Antibody-drug conjugates of vincristine, processes for preparing the same and uses thereof

Abstract

The present invention relates to new vincristine antibody-drug conjugates and to their use in the treatment of proliferative diseases.

Description

[0001] ANTIBODY-DRUG CONJUGATES OF VINCRISTINE, PROCESSES FOR PREPARING THE SAME AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] The Invention relates to the development of antibody-drug conjugates (ADCs) of vincristine and to their use for the treatment of proliferative diseases, in particular aggressive cancers.

[0004] TECHNICAL BACKGROUND

[0005] The treatment of proliferative diseases, such as cancers, represents nowadays a major social issue. Numerous treatments have been developed until today, however most treatments are specific of one type of cancer, and their efficacy is not always satisfying despite harsh treatment conditions and numerous side effects. There thus remains a need for developing new efficient methods for treating proliferative diseases, such as cancers.

[0006] One problem of cancer treatment is to achieve a localized delivery of drugs to target cells tissues and tumors to achieve maximal efficacy and minimal toxicity. In this regard, macromolecules composed of an anticancer agent conjugated to an antibody targeting a specific antigen, called antibody-drug conjugates (ADCs), have been developed and proven to be efficient so far for the treatment of some cancers.

[0007] Antibody-drug conjugates (ADCs) were developed to selectively deliver highly potent cytotoxic compounds into cancer cells without causing damage to healthy cells. Thus, loaded on ADCs, DNA-binding agents such as calicheamicins and microtubule polymerization inhibitors including auristatin or maytansinoids, with potency in the picomolar to tens of picomolar range, otherwise unsuitable for systemic delivery, were applied in cancer therapy. However, while the ADC approach increases the therapeutic index of these compounds, one of its main limitations is premature payload deconjugation, mainly due to chemical instability of the linker, leading to off-target, off-tumor cytotoxicity. Consequently, many ADCs are associated with severe adverse effects, such as neutropenia, thrombocytopenia and hepato-or cardiotoxicity.

[0008] Among other developments in the field of ADCs, recent advances in linker design allowed to consider the conjugation of higher amounts of less toxic payloads, such as some Topoisomerase I inhibitors, on the same antibody moiety. Indeed, the addition of chemical functions such as polyethylene glycol (PEG) or sulfonate moieties to obtain more hydrophilic linkers allows to counterbalance hydrophobicity of payloads and synthesize ADCs with higher drug-to-antibody ratios (DAR) and lower risk of aggregation. These developments increase the stability and pharmacokinetics of ADCs but also lower their immunogenicity. Recently, two ADCs with high DARs (DAR ~ 8), Sacituzumab govitecan (Trodelvy®), an anti-TROP2 antibody conjugated to the topoisomerase I inhibitor SN38 and indicated in the treatment of triplenegative breast cancer (TNBC), and Trastuzumab deruxtecan (Dxd, topoisomerase I inhibitor) (Enhertu®) for HER2 positive (HER2+) cancers, have been marketed.

[0009] Finally, trastuzumab emtansine (Kadcyla®), an anti-HER2 antibody conjugated to the cytotoxic agent DM1, is indicated in the treatment of HER2 positive breast cancer.

[0010] These ADCs involve payloads such as SN38, Dxd and DM1, whose cytoxicity is quite high in their free form. Even if their conjugation to an antibody may lower their toxicity, their free form remains toxic in case of deconjugation. There is thus a risk of systemic toxicity.

[0011] It would thus be useful to develop potent ADCs incorporating payloads that are less toxic in their free form, in order to limit the risks of systemic toxicity, especially in case of deconjugation. Advantageously, the ADC toxicity should be specific to the targeted cells.

[0012] SUMMARY OF THE INVENTION

[0013] In this respect, the Inventors have evidenced that specific vincristine ADCs exhibit a higher toxicity than that of free vincristine, and a toxicity at least as high as that of reference ADCs such as Enhertu®, Trodelvy® or Kadcyla®, in specific cell lines. Such result is unexpected because the toxicity of free vincristine is known to be far lower than that of the free payloads of these ADCs (Dxd, SN-38, and DM1, respectively). Furthermore, the toxicity of the developed ADCs is specific to the targeted cells.

[0014] A first object of the present invention is thus an ADC of formula (Z) or a pharmaceutically acceptable salt thereof wherein - MAB is a monoclonal antibody comprising at least one, preferably at least four, cysteine residues and which targets at least one protein which is expressed in a cancer cell,

[0015] - LINK is a cleavable linker which is covalently bound to MAB via the sulphur atom of one of its cysteine residues, and

[0016] - n is comprised between 1 and 8.

[0017] In some embodiments, the ADC is of formula (A), or a pharmaceutically acceptable salt thereof: wherein MAB, LINK and n are as defined above.

[0018] In some embodiments, the cleavable linker LINK is of formula (XI): wherein

[0019] - Y is a moiety bound to the sulphur atom of a cysteine residue of MAB, preferably Y is a maleimide moiety;

[0020] - X is a saturated or unsaturated, linear, branched or cyclic, C1-C20 hydrocarbon chain, optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NR-, -C(=O)-, -O(C=O)-, -C(=O)O-, -NR(C=O)-, -C(=O)NR-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, - NRIC(=0)NR2-, and / or one or more aryl and / or heteroaryl groups optionally substituted by one or more functional groups selected from the group consisting of - OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain, - PP is a polypeptide chain comprising 2 to 10 amino-acids, and

[0021] - Ar is an aryl or a heteroaryl group optionally substituted by one or more functional groups selected from the group consisting of -OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain.

[0022] In some embodiments,

[0023] - Y is a maleimide moiety,

[0024] - X is a saturated C1-C20 hydrocarbon chain, optionally interrupted by -O-, -S-, -SO-, - SO2-, -NR-, -C(=O)-, -O(C=O)-, -C(=O)O-, -NR(C=O)-, -C(=O)NR-, or an unsubstituted phenyl group, where R is H or a C1-C3 alkyl chain,

[0025] - PP is a polypeptide chain of 2 to 10 amino acids consisting of proteinogenic amino acids and / or biologically active amino acids, and

[0026] - Ar is an unsubstituted phenyl group.

[0027] In some embodiments, each amino acid of the polypeptide PP is independently selected from the group consisting of Glycine, Alanine, Valine, Glutamic acid, Phenylalanine and Citrulline.

[0028] In some embodiments, the polypeptide PP is selected from the group consisting of Val-Cit, Vai-Ala, Ala-Ala and Gly-Gly-Phe-Gly, preferably the polypeptide PP is Val-Cit.

[0029] In some embodiments, X is a saturated C1-C20 hydrocarbon chain, preferably a n-pentylene chain.

[0030] In some embodiments, Ar is an unsubstituted phenylene, preferably an unsubstituted 1,4- phenylene.

[0031] In some embodiments, MAB is an antibody which targets an antigen selected from the group consisting of TROP-2, HER-2, HER-3, DLL-3, CD20 and PD-L1, preferably TROP-2 or HER- 2.

[0032] In some embodiments, MAB is an antibody selected from the group consisting of Sacituzumab, Trastuzumab, Avelumab and Durvalumab, preferably Sacituzumab or Trastuzumab.

[0033] In some embodiments, the ADC is of formula (C):

[0034] wherein MAB is Sacituzumab or Trastuzumab and wherein n is comprised between 6 and 8, preferably n is about 8.

[0035] Another object of the invention is a process for preparing an antibody-drug conjugate as defined in the present invention, which comprises the following steps:

[0036] (A) reacting a compound of formula (I) with vincristine, or a salt of vincristine, in presence of a carbonate salt, preferably potassium carbonate; wherein Ar, X and PP are as defined above, so as to produce a compound of formula (II), wherein X, PP and Ar are as defined above;

[0037] (B) at least partially, preferably totally, reducing the disulfide bridges between the cysteine residues of a monoclonal antibody MAB as defined above, preferably by incubating said antibody in presence of a reducing agent, such as tris(2- carboxyethyl)phosphine (TCEP), supported TCEP or Dithiothreitol (DTT), so as to produce an at least partially, preferably totally, reduced antibody, and

[0038] (C) coupling compound (II) obtained in step (A) to the at least partially, preferably totally, reduced antibody obtained in step (B), so as to produce an antibody-drug conjugate as defined above.

[0039] Another object of the invention is a pharmaceutical composition comprising an ADC according to the invention and a pharmaceutically acceptable excipient.

[0040] A last object of the invention is an ADC according to the invention for use in the treatment of a proliferative disease such as a cancer.

[0041] In some embodiments, the cancer is a cancer overexpressing TROP2 or HER2, preferably selected from the group consisting of a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a non-small cell lung cancer, a lung adenocarcinoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a prostate cancer, a bladder cancer, a gallbladder cancer, a cervical cancer, an uterine serous papillary carcinoma, an endometrial cancer, a nasopharyngeal cancer, a hilar cholangiocarcinoma, an oral squamous cell carcinoma, an oesophageal squamous cell carcinoma, a head-and-neck squamous cell carcinoma, and a laryngeal squamous cell carcinoma, preferably a breast cancer, more preferably a triple negative breast cancer (TNBC).

[0042] FIGURES

[0043] Figure 1: In vitro cytotoxic effect of trastuzumab -vincristine (T-vincri stine; av. DAR = 7.8), rituximab -vincristine (R- vincristine; av. DAR = 7.4), vincristine alone and trastuzumab alone on different HER2+and HER2very lowcancer cell lines. (A-D): Cells were seeded at an appropriate density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 120 h later using the WST-1 cell proliferation assay. Graphs represent mean values ± SEM from at least three independent experiments and are expressed as the percentage of living cells relatively to untreated SKBR3 (A), BT474 (B), NCI-N87 (C) and MDA-MB-231 (D) cells. (E): Table recapitulating IC50 values ± SEM of vincristine and T-vincristine for the different cell lines. Curve-fits and IC50 values were obtained using the GraphPad Prism software. NA: not applicable. Figure 2: In vitro cytotoxic effect of trastuzumab -vincristine (T-vincri stine; av. DAR = 7.8) on HER2+and HER2very lowcancer cell lines in comparison with standard trastuzumab-based ADCs T-DM1 (DAR = 3.5) and T-MMAE (DAR = 8). (A-B): Cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 72 h later using the WST-1 cell proliferation assay. Graphs represent mean values ± SEM from at least three independent experiments and are expressed as the percentage of living cells relatively to untreated SKBR3 (A) and MDA-MB-231 cells (B). (C): Table recapitulating IC50 values ± SEM of vincristine, T-vincristine, T-MMAE and T-DM1 for both cell lines. Curve-fits and IC50 values were obtained using the GraphPad Prism software. NA: not applicable.

[0044] Figure 3: In vivo antitumoral activity of trastuzumab -vincristine (T-vincristine; av. DAR = 7.8) conjugate in comparison with standard trastuzumab-based ADCs T-DM1 (DAR = 3.5) (Kadcyla®) and trastuzumab-deruxtecan T-Dxd (DAR = 7.8) (Enhertu®). (A-C): NOD-SCID mice with subcutaneous heterotopic SKBR3 tumor cell xenografts were subjected to a unique intravenous injection of either PBS IX (Vehicle) or 5 mg. kg'1of T-vincristine, T-Dxd (Enhertu®), or T-DM1 (Kadcyla®). (A): tumor volume was measured every 2-3 days for 60 days. Experimental results are expressed as means of the percentage of remaining tumor volume ± SEM (n = 5 for T-DM1, n = 6 for all other groups). (B): Percentage of mice without complete tumor regression post-injection is represented as a function of time. Statistical analysis of differences between groups was performed using a Log-rank (Mantel-Cox) test with GraphPad Prism 7.0. Statistically significant differences were observed between T-vincristine and T-Dxd (p value 0.0235 = < 0.05 (*)) and T-vincristine and T-DM1 (p value 0.0087 = < 0.01 (**)). (C): Percentage of surviving mice post-injection is represented as a function of time. Statistically significant differences were observed between groups (Log-rank (Mantel-Cox) test, p value 0.0165 = < 0.05 (*)).

[0045] Figure 4 (A-C): Binding assays of sacituzumab-FITC (A), sacituzumab-vinscri stine (S- vincristine)-FITC (B) and Trodelvy® (sacituzumab-SN38 or S-SN38)-FITC (C) in HCC 1937 BRCA1 restored (TROP2+), MDA-MB-231 (TROP2low) and MDA-MB-436 (TROP2very low) cells. The percentage of FITC-positive cells is represented relative to the concentration of each compound (n=3, mean ± SEM). (D): Binding assays of sacituzumab-FITC, S-vinscristine- FITC and S-SN38-FITC (Trodelvy®-FITC) in the HCC 1937 BRCA1 restored cell line. Results are presented as in (A-C). (n=3, mean ± SEM). Figure 5: In vitro cytotoxic effect of sacituzumab -vincristine S-vincristine (av. DAR = 7.8) on TROP2+, TROP2lowand TROP2very lowcancer cell lines in comparison with vincristine alone, SN38 alone, sacituzumab alone and sacituzumab-SN38 (S-SN38; DAR = 7.8; Trodelvy®) ADC. (A-D): Cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 72 h later using the WST-1 cell proliferation assay. Graphs represent mean values ± SEM from at least three independent experiments and are expressed as the percentage of living cells relatively to untreated HCC 1937 BRCA restored (A), MDA-MB- 468 (B), MDA-MB-231 (C) and MDA-MB-436 (D) cells. (E): Table recapitulating IC50 values ± SEM of vincristine, S-vincristine, Trodelvy® (S-SN38) and SN38 for cell lines included in this study. Curve-fits and IC50 values were obtained using the GraphPad Prism software. NA: not applicable.

[0046] Figure 6: In vitro cytotoxic effect of trastuzumab -vincristine (T-vincri stine; av. DAR = 7.8), trastuzumab-vinblastine (T-vinblastine av. DAR = 8), trastuzumab -vinorelbine (T-vinorelbine av. DAR = 6), vincristine alone, vinblastine alone and vinorelbine alone on SKBR3 (HER2+) and MDA-MB-231 (HER2veiy low) cancer cells. Cells were seeded at an appropriate density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 120 h later using the WST-1 cell proliferation assay. Graphs represent mean values ± SEM from three independent experiments and are expressed as the percentage of living cells relatively to untreated SKBR3 (A) and MDA-MB-231 (B) cells. (C): Table recapitulating IC50 values ± SEM of vincristine, T-vincri stine, vinblastine, T-vinblastine, vinorelbine and T-vinorelbine for the different cell lines. Curve-fits and IC50 values were obtained using the GraphPad Prism software.

[0047] Figure 7 In vitro cytotoxic effect of trastuzumab-vincristine ADCs comprising different linkers (Val-Cit, Vai-Ala, Ala-Ala and Gly-Gly-Phe-Gly polypeptide moiety) on SKBR3 (HER2+) and MDA-MB-23 1 (HER2very low) cancer cells.

[0048] Figure 8 In vitro cytotoxic effect of ADCs according to the invention (T-Val-Cit-PAB-VCR+(DAR4) and (DAR8)) in comparison with corresponding ADCs comprising a carbonate linker (T-Val-Cit-PABC-VCR (DAR4) and (DAR8)) on SKBR3 (HER2+) and MDA-MB-231 (HER2very low) cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0049] The Inventors have conceived a new family of antibody-drug conjugates (ADCs) of vincristine, which has a higher biological activity than free vincristine or other reference ADCs, such as Sacituzumab govitecan (Trodelvy®). This new family of antibody-vincristine conjugates features an enzymatically cleavable linker binding the antibody to vincristine. This cleavable linker allows the ADC of the Invention to present an improved therapeutic index compared to free vincristine and other ADCs. The linker of the ADC of the Invention preferably features a peptide carefully selected to be cleaved in conditions which can be met in cancer cells, in order to release the vincristine inside the cancer cell.

[0050] The toxicity of the ADCs of the invention is furthermore specific to the targeted cells.

[0051] Accordingly, the Invention relates to an ADC of formula (Z) or a pharmaceutically acceptable salt thereof:

[0052] [MAB] — S LINK] — [vincristine] wherein

[0053] - MAB is a monoclonal antibody comprising at least one, preferably at least four, cysteine residues and which targets at least one protein which is expressed in a cancer cell,

[0054] - LINK is a cleavable linker which is covalently bound to MAB via the sulphur atom of one of its cysteine residues, and

[0055] - n is comprised between 1 and 8.

[0056] In some embodiments, the antibody-drug conjugate is of formula (A): wherein MAB, LINK and n are as defined above. In the present inventions, the terms “ADC” and “conjugate ” are used indifferently.

[0057] In the present invention, the term "vincristine" also encompasses analogues of vincristine, such as those disclosed in Boger et al. Acc. Chem. Res. 2015, 48, 653-662. Such analogues may be modified at position 10’, for instance with a fluorine atom; at position 20’, for instance with the corresponding diastereoisomer or with H, an amide group NH-COR, an urea group NH- CO-NHR, a thiourea group NH-CS-NHR or a carbamate group NH-CO-OR; at position 5, for instance with H, an alkyl group, an alkene group, or a aldehyde; and / or at position 16’, for instance with a different ester group, a nitrile group, a primary alcohol group or an amide group. In some embodiments, vincristine is not modified.

[0058] Monoclonal antibody (MAB)

[0059] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, z.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, analogues and biosimilar compounds) of antibodies and antibody fragments. Preferably, the antibody according to the invention is a monoclonal antibody (e.g. a chimeric, humanized or human antibody), or a fragment of a 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.

[0060] Antibody fragments that recognize 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.

[0061] Antibodies 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.

[0062] The antibody 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. Preferably, the antibody according to the invention is an immunoglobulin G IgG, preferably of subtype IgGl or IgG4.

[0063] As used herein, “ monoclonal antibodies’" refer to antibodies that may be produced by a single clone of B-cells and bind to the same epitope. Monoclonal antibodies may be recombinant monoclonal antibodies.

[0064] The monoclonal antibody targets at least one protein which is expressed in a cancer cell, preferably at least one protein expressed in the target cancer cell. Preferably, the monoclonal is at least bivalent. In some embodiments, the monoclonal antibody is hetero-bivalent.

[0065] In some embodiments, the monoclonal antibody targets an antigen selected from the group consisting of TROP-2, HER-2, HER-3, DLL-3, CD20 and PD-L1, preferably from the group consisting of TROP-2, HER-2, CD20 and PD-L1. In a preferred embodiment, the monoclonal antibody targets TROP-2 and / or HER-2.

[0066] In some embodiments, the monoclonal antibody is selected from the group consisting of Sacituzumab, Trastuzumab, Rituximab, Avelumab, Durvalumab, Datopotamab, Zenocutuzumab, Zanidatamab, Margetuximab, Pertuzumab, Odronextamab, Epcoritamab, Glofitamab, Ublituximab, Mosunetuzumab, Ocrelizumab, Obinutuzumab, Ofatumumab, Tositumomab, Ibritumomab, Sugemalimab, Cosibelimab, Atezolizumab, Rovalpituzumab, Tarlatamab, SC-002, BI 764532, AMG 119, ZG006, Patritumab, Lumretuzumab, Seribantumab and GSK2849330.

[0067] In some embodiments, the monoclonal antibody is selected from the group consisting of Sacituzumab, Trastuzumab, Rituximab, Avelumab, Durvalumab, Datopotamab, Zenocutuzumab, Zanidatamab, Margetuximab, Pertuzumab, Odronextamab, Epcoritamab, Glofitamab, Ublituximab, Mosunetuzumab, Ocrelizumab, Obinutuzumab, Ofatumumab, Tositumomab, Ibritumomab, Sugemalimab, Cosibelimab and Atezolizumab. In a preferred embodiment, the monoclonal antibody is selected from the group consisting of Sacituzumab, Trastuzumab, Avelumab and Durvalumab. In a more preferred embodiment, the monoclonal antibody is selected from the group consisting of Sacituzumab and Trastuzumab.

[0068] TROP-2 refers to trophoblast cell-surface antigen 2, a membrane glycoprotein known to be implied in different cancers. In some embodiments, the monoclonal antibody targets TROP-2 and is preferably selected from the group consisting of Sacituzumab and Datopotamab, preferably Sacituzumab. HER-2 refers to Human Epidermal Growth Factor Receptor-2, a membrane receptor known to be implied in different cancers. In some embodiments, the monoclonal antibody targets HER- 2 and is preferably selected from the group consisting of Trastuzumab, Zenocutuzumab, Zanidatamab, Margetuximab and Pertuzumab, preferably Trastuzumab.

[0069] CD20 refers to B-lymphocyte antigen CD20, which is expressed on the surface of all B-cells. In some embodiments, the monoclonal antibody targets CD20 and is preferably selected from the group consisting of Odronextamab, Epcoritamab, Glofitamab, Ublituximab, Mosunetuzumab, Ocrelizumab, Obinutuzumab, Ofatumumab, Tositumomab and Ibritumomab.

[0070] PD-L1 refers to Programmed death-ligand 1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and which is a 40kDa type 1 transmembrane protein. In some embodiments, the monoclonal antibody targets PD-L1 and is preferably selected from the group consisting of Sugemalimab, Cosibelimab and Atezolizumab.

[0071] DLL3 refers to Delta-like ligand 3, a member of the Notch ligand family, which is an atypical inhibitory ligand predominantly localized in the Golgi apparatus of normal cells but aberrantly expressed on the cell surface of certain tumor cells. In some embodiments, the monoclonal antibody targets DLL3 and is preferably selected from the group consisting of Rovalpituzumab, Tarlatamab, SC-002, BI 764532, AMG 119 and ZG006.

[0072] HER3 refers to Human epidermal growth factor receptor 3, also known as ERBB3, which is a member of the epidermal growth factor receptor (EGFR / ERBB) family of receptor tyrosine kinases. HER3 lacks intrinsic kinase activity but plays a critical role in heterodimerization with other ERBB receptors to activate downstream signaling pathways, such as PI3K / AKT. In some embodiments, the monoclonal antibody targets HER3 and is preferably selected from the group consisting of Patritumab, Lumretuzumab, Seribantumab, and GSK2849330.

[0073] Cleavable Linker (LINK)

[0074] The cleavable linker is a chemical group which links the monoclonal antibody and the vincristine together, and which breaks under specific biological or chemical conditions.

[0075] As showed by the Applicant, the use of a cleavable linker to bind vincristine to a monoclonal antibody MAB allows to obtain an ADC with a higher biological activity, compared to the use of a non-cleavable linker. The cleavable linker LINK is covalently bound to the monoclonal antibody MAB via a sulphur atom of one of its cysteine residues, and to the vincristine preferably via a quaternary amine function. Preferably, the cleavable linker LINK is bound to the tertiary amine of the catharanthine subunit of vincristine. In some other embodiments, the cleavable linker LINK may be bound to the other tertiary amine, to the indole ring and / or to a tertiary alcohol of vincristine.

[0076] Typically, the precursor used to link the cleavable linker to the MAB and to vincristine may comprise at one end a reactive group allowing to form a covalent bond with the sulphur atom of a cysteine residue of the monoclonal antibody MAB, such as a maleimide moiety or any other thio-selective compound, such as 3-arylpropiolonitriles (APN) or rebridging agents, and at the other end a reactive group allowing to form a quaternary amine with an amine function of the vincristine, such as a halogen or hydroxy group.

[0077] In some embodiments, the cleavable linker is an enzymatically cleavable linker. In a preferred embodiment, the cleavable linker is cleaved by cytoplasmic peptidases. In a more preferred embodiment, the cleavable linker is cleaved by a beta-galactosidase or a cathepsin, preferably a cathepsin, such as cathepsin B.

[0078] In other embodiments, the cleavable linker is a physically and / or chemically cleavable linker. Such linkers may be cleaved by stimuli such as reductive conditions, pH, temperature, the presence of a specific compound, light of a certain wavelength, etc.

[0079] In some embodiments, the cleavable linker comprises a polypeptide moiety, preferably selected from the group consisting of a Val-Cit moiety, a Vai-Ala moiety, an Ala- Ala moiety and a Gly- Gly-Phe-Gly moiety, more preferably the polypeptide moiety is a Val-Cit moiety.

[0080] In some embodiments, the cleavable linker is of formula (XI) wherein

[0081] - Y, present or absent, is a moiety covalently bound to the sulphur atom of a cysteine residue of the MAB, preferably Y is a maleimide moiety,

[0082] - the Ar-CH2- moiety, preferably a benzylene moiety, more preferably a 1,4-benzylene or p- benzylene moiety, is covalently bound to a nitrogen atom of vincristine, - X is a saturated or unsaturated, linear, branched or cyclic, C1-C20 hydrocarbon chain, optionally interrupted by one or more -O-, -S-, -SO-, -SO2- -NR-, -C(=O)-, -O(C=O)-, - C(=O)O-, -NR(C=O)-, -C(=O)NR-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, - NRIC(=O)NR2-, and / or one or more aryl and / or heteroaryl groups optionally substituted by one or more functional groups selected from the group consisting of -OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C 1 -C3 alkyl chain,

[0083] - PP is a polypeptide chain comprising 2 to 10 amino acids, and

[0084] - Ar is an aryl or a heteroaryl group optionally substituted by one or more functional groups selected from the group consisting of -OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain.

[0085] The wavy lines represent the positions where the linker is bound to the remainder of the ADC.

[0086] In some embodiments, X is a saturated C1-C20 hydrocarbon chain, optionally interrupted by -O-, -S-, -SO-, -SO2-, -NR-, -C(=O)-, -O(C=O)-, -C(=O)O-, -NR(C=O)-, -C(=O)NR-, or an unsubstituted phenyl group, where R is H or a C1-C3 alkyl chain.

[0087] In a preferred embodiment, X is a C1-C20 linear alkyl chain, optionally interrupted by one or more -O-, preferably a C2-C10 linear alkyl chain, optionally interrupted by one or more -O-, more preferably a C4-C6 linear alkyl chain, optionally interrupted by one or more -O-.

[0088] In a particular embodiment, X is a polyethylene glycol chain comprising 2 to 20 carbon atoms or a polypropylene glycol chain comprising 3 to 18 carbon atoms.

[0089] In a preferred embodiment, X is a C5 linear alkyl chain, ie a n-pentylene chain.

[0090] The terms mentioned herein with prefixes such as for example C1-C20 or C2-C10 can also be used with lower numbers of carbon atoms such as C1-C2, C1-C5, or C2-C5. If, for example, the term C1-C3 is used, it means that the corresponding hydrocarbon chain may comprise from 1 to 3 carbon atoms, especially 1, 2 or 3 carbon atoms. If, for example, the term C2-C6 is used, it means that the corresponding hydrocarbon chain may comprise from 2 to 6 carbon atoms, especially 2, 3, 4, 5 or 6 carbon atoms.

[0091] The term “alkyl” refers to a saturated, linear or branched aliphatic group. The term “(Cl- C5)alkyl” more specifically means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or pentyl.

[0092] The term “aryl” corresponds to a mono- or bi-cyclic aromatic hydrocarbon having from 5 to 14 carbon atoms. Preferably, the term “aryl” refers to phenyl, biphenyl, or naphthalenyl. The term “heteroaryF as used herein corresponds to an aromatic, mono- or poly-cyclic group comprising between 5 and 14 atoms and comprising one or more heteroatoms, such as nitrogen (N), oxygen (O) or sulphur (S) atom. Examples of such mono- and poly-cyclic heteroaryl groups may be: pyridinyl, thiazolyl, thiophenyl, furanyl, pyrrolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthal enyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, triazinyl, thiadiazolyl, thianthrenyl, isobenzofuranyl, phenoxanthinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, phtalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, P- carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, benzotriazolyl, benzoxazolyl, benzisoxazolyl, oxindolyl, benzothienyl, benzothiazolyl, s-triazinyl, oxazolyl, or thiofuranyl.

[0093] The term “halogen” corresponds to a fluorine, chlorine, bromine, or iodine atom, preferably a fluorine, chlorine or bromine atom.

[0094] The term “heteroatom” refers to a non-metallic atom, different from carbon and hydrogen, and having at least one pair of electrons. Preferred heteroatoms are N, O, S, Se, Si, and P.

[0095] The expression “substituted by at least one” or “comprising at least one” means that the radical or group is substituted by or comprises one, two, three or several groups of the list, preferably one or two groups of the list.

[0096] In some embodiments, PP is a polypeptide chain of 2 to 8 amino acids, preferably 2 to 5 amino acids, in particular 2 to 4 amino acids.

[0097] An "amino acid" is a molecule comprising at least one carboxylic acid function (COOH) and one amine function (NH2), and at least one carbon atom linking this carboxylic acid function and this amine function. Amino acids include the 22 proteinogenic a-amino acids, but also non- proteinogenic a-amino acids, such as citrulline, preferably L-citrulline, and P and y- amino acids.

[0098] A “proteinogenic amino acid' is an amino acid that is incorporated biosynthetically into proteins during translation. The word "proteinogenic" means "protein creating" . There are 22 genetically encoded (proteinogenic) amino acids, 20 in the standard genetic code and an additional 2 (selenocysteine and pyrrolysine) that can be incorporated by special translation mechanisms. Proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine and pyrrolysine.

[0099] In some embodiments, each amino acid is L or D, preferably L

[0100] In a preferred embodiment, PP is a polypeptide chain comprising at least one amino acid selected from the group consisting of Glycine, Alanine, Valine, Glutamic acid, Phenylalanine and Citrulline.

[0101] In an embodiment, PP is a polypeptide chain consisting of amino acids selected from the group consisting of Glycine, Alanine, Valine, Glutamic acid, Phenylalanine and Citrulline.

[0102] In a particular embodiment, PP is a polypeptide selected from the group consisting of Val-Cit, Vai-Ala and Gly-Gly-Phe-Gly.

[0103] In some embodiments, Ar is a 6-membered aryl or heteroaryl group, preferably a phenyl group, optionally substituted by one or more functional groups selected from the group consisting of -OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain. In a preferred embodiment, Ar is an unsubstituted phenyl group, preferably positioned so that the moieties situated on both sides of the phenyl group in the ADC structure are in para position towards each other.

[0104] In some embodiments, -NH-Ar- is a / / ra-aminobenzene moiety.

[0105] In some embodiments, the cleavable linker is of formula (X2): wherein X and Ar are as defined above.

[0106] In a preferred embodiment, the linker is of formula (X3): wherein X is as defined above.

[0107] In a particular embodiment, the linker is of formula (X4): Antibody-Drug Conjugate (ADC)

[0108] As demonstrated by the Applicant, the biological activity of the ADCs according to the Invention is unexpectedly higher than that of free vincristine.

[0109] In some embodiments, the ADC is of formula (X5) wherein

[0110] - MAB, X, PP and Ar are as defined above,

[0111] - n is comprised between 1 and 8, preferably between 2 and 8, more preferably between 4 and 8.

[0112] In some embodiments, the ADC is of formula (X6):

[0113] wherein

[0114] - MAB, X and Ar are as defined above,

[0115] - n is comprised between 1 and 8, preferably between 2 and 8, more preferably between 4 and 8.

[0116] In a preferred embodiment, the ADC of the Invention is of formula (X7): wherein

[0117] - MAB is as defined above, - X is a C1-C20 linear alkyl chain, optionally interrupted by one or more -O-, preferably, X is a C5 linear alkyl chain, and

[0118] - n is comprised between 1 and 8, preferably between 2 and 8, more preferably between 4 and

[0119] 8. In the context of the invention, a “pharmaceutically acceptable salt" refers to a salt of a conjugate according to the invention, and of an alkali metal, of an alkaline-earth metal, or of ammonium, comprising the salts obtained with organic ammonium bases, or salts of a conjugate according to the invention, and of an organic or inorganic acid.

[0120] Salts which are more particularly suitable for the invention may be sodium, potassium, calcium, magnesium salts, quaternary ammonium salts such as tetramethylammonium or tetraethylammonium, and addition salts with ammonia and pharmaceutically acceptable organic amines, such as methylamine, dimethylamine, trimethylamine, ethylamine, triethylamine, ethanolamine or / / 7.s(2-hydroxyethyl)amine.

[0121] Salts of a conjugate according to the invention, and of an inorganic acid that are suitable for the invention may be obtained with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid.

[0122] Salts of a conjugate and of an organic acid that are suitable for the invention may be obtained with carboxylic acids and sulfonic acids, such as formic acid, acetic acid, oxalic acid, citric acid, lactic acid, malic acid, succinic acid, malonic acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid or / ?-toluenesulfonic acid.

[0123] Drug-Antibody Ratio

[0124] The drug-antibody ratio (DAR), which corresponds to n in the above formulae, is the average drug to antibody ratio. Methods for determining the average DAR (avDAR) are known in the art. For instance, avDAR may be measured according to the following equation:

[0125] DAR= (E(8)(fc=0)(fcx intensity DARfc) / E(8)(fc=0)intensity DARfc.

[0126] The intensity corresponds to the area under the curve of the peaks for each measured specie in the sample deconvoluted mass spectrum. The method was reported in publication Debaene et al. Anal Chem 2014, 86, 21, 10674-10683.

[0127] In the present invention, n is at least 1, preferably at least 2, more preferably at least 4, in particular at least 6. ADCs with higher DAR allow delivery of higher amounts of vincristine at the targeted cells. In the present invention, n does preferably not exceed 8. If n is higher than 8, the selectivity of conjugation may be lowered during the preparation process.

[0128] Process for preparing an ADC

[0129] The Invention also relates to a process for preparing an ADC according to the invention. The process of the Invention characterizes the conjugation of the monoclonal antibody MAB and the drug vincristine via a cleavable linker LINK of formula (XI) wherein Y, X, PP and Ar are as defined above.

[0130] The Applicant demonstrated that the process of the Invention allows to obtain an ADC of vincristine with high yield and high purity.

[0131] The process of the Invention also allows to obtain an ADC of vincristine with a high drug:antibody ratio (DAR), typically of 1 to 8, preferably of 4 to 8.

[0132] Typically, the process of the Invention comprises at least 3 steps: bonding the cleavable linker LINK to vincristine (step (A)), reducing the disulfide bridges of monoclonal antibody MAB (step B)), and conjugating the cleavable linker LINK to the reduced monoclonal antibody MAB (step (C)). Steps (A) to (C) may be implemented in any suitable conditions, such as any temperature and / or duration conditions, and in any suitable order.

[0133] In some embodiments, the process of the Invention comprises the following steps:

[0134] (A) reacting a compound of formula (I) with vincristine, or a salt of vincristine, such as vincristine sulfate, in presence of a carbonate salt, wherein X, PP and Ar are as defined above, so as to produce a compound of formula (II) wherein X, PP and Ar are as defined above, (B) at least partially, preferably totally, reducing the disulfide bridges between the cysteine residues of a monoclonal antibody as defined above, and

[0135] (C) coupling compound (II) obtained in step (A) to the at least partially, preferably totally, reduced antibody obtained in step (B), so as to produce the ADC of the Invention.

[0136] The process of the invention is described with Y being a maleimide moiety, but it can of course be implemented similarly with Y being any other thiol -selective moiety Y.

[0137] Typically, the carbonate salt used in step (A) of the process can be lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, preferably potassium carbonate.

[0138] In some embodiments, the reaction in step (A) is carried out in an aprotic polar organic solvent, such as dimethylformamide (DMF), dimethylacetamide (DMA), A-methylpyrrolidone (NMP), acetonitrile, A,A'-dimethylpropylene urea (DMPU), tetrahydrofuran (THF) or methyltetrahydrofuran (Me-THF), or in a mixture of such aprotic polar organic solvent with water. In a preferred embodiment, the reaction in step (A) is carried out in DMF or in a DMF / water mixture, preferably a DMF / water mixture comprising about 3v / v% water.

[0139] The use of a carbonate salt instead of diisopropylethylamine as disclosed in prior art for vinblastine (Staben et al. 2016) in step (A) afforded a substantial increase of the yield of the quaternary amination step (74% instead of 18%). Furthermore, compound (II) is obtained with high purity.

[0140] In a preferred embodiment, in step (B), the disulfide bridges between the cysteine residues of the antibody as defined above are totally reduced.

[0141] In some embodiments, step (B) is carried out by incubating said antibody in presence of a reducing agent, such as / / 7.s(2-carboxyethyl)phosphine (TCEP), supported TCEP or dithiothreitol (DTT). In a preferred embodiment, the reducing agent is TCEP.

[0142] In some embodiments, step (C) is carried out by incubating compound (II) obtained in step (A) with the at least partially reduced antibody obtained in step (B) at room temperature. In a preferred embodiment, step (B) and step (C) are carried out in a one-pot process.

[0143] The process according to the invention may comprise any suitable additional steps, before step (A), between step (A) and step (B), between step (B) and step (C), and / or after step (C).

[0144] For instance, each synthesized compound may be separated from the obtained reaction mixtures by any suitable technique known in the art. Each synthesized compound may also be purified by any suitable technique known in the art, such as by chromatography. Pharmaceutical composition

[0145] Another object of the invention is a pharmaceutical composition comprising an ADC according to the invention and a pharmaceutically acceptable excipient.

[0146] The pharmaceutical composition comprising the ADC of the Invention is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. The pharmaceutical composition according to the Invention may notably be formulated to release the ADC of the Invention immediately upon administration or at any predetermined time or time period after administration.

[0147] The pharmaceutical composition may further comprise a pharmaceutically acceptable vehicle and / or a pharmaceutically excipient.

[0148] As used herein, the term “pharmaceutically acceptable excipient' means an inactive or inert, and therefore nontoxic, component, as it has no pharmacological action itself, which can be used to improve properties of a composition, such as shelf-life, retention time at the application site, consumer acceptance, etc. It includes, without limitation, surfactants (cationic, anionic, or neutral); surface stabilizers; other enhancers, such as preservatives, wetting or emulsifying agents; solvents; buffers; salt solutions; dispersion medium; isotonic and adsorption delaying agents, and the like; that are physiologically compatible.

[0149] In some embodiments, the pharmaceutical composition is a liquid or a gel, such as a hydrogel.

[0150] Possible pharmaceutical compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intratumoral, intrathecal, intraspinal, intravenous and intradermal) administration. For these formulations, conventional excipients can be used according to techniques well known by those skilled in the art. In some embodiments, the pharmaceutical composition is suitable for intravenous and / or subcutaneous administration.

[0151] The compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs and concentrated drops. Non-toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials, are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablet. Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as glidant and compounds such as talc or stearic acids are most commonly used as lubricants.

[0152] For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

[0153] For transmucosal administration, nasal spray, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.

[0154] The concentration of the ADC in the pharmaceutical composition according to the invention may vary in a wide range depending among others on the ADC structure, the used pharmaceutically acceptable excipient and the route of administration.

[0155] In some embodiments, the ADC is present in the pharmaceutical composition at a concentration comprised between 100 mg and 500 g / L, preferably comprised between 500 mg / L and 50 g / L, more preferably comprised between 1 g / L and 20 g / L, even more preferably between 2 g / L and 10 g / L.

[0156] In some embodiments, the pharmaceutical composition according to the invention comprises from 0.01 wt% to 50 wt% of ADC and from 50 wt% to 99.99 wt% of pharmaceutically acceptable excipient, preferably from 0.2 wt% and 1 wt% of ADC and from 99 wt% to 99.8 wt% of pharmaceutically acceptable excipient.

[0157] Use o f the ADC

[0158] The Invention also relates to an ADC 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.

[0159] The term "cancer", as used herein, refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, and / or immortality, and / or metastatic potential, and / or rapid growth and / or proliferation rate, and / or certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases) in any type of subject. It may refer to solid tumor as well as hematopoietic tumor.

[0160] In some embodiments, the proliferative disease is a cancer overexpressing TROP-2. In a preferred embodiment, the cancer overexpressing TROP-2 is selected from the group consisting of a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a non-small cell lung cancer, a lung adenocarcinoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a prostate cancer, a bladder cancer, a gallbladder cancer, a cervical cancer, an uterine serous papillary carcinoma, an endometrial cancer, a nasopharyngeal cancer, a hilar cholangiocarcinoma, an oral squamous cell carcinoma, an esophageal squamous cell carcinoma, a head-and-neck squamous cell carcinoma, and a laryngeal squamous cell carcinoma. In a more preferred embodiment, the cancer is a breast cancer. In an even more preferred embodiment, the cancer is a triple negative breast cancer (TNBC). In some embodiments, the BRCA gene of the TNBC to be treated according to the invention is mutated. In other embodiments, the BRCA gene of the TNBC to be treated according to the invention is wild-type.

[0161] In some embodiments, the cancer is a cancer overexpressing HER-2. In a preferred embodiment, the cancer overexpressing HER-2 is a breast cancer and / or a stomach cancer. In a more preferred embodiment, the cancer overexpressing HER-2 is a breast cancer.

[0162] In some embodiments, the cancer overexpresses TROP-2 and HER-2.

[0163] In some embodiments, the cancer is a cancer overexpressing CD-20. Examples of cancers overexpressing CD-20 include mature B cell-derived malignancies, such as chronic lymphocytic leukemia (CLL) and various B cell-derived nonHodgkin lymphomas (B-NHL), including follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). CD20 is also present in multiple subtypes of B cell precursor acute lymphoblastic leukemia (B-ALL).

[0164] In some embodiments, the cancer is a cancer overexpressing PDL-1. Examples of cancers overexpressing PDL-1 include renal cell carcinoma (RCC), breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC), papillary thyroid cancer and testicular cancer.

[0165] In some embodiments, the cancer is a cancer overexpressing DLL3. Examples of cancers overexpressing DLL3 include small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), neuroendocrine prostate cancer (NEPC) and other high-grade neuroendocrine tumors.

[0166] In some embodiments, the cancer is a cancer overexpressing HER3. Examples of cancers overexpressing HER3 include breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer (NSCLC), ovarian cancer, and head and neck squamous cell carcinoma (HNSCC). HER3 is frequently implicated in tumor progression and therapeutic resistance, particularly in cancers driven by HER2 amplification or EGFR mutations.

[0167] The conjugate according to the invention or the pharmaceutical composition according to the invention may be administered by any convenient route to a subject in need thereof. For instance, it can be administered by a systemic route, in particular by subcutaneous, intramuscular, intravenous or intradermal, preferably by intravenous, injection. The conjugate according to the invention or the pharmaceutical composition according to the invention may be administered as a single dose or in multiple doses. The conjugate according to the invention or the pharmaceutical composition according to the invention may be administered between every day and every month, preferably every week or every two weeks, more preferably every week. The duration of treatment with a conjugate according to the invention or a pharmaceutical composition according to the invention is preferably comprised between 1 and 20 weeks, preferably between 1 and 10 weeks. Alternatively, the treatment may last as long as the symptoms of the disease persist.

[0168] The amount of conjugate according to the invention or pharmaceutical composition according to the invention to be administered has to be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient. Typically, the dosage is similar to that of other ADCs, such as commercialized ADCs.

[0169] In some embodiments, the ADC is administered in a dose comprised between 0.1 and 1000 mg / kg, preferably between 0.5 and 500 mg / kg, more preferably between 1 and 100 mg / kg, more preferably between 2 and 50 mg / kg, more preferably between 2.5 and 10 mg / kg, in particular a dose of about 5 mg / kg.

[0170] The ADCs may be administered as single therapeutic agents (monotherapy) or adjunctively with or to other anti-cancer treatments and / or therapeutic agents, typically but not necessarily those used to treat the type of cancers being treated. Adjunctive therapies and / or therapeutic agents typically will be used at their approved dose, route of administration, and frequency of administration, but may be used at lower dosages and / or less frequently. When administered as monotherapy, the ADC will typically be administered on a schedule that provides therapeutic benefit. When administered adjunctive to or with another therapy and / or agent, the ADC may be administered before (neoadjuvant), after (adjuvant) or concurrently with the other therapy or agent.

[0171] Another object of the present invention is a method for the treatment of a proliferative disease, such as a cancer, comprising administering to a subject in need thereof of a therapeutically effective amount of an ADC according to the invention or of a pharmaceutical composition according to the invention.

[0172] As used herein, the term “ subject” refers to any animal including, but not limited to mammals, including humans. It is not intended that the term be limited to a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are encompassed by the term. In preferred embodiments, the subject is a human subject, and doses disclosed herein are for adult humans, but can be adjusted for other mammals, as well as children. Where an ADC is administered to a human subject, the person of ordinary skill will realize that the target antigen to which the ADC binds will be a human antigen, such as human TROP-2 or human HER-2.

[0173] Preferably, the subject is a subject suffering from a proliferative disease, such as a cancer, or at risk of developing a proliferative disease, such as a cancer.

[0174] A "therapeutically effective amount" refers to the necessary and sufficient amount of a therapeutic agent to be administered to a subject to slow down or halt the progression, aggravation or deterioration of at least one of the symptoms of a disease. This administered amount can allow the relief of the symptoms of a disease or the cure of this disease.

[0175] "Treating" or "treatment" refer 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.

[0176] Another object of the present invention is the use of an ADC according to the invention or of a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a proliferative disease, such as a cancer.

[0177] All features and embodiments disclosed for the ADCs and pharmaceutical compositions for use in the treatment of a proliferative disease apply similarly to the method of treatment of a proliferative disease and to the use of the ADCs and pharmaceutical compositions in the manufacture of a medicament for the treatment of a proliferative disease.

[0178] In the present application, the term "about" (or ca.) preceding a value is well-known to the skilled artisan and means that said value may vary to a certain extent depending on the context in which the term is used. If certain uses of this term are not clear to the skilled artisan depending on the context, then "about" means ± 20%, preferably ± 10% of said value.

[0179] Unless otherwise indicated, when a range is expressed by means of the expression "comprised between", the limit values are included within the range described.

[0180] 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. EXAMPLES

[0181] Example 1: Synthesis of an ADC according to the Invention

[0182] 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. Air and / or moisture-sensitive liquids were transferred via a syringe. Concentration in vacuo refers to distillation on a Biichi rotary evaporator, and where appropriate, under high vacuum. Volume ratios (v / v) are indicated when referring to mixtures of solvents (e.g. DCM / MeOH 95:5). a) Synthesis of MC-ValCit-PAB-Cl 2 Mc-Val-Cit-PAB-Cl was synthesized according to the procedure described by Staben et al. Analytical data were found to be in agreement with those published. b) Synthesis of MC-ValCit-PAB-Vincristine 11 To a solution of vincristine 1 (1 equiv., 10 mg, 0.0108 mmol) and mc-Val-Cit-PAB-Cl 2 (1.1 equiv., 7 mg, 0.0119 mmol) in DMF / H2O (97:3; 0.5 mL) was added K2CO3 (3.0 equiv., 5.4 mg, 0.0324 mmol) and the mixture was stirred at 37 °C for 24 h. The reaction medium was then concentrated in vacuo and the crude product was purified by column chromatography (CH2Cl2 / MeOH 9: 1, isochratic) to afford the title compound (11 mg, 0.0080 mmol, 74%) as a light yellow solid.

[0183] Rf 0.10 (9:1, CH2Cl2 / MeOH).

[0184] ’H NMR (500 MHz, CDCh) 8H 10.46 (s, 1H, NHs), 10.01 (s, 1H, NH1’), 9.05 (s, 1H, N1CHO), 8.55 (m, 1H, OH16), 8.21 (s, 1H, NH1), 7.88 (s, 1H, NHg), 7.78 (d, J= 7.1 Hz, 2H, Ht), 7.66 (d, J= 7.7 Hz, 1H, H9’), 7.49 (d, J= 7.1 Hz, 2H, Hu), 7.41 (m, 2H, H12 + H12’), 7.09 (m, 1H, HU’), 7.03 (m, 1H, H10’), 6.98 (s, 2H, Ha), 6.86 (s, 1H, H9), 6.34 (m, 1H, Hq), 5.82 (m, 2H, H14), 5.50 (s, 2H, Hr), 5.32 (d, J= 9.4 Hz, 1H, H15), 5.26 (s, 1H, OH20’), 4.96 (s, 1H, H17), 4.63 (m, 1H, Hv), 4.48 (m, 1H, Hv), 4.45 (m, 2H, H5’ + H2), 4.40 (m, 1H, Hm), 4.25 (m, 1H, H6’), 4.18 (t, J = 7.1 Hz, 1H, Hh), 4.04 (br d, J = 12.3 Hz, H3’), 3.86 (s, 3H, OCH311), 3.68 (m, 1H, H17’), 3.66 (s, 3H, CO2CH316’), 3.59 (m, 1H, H5’), 3.50 (m, 1H, H6’), 3.46 (s, 3H, CO2CH316), 3.36 (m, 1H, H21’), 3.32 (m, 3H, H3 + 2Hb), 3.27 (m, 1H, H5), 3.14 (m, 1H, H21’), 3.04 (m, 1H, H21), 2.97 (m, 2H, Hp), 2.93 (m, 1H, H3’), 2.86 (m, 1H, H3), 2.61 (m, 1H, H5), 2.35 (m, 1H, H17’), 2.17 (m, 2H, Hf), 2.04 (m, 1H, H6), 1.97 (m, 1H, Hi), 1.93 (s, 3H, OCOCH317), 1.84 (m, 1H, H6), 1.73 (m, 1H, Hn), 1.62 (m, 1H, Hn), 1.49 (m, 3H, He + 2H15’), 1.45 (m, 2H, He), 1.38 (m, 2H, Ho), 1.38 (m, 2H, H19), 1.37 (m, 2H, H19’), 1.31 (m, 1H, H14’), 1.19 (m, 2H, Hd), 0.85 (m, 3H, Hj / k), 0.82 (m, 3H, Hk / j), 0.80 (m, 3H, H18’), 0.58 (t, J= 7.0 Hz, 3H, H18).

[0185] HRMS (ESI+) ealed for C74H96NIOOI62+[(M+H) / 2]+690.3497, found 690.3501.

[0186] Analytical data were consistent with those reported by by Staben et al.

[0187] Bioconjugation of MC-ValCit-PAB-vincristine 11. a) On partially reduced antibodies

[0188] To a solution of trastuzumab 12 or rituximab 13 (1 equiv., 5 mg / mL, 200 pL, 1 mg, 6.9 nmol) in PBS at pH 6.5 was added a solution of TCEP (2 equiv., 10 mM in H2O mQ, 1.38 pL, 13.8 nmol) and EDTA (1%; 0.5 M) and the mixture was incubated at 37 °C for 2 h. Excess of reagent was then removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) pre-equilibrated with PBS pH 6.5 to give a solution of partially reduced antibody. Directly after, a solution of MC-ValCit-PAB-Vincristine 11 (20 equiv., 10 mM in DMSO) was added and the mixture was incubated at 25 °C for 2 h under argon atmosphere. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) pre-equilibrated with PBS pH 7.5 to give a solution of antibodydrug conjugates. Buffer was then exchanged on viva spin to afford the final solution in PBS at pH 7.5 with a yield varying from 60 to 90% (concentrations measured by BCA).

[0189] DoC was determined by native ESI-MS (avDAR = 3.6 - 3.8).

[0190] This protocol was used to obtain T-vincristine D3.6 14 and R-vincristine D3.4 16. b) On fully reduced antibodies

[0191] To a solution of trastuzumab 12 or rituximab 13 (1 equiv., 5 mg / mL, 200 pL, 1 mg, 6.9 nmol) in PBS at pH 6.5 was added a solution of TCEP (10 equiv., 10 mM in H2O mQ, 6.9 pL,

[0192] 69 nmol) and the mixture was incubated at 37 °C for 2 h. Directly after, a solution of MC- ValCit-PAB-Vincristine 11 (20 equiv., 10 mM in DMSO) was added and the mixture was incubated at 25 °C for 2.5 h under argon atmosphere. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) preequilibrated with PBS pH 7.5 to give a solution of antibody-drug conjugates. Buffer was then exchanged on viva spin to afford the final solution in PBS at pH 7.5 with a yield varying from

[0193] 70 to 90% (concentrations measured by BCA).

[0194] DoC was determined by native ESI-MS (DAR = 7.8 for T-vincristine and 7.4 for R-vincristine).

[0195] This protocol was used to obtain T-vincristine D7.8 15 and R-vincristine D7.4 17 used in example 2.

[0196] Vincristine was similarly bioconjugated with Sacituzumab to obtain Sacituzumab vincristine D7.8 ADC used in example 3.

[0197] The same process was efficiently implemented to bioconjugate vincristine to avelumab and durvalumab. Bioconiugation of MC-ValCit-PAB-MMAE a) Bioconiugation of MC-ValCit-PAB-MMAE 20 on partially reduced antibodies

[0198] TCEP (2 equiv., 10 mM in H2O mQ, 0.69 pL, 6.9 nmol) was added to a solution of trastuzumab 12 (1 equiv., 5 mg / ml, 100 pL, 0.5 mg, 3.45 nmol) in PBS pH 6.5 with EDTA 1% v / v (solution of 0,5 M) and the mixture was incubated at 37 °C for 2h. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) preequilibrated with PBS pH 6.5 to give a solution of partially reduced antibody. Directly after, MC-ValCit-MMAE 20 (20 equiv., 10 mM in DMSO, 6.9 pL, 69 nmol) was added and the mixture was incubated at 25 °C for 2h under argon atmosphere. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) pre-equilibrated with PBS pH 7.5 to give a solution of T-MMAE D2 18. Buffer was exchanged on viva spin to afford the final solution in PBS pH 7.5 with 70 - 90% yield (concentrations were measured by BCA assays).

[0199] DoC was determined by native ESI-MS (avDAR = 2.3). b) Bioconiugation of MMAE on totally reduced antibodies

[0200] TCEP (20 equiv., 10 mM in H2O mQ, 6.9 pL, 69 nmol) was added to a solution of trastuzumab 12 (1 equiv., 5 mg / ml, 100 pL, 0.5 mg, 3.45 nmol) in PBS pH 6.5 with EDTA 1 % v / v (solution of 0,5 M) and the mixture was incubated at 37 °C for 2h. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) preequilibrated with PBS pH 6.5 to give a solution of partially reduced antibody. Directly after, MC-ValCit-MMAE 20 (20 equiv., 10 mM in DMSO, 6.9 pL, 69 nmol) was added and the mixture was incubated at 25 °C for 2h under argon atmosphere. Excess of reagent was removed by gel filtration chromatography using Bio-spin P-30 columns (Bio-Rad, Hercules, U.S.A.) pre-equilibrated with PBS pH 7.5 to give a solution of T-MMAE D8 19. Buffer was exchanged on viva spin to afford the final solution in PBS pH 7.5 with a 70 - 90 % yield (concentrations were measured by BCA assays).

[0201] DoC was determined by native ESI-MS (avDAR ~ 8).

[0202] Example 2: In vitro cytotoxic effect of trastuzumab vincristine (T-vincristine, av. DAR = 7.8)) 15 on HER2+and HER2lowcancer cell lines.

[0203] Biomolecules

[0204] All enzymes, reagents and solvents were obtained from commercial sources - Sigma Aldrich France, Fischer Scientific France, VWR France, New England Biolabs -, and used without prior purification. Monoclonal antibodies were provided by the Institut de Cancerologie Strasbourg Europe (Strasbourg, France). Commercial ADC T-DM1 and T-Dxd were purchased from MedChemExpress. Concentrations in antibody solutions were determined by UV absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch, France) at 280 nm at ambient temperature. Sample buffer (DPBS, calcium and magnesium free, Merck, Ref. D8537-6X500ML) was used as blank for baseline correction. In case of impaired absorbance at 280 nm or unknown extinction coefficient, BCA assay was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Illkirch, France, Ref. 23225), using BSA diluted in PBS (IX, pH 7.5) as ladder concentration. Incubation during reduction and conjugation experiments took place either in an Eppendorf thermomixer comfort (catalog #5355) or in a digital heated shaker dry bath (Ref.: 88880027).

[0205] Biomolecule purification

[0206] Antibody conjugates were purified by gel filtration chromatography either on Bio-spin 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 microconcentrators (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) with protein samples at 10 nL / pg.

[0207] Cell lines and cell culture

[0208] All the cell lines used in this study were purchased from ATCC and are listed in Table 1 with their characteristics. SKBR3, BT474 and NCI-N87 cells were cultured in 4.5 g / L glucose DMEM (Gibco Life Technologies 41965-062) supplemented with 10% heat inactivated fetal calf serum (FBS) and 1% gentamicin. MDA-MB-231 cells were cultured in RPMI 1640 (Gibco 21875-034) supplemented with 10% fetal calf serum (FBS) and 1% gentamicin. Cells were maintained at 37 °C in a humidified 5% CO2 and 20% O2 atmosphere.

[0209] Table 1 presents the characteristics of the cell lines used in this study. (Adapted from Keung et al., 2020)

[0210] Cell viability assays

[0211] Cells were seeded in 96-well plates at a density of 2 000 cells / well for MDA-MB-231 cells and 5 000 cells / well for all the other cell lines and allowed to grow under normal oxygen conditions (20% O2). Cells were treated with the appropriate compound 24 h after plating and maintained at 37 °C in a humidified 5% CO2 and 20% O2 atmosphere. Cell viability was measured 72 h or 120 h later with the WST-1 cell proliferation assay (Takara®) according to the manufacturer’s instructions. To represent the relative cell proliferation rate, results were normalized against the untreated condition.

[0212] Statistical analysis

[0213] All experiments were performed at least in 3 independent biological replicates, unless otherwise indicated. Curve fits were obtained and means, SEM and IC50 were calculated and analyzed using the GraphPad Prism software.

[0214] Experiment

[0215] Cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24h prior to treatment with different concentrations of each compound. Cell viability was measured 120h later using the WST-1 cell proliferation assay.

[0216] Figure 1 presents the in vitro cell viability of each cell line in presence of different concentrations of trastuzumab-vincristine (T-vincri stine) (av. DAR = 7.8), an ADC according to the invention, trastuzumab alone, vincristine alone and rituximab-vincristine (R- vincristine) (av. DAR = 7.4) ADC. IC50 values of 0.27 nM or lower (as low as 0.025 nM) were obtained for T-vincristine in HER2+ cell lines. Said IC50 values are lower than those of free vincristine.

[0217] Rituximab is an anti-CD20 antibody. Said antigen is not expressed is the different used cells. The low activity of rituximab-vincristine ADC assesses that internalizing the T-vincristine ADC following its binding to HER2 is necessary for the ADC to be efficient on the cell.

[0218] The cytotoxic effect of T-vincristine (av. DAR = 7.8) 15 on HER2+and HER2lowcancer cell lines was further compared to that of reference ADCs, namely trastuzumab -MMAE (T- MMAE) and trastuzumab-DMl (Kadcyla®, trastuzumab emtansine or T-DM1) (Roche).

[0219] Cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 72 h later using the WST-1 cell proliferation assay.

[0220] Figure 2 presents the in vitro cell viability of SKBR3 (HER2+) and MDA-MB-231 (HER2verylow) cell lines in the presence of different concentrations of each ADC.

[0221] Despite the much lower cytotoxicity of free vincristine compared to free DM1 or MMAE, vincristine-loaded ADCs display similar or even higher potency compared to these reference compounds.

[0222] Example 3: In vivo antitumoral activity of trastuzumab-vincristine (T-vincristine, av. DAR = 7.8) conjugate in comparison with standard trastuzumab-based ADC T-DM1 (Kadcyla®) and T-Dxd (Enhertu®).

[0223] In vivo assays

[0224] The right flanks of a cohort of 32 seven-week-old immunodeficient NOD-SCID mice were injected subcutaneously with SKBR3 breast cancer cells and 25% Matrigel® HC (Corning, ref. 354248) (5 x 106 cells / 200 pL, 81% viability as determined with "count and viability" kit, Muse®, Millipore). Treatment was initiated when the tumors reached an average size of 140 mm3, ca. 8 weeks following injection of SKBR3 cells. Four homogeneous groups of 5 to 6 animals were then formed. Each group received a single intravenous injection (5 mg.kg-1) of the tested compounds: trastuzumab-vincristine T-vincristine (av. DAR = 7.8), trastuzumab- emtansine T-DM1 (Kadcyla®), trastuzumab -deruxtecan T-Dxd (Enhertu®), or vehicle (PBS IX). Tumor growth was monitored using a caliper (Helios Preisser, ref. 821498) every 2-3 days. Measurements of tumor volume are presented as mean ±SEM. Statistical analysis for tumor removal proportions (% of mice without complete tumor regression) and survival curves (% of surviving mice as a function of time postinjection) was performed using a Log-rank (Mantel-Cox) test with GraphPad Prism 7.0. Data are considered as statistically reliable when p < 0.05 (*) and p < 0.01 (**). Animal welfare was monitored each day to highlight any behavior trouble, pain or bodyweight loss according to rodent protection tests.

[0225] Figure 3 presents the in vivo antitumoral activity of trastuzumab -vincristine (T -vincristine, av. DAR = 7.8) conjugate in comparison with standard trastuzumab-based ADC T-DM1 (Kadcyla®) and T-Dxd (Enhertu®).

[0226] Experiment

[0227] The activity of T-vincristine (av. DAR = 7.8) was assayed in immunodeficient NOD-SCID mice xenografted subcutaneously with SKBR3 cells. The animals were bred for eight weeks until sufficient tumor growth occurred before being split into four groups of five to six mice each. Each animal of a given group received a single intravenous injection of either the vehicle (PBS IX, negative control) or one of three following ADC, all at the same concentration of 5 mg. kg'1: T-vincristine, T-DM1 (Kadcyla®, positive control), and T-Dxd (Enhertu®, positive control). Tumor volume measurements revealed a remarkable antitumor activity of the T- vincristine conjugate, with complete tumor regression observed only nine days after injection, whereas complete tumor regression was only observed at later time points (> 20 days) in mice treated with any of the two positive control ADC (Figure 3A and B). Moreover, no apparent toxicity was observed up to 60 days post-injection in mice receiving T-vincristine, as determined by bodyweight loss, and neither tumor relapse nor metastasis, as shown by the survival curves in Figure 3C. Conversely, toxicity was observed in half the mice of the T-Dxd group, materialized by a bodyweight loss over 10%, and late (i.e., after more than 30 days) deterioration of mice health was witnessed in all three groups (i.e., vehicle, T-Dxd, and T- DM1), probably due to metastases. Overall, these results illustrate and confirm the strong potential of vincristine immunoconjugates as safe and effective ADC for in vivo anti-cancer applications.

[0228] Example 4: Capacity of sacituzumab-vincristine (S-vincristine, av. DAR = 7.8) to link TROP2+cell lines.

[0229] Biomolecules

[0230] Sacituzumab, ADC Sacituzumab-SN38 (Trodelvy®) vincristine and SN38 were purchased by MedChemExpress.

[0231] Cell lines and cell culture HCC 1937 BRCA1 restored cells were provided by the team of Nicola J Curtin (Newcastle University NCL Northern Institute for Cancer Research). The other cell lines used in this study were purchased from ATCC. All the cell lines used are listed in Table 2 with their characteristics. MDA-MB-231, MDA-MB-436 and HCC 1937 BRCA1 restored cells were cultured in RPMI 1640 (Gibco 21875-034) supplemented with 10 % fetal calf serum (FBS) and 1 % gentamicin. MDA-MB-468 cells were cultured in 1 g / L glucose DMEM (Gibco Life Technologies 31885-049) supplemented with 10 % fetal calf serum (FBS) and 1 % gentamicin. Cells were maintained at 37 °C in a humidified 5 % CO2 and 20 % 02 atmosphere.

[0232] Table 2 presents the characteristics of the cell lines used in this study.

[0233] Table 2

[0234] In vitro binding assays

[0235] Fluorescein isothiocyanate (FITC) conjugated antibody or ADCs were obtained using 10 equivalents of FITC in a pH 7.4 phosphate buffer saline (PBS) solution. The conjugation reactions were performed in the same solvent, for 2 h at 25 °C before antibody conjugates were purified by gel filtration chromatography on Bio-spin P-6 columns obtained from Bio-rad (Hercules, U.S.A). Concentrations of antibody solutions were evaluated with the BCA Protein Assay Kit (Thermo Fisher Scientific, Illkirch, France, Ref. 23225), using BSA diluted in PBS (IX, pH 7.5) as ladder concentration. Incubation during reduction and conjugation experiments took place either in an Eppendorf thermomixer comfort (catalog #5355) or in a digital heated shaker dry bath (Ref.: 88880027). The relative FITC conjugation ratio was evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch, France) at 280 nm at room temperature. Sample buffer (DPBS, calcium and magnesium free, Merck, Ref. D8537- 6X500ML) was used as blank for baseline correction. The FITC conjugation degree was 4-fold lower for S-vincristine-FITC and S-SN38-FITC (Trodelvy-FITC) in comparison to Sacituzumab-FITC.

[0236] Following FITC conjugation, cells were seeded in 6-well plates at an appropriate density and allowed to grow under normal oxygen conditions (20% O2). 48-72h after plating when 70-80% of confluency was reached, cells were harvested and filtered as single cells through a 40 pm cell strainer and washed in ice cold FACS buffer (IX PBS; 1 mM EDTA; 50 mM Hepes Buffer; 0.5% BSA). They were then blocked with FACS blocking buffer (IX PBS; 1 mM EDTA; 50 mM Hepes Buffer; 2 % BSA), for 15 minutes at room temperature. Following the blocking step, cells were washed once with FACS buffer and incubated for 30 minutes at room temperature with the FITC conjugates at an appropriate concentration in FACS buffer. Cells were subsequently washed three times before being analyzed using the BD CSampler® Plus. The number of viable and single cells and the percentage of FITC positive cells were calculated with the BD CSampler® Plus software.

[0237] Cell viability assays

[0238] Cells were seeded in 96-well plates at a density of 2 000 cells / well for MDA-MB-231 cells and 5 000 cells / well for all the other cell lines and allowed to grow under normal oxygen conditions (20% O2). 24h after plating, cells were treated with the appropriate compound and maintained at 37 °C in a humidified 5 % CO2 and 20 % atmosphere. Cell viability was measured 72h later with the WST-1 cell proliferation assay (Takara®) according to the manufacturer’s instructions. To represent the relative cell proliferation rate, results were normalized against the untreated condition.

[0239] Statistical analysis

[0240] All experiments were performed at least in 3 independent biological replicates, unless otherwise indicated. Curve fits were obtained and means, SEM and IC50 were calculated and analyzed using the GraphPad Prism software.

[0241] The capacity of Sacituzumab vincristine (S -vincristine) D7.8 to link specifically cell lines expressing TROP-2 was assessed. FITC fluorophore was conjugated to Sacituzumab vincristine D7.8 ADC, and the proportion of FITC+cells was determined by flow cytometry in different cell lines. The following cell lines were used: HCC 1937 BRCA1 restored cell line, which is TROP2+, MDA-MB-231 cell line, which is TROP2low, and MDA-MB-436 cell line, which is TROP2very low

[0242] Sacituzumab alone and Trodelvy® (Sacituzumab-SN38) were also assessed in the same conditions.

[0243] Figure 4 presents the percentage of FITC+cells for sacituzumab and the different ADCs in the different cell lines. Sacituzumab-vincristine (S-vincristine) maintains the capacity of sacituzumab to bind to TROP2. The difference between sacituzumab and sacituzumab- vincristine may be due to a higher conjugation degree of FITC on sacituzumab than on sacituzumab-vincristine due to steric hindrance. A similar result was observed for the sacituzumab-SN38-FITC.

[0244] Example 5: In vitro cytotoxic effect of Sacituzumab-vincristine (S-vincristine, av. DAR = 7.8) on TROP2+and TROP2lowcancer cell lines.

[0245] Measurements of cell proliferation / viability were implemented in the presence of increasing concentrations of the compound to be assessed with WST-1 tests or violet crystal tests. The TROP2+cell lines TNBC HCC 1937 restored and MDA-MB-468 were used as positive controls and cytotoxic activity on these cell lines was compared to the activity of compounds and ADCs on TROP2lowMDA-MB-431 and TROP2veiy lowMDA-MB-436 cells.

[0246] In all experiments, the activity of the sacituzumab-ValCit-vincristine (S-vincristine, av. DAR = 7.8) ADC was compared to that of free vincristine, free SN38 (drug associated with sacituzumab in the pharmaceutical specialty Trodelvy®), sacituzumab and Trodelvy®. In HCC 1937 BRCA1 restored cells, trastuzumab-ValCit-vincristine (T -vincristine, av. DAR = 7.8) obtained and previously evaluated by our teams were evaluated in parallel. The results obtained are presented in Figure 5.

[0247] These show that S-vincristine conjugates exhibit strong and selective cytotoxicity following vincristine internalization in several TROP2 overexpressing cancer lines with IC50s in the subnanomolar range, conditions in which sacituzumab alone is not active. The IC50 values obtained in TROP2+lines are, respectively: TNBC HCC 1937 BRCA restored: 0.014 nM; TNBC MDA-MB-468: 0.005 nM. No effect of this ADC is observed in the TNBC cell line MDA-MB-231 expressing low levels of TROP2 and in TROP2veiy lowTNBC MDA-MB-436 cells. The selectivity of action of sacituzumab-ValCit vincristine (S-vincristine) on cells overexpressing TROP2 is reinforced by the absence of effect of another vincristine conjugate that was developed using the same methods, trastuzumab (anti-HER2 antibody)-ValCit- vincristine (T-vincri stine) under the same conditions in HCC 1937 BRCA1 restored (TROP2+HERZ) cells.

[0248] Moreover, in all TROP2+lines, the biological activity of vincristine conjugated to the antibody is, depending on the lines, 10 to 50 times greater than that of free vincristine. This is not the case for the vast majority of cytotoxic molecules present on ADCs currently on the market, such as MMAE or DM1, which are inhibitors of microtubule polymerization in the same way as vincristine but, present a cytotoxicity of the order of ten picomolars in their free form. This is also not the case for SN38, the topoisomerase inhibitor conjugated to sacituzumab in the composition of Trodelvy®, which has similar activity to the corresponding ADC in the HCC 1937 BRCA1 restored and the MDA-MB-468 (TROP2+) cell lines as can be seen in Figure 5.

[0249] Furthermore, in vitro, in TROP2+lines, S-vincristine is 80 to 340 times more cytotoxic than Trodelvy®, the reference anti-TROP2 ADC (sacituzumab-SN38) for the treatment of unresectable or metastatic TNBC. In addition, Trodelvy® does not have any specificity related to the level of expression of TROP2, which is the case for the S-vincristine ADC.

[0250] Example 6: Comparison with ADCs comprising other Vinca alkaloids

[0251] The in vitro cytotoxic effect of trastuzumab-vincristine (T-vincri stine; av. DAR = 7.8), trastuzumab-vinblastine (T-vinblastine av. DAR = 8), trastuzumab -vinorelbine (T-vinorelbine av. DAR = 6), vincristine alone, vinblastine alone and vinorelbine alone on SKBR3 (HER2+) cancer cells was assessed like in example 2.

[0252] Figure 6 discloses the obtained results.

[0253] In all experiments including in the one whose results are presented on Figure 6, trastuzumabvincristine presented much higher cytotoxicity on SKBR3 HER2+cells compared to free vincristine, whereas trastuzumab conjugated with other Vinca alkaloids, namely trastuzumabvinblastine and trastuzumab-vinorelbine, presented a cytotoxic affect in the same order of magnitude than that of the corresponding Vinca alkaloid alone. In addition, altogether these results suggest that whereas trastuzumab -vincristine and trastuzumab-vinblastine conjugates present similar IC50 values in the subnanomolar range on HER2+cell lines, trastuzumab- vinorelbine conjugates are much less effective. Example 7: Synthesis and in vitro assays of further ADCs according to the invention

[0254] Synthesis

[0255] Quaternary ammonium linker-payloads with various peptide linkers were synthesized with the following general procedure:

[0256] Commercially available benzyl alcohol linkers were reacted with thionyl chloride (2.0 equiv.) in acetonitrile at 0 °C. The reaction mixture was then allowed to warm up to room temperature and was stirred for one hour to obtain the various benzyl chloride linkers in quantitative yields.

[0257] To a solution of vincristine (VCR) sulfate (1.0 equiv.) and the benzyl chloride linker (1.1 equiv.) in dimethylformamide DMF / H2O (97:3) was added K2CO3 (3.0 equiv.) and the mixture was stirred at 37 °C for 24 hours. The reaction medium was then concentrated in vacuo and the crude product was purified by column chromatography (CFCCb / MeOH 9: 1, isocratic) to afford the title compound as a white solid (Mc-Val-Ala-PAB-VCR+ 65%; Mc-Ala-Ala-PAB-VCR+ 63%, Mc-Gly-Gly-Phe-Gly-PAB-VCR+ 68%).

[0258] The corresponding ADCs with trastuzumab ADCs were prepared following the conjugation procedures disclosed in previous examples with partial or full reduction to reach DAR4 and DAR8 ADCs respectively.

[0259] Biological assays

[0260] Cells were seeded in 96-well plates at a density of 5,000 cells / well and allowed to grow under normal oxygen conditions (20% O2). Cells were treated with the appropriate compounds 24 h after plating and maintained at 37 °C in a humidified 5% CO2 and 20% O2 atmosphere. Cell viability was measured 96 h later with the CellTiter Gio viability assay (Promega) according to the manufacturer’s instructions. To represent the relative cell viability rate, results were normalized against the untreated condition.

[0261] Figure 7 presents the in vitro efficacy of the 4 vincristine ADCs in HER2+and HER2veiy lowcells respectively. All 4 ADCs show to be efficient at selectively affecting the viability of HER.2 cells. Example 8: Comparison with vincristine ADCs comprising a carbonate linker

[0262] Synthesis

[0263] V Vincristine VCR chloroformate.

[0264] VCR (5 mg, 6 pmol, 1.0 equiv.), 4-dimethylaminopyridine DMAP (2.2 mg, 18 pmol, 3.0 equiv.) and triphosgene (0.9 mg, 3 pmol, 0.5 equiv.) were taken in an eppendorf tube and the reaction was initiated by adding dichloromethane (0.12 mL, 50 mM). The mixture turned to clear light yellow solution within a minute. The formation of the chloroformate was monitored by quenching an aliquot with anhydrous methanol and comparing the thin layer chromatography TLC mobility with that of VCR (TLC condition: toluene / methanol / triethylamine 8.75:0.75:0.5, Rf (VCR): 0.27, Rf (VCR chloroformate): 0.35). The formation of the chloroformate was complete within a few minutes, and the product was reacted in situ with the linker after 5 min from the start-up of the reaction.

[0265] V Mc-Val-Cit-PABC-VCR.

[0266] In one run, VCR chloroformate generated from VCR was reacted in situ with the Mc-Val-Cit- PABOH linker (5.2 mg, 9 pmol, 1.5 equiv.) dissolved in dimethylformamide DMF (0.12 mL) for one hour. The reaction mixture was then purified by preparative HPLC using a water / acetonitrile (TFA 0.1%) gradient from 0 to 30% of acetonitrile in 20 minutes to obtain the carbonate linker-payload Mc-Val-Cit-PABC-VCR (white solid; 1 mg; 11.7%).

[0267] The corresponding ADCs with Trastuzumab were prepared following described conjugation procedures with partial or full reduction to reach DAR4 and DAR8 ADCs respectively.

[0268] Biological assays

[0269] Cells were seeded in 96-well plates at a density of 5,000 cells / well and allowed to grow under normal oxygen conditions (20% O2). Cells were treated with the appropriate compounds 24 h after plating and maintained at 37 °C in a humidified 5% CO2 and 20% O2 atmosphere. Cell viability was measured 96 h later with the CellTiter Gio viability assay (Promega) according to the manufacturer’s instructions. To represent the relative cell viability rate, results were normalized against the untreated condition.

[0270] Figure 8 presents the in vitro efficacy of vincristine ADCs according to the invention and of the corresponding ADCs comprising a carbonate linker instead of a quaternary ammonium linker in HER2+and HER2very lowcells respectively.

[0271] Contrary to ADCs according to the invention, the corresponding ADCs comprising a carbonate linker do not present a selective toxicity towards HER2+SKBR3 cells in comparison to HER2veiy lowMDA-MB-231 cells.

Claims

CLAIMS1. An antibody-drug conjugate of formula (Z) or a pharmaceutically acceptable salt thereof:wherein- MAB is a monoclonal antibody comprising at least one, preferably at least four, cysteine residues and which targets at least one protein which is expressed in a cancer cell,- LINK is a cleavable linker which is covalently bound to MAB via the sulphur atom of one of its cysteine residues, and- n is comprised between 1 and 8.

2. The antibody-drug conjugate according to claim 1, wherein it is of formula (A), or a pharmaceutically acceptable salt thereof:wherein MAB, LINK and n are as defined in claim 1.

3. The antibody-drug conjugate according to claim 1 or claim 2, wherein the cleavable linker LINK is of formula (XI):wherein44- Y is a moiety bound to the sulphur atom of a cysteine residue of MAB, preferably Y is a maleimide moiety;- X is a saturated or unsaturated, linear, branched or cyclic, C1-C20 hydrocarbon chain, optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NR-, -C(=O)-, -O(C=O)-, -C(=O)O-, -NR(C=O)-, -C(=O)NR-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, - NRIC(=O)NR2-, and / or one or more aryl and / or heteroaryl groups optionally substituted by one or more functional groups selected from the group consisting of - OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain,- PP is a polypeptide chain comprising 2 to 10 amino-acids, and- Ar is an aryl or a heteroaryl group optionally substituted by one or more functional groups selected from the group consisting of -OR, -NR1R2, -C(=O)OR, -NO2 and a halogen atom, where R, Ri and R2 are each independently H or a C1-C3 alkyl chain.

4. The antibody-drug conjugate according to claim 3, wherein- Y is a maleimide moiety,- X is a saturated C1-C20 hydrocarbon chain, optionally interrupted by -O-, -S-, -SO, - SO2-, -NR-, -C(=O)-, -O(C=O)-, -C(=O)O-, -NR(C=O)-, -C(=O)NR-, or an unsubstituted phenyl group, where R is H or a C1-C3 alkyl chain,- PP is a polypeptide chain of 2 to 10 amino acids consisting of proteinogenic amino acids and / or biologically active amino acids, and- Ar is an unsubstituted phenyl group.

5. The antibody-drug conjugate according to claim 3 or claim 4, wherein each amino acid of the polypeptide PP is independently selected from the group consisting of Glycine, Alanine, Valine, Glutamic acid, Phenylalanine and Citrulline.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the polypeptide PP is selected from the group consisting of Val-Cit, Vai-Ala, Ala-Ala and Gly-Gly-Phe-Gly, preferably the polypeptide PP is Val-Cit.

7. The antibody-drug conjugate according to any one of claims 3 to 6, wherein X is a saturated C1-C20 hydrocarbon chain, preferably a n-pentylene chain.

8. The antibody-drug conjugate according to any one of claims 3 to 6, wherein Ar is an unsubstituted phenylene, preferably an unsubstituted 1,4-phenylene.

9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein MAB is an antibody which targets an antigen selected from the group consisting of TROP-2, HER- 2, HER-3, DLL-3, CD20 and PD-L1, preferably TROP-2 or HER-2.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein MAB is an antibody selected from the group consisting of Sacituzumab, Trastuzumab, Avelumab and Durvalumab, preferably Sacituzumab or Trastuzumab.

11. The antibody-drug conjugate according to any one of claims 1 to 10, which is of formula(C):wherein MAB is Sacituzumab or Trastuzumab and wherein n is comprised between 6 and 8, preferably n is about 8.

12. A process for preparing an antibody-drug conjugate as defined in any one of claims 1 to 11, which comprises the following steps:(A) reacting a compound of formula (I) with vincristine, or a salt of vincristine, in presence of a carbonate salt, preferably potassium carbonate;wherein Ar, X and PP are as defined in any one of claims 3 to 8, so as to produce a compound of formula (II),wherein X, PP and Ar are as defined in any one of claims 3 to 8;(B) at least partially, preferably totally, reducing the disulfide bridges between the cysteine residues of a monoclonal antibody MAB as defined in any one of claims 1 to 11, preferably by incubating said antibody in presence of a reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), supported TCEP or Dithiothreitol (DTT), so as to produce an at least partially, preferably totally, reduced antibody, and(C) coupling compound (II) obtained in step (A) to the at least partially, preferably totally, reduced antibody obtained in step (B), so as to produce an antibody-drug conjugate.

13. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 10, and a pharmaceutically acceptable excipient.

14. An antibody-drug conjugate according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 13, for use in the treatment of a proliferative disease such as a cancer.

15. An antibody-drug conjugate or a pharmaceutical composition for use according to claim 14, wherein the cancer is a cancer overexpressing TROP2 or HER2, preferably selected from the group consisting of a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a non-small cell lung cancer, a lung adenocarcinoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a prostate cancer, a bladder cancer, a gallbladder cancer, a cervical cancer, an uterine serous papillary carcinoma, an endometrial cancer, a nasopharyngeal cancer, a hilar cholangiocarcinoma, an oral squamous cell carcinoma, an oesophageal squamous cell carcinoma, a head-and-neck squamous cell carcinoma, and a laryngeal squamous cell carcinoma, preferably a breast cancer, more preferably a triple negative breast cancer (TNBC).

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