopeptides from Dolabella auricularia. and are used in research on various cancers.

[0166] Tubulysins are a class of natural products extracted from myxobacteria, which can effectively inhibit the polymerization of tubulin and therefore have anti-mitotic activity. Among them, Tubulysin D has the best activity. Tubulysin D is a complex tetrapeptide compound with O- acyl / N,O-acetal functional group in its structure, so it is unstable under acidic and alkaline conditions. US2011 / 0021568 and US2013 / 0224228 respectively disclose a series of analogues of tubulysin, which have the above unstable functional groups removed from the structure and at the same time have high cell activity.

[0167] Examples of tubulysins include tubulysin D, tubulysin M and tubulysin Pr.

[0168] Examples of tubulysin derivatives include metabolites of above-listed tubulysins.

[0169] Camptothecin is a topoisomerase inhibitor isolated from the bark and stem of Camptotheca acuminata.

[0170] Examples of camptothecin derivatives include irinotecan, SN-38, topotecan, deruxtecan, belotecan, 9-aminocamptothecin and exatecan.

[0171] Maytansine compounds inhibit cell proliferation by inhibiting the formation of microtubules of tubulin. Maytansinoids are derivatives of maytansine. Maytansinoids have high-efficiency cytotoxicity, but their clinical application in cancer treatment has great limitations. This is mainly due to the low selectivity of such molecules to tumours. However, this high cytotoxicity makes them the preferred drug part of antibody-drug conjugates.

[0172] Examples of mertansines include maytansine derivatives DM1 (Mertansine derivatives Ml), maytansine derivatives DM4 (Mertansine derivatives M4), and analogues thereof. Nonribosomal peptides (NRP) are a class of peptide secondary metabolites, usually produced by microorganisms like bacteria and fungi.

[0173] Examples of non-ribosomal peptides include bleomycin, epothilone and fabclavine, preferably bleomycin.

[0174] Examples of chemotherapeutic antibiotics include calicheamicins, such as ozogamicin, anthracyclins, such as doxorubicins and daunorubicins, benzodipyrrole antibiotics, such as duocarmycins, CC-1065, and other cyclopropapyrroloind-4-one derivatives, and peptidic antibiotics, such as dactinomycin.

[0175] Examples of nitrogen mustards include cyclophosphamide, chlorambucil, uramustine, melphalan, and bendamustine, preferably melphalan.

[0176] Examples of taxane derivatives include cabazitaxel, docetaxel and paclitaxel.

[0177] In an embodiment, the molecule of interest is a camptothecin, preferably exatecan, and the isocyanide compound is an isocyanide derivative of this molecule of interest.

[0178] In an embodiment, the molecule of interest is an auristatin, preferably MMAE, and the isocyanide compound is an isocyanide fragment of this molecule of interest.

[0179] Pharmaceutically acceptable salts of drugs and of drug analogues and derivatives are also encompassed by the term “drugs”, “drugs analogues” and “drugs derivatives” .

[0180] Protein

[0181] The protein implied in the process of the present invention may be any protein or fragment thereof, provided that it comprises at least one primary amine group and at least one carboxylic acid group.

[0182] In particular, the protein is selected from those which bind specifically a molecule present at the membrane of a cancer cell, preferably a molecule selected from the group consisting of proteins, glycoproteins, glycolipids, carbohydrates, or a combination thereof, even more preferably the protein binds specifically a protein present at the membrane of a cancer cell. The membrane molecule to which the protein is capable to bind to is a molecule mainly or exclusively present at the membrane of a cancer cell. In a particular embodiment, the membrane molecule recognized by the protein is a protein overexpressed at the membrane of a cancer cell.

[0183] In some embodiments, the protein is selected from the group consisting of lipocalins, anticalins, antibodies, and fragments thereof. Antibodies may be polyclonal antibodies or monoclonal antibodies, preferably monoclonal antibodies (mAb). Antibodies also include nanobodies.

[0184] Antibody fragments include fragment antigen-binding regions (Fab).

[0185] In some embodiments, the antibodies may be a mAb, a nanobody, or a fragment such as a Fc fragment, a Fv fragment, a Fab fragment, a F(ab’)2 fragment, a scFv fragment, or any other fragment of an antibody.

[0186] In some embodiments, the antibody is a recombinant antibody or a mutant antibody.

[0187] In some embodiments, the antibody is an IgGl antibody, an IgG2 antibody, or an antibody of any other subtype.

[0188] In some embodiments, the protein is an anticalin or a monoclonal antibody.

[0189] In an embodiment, the antibody is selected from the group consisting of trastuzumab, bevacizumab, ramucirumab and daratumumab.

[0190] In an embodiment, the antibody is selected from the group consisting of trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab, sacituzumab, gemtuzumab, and cetuximab.

[0191] In an embodiment, the antibody is selected from the group consisting of trastuzumab, ramucirumab, rituximab, sacituzumab cetuximab and gemtuzumab.

[0192] In some embodiments, the antibody comprises a N-terminal aspartic acid residue or a N- terminal glutamic acid residue. In some embodiments, the antibody does not comprise a C- terminal carboxylic acid.

[0193] Antibodies used according to the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. The antibodies of the invention can be obtained by producing and culturing hybridomas.

[0194] The antibody used according to the invention may be a monomeric antibody or a multimeric antibody and it may comprise at least a variable domain, in particular when the antibody is multimeric.

[0195] In some embodiments, the antibody is selected from the group consisting of hRl (anti -IGF -1R), hPAM4 (anti-MUC5ac), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLLl (anti-CD74), hLL2 (anti-CD22), hRFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA- DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-TROP-2), hMN-3 (anti- CEACAM6), Ab 124 and Ab 125 (anti-CXCR4). More preferably, the antibody is IMMU- 31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti- CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLLl (anti-CD74), hLL2 (anti-CD22), hL243 or IMMU-114 (anti- HLA-DR), hA19 (anti-CD19) or hA20 (anti- CD20). Alternative antibodies of use include abciximab (anti- glycoprotein Ilb / IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti- EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti- ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti- CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20), CC49 (anti- -5- CA 02961774), AB-PG1-XG1-026 (anti-PSMA), D2 / B (anti-PSMA), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CDl l a), GAI 01 (anti-CD20), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-a4 integrin), omalizumab (anti-IgE); anti-TNF-a antibodies such as CDP571, MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303, infliximab, certolizumab pegol, anti-CD4OL, adalimumab, belimumab and variants thereof.

[0196] Preferably, the antibody used according to the invention is an immunoglobulin G (IgG), preferably subtypes IgGl or IgG4, for example trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab sacituzumab, gemtuzumab or cetuximab.

[0197] Primary amine and carboxylic acid groups o f the protein

[0198] The primary amine group of the protein and the carboxylic acid group of the protein that are implied in the conjugation may be any primary amine group and any carboxylic acid group present at the surface of the protein.

[0199] Preferably, the implication of the primary amine group and the carboxylic acid group in the conjugation does not alter the biological activity of the protein. For instance, when the protein is an antibody or a fragment thereof, the implication of the primary amine group and the carboxylic acid group in the conjugation does not alter the antibody affinity with the corresponding targets, such as the target cells.

[0200] In some embodiments, the primary amine group and the carboxylic acid group belong to the same amino acid of the protein. In other embodiments, the primary amine group and the carboxylic acid group belong to different amino acids of the protein. Without wishing to be bound by any theory, the Inventors believe that it is advantageous that the primary amine group and the carboxylic acid group are not too distant from each other when the protein is its functional 3D-configuration.

[0201] In some embodiments, the primary amine group and the carboxylic acid group are separated by a moiety which length corresponds to that of a moiety comprising from 2 to 20 atoms. In some embodiments, the primary amine group and the carboxylic acid group are separated by a distance of 20 angstroms or less, preferably a distance comprised between 1 and 10 angstroms.

[0202] In some embodiments, the primary amine group and / or the carboxylic acid group is part of a side-chain of and amino acid.

[0203] In some embodiments, the primary amine group is part of a lysine, an arginine, a glutamine, an asparagine, an aspartic acid or a glutamic acid. When the primary amine group is part of a lysine, it is preferably part of a side-chain of lysine. In some embodiments, the primary amine group is part of a N-terminal lysine, arginine, glutamine, asparagine, aspartic acid, or glutamic acid amino acid, more preferably a N-terminal arginine, glutamine asparagine, aspartic acid or glutamic acid amino acid. In a particular embodiment, the primary amine group is part of a sidechain of a lysine or part of a N-terminal aspartic acid or glutamic acid.

[0204] In some embodiments, the carboxylic acid group of the protein is part of an aspartate or a glutamate amino acid, preferably a N-terminal aspartate or glutamate amino acid. Conjugation at these positions did not affect the antibody affinity towards its target(s).

[0205] In some embodiments, the primary amine group and / or the carboxylic acid group are part of a C -terminal amino acid.

[0206] Conjugate

[0207] A second object of the invention is a conjugate of a protein and a molecule of interest of formula wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=0 moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein Ri is a molecule of interest or a fragment thereof.

[0208] The invention also pertains to a conjugate of a protein and a molecule of interest of formula wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and

[0209] O

[0210] (Io) wherein , wherein R is a substituted nitrogen atom, is a molecule of interest.

[0211] The conjugates according to the invention, and the conjugates obtainable and / or obtained, preferably obtained, by the process according to the invention, comprise a molecule of interest, in particular a drug or a drug analogue, covalently linked to a protein, in particular an antibody, without a linker as classically defined in the field of conjugates.

[0212] The ratio of the molecule of interest to the protein in the conjugate, in particular the drugantibody ratio (DAR), may vary in a wide range, depending on the used reagents and the coupling conditions. In some embodiments, the ratio of the molecule of interest to the protein in the conjugate is comprised between 1 and 20, preferably between 1 and 10.

[0213] The conjugate according to the invention may be obtained and / or used in admixture with conjugates of the same protein with fragments of the same drug, which are obtained by a Passerini coupling (Somay et al., Chem. Eur. J. 2020, 26, 61, p.13797-13805). Said multicomponent coupling involves similar reagents and similar conditions to those of the Ugi coupling which is used to prepare the conjugates according to the invention. Ugi adducts and Passerini adducts may be present on the same protein and / or on different proteins. In some embodiments, the toxicity of the conjugate of formula (I) is higher than a first threshold, and the toxicity of the isocyanide compound and of the carbonyl compound is lower than a second threshold, the second threshold being lower than the first threshold. Preferably, the toxicity is expressed as an ICso value.

[0214] In some embodiments, the therapeutic index of the conjugate of formula (I) is higher than a first threshold, and the therapeutic index of the isocyanide compound and of the carbonyl compound is lower than a second threshold, the second threshold being lower than the first threshold.

[0215] Another object of the present invention is a pharmaceutical composition comprising at least one conjugate according to the invention and a pharmaceutically acceptable excipient.

[0216] In some embodiments, the conjugate according to the invention is present in a concentration comprised between Ig / L and 200g / L, preferably between 5g / L and 20g / L in the pharmaceutical composition according to the invention.

[0217] The conjugates according to the invention and the pharmaceutical compositions according to the invention may be used in the treatment and / or diagnosis of proliferative diseases, such as cancer.

[0218] Another object of the present invention is thus a conjugate according to the invention, or a pharmaceutical composition according to the invention, for use as a medicament.

[0219] Another object of the present invention is thus a conjugate according to the invention, or a pharmaceutical composition according to the invention, for use in the treatment of a proliferative disease such as a cancer.

[0220] The cancer to be treated according to the invention may be selected from the group consisting of lung cancer, colorectal cancer, head and neck cancer, pancreatic cancer, gastric cancer, bladder cancer, glioblastoma, ovarian, breast, prostate, cervical, leukaemia, lymphoma, myeloma, bone cancer, skin cancer, melanoma, neuroblastoma, liver cancer and oesophageal cancer. In some embodiments, the cancer is selected from the group consisting of haematopoietic and lymphoid tissues cancers, gynaecological cancers, brain cancers and urologic cancers.

[0221] In some embodiments, the cancer is a HER2 positive cancer.

[0222] Another object of the present invention is the use of a conjugate according to the invention in the manufacture of a medicament for the treatment of a proliferative disease such as a cancer. Another object of the present invention is a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a conjugate according to the invention or a pharmaceutical composition according to the invention.

[0223] The subject in need thereof is preferably a mammal, in particular a human.

[0224] The dose of conjugate according to the invention to be administered can be adapted by the practician depending on the cancer to be treated and the structure of the conjugate among others. It may vary in a wide range. In some embodiments, the dose of conjugate is comprised between 1 mg / kg and 100 mg / kg, more preferably between 1 mg / kg and 5 mg / kg.

[0225] The invention will also be described in further detail in the following examples, which are not intended to limit the scope of this invention, as defined by the attached claims.

[0226] EXAMPLES

[0227] Materials and methods SYNTHETIC CHEMISTRY

[0228] All reagents were obtained from commercial sources and used without prior purification. Dry solvents were obtained from Merck. All reactions were carried out under an atmosphere of argon in flame-dried glassware with magnetic stirring. Reactions performed at 0 °C were cooled with an ice and H2O bath, while those at - 78 °C were cooled with an acetone / dry ice colling bath. Concentration in vacuo refers to distillation on a Biichi rotary evaporator, and where appropriate, under high vacuum.

[0229] Analytical thin layer chromatography (TLC) was performed using plates cut from aluminium sheets (ALUGRAM Xtra SIL G / UV254) purchased from Macherey -Nagel. Visualisation was achieved under a 254 or 365 nm UV light and by immersion in an appropriate staining solution.

[0230] Column chromatography was carried out as “Flash Chromatography” using silica gel G-80, G- 40, G-25, G-12 or G-4 (40-63 pm) columns from Buchi on a Buchi Reveleris X2.

[0231] Reverse-phase chromatography was performed on a semi-preparative Waters Delta 600 HPLC (pump: Waters 600 Controller, UV-Vis detector: Waters 2489, detection at 214 nm) using a Sunfire C18 (150 mm x 2 mm, 5 pM, Waters) at a flow of 17.0 mL / min. Unless otherwise indicated, the eluent system used was water + trifluoroacetic acid TFA (0.1 %) / acetonitrile ACN. The gradient applied was 5% to 95% ACN in 25 min followed by 5 min of reequilibration. BIOMOLECULES

[0232] All reagents, proteins (besides monoclonal antibodies), enzymes and solvents were obtained from commercial sources - Sigma Aldrich France, Fischer Scientific France or VWR France - , and used without prior purification. Monoclonal antibodies and T-DM1 samples were provided by the Institut de Cancerologie Strasbourg Europe (Strasbourg, France). Concentrations in protein, antibody or fragment antigen-binding (Fab) solutions were determined by UV absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch, France) at 280 nm at ambient temperature. Sample buffer was used as blank for baseline correction. The concentration of antibody conjugates was also measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Illkirch, France, Ref. 23225). Incubation during digestion, reduction and conjugation experiments took place either in an Eppendorf thermomixer comfort (catalogue # 5355) equipped with a 2-mL block, or in a digital heated shaker dry bath (Ref.: 88880027). BUFFERS LIST

[0233] Buffers were prepared with double-deionised water and filter sterilised (0.20 pm).

[0234] Phosphate Buffer (PB; 1 M, pH 7.4) contains a mixture of 1 M NaftPCh and 1 M Na2HPO4 in a 2:21 v / v ratio.

[0235] Borate Buffer Saline (BBS) contains 25 mM boric acid (H3BO3), 25 mM sodium chloride (NaCl), and the pH was adjusted accordingly.

[0236] Dulbecco’s Phosphate Buffer Saline IX; DPBS IX (calcium and magnesium free, Merck, Ref. D8537-6 x 500 mL) contains 10 mM phosphate, 138 mM NaCl, pH 7.4.

[0237] Tris buffer contains 1000 mM Tris base, pH 8.

[0238] Conjugation buffer contains 40 mM PB, 20 mM NaCl, 6 mM EDTA, pH 7.4.

[0239] Sodium acetate buffer contains 20 mM NaOAc, pH 3.1.

[0240] Digestion buffer 1 contains 50 mM PB, 150 mM NaCl, 1 mM EDTA, pH 6.8.

[0241] Digestion buffer 2 contains 50 mM PB, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, pH 6.8.

[0242] Digestion buffer 3 contains 20 mM NaH2PO4, 10 mM EDTA, 80 mM cysteine HCl, pH 7. BIOMOLECULES PURIFICATION

[0243] Protein, antibody, or Fab conjugates were purified by gel filtration chromatography either on Bio-spin P-30 and P-6 columns obtained from Bio-rad (Hercules, U.S.A) or on Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mL (Thermo Fisher Scientific, Pierce Biotechnology, USA). Vivaspin micro-concentrators (500 pL, 50 kDa, 30 kDa, 10 kDa and 3 kDa cutoff) from Sartorius (Gottingen, Germany) were used for buffer exchange. Antibody deglycosylation was achieved by incubating Remove-iT® Endo S (New England Biolabs, Ipswich, USA).

[0244] Purification by steric exclusion chromatography (SEC) was performed on an AKTA Pure system (GE Healthcare) with a Superdex column (S200 10 / 300 GL), using DPBS IX + 5 mM EDTA (pH 7.4) as eluent at a flow rate of 0.5 mL / min.

[0245] V SDS-PAGE analysis

[0246] Reducing or non-reducing SDS-PAGE was performed on 5 - 15% Mini- PROTRIETHYLAMINEN® TGX™ gel (Bio-Rad, Hercules, U.S.A., Ref. 4561094) following standard lab procedures. To the samples containing antibody conjugates (11 pL, 0.2 mg / mL solution in DPBS IX) was added 3 pL of loading buffer (either reducing or non-reducing Laemmli SDS sample buffer 4X, Alfa Aesar, Ref., J63615.AC) and heated at 90 °C for 10 min. The gel was run at constant voltage (200 V) for 35 min using TRIS 2.5 mM - Glycine 19.2 mM - SDS 0.01% as a running buffer (Bio-rad, Hercules, U.S.A., Ref. 1610772). The fluorescence was visualized on a ImageQuant™ LAS4000 (GE Healthcare) prior to staining with InstantBlue® Protein Stain (Merck, Ref. ISB1L).

[0247] V In vitro ADC cytotoxicity assay

[0248] SKBR-3 (HER2-positive) and MDA-MB-231 (HER2 -negative) cell lines were grown in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS), Penicillin (100 units / mL), and Streptomycin (100 pg / mL). Cell lines were maintained in a 5% CO2 humidified atmosphere at 37 °C. The day before experiment, both cell lines were seeded in 96-well plates at 6 000 cells / well in 100 pL fresh cell medium. The day of experiment, the medium was removed carefully and cells were incubated with conjugates in fresh cell medium, 100 pL / well, in triplicate for 96 h. 10 pL of resazurin (Sigma- Aldrich R7017, 0.15 mg / mL in PBS) was added into each well and incubated for 2 - 4 h at 37 °C. Cell viability was measured by quantifying fluorescence (excitation 531 / 25 nm, emission 595 / 60 nm) using a plate reader (Perkin Elmer, Victor 2030). EC50 values were determined using four-parameter logistic fitting in GraphPad Prism 8.0.

[0249] V Spectroscopy and spectrometry

[0250] 1H and13C NMR spectra were recorded at 23 °C on Bruker Advance III - 400 MHz / 500 MHz spectrometers. Recorded shifts are reported in parts per million (5) and calibrated using residual nondeuterated solvent. Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app = apparent), coupling constant (J, Hz), integration and assignment for1H NMR data.

[0251] Analytical LC-MS analyses were carried out on Waters ARC separations module equipped with Waters 2998 PDA UV detector, Waters Acquity QDa mass detector and XB ridge®, 3.5 pm, Cl 8, 50 x 4.6 mm column. The flow rate was 1 mL / min and the solvent system was composed as follows: solvent A: 0.05% TFA in H2O; solvent B: acetonitrile. The gradient run was: 0 - 5 min. - 5% to 95% B; 5 - 6 min. - 95% B; 6 - 7 min. - 5% B. Mass detector was operated in positive MS Scan mode with 600 °C probe temperature, 1.5 kV capillary voltage and 10 V cone voltage.

[0252] High resolution mass spectra (HRMS) were obtained using an Agilent Q-TOF 6520.

[0253] Infrared (IR) spectra were recorded in a Thermo-Nicolet FT / IR-380 spectrometer. Spectra were interpreted with OMNIC 9 software and are reporter in cm-1. The abbreviations used are w (weak), m (medium), s (strong).

[0254] V Native mass spectrometry analyses (Native MS, nMS)

[0255] Native MS analysis on the LCT (Micromass, UK). 20 pg of mAb samples buffer exchanged to 150 mM NH4OAc at pH 7.5 using vivaspin, were analysed on a time-of-flight (ToF) LCT mass spectrometer upgraded for high m / z values by MS vision. The MS was coupled to an automated chip-based nanoESI device (Tri Versa NanoMate, Advion, USA). The LCT was operated in the positive mode with a sample cone and pressure in the interface region set to Vc = 180 V and Pi = 6 mbar, respectively to ensure good desolvation and transmission of the native structure of the antibodies. Acquisitions of mass spectra were carried out over an m / z range of 1,000- 10,000with a 1.5 s scan time. External calibration was performed using singly charged ions produced by a 2 g / L solution of caesium iodide in 2-propanol / water (50 / 50 v / v). MS data interpretations were performed using Mass Lynx V4.1 (Waters, Manchester, UK).

[0256] Size-exclusion chromatography (SEC) coupled to native MS (SEC-nMS) on the BioAccord LC-MS system (Waters, Manchester, UK). 5-20 pg of samples were injected through a MaxPeak Premier Protein SEC 250 A, 1.7 pm, 4.6 * 150 mm (Waters, Manchester, UK) using 150 mM NH4OAc (pH 6.9) at a flowrate of 250 mL / min over 6 min, into the LC- MS system. The BioAccord platform comprises an Acquity UPLC M-Class system; including a binary solvent manager, a sample manager at 4 °C, a column oven at room temperature and a UV detector operating at 214 nm and 280 nm, coupled to an RDa ToF detector. The mass spectrometer was calibrated in the 400-7,000 m / z range in the positive mode using a solution containing, 50 ng / pL of sodium iodide in isopropanol / water (80 / 20 v / v) and 0.5 ng / pL of rubidium iodide in isopropanol / water (80 / 20 v / v). A LockMass solution containing 3.75 ng / pL of leucine encephalin, 12.5 ng / pL of caffeine and 2.5 ng / pL of 1 -pentanesulfonic acid in ACN / water (80 / 20 v / v) was inj ected automatically prior to each inj ection. The MS was operated with a capillary voltage of 3.5 kV and a pressure of 2 mbar. The cone voltage was set to 80 V. Acquisitions were performed on the m / z range 400-7,000 with a 1 s scan time. Data processing

[0257] MS data interpretations were performed using MassLynx V4.1 (Waters, Manchester, UK) for the LCT analyses and using UNIFI vl.913.9 (Waters, Manchester, UK) for the BioAccord analyses. The avDoC values were calculated based on the relative peak intensities measured from the raw mass spectra (four charge states) using the equation below: where k is the number of modifications and Ik is the relative peak intensity of DoCk.

[0258] Trypsin digestion. 20 pg of sample were solubilized in 150 mM ammonium hydrogen carbonate, 0.1% RapiGest (Waters, Manchester, UK) at pH 7.8, to obtain a final volume of 24 pL. Disulfide reduction was performed by incubating the solution with 5 mM dithiothreitol for 30 min at 57°C. Alkylation of cysteine residues was performed in 10 mM iodoacetamide in the dark at room temperature for 40 min. The enzyme was prepared by suspending 20 pg of trypsin (Promega, V5111) in 100 pL of water. Digestion was performed by adding 1 pL of trypsin which corresponds to a 1 : 100 enzyme: substrate ratio. Samples were incubated overnight at 37°C. The reaction was stopped by 1% of trifluoroacetic acid. RapiGest was eliminated by incubation at 37°C for 30 min and centrifugation at 10,000 g for 5 min.

[0259] NanoLC-MS / MS. The analyses were performed using a nanoACQUITY Ultra-Performance- LC (Waters, Manchester, UK) coupled to a Q ExactiveTM Plus Quadrupole-OrbitrapTM Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). A volume equivalent to 140 ng of digest were trapped on a Symmetry C18 pre-column (180 pm x 20 mm, 5 pm particle size, Waters) and the peptides were separated on an ACQUITY UPLC® BEH130 C18 separation column (75 pm x 250 mm, 1.7 pm particle size, Waters). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptide trapping was performed during 3 min at a flow rate of 5 pL / min with 99% A and 1% B and elution was performed at 60 °C at a flow rate of 350 nL / min from 6% to 40% of B in 43 minutes. MS and MS / MS acquisition were performed in positive mode, with the following settings: spray voltage 1800 V and capillary temperature 250°C. The MS scan had a resolution of 70000, the AGC target was 3x106 and the maximum IT was 50 ms on m / z [300-1800] range. The MS / MS scans were acquired at a resolution of 17500, the AGC target was 1x105 and the maximum IT was 100 ms with fixed first mass of 100 m / z and Isolation window of 2 m / z. Top 10 HCD was selected with intensity threshold of 5x104 and dynamic exclusion of 3 s. The normalised collision energy (NCE) was fixed at 27 V. The complete system was fully controlled by Thermo Scientific™ Xcalibur™ software. Raw data collected were processed and converted with MSConvert into .mgf peak list format. identification for antibody . Identification of peptides was performed by using the search engine MASCOT 2.6.2 algorithm (Matrix Science, London, UK) and Byos® 5.0 software (Protein Metrics, Cupertino, USA). The search was performed against the amino acid sequence of trastuzumab. Spectra were searched with a mass tolerance of 10 ppm for MS and 0.05 Da for MS / MS data. The search was made without enzyme specified for MASCOT search, in order to allow the identification of any non-specific peptide cleavage. For Byos® search, trypsin was specified as enzyme with a maximum of three missed cleavages. Variable modifications were specified: carbamidomethylation of cysteine residues, oxidation of methionine residues and adduct of Ugi (769.431 Da for ik-025-02 and 773.389 for ik-025-05) and Passerini (787.442 Da for ik-025-02 and 791.400 for ik-025-05) payload on lysine, aspartate and glutamate residues. Peptide identifications were validated with a minimal ion score of 25 for Mascot and 300 for Byos.

[0260] Peptides containing Ugi or Passerini payload were validated with the following criteria: i) tryptic peptide (no unspecific cleavage); ii) retention time higher than that of the unmodified peptide; iii) identification of signature fragment ions at m / z 286.172 and 637.343 (characteristic of payload fragmentation); and iv) identification with both search engines Byos® and MASCOT. Example 1. Synthesis of isonitrile compounds according to the invention la. Compound 19

[0261] Compound 19 was synthesized according to the scheme of Figure 1.

[0262] • Synthesis of dolaproine- norephedrine fragment 11

[0263] Diethyl phosphorocyanidate DEPC-mediated amide coupling between Boc-dolaproine and norephedrine afforded the corresponding coupling product in good yield. The coupling product was deprotected by exposition to a solution of HC1 4 N in dioxane at 0 °C, allowing to obtain 11 in quantitative yield, with no need for further purification.

[0264] • Synthesis of MMAE-building block 13

[0265] Cbz-L-isoleucine was first exposed to classical methylation conditions, using sodium hydride and methyl iodide, delivering the TV-methylated product 2, whose carboxylic acid was fully reduced to the corresponding primary alcohol 3, isolated in 87% yield.

[0266] Next, a Parikh-Doering oxidation smoothly led to aldehyde 4 in 85% yield, which was engaged in an aldol addition with tert-butyl acetate in the presence of lithium diisopropylamide LDA. This reaction led to a mixture of the two diastereomers (37?, 45, 55)-5 and (35, 45, 55)-6 in a 55:45 ratio, respectively. 5 was separated by column chromatography, leading to diastereopure 5, isolated in 40% yield. Successive O-methylation of 5 with proton sponge and Meerwein’s salt resulted in product 7 in 70% yield, prior to the removal of the tert-butyl ester, leading to 8 in quantitative yield. The resulting acid was then engaged in the amide coupling with the dolaproine-norephedrine fragment 11, obtained as detailed above, to obtain 12. A final hydrogenolysis of the Cbz protecting group delivered the MMAE precursor 13 in quantitative yield.

[0267] • Synthesis of valine isocyanide 18

[0268] Starting from L-valine 14, methyl ester product 15 was accessed by prolonged exposition to thionyl chloride at room temperature. TV-Terminal formylation was realised by heating 15 in refluxing acetonitrile in the presence of formic acid to deliver formamide 16 in 72% yield. Full conversion to valine isocyanide was obtained by one pot addition of I2, PPhs and N- methylmorpholine NMM in dichloromethane at -20 °C. The purification was accomplished by flash chromatography in isocratic CH2CI2, which led to 17 in 93% yield. Final saponification step with LiOH afforded valine isocyanide lithium carboxylate 18 in 97% yield.

[0269] • Coupling of 13 and 18

[0270] Benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was used for the amide coupling between valine isocyanide 18 and MMAE-precursor 13, which was carried out in dichloromethane in the presence of EtsN. The reaction was monitored by LC-MS. Two diastereoisomers were formed, in identical amounts. Carefully designed purification conditions by preparative RP-HPLC led to both pure 19-DB (17% yield) and 19-DC (18% yield). lb. Compound 21

[0271] Compound 21 ((95)-9-ethyl-5-fluoro-9-hydroxy-l-isocyano-4-methyl-l,2,3,9,12,15- hexahydro- 1 OH, 13 J / -benzo[t / e]pyrano[3 ',4' : 6,7]indolizino[ 1 ,2-b] quinoline- 10,13 -di one, also named Exatecan Isocyanide) was synthesized according to the following steps.

[0272] In a Schlenk tube containing Exatecan mesylate (1.0 eq., 50 mg, 0.11 mmol), sodium chlorodifluoroacetate (3.0 eq., 54 mg, 0.34 mmol) and K2CO3 (3.0 eq., 47 mg, 0.34 mmol) was made inert atmosphere per argon, the system was sealed, and dimethylformamide DMF (1.3 mL) was added prior heating up to 100 °C under vigorous stirring. After 3 h, the reaction mixture was concentrated under reduced pressure and the crude mixture was purified by flash chromatography (CEECh / MeOH 100:0 to 0:20, in 30 column volumes CV, elution 14% MeOH) to afford the compound 21 (10 mg, 0.02 mmol, 19%) as a brown solid.

[0273] Various analytical techniques provided the following characteristics of compound 21:

[0274] Rf: 0.52 (CH2Cl2 / MeOH 95:5).

[0275] 19F NMR (500 MHz, DMSO-6-298 K): 5 -110.44, -110.46 HRMS (ESI+): calc, for C25H21FN3O4+[M+H+] 446.1456, found 446.1530.

[0276] Example 2. Preparation of conjugates according to the invention

[0277] 2a. Coupling of compound 19 or 21 and isobutyraldehyde to trastuzumab

[0278] 1 equivalent (10 mg / mL or 15 mg / mL) trastuzumab was contacted with 45 equivalents of isobutyraldehyde and 45 equivalents of compound 19 as a single diastereoisomer.

[0279] Conjugate Cl was obtained in 100% yield, without any precipitation.

[0280] For a 10 mg / mL concentration of trastuzumab, mass spectra analysis and deconvolution thereof provided an average drug-antibody ratio of 1.9 with diastereoisomer A (probably R configuration) of compound 19, and an average drug-antibody ratio of 2.7 with diastereoisomer B (probably S configuration) of compound 19. The corresponding spectra are provided on figures 2 and 3.

[0281] For a 15 mg / mL concentration of trastuzumab, mass spectra analysis and deconvolution thereof provided an average drug-antibody ratio of 4.0 with diastereoisomer A of compound 19, and an average drug-antibody ratio of 5.1 with diastereoisomer B of compound 19. The corresponding spectra are provided on figures 4 and 5.

[0282] The same coupling reaction of compound 19 and isobutyraldehyde with trastuzumab was implemented with 10 mg / mL trastuzumab, 90 equivalents of isobutyraldehyde and 90 equivalents of each diastereoisomer of compound 19. Conjugate Cl was obtained in 100% yield, without any precipitation.

[0283] Mass spectra analysis and deconvolution thereof provided an average drug-antibody ratio of 6.7 with diastereoisomer A of compound 19, and an average drug-antibody ratio of 7.7 with diastereoisomer B of compound 19. The corresponding spectra are provided on figures 6 and 7.

[0284] Table 1 below presents the different conditions used and the obtained DAR.

[0285] Table 1 Conjugate Cl is thus obtained in one-step, with a quantitative yield, from trastuzumab, isobutyraldehyde, and isonitrile 19.

[0286] The DAR can be easily controlled by adapting the trastuzumab concentration, the number of aldehyde and isonitrile equivalents, and / or the used diastereoisomer of compound 19.

[0287] As for Cl conjugate, compound 21 obtained in example lb was coupled to trastuzumab. 2b. Coupling with different aldehydes

[0288] The process of example 2a was reproduced by replacing isobutyraldehyde with different other aldehydes, and with 10 mg / mL trastuzumab. The obtained yields and average DAR are presented in Table 2 below.

[0289] Table 2 2c. Coupling with a different isonitrile

[0290] The process of example 2a was reproduced by replacing isonitrile 19 with isonitrile 20.

[0291] Conjugate C4 was obtained, with an average DAR of 7.1 for 10 mg / mL trastuzumab, and an average DAR of 12.9 for 15 mg / mL trastuzumab.

[0292] 2d. Coupling of compound 19 and isobutyraldehyde to different antibodies

[0293] Four conjugates (C5-C9), each comprising a different mAb, are obtained according to the steps below. Isobutyraldehyde (100 mM, in dimethylsulfoxide DMSO, 90 eq) and compound 19 (65 mM, in DMSO, 90 eq) were added to a mAb solution (10 mg / mL, in PBS IX, pH 7.4, 1 eq). The reaction mixture was then incubated for 16 h at 25 °C, after which a 50 wt.% solution of hydroxylamine in H2O (10 % in volume) was added. The resulting mixture was then incubated for 1 h at 25 °C, before the excess of reagent was removed by gel filtration chromatography using Biospin P-30 pre-equilibrated with PBS IX, pH 7.4 to give a solution of conjugate.

[0294] Mass spectra analysis and deconvolution thereof provided an average DAR for each of the C5- C9 conjugates. These average DAR are presented in Table 3 below. The corresponding spectra of each of the C5-C9 conjugates are provided on figures 8-12, which demonstrate the efficient coupling of compound 19 and isobutyraldehyde to each mAb.

[0295] Table 3

[0296] Example 3: In vitro toxicity assays

[0297] 3a. Cytotoxicity of conjugate Cl

[0298] Cytotoxicity of conjugate Cl obtained at example 2 was assessed on two different types of cells, SKBR3 cells which are positive to Human Epidermal Growth Factor Receptor 2 (HER2+), and MDA-MB-231 which are negative to Human Epidermal Growth Factor Receptor 2 (HER2-). Cl with diastereoisomer 19A was tested with DAR 1.9 and 6.7. Cl with diastereoisomer 19B was tested with DAR 2.7 and 7.7.

[0299] The results are provided in Figure 13. Conjugate Cl exhibited toxicity on HER2+ cells, and no toxicity on HER2- cells. Low ICso values were obtained. For instance, an ICso of 8 nM was obtained for the conjugate with a DAR of 7.7.

[0300] Comparatively, the cytotoxicity of both diastereoisomers of isonitrile 19 was assessed in the same conditions. Results are presented on Figure 14. Both diastereoisomers of isonitrile 19 are nontoxic up to 10'5M, on both types of cells (HER2+ and HER2-).

[0301] Consequently, the conjugation of non-toxic drug fragments (isonitrile 19) to a protein with a process according to the invention afforded a highly potent antibody-drug conjugate, the toxicity being generated only upon conjugation.

[0302] 3b. Cytotoxicity of conjugate with different antibody

[0303] Conjugate C2 was prepared in the same conditions as those implemented for conjugate Cl, by replacing trastuzumab with ramucirumab.

[0304] Cytotoxicity of diastereoisomer B of conjugate C2 was assessed as detailed above for conjugate Cl. An ICso value of 850 nM on MDA-MB-231 cells and an IC50 value of 390 nM on SKBR3 were obtained. 3c. Cytotoxicity of conjugates with different aldehydes

[0305] Cytotoxicity of the conjugates obtained at example 2b was assessed similarly. Obtained ICso values are presented in Table 4 below.

[0306] Table 4

[0307] 3d. Cytotoxicity of conjugate C4

[0308] Cytotoxicity of conjugate C4 obtained at example 2c was assessed similarly. Obtained ICso values are presented in Table 5 below.

[0309] Table 5

[0310] Example 4 : In vivo studies with xenografted mice

[0311] Conjugate C3 was prepared in the same conditions as those implemented for conjugate Cl, by using 15 mg / mL trastuzumab. An average DAR of 9.6 was obtained for diastereoisomer B of conjugate C3. Diastereoisomer B of conjugate C3 was used for the present assessment.

[0312] In vitro cytotoxicity of diastereoisomer B of conjugate C3 was assessed, providing a 40 nM ICso value for SKBR3 cells, and a 480 nM IC50 value for MDA-MB-231 cells.

[0313] In vitro cytotoxicity of diastereoisomer B of conjugate C3 was assessed in xenografted mice at 4 different doses (10.2 mg / kg, 3.2 mg / kg, 1.4 mg / kg and 0.32 mg / kg) and compared to that of positive control T-mc-VC-PAB-MMAE antibody-drug conjugate (9.9 mg / kg).

[0314] EC50 values of positive control were of 24 pM on SKBR3 cells, and 40 nM on MDA-MB-231 cells.

[0315] Body weight, tumour volume and tumour weight were studied over 20 days. Results are presented in Figure 15. Conjugate C3 exhibited a dose-dependent toxicity effect on the tumour. The effect obtained for the highest dose, 10.2 mg / kg, is of the same order of magnitude as that obtained with the positive control at 9.9 mg / kg.

[0316] Example 5: Peptide mapping

[0317] Peptide mapping was implemented with conjugate Cl as obtained in example 2 with 10 mg / mL trastuzumab and 45 equivalents of isobutyraldehyde and of isonitrile 19, and with a DAR of

[0318] 2.7.

[0319] The analysis of extracted-ion chromatograms evidenced that lysine residues, aspartic acid residues, and glutamic acid residues were implied in the conjugation. Moreover, this analysis also evidenced that N-terminal residues, especially light chain N-terminal aspartic acid residue and heavy chain N-terminal glutamic acid residue, were implied in the conjugation.

Claims

CLAIMS1. Process for preparing a conjugate of a protein and a molecule of interest of formulathe process comprising the step of reacting a protein with an isocyanide compound0(lb) of formulaR-i- NCan(ja carbOnyl compound of formula; wherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom, wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein the isocyanide compound of formulala)R1— NCis an isocyanide derivative of a molecule of interest or a fragment thereof.

2. Process for preparing a conjugate according to claim 1, wherein the step of reacting the protein with the isocyanide compound and the carbonyl compound comprises contacting the protein with the isocyanide compound and the carbonyl compound in conditions suitable for an Ugi multicomponent reaction.

3. Process for preparing a conjugate according to claim 1 or claim 2, wherein the molecule of interest is a drug, a drug analogue or a fragment thereof.

4. Process for preparing a conjugate according to claim 3, wherein the drug is a chemotherapeutic drug and / or a cytotoxic drug.

5. Process for preparing a conjugate according to claim 3 or 4, wherein the drug is selected from the group consisting of auristatins, dolastatins, tubulysins, camptothecins, mertansines, non-ribosomal peptides, antibiotics, nitrogen mustards, taxanes, analogues thereof and derivatives thereof.

6. Process for preparing a conjugate according to claim 5, wherein the drug is an auristatin, preferably selected from the group consisting of monomethyl dolastatin 10 (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and monomethyl soblidotin (MMAPE).

7. Process for preparing a conjugate according to claim 5, wherein the drug is a camptothecin, preferably selected from the group consisting of irinotecan, SN-38, topotecan, deruxtecan, belotecan, 9-aminocamptothecin and exatecan, and more preferably, the drug is exatecan.

8. Process for preparing a conjugate according to any one of claims 1 to 7, wherein the primary amine group of the protein and the carboxylic acid group of the protein are separated by a moiety comprising from 2 to 20 atoms.

9. Process for preparing a conjugate according to any one of claims 1 to 8, wherein the primary amine group of the protein is part of a lysine side-chain or a N-terminal residue, preferably a N-terminal aspartic acid or glutamic acid, and / or the carboxylic acid group of the protein is part of an aspartate or a glutamate amino acid, preferably a N-terminal aspartate or glutamate amino acid, or a C-terminal amino acid, preferably an aspartate or glutamate amino acid side chain.

10. Process for preparing a conjugate according to any one of claims 1 to 9, wherein the primary amine group of the protein and the carboxylic acid group of the protein are part of a single amino acid or of two different amino acids.

11. Process for preparing a conjugate according to any one of claims 1 to 10, wherein the protein is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof, in particular trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab, sacituzumab, gemtuzumab, cetuximab, a variant thereof or a fragment thereof.

12. Process for preparing a conjugate according to any one of claims 1 to 11, wherein one of R2and R3 is a hydrogen atom, and the other of R2 and R3 is an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkylaryl group, preferably selected from the group consisting of amethyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group.

13. Process for preparing a conjugate according to any one of claims 1 to 6 and 8-12, wherein the isocyanide compound is a compound of formula, or a diastereoisomer thereof, wherein R’ is a C1-C7 alkyl group or arylalkyl group, wherein the alkyl and arylalkyl groups may be terminated and / or interrupted by at least one heteroatom, wherein R4 is a terminal group preferably selected from the group consisting of (+)-norephedrine, (S)- dolaphenine, L-phenylalanine and phenethylamine and wherein R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L-phenylalanine or phenethylamine.

14. Process for preparing a conjugate according to claim 13, wherein R4 is (+)- norephedrine and / or R’ is an ethyl or an isopropyl group.

15. Process for preparing a conjugate according to any one of claims 1 to 14, wherein the step of reacting the protein with the isocyanide compound and the carbonyl compound is the only step of the process.

16. Process for preparing a conjugate according to any one of claims 1 to 15, wherein the toxicity of the conjugate of formula (I) is higher than a first threshold, and the toxicity of the isocyanide compound and of the carbonyl compound is lower than a second threshold, the second threshold being lower than the first threshold.

17. Conjugate of a protein and a molecule of interest of formulawherein Ri is an organic moiety different from a hydrogen atom, wherein each of R2 and R3 is independently an organic moiety or a hydrogen atom,wherein the N and C=O moieties which are covalently linked to the remainder of the protein in formula (I) respectively originate from a primary amine group of the protein and a carboxylic acid group of the protein, and wherein Ri is a molecule of interest or a fragment thereof.

18. Conjugate according to claim 17, wherein one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 is selected from the group consisting of a methyl group, an ethyl group, a tert-butyl group, an isopropyl group, and a benzyl group, and whereinwherein R’ is an alkyl group, wherein R4 is a terminal group preferably selected from the group consisting of (+)- norephedrine, (S)-dolaphenine, L-phenylalanine and phenethylamine and R4 is bonded to the remainder of the molecule through the primary amine group of (+)- norephedrine, (S)-dolaphenine, L-phenylalanine or phenethylamine, and whereinrepresents the position of the bond to the nitrogen atom of the remainder of the protein conjugate of formula (I).

19. Conjugate according to claim 18, wherein R4 is (+)-norephedrine and / or R’ is an ethyl or an isopropyl group.

20. Conjugate according to any one of claims 17 to 19, wherein the protein is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof, in particular trastuzumab, bevacizumab, ramucirumab, daratumumab, rituximab, sacituzumab, gemtuzumab, cetuximab or a fragment thereof.

21. Conjugate according to any one of claims 17 to 20, wherein the molecule of interestprotein ratio is comprised between 1 and 20, preferably between 1 and 10.

22. Conjugate obtained with a process according to any one of claims 1 to 16, or conjugate according to any one of claims 17 to 21, for use as a medicament.

23. Conjugate obtained with a process according to any one of claims 1 to 16, or protein conjugate according to any one of claims 17 to 21, for use in the treatment of a proliferative disease such as a cancer.

24. Isocyanide compound of formuladiastereoisomer thereof, wherein R’ is an alkyl group, wherein R4 is a terminal group preferably selected from the group consisting of (+)-norephedrine, (S)-dolaphenine, L-phenylalanine and phenethylamine and wherein R4 is bonded to the remainder of the molecule through the primary amine group of (+)-norephedrine, (S)-dolaphenine, L-phenylalanine or phenethylamine.

25. Process for preparing an antibody-drug conjugate by a one-step four-centre three- component Ugi reaction, wherein the active pharmaceutical ingredient of the drug is an isocyanide derivative thereof or an isocyanide fragment thereof.

Images