Mutant fab fragment for obtaining site-specific mono-or bifunctionalised conjugates

Abstract

The present invention relates to an antigen-binding mutant antibody fragment comprising a mutated heavy chain variable domain, wherein the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is substituted by a cysteine, provided that this fragment is not a Fab fragment in which the mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO: 2. The present invention also relates to nucleic acid molecules, vectors and host cells that can be used for the production of the mutant antibody fragment, final and intermediate conjugates comprising same and methods for the production of same, as well as various uses thereof.

Description

[0001] Mutant Fab fragment for obtaining site-specific mono- or bi-functionalized conjugates FIELD OF THE INVENTION The present invention relates to a mutant Fab fragment, characterized in that the amino acid residue at position 128 of the heavy chain according to the IMGT nomenclature is substituted by a cysteine, provided that said fragment is not a Fab fragment whose mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO: 2, as well as its use for obtaining site-specific mono- or bi-functionalized conjugates thereof, an intermediate conjugate, methods for preparing the final or intermediate conjugate and mono- or bi-functionalized conjugates, as well as various uses of these conjugates.STATE OF THE ART The use of isotopic imaging agents in which an imaging radioisotope (positron emitter or gamma emitter) is attached to a specific vector of receptors overexpressed by tumor cells is today a very effective approach for the diagnosis of many cancers. Moreover, the recent clinical successes of Lutathera and PSMA-617, or the older example of zevalin, confirm the effectiveness of such radiopharmaceuticals for therapy (respectively of neuroendocrine tumors, prostate cancer and non-Hodgkin's lymphoma) when the radioisotope is a beta- (eg Lu-177 or Y-90) or alpha (eg Ac-225 or Pb-212) emitter. In addition, the use of fluorescent conjugates is of growing interest for surgical assistance (FGS for Fluorescence Guided Surgery).Indeed, the presence of a fluorescent molecule helps the surgeon visualize tumor tissues invisible to the naked eye, thus allowing better delineation of tumor areas, and therefore better tumor resection, thus reducing the risk of recurrence. The development of these approaches requires modifying the biological vector in order to introduce the desired functionality (cytotoxic molecule, chelating agent for a radiometal, fluorophore, etc.). Many bioconjugation methods have thus been developed, including certain so-called "site-specific" approaches to control the attachment site on proteins. However, there are few examples of strategies that are sufficiently modular to allow the customized introduction of several functionalities.Thus, some examples of conjugates containing both a radiometal chelating agent and a fluorophore have been described, particularly in the case of vectors based on a whole antibody, but these are often constructed using sequential functionalization methods that do not allow precise control of the ratio of the two imaging probes and good reproducibility of the syntheses. Thus, a trastuzumab conjugated to a bifunctionalized tetrazine platform with a chelating agent (DOTAGA) and a fluorophore (disulphonated Cy5) has been described, but the grafting of the bifunctionalized tetrazine platform was carried out in a random and non-site-specific manner, by reaction of a bicyclo[6.1.0]nonyne (BCN) group on the amine functions of the antibody (WO2018172543A1 and Coline Canovas, et al.. Modular Assembly of Multimodal Imaging Agents through an Inverse Electron Demand Diels–Alder Reaction.Bioconjugate Chemistry, American Chemical Society, 2019, 30 (3), pp.888-897). Similarly, a diabody modified by the addition of an H6-GGC tag (6 histidines followed by two glycines and a cysteine) at the C-terminus and conjugated on these two cysteines by a fluorophore (IRDye800CW) and randomly by a radioisotope (. 124Iodine) was synthesized (Zettlitz, KA, et al., 2018. Dual-modality immunoPET and near-infrared fluorescence (NIRF) imaging of pancreatic cancer using an anti-prostate cancer stem cell antigen (PSCA) cys-diabody. J Nucl Med jnumed.117.207332). However, random grafting does not guarantee the maintenance of affinity for the antigen, nor does it allow good reproducibility of the conjugate obtained in each batch produced, which is of course not acceptable for conjugates intended to be administered to patients. A site-specific bifunctionalized trastuzumab with a chelating agent (DFO) and a fluorophore (IRDye800CW) has been described, the conjugate being based on a lysine platform trifunctionalized with DFO, IRDye800CW and a BCN group, and its grafting onto the antibody at the level of the two N-glycans, previously modified to include an azide group reacting with the BCN group of the lysine platform (Pierre Adumeau, et al.Site-Specific Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bi-modal PET / NIRF Imaging of HER2-Positive Tumors. Bioconjugate Chemistry, American Chemical Society, 2022, 33 (3), pp.530-540). However, in the context of brain tumors, conjugates must also be able to cross the blood-brain barrier (BBB). However, whole-antibody conjugates have difficulty crossing the BBB and are therefore not well suited for brain tumors. In addition, whole-antibody antibodies have relatively long in vivo lifetimes (especially IgG) due to their Fc domain, which are undesirable, especially for diagnostics. Both of these issues of highly selective barrier and in vivo lifetime could be circumvented using smaller antibody formats.Thus, a conjugate between an anti-EGFR VHH, a photosensitizing fluorophore (IRDye700DX) and a chelating agent (diethylenetriamine-pentaacetic acid or ADTP) via a tetrazine platform was synthesized. The grafting of the VHH onto the tetrazine platform was done by grafting onto a cysteine ​​added to the C-terminus of the VHH (Renard, E. et al. Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors. Cancers 2021, 13, 428). WDO2011 / 003622A1 describes a bivalent anti-HER2 nanobody comprising two VH domains fused via a linker (Nanobody 1-(VVTS)C of sequence SEQ ID NO: 17). In the 2. èmeVH domain of the nanobody, the amino acid at position 128 IMGT was replaced by a cysteine. If the nanobody was produced, its ability to bind its antigen with the same affinity as without the substitution at position 128 IMGT has not been verified. In addition, no conjugate of this nanobody with another chemical group at the thiol group of the cysteine ​​inserted at position 128 IMGT is described. WO2011 / 006914A2 describes a variant called DSM0162 of a single domain anti-TNFR1 antibody (called DOM1h-574-16), in which the amino acid at position 128 IMGT was substituted by a cysteine, as well as a conjugate of this fragment to polyethylene glycol (PEG) via a maleimide linker, with the aim of increasing its in vivo half-life. A half-life was calculated based on an ELISA assay using TNFR1 capture. However, no coupling yield is reported and it is not demonstrated that the affinity for the antigen is unaffected by the substitution.US2006 / 062784A1 describes a variant (called DOM8-24cys of sequence SEQ ID NO: 85) of a single domain anti-CD40 antibody (DOM-24, SEQ ID NO: 26), in which the amino acid at position 128 IMGT has been substituted by a cysteine, as well as the possibility of conjugating it to polyethylene glycol (PEG). However, no precise conjugation method is described, no coupling yield is mentioned and no data confirming that the variant and the conjugate maintain the same affinity for their CD40 antigen are presented. WO2018 / 106895A1 describes two fusion proteins of sequences SEQ ID NO: 816 and 817 (Table 4 of Example 2) comprising the Shiga toxin A subunit fused to a VHH of unknown antigenic specificity, in which the amino acid at position 128 IMGT has been substituted by a cysteine. However, the activity of these two fusion proteins has not been tested, and therefore it has not been demonstrated that they maintain their affinity for their antigen.Furthermore, no conjugates of these specific fusions are described. WO2022 / 133089A1 describes a single-chain antibody (sdAb) directed against CEA6 named Cap03-04-CEA6-R3-39 / 19 of sequence SEQ ID NO: 154, in which the amino acid at position 128 IMGT has been substituted by a cysteine. However, no functional data confirming that this sdAb maintains equivalent affinity for its antigen are presented. No conjugates of this specific sdAb are described either. Nevertheless, VHHs are not necessarily available for all targets of interest, while many antibodies against targets of interest are available.The use of a small antibody fragment, such as a Fab or scFv fragment (or any other antibody fragment format with a heavy chain and a light chain), could therefore make it possible to benefit from all existing antibodies targeting targets of interest while limiting the size sufficiently to allow crossing the BBB, and limiting the in vivo lifespan, due to the absence of an Fc domain. WO2010 / 115866A1 describes an anti-GPIIb / IIIa scFv (AP3 LC-HC scFV C39S S248C). In the part of the scFv corresponding to the VH domain, the amino acid at position 128 IMGT has been substituted by a cysteine. Conjugates of scFv with a chemical moiety comprising polyethylene glycol (PEG) (Example 13) or a FVIII variant (Example 14), the conjugation involving the thiol group of cysteine, are also described. However, no coupling efficiencies are reported.Furthermore, this paper does not present any data confirming that the conjugates are functional and retain their affinity for the GPIIb / IIIa antigen. On the contrary, in both cases, the raw conjugate analysis data suggest that the resulting material contains a large proportion of aggregated material, unlikely to retain its affinity for the antigen. A Fab fragment would be particularly suitable, due to its intermediate molecular weight (neither too high nor too low), allowing it to cross the BBB and having an in vivo lifetime that is neither too long nor too short. In addition, the presence of two partial heavy chains may allow double conjugation, which would be absent in VHH or ScFv-type fragments, thus allowing better specific activity in both fluorescence and isotopic imaging. However, the preparation of bioconjugates based on a Fab fragment poses challenges.Notably, a Fab fragment generally does not comprise an N-glycan that can be modified to have an azide group capable of reacting with the BCN group of a lysine further functionalized with a chelating agent and a fluorophore as described in Adumeau et al (Site-Specific Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bi-modal PET / NIRF Imaging of HER2-Positive Tumors. Bioconjugate Chemistry, American Chemical Society, 2022, 33 (3), pp.530-540). WO2018172543A1 describes a Fab' fragment bifunctionalized via a tetrazine platform. Conjugation is carried out on the cysteines of the hinge region, which has the disadvantage of causing problems of homogeneity and reproducibility. Additionally, this method requires the presence of a hinge region, which is not present in many antibody fragments, such as a Fab fragment, scFv, Fv fragment, diabody, tribody, or tetrabody.This method is therefore limited to the conjugation of Fab' fragments, with limited homogeneity and reproducibility. Junutula JR et al tested the possibility of replacing some residues of the heavy or light chain fragment of the Hu4D antibody Fab fragment with a cysteine ​​to allow site-specific conjugation (Junutula JR et al. Rapid identification of reactive cysteine ​​residues for site-specific labeling of antibody-Fabs, Journal of Immunological Methods, Volume 332, Issues 1–2, 2008, Pages 41–52, https: / / doi.org / 10.1016 / j.jim.2007.12.011). The selected position in the heavy chain corresponds to the first amino acid of the constant part, which is position 129 of the heavy chain according to the IMGT nomenclature.Such a position, located in the constant region, can again only be used for antibody fragments comprising part of the constant region, but not for antibody fragments comprising only the variable regions of the antibody, such as an scFv fragment, an Fv fragment, a diabody, a tribody, or a tetrabody. The method used is therefore limited to fragments comprising at least part of the constant region. Other positions in the VH domain allowing site-specific conjugation have been identified, but these are positions that are not very conserved within the murine and human VH genes and play a crucial role in the correct folding of the antibody heavy chain (see Example 1 below), which does not allow them to be used in a general method that can be applied to any type of initial antibody and to generate any type of antibody fragment comprising a VH domain.Finally, this paper illustrates how difficult it is to predict the effect of mutation of a cysteine ​​position on conjugation capacity, as the reactivity of thiol groups obtained by mutation of residues very close to each other to cysteine ​​is widely variable (see Table 2 of Junutula JR et al). There is therefore a need for new methods to easily obtain bioconjugates for any type of antibody fragment whose stability and affinity for the antigen are guaranteed by site-specific grafting. Such a need exists particularly for Fab fragments. The role of the endothelin axis in tumor development is widely described in the scientific literature and responds to many pro-tumor processes.Endothelin 1 acts as a true growth factor in tumor development, which results in changes in the expression of ETA, ETB and ET-1 in different tumors such as colon, breast, ovarian, prostate, kidney, bladder, lung, ENT cancer, gliomas and melanoma. More generally, stimulation of the endothelin type A receptor by endothelin 1 would induce the proliferation of epithelial tumors. This is for example the case of tumor cells from ovarian, cervical, prostate and colon cancer. ET. Bis expressed in other tumor types such as skin cancer and particularly in melanoma, Kaposi's sarcoma, breast cancer, vulvar cancer and certain gliomas. In these different tumors ETB plays a role in tumor progression and proliferation. When an antagonist of this receptor is used, there is then an inhibition of cell proliferation. Therefore, these antagonists seem to be prime candidates for the therapy of these tumors. Various clinical trials of chemical antagonists of endothelin receptors have been or are being conducted in the treatment of cancer, particularly for prostate cancer (metastatic or not), lung cancer, glioma, metastatic renal cell carcinoma, gallbladder cancer, metastatic melanoma, pancreatic cancer, and glioblastoma. These chemical antagonists are well tolerated by the body and have a completely classic biodistribution.However, they have demonstrated mixed clinical efficacy, which may suffer from the presence of high concentrations of the endogenous ligand in the tumor. In order to target ETRs and act on the endothelin axis, which is overactivated at the tumor level, exploring other therapeutic alternatives than ETR antagonists seems to be more judicious. Antibodies specifically binding to an endothelin receptor (ETA or ETB) are therefore candidates of interest, particularly in the form of conjugates with either a therapeutic molecule (for therapy) or with a molecule useful in imaging (for diagnosis and assistance in surgical tumor resection). However, endothelin receptors belong to class 1 or family A of G Protein-Coupled Receptors (GPCRs). As a result, they comprise 7 transmembrane domains (TM).Coupled to heterotrimeric G proteins at the carboxyl terminus of the GPCR, they enable signal transduction inside the cell. The TM domains consist of alpha helices connected by intracellular (i1, i2, and i3) and extracellular (e1, e2, and e3) loops. It is also common to find 2 conserved cysteines between the extracellular regions e1 and e2 (Figure 9). The extracellular loops e1, e2, and e3, to which antibodies specifically binding to ETA or ETB typically bind, are particularly short, with a length of only 5 amino acids (e1 of ETA and ETB), 20 or 22 amino acids (e2 of ETA and ETB, respectively), and 6 amino acids (e3 of ETA and ETB) (see Figure 10). This feature is not shared by many cancer antigens, which have much larger extracellular domains.This is particularly the case for HER-2 (also called ERBB2, its extracellular domain corresponds to amino acids 23-652, or 630 amino acids, see Uniprot P04626), TNFR1 (also called TNR1A or TNFRSF1A, its extracellular domain corresponds to amino acids 30-211, or 182 amino acids, see Uniprot P19438), CD40 (also called TNR5, its extracellular domain corresponds to amino acids 21-193, or 172 amino acids, see Uniprot P25942), CEA6 (also called CEAM6 or CEACAM6, its extracellular domain corresponds to amino acids 35-142, or 108 amino acids, see Uniprot P40199), and GPIIb / IIIa (GPIIb is also called ITA2B or ITGA2B, its extracellular domain corresponds to amino acids 32-993, or 962 amino acids, see Uniprot P08514; GPIIIa is also called ITB3 or ITGB3, its extracellular domain corresponds to amino acids 27-718, or 692 amino acids, see Uniprot P05106).Due to the very short size of the extracellular loops (e1, e2 and e3), antigen binding constraints are strong in the case of endothelin A and B receptors. These constraints are notably illustrated by the fact that the binding of ET-1 to ETB leads to allosteric modifications of the ETB receptor (Shihoya, W., Nishizawa, T., Okuta, A. et al. Activation mechanism of endothelin ETB receptor by endothelin-1. Nature 537, 363–368 (2016)). Therefore, much more than for other antigens, the skilled person would expect that the slightest structural modification of an antibody specifically binding to ETA or ETB that affects its conformation is likely to lead to the abolition of antigen recognition.This is further illustrated by the fact that a simple chimerization (replacement of mouse constant regions by human constant regions) of the RB49 antibody led to the production of a non-functional antibody having lost the ability to bind to the endothelin B receptor (Marie Hautiere et al., (2023) The functionality of a therapeutic antibody candidate restored by a single mutation from proline to threonine in the variable region, Human Vaccines & Immunotherapeutics, 19:3). However, there is a need for antibody fragments capable of binding to ETA or ETB, in particular after conjugation with a therapeutic molecule (for therapy) or with a molecule useful in imaging (for diagnosis and assistance in surgical tumor resection). SUMMARY OF THE INVENTION The invention meets this need.Indeed, in the context of the invention, the inventors have developed mutant Fab fragments from antibodies specifically binding to ETA or ETB, in which the amino acid residue at position 128 of the heavy chain according to the IMGT nomenclature (i.e. the last amino acid of the VH domain) is substituted by a cysteine, and shown that this mutation does not alter the affinity of the Fab for the antigen, and allows site-specific grafting (therefore with good homogeneity and good reproducibility): a) of a bi-functionalized tetrazine platform, without alteration of the disulfide point guaranteeing the stability of the Fab fragment and without alteration of the binding to the antigen (see Example 2 below) or b) of a chelating agent via a maleimide group (see Example 3 below).Furthermore, because the mutation is located in the VH domain of the heavy chain, it can be used broadly to obtain any other antibody fragment having a VH domain, without requiring further development, making the method much more versatile than those described in the prior art. Finally, the last amino acid of the VH domain is widely conserved in both humans and mice (see Example 1), which makes the proposed mutation usable in a large number of antibodies.According to a first aspect, the invention therefore relates to an antigen-binding mutant antibody fragment comprising a mutated heavy chain variable domain, the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain being substituted by a cysteine, provided that said fragment is not a Fab fragment whose mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO: 2. According to a second aspect, the invention also relates to a nucleic acid molecule or pair of nucleic acid molecules encoding the mutant antibody fragment according to the invention. According to a third aspect, the invention also relates to a vector comprising the nucleic acid molecule or pair of nucleic acid molecules according to the invention.According to a fourth aspect, the invention also relates to a host cell, a transgenic non-human animal or a transgenic plant comprising the nucleic acid molecule or the pair of nucleic acid molecules according to the invention or the vector according to the invention. According to a fifth aspect, the invention also relates to a conjugate comprising the mutant antibody fragment according to the invention, linked to at least one, in particular one or two, molecules of interest, via the cysteine, and more particularly the thiol function of the cysteine, at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment.According to a sixth aspect, the invention also relates to a method for preparing a conjugate according to the invention comprising the following steps: a) reduction of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of tris(2-carboxyethyl)phosphine (TCEP), to give a reduced mutant antibody fragment, b) reoxidation of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of dehydroascorbic acid, to give a partially reduced mutant antibody fragment, c) conjugation with at least one molecule of interest of the thiol function of the cysteine ​​at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the partially reduced mutant antibody fragment to give a conjugate according to the invention.According to a seventh aspect, the invention also relates to an intermediate conjugate comprising the mutant antibody fragment according to the invention, linked to a reactive chemical group, via cysteine, and more particularly the thiol function of cysteine, at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment. According to an eighth aspect, the invention also relates to a conjugate according to the invention, for use in: a) a method for diagnosing a tumor by imaging, b) a method for surgical treatment by tumor resection guided by fluorescence or nuclear imaging probe, c) a method for monitoring by imaging the efficacy of an antitumor treatment, or d) any combination of methods a), b) and c). DESCRIPTION OF THE FIGURES Figure 1: Figure 1 represents the antigen binding curves on CHO-ET cells. Aof the unmutated Fab fragment of the xiRA63 antibody and a conjugate obtained by random grafting of a tetrazine bifunctionalized with a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800). Figure 2: Figure 2 represents the antigen binding curves on CHO-ETA cells of the mutant Fab fragment with a cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature of the xiRA63 antibody and a conjugate obtained by site-specific grafting of a tetrazine bifunctionalized with a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800). Figure 3: Figure 3 represents the validation by MALDI-TOF mass spectrometry of the conjugation of the DFO group to the ThioFab-xiRA63 fragment (light gray line: native ThioFab-xiRA63, dark gray line: ThioFab-xiRA63-DFO). Figure 4: Figure 4 represents the validation of the affinity of the ThioFab-xiRA63-DFO fragment conjugate by flow cytometry.Comparison of the binding curve obtained by flow cytometry between ThioFab-xiRA63 (orange) and ThioFab-xiRA63-DFO (blue). To test their specificity, the antibodies were tested on CHO-ETA (solid line) and CHO-WT (dotted line) not visible in the figure. Data are presented as mean ± SEM. Statistical comparisons were performed with the paired two-tailed Student's t-test (* p < 0.05, ** p < 0.01, **** p < 0.0001). MFI: Median Fluorescence Intensity. Figure 5: Figure 5 represents the evaluation of the radiolabeling of [. 89 Zr]Zr-ThioFAb-xiRA63- DFO: (A.) HPLC-UV chromatograms of ThioFab-xiRA63-DFO detected at 280 nm before and after radiolabeling with 89 Zr with a retention time of 18 minutes for ThioFab-xiRA63-DFO. The second peak after radiolabeling at 25 minutes corresponds to gentisic acid. (B.) HPLC-radiochromatogram of [ 89[Zr]-ThioFab-xiRA63. AU: arbitrary unit. Figure 6: Figure 6 represents a summary of the in vivo experiment: (A.) Experiments and animal sacrifices. 6 mice were implanted with gli7 cells and 5 of them were injected with [89Zr]Zr-ThioFab-xiRA63 (n=5). One mouse was excluded from the experiment and another mouse had no tumor on MRI. At the end, 4 mice with a tumor were injected with [89Zr]Zr-ThioFab-xiRA63. Regarding the biodistribution analysis, all mice were used, including those without tumor. (B.) Summary table of [89Zr]Zr-ThioFab-xiRA63 injection on mice bearing a human glioblastoma tumor. Data are presented as mean ± SD Figure 7: Figure 7 represents the biodistribution of the radioligand [89Zr]Zr-ThioFab-xiRA63 over time in a nude mouse model.Biodistribution [89Zr]Zr-ThioFab-xiRA63 extrapolated from PET imaging at different time points and in key organs. (n=6 mice). Data are presented as mean ± SD, N = 6 mice, %DI.cm. -3 : percentage of injected dose per tissue volume. Figure 8: Figure 8 represents a brain tumor imaged over time by PET with the radioligand [ 89 Zr]Zr-ThioFab-xiRA63: (A.) Quantification of activity in the tumor obtained with the antibody [ 89 Zr]Zr-ThioFab-xiRA63 at 1h, 5h, 24h, 48h, 72h, D7 pi (B.) Example of a PET scan of one of the brain tumors imaged with the [ 89 Zr]Zr- ThioFab-xiRA6348h pi with the 3 visions (transverse, axial and coronal sections). DI.cm- 3: percentage of injected dose per tissue volume. Figure 9: Classical structure of GPCRs (after Bockaert et al. Bull. Acad. Natle Méd., 2012, 196, no. 9, 1765-1775). Receptors with 7 TM domains linked by 3 extracellular loops (e1, e2 and e3) and 3 intracellular loops (i1, i2, i3). The N-terminal domain is located in the extracellular region while the C-terminal domain composed of an 8 èmehelix is ​​located on the intracellular side and can interact with intracellular proteins (GIPs for "GPCR Interacting Proteins"). Figure 10: 2-dimensional (2D) structure of human ETA and ETB obtained by GPCRdb. Figure 11: Competition assay protocol. Cells were pre-incubated overnight at 4°C in the presence of a molar excess (500nM) of Fab-xiRB49-P125T or ThioFab-xiRB49-P125T antibody fragments. Subsequently, the control range of xiRB49-P125T antibodies was added to each cell point. The ranges read by flow cytometry were revealed by the secondary polyclonal goat anti-human IgG antibody coupled to Alexa Fluor488 (1110120) targeting only the Fc region of the antibodies. Throughout this experiment, three washes with 1X PBS (pH: 7.4) were performed between each step to remove non-specific signal.Figure 12: Thermal denaturation curves and inflection temperatures Ta and T12 of the xiRB49-P125T, Fab-xiRB49-P125T, and ThioFab-xiRB49-P125T antibodies. Figure 13: Flow cytometric binding curves of the Fab-xiRB49-P125T and ThioFab-xiRB49-P125T antibodies allowing the determination of their apparent Kd and Bmax. Figure 14: Flow cytometric binding curves of the competition assay between xiRB49-P125T and Thio-Fab-xiRB49-P125T or Fab-xiRB49-P125T. Figure 15: Flow cytometric binding curves with an anti-Fd secondary antibody. Summary diagram of secondary antibodies recognizing Fab-xiRB49-P125T and ThioFab-xiRB49-P125T. DETAILED DESCRIPTION OF THE INVENTION General Definitions By "a" or "an", we mean one or more.In other words, when "a" or "an" is used with respect to a characteristic, it covers both embodiments with the characteristic of interest occurring only once and those with the characteristic of interest occurring several times. For example, an antibody fragment that comprises a mutated heavy chain variable domain may comprise a single mutated heavy chain variable domain (in the case of an Fv or scFv fragment in particular) or several mutated heavy chain variable domains (in the case of a Fab fragment, a diaboby, tribody or tetrabody in particular).As used herein to define products, compositions, and methods, the terms "comprising" (and any form of "comprising," such as "comprises" and "includes"), "having" (and any form of "having," such as "has" and "has"), "including" (and any form of inclusion, such as "includes" and "includes"), or "containing" (and any form of "containing," such as "contains" and "contains") are open-ended and do not exclude additional, unmentioned method elements or steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence is part of the final amino acid sequence of the polypeptide. Such a polypeptide may have up to several hundred additional amino acid residues. "Consisting essentially of" or "consisting essentially of" is intended to exclude other components or steps of any essential importance.Thus, a polypeptide "consists essentially of" an amino acid sequence when such an amino acid sequence is present with possibly only a few additional amino acid residues (e.g., a peptide of at most 20 amino acids, such as a 6-histidine Hisx6 tag, may additionally be present). "Consisting of" or "consisting of" means excluding more than trace amounts of other components or steps. For example, a polypeptide "consists of" an amino acid sequence when the polypeptide contains no other amino acids than the stated amino acid sequence. "Antibody" or "immunoglobulin" or "Ig" means a molecule comprising at least one binding domain for a given antigen and a constant domain comprising an Fc moiety capable of binding to FcR receptors.In most mammals, such as humans and mice, an antibody is composed of 4 polypeptide chains: 2 heavy chains and 2 light chains linked together by a variable number of disulfide bridges ensuring flexibility to the molecule. Each light chain consists of a constant domain (CL) and a variable domain (VL); the heavy chains being composed of a variable domain (VH) and 3 or 4 constant domains (CH1 to CH3 or CH1 to CH4) depending on the antibody isotype. In a few rare mammals (camelidae, notably camels and llamas) and in cartilaginous fish (notably sharks), antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (VH) and a constant region. The variable domains are involved in antigen recognition, while the constant domains are involved in the biological, pharmacokinetic and effector properties of the antibody.The variable region differs from one antibody to another. Indeed, the genes coding for the heavy and light chains of antibodies are generated by recombination of three and two distinct gene segments, respectively, called VH, DH, and JH-CH for the heavy chain and VL and JL-CL for the light chain. The CH and CL segments do not participate in recombination and form the constant regions of the heavy and light chains, respectively. Recombination of the VH-DH-JH and VL-JL segments forms the variable regions of the heavy and light chains, respectively. The VH and VL regions each have three hypervariable zones or complementarity-determining regions (CDRs), called CDR1, CDR2, and CDR3, with CDR3 being the most variable, since it is located at the recombination zone.These three CDR regions, and particularly the CDR3 region, are located in the part of the antibody that will be in contact with the antigen and are therefore very important for antigen recognition. Thus, antibodies retaining all three CDR regions and each of the heavy and light chains of an antibody largely retain the antigenic specificity of the original antibody. In a number of cases, an antibody retaining only one of the CDRs, and particularly the CDR3, also retains the specificity of the original antibody. The CDR1, CDR2, and CDR3 regions are each preceded by the FR1, FR2, and FR3 regions, respectively, corresponding to the framework regions (FR) that vary the least from one VH or VL segment to another. The CDR3 region is also followed by an FR4 framework region. The CDRs of an antibody are defined from the amino acid sequence of its heavy and light chains in relation to criteria known to those skilled in the art.Different methods for determining CDRs have been proposed, and the portion of the amino acid sequence of an antibody heavy or light chain variable region defined as a CDR varies depending on the method chosen. A first method of determination is that proposed by Kabat et al (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In this method, CDRs are defined by searching for the amino acids responsible for binding to the antibody antigen. A 2. èmeThis method was proposed by the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT), based this time on the determination of hypervariable regions. In this method, a unique numbering was defined to compare variable regions regardless of the antigen receptor, chain type or species (Lefranc et al. 2003). This numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementarity determining regions (CDR1-IMGT: positions 27 to 38, CDR2-IMGT: positions 56 to 65 and CDR3-IMGT: positions 105 to 117). There are still other methods of determination, which will not be detailed here, but are known to those skilled in the art.A program for determining CDRs from the amino acid sequence of an antibody heavy or light chain according to different nomenclatures (notably Kabat and IMGT) is the abYsis “Annotate” tool available at http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi. For CDR determination according to IMGT nomenclature only, the IMGT “DomainGapAlign” tool available at https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi described in two articles by Ehrenmann F. et al (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6:737-749 (2011)) can also be used.Unlike variable regions, the sequence of which varies greatly from one antibody to another, constant regions are characterized by an amino acid sequence that is very similar from one antibody to another, characteristic of the species and the isotype, with possibly some somatic mutations. The heavy chain constant region is composed of the N C-terminal of a CH1 domain, a hinge region and then the CH2 and CH3 or CH2 to CH4 domains (depending on the isotype). The "Fc fragment" is naturally composed of the constant region of the heavy chain excluding the CH1 domain, i.e. the lower hinge region and the CH2 and CH3 or CH2 to CH4 constant domains (depending on the isotype). The positions of the CH1 to CH3 or CH1 to CH4 domains and the hinge region of the different antibody isotypes of many species are known to those skilled in the art, and can be found in particular on the IMGT website.The Fc fragment is glycosylated at the CH2 domain with the presence, on each of the 2 heavy chains, of an N-glycan linked to the asparagine residue at position 297 (Asn 297). The following binding domains, located in the Fc, are important for the biological properties of the antibody: - FcRn receptor binding domain, involved in the pharmacokinetic properties (in vivo half-life) of the antibody: Various data suggest that certain residues located at the interface of the CH2 and CH3 domains are involved in binding to the FcRn receptor. - C1q complement protein binding domain, involved in the CDC response (for "complement-dependent cytotoxicity"): located in the CH2 domain; - FcR receptor binding domain, involved in phagocytosis or ADCC responses (for "antibody-dependent cellular cytotoxicity"): located in the CH2 domain.The light chain constant region (for antibodies having two heavy chains and two light chains) is composed of a single domain called CL. A "monoclonal antibody" or "monoclonal antibody composition" means a composition comprising antibody molecules having an identical and unique antigenic specificity. The antibody molecules present in the composition may vary in their post-translational modifications, and in particular in their glycosylation structures or their isoelectric point, but have all been encoded by the same heavy and light chain sequences and therefore have, before any post-translational modification, the same protein sequence.Some differences in protein sequences, linked to post-translational modifications (such as the cleavage of the C-terminal lysine of the heavy chain, the deamidation of asparagine residues and / or the isomerization of aspartate residues), may nevertheless exist between the different antibody molecules present in the composition. Antibodies can be of several "isotypes", depending on the nature of their constant region: the constant regions γ, α, µ, ɛ and δ correspond respectively to the immunoglobulins IgG, IgA, IgM, IgE and IgD. The constant regions γ include several subtypes: γ1, γ2, γ3, these three types of constant regions having the particularity of fixing human complement, and γ4, thus creating the subisotypes IgG1, IgG2, IgG3, and IgG4.By "chimeric" antibody is meant an antibody which contains a natural variable region (light chain and heavy chain) derived from an antibody of a given species in association with the constant regions of light chain and heavy chain of an antibody of a species heterologous to said given species. Advantageously, if the monoclonal antibody composition for use as a medicament according to the invention comprises a chimeric monoclonal antibody, the latter comprises human constant regions. Starting from a non-human antibody, a chimeric antibody can be prepared using genetic recombination techniques well known to those skilled in the art.For example, the chimeric antibody may be produced by cloning for the heavy chain and the light chain a recombinant DNA comprising a promoter and a sequence coding for the variable region of the non-human antibody, and a sequence coding for the constant region of a human antibody. For methods of preparing chimeric antibodies, one may for example refer to the document Verhoeyen et al (Verhoeyen et al. BioEssays, 8: 74, 1988). By "humanized" antibody is meant an antibody which contains CDR regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies. In addition, some of the residues in the backbone segments (referred to as FRs) can be modified to maintain binding affinity (Jones et al. Nature, 321:522-525, 1986; Verhoeyen et al. Science, 239:1534-1536, Verhoeyen et al. BioEssays, 8:74, 1988; 1988; Riechmann et al.Nature, 332: 323-327, 1988). The humanized antibodies according to the invention can be prepared by techniques known to those skilled in the art such as “CDR grafting”, “resurfacing”, SuperHumanization, “Human string content”, “FR libraries”, “Guided selection”, “FR shuffling” and “Humaneering” technologies, as summarized in the review by Almagro et al (Almagro et al. Frontiers in Bioscience 13, 1619-1633, January 1, 2008). By “human” antibody is meant an antibody that contains only amino acid sequences of human origin. A human antibody recognizing a given antigen can be obtained either by immunizing a healthy human volunteer or by other methods, such as phage display (see for example Hammers CM, Stanley JR. Antibody phage display: technique and applications. J Invest Dermatol. 2014 Feb;134(2):1-5. doi: 10.1038 / jid.2013.521) or the immunization of mammals (especially mice) in which the native immunoglobulin gene loci have been deleted and the human immunoglobulin gene loci have been inserted (see e.g. Brüggemann, M., Osborn, M.J., Ma, B. et al. Human Antibody Production in Transgenic Animals. Arch. Immunol. Ther. Exp. 63, 101–108 (2015). https: / / doi.org / 10.1007 / s00005-014-0322-x). An "antigen-binding antibody fragment" means a fragment of an antibody that retains the antigen-binding domain and thus has the same antigenic specificity as the original antibody.Examples of such antigen-binding antibody fragments include: - for double-chain antibodies (from most mammals, including mice and humans): Fv, scFv, Fab, F(ab')2, Fab' fragments, diabodies (generally referred to as "diabodies"), triantibodies (generally referred to as "tribodies"), tetraantibodies (generally referred to as "tetrabodies"); and - for single-chain antibody fragments (from camelids or cartilaginous fish), VHH (VH domain of a camelid antibody) or VNAR (VH domain of a cartilaginous fish antibody) fragments. By "Fv fragment" or "variable fragment" is meant an antibody fragment formed from the VH and VL domains associated non-covalently.By "scFv fragment" or "scFv" or "single chain variable fragment" or "single chain FV" is meant a fragment formed by the fusion of the VH domain to the VL domain of an antibody via a peptide called a "linker" consisting of a reduced number of amino acids (generally 15 to 20), either in the VH-linker-VL format or in the VL-linker-VH format. The amino acids of the linker are most often chosen from glycine, serine, threonine, asparagine, alanine and proline, with glycine and serine being most often used in the majority. A common scFv format is of the formula VH-linker-VL where the linker is of the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23). A scFv generally has a molar mass of approximately 25 kDa.A "Fab fragment" is a fragment formed by joining the portion of the heavy chain upstream of the papain cleavage site (the CH1 domain is included, but not the hinge region or the other CH domains) and the entire light chain of the antibody of interest. The two chains are linked by a disulfide bridge as in whole Ig. Fab fragments are monomers of approximately 50 kDa. A "Fab' fragment" is a fragment formed by joining the portion of the heavy chain upstream of the pepsin cleavage site (the CH1 domain and the hinge region are included, but not the other CH domains) and the entire light chain of the antibody of interest. A "F(ab')2 fragment" is a fragment formed by joining two Fab' fragments by disulfide bridges at the cysteines of the hinge region. By "diabody" or "diaantibody" we mean a dimer composed of two scFv fragments.The two scFv fragments can be identical (the diabody then has a single antigenic specificity) or different (the diabody can then be bispecific if the two scFvs recognize different antigens). A diabody generally has a molar mass of approximately 50 kDa (twice that of a scFv). By "triantibody" or "tribody" or "triabody" is meant a trimer composed of three scFv fragments. The three scFv fragments can be identical (the tribody then has a single antigenic specificity) or different (the tribody can then be bi- or tri-specific depending on the antigenic specificity of the three scFvs). A tribody generally has a molar mass of approximately 75 kDa (three times that of a scFv). By "tetraantibody" or "tetrabody" is meant a quadrimer composed of four scFv fragments.The four scFv fragments can be identical (the tetrabody then has a single antigenic specificity) or different (the tetrabody can then be bi-, tri- or quadri-specific depending on the antigenic specificity of the four scFvs). A tetrabody generally has a molar mass of approximately 100 kDa (4 times that of a scFv). By "miniantibody" or "minibody", we mean a fragment formed by the fusion of a scFv to a domain that tends to dimerize, and in particular to a CH3 domain. A minibody generally has a molar mass of approximately 75 kDa. By "nanobody" or "single domain antibody", we mean the variable domain of the heavy chain of a single chain antibody. A nanobody can in particular be a VHH or a VNAR. By "VHH", we mean the variable domain of the heavy chain of a single chain antibody of camelid (in particular llama and alpaca).The term "VNAR" refers to the variable domain of the heavy chain of a single-chain antibody from cartilaginous fish (especially shark). A VHH or VNAR generally has a molecular weight of about 12 to 15 kDa. An antibody or antibody fragment "specifically binds to" an antigen when it binds to that antigen with a significantly higher binding affinity (e.g., at least 10 times higher) than it binds to other antigens. There are various techniques for measuring the affinity of an antibody for an antigen, well known to those skilled in the art. The term "antigen" refers to a substance that, when administered to a subject, can stimulate the immune system and cause the production of antibodies recognizing that substance.In terms of composition, an antigen can be a peptide or a protein, but can also be a nucleic acid, a lipid, a sugar, etc. In terms of origin, an antigen can be a cancer antigen, an antigen of a pathogenic microorganism (for example, a virus, a bacterium, a parasite), or a self-antigen. By "cancer antigen" is meant an antigen expressed on the surface of a cancer cell. This cancer antigen is preferably expressed specifically on the surface of cancer cells (for example, due to a tumor-specific mutation), to the exclusion of the subject's healthy cells, expressed predominantly on the surface of cancer cells (i.e., the majority of the subject's healthy cells do not express the cancer antigen), or overexpressed on the surface of cancer cells (i.e.,its level of expression on the surface of cancer cells is significantly, for example at least 2, at least 5 or at least 10 times, higher than the level of expression on the surface of healthy cells). Many cancer antigens are known in the prior art. By "endothelin" is meant a neuropeptide secreted by the vascular endothelium, having a potent vasoconstrictor effect on smooth muscle cells. In humans, there are three endothelins called ET-1, ET-2 and ET-3 composed of 21 amino acids and comprising two disulfide bridges between cysteines 1 and 15 and 3 and 11. ET-1, ENT-2 and ET-3 are derived from three much larger precursors: preproendothelin 1, 2 and 3 (polypeptides of approximately 200 amino acids).Endothelins or ETs are a family of 3 peptides of 21 amino acids, ET-1, ET-2 and ET-3, which bind to two distinct receptors with 7 transmembrane domains: ETA (for "Endothelin subtype A receptor", also called EDNRA, information on the human gene coding for ETA is available on the NCBI Entrez Gene database under accession number Gene ID: 1909) and ETB (for "Endothelin subtype B receptor", also called EDNRB, information on the human gene coding for ETB is available on the NCBI Entrez Gene database under accession number Gene ID: 1910), these two receptors belonging to the family of G protein-coupled receptors (GPCR). The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes.ET-1 plays a crucial role in regulating physiological smooth muscle motility, but ET-1 is also implicated in a wide variety of pathologies, including hypertension, heart failure, renal disorders, and infectious diseases. In addition, the ET axis is overexpressed in cancer of different organs contributing to tumor growth by acting on cell proliferation, survival, migration, differentiation, angiogenesis, and recruitment of inflammatory cells. ETAs are upregulated in prostate, ovarian and breast cancers while ETBs are overexpressed and deregulated in melanoma, lung, kidney and vulvar cancers ref (Rosanò, L., Spinella, F., Bagnato, A., 2013. Endothelin 1 in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 13, 637–651).Pathogenic microorganism antigen means an antigen expressed on the surface of a pathogenic microorganism or cells infected by a pathogenic microorganism. Self-antigen means an antigen naturally expressed by certain healthy cells of a subject. Self-tolerance mechanisms exist, and the immune system does not normally generate antibodies that bind specifically to a self-antigen. However, sometimes the control mechanisms are insufficient and a subject produces antibodies against one or more self-antigens. This is called an autoimmune disease.The "identity percentages" referred to in the context of the disclosure of the present invention are determined on the basis of a global alignment of the sequences (nucleic or protein) to be compared, that is to say on an alignment of the sequences taken in their entirety over their entire length using any algorithm well known to those skilled in the art such as the Needleman and Wunsch algorithm (Needleman and Wunsch. J.Mol. Biol. 48,443-453, 1970). This sequence comparison can be carried out using any software well known to those skilled in the art, for example the needle software using the "Gap open" parameter equal to 10.0, the "Gap extend" parameter equal to 0.5 and a "Blosum 62" matrix. The needle software is for example available on the website ebi.ac.uk worldwide under the name "Align".An "amino acid equivalent" to another amino acid means any amino acid whose structure is close to that of the original amino acid and is therefore unlikely to alter the biological activities of the antibody. Examples of equivalent amino acids are presented in the following Table 1: [Table 1] Original amino acid Substitution(s) Ala (A) Val, Gly, Pro Arg (R) Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (G) Asp Gly (G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met (M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) Tyr Tyr (Y) Phe, Trp Val (V) Leu, Ala Table 1. Substitutions by equivalent amino acids By "molecule of interest" is meant a molecule useful for an application of the conjugate according to the invention, in particular in the field of diagnosis or in vitro research tools.Such molecules of interest include in particular molecules that are detectable or likely to become detectable, affinity molecules (and pharmacomodulatory molecules. By "molecule that is detectable or likely to become detectable" is meant a molecule that can be detected, directly (detectable molecule) or indirectly after modification (molecule likely to become detectable), using medical imaging techniques known to those skilled in the art, in particular isotopic imaging (such as positron emission tomography (PET), single-photon emission tomography (SPECT) and Cerenkov Luminescence imaging (CLI)); optical imaging; fluorescence imaging. These different medical imaging techniques use different types of detectable molecules."Isotope imaging" or "nuclear imaging" detects "radionuclides" or "radioisotopes," i.e., radioactive isotopes of a given atom. In positron emission tomography (PET) and single-photon emission computed tomography (SPECT), gamma rays from a radionuclide with direct or indirect emission of gamma photons are detected by gamma cameras. In SPECT, the radionuclides used directly emit a gamma ray when they decay, while in PET, the radionuclides used emit a positron during their decay, which, after annihilation with a surrounding electron, leads to the emission of 2 gamma rays, thus obtained indirectly. In Cerenkov Luminescence Imaging (CLI), Cerenkov radiation (CR) is detected with optical imaging instruments (we are therefore at the junction of isotopic imaging and optical imaging).Radioisotopes can be present directly in the probe (detectable molecule), or a probe with a chelating agent (molecule likely to become detectable) can also be used, which is coupled just before administration with a radiometal. A "chelating agent" means a substance that has the ability to bind metal cations by forming a stable complex. A "radiometal" means a radioactive isotope of a metal atom. Optical imaging can detect absorbance, particularly that of a chromophore. A "chromophore" means a group of atoms containing one or more double bonds, and forming with the rest of the molecule a sequence of conjugated double bonds, thus giving its color to the molecule containing it. Optical imaging can also detect Cerenkov radiation (CR). Fluorescence imaging detects the fluorescence emission of a probe containing a fluorescent molecule or fluorophore.A "fluorescent molecule" or "fluorophore" means a molecule that emits light when exposed to light or other radiation, the emitted light having a longer wavelength than the light to which the molecule was exposed. Many fluorophores are molecules comprising several conjugated aromatic rings or planar, cyclic molecules possessing one or more π bonds. An "affinity molecule" means a molecule that can be selectively and non-covalently linked to another partner chemical group, thus forming a complex. A "pharmacomodulatory molecule" means a chemical molecule, whether synthetic or natural, capable of modifying the pharmacokinetic properties of another molecule to which it is linked. "Halo" means bromo, chloro, iodo, or fluoro. "(C1-C. x)alkyl”, means a saturated, linear or branched hydrocarbon chain comprising 1 to x carbon atoms. A (C1-C6)alkyl group is therefore a saturated, linear or branched hydrocarbon chain comprising 1 to 6, preferably 1 to 4, carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl groups. “Cycloalkane” means a saturated hydrocarbon monocycle or polycycle comprising one or more, in particular 1 or 2, cycles, the cycles being able to be joined (i.e. they share a bond between two carbon atoms) or bridged (i.e. they share a sequence of at least three carbon atoms). Preferably, the cycloalkane will comprise 3 to 9 carbon atoms. It will preferably be cyclopropane, cyclohexane, cyclooctane, bicyclononane, norbonane. By "heterocycloalkane" is meant a cycloalkane in which one or more, in particular 1, 2, 3 or4, carbon atoms have been replaced respectively by a heteroatom chosen from O, S and N, in particular O or N. It may in particular be piperidine, 1,2-piperazine, 1,4-piperazine, pyrrolidine, morpholine. By "cycloalkene" is meant a hydrocarbon monocycle or polycycle comprising at least one double bond and comprising one or more, in particular 1 or 2 cycles, the cycles being able to be joined (i.e. they share a bond between two carbon atoms) or bridged (i.e. they share a sequence of at least three carbon atoms). Preferably, the cycloalkene will comprise 3 to 10 carbon atoms. It will preferably be cyclopropene, cyclohexene, cyclooctene, bicyclononene, norbornene. By "heterocycloalkene" is meant a cycloalkene in which one or more, in particular 1, 2, 3 or 4, carbon atoms have been replaced respectively by a heteroatom chosen from O, S and N, in particular O or N. This may in particular be 3-pyrroline,1,2,3,6-tetrahydropyridazine. By "cycloalkyne" is meant a hydrocarbon monocycle or polycycle comprising at least one triple bond and comprising one or more, in particular 1 or 2 cycles, the cycles being able to be joined (i.e. they share a bond between two carbon atoms) or bridged (i.e. they share a sequence of at least three carbon atoms). Preferably, the cycloalkyne will comprise 3 to 9 carbon atoms. It will preferably be cyclooctyne, bicyclononyne. By "heterocycloalkyne" is meant a cycloalkyne in which one or more, in particular 1, 2, 3 or 4, carbon atoms have been replaced respectively by a heteroatom chosen from O, S and N, in particular O or N. It may in particular be azacyclooctyne. By "aromatic cycle" is meant an aromatic hydrocarbon monocycle or polycycle comprising one or more, in particular 1 or 2, joined cycles. It will advantageously comprise 6 to 14, in particular 6 to 10 atoms ofcarbon. It can be a benzene or a naphthalene. When an aromatic ring is joined with another ring, this means that the two rings share a bond between two carbon atoms. By "heteroaromatic ring" is meant an aromatic monocycle or polycycle comprising one or more, in particular 1, 2 or 3, joined rings, the atoms constituting the ring or rings comprising one or more, in particular 1, 2, 3 or 4 heteroatoms chosen from O, S and N, in particular O and N, the other atoms constituting the ring or rings being carbon atoms. The ring or rings will advantageously be formed from 5 to 15, in particular 5 to 10 atoms. Preferably, each ring will be a 5- or 6-membered ring. It can in particular be tetrazine, triazine, 1,2,3-triazole, pyridazine, thiophene, thiazole, thiadiazole, pyridine. Mutant Antibody Fragment In a first aspect, the invention relates to an antigen-binding mutant antibody fragment comprising a variable domain ofmutated heavy chain, the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain being substituted by a cysteine, provided that said fragment is not a Fab fragment whose mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO: 2. Type of fragment The invention covers any type of mutant antibody fragment binding to the antigen and comprising a mutated heavy chain variable domain as defined herein. In particular, the invention covers both a fragment: a) which further comprises a light chain variable domain (derived from antibodies having two heavy chains and two light chains) and b) which does not comprise a light chain variable domain (derived from single-chain antibodies comprising only one heavy chain). Fragments which further comprise a light chain variable domain (derived from antibodieshaving two heavy chains and two light chains) are however preferred, because the amino acid sequences of the heavy and light chains of many antibodies having two heavy chains and two light chains against many antigens are available in the prior art and therefore accessible to those skilled in the art. Among the fragments which further comprise a light chain variable domain (derived from antibodies having two heavy chains and two light chains), the mutant antibody fragment according to the invention can be any type of fragment having a mutated heavy chain variable domain (VH) and a light chain variable domain (VH), but can advantageously be chosen from a Fab fragment, an scFv fragment, an Fv fragment, a diabody, a tribody, a tetrabody, and a minibody. In particular, the Fab, scFv and diabody fragments are preferred (especially the Fab fragment), because they are those with the lowest molecular weight, and therefore the mosteven to cross the blood-brain barrier and with a short in vivo lifespan suitable for diagnosis. Among the fragments which do not include a light chain variable domain (derived from single-chain antibodies comprising only a heavy chain), the mutant antibody fragment according to the invention may in particular be a nanobody, advantageously chosen from the VHH and VNAR fragments. The substitution of the amino acid residue in position 128 according to the IMGT nomenclature of the heavy chain variable domain by a cysteine ​​aims to allow selective conjugation on this cysteine. Indeed, the inventors have shown that, subject to using a particular method, it is possible to carry out specific conjugation on this cysteine ​​in a Fab fragment, without affecting the other cysteines present in this fragment. The mutant antibody fragments according to the invention are intended to be conjugated to groups useful for in vivo use (diagnosticin particular), in particular in humans. Consequently, the mutant antibody fragment according to the invention is advantageously chimeric (if it contains part of the constant region, such as a Fab, then this part is human), humanized or human. For use in a non-human animal, a mutant antibody fragment according to the invention will be used by analogy (if it contains part of the constant region, such as a Fab, then this part is from the species of the animal in which the fragment is used), modified to replace the framework regions of the variable domains with regions from the species of the animal in which the fragment is used, or entirely from the species in which the fragment is used. Antigen recognized by the mutant antibody fragment The antigen recognized by the antibody fragment (in particular Fab, scFv or diabody, in particular Fab) mutant according to the invention is of little importance, the invention being applicable regardless of the antigen to which the original antibodybinds specifically. However, the mutant antibody fragments according to the invention are intended to be conjugated to groups useful for in vivo use (diagnosis in particular), and the antigen will therefore advantageously be chosen from antigens of diagnostic interest. In particular, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to a cancer antigen, an antigen of a pathogenic microorganism (in particular a viral, bacterial or parasitic antigen), or a self-antigen. A mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binding specifically to a cancer antigen will be useful for in vivo uses in the context of the diagnosis, and possibly the treatment, of cancers, and is particularly preferred. A mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention can bind specifically toany cancer antigen of interest. Cancer antigens of interest include, in particular, CD19, CD20, CD22, CD38, HER2, CEA, PD1, PD-L1, CTLA-4, B7-H3 (also known as CD276), EpCAM, Folate receptor alpha, BCMA, LAG-3, gp100, GD2, TROP-2, Nectin-4, CD79b, CCR4, PDGRFα, SLAMF7, EGFR, and endothelin receptor A or receptor B. The mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention may in particular bind specifically to endothelin receptor A or receptor B. A mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention specifically binding to an antigen of a pathogenic microorganism will be useful for in vivo uses in the context of the diagnosis, and possibly the treatment, of an infection by the pathogenic microorganism from which the antigen originates. A mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention specifically binding to an antigen of a pathogenic microorganism will be useful for in vivo uses in the context of the diagnosis, and possibly the treatment, of an infection by the pathogenic microorganism from which the antigen originates.specifically to a self-antigen will be useful for in vivo uses in the diagnosis, and possibly treatment, of an autoimmune disease involving the production of antibodies against a self-antigen. Preferred antibodies from which the mutant antibody fragment is derived The inventors have demonstrated that it is possible to specifically conjugate a mutant Fab fragment derived from an anti-endothelin receptor A RA63 antibody whose alanine residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain has been substituted by a cysteine. As explained in Example 1 below, the residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain of antibodies having a murine or human FR4 region is, unless there is a somatic mutation at this position, a serine (in the majority of cases) or an alanine. The mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention is therefore advantageouslyderived (by substitution of the residue at position 128 according to the IMGT nomenclature of the antibody heavy chain variable domain by a cysteine) from an antibody fragment whose amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is a serine or an alanine. The mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention can therefore be derived (by substitution of the residue at position 128 according to the IMGT nomenclature of the antibody heavy chain variable domain by a cysteine) from an antibody fragment whose amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is a serine. Alternatively, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention can therefore be derived (by substitution of the residue in position 128 according to the IMGT nomenclature of the variable domain of the heavy chain of the antibody by acysteine) of an antibody fragment whose amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is an alanine. In this case, the mutant antibody fragment according to the invention may be derived (by substitution of the residue at position 128 according to the IMGT nomenclature of the antibody heavy chain variable domain by a cysteine) from a fragment of the antibody RB49 or RA63 or a variant thereof, which bind specifically to the endothelin receptor B. The RB49 antibody specifically binds to endothelin receptor B and has 6 CDRs defined according to the IMGT nomenclature as: - heavy chain: GYTFISYW (SEQ ID NO: 3, CDR1-H), IDPDSGGT (SEQ ID NO: 4, CDR2-H) and AREGDYAWFAY (SEQ ID NO: 5, CDR3-H), and - light chain: QSIVHSNGNTY (SEQ ID NO: 6, CDR1-L), KVS (SEQ ID NO: 7, CDR2-L) and FQGSHVPWT (SEQ ID NO: 8, CDR3-L). The RA63 antibody specifically binds to endothelin receptor A and has 6 CDRs defined according to theIMGT nomenclature as: - heavy chain: GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) and VSSYYSGSFFAY (SEQ ID NO: 11), and - light chain: SQSIVYSNGKIYL (SEQ ID NO: 12), KVS (SEQ ID NO: 13) and FQGSHLPLT (SEQ ID NO: 14). Thus, in a preferred embodiment, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor B, and comprises (in addition to the substitution of the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain by a cysteine): - a heavy chain variable domain (VH) comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having according to the IMGT nomenclature for respective sequences GYTFISYW (SEQ ID NO: 3), IDPDSGGT (SEQ ID NO: 4) and AREGDYAWFAY (SEQ ID NO: 5) or a sequence with at least 80% identity with GYTFISYW (SEQ ID NO: 3), IDPDSGGT (SEQ ID NO: 4) or AREGDYAWFAY (SEQ ID NO: 5), and ...), and - a heavy chain variable domain (VH) comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having according to thelight chain (VL) comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having according to the IMGT nomenclature for respective sequences QSIVHSNGNTY (SEQ ID NO: 6), KVS (SEQ ID NO: 7) and FQGSHVPWT (SEQ ID NO: 8) or a sequence with at least 80% identity with QSIVHSNGNTY (SEQ ID NO: 6), KVS (SEQ ID NO: 7) or FQGSHVPWT (SEQ ID NO: 8). When at least one of the 6 CDRs defined in the paragraph above does not have 100% identity with SEQ ID NO: 3 to 8, the amino acid(s) which differ from the native sequence are preferably substituted by an equivalent amino acid, so as to maintain the affinity for the ETB antigen. Even more preferably, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor B, and further comprises (in addition to the CDRs indicated above which correspond to that of the RB49 antibody or to variants thereof): - a VH domain having the sequence SEQ IDNO: 15 (QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVPVSA) or a sequence having at least 80% identity with SEQ ID NO: 15 such as the sequence SEQ ID NO: 27 (QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVTVSC), and - a VL domain having the sequence SEQ ID NO: 16 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK) or a sequence having at least 80% identity with SEQ ID NO: 16. The sequences of the VH and VL domains most important for antigen binding are those of the 6 CDRs. Therefore, in a preferred embodiment, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor B, and comprises: - a VH domain having a sequence having at least 80% identity with SEQID NO: 15 and comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having, according to the IMGT nomenclature, the respective sequences GYTFISYW (SEQ ID NO: 3), IDPDSGGT (SEQ ID NO: 4) and AREGDYAWFAY (SEQ ID NO: 5); and - a VL domain having a sequence having at least 80% identity with SEQ ID NO: 16 comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having, according to the IMGT nomenclature, the respective sequences QSIVHSNGNTY (SEQ ID NO: 6), KVS (SEQ ID NO: 7) and FQGSHVPWT (SEQ ID NO: 8). Particularly advantageously, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor B, and comprises: - a VH domain having the sequence SEQ ID NO: 15 or SEQ ID NO: 27, and - a VL domain having the sequence SEQ ID NO: 16. In another preferred embodiment, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention bindsspecifically to the endothelin A receptor, and comprises (in addition to the substitution of the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain by a cysteine): - a VH domain comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having according to the IMGT nomenclature for respective sequences GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) and VSSYYSGSFFAY (SEQ ID NO: 11) or a sequence with at least 80% identity with GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) or VSSYYSGSFFAY (SEQ ID NO: 11), and - a VL domain comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having according to the IMGT nomenclature for respective sequences SQSIVYSNGKIYL (SEQ ID NO: 12), KVS (SEQ ID NO: 13) and FQGSHLPLT (SEQ ID NO: 14) or a sequence with at least 80% identity with SQSIVYSNGKIYL (SEQ ID NO: 12), KVS (SEQ ID NO: 13) or FQGSHLPLT (SEQ ID NO: 14). When at least one of the 6 CDRs defined in the paragraph above does not have 100%of identity with SEQ ID NO: 9 to 14, the amino acid(s) which differ from the native sequence are preferably substituted by an equivalent amino acid, so as to maintain the affinity for the ETA antigen. Even more preferably, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor A, and further comprises (in addition to the CDRs indicated above which correspond to that of the RA63 antibody or to variants thereof): - a VH domain having the sequence SEQ ID NO: 17 (EVQLVESGGGLVQPKGSLKLSCAASGFTFNIYAMNWIRQAPGKGLEWIARIRSKSNNYATYYADSVKDRFTI SRDDSQNMVYLQMNNLKTEDTAMYYCVSSYYSGSFFAYWGQGTLVTVSA) or a sequence having at least 80% identity with SEQ ID NO: 17 such as the sequence SEQ ID NO: 28 (EVQLVESGGGLVQPKGSLKLSCAASGFTFNIYAMNWIRQAPGKGLEWIARIRSKSNNYATYYADSVKDRFTI SRDDSQNMVYLQMNNLKTEDTAMYYCVSSYYSGSFFAYWGQGTLVTVSC), and - a VL domain having the sequence SEQ ID NO: 18(DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKIYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG TDFTLKISRVEAEDLGVYYCFQGSHLPLTFGAGTKLELKR) or a sequence having at least 80% identity with SEQ ID NO: 18. The sequences of the VH and VL domains most important for antigen binding are those of the 6 CDRs. Therefore, in a preferred embodiment, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin receptor A, and comprises: - a VH domain having a sequence having at least 80% identity with SEQ ID NO: 17 and comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having, according to the IMGT nomenclature, the respective sequences GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) and VSSYYSGSFFAY (SEQ ID NO: 11); and - a VL domain having a sequence having at least 80% identity with SEQ ID NO: 18 comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having, according to the IMGT nomenclature, the sequences GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) and VSSYYSGSFFAY (SEQ ID NO: 11);IMGT for respective sequences SQSIVYSNGKIYL (SEQ ID NO: 12), KVS (SEQ ID NO: 13) and FQGSHLPLT (SEQ ID NO: 14). Particularly advantageously, the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention binds specifically to the endothelin B receptor, and comprises: - a VH domain having the sequence SEQ ID NO: 17 or SEQ ID NO: 28, and - a VL domain having the sequence SEQ ID NO: 18. The RB49 and RA63 antibodies from which the mutant antibody fragment according to the invention is advantageously derived are chimeric antibodies, and when the mutant antibody fragment according to the invention contains a part of the constant regions (for example for a Fab fragment), this is preferably human. In particular, the mutant antibody fragment according to the invention may in particular be chosen from: a) a mutant Fab fragment binding specifically to the endothelin B receptor and comprising a heavy chain fragment ofsequence SEQ ID NO: 19 (QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVTVSCASTKGPSVFPLAPSSKTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTH) and a light chain of sequence SEQ ID NO: 20 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC); and b) a mutant Fab fragment specifically binding to the endothelin receptor A and comprising a heavy chain fragment of sequence SEQ ID NO: 21 (EVQLVESGGGLVQPKGSLKLSCAASGFTFNIYAMNWIRQAPGKGLEWIARIRSKSNNYATYYADSVKDRFTI SRDDSQNMVYLQMNNLKTEDTAMYYCVSSYYSGSFFAYWGQGTLVTVSCASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTH) and a light chain of sequence SEQ ID NO: 22(DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKIYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG TDFTLKISRVEAEDLGVYYCFQGSHLPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC). Alternatively, the mutant antibody fragment according to the invention may in particular be chosen from. a) an scFv fragment specifically binding to the endothelin receptor B comprising or consisting of the amino acid sequence SEQ ID NO: 25 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGGSQVQLQQPG AALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATLTVDKSSSTA YMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVTVSC); and b) an endothelin receptor A-specifically binding scFv fragment comprising or consisting of the amino acid sequence SEQ ID NO: 26 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKIYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGExcluded fragments The mutant antibody fragment according to the invention is not a Fab fragment whose mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO: 2. Such a Fab fragment would correspond to the Fab fragment of the antibody Hu4D5 (humanized antibody specifically binding to HER2), in which the serine residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is substituted by a cysteine. A mutant antibody fragment according to the invention may more broadly not be an antibody fragment whose heavy chain variable (VH) domain corresponds to the amino acid sequence SEQ ID NO: 1. This excludes all mutant antibody fragments whose VH domaincorresponds to the VH domain of the Hu4D5 antibody (humanized antibody specifically binding to HER2), in which the serine residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is substituted by a cysteine ​​(regardless of the type of fragment or the sequence of the possible light chain). Even more broadly, a mutant antibody fragment according to the invention may not be a fragment of an antibody binding to the human HER2 antigen. Alternatively, the mutant antibody fragment according to the invention is not derived (by substitution of the residue at position 128 according to the IMGT nomenclature of the antibody heavy chain variable domain by a cysteine) from an antibody fragment whose amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain is a serine. Nucleic acid molecule or pair of nucleic acid molecules encoding the mutant antibody fragment according to the invention The present inventionalso relates to a nucleic acid molecule or pair of nucleic acid molecules encoding the mutant antibody fragment according to the invention. Such a molecule or pair of molecules is useful in particular for producing the mutant antibody fragment according to the invention. Depending on the desired mutant antibody fragment, it may comprise a single chain (for example, fragments of the scFv, diabody, tribody, tetrabody, minibody, VHH, VNAR type) or two chains (for example, fragments of the Fv, Fab, Fab', F(ab')2 type). When the mutant antibody fragment according to the invention comprises a single chain, a single nucleic acid molecule is preferably used to encode this chain. When the mutant antibody fragment according to the invention comprises two chains, the two chains may be encoded either by a single nucleic acid molecule or by two nucleic acid molecules. Due to the degeneracy of the genetic code, all the sequencesdifferent nucleic acids, coding for a particular amino acid sequence of a mutant antibody fragment according to the invention are within the scope of the invention. In particular, the sequence of a nucleic acid according to the invention may have been optimized to promote its expression in a host cell, a transgenic non-human animal or a transgenic plant of interest. Indeed, there are generally several combinations of three nucleotides coding for the same amino acid (except for methionine and tryptophan), called synonymous codons. However, some of these combinations are generally used preferentially by a given cell or organism (this is then referred to as genetic code usage bias). This preference depends in particular on the producing organism or from which the cell originates. Consequently, when a protein derived from one or more organisms is produced in a heterologous organism or in a cell of such a heterologous organism, it mayIt may be useful to modify the nucleic acid sequence coding for the protein so as to primarily use the preferred codons of the heterologous organism. Data are available in the literature concerning the preferred codon usage by different species and a person skilled in the art knows how to optimize the expression of a given protein in an organism or a cell of a heterologous organism. The nucleic acid molecule(s) coding for the mutant antibody fragment according to the invention may further comprise a nucleic acid sequence coding for a signal peptide at the N-terminal of the chain(s) of the mutant antibody fragment according to the invention. Vector The present invention also relates to a vector comprising the nucleic acid molecule or the pair of nucleic acid molecules according to the invention, as described above. Such a vector is also useful for producing the mutant antibody fragment according to the invention, which may be used fortransform (in a stable or transient manner) a host cell which will then produce the mutant antibody fragment according to the invention. Such a vector comprises the elements necessary for the expression of said nucleic sequence, and in particular a promoter, a transcription initiation codon, termination sequences, and appropriate transcription regulatory sequences. These elements vary depending on the host used for expression and are easily chosen by a person skilled in the art in light of his general knowledge. In particular, for a vector intended for expression in eukaryotic cells, the vector advantageously comprises a “Kozak sequence”, that is to say a conserved sequence found on eukaryotic messenger RNAs at the translation start site, around the start codon AUG (generally GCCGCCRCCATGG (SEQ ID NO: 24), the translation initiation codon being underlined, R=A or G). The vector may in particularbe plasmidic or viral. Examples of plasmid and viral vectors suitable for expressing an antibody fragment are known to those skilled in the art (see in particular Sandomenico, A., Sivaccumar, JP, Ruvo, M., 2020. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. International Journal of Molecular Sciences 21, 6324; Spadiut, O., Capone, S., Krainer, F., Glieder, A., Herwig, C., 2014. Microbials for the production of monoclonal antibodies and antibody fragments. Trends in Biotechnology 32, 54–60). Host cell, transgenic non-human animal or transgenic plant The present invention also relates to a host cell, a transgenic non-human animal or a transgenic plant comprising the nucleic acid molecule or the pair of nucleic acid molecules according to the invention or the vector according to the invention as described above. Such a host cell, such a transgenic non-human animal and such a plantTransgenic cells are also useful for producing the mutant antibody fragment according to the invention, these expressing the mutant antibody fragment according to the invention. The host cell may be of prokaryotic or eukaryotic origin, and may in particular be chosen from bacterial cells, insect cells, plant cells, yeast cells or mammalian cells. The mutant antibody fragment according to the invention may then be produced by culturing the host cell under appropriate conditions. A host cell according to the invention may in particular be obtained by transforming a cell line with a vector according to the invention and separating the different cell clones obtained. The transformed cell line is preferably of eukaryotic origin, and may in particular be chosen from insect cells, plant cells, yeast cells or mammalian cells. Suitable cell lines for producing the mutant antibody fragment according to the invention include in particular Escherichia coliamong bacteria, Saccharomyces cerevisiae and Pichia pastoris among yeasts, and the cell lines CHO, NS0, Sp2 / 0, HEK293, and PER.C6 among mammalian cells. A transgenic non-human animal according to the invention can be obtained by direct injection of the gene(s) of interest (here the nucleic acid molecule or the pair of nucleic acid molecules according to the invention) into a fertilized egg (Gordon et al., 1980 Proc Natl Acad Sci US A.;77:7380-4). A transgenic non-human animal may also be obtained by introducing the gene(s) of interest (here the nucleic acid molecule or the pair of nucleic acid molecules according to the invention) into an embryonic stem cell and preparing the animal by a chimera aggregation method or a chimera injection method (see Manipulating the Mouse Embryo, A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press atOxford University Press (1993)). A transgenic non-human animal can also be obtained by a cloning technique in which a nucleus, into which the gene(s) of interest (here the nucleic acid molecule or pair of nucleic acid molecules according to the invention) has been introduced, is transplanted into an enucleated egg (Ryan et al, 1997 Science; 278: 873 – 876; Cibelli et al., 1998 Science, 280: 1256-1258; WO00 / 26357). The mutant antibody fragment according to the invention can then be accumulated in the transgenic animal and harvested, in particular from the animal's milk or eggs. For the production of antibodies in the milk of transgenic non-human animals, preparation methods are described in particular in WO90 / 04036, WO95 / 17085, WO01 / 26455, WO2004 / 050847, WO2005 / 033281, WO2007 / 048077. Methods for purifying proteins of interest from milk are also known (see WO01 / 26455, WO2007 / 106078). Transgenic non-human animals of interestinclude in particular mouse, rabbit, rat, goat, cattle (in particular cow), and poultry (in particular chicken). A transgenic plant according to the invention can be chosen from any plant allowing the production of antibody fragments. Many antibodies have already been produced in transgenic plants and the technologies necessary for obtaining a transgenic plant expressing an antibody of interest and for recovering the antibody are well known to those skilled in the art (see Stoger E, et al. Molecular Breeding 9: 149–158, 2002, Fisher R, et al. Vaccine 21 (2003) 820–825, Ma JK, et al. Nat Rev Genet. 2003 Oct;4(10):794-805, Schillberg S, et al. Vaccine 23 (2005) 1764–1769). Conjugate The invention also relates to a conjugate comprising the mutant antibody fragment according to the invention, linked to at least one, in particular one or two, molecules of interest, advantageously via cysteine, and more particularly the thiol function of cysteine, inposition 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment. Molecule(s) of interest conjugated to the mutant antibody fragment according to the invention Different types of molecules of interest are likely to be conjugated to the mutant antibody fragment according to the invention, depending on the use envisaged for the conjugate. The mutant antibody fragment according to the invention may in particular be conjugated to one or more molecules chosen from a molecule that is detectable or likely to become detectable, an affinity molecule, and a pharmacomodulatory molecule. In one embodiment, the mutant antibody fragment according to the invention may in particular be conjugated to one or more molecule(s) that is detectable or likely to become detectable, in particular in the context of diagnostic uses of the conjugate. The mutant antibody fragment according to the invention may in particular be conjugated to a (conjugate intended to be used ina single type of imaging) or two (conjugate intended for use in several types of imaging detecting different physical phenomena carrying two molecules detectable or likely to become detectable of different type, or conjugate intended for use in a single type of imaging carrying two copies of the same molecule detectable or likely to become detectable in order to increase the signal) molecule(s) detectable or likely to become detectable. Each molecule detectable or likely to become detectable is chosen according to the type of imaging for which the conjugate is intended. Examples of molecules detectable or likely to become detectable that can be conjugated to the mutant antibody fragment according to the invention are: a) a radioisotope (detectable molecule), a chelating agent (molecule likely to become detectable, which becomes detectable after contact with a radiometal), or an affinity molecule(molecule capable of becoming detectable, which becomes detectable after contacting the mutant antibody fragment conjugated to the affinity molecule with a radioisotope conjugated to a binding partner of the affinity molecule, or with a chelating agent conjugated to a binding partner of the affinity molecule and then a radiometal). Such detectable molecules are useful first of all when the conjugate is intended to be used in isotopic imaging. The radioisotope can be covalently conjugated to the mutant antibody fragment according to the invention, or non-covalently by complexing with a chelating agent covalently conjugated to the mutant antibody fragment according to the invention. Radioisotopes or radiometals emitting directly (direct emission of a gamma photon during decomposition) or indirectly (emission of a positron during its disintegration, which after annihilation with a surrounding electron, leads to the emission of 2gamma rays) are preferred because they are detectable by positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Some of the radioisotopes used in isotopic imaging are radiometals, generally indirectly conjugated to the probe used (here the mutant antibody fragment according to the invention), the probe comprising a chelating agent which binds the radiometal. Examples of chelating agents include linear or cyclic chelating agents, such as DOTA, NOTA, deferoxamine B, and their derivatives. Preferred derivatives of DOTA and NOTA are, for example, DOTAGA and (R)-NODAGA, respectively. Examples of radiometals that emit gamma rays directly or indirectly (via the emission of a positron, which after annihilation with a surrounding electron, leads to the emission of 2 gamma rays) include 99m Tc, 111 In, 64Cu, 68 Ga, 89 Zr, and 44 Sc. 99m Tc, 111 In are direct emitters of gamma rays detectable by single-photon emission computed tomography (SPECT), while 64 Cu, 68 Ga, 89 Zr, and 44Sc are indirect emitters of gamma rays detectable by positron emission tomography (PET). The use of a chelating agent can be advantageous because it allows, in particular, to store the conjugate ready to chelate and to add the radioisotope, which can have a very short half-life, only at the last moment before use. In this case, the complexation can be carried out just before using the conjugate of the invention for an isotopic imaging application. Although the conjugation to the chelating agent can be indirect via an affinity molecule and its binding partner, the mutant antibody fragment according to the invention is advantageously conjugated directly to a chelating agent. Another radioisotope that can be used in PET imaging is 18F, which is a positron emitter (and therefore an indirect emitter of gamma photons) and which can in particular be introduced onto so-called prosthetic groups, known to those skilled in the art, which can be conjugated to the mutant antibody fragment according to the invention. b) a fluorophore (detectable molecule) or an affinity molecule (molecule capable of becoming detectable, the fluorophore is then detected after bringing the mutant antibody fragment conjugated to the affinity molecule into contact with a fluorophore conjugated to a binding partner of the affinity molecule). Fluorophores are useful when the conjugate is intended to be used in fluorescence imaging.As for radiometals in the case of radioisotopes, the fluorophore may be covalently conjugated to the mutant antibody fragment according to the invention, or non-covalently when it is previously conjugated to a binding partner of the affinity molecule which complexes with the affinity molecule covalently conjugated to the mutant antibody fragment according to the invention. Examples of fluorophores capable of being detected by fluorescence imaging and therefore of being conjugated to the mutant antibody fragment according to the invention include compounds of the cyanine family (in particular Cyanine3, Cyanine5, Cyanine5.5, Cyanine7, Cyanine7.5, carrying or not water-solubilizing groups, such as sulfones); compounds of the xanthene family (in particular compounds of the rhodamine family such as X-rhodamine, rhodamine B and compounds of the fluorescein family, carrying or not water-solubilizing groups, such as sulfones); compounds of the coumarin family (in particular, hydroxycoumarin, aminocoumarin, methoxycoumarin, carrying or not water-solubilizing groups, such as sulfones); and compounds of the boron-dipyrromethene family (BODIPY, including azaBODIPY derivatives). Preferably, the fluorophore is selected from Cyanine5, Cyanine 7, a rhodamine or an azaBODIPY. Examples of (affinity molecule / binding partner) pairs include (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin).c) a chromophore (detectable molecule) or an affinity molecule (molecule capable of becoming detectable, the chromophore is then detected after contacting the mutant antibody fragment conjugated to biotin with a chromophore conjugated to a streptavidin molecule). Chromophores are useful when the conjugate is intended to be used in optical imaging by absorbance measurement.As with radiometals in the case of radioisotopes, the chromophore may be covalently conjugated to the mutant antibody fragment of the invention, or non-covalently when it is previously conjugated to a binding partner of the affinity molecule that complexes with the affinity molecule covalently conjugated to the mutant antibody fragment of the invention. Examples of chromophores that may be conjugated to the mutant antibody fragment of the invention for use in optical imaging include phenolphthalein, gentian violet, or Congo Red. Examples of (affinity molecule / binding partner) pairs include (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin). Thus, the molecules detectable or likely to become detectable can be chosen from a fluorophore, a chromophore, an affinity molecule, a radioisotope and a chelating agent.When the mutant antibody fragment according to the invention is conjugated to two detectable or detectable molecules, it may in particular be conjugated to: 1) a radioisotope or a chelating agent for use in isotopic imaging and a fluorophore or an affinity molecule for use in fluorescence imaging; 2) two detectable or detectable molecules of the same type and preferably identical, to increase the detected signal. In this embodiment, the detectable or detectable molecule may in particular be chosen from: a) radioisotopes or chelating agents for use in isotopic imaging, or b) fluorophores or affinity molecules for use in fluorescence imaging. The mutant antibody fragment according to the invention may further or alternatively be conjugated to an affinity molecule.Such a conjugate may then be complexed to a detectable molecule or a molecule capable of being detectable (e.g., a chelating agent) capable of forming a complex with the affinity molecule that is conjugated to the mutant antibody fragment of the invention. Examples of affinity molecules include a biotin (capable, in particular, of forming a complex with a streptavidin previously conjugated to a fluorophore, a chromophore, a radioisotope, or a chelating agent), an avidin (capable, in particular, of forming a complex with a biotin previously conjugated to a fluorophore, a chromophore, a radioisotope, or a chelating agent), and a streptavidin (capable, in particular, of forming a complex with a biotin previously conjugated to a fluorophore, a chromophore, a radioisotope, or a chelating agent). The affinity molecule may alternatively be used to readily purify the mutant antibody fragment of the invention.In this case, in addition to the affinity molecules mentioned in the previous paragraph, a hexahistidine peptide can also be used, making it possible to easily purify the fragment on a column comprising nickel (a binding partner to which the hexahistidine peptide complexes). The affinity molecule can therefore be chosen from biotin, avidin, streptavidin and a hexahistidine peptide. The mutant antibody fragment according to the invention can additionally or alternatively be conjugated to a pharmacomodulatory molecule. Such conjugation can make it possible to improve the pharmacokinetics of the mutant antibody fragment according to the invention.Examples of pharmacomodulatory molecules include linear or branched chains of poly(ethylene glycol), linear or branched chains of poly(glutamic acid), cholesterol, and molecules capable of binding to albumin (e.g. fatty acids, metal ions, bilirubin, a toxin, the p-iodophenyl butyryl group, the biphenyl group). Structure of the conjugates The conjugate according to the invention is linked to at least one, in particular one or two, molecules of interest, advantageously via cysteine, and more particularly the thiol function of cysteine, at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment. According to a first embodiment, the conjugate is linked to two molecules of interest, advantageously via cysteine, and more particularly the thiol function of cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment.Advantageously, at least one of these molecules is a molecule that is detectable or capable of becoming detectable, and preferably the two molecules each independently represent a molecule that is detectable or capable of becoming detectable, such as a fluorophore, a chromophore, an affinity molecule, a radioisotope, or a chelating agent. For example, one of these detectable molecules may be a fluorophore and the other may be a radioisotope or a chelating agent. For example, one of the molecules may be the chelating agent DOTA (allowing the chelation of a metal radioisotope such as 68Ga) and the other a tetrasulfonated cyanine 7 (fluorophore emitting in the near infrared). Such a conjugate may correspond to the following formula (I):. in which: R 1 represents a mutant antibody fragment according to the invention linked to X 1via cysteine, and more particularly the thiol function of cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature; with X 4 representing a single bond, O, S, or NR 4 , and R 4 representing H or (C1-C6)alkyl, the wavy bond indicating the point of attachment to R 1 and the dotted line indicating the point of attachment to L 1 ; L 1 , L 2 and L 3 each independently represent a single bond or a spacer, the spacer being a (C1-C30)-alkyl chain, for example (C1-C20)-alkyl, optionally preceded and / or interrupted and / or followed by one or more units chosen from the group consisting of aromatic, heteroaromatic, cycloalkane, cycloalkene, heterocycloalkane, heterocycloalkene, -O-, -S-, -NR rings 5 -, -C(O)-, -C(S)-, -C≡C-, and - C(R 6 )=C(R 7 )-, with R 5representing H or (C1-C6)alkyl, R 6 and R 7 each independently representing H or (C1-C6)alkyl, and the aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, heterocycloalkyl and heterocycloalkenyl rings being optionally substituted by one or more (C1-C6)alkyl groups; represents a double bond or a single bond, preferably a double bond; ring A 1 represents a cycloalkane, cycloalkene, heterocycloalkane or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy; X 2 and X 3 , identical or different, each represent O, S or NR 8 , with R 8 representing H or (C1-C6)alkyl, preferably H; R 2 and R 3, identical or different, preferably different, each represent a residue of a molecule of interest advantageously chosen from molecules detectable or likely to become detectable, affinity molecules and pharmacomodulatory molecules. Preferably, X 1 represents . Preferably, L 1 , L 2 and L 3 each independently represent a spacer. According to a first embodiment, the spacer is a (C1-C30)-alkyl chain, in particular (C1-C20)-alkyl, such as (C1-C10)-alkyl or (C1-C6)-alkyl, and in particular an ethyl or hexyl group (eg n-hexyl). According to a second embodiment, the spacer is a PEG chain of formula –(CH2CH2O) x - or –(CH2CH2O) x-CH2CH2- with x an integer from 1 to 12, in particular from 2 to 8 and in particular 2, 4 or 8. According to a third embodiment, the spacer is a (C1-C30)-alkyl group, in particular (C1-C20)-alkyl, such as (C1-C10)-alkyl or (C1-C6)-alkyl, preceded or followed by a C(O) group such as a propanoyl group. Preferably, at least one of X 2 and X 3 represents S. Advantageously, X 2 and X 3 are chosen independently from S and NR 8 . Preferably one of X 2 and X 3 is S and the other is S or NR 8 . Advantageously, R 8 is H. , , , dotted line indicating the point of attachment to L 1 and the wavy bonds indicating the points of attachment to the carbon atoms bearing X 2 or X 3 . Preferably, at least one of R 2 and R 3is a residue of a molecule that is detectable or likely to become detectable, and in particular R 2 and R 3 each independently represent a residue of a molecule that is detectable or likely to become detectable, advantageously R 2 and R 3 represent respectively a residue of a fluorophore and a residue of a radioisotope or a chelating agent. In particular, R 2 and R 3may respectively represent a residue of a sulfonated Cyanine 7 and a residue of R-NODAGA (chelating agent). According to a second embodiment, the conjugate is linked to a single molecule of interest which is advantageously a detectable molecule or likely to become detectable, such as a fluorophore, a chromophore, an affinity molecule, a radioisotope, or a chelating agent; and this advantageously via the cysteine, and more particularly the thiol function of the cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment. Such a conjugate may correspond to the following formula (IIa) or (IIb): R 1 -X 1 -L 1 -R 2 (IIa) or R 1 -X 1 -L 1 -X 1 '-L 4 -R 2 (IIb) in which: R 1 represents a mutant antibody fragment according to the invention linked to X 1via cysteine, and more particularly the thiol function of cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature; with X 4 representing a single bond, O, S, or NR 4 , and R 4 representing H or (C1-C6)alkyl, the wavy bond indicating the point of attachment to R 1 and the dotted line indicating the point of attachment to L 1 ; as defined above and cycle A 2 representing a cycloalkene or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy, the wavy bond indicating the point of attachment to L 4 , and the dotted line indicating the point of attachment to L 1 ; L 1 and L 4each independently represent a single bond or a spacer, the spacer being a (C1-C30)-alkyl chain, for example (C1-C20)-alkyl, optionally preceded and / or interrupted and / or followed by one or more units chosen from the group consisting of aromatic, heteroaromatic, cycloalkane, cycloalkene, heterocycloalkane, heterocycloalkene, -O-, -S-, -NR rings 5 -, -C(O)-, -C(S)-, -C≡C-, and -C(R 6 )=C(R 7 )-, with R 5 representing H or (C1-C6)alkyl, R 6 and R 7 each independently representing H or (C1-C6)alkyl, and the aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, heterocycloalkyl and heterocycloalkenyl rings being optionally substituted by one or more (C1-C6)alkyl groups; R 2represents a residue of a molecule of interest advantageously chosen from molecules that are detectable or likely to become detectable, affinity molecules and pharmacomodulatory molecules. Preferably, X 1 represents . Preferably, L 1 and L 4 each independently represent a spacer. According to a first embodiment, the spacer is a chain (C1-C 30 )-alkyl, in particular (C1-C 20 )-alkyl, such as (C1-C10)-alkyl or (C1-C6)-alkyl, and in particular an ethyl or hexyl group (eg n-hexyl). According to a second embodiment, the spacer is a PEG chain of formula –(CH2CH2O)x- or –(CH2CH2O)x-CH2CH2- with x an integer from 1 to 12, in particular from 2 to 8 and in particular 2, 4 or 8. According to a third embodiment, the spacer is a (C1-C 30 )-alkyl, in particular (C1-C 20 )-alkyl, such as (C1-C 10)-alkyl or (C1-C6)-alkyl, preceded or followed by a C(O) group such as a propanoyl group. Preferably, X 1 ' represents , the dotted line indicating the point of attachment to L 1 or L 4 and the wavy bonds indicating the points of attachment to the NN=N or ON=N motif. Preferably, R 1is a residue of a molecule that is detectable or capable of becoming detectable, advantageously chosen from a fluorophore, a chromophore, an affinity molecule, a radioisotope, and a chelating agent, and in particular from a fluorophore, a radioisotope and a chelating agent. Conjugation methods and synthetic intermediates When the mutant antibody fragment according to the invention comprises one or more disulfide bridges (in particular Fab, minibody, scFv, diabody, tribody and tetrabody fragment), the conjugates according to the invention can advantageously be prepared by a preparation method comprising the following steps in the order indicated: a) reduction of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of tris(2-carboxyethyl)phosphine (TCEP), to give a reduced mutant antibody fragment, b) reoxidation of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of dehydroascorbic acid,to give a partially reduced mutant antibody fragment, c) conjugation with at least one molecule of interest of the thiol function of the cysteine ​​in position 128 of the heavy chain variable domain according to the IMGT nomenclature of the partially reduced mutant antibody fragment to give a conjugate according to the invention. Indeed, this method comprising steps a) and b) as described here prior to step c) of conjugation allows an extremely specific conjugation at the level of the cysteine, and more particularly the thiol function of the cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment. Step a) aims to reduce all the disulfide bridges of the mutant antibody fragment, whether the disulfide bridges connecting the heavy chains of the mutant antibody fragment (also called inter-chain disulfide bridges),than the disulfide bridge formed with the thiol function of the cysteine ​​at position 128. Step b) aims to reoxidize only the inter-chain disulfide bridges, so as to obtain a partially reduced mutant antibody fragment, i.e. in which only the thiol of the cysteine ​​at position 128 is in reduced form. This thiol is then in the form of an SH group which can serve as a reactive chemical group to covalently conjugate at least one molecule of interest. Step c) aims to conjugate the at least one molecule of interest to the SH function of the cysteine ​​at position 128. For this, the partially reduced mutant antibody fragment obtained in step b) will be coupled with a molecule carrying, on the one hand, at least one molecule of interest and, on the other hand, a chemical group capable of reacting with an SH thiol function. Such a chemical group capable of reacting with an SH function may be in particular, , with Hal representing Br or I and X 4 representing a single bond, O, S, or NR 4 , R 4 representing H or (C1-C6)alkyl. The method described above comprising steps a) and b) as described here prior to step c) of conjugation is also preferred for antibody fragments, in particular the Fv, VHH, VNAR fragments, because these fragments are often isolated in the form of dimers or adducts with glutathione, cysteines, etc. Steps a) of reduction and b) of reoxidation before conjugation are therefore also advantageously used. According to a first particular embodiment of the invention, the conjugate according to the invention is a conjugate of formula (I) as defined above and step c) can be carried out by coupling between the partially reduced mutant antibody fragment (corresponding to the R1H molecule) and a compound of the following formula (III): for which: L 1 is as defined above, cycle A3 represents a cycloalkyne, cycloalkene, heterocycloalkyne or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy, and X 1a represents a chemical group reactive with an SH function, to give a compound of the following formula (IV): X1 R1 (IV) for which R 1 , X 1 , L 1 and cycle A 3 are as defined above, followed by the coupling between the compound of formula (IV) and a compound of the following formula (V): for which R 2 , R 3 , L 2 , L 3 , X 2 and X 3 are as defined above.

[0002] Preferably, X 1a , which represents a chemical group reactive with an SH function, representing a single bond, O, S, or NR 4 , and R 4 representing H or (C1-C6)alkyl. The reaction of X 1a with the SH function of cysteine ​​at position 128 will form the X group 1 and covalently linking the compound of formula (III) to the partially reduced mutant antibody fragment. Preferably, such a reaction between the SH function of a cysteine ​​and a chemical group reactive with an SH function is well known in the art. Ring A 3 carried by the compound of formula (IV) thus formed can then be used to graft two molecules of interest via the reaction of this cycle A 3 with the tetrazine group of the compound of formula (V) which carries the two molecules of interest (R 2 and R 3). This coupling reaction is well known to those skilled in the art and described in particular in WO2018 / 172543. The compound of formula (V) bearing the tetrazine group can also be prepared according to a method described in WO2018 / 172543. According to a second particular embodiment of the invention, the conjugate is a conjugate of formula (IIa) or (IIb) as defined above. Step c) will advantageously involve coupling of the partially reduced mutant antibody fragment (R 1 H) with a compound bearing a chemical group reactive with respect to an SH function (X 1a ). Such a coupling reacting a thiol SH function with a chemical group reactive with respect to an SH function (X 1 a) is well known in the art. The chemical group X 1a reactive with an SH function can be grafted directly onto a molecule of interest via an L spacer 1 or not (when L 1represents a single bond). This allows the preparation of a conjugate of formula (IIa). In this case, step c) can be carried out by coupling the partially reduced mutant antibody fragment (R 1 H) with a compound of formula X 1a -L 1 -R 2 in which L 1 , X 1a and R 2 are as defined above. On the contrary, a different reactive chemical group (G 2 ) can be grafted to the molecule of interest via an L spacer 4 or not (when L 4 represents a single bond). In this case, the partially reduced mutant antibody fragment (R 1 H) is conjugated to the molecule of interest (carrying a reactive group G 2 ) using a bifunctional compound carrying: ^ at one end a chemical group reactive with an SH function (X 1a ) to be able to couple to the partially reduced mutant antibody fragment (R 1H), and ^ at the other end a chemical group G 1 able to react with G 2 so as to allow coupling with the molecule of interest. This makes it possible to prepare a conjugate of formula (IIb). According to a first aspect, step c) can be carried out by coupling the partially reduced mutant antibody fragment (R 1 H) with a compound of formula X 1a -L 1 -X 1 '-L 4 -R 2 respectively in which L 1 , X 1 ', X 1a , L 4 and R 2 are as defined above. The compound of formula X 1a -L 1 -X 1 '-R 2 can then be prepared by coupling between a compound of formula X 1a -L 1 -G 1 and a compound of formula G 2 -L 4 -R 2 in which X 1a , L 1 , L 4 and R 2 are as defined above and G 1 and G 2are different and each represents a reactive chemical group capable of reacting together to form a group X 1 '. The chemical groups G 1 and G 2 can be chosen according to Table 2 below, cycle A 3 being as defined above and cycle A 4 representing a cycloalkyne or heterocycloalkyne ring optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy: [Table 2] 1 er reactive group 2 ème reactive group (G 1 or G 2 ) (G 1 or G 2 ) capable of reacting with 1 er reactive group N3A4 N+ O- A4 NN A3 NN According to a second aspect, step c) can be carried out by coupling the partially reduced mutant antibody fragment (R 1 H) with a compound of formula X 1a -L 1 -G 1in which L 1 , G1 and X 1a are as defined above, to give a conjugate of formula R 1 -X 1 -L 1 -G 1 , followed by the coupling between the conjugate of formula R 1 -X 1 -L 1 -G 1 and a compound of formula G 2 -R 2 in which G 2 and R 2are as defined above. The present invention therefore also relates to intermediate conjugates useful as a synthesis intermediate during the preparation of the conjugates according to the invention. The present invention thus also relates to an intermediate conjugate comprising the mutant antibody fragment according to the invention, linked, via cysteine, and more particularly the thiol function of cysteine, in position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment, to a reactive chemical group. Reference, the reactive chemical group is chosen from N, N+ - Preferably 3O, , , , the A cycles 3 and A 4 being as defined above. Preferably, cycle A 3 will be chosen from , , advantageously chosen from 5

[0003] , According to a particular embodiment, the intermediate conjugate according to the invention corresponds to the formula R 1 -X 1-L 1 -Z in which R 1 , X 1 and L 1are as defined above, and Z represents a reactive chemical group as defined above. Uses of the conjugate Depending on the molecule(s) conjugated to the mutant antibody fragment according to the invention at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature, the conjugate according to the invention may be used in different in vivo applications. In particular, the present invention also relates to the conjugate according to the invention, for its use in: a) a method for diagnosing a tumor by imaging, b) a method for surgical treatment by tumor resection guided by fluorescence or isotopic imaging probe, c) a method for monitoring by imaging the effectiveness of an antitumor treatment, or d) any combination of methods a), b) and c) (such as a) and b); a) and c); b) and c); or a), b) and c)).Method for diagnosing a tumor by imaging The invention therefore relates to the conjugate according to the invention, for its use in a method for diagnosing a tumor by imaging. The present invention also relates to the use of a conjugate according to the invention in a method for diagnosing a tumor by imaging. The present invention also relates to a method for detecting tumor cells in a subject by imaging, comprising: a) administering to a subject suspected of suffering from a tumor in an organ a conjugate according to the invention whose antibody fragment binds specifically to an antigen of the suspected tumor, b) obtaining by imaging one or more images of the organ suspected of comprising a tumor, and c) detecting the presence or absence of tumor cells labeled by the conjugate in the image(s) obtained in step b).The present invention also relates to a method for determining the tumor burden in a patient suffering from a tumor by imaging, comprising: a) administering to the patient suffering from a tumor a conjugate according to the invention whose antibody fragment binds specifically to an antigen of the patient's tumor, b) obtaining by imaging one or more images of one or more organs of the patient, and c) determining the tumor burden of the patient from the image(s) obtained in step b). In this application, the molecule(s) conjugated to the mutant antibody fragment according to the invention at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature is advantageously chosen from molecules that are detectable or likely to become detectable. The molecule(s) detectable or likely to become detectable will be chosen according to the type(s) of imaging envisaged.For use in a method for diagnosing a tumor by isotopic imaging, the mutant antibody fragment according to the invention is advantageously conjugated at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature to a radioisotope or a chelating agent (a complex with a radiometal is then formed just before administration to the patient). In particular, for positron emission tomography (PET) or single-photon emission tomography (SPECT), the mutant antibody fragment according to the invention is advantageously conjugated at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature to a radioisotope emitting direct or indirect gamma rays (in particular. 18 F) or to a chelating agent (a complex with a radiometal emitting direct or indirect gamma rays, in particular chosen from 99m Tc, 111 In, 64 Cu, 68Ga, 89 Zr, and 44 Sc, is then formed just before administration to the patient). For Cerenkov Luminescence Imaging (CLI), the mutant antibody fragment according to the invention is advantageously conjugated to the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature at 90 Y, 68 Ga, 225Ac. For use in a method for diagnosing a tumor by optical imaging, the mutant antibody fragment according to the invention is advantageously conjugated at the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature to a chromophore or an affinity molecule (a complex with a chromophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), and (avidin / biotin), (streptavidin / biotin)). For use in a method of diagnosing a tumor by fluorescence imaging, the mutant antibody fragment according to the invention is advantageously conjugated at the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature to a fluorophore or an affinity molecule (a complex with a fluorophore conjugated to a binding partnerof the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin)). The mutant antibody fragment according to the invention can also be conjugated at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature to two different detectable molecules or molecules likely to become detectable, allowing its use in two different types of imaging. For example, the mutant antibody fragment according to the invention can be conjugated at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature to two molecules detectable or capable of being detectable by different imaging techniques, and in particular to: a1) a radioisotope emitting direct or indirect gamma rays or a chelating agent for its use in a method of diagnosing atumor by isotopic imaging (in particular by positron emission tomography (PET) or by single-photon emission tomography (SPECT)) and a fluorophore for its use in a method of diagnosing a tumor by fluorescence imaging; a2) a radioisotope emitting direct or indirect gamma rays or a chelating agent for its use in a method of diagnosing a tumor by isotopic imaging (in particular by positron emission tomography (PET) or by single-photon emission tomography (SPECT)) and an affinity molecule (a complex with a fluorophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and(streptavidin / biotin)) for use in a method of diagnosing a tumor by fluorescence imaging; b1) a direct or indirect gamma-ray emitting radioisotope or a chelating agent for use in a method of diagnosing a tumor by isotopic imaging (in particular by positron emission tomography (PET) or by single-photon emission tomography (SPECT)) and a chromophore for use in a method of diagnosing a tumor by optical imaging; (b2) a direct or indirect gamma-emitting radioisotope or a chelating agent for use in a method of diagnosing a tumor by isotopic imaging (in particular by positron emission tomography (PET) or by single-photon emission tomography (SPECT)) and an affinity molecule (a complex with a chromophore conjugated to abinding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin)) for its use in a method of diagnosing a tumor by optical imaging; c1) a fluorophore or an affinity molecule (a complex with a fluorophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin)) for its use in a method of diagnosing a tumor by fluorescence imaging and a chromophore for its use in a method of diagnosing a tumor by optical imaging; or c2) a fluorophore for its use in a method ofdiagnosis of a tumor by fluorescence imaging and an affinity molecule (a complex with a fluorophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin)) for its use in a method of diagnosing a tumor by optical imaging. Among the conjugates with two molecules detectable or likely to be detectable by different imaging techniques described above, the conjugates a1 and b1) are preferred. Alternatively, the mutant antibody fragment according to the invention can be conjugated at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature to two molecules detectable or likely to be detectable by the same imaging technique (this making it possible to increase the signal and therefore thedetection sensitivity), and in particular to: a) two radioisotopes emitting direct or indirect gamma rays (different or preferably identical) or two chelating agents (different or preferably identical) for its use in a method of diagnosing a tumor by isotopic imaging (in particular by positron emission tomography (PET or PET for the English acronym) or by single-photon emission tomography (SPECT or TEMP for the English acronym)), b) two fluorophores (different or preferably identical) for its use in a method of diagnosing a tumor by fluorescence imaging, c) two chromophores (different or preferably identical) for its use in a method of diagnosing a tumor by optical imaging, or d) two affinity molecules (different or preferably identical) for its use in a method of diagnosing a tumor by optical or fluorescence imaging. Among the conjugates with twomolecules detectable or capable of being detectable by the same imaging technique described above, the conjugates a) (two radioisotopes emitting direct or indirect gamma rays (different or preferably identical) or two chelating agents, different or preferably identical) and b) (two fluorophores, different or preferably identical) are preferred. Alternatively, the mutant antibody fragment according to the invention can also be conjugated at the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature to a molecule detectable or capable of becoming detectable and a pharmacomodulatory molecule, the latter making it possible to modify the pharmacokinetic properties of the diagnostic conjugate. In this embodiment, the mutant antibody fragment according to the invention can also be conjugated at the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature to: a) a pharmacomodulatory molecule and a radioisotopedirect or indirect emitter of gamma rays or a chelating agent for its use in a method of diagnosing a tumor by isotopic imaging (in particular by positron emission tomography (PET) or by single-photon emission tomography (SPECT)); b) a pharmacomodulatory molecule and a fluorophore for its use in a method of diagnosing a tumor by fluorescence imaging; c) a pharmacomodulatory molecule and a chromophore for its use in a method of diagnosing a tumor by optical imaging; d) a pharmacomodulatory molecule and an affinity molecule (a complex with a fluorophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and(streptavidin / biotin)) for use in a method of diagnosing a tumor by optical or fluorescence imaging. Among the conjugates with a detectable or capable of becoming detectable molecule and a pharmacomodulatory molecule described above, the conjugates a) (a pharmacomodulatory molecule and a radioisotope emitting direct or indirect gamma rays or a chelating agent) and b) (a pharmacomodulatory molecule and a fluorophore) are preferred. Method of surgical treatment by tumor resection guided by fluorescence or nuclear imaging probe The invention also relates to the conjugate according to the invention, for use in a method of surgical treatment by tumor resection guided by fluorescence or isotopic imaging probe. The present invention also relates to a method of surgical treatment by tumor resection guided by fluorescence or isotopic imaging probe, comprising: a) administering to apatient of a conjugate according to the invention whose antibody fragment binds specifically to an antigen of the patient's tumor, b) obtaining by real-time imaging images of the tumor labeled by the conjugate, and surgical resection of the entire tumor using the real-time images obtained. In this application, the mutant antibody fragment according to the invention is conjugated at the cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature to: a1) a fluorophore for its use in a method of surgical treatment by fluorescence-guided tumor resection; a2) an affinity molecule (a complex with a fluorophore conjugated to a binding partner of the affinity molecule is then formed before administration to the patient, the pair (affinity molecule / binding partner) being advantageously chosen from (biotin / avidin), (biotin / streptavidin), (avidin / biotin), and (streptavidin / biotin)) for its use ina method of surgical treatment by tumor resection guided by fluorescence imaging; b1) a chelating agent (in this case, a complex with a radiometal, direct or indirect emitter of gamma rays, is formed just before administration to the patient) for its use in a method of surgical treatment by tumor resection guided by an isotopic imaging probe, in particular by a single-photon emission computed tomography (SPECT) imaging probe, the radiometal is then a direct emitter of gamma rays, such as 99m Tc and 111 In) or by positron emission tomography (PET), the radiometal is then an indirect emitter of gamma rays, such as 64 Cu, 68 Ga, 89 Zr, and 44Sc), with a preference for use in a surgical treatment method by tumor resection guided by single-photon emission computed tomography (SPECT) imaging probe, the radiometal is then a direct emitter of gamma rays, such as 99m Tc and 111 In); b2) a direct or indirect emitting radioisotope (for example 18F) of gamma rays for its use in a method of surgical treatment by tumor resection guided by isotopic imaging probe; c1) a molecule defined in a1) and a molecule defined in b), thus allowing its use in a method of surgical treatment by tumor resection guided by fluorescence or by isotopic imaging probe; or c2) a molecule defined in a2) and a molecule defined in b), thus allowing its use in a method of surgical treatment by tumor resection guided by fluorescence or by isotopic imaging probe. In this method, the conjugates a1), b1) and c1) are preferred. Method for monitoring by imaging the effectiveness of an antitumor treatment The invention also relates to the conjugate according to the invention, for its use in a method for monitoring by imaging the effectiveness of an antitumor treatment. The present invention also relates to a method for treating a cancer patient,comprising: a) determining the tumor burden of the patient by the tumor burden determination method according to the invention before or concomitantly with the start of the administration of the antitumor treatment, b) administering the antitumor treatment to the patient, c) determining the tumor burden of the patient by the tumor burden determination method according to the invention after the start of the administration of the antitumor treatment, d) comparing the tumor burdens determined in steps a) and c), and e) administering an antitumor treatment to the patient, wherein: - if the tumor burden determined in step c) is lower than or equal to that determined in step a), then the antitumor treatment administered in step e) is the same as in step b); - if the tumor burden determined in step c) is higher than that determined in step a),then the antitumor treatment administered in step e) is different from that administered in step b). In this application, the molecule(s) conjugated to the mutant antibody fragment according to the invention at the cysteine ​​in position 128 of the heavy chain according to the IMGT nomenclature is advantageously chosen from molecules which are detectable or likely to become detectable. The molecule which is detectable or likely to become detectable will be chosen according to the type(s) of imaging envisaged, as described above in the section relating to the use of the conjugate according to the invention in a method for diagnosing a tumor by imaging. To determine the effectiveness of an antitumor treatment, the conjugate according to the invention is advantageously used: a) before or concomitantly with the start of the administration of the antitumor treatment, and b) at least once after the start of the administration of the antitumor treatment,and the tumor burden detected after the start of the administration of the antitumor treatment is compared to that detected before or concomitantly with the start of the administration of the antitumor treatment. If the tumor burden has decreased or remains stable after the start of the administration of the antitumor treatment, then the antitumor treatment is considered effective. If the tumor burden has increased after the start of the administration of the antitumor treatment, then the antitumor treatment is considered ineffective. The present invention is now illustrated by examples below. EXAMPLES Example 1: Comparison of the conservation of residues at positions 5, 24 and 128 of the heavy chain according to the IMGT nomenclature Junutula JR et al showed for a Fab Hu4D5 (Fab of a human anti-HER2 antibody) that the substitution of positions 5 and 23 of the heavy chain (according to a sequential numbering,each position number corresponding to the amino acid number in the amino acid sequence) by a cysteine ​​allows good reactivity of the thiol group of the cysteine ​​(see Table 2 of Junutula JR et al. Rapid identification of reactive cysteine ​​residues for site-specific labeling of antibody-Fabs, Journal of Immunological Methods, Volume 332, Issues 1–2, 2008, Pages 41-52, https: / / doi.org / 10.1016 / j.jim.2007.12.011). These positions correspond to positions 5 and 24 according to the IMGT nomenclature. In the Hu4D5 Fab, the amino acids at positions 5 and 24 of the heavy chain according to the IMGT nomenclature are a valine (V) and an alanine (A) respectively. The conservation of amino acids present in murine or human antibodies at positions 5, 24, and 128 of the heavy chain according to the IMGT nomenclature was analyzed. Materials and methods The heavy chain of an antibody is generated by recombination between 3 gene segments called V,D and J. Positions 5 and 24 of the heavy chain according to the IMGT nomenclature are located in the V segment. Therefore, to analyze their conservation within murine and human antibodies, the alignments of the murine V segments, on the one hand, and the human V segments, on the other hand, were obtained from the IMGT database. Within these alignments, the different residues present in position 5 or 24 of the heavy chain according to the IMGT nomenclature were identified and their frequency (number of occurrences / number of V segments) determined. Position 128 of the heavy chain according to the IMGT nomenclature is located in the J segment. Therefore, to analyze its conservation within murine and human antibodies, the alignments of the murine J segments, on the one hand, and the human J segments, on the other hand, were obtained from the IMGT database. Within these alignments,The different residues present at position 128 of the heavy chain according to the IMGT nomenclature were identified and their frequency (number of occurrences / number of V segments) determined. Results The results concerning position 5 of the heavy chain according to the IMGT nomenclature are presented in Table 3 below, and show that this position is not very conserved and varies significantly depending on the V segment which is used in the VH domain. [Table 3] Murine V segments Human V segments Amino acids at position 5 Q (60%), V (17%), K (17%), LV (63%), Q (19%), I (6%), heavy chain (IMGT) (2%), E (2%), P (0.7%), HL (5%), K (4%), G (1%), (0.4%), N (0.4%), D (0.4%) MR (1%), T (1%) (0.4%) Thus, a large number of different amino acids can be present at this position, depending on the V segment used in the VH domain of the original antibody fragment, some of which have quite different properties (e.g.,valine (V) is one of the amino acids with a slightly hydrophobic uncharged side chain, while arginine (R) and lysine (K) are among the amino acids with a positively charged side chain, and glutamine (Q) and threonine (T) are among the amino acids with a polar uncharged side chain). In addition, arginine (R), lysine (K), and glutamine (Q) have significantly longer side chains than valine. While replacing a valine (V) with a cysteine ​​allows for good reactivity of the cysteine ​​thiol, it is not clear whether replacing an arginine (R), lysine (K), glutamine (Q), or threonine (T) with a cysteine ​​would lead to the same result. In addition,Position 5 of the heavy chain according to the IMGT nomenclature is not indicated for introducing a substitution because positions 6 and 7 located immediately next to it are known to play a crucial role in the correct folding of the antibody chain. Introducing a C cysteine ​​at this position could therefore disrupt the correct folding of the heavy chain (Honegger, A., Pluckthun, A., 2001. The Influence of the Buried Glutamine or Glutamate Residue in Position 6 on the Structure of Immunoglobulin Variable Domains. Journal of Molecular Biology 309, 687–699. https: / / doi.org / 10.1006 / jmbi.2001.4664; Jung S, Spinelli S, Schimmele B, Honegger A, Pugliese L, Cambillau C, et al. 2001;309:701–16). Therefore, replacing the residue at position 5 with a cysteine,then conjugating it to a chemical group, could generate instability in the conformation of the variable region. Position 5 of the heavy chain according to the IMGT nomenclature is therefore not suitable for a general method of substituting this position with a cysteine ​​to allow site-specific conjugation. The results concerning position 24 of the heavy chain according to the IMGT nomenclature are presented in Table 4 below, and also show that this position is poorly conserved and varies significantly depending on the V segment that is used in the VH domain. [Table 4] Murine V segments Human V segments Amino acids at position 24 K (48%), A (16%), V (15%), SA (68%), K (22%), T (13% 6%), of the heavy chain (IMGT) (8%), T (7.6%), E (1.8%), QV (5%), S (1%) and their frequency (%) (1.3%), M (1.3%), P (0.4%), D (0.4%), I (0.4%) Thus,A number of distinct amino acids are used at this position depending on the V segment used in the VH domain of the original antibody fragment, some of which have quite different properties (e.g., valine V and alanine A are among the amino acids with a slightly hydrophobic uncharged side chain, while lysine K is among the amino acids with a positively charged side chain, and threonine T is among the amino acids with a polar uncharged side chain). In addition, lysine K has a significantly longer side chain than alanine. While replacing an alanine (A) with a cysteine ​​allows for good reactivity of the cysteine ​​thiol, it is not clear whether replacing a lysine K or a threonine T with a cysteine ​​would lead to the same result. Furthermore,Position 24 of the heavy chain according to the IMGT nomenclature is not suitable for introducing a substitution because it plays a crucial role in the correct folding of the antibody chain, being located right next to the cysteine ​​at position 23 IMGT making the SS bridge between the position of C23 and C104 (IMGT). Introducing a C at position 24 could therefore disrupt the correct folding and stabilization of the heavy chain. The proximity of CDR1-H (which starts only 3 amino acids further) could further disrupt the interaction with the antigen once the conjugation of the chemical group has been carried out at this position. Position 24 of the heavy chain according to the IMGT nomenclature is therefore not suitable for a general method of substituting this position with a cysteine ​​to allow site-specific conjugation. Finally, the results concerning position 128 of the heavy chain according to the IMGT nomenclature are presented in Table 5 below,and show instead that this position is highly conserved, with only one amino acid (S) being used by all human J segments, and only two distinct amino acids (S or A), one of which is common to human J segments, being used by mouse J segments. [Table 5] Mouse V segments Human V segments Amino acids in position S (75%), A (25%), S (100%) 128 of the heavy chain (IMGT) Thus, only one amino acid (S) is present at position 128 IMGT of the heavy chain in human J segments, and the majority of amino acids present at position 128 IMG of the heavy chain in mouse J segments are also serine, the only other amino acid used at this position is alanine. In addition, serine and valine have in common that they are amino acids with a short side chain, and therefore with similar steric hindrance,which makes it plausible that what works by replacing an alanine at position 128 IMGT of the heavy chain with a cysteine ​​will also work by replacing a serine at position 128 IMGT of the heavy chain with a cysteine. Furthermore, position 128 IMGT of the heavy chain corresponds to the last amino acid of the VH domain and is therefore not involved in antigen recognition or heavy chain folding. For all these reasons, position 128 IMGT of the heavy chain is therefore much more suitable than positions 5 and 24 IMGT of the heavy chain for a general method of substituting this position with a cysteine ​​to allow site-specific conjugation. Conclusion In summary,heavy chain IMGT positions 5 and 24 are poorly conserved and involved in the correct folding of the heavy chain (position 24 IMGT is also located near CDR1-H and also near the cysteine ​​at position 23 and their replacement by a cysteine ​​could therefore interfere with the canonical disulfide bridge between the cysteines at positions 23 and 104. These positions are therefore not suitable for a general method of substituting this position with a cysteine ​​to allow site-specific conjugation. In contrast, position 128 IMGT of the heavy chain, the last amino acid before the constant region, is highly conserved and is not involved in either antigen recognition or heavy chain folding. It is therefore particularly well suited to a general method of substituting this position with a cysteine ​​to allow site-specific conjugation. In addition,its position in the VH domain allows it to be used regardless of the type of antibody fragment of interest, provided that it includes a VH domain. This is not the case for position 129 IMGT identified in Junutula JR et al. as the best position of the Hu4D5 Fab heavy chain for site-specific conjugation (Junutula JR et al. Rapid identification of reactive cysteine ​​residues for site-specific labeling of antibody-Fabs, Journal of Immunological Methods, Volume 332, Issues 1–2, 2008, Pages 41-52, https: / / doi.org / 10.1016 / j.jim.2007.12.011). This position is indeed the first amino acid of the constant region. Thus, no fragment without a constant region (e.g., an scFv fragment, an Fv fragment, a diabody, a tribody,or a tetrabody) cannot be conjugated using this position. Example 2: Preparation of a mutant Fab of the chimeric antibody xiRA63 with a cysteine ​​at position 128 (IMGT nomenclature) of the heavy chain and random or site-specific grafting onto a tetrazine already bifunctionalized by a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800) Materials and methods Production of ThioFab-xiRA63: The heavy and light chains of ThioFab-xiRA63 or Fab-xiRA63 were cloned into the eukaryotic expression vector pTT5 (Durocher et al., 2002). The plasmids were co-transfected into ExpiCHO-S cells (ThermoFisher) according to the manufacturer's instructions "ExpiCHO™ Expression System User Guide" (MAN0014337 ThermoFisher Scientific). Day 12 post-transfection, the supernatant was clarified by centrifugation at 4000-5000xg for 30 min at 4°C and filtered through a 0.22 pm. ThioFab xiRA63 was then affinity purified on a HiTrap KappaSelect column (GE Healthcare). After elution, the antibody solutions were dialyzed with the Slide-A-Lyzer™ G2 Dialysis Cassette (Thermoscientific) with a 10kDa cutoff in 1 liter of PBS. Conjugation of ThioFab-DFO-IRDye800 ThioFab xiRA63 carrying a cysteine ​​is reduced in the presence of 15 equivalents of TCEP (20mM, PBS) at 37°C for 1h. After purification by ultrafiltration (Amicon 10kDa cut-off), 20 equivalents of dehydroascorbic acid (dhAA, 50mM, carbonate-bicarbonate buffer pH 9) are added and the reaction medium is stirred at 25°C for 1h in order to specifically reoxidize the disulfide bridges. The excess dhAA is removed by ultrafiltration then 20 equivalents of TCO-PEG3-maleimide (15 mM, DMSO) are added before stirring for 2h at 25°C. Purification is then carried out by centrifugation. Finally, 15 equivalents of DFO-Tz-IRDye800 (30mM,DMSO) are added and the IEDDA reaction takes place overnight at 25°C. The conjugated ThioFab is then purified by size exclusion chromatography (Superdex 7510 / 300 GL). Analysis of the fragment [, 89 Zr]-ThioFab-xiRA63-DFO by flow cytometry: We used the protocol of Herbet et al., 2018 (Herbet A. et al. Antibodies Targeting Human Endothelin-1 Receptors Reveal Different Conformational States in Cancer Cells. Physiol. Res. 67 (Suppl. 1): S257-S264, 2018. https: / / doi.org / 10.33549 / physiolres.933848). Two cell lines were used to assess the apparent affinity of the different antibody formats: Chinese Hamster Ovary Wild-Type (CHO-WT) and Chinese Hamster Ovary stably transfected with a eukaryotic expression vector encoding human ETA (CHO-ETA). Cells were resuspended in saturation buffer (1X PBS, 5% normal goat serum, 0.1% bovine serum albumin) and 3.10 5Live cells were seeded in each tube. Apparent affinity was determined with a dilution range of 0.1 nM to 500 nM for the fragments ThioFab-xiRA63, ThioFab-xiRA63-DFO-IRDye800 and Fab-xiRA63 and Fab-xiRA63-DFO-IRDye800. Samples were incubated at 4°C overnight. The next day, the system was visualized by a secondary antibody Fd anti-human goat IgG (CliniSciences 2046-02) conjugated to a fluorescent molecule, FITC, diluted 500 emeand incubated for 4 hours at 4°C. Three PBS washes of the samples were performed between each step. Sample fluorescence was measured at a wavelength of 488nm with the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and expressed as a percentage of the average fluorescence intensity per sample. The "one site specific binding" function on GraphPad was applied to obtain the binding curve, Bmax and apparent Kd ±SEM. Results Figure 1 presents antigen binding curves comparing the unmutated Fab fragment of the xiRA63 antibody and a conjugate obtained by random grafting of a tetrazine bifunctionalized with a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800).Figure 2 presents antigen binding curves comparing the mutant Fab fragment with a cysteine ​​at position 128 (IMGT) of the heavy chain according to the IMGT nomenclature of the xiRA63 antibody and a conjugate obtained by site-specific grafting of a tetrazine bifunctionalized with a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800). Tables 6 and 7 below further present the Bmax and Kd values ​​obtained from the curves.[Table 6] Unmutated Fab Random conjugate Bmax 70.39 28.43 Kd 1.811 2.9712 [Table 7] Mutant Fab Site-specific conjugate Bmax 63.64 55.27 Kd 0.9418 0.4562 While random conjugation of unmutated Fab results in a loss of target affinity and recognition (see Figure 1 and Table 6), site-specific conjugation of mutant Fab with a cysteine ​​at position 128 of the heavy chain according to IMGT nomenclature does not induce a significant change in antigen binding compared to native mutant Fab (see Figure 2 and Table 7). Conclusion The site-specific conjugate obtained from the mutant Fab with a cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature presents better conjugation characteristics than the random conjugate obtained from the classical Fab which is extremely perturbed.Due to its much better properties, the clinical use of the site-specific conjugate obtained from the mutant Fab with a cysteine ​​at position 128 of the heavy chain according to the IMGT nomenclature can then be considered. Example 3: Preparation of a mutant Fab of the chimeric antibody xiRA63 with a cysteine ​​at position 128 (IMGT nomenclature) of the heavy chain, grafting, in vitro characterization and in vivo use for imaging in a glioblastoma model Materials and methods Production of ThioFab-xiRA63: The heavy and light chains of ThioFab-xiRA63 were cloned into the eukaryotic expression vector pTT5 (Durocher et al., 2002). Plasmids were co-transfected into ExpiCHO-S cells (ThermoFisher) according to the manufacturer's instructions "ExpiCHO™ Expression System User Guide" (MAN0014337 ThermoFisher Scientific).Day 12 post-transfection, the supernatant was clarified by centrifugation at 4000-5000xg for 30 min at 4°C and filtered through a 0.22 µm filter. ThioFab xiRA63 was then affinity purified on a HiTrap KappaSelect column (GE Healthcare). After elution, the antibody solutions were dialyzed with the Slide-A-Lyzer™ G2 Dialysis Cassette (Thermoscientific) with a 10kDa cutoff in 1 L of PBS. ThioFab-xiRA63-DFO conjugation: ThioFab-xiRA63 (500 µg, 459 µL at 1.1 g / L) was reduced with 30.8 µL of TCEP (20mM) at 37°C for 1 hour. After purification using centrifuge filter units with a molecular weight cutoff of 10,000 Da, reoxidation of the native interchain disulfide bond was performed with 6.75 µL of dehydroascorbic acid (dhAA, 20 equiv., 50 mmol / L in carbonate / bicarbonate buffer pH 9) for 1 hour at 37°C.After removal of excess dhAA with centrifugal filtration units, 3.62 µL of deferoxamine-maleimide (10 equiv., 15 mmol / L in DMSO) was added to the reaction mixture and stirred at 37 °C for 2 hours. The conjugate was then purified by size exclusion chromatography (Superdex 7510 / 300 GL) to remove potential dimers. Validation of DFO conjugation on ThioFab-xiRA63 was performed by mass spectrometry (MALDI-TOF). Radiolabeling of ThioFab-xiRA63-DFO with Zirconium 89 [. 89Zr]: To radiolabel this antibody fragment with zirconium-89 (PerkinElmer), a previously described protocol from Vosjan et al., 2010 (Vosjan, M., Perk, L., Visser, G. et al. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine. Nat Protoc 5, 739–743 (2010). https: / / doi.org / 10.1038 / nprot.2010.13) was used. The day before injection 0.65 mg of ThioFab-xiRA63-DFO, pH adjusted to 7.2, was incubated with the radioelement 89 Zr for 1 h at 37 °C with stirring at 300 rpm. The oxalic acid from the solution of 89 Zr (274 µL, 11.17 mCi) was neutralized with 90 µL of Na2CO3 (2 M) before adding them to the antibody solution. The [ 89Zr]Zr-ThioFab-xiRA63-DFO was then purified on a PD-10 column with gentisic acid solution (5 mg / mL in 0.25 M sodium acetate, pH 5.5) as the mobile phase and concentrated with Vivaspin® ultrafiltration tubes (Satorius) with a 10 kDa cutoff. Evaluation of the radiochemical purity of the fragment [ 89 Zr]Zr-ThioFab-xiRA63-DFO by HPLC: The radiochemical purity of the radioligand was verified via size exclusion high performance liquid chromatography (HPLC) coupled with a UV (UVD 170U UV / VIS) and gamma scintillation detector (Packard). Measurements were performed on a bioZen 1.8 µm SEC-2 LC analytical column (Phenomenex) coupled to a DIONEX HPLC system (Thermo-Fisher). Elution was performed by a linear gradient with a solution of KH2PO4 (50 mM) and KCl (250 mM) (pH 6.8), at a flow rate of 0.2 mL / min. Analysis of the fragment [ 89Zr]Zr-ThioFab-xiRA63-DFO by flow cytometry: We used the protocol of Herbet et al., 2018 (Herbet A. et al. Antibodies Targeting Human Endothelin-1 Receptors Reveal Different Conformational States in Cancer Cells. Physiol. Res. 67 (Suppl. 1): S257-S264, 2018. https: / / doi.org / 10.33549 / physiolres.933848). Two cell lines were used to assess the apparent affinity of the different antibody formats: Chinese Hamster Ovary Wild-Type (CHO-WT) and Chinese Hamster Ovary stably transfected with human ETA (CHO-ETA). The cells were resuspended in saturation buffer (1X PBS, 5% normal goat serum, 0.1% bovine serum albumin) and 3.10 5Live cells were seeded into each tube. Apparent affinity was determined with a dilution range of 0.01 nM to 50 nM for the ThioFab-xiRA63-DFO and ThioFab-xiRA63 fragments. Samples were incubated at 4°C overnight. The next day, the system was visualized by a secondary antibody Fd anti-human goat IgG (CliniSciences 2046-02) conjugated to a fluorescent molecule, FITC, diluted 500 emeand incubated for 4 hours at 4°C. Three PBS washes of the samples were performed between each step. Sample fluorescence was measured at a wavelength of 488nm with the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and expressed as a percentage of the average fluorescence intensity per sample. The "one site specific binding" function on GraphPad was applied to obtain the binding curve, Bmax and apparent Kd ±SEM. Preclinical model: All animal experiments were carried out in accordance with the European Directive 2010 / 63 / EU and its transposition into French law (Decree No. 2013-118). Six four-week-old female NMRI nude mice (Janvier Labs) were orthotopically implanted with 5.10 5 glioblastoma cells (Gli7; ETA +), in the striatum (2 mm to the right of bregma and 2.5 mm deep from the dura mater) under isoflurane anesthesia combined with local xylocaine. A 10 µL Hamilton syringe was used with a flow rate of 0.25 µL / min. During the injection, 0.05 mg.kg -1 of buprenorphine was administered subcutaneously to each mouse. The mice were housed four at a time in ventilated cages with 40% humidity and a temperature-controlled room at 22°C. Antibody injection: 78 days after glioblastoma cell implantation (Herbet A. et al. Antibodies Targeting Human Endothelin-1 Receptors Reveal Different Conformational States in Cancer Cells. Physiol. Res. 67 (Suppl. 1): S257-S264, 2018. https: / / doi.org / 10.33549 / physiolres.933848), a total of 5 mice received an intravenous bolus of [ 89Zr]Zr-ThioFab-xiRA63 (100 µC ± 5 µC). PET-CT image acquisition: We performed a first dynamic acquisition of 60 minutes immediately after the injection of the antibody using an Inveon microPET scanner or an Inveon microPET / CT scanner (Siemens). Then, a 20-minute PET acquisition was performed at times: 5h, 24h, 48h, 72h and 7 days after the injection. The imaging sessions of the mice were performed under anesthesia. PET-CT analysis: We used PMOD software (Version 3.9) to analyze the different acquisitions. Spherical volumes of interest (VI) with a fixed size of 8mm 3 were created in several organs: heart, liver, kidneys, spleen, and muscle. For bone and brain tumor, VIs were plotted using the "iso-contour" tool. We applied the same VIs in the contralateral side to the tumor (used as a negative control) as for the tumor. The mean volume activity (kBq.cm -3) in each VI was corrected for the half-life of the 89 Zr (t1 / 2=3.3 days) following the radioactive decay law. Then, to quantify the antibody, we divided it by the total injected activity to obtain the percentage of injected dose per tissue volume (%DI.cm -3). Statistics: GraphPad Prism software (v9.0.1) was used for statistical analyses. Student's t-test (two-tailed) was performed to compare two groups of data (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Results Mass spectrometry validation of DFO antibodies: ThioFab-xiRA63 has an m / z equivalent to 48631.5 Dalton while ThioFab-xiRA63-DFO has an m / z of 49342.5 Dalton. The difference of 711 Dalton corresponds perfectly to the molecular weight of DFO-maleimide (Figure 3). Flow cytometry validation of DFO antibodies: ThioFab-xiRA63 with and without DFO show high affinity for human ETA receptors (see Figure 4), with similar apparent affinity constants in the nanomolar range (Kd) showing that DFO conjugation on the additional free thiol does not alter the affinity of our fragment for its target (p-value > 0.1).Their specificity was tested with negative control cells CHO-WT and positive control cells CHO-ETA. The signal obtained on CHO-WT is in the background noise, i.e. less than 2% of the MFI obtained with the concentration of ThioFab xiRA63-DFO. PET imaging For PET imaging experiments, ThioFab-xiRA63-DFO was radiolabeled with Zirconium 89 [. 89 Zr] according to the procedure previously published by Vosjan et al. (Vosjan, M., Perk, L., Visser, G. et al. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine. Nat Protoc 5, 739–743 (2010). https: / / doi.org / 10.1038 / nprot.2010.13). Their radiochemical purity was assessed by size exclusion high-performance liquid chromatography (HPLC) and was estimated to be 100% (Figure 5). Preclinical model Once the antibody was radiolabeled ([ 89Zr]Zr-ThioFab-xiRA63) validated, we injected it (5.1 ± 0.13 MBq, 3.3 ± 0.1 nmol) into mice (n=5) previously orthotopically xenografted with glioblastoma tumor cells named Gli7 expressing ETA at their membrane (ETA+, Herbet A. et al. Antibodies Targeting Human Endothelin-1 Receptors Reveal Different Conformational States in Cancer Cells. Physiol. Res. 67 (Suppl. 1): S257- S264, 2018. https: / / doi.org / 10.33549 / physiolres.933848) (Figure 6). Subsequently, the mice were imaged whole-body by microPET / CT at different times (for 1h after injection, then at 5h, 24h, 48h, 72h, and 168h or 7 days pi) allowing us to study the tissue distribution (biodistribution) of our PET immunotracer over time. In this experiment (Figure 7), the [ 89 Zr]Zr-ThioFab-xiRA63 (<60kDa) appears to follow a renal elimination pathway (70% ID.cm -3accumulation in the kidney in the first hour pi). Indeed, small molecules are rapidly eliminated from the bloodstream due to glomerular filtration and renal excretion. Our tracer follows one of the classic elimination pathways due to its low molecular weight, without targeting non-specific organs in which we could find murine endothelin A receptor (muscle, bone, bladder, ovaries, etc.). Regarding the accumulation of [ 89 Zr]Zr-ThioFab-xiRA63 in Gli 7 brain tumors (Figure 8A), its detection is visualized from 1h pi (~1%DI.cm -3 ). Due to its rapid plasma and blood elimination, its signal weakens rapidly and is close to ~0.3%DI.cm -3after 5 hours p.i. However, this rapid elimination allows us a better contrast to visualize the tumor due to the low parasitic signals and the almost absence of background noise (Figure 8B). Conclusion We have demonstrated the functionality of the conjugated fragment [Zr 89]Zr-ThioFab-xiRA63-DFO in vivo. The latter is well able to recognize the brain tumor expressing endothelin receptors. Its biodistribution is also consistent with a Fab fragment (mutant or not) and molecules with a molecular weight below 60kDa, i.e. rapid elimination by the kidneys. Example 4: Preparation of a thioFab (thioFab-xiRB49) of the chimeric RB49 antibody (xiRB49) Unexpectedly, simple chimerization (substitution of mouse constant regions by human constant regions) of RB49 led to a non-functional chimeric antibody, unable to bind specifically to the ETB receptor. In silico studies have shown that the presence of an unusual proline at position 125 of the variable region of the heavy chain of RB49 could induce a different folding of the variable region during its chimerization.The CDR3s of the heavy and light chains then come into prolonged contact, making them unavailable for interaction with the ET antigen. B . A mutation of P125 by a canonical threonine allowed to restore the functionality of the chimeric antibody (xiRB49) (Marie Hautiere et al ., (2023) The functionality of a therapeutic antibody candidate restored by a single mutation from proline to threonine in the variable region, Human Vaccines & Immunotherapeutics, 19:3). These results suggest that the affinity of the RB49 antibody for its ET antigen Bcan be affected by a single mutation in the VH domain (FR4 part). They indeed highlight the structural complexity of the RB49 antibody, making the effect of any modification of it difficult to predict. Despite a modification of the conformation of the Kappa constant region associated with the substitution of alanine at position 128 IMGT by a cysteine ​​(A128C IMGT), it is demonstrated here that a Fab of this antibody further comprising a mutation of the cysteine ​​at position 128 IMGT of the VH domain (thioFab-xiRB49) retains its affinity for its antigen ET B. Materials and methods Production and purification of chimeric antibody xiRB49-P125T, Fab-xiRB49-P125T and ThioFab-xiRB49-P125T We generated a chimeric version of RB49 called xiRB49. The heavy and light chains encoding IgG-xiRB49, Fab-xiRB49-P125T and ThioFab-xiRB49-P125T were subcloned into the expression plasmid pTT5. The vectors were co-transfected into ExpiCHO-S cells (ThermoFisher Scientific) with the ExpiCHO Expression System kit (ThermoFisher Scientific) according to the manufacturer's instructions (MAN0014337 ThermoFisher Scientific). At day 12 post-transfection, the supernatant was clarified by centrifugation at 4000-5000 × g for 30 min at 4 °C and filtered through a 0.22 μm filter. xiRA63 was purified on a HiTrap Protein A HP column (GE HealthCare) and Fab and ThioFab on the HiTrap KappaSelect (GE HealthCare).After elution, the antibody solutions were dialyzed with the Slide-A-Lyzer™G2 dialysis cassette (ThermoFisher Scientific) into 1 L of phosphate-buffered saline (PBS). Thermal stability study Samples at 1 mg⋅mL-1 were deposited in the capillaries to determine the antibody denaturation temperature curves (Tycho NT6 instrument, NanoTemper). The ratio A350 nm / A330 nm was calculated, and its first derivative gave us an inflection temperature (Ti) corresponding to the conformational change of the protein domain from the native to the denatured state. Determination of antibody binding curves on ET. Bwith a goat polyclonal anti-human Kappa secondary antibody Binding experiments were performed on CHO cells expressing or not endothelin receptors. Cells were incubated overnight at 4°C with Fab-xiRB49-P125T or ThioFab-xiRB49-P125T ranging from 0.01 nM to 150 nM and detected with a goat polyclonal anti-human Kappa secondary antibody conjugated to Alexa Fluor 488 (A18854). Throughout this experiment, three washes with 1X PBS (pH: 7.4) were performed between each step to eliminate non-specific signal. Competition assay Cells were pre-incubated overnight at 4°C in the presence of a molar excess (500nM) of Fab-xiRB49-P125T or ThioFab-xiRB49-P125T antibody fragments. Subsequently, a control panel of xiRB49-P125T antibodies was added for each condition (see Figure 11).The ranges read by flow cytometry were revealed by the goat anti-human IgG polyclonal secondary antibody coupled to Alexa Fluor488 (1110120) targeting only the Fc region of the antibodies. Throughout this experiment, three washes with 1X PBS (pH: 7.4) were performed between each step to eliminate the non-specific signal. Determination of antibody binding curves on ETB with an anti-Fd secondary antibody Binding experiments were performed on CHO cells expressing or not endothelin receptors. Cells were incubated overnight at 4°C with Fab-xiRB49-P125T or ThioFab-xiRB49-P125T ranging from 0.01 nM to 150 nM and detected with a FITC-conjugated goat anti-human Fd-IgG polyclonal secondary antibody (Southern Biotech™ 204602). Throughout this experiment, three washes with 1X PBS were performed. (pH:7.4) were performed between each step to eliminate the non-specific signal. Results Physicochemical parameters of ThioFab-xiRB49-P125T After the construction of the vectors necessary for its production and purification, the physicochemical properties of ThioFab-xiRB49-P125T were determined. The ThioFab-xiRB49-P125T fragment, like the ThioFab-xiRA63 fragment, had an expected molecular weight of 50kDa with a completely acceptable purity (> 95%) assessed by SDS PAGE. The hydrodynamic radius obtained by DLS was approximately 2.6 nm ± 0.1 nm and identical to that of all the Fabs obtained (results not shown). We studied the thermal stability of the antibodies and obtained thermal denaturation curves (Tycho NT, Nanotemper). Figure 12 presents the first derivative of the A350 nm / A330 nm ratio of the xiRB49-P125T, Fab-xiRB49-P125T and ThioFab-xiRB49-P125T antibodies, allowing the inflection temperatures (T i). The three antibodies show the same T i2, reflecting the Fab domain of the order of 90°C. After confirming the physicochemical properties of the different fragments, their functionality was evaluated. Antibody functional studies: first determination of antibody binding curves on ETB The functionality of Fab-xiRB49-P125T and ThioFab-xiRB49-P125T was evaluated by performing flow cytometry binding curves on CHO-ETB cells (Figure 13). The results show that Fab-xiRB49-P125T has a high apparent affinity in the nanomolar range and a Bmax close to 83% MFI (Figure 13). Surprisingly, no binding curve was obtained for ThioFab-xiRB49-P125T (Figure 13) while its Ti2 indicates that its Fab domain appears to be folded in the same way as the corresponding antibody and Fab suggesting that ThioFab should be functional. It was hypothesized that ThioFab-xiRB49-P125T was not recognized by the secondary antibody.Indeed, in this binding experiment, Fabs are revealed by a secondary goat polyclonal antibody anti-human Kappa-AlexaFluor 488 (ref A18854). It is possible that the cysteine ​​added at the end of the variable region, position A128C IMGT, could have modified this interaction necessary for its detection. Functional validation of ThioFab-xiRB49-P125T In order to verify this hypothesis, a competition test between xiRB49-P125T and Fab-xiRB49-P125T or ThioFab-xiRB49-P125T was developed (see Appendix - Supplementary material and method). CHO-ETB cells were pre-incubated overnight in the presence of Fab-xiRB49-P125T or ThioFab-xiRB49-P125T fragments at a concentration of 500 nM. The next day and after washing, xiRB49-P125T is added at increasing concentrations. Thus, 3 conditions are tested (Figure 11): 1.A reference condition (positive control) with CHO-ETB cells pre-incubated in the presence of PBS (vehicle) and therefore without signal displacement when adding xiRB49-P125T at different concentrations; 2. A control condition for the validity of the competition test: the Fab-xiRB49-P125T fragment being functional, it should interfere with the fixation of xiRB49-P125T on ETB. Thus, a higher concentration of xiRB49-P125T antibody will be necessary to shift this signal, which will result in the modification of an apparent Kd. 3. A test condition with the ThioFab-xiRB49-P125T fragment. If the effect observed with the Fab-xiRB49-P125T condition is similar to that obtained with ThioFab-xiRB49-P125T then this antibody is functional but not recognized by the anti-human Kappa- secondary antibody. Figure 14 shows the results of the binding curves obtained with the competition assay.As expected, Fab-xiRB49-P125T effectively displaces the signal emitted by xiRB49-P125T with an apparent affinity increasing from 2.49 nM to 16.62 nM. Similarly, ThioFab-xiRB49-P125T induces an equivalent displacement of the signal emitted by xiRB49-P125T with an apparent Kd increasing to 11.69 nM. No significant difference is observed between the two competition conditions involving Fab-xiRB49-P125T and ThioFab-xiRB49-P125T. This result proves that the ThioFAb-xiRB49-P125T antibody is indeed functional as suggested by its thermal denaturation curve and its Ti2 inflection temperature. Indirectly, the lack of recognition of ThioFab-xiRB49-P125T by a secondary anti-human polyclonal Kappa antibody strongly suggests that the addition of cysteine ​​nevertheless modifies the conformation of the Kappa constant region.A new secondary antibody targeting this time the Fd region of the antibodies (CH1+VH) was used to validate the functionality of ThioFab-xiRB49-P125T. Under these conditions, the binding to the ETB antigen of ThioFAb-xiRB49-P125T is clearly demonstrated (Figure 15), with an apparent Kd and a Bmax similar to Fab-xiRB49-P125T. All these results corroborate the hypothesis that the Cysteine ​​at position 128 IMGT modifies the Kappa region in ThioFab-xiRB49-P125T, which is then no longer recognized by a secondary anti-Kappa-Human-polyclonal antibody. ThioFab-xiRB49-P125T, however, retains an intact capacity for binding to the ETB antigen. Conclusion Despite the effect of abolishing the affinity for the ETB antigen linked to simple chimerization, a substitution of proline at position 125 IMGT by a threonine (P125T IMGT) allowed the affinity for the ET antigen to be restored. Bof the chimeric antibody (xiRB49-P125T) and a Fab fragment of such a chimeric antibody (Fab-xiRB49-P125T). In addition, despite a partial destructuring of the Kappa region (which is then no longer recognized by a polyclonal anti-human Kappa secondary antibody) associated with the additional substitution in Fab-xiRB49-P125T of alanine at position 128 IMGT by a cysteine ​​(A128C IMGT), this substitution does not affect the affinity for the ETB antigen of ThioFab-xiRB49-P125T. Strangely, this destructuring of the Kappa region is not found for ThioFab-xiRA63 following the insertion of C121 suggesting a phenomenon specific to the variable regions of RB49.

Claims

CLAIMS 1. Mutant antibody fragment specifically binding to endothelin receptor A or receptor B comprising a mutated heavy chain variable domain, the amino acid residue at position 128 according to the IMGT nomenclature of the heavy chain variable domain being substituted by a cysteine, provided that said fragment is not a Fab fragment whose mutated heavy chain fragment corresponds to the amino acid sequence SEQ ID NO: 1 and the light chain fragment corresponds to the amino acid sequence SEQ ID NO:

2.

2. Mutant antibody fragment according to claim 1, characterized in that it further comprises a light chain variable domain and is advantageously chosen from a Fab fragment, an scFv fragment, an Fv fragment, a diabody, a tribody, a tetrabody, and a minibody; or 3. Mutant antibody fragment according to claim 2, characterized in that it is a Fab fragment. 4.Mutant antibody fragment according to claim 1, characterized in that it does not comprise a light chain variable domain and is advantageously a nanobody.

5. Mutant antibody fragment according to any one of claims 1 to 4, characterized in that it is chimeric, humanized or human. 6.Mutant antibody fragment according to any one of claims 1 to 5, characterized in that the antibody fragment binds specifically to the endothelin receptor B, and comprises: - a heavy chain variable domain (VH) comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having according to the IMGT nomenclature for respective sequences GYTFISYW (SEQ ID NO: 3), IDPDSGGT (SEQ ID NO: 4) and AREGDYAWFAY (SEQ ID NO: 5) or a sequence with at least 80% identity with GYTFISYW (SEQ ID NO: 3), IDPDSGGT (SEQ ID NO: 4) or AREGDYAWFAY (SEQ ID NO: 5), and - a light chain variable domain (VL) comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having according to the IMGT nomenclature for respective sequences QSIVHSNGNTY (SEQ ID NO:6), KVS (SEQ ID NO:7) and FQGSHVPWT (SEQ ID NO:8) or a sequence with at least 80% identity with QSIVHSNGNTY (SEQ ID NO:6), KVS (SEQ ID NO:7) or FQGSHVPWT (SEQ ID NO:8). 7.Mutant antibody fragment according to any one of claims 1 to 5, characterized in that the antibody fragment binds specifically to endothelin receptor A. and comprises: - a VH domain comprising three heavy chain CDRs CDR1-H, CDR2-H and CDR3-H having according to the IMGT nomenclature for respective sequences GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) and VSSYYSGSFFAY (SEQ ID NO: 11) or a sequence with at least 80% identity with GFTFNIYA (SEQ ID NO: 9), IRSKSNNYAT (SEQ ID NO: 10) or VSSYYSGSFFAY (SEQ ID NO: 11), and - a VL domain comprising three light chain CDRs CDR1-L, CDR2-L and CDR3-L having according to the IMGT nomenclature for respective sequences SQSIVYSNGKIYL (SEQ ID NO: 12), KVS (SEQ ID NO: 13) and FQGSHLPLT (SEQ ID NO: 14) or a sequence with at least 80% identity with SQSIVYSNGKIYL (SEQ ID NO:12), KVS (SEQ ID NO:13) or FQGSHLPLT (SEQ ID NO:14). 8.Mutant antibody fragment according to any one of claims 1 to 6, characterized in that the antibody fragment binds specifically to the endothelin receptor B, and comprises: - a VH domain having the sequence SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15, and - a VL domain having the sequence SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO:

16.

9. Mutant antibody fragment according to any one of claims 1 to 5 and 7, characterized in that the antibody fragment binds specifically to the endothelin receptor A, and comprises: - a VH domain having the sequence SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17, and - a VL domain having the sequence SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO:

18. 10.Nucleic acid molecule or pair of nucleic acid molecules encoding the mutant antibody fragment according to any one of claims 1 to 9.

11. Vector comprising the nucleic acid molecule or pair of nucleic acid molecules according to claim 10.

12. Host cell, transgenic non-human animal or transgenic plant comprising the nucleic acid molecule or pair of nucleic acid molecules according to claim 10 or the vector according to claim 11.

13. Conjugate comprising the mutant antibody fragment according to any one of claims 1 to 9, linked to at least one, in particular one or two, molecules of interest, via cysteine, and more particularly the thiol function of cysteine, at position 128 of the. heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment.

14. Conjugate according to claim 13, corresponding to the following formula (I), (IIa) or (IIb): R 1 -X 1 -L 1 -R 2 (IIa) R 1 -X 1 -L 1 -X 1 '-L 4 -R 2 (IIb) in which: R 1 represents a mutant antibody fragment according to any one of claims 1 to 6 linked to X 1 via cysteine, and more particularly the thiol function of cysteine, at position 128 of the heavy chain variable domain according to the IMGT nomenclature; with X 4 representing a single bond, O, S, or NR 4 , and R 4 representing H or (C1-C6)alkyl, the wavy bond indicating the point of attachment to R 1 and the dotted line indicating the point of attachment to L 1 ; wavy indicating the attachment point to L 4 , and the dotted line indicating the point of attachment to L 1 ; L 1 , L 2 , L 3 and L 4each independently represent a single bond or a spacer, the spacer being a chain (C1-C 30 )-alkyl, for example (C1-C 20 )-alkyl, optionally preceded and / or interrupted and / or followed by one or more units chosen from the group consisting of aromatic, heteroaromatic, cycloalkane, cycloalkene, heterocycloalkane, heterocycloalkene, -O-, -S-, -NR rings 5 -, -C(O)-, -C(S)-, -C≡C-, and - C(R 6 )=C(R 7 )-, with R 5 representing H or (C1-C6)alkyl, R 6 and R 7 each independently representing H or (C1-C6)alkyl, and the aromatic, heteroaromatic, cycloalkyl, cycloalkenyl, heterocycloalkyl and heterocycloalkenyl rings being optionally substituted by one or more (C1-C6)alkyl groups; represents a double bond or a single bond, preferably a double bond; ring A 1represents a cycloalkane, cycloalkene, heterocycloalkane or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy; ring A 2 represents a cycloalkene or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy; X 2 and X 3 , identical or different, each represent O, S or NR 8 , with R 8 representing H or (C1-C6)alkyl, preferably H; R 2 and R 3, identical or different, preferably different, each represent a residue of a molecule of interest advantageously chosen from molecules which are detectable or capable of becoming detectable, affinity molecules and pharmacomodulatory molecules.

15. Conjugate according to claim 14, in which the molecule of interest is: - a molecule which is detectable or capable of becoming detectable chosen from a fluorophore, a chromophore, an affinity molecule, a radioisotope and a chelating agent; - an affinity molecule chosen from biotin, avidin, streptavidin and a hexahistidine peptide; and / or - a pharmacomodulatory molecule chosen from compounds bearing linear or branched chains of poly(ethylene glycol), linear or branched chains of poly(glutamic acid), cholesterol, and molecules capable of binding to albumin. 16.A conjugate according to any one of claims 14 or 15, having formula (I) and wherein at least one of R. 1 and R 2 is a residue of a molecule that is detectable or likely to become detectable, and in particular R 1 and R 2 each independently represent a residue of a molecule that is detectable or likely to become detectable, advantageously R 1 and R 2represent respectively a residue of a fluorophore and a residue of a radioisotope or a chelating agent.

17. A method for preparing a conjugate according to any one of claims 13 to 16 comprising the following steps: a) reduction of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of tris(2-carboxyethyl)phosphine (TCEP), to give a reduced mutant antibody fragment, b) reoxidation of the disulfide bridge(s) of the mutant antibody fragment, in particular in the presence of dehydroascorbic acid, to give a partially reduced mutant antibody fragment, c) conjugation with at least one molecule of interest of the thiol function of the cysteine ​​at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the partially reduced mutant antibody fragment to give a conjugate according to any one of claims 13 to 16.

18. A method according to claim 17, wherein: - the conjugate is a conjugate of formula (I) according to any one of claims 14 to 16 and step c) is carried out by coupling between the partially reduced mutant antibody fragment and a compound of the following formula (III): for which L 1 is as defined in claim 14, cycle A 3 represents a cycloalkyne, cycloalkene, heterocycloalkyne or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy, and X 1a represents a chemical group reactive with respect to an SH function, advantageously , with Hal representing Br or I, X 4 representing a single bond, O, S, or NR 4 , and R 4 representing H or (C1-C6)alkyl, to give a compound of the following formula (IV): for which R1 , X 1 and L 1 are as defined in claim 14, and ring A 3 is as defined above, followed by the coupling between the compound of formula (IV) and a compound of the following formula (V): for which R 2 , R 3 , L 2 , L 3 , X 2 and X 3 are as defined in claim 14; or - the conjugate is a conjugate of formula (IIa) according to any one of claims 14 to 16 and step c) is carried out by coupling the partially reduced mutant antibody fragment with a compound of formula X 1a -L 1 -R 2 in which L 1 and R 2 are as defined in claim 14 and X 1ais as defined above; or - the conjugate is a conjugate of formula (IIb) according to any one of claims 14 to 16 and step c) is carried out: i) by coupling the partially reduced mutant antibody fragment with a compound of formula X 1a -L 1 -X 1 '-L 4 -R 2 in which L 1 , L 4 , X 1 ' and R 2 are as defined in claim 14 and X 1a is as defined above; or ii) by coupling the partially reduced mutant antibody fragment with a compound of formula X 1a -L 1 -G 1 in which X 1a is as defined above, L 1 is as defined in claim 14 and G 1 represents a reactive chemical group, to give a conjugate of formula R 1 -X 1 -L 1 -G 1 , followed by the coupling between the conjugate of formula R 1 -X 1 -L1 -G 1 and a compound of formula G 2 -L 4 -R 2 in which L 4 and R 2 are as defined in claim 14 and G 2 is a reactive chemical group different from G 1 and able to react with G 1 to form a group X 1 '.

19. Intermediate conjugate comprising the mutant antibody fragment according to any one of claims 1 to 9, linked to a reactive chemical group, via cysteine, and more particularly the thiol function of cysteine, at position 128 of the heavy chain variable domain according to the IMGT nomenclature of the mutant antibody fragment.

20. Intermediate conjugate according to claim 19, corresponding to the formula R 1 -X 1 -L 1 -Z in which R 1 , X 1 and L 1are as defined in claim 14, and Z represents a reactive chemical group.

21. An intermediate conjugate according to claim 19 or 20, wherein the reactive chemical group is selected from , with cycle A 3 representing a cycloalkyne, cycloalkene, heterocycloalkyne or heterocycloalkene ring, optionally joined with one or more, in particular one or two, aromatic rings and optionally substituted by one or more groups chosen from (C1-C6)alkyl, halogeno, and hydroxy.

22. Conjugate according to any one of claims 13 to 16, for its use in: a) a method of diagnosing a tumor by imaging, b) a method of surgical treatment by tumor resection guided by fluorescence or nuclear imaging probe, c) a method of monitoring by imaging the effectiveness of an antitumor treatment, or d) any combination of methods a), b) and c).

Images