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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0058—Antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2869—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3053—Skin, nerves, brain
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

• 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.
• fluorescent conjugates are of growing interest for surgical assistance (FGS for Fluorescence Guided Surgery).
• FGS Fluorescence Guided Surgery
• 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 resection of tumors, 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 some so-called "site-specific" approaches to control the attachment site on proteins.
• strategies that are sufficiently modular to allow the customized introduction of several functionalities.
• 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.
• a trastuzumab conjugated to a bifunctionalized tetrazine platform by a chelating agent (DOTAGA) and a fluorophore (disulphonated Cy5) has been described, but the grafting of the bifunctionalized tetrazine platform was carried out randomly and non-site-specifically, 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).
• BCN bicyclo[6.1.0]nonyne
• a diabody modified by the addition of a H6-GGC tag (6 histidines followed by two glycines and a cysteine) at the C-terminal and conjugated on these two cysteines by a fluorophore (IRDye800CW) and randomly by a radioisotope ( 124 Iodine) has been synthesized (Zettlitz, K.A., 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).
• a site-specific bifunctionalized trastuzumab with a chelating agent (DFO) and a fluorophore (IRDye800CW) has been described, the conjugate 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.
• 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 at the C-terminal 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).
• a linker Nabody 1-(VVTS)C of sequence SEQ ID NO:17.
• 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.
• 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), whose amino acid at position 128 IMGT has been substituted by a cysteine, as well as a conjugate of this fragment to polyethylene glycol (PEG) via a maleimide linker, in order to increase its half-life in vivo.
• 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 not affected 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).
• PEG polyethylene glycol
• 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.
• SEQ ID NO: 816 and 817 Table 4 of Example 2
• 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.
• no conjugate of these specific fusions is 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.
• sdAb single chain antibody directed against CEA6
• 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.
• no functional data confirming that this sdAB maintains an equivalent affinity for its antigen is presented. No conjugate of this specific sdAb is described either.
• VHHs are not necessarily available for all targets of interest, while many antibodies against targets of interest are available.
• 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 by 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.
• 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 to have an in vivo lifetime that is neither too long nor too short.
• the presence of two partial heavy chains can allow double conjugation, which would be absent in VHH or ScFv type fragments, thus allowing better specific activity in both fluorescence and isotopic imaging.
• the preparation of bioconjugates based on a Fab fragment poses difficulties.
• a Fab fragment generally does not include an N-glycan that can be modified to have an azide group capable of reacting with the BCN group of a lysine further functionalized by 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 bifunctionalized Fab’ fragment via a tetrazine platform.
• Conjugation is performed on the cysteines of the hinge region, which has the disadvantage of causing problems of homogeneity and reproducibility.
• this method requires the presence of a hinge region, which is not present in many antibody fragments, such as a Fab fragment, scFv, an Fv fragment, a diabody, a tribody, or a 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.
• 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 chosen 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 heavy chain of the antibody (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.
• this paper illustrates how difficult it is to predict the effect of mutation of a cysteine position on conjugation capacity, since 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).
• 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 or melanoma.
• endothelin type A receptor stimulates the proliferation of epithelial tumors.
• This is for example the case of tumor cells from ovarian, cervical, prostate or colon cancer.
• ET B is expressed in other tumor types such as skin cancer and in particular in melanoma, Kaposi's sarcoma, breast cancer, vulvar cancer and certain gliomas.
• ETB plays a role in tumor progression and proliferation.
• an antagonist of this receptor is used, there is then an inhibition of cell proliferation. Therefore, these antagonists seem to be candidates of choice for the therapy of these tumors.
• Antibodies that specifically bind to an endothelin receptor 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 with surgical tumor resection).
• endothelin receptors belong to class 1 or family A of G Protein Coupled Receptors (GPCRs). As a result, they include 7 transmembrane domains (TM). Coupled to heterotrimeric G proteins at the carboxyl end of the GPCR, they allow signal transduction inside the cell. TM domains consist of alpha helices connected by loops intracellular (i1, i2 and i3) and extracellular (e1, e2 and e3).
• the mutation is 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.
• the last amino acid of the VH domain is widely conserved in both humans and mice (see Example 1), making the proposed mutation usable in a large number of antibodies.
• 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.
• TCEP tris(2-carboxyethyl)phosphine
• Figure 1 shows the antigen binding curves on CHO-ET cells HAS of the unmutated Fab fragment of the xiRA63 antibody and a conjugate obtained by random grafting of a tetrazine bifunctionalized by a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800).
• DFO Zirconium-89 chelating agent
• IRSe800 a fluorophore
• Figure 2 shows 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 by a Zirconium-89 chelating agent (DFO) and a fluorophore (IRDye800).
• Figure 3 shows 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 shows 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 represents the evaluation of 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 shows a summary of the in vivo experiment: (A.) Experiments and animal sacrifices.
• B. Summary table of [89Zr]Zr-ThioFab-xiRA63 injection in mice bearing a human glioblastoma tumor.
• 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 p.i.
• B. Example of a PET scan of one of the brain tumors imaged with the [ 89 Zr]Zr- ThioFab-xiRA6348h p.i. 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 th helix 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.
• 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 Binding curves obtained by flow cytometry of the Fab-xiRB49-P125T and ThioFab-xiRB49-P125T antibodies allowing the determination of their apparent Kd and their Bmax.
• Figure 14 Binding curves obtained by flow cytometry of the competition test between xiRB49-P125T and Thio-Fab-xiRB49-P125T or Fab-xiRB49-P125T.
• Figure 15 Binding curves obtained by flow cytometry with an anti-Fd secondary antibody.
• 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).
• 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.
• 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 20 or fewer amino acids, such as a 6-histidine tag Hisx6, may additionally be here).
• Consisting of” or “consisting of” means excluding more than trace amounts of other components or steps.
• a polypeptide "consists of" a sequence of amino acids when the polypeptide does not contain any amino acids other than the stated amino acid sequence.
• Antibody or “immunoglobulin” or “Ig” means a molecule comprising at least one antigen-binding domain and a constant domain comprising an Fc moiety capable of binding to FcR receptors.
• an antibody is composed of 4 polypeptide chains: 2 heavy chains and 2 light chains linked together by a variable number of disulfide bridges providing 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.
• 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.
• the genes coding for the heavy and light chains of antibodies are generated by recombination of respectively three and two distinct gene segments 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.
• VH-DH-JH and VL-JL segments form the variable regions of the heavy and light chains respectively.
• the VH and VL regions each have 3 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.
• CDRs complementarity determining regions
• These three CDR regions, and particularly CDR3, are found in the part of the antibody that will be in contact with the antigen and are therefore very important for antigen recognition.
• antibodies retaining all three CDR regions and each of the heavy and light chains of an antibody retain the antigenic specificity of the original antibody for the most part.
• an antibody retaining only one of the CDRs, and in particular 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 (framework region FR) that vary the least from one VH or VL segment to another.
• the CDR3 region is also followed by a framework region FR4.
• 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 a variable region of the heavy or light chain of an antibody 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)).
• the CDRs are defined by searching for the amino acids responsible for binding to the antibody antigen.
• a 2 th method was proposed by the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT), this time based on the determination of hypervariable regions.
• IMGT INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®
• 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).
• FR1-IMGT positions 1 to 26
• FR3-IMGT 66 to 104
• FR4-IMGT 118 to 128
• CDR1-IMGT positions 27 to 38
• CDR2-IMGT positions 56 to 65
• CDR3-IMGT positions 105 to 117
• a program for determining the CDRs from the amino acid sequence of an antibody heavy or light chain according to different nomenclatures is the abYsis “Annotate” tool available at http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi.
• 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.
• variable regions whose sequence varies greatly from one antibody to another
• constant regions are characterized by an amino acid sequence very close 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 N at the C-terminus of a CH1 domain, a hinge region then 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 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 in particular be found 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).
• 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 (for “antibody-dependent cellular cytotoxicity”) responses: located in the CH2 domain.
• the light chain constant region for antibodies with two heavy chains and two light chains is composed of a single domain called CL.
• “monoclonal antibody” or “monoclonal antibody composition” is meant a composition comprising antibody molecules with identical and unique antigenic specificity.
• the antibody molecules present in the composition are likely to 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.
• Certain differences in protein sequences, linked to post-translational modifications (such as for example 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 ⁇ , ⁇ , ⁇ , ⁇ and ⁇ correspond respectively to the immunoglobulins IgG, IgA, IgM, IgE and IgD.
• the constant regions ⁇ include several subtypes: ⁇ 1, ⁇ 2, ⁇ 3, these three types of constant regions having the particularity of fixing human complement, and ⁇ 4, thus creating the subisotypes IgG1, IgG2, IgG3, and IgG4.
• chimeric antibody we mean an antibody that 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.
• the monoclonal antibody composition for use as a medicament according to the invention comprises a chimeric monoclonal antibody, the latter comprises human constant regions.
• a chimeric antibody can be prepared using genetic recombination techniques well known to those skilled in the art.
• the chimeric antibody can 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.
• 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.
• Verhoeyen et al Verhoeyen et al. BioEssays, 8: 74, 1988.
• a "humanized" antibody is defined as an antibody that contains CDR regions derived from an antibody of non-human origin, with the other parts of the antibody molecule being derived from one (or more) human antibodies.
• FRs residues of the backbone segments
• FRs residues of the backbone segments
• humanized antibodies according to the invention can be prepared by techniques known to those skilled in the art such as the technologies of “CDR grafting”, “resurfacing”, SuperHumanization, “Human string content”, “FR libraries”, “Guided selection”, “FR shuffling” and “Humaneering”, as summarized in the review by Almagro et al (Almagro et al. Frontiers in Bioscience 13, 1619-1633, January 1, 2008).
• 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.
• antigen-binding antibody fragment is meant a fragment of an antibody retaining the antigen-binding domain and thus having the same antigenic specificity as the original antibody.
• antigen-binding antibody fragments include: - for double-chain antibodies (derived from most mammals, including mouse and human): Fv, scFv, Fab, F(ab') fragments 2 , Fab’, diabodies (generally called “diabodies”), triantibodies (generally called “tribodies”), tetraantibodies (generally called “tetrabodies”); and - for single-chain antibody fragments (from camelids or cartilaginous fish), VHH fragments (VH domain of a camelid antibody) or VNAR fragments (VH domain of a cartilaginous fish antibody).
• Fv fragment or “variable fragment” is meant an antibody fragment formed from the VH and VL domains associated non-covalently.
• 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.
• Fab fragment we mean a fragment formed by the assembly of the part of the heavy chain located upstream of the papain cleavage site (the CH1 domain is included, but the hinge region and the other CH domains) and the entire light chain of the antibody of interest.
• Fab fragments are monomers of approximately 50 kDa.
• Fab fragment
• F(ab’) fragment 2 we mean a fragment formed by the pairing of 2 Fab' fragments by disulfide bridges at the cysteines of the hinge region.
• 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 (2 times that of a scFv).
• trimer or “tribody” or “triabody”, we mean 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 (3 times that of a scFv).
• 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).
• minibody or “minibody” is meant 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.
• nanobody or “single domain antibody” is meant the variable domain of the heavy chain of a single chain antibody.
• a nanobody can in particular be a VHH or a VNAR.
• VHH means the variable domain of the heavy chain of a single-chain antibody from a camelid (including llama and alpaca).
• VNAR means the variable domain of the heavy chain of a single-chain antibody from a cartilaginous fish (including shark).
• a VHH or VNAR typically 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., than at least 10 times greater) than that with which it binds to other antigens.
• a significantly higher binding affinity e.g., than at least 10 times greater
• antigen is meant a substance which, when administered to a subject, can stimulate the immune system and cause the production of antibodies recognizing this substance.
• an antigen can be a peptide or a protein, but can also be a nucleic acid, a lipid, a sugar, etc.
• an antigen can in particular be a cancer antigen, an antigen of a pathogenic microorganism (for example of a virus, a bacterium, a parasite), or a self-antigen.
• 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 healthy cells of the subject, expressed predominantly on the surface of cancer cells (i.e. the majority of healthy cells of the subject do not express the cancer antigen), or overexpressed on the surface of cancer cells (i.e.
• endothelin a neuropeptide secreted by the vascular endothelium, having a powerful vasoconstrictor effect on smooth muscle cells.
• ET-1, ET-2 and ET-3 composed of 21 amino acids and including 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 about 200 amino acids).
• ETDs endothelins
• EDNRA Endothelin subtype A receptor
• ETB Endothelin subtype B receptor
• GPCRs G protein-coupled receptors
• ET-1 endothelins and their receptors
• ET-1 plays a crucial role in the regulation of physiological smooth muscle motility, but ET-1 is also involved in a wide variety of pathologies including hypertension, heart failure, renal disorders and infectious diseases.
• 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 deregulated in melanoma, lung, renal and vulvar cancers ref (Rosan ⁇ , L., Spinella, F., Bagnato, A., 2013.
• 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. There are self-tolerance mechanisms and the immune system does not normally generate antibodies that bind specifically to a self antigen, but sometimes the control mechanisms are insufficient and a subject produces antibodies against one or more self antigens. This is called an autoimmune disease.
• identity percentages 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.
• 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.
• molecules of interest include in particular molecules that are detectable or likely to become detectable, affinity molecules (and pharmacomodulatory molecules.
• 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; imaging by fluorescence.
• PET positron emission tomography
• SPECT single-photon emission tomography
• CLI Cerenkov Luminescence imaging
• Isotopic imaging or “nuclear imaging” detects "radionuclides” or “radioisotopes”, i.e. radioactive isotopes of a given atom.
• PET positron emission tomography
• SPECT single-photon emission tomography
• the gamma rays of a radionuclide with direct or indirect emission of gamma photons are detected by gamma cameras.
• the radionuclides used In SPECT, the radionuclides used directly emit a gamma ray when they disintegrate, while in PET, the radionuclides used emit a positron during their disintegration, which after annihilation with a surrounding electron, leads to the emission of 2 gamma rays, thus obtained indirectly.
• Cerenkov Luminescence Imaging CLI
• Cerenkov radiation CLI
• 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, in particular 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 comprising 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 multiple conjugated aromatic rings or planar, cyclic molecules having one or more ⁇ bonds.
• An “affinity molecule” means a molecule that can be selectively and non-covalently bound to another chemical partner group, thereby forming a complex.
• pharmacomodulatory molecule is meant a chemical molecule, synthetic or natural, that can modify the pharmacokinetic properties of another molecule to which it is linked.
• halogeno is meant bromo, chloro, iodo, or fluoro.
• (C 1 -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.
• the cycloalkane will comprise 3 to 9 carbon atoms. It will preferably be cyclopropane, cyclohexane, cyclooctane, bicyclononane, norbonane.
• heterocycloalkane we mean a cycloalkane 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 piperidine, 1,2-piperazine, 1,4-piperazine, pyrrolidine, morpholine.
• cycloalkene we mean 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).
• the cycloalkene will comprise 3 to 10 carbon atoms. It will preferably be cyclopropene, cyclohexene, cyclooctene, bicyclononene, norbornene.
• 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. It may in particular be 3-pyrroline, 1,2,3,6-tetrahydropyridazine.
• 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). carbon).
• the cycloalkyne will comprise 3 to 9 carbon atoms. It will preferably be cyclooctyne, bicyclononyne.
• 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.
• 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 carbon atoms.
• heteromatic cycle an aromatic monocycle or polycycle comprising one or more, in particular 1, 2 or 3, attached cycles, the atoms constituting the cycle or cycles 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 cycle or cycles being carbon atoms.
• the cycle or cycles will advantageously be formed from 5 to 15, in particular 5 to 10 atoms.
• each cycle will be a 5- or 6-membered cycle.
• the invention 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.
• the invention covers any type of antigen-binding mutant antibody fragment comprising a mutated heavy chain variable domain as defined herein.
• the invention covers both a fragment: a) which further comprises a light chain variable domain (from antibodies having two heavy chains and two light chains) and b) which does not comprise a light chain variable domain (from single chain antibodies comprising only one heavy chain).
• Fragments which further comprise a light chain variable domain are however preferred, since 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.
• the mutant antibody fragment according to the invention may be any type of fragment having a mutated heavy chain variable domain (VH) and a light chain variable domain (VH), but may advantageously be chosen from a Fab fragment, an scFv fragment, an Fv fragment, a diabody, a tribody, a tetrabody, and a minibody.
• VH mutated heavy chain variable domain
• VH light chain variable domain
• the Fab, scFv and diabody fragments are preferred (in particular the Fab fragment), because they are those with the lowest molecular weight, and therefore the most capable of crossing the blood-brain barrier and with a short in vivo lifespan suitable for diagnosis.
• 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 at position 128 according to the IMGT nomenclature of the heavy chain variable domain by a cysteine aims to allow selective conjugation on this cysteine.
• 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.
• mutant antibody fragments according to the invention are intended to be conjugated to groups useful for in vivo use (diagnosis in 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.
• a mutant antibody fragment according to the invention if it contains a 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 mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) according to the invention is of little importance, the invention being applicable regardless of the antigen to which the original antibody binds specifically.
• 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.
• 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 to any 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, PDGRF ⁇ , SLAMF7, EGFR, and endothelin receptor A or receptor B.
• the mutant antibody fragment in particular Fab, scFv or diabody, in particular Fab
• Fab fragment according to the invention can 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 a self-antigen will be useful for in vivo uses in the context of the diagnosis, and possibly the treatment, of an autoimmune disease involving the production of antibodies against a self-antigen.
• mutant antibody fragment 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.
• 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 (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 advantageously 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 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.
• 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 variable domain of the heavy chain of the antibody by a cysteine) from an antibody fragment whose amino acid residue at position 128 according to the IMGT nomenclature of the variable domain of the heavy chain is an alanine.
• the mutant antibody fragment according to the invention can be derived (by substitution of the residue at position 128 according to the IMGT nomenclature of the variable domain of antibody heavy chain by a cysteine) of a fragment of the antibody RB49 or RA63 or a variant thereof, which specifically bind to the endothelin receptor B.
• the antibody RB49 specifically binds to the 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).
• IMGT 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,
• the RA63 antibody specifically binds to endothelin receptor A and has 6 CDRs defined according to the IMGT 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).
• the mutant antibody fragment (in particular Fab, scFv or diabody, in particular Fab) binds specifically to the endothelin B 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 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
• 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.
• 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 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 ID NO: 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 TVDKSSSTAYMQLSSLTSED
• 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 a sequence having at least 80% identity with SEQ ID NO: 15 and 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); 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 for respective sequences QSIVHS
• 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: 15 or SEQ ID NO: 27, and- a VL domain having the sequence SEQ ID NO: 16.
• 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 (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 heavy chain CDRs CDR1-H, C
• 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.
• 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 SRDDSQNMVYLQMNNLKTEDTAMYYCVSSYYSGSFF
• 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 for 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 for respective sequences
• 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.
• mutant antibody fragment according to the invention may in particular be chosen from: a) a mutant Fab fragment specifically binding to the endothelin B receptor and comprising a heavy chain fragment of sequence SEQ ID NO: 19 (QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVTVSCASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTH) and a light chain of sequence SEQ ID NO: 20 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDL
• an scFv fragment specifically binding to the endothelin receptor B comprising or consisting of the amino acid sequence SEQ ID NO: 25 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGGSQVQLQPG AALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATLTVDKSSSTA YMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVTVSC); and b) a scFv fragment specifically binding to the endothelin receptor A comprising or consisting of the amino acid sequence SEQ ID NO: 26 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKIYLE
• 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 'amino acids SEQ ID NO:2.
• Such a Fab fragment would correspond to the Fab fragment of the antibody Hu4D5 (humanized antibody binding specifically 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 domain (VH) corresponds to the amino acid sequence SEQ ID NO: 1.
• mutant antibody fragments whose VH domain corresponds to the VH domain of the Hu4D5 antibody (humanized antibody binding specifically 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.
• 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 invention also 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.
• mutant antibody fragment 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).
• a single nucleic acid molecule is preferably used to encode this chain.
• 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.
• nucleic acid sequences coding for a particular amino acid sequence of a mutant antibody fragment according to the invention are within the scope of the invention.
• 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.
• some of these combinations are generally used preferentially by a given cell or organism (this is then referred to as a bias in the use of the genetic code). This preference depends in particular on the producing organism or from which the cell originates.
• nucleic acid molecule(s) coding for the mutant antibody fragment according to the invention may further comprise a nucleic sequence coding for a signal peptide at the N-terminus of the chain(s) of the mutant antibody fragment according to the invention.
• 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.
• a vector is also useful for producing the mutant antibody fragment according to the invention, the latter being able to be used to transform (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 regulation sequences. These elements vary depending on the host used for the expression and are easily chosen by a person skilled in the art in the light of his general knowledge.
• the vector may in particular be 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, J.P., Ruvo, M., 2020. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments.
• 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 transgenic plant 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 the production of the mutant antibody fragment according to the invention include in particular Escherichia coli among 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 may 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 U S A.;77:7380-4).
• a transgenic non-human animal may also be obtained by introduction of 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 preparation of 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 at Oxford University Press (1993)).
• the gene(s) of interest here the nucleic acid molecule or the pair of nucleic acid molecules according to the invention
• a transgenic non-human animal may also be obtained by a cloning technique in which a nucleus, into which the gene(s) of interest (here the nucleic acid molecule or the 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 may then be accumulated in the transgenic animal and harvested, in particular from the animal's milk or eggs.
• transgenic non-human animals 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 interest include in particular mice, rabbits, rats, goats, cattle (in particular cows), and poultry (in particular chickens).
• a transgenic plant according to the invention may be chosen from any plant allowing the production of antibody fragments.
• 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, in position 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.
• the mutant antibody fragment according to the invention may in particular be conjugated to one or more molecule(s) that are 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 one (conjugate intended to be used in a single type of imaging) or two (conjugate intended to be used in several types of imaging detecting different physical phenomena carrying two molecules detectable or likely to become detectable of different type, or conjugate intended to be used 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 that are detectable or capable of becoming detectable that can be conjugated to the mutant antibody fragment according to the invention are: a) a radioisotope (detectable molecule), a chelating agent (molecule capable of becoming detectable, which becomes detectable after contacting 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).
• the radioisotope can be covalently conjugated to the mutant antibody fragment according to the invention, or non-covalently by complexation with a chelating agent covalently conjugated to the mutant antibody fragment according to the invention.
• Radioisotopes or radiometals emitting gamma rays 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 2 gamma rays) are preferred because they are detectable by positron emission tomography (PET) or by single-photon emission tomography (SPECT).
• radiometals generally indirectly conjugated to the probe used (here the mutant antibody fragment according to the invention), the probe comprising a chelating agent which fixes the radiometal.
• chelating agents include linear or cyclic chelating agents, such as DOTA, NOTA, deferoxamine B, and their derivatives.
• DOTA and NOTA are for example respectively DOTAGA and (R)-NODAGA.
• Examples of radiometals emitting directly or indirectly (via the emission of a positron, which after annihilation with a surrounding electron, leads to the emission of 2 gamma rays) gamma rays include 99m Tc, 111 In, 64 Cu, 68 Yes, 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 Yes, 89 Zr, and 44 Sc are indirect emitters of gamma rays detectable by positron emission tomography (PET).
• a chelating agent can be advantageous because it allows in particular to be able 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.
• the complexation can be carried out just before use of the conjugate of the invention for an isotopic imaging application.
• 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 18 F, which is a positron emitter (and therefore an indirect emitter of gamma photons) and which can notably be introduced on so-called prosthetic groups, known to those skilled in the art, which can be conjugated to the mutant antibody fragment according to the invention.
• a fluorophore detecttable molecule
• 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.
• the fluorophores are useful when the conjugate is intended to be used in fluorescence imaging.
• the fluorophore can 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.
• 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).
• BODIPY boron-dipyrromethene family
• the fluorophore is selected from Cyanine5, Cyanine7, a rhodamine or an azaBODIPY.
• Examples of (affinity molecule/binding partner) pairs include (biotin/avidin), (biotin/streptavidin), (avidin/biotin), and (streptavidin/biotin).
• a chromophore detecttable molecule
• 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.
• the chromophore can 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.
• chromophores capable of being conjugated to the mutant antibody fragment according to the invention for use in optical imaging include phenolphthalein, gentian violet or Congo Red.
• (affinity molecule/binding partner) pairs include (biotin/avidin), (biotin/streptavidin), (avidin/biotin), and (streptavidin/biotin).
• the molecules detectable or capable of becoming detectable can be chosen from a fluorophore, a chromophore, an affinity molecule, a radioisotope and a chelating agent.
• the mutant antibody fragment according to the invention 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.
• 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 can then be complexed to a detectable molecule or to a molecule capable of being detectable (for example, a chelating agent) capable of forming a complex with the affinity molecule which is conjugated to the mutant antibody fragment according to the invention.
• 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).
• 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
• the affinity molecule can alternatively be used to easily purify the mutant antibody fragment according to the invention.
• it is also possible to use a hexahistidine peptide making it possible to easily purify the fragment on a column comprising nickel (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 may additionally or alternatively be conjugated to a pharmacomodulatory molecule. Such conjugation may 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 (for example fatty acids, metal ions, bilirubin, a toxin, the p-iodophenyl butyryl group, the biphenyl group).
• albumin for example fatty acids, metal ions, bilirubin, a toxin, the p-iodophenyl butyryl group, the biphenyl group.
• 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.
• 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.
• one of these detectable molecules may be a fluorophore and the other may be a radioisotope or a chelating agent.
• 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).
• DOTA chelating agent
• 68Ga metal radioisotope
• 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 1 via 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
• X 1 represents .
• L 1 , L 2 and L 3 each independently represent a spacer.
• 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 (e.g. n-hexyl).
• X 2 and X 3 are chosen independently from S and NR 8 .
• one of X 2 and X 3 is S and the other is S or NR 8 .
• 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 .
• R 2 and R 3 is a residue of a molecule detectable or likely to become detectable, and in particular R 2 and R 3 each independently represent a residue of a molecule 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.
• R 2 and R 3 may represent respectively a residue of a sulfonated Cyanine 7 and a residue of R-NODAGA (chelating agent).
• X 1 represents .
• L 1 and L 4 each independently represent a spacer.
• the spacer is a chain (C 1 -C 30 )-alkyl, especially (C 1 -C 20 )-alkyl, such as (C1-C10)-alkyl or (C1-C6)-alkyl, and in particular an ethyl or hexyl group (e.g. n-hexyl).
• 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.
• R 1 is a residue of a molecule which 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.
• 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 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.
• TCEP tris(2-carboxyethyl)phosphine
• 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), or 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, that is to say 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 in position 128.
• 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 can 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 process 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 to glutathione, cysteines, etc. Steps a) of reduction and b) of reoxidation before conjugation are therefore also advantageously used.
• 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 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 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
• 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 the cysteine at position 128 will form the group X 1 and covalently link the compound of formula (III) to the antibody fragment partially reduced mutant.
• such a reaction between the SH function of a cysteine and a chemical group reactive with respect to an SH function is well known in the art.
• 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.
• a different reactive chemical group (G 2 ) can be grafted to the molecule of interest via a spacer L 4 or not (when L 4 represents a single bond).
• 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 2 are different and each represent a reactive chemical group capable of reacting together to form a group X 1 '.
• 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 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, to a reactive chemical group.
• the reactive chemical group is chosen from N, N+ - Preferably 3 O, , , , cycles A 3 and A 4 being as defined above.
• cycle A 3 will be chosen from , , advantageously chosen from 5
• 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 1 are as defined above, and Z represents a reactive chemical group as defined above.
• the conjugate according to the invention may be used in different in vivo applications.
• 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).
• the molecule(s) conjugated to the mutant antibody fragment according to the invention at the cysteine at 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.
• 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 radioisotope or a chelating agent (a complex with a radiometal is then formed just before administration to the patient).
• 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 two different detectable molecules or molecules likely to become detectable, allowing its use in two different types of imaging.
• the mutant antibody fragment according to the invention can be conjugated at the cysteine at 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 a tumor 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 direct or indirect gamma-ray emitting radioisotope or a chelating agent for use in
• the conjugates a1 and b1) are preferred.
• the mutant antibody fragment according to the invention may be conjugated at the cysteine at position 128 of the heavy chain according to the IMGT nomenclature to two molecules detectable or capable of being detectable by the same imaging technique (this making it possible to increase the signal and therefore the detection 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 by single-photon emission tomography (SPECT)), 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 use in
• the mutant antibody fragment according to the invention may 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 radioisotope emitting direct or indirect gamma rays or a chelating agent for its use in a method for 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 use in a method of diagnosing a tumor by fluorescence imaging; c) a pharmacomodulatory molecule and a chromophore for 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
• the conjugates with a detectable or detectable molecule and a pharmacomodulatory molecule described above, the conjugates a) (a pharmacomodulatory molecule and a direct or indirect gamma-ray emitting radioisotope 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 fluorescence-guided tumor resection or isotopic imaging probe, comprising: a) administering to a patient 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 surgically resecting the entire tumor using the real-time images obtained.
• 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 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 in a 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 fluorescence
• 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 of 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 patient's tumor burden 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 to the patient an antitumor treatment, 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).
• 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 that 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.
• 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.
• 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 for 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.
• 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 indicated for introducing a substitution because it plays a crucial role in the proper folding of the antibody chain, being located right next to the cysteine at position 23 IMGT performing the SS bridge between the C23 and C104 position (IMGT). Introducing a C at position 24 could therefore disrupt the proper folding and stabilization of the heavy chain.
• Murine V segments Human V segments Amino acids at 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 murine J segments are also serine, the only other amino acid used at this position is alanine.
• 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 substitution of this position with a cysteine to allow site-specific conjugation.
• heavy chain IMGT positions 5 and 24 are poorly conserved and involved in proper heavy chain folding (in addition, IMGT position 24 is 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 cysteines at positions 23 and 104. These positions are therefore not suitable for a general method of substituting this position by a cysteine to allow site-specific conjugation.
• heavy chain IMGT position 128, 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 for a general method of substituting this position by a cysteine to allow site-specific conjugation.
• 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.
• 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)
• IMGT nomenclature a cysteine at position 128
• DFO Zirconium-89 chelating agent
• IRDye800 fluorophore
• Plasmids were co-transfected into ExpiCHO-S cells (ThermoFisher) according to the manufacturer's instructions "ExpiCHOTM 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 ⁇ m filter. Thiofab xiRA63 was then affinity purified on a HiTrap KappaSelect column (GE Healthcare). After elution, antibody solutions were dialyzed with the Slide-A-LyzerTM G2 dialysis cassette (Thermoscientific) with a cut-off threshold of 10kDa in 1 liter of PBS.
• 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 by 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.
• the site-specific conjugate obtained from the mutant Fab with a cysteine at position 128 of the heavy chain according to the IMGT nomenclature exhibits 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 envisaged.
• 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
• IMGT nomenclature a cysteine at position 128
• the plasmids were co-transfected into ExpiCHO-S cells (ThermoFisher) according to the manufacturer's instructions "ExpiCHOTM 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 ⁇ m 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-LyzerTM G2 Dialysis Cassette (Thermoscientific) with a 10kDa cutoff in 1 L of PBS.
• ThioFab-xiRA63-DFO conjugation ThioFab-xiRA63 (500 ⁇ g, 459 ⁇ L at 1.1 g/L) was reduced with 30.8 ⁇ L of TCEP (20mM) at 37°C for 1 hour. After purification using centrifugal filter units with a molecular weight cutoff of 10,000 Da, reoxidation of the native interchain disulfide bond was performed with 6.75 ⁇ L of dehydroascorbic acid (dhAA, 20 equiv., 50 mmol/L in carbonate/bicarbonate buffer pH 9) for 1 hour at 37°C.
• dehydroascorbic acid dhAA, 20 equiv., 50 mmol/L in carbonate/bicarbonate buffer pH 9
• Radiolabeling of ThioFab-xiRA63-DFO with Zirconium 89 [ 89 Zr] To radiolabel this antibody fragment with zirconium-89 (PerkinElmer), a protocol previously described by 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 [ 89 Zr]Zr-ThioFab-xiRA63-DFO was then purified on a PD-10 column with a solution of gentisic acid (5 mg/mL in 0.25 M sodium acetate, pH 5.5) as mobile phase and concentrated with Vivaspin® ultrafiltration tubes (Satorius) with a cutoff of 10 kDa.
• mice 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 associated with local xylocaine.
• a 10 ⁇ L Hamilton syringe was used with a flow rate of 0.25 ⁇ L/min.
• 0.05 mg.kg -1 of buprenorphine was administered subcutaneously to each mouse. Mice were housed in fours 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 [ 89 Zr]Zr-ThioFab-xiRA63 (100 ⁇ C ⁇ 5 ⁇ C).
• PET-CT image acquisitions We performed a first 60-minute dynamic acquisition immediately after antibody injection using an Inveon microPET scanner or an Inveon microPET/CT scanner (Siemens). Then, a 20-minute PET acquisition was performed at the following times: 5h, 24h, 48h, 72h and 7 days after 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 8mm3 were created in several organs: heart, liver, kidneys, spleen and muscle. For bone and brain tumor, VIs were drawn using the "iso-contour" tool.
• 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.
• [ 89Zr ]Zr-ThioFab-xiRA63 ( ⁇ 60kDa) appears to follow a renal elimination pathway (70% ID.cm -3 accumulation in the kidney in the first hour pi). Indeed, small molecules are rapidly eliminated from the bloodstream due to glomerular filtration and renal excretion.
• Example 4 Preparation of a thioFab (thioFab-xiRB49) of the chimeric RB49 antibody (xiRB49)
• simple chimerization substitution of mouse constant regions by human constant regions
• RB49 led to a non-functional chimeric antibody, unable to bind specifically to the ETB receptor.
• 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 CDR3 of the heavy and light chains then come into prolonged contact, making them unavailable for interaction with the ETB antigen.
• the heavy and light chains encoding IgG-xiRB49, Fab-xiRB49-P125T and ThioFab-xiRB49-P125T were subcloned into the expression plasmid pTT5.
• 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).
• the supernatant was clarified by centrifugation at 4000-5000 ⁇ g for 30 min at 4 °C and filtered through a 0.22 ⁇ m 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-LyzerTMG2 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).
• Figure 12 shows 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 ) to be obtained.
• the three antibodies show the same T i 2 , reflecting the Fab domain of the order of 90°C. After confirming the physicochemical properties of the different fragments, their functionality was evaluated.
• the ThioFab-xiRB49-P125T induces an equivalent shift of the signal emitted by the xiRB49-P125T with an apparent Kd then 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.

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