EP4110405A1 - Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie - Google Patents

Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie

Info

Publication number
EP4110405A1
EP4110405A1 EP21714002.9A EP21714002A EP4110405A1 EP 4110405 A1 EP4110405 A1 EP 4110405A1 EP 21714002 A EP21714002 A EP 21714002A EP 4110405 A1 EP4110405 A1 EP 4110405A1
Authority
EP
European Patent Office
Prior art keywords
antibody
drug conjugate
drug
mmae
less
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21714002.9A
Other languages
German (de)
English (en)
French (fr)
Inventor
Ludovic JUEN
Christine BALTUS
Audrey DESGRANGES
Antoine TOUZÉ
Thibault KERVARREC
Valérie LEBLOND
Mahtab SAMIMI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mcsaf Inside Onocology
Original Assignee
Mcsaf Inside Onocology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mcsaf Inside Onocology filed Critical Mcsaf Inside Onocology
Publication of EP4110405A1 publication Critical patent/EP4110405A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6865—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from skin, nerves or brain cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel antibody-drug conjugates comprising an antibody directed against the CD56 antigen coupled to a cytotoxic drug and their use as a drug, in particular in anti-cancer therapy.
  • An antibody-drug conjugate (in English "Antibody Drug Conjugate” or “ADC”) constitutes a means of selective delivery of a cytotoxic drug.
  • the antibody-drug conjugate therefore makes it possible to combine the specificity of targeting by the antibodies with new powerful effector functions by the agents which are conjugated to them.
  • the general structure of an antibody-drug conjugate is that of formula (I).
  • the part that binds the antibody and the drug is called the linker arm or linker. It can be grafted onto the antibody via at least one of the eight cysteines forming the 4 inter-chain disulfide bridges.
  • the number of cytotoxic drug molecules grafted onto the antibody determines a ratio called the Drug-to-Antibody Ratio (DAR).
  • DAR Drug-to-Antibody Ratio
  • the antibody After binding to its target antigen, the antibody is internalized in the cell by endocytosis mediated by receptors. The vesicles fuse with lysosomes where the cytotoxic drug is released from the antibody via different mechanisms. The active cytotoxic drug then acts on the cell by inducing its death and sometimes on neighboring cancer cells by transport or diffusion in the environment.
  • the antibody is therefore mainly used as a vector and delivers the cytotoxic drug into the targeted cell.
  • Merkel cell carcinoma is an aggressive skin cancer occurring in the elderly.
  • the treatment of inoperable metastatic patients relied on polychemotherapy based on platinum salts, with no benefit on survival.
  • the use of immunotherapy targeting PD-L1 (avelumab) in the first line allows an objective response to be obtained in approximately 50% of patients with inoperable MCC with a sustained response observed in half of responder patients.
  • the persistence of non-responder patients encourages the development of new therapeutic strategies.
  • the development of targeted therapy in CCM appears to be a promising strategy.
  • CD56 has been identified as a therapeutic target strongly expressed by the majority of TLCs.
  • IMGN901 which is an anti-CD56 ADC coupled to mertansine (DM1) by a first generation technology, inhibits the polymerization of tubulin.
  • DM1 mertansine
  • An acceptable tolerance of IMGN901 has been demonstrated by several phase I studies in patients with solid tumors expressing CD56 including cases of TLC.
  • a phase II study has been proposed with the same molecule in small cell carcinoma of the lung, in combination with chemotherapy (cisplatin etoposide).
  • a first subject of the invention relates to an antibody-drug conjugate of formula (I) below: in which :
  • A is an anti-CD56 antibody or antibody fragment;
  • the attachment head is represented by one of the following formulas:
  • the linkage arm is a cleavable linkage arm chosen from the following formulas:
  • the spacer is represented by the following formula: m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 4.
  • a second object of the invention relates to a composition comprising one or more antibody-drug conjugate (s) according to the invention.
  • a third subject of the invention relates to an antibody-drug conjugate according to the invention or a composition according to the invention, for use as a drug.
  • a fourth subject of the invention relates to an antibody-drug conjugate according to the invention or a composition according to the invention, for use in the treatment of CD56 + cancer.
  • a fifth object of the invention relates to a process for preparing an antibody-drug conjugate according to the invention comprising the following steps:
  • the linkage arm is a cleavable linker arm chosen from the following formulas:
  • the spacer is represented by the following formula: X is Br, Cl, I or F; m is an integer ranging from 1 to 10, advantageously ranging from 2 to 7, from 3 to 6, advantageously equal to 4 or 5; and
  • step (ii) reacting the cytotoxic conjugate obtained in step (i) with an anti-CD56 antibody or an anti-CD56 antibody fragment.
  • the process for preparing an antibody-drug conjugate according to the invention comprises a step which consists in reacting 6- (2,6-bis (bromomethyl) pyridin-4-yl) amido- / V-hexanamide-valine-citrulline-p- aminobenzoyl carbamate from MMAE or 6 - ((2,6-bis (bromomethyl) pyridin-4-yl) amino) -6-oxohexanamide-valine-citrulline-p-aminobenzoyl carbamate from 6- (2,6-bis (bromomethyl) pyridin-4-yl) amido- / V-hexanamide-valine-citrulline-p- aminobenzoyl carbamate from MMAE or 6 - ((2,6-bis (bromomethyl) pyridin-4-yl) amino) -6-oxohexanamide-valine-citrulline-p-aminobenzoyl carbamate from
  • MMAE with an anti-CD56 antibody or an anti-CD56 antibody fragment.
  • cytotoxic conjugate denotes a conjugate which comprises a cytotoxic drug.
  • a cytotoxic conjugate denotes a conjugate of formula (II) below: in which: the attachment head is represented by one of the following formulas: the linkage arm is a cleavable linker arm chosen from the formulas following: the spacer is represented by the following formula: X is Br, Cl, I or F; m is an integer ranging from 1 to 10.
  • cytotoxic drug denotes any natural or synthetic molecule capable of inhibiting or preventing the function and / or development of cells.
  • cytotoxic is understood to mean the property for a chemical or biological agent of altering cells, possibly to the point of destroying them.
  • the cytotoxic drug is chosen from any compound having obtained Marketing Authorization and which is used in anti-cancer or anti-inflammatory therapy, any molecule under clinical evaluation in therapy anti-cancerous or anti-inflammatory.
  • the cytotoxic drug will be chosen, for example, from paclitaxel (Taxol®) or docetaxel (Taxotère®) or one of its derivatives, topotecan, bortezomib, daunorubicin, daunorubicin analogues, vincristine, mitomycin C, retinoic acid, methotrexate, ilomedine, aspirin, IMIDs, lenalidomide, pomalidomide.
  • the cytotoxic drug is selected from the group consisting of duocarmycin and its analogues, dolastatins, combretastatin and its analogues, calicheamicin, N-acetyl-y-calicheamycin (CMC), a derivative of calicheamycin, maytansin and its analogues, such as a maytansinoid derivative, for example DM1 and DM4, auristatins and their derivatives, such as auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin, disorazole, epothilones, echinomycin, estramustine, cemadotine, eleutherobin, methopterin, actinomycin, mitomycin A, camptothecin, a camptothecin derivative,
  • the cytotoxic drug is chosen from methotrexate, IMIDs, duocarmycin, combretastatin, calicheamicin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, DM1, DM4, SN38, amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, pyrrolopyridodiazepine, pyrrolopyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, and ricin, preferably MMAE, represented by the following formula:
  • the cytotoxic conjugate corresponds to the following formula (IIa) or to the following formula (lia '):
  • the term "antibody”, also called “immunoglobulin” denotes a heterotetramer consisting of two heavy chains of about 50-70 kDa each (called the H chains for Heavy) and two light chains of about 25 kDa each ( say L chains for Light), linked together by intra- and inter-chain disulfide bridges.
  • Each chain consists, in the N-terminal position, of a region or variable domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, consisting of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.
  • VL region or variable domain
  • CL constant region
  • chimeric antibody is meant an antibody in which the sequences of the variable regions of the light chains and of the heavy chains belong to a different species from that of the sequences of constant regions of the light chains and of the heavy chains.
  • the sequences of the variable regions of the heavy and light chains are preferably of murine origin while the sequences of the constant regions of the heavy and light chains belong to a non-murine species.
  • all species of non-murine mammals are likely to be used, and in particular man, monkey, suidae, bovidae, animals. equines, felids, canids or even birds, this list not being exhaustive.
  • the chimeric antibodies according to the invention contain sequences of constant regions of heavy and light chains of human origin and sequences of variable regions of heavy and light chains of murine origin.
  • humanized antibody is meant an antibody of which all or part of the sequences of the regions involved in the recognition of the antigen (the hypervariable regions or CDR: Complementarity Determining Region) and sometimes certain amino acids of the FR regions (regions Framework) are of non-human origin while the sequences of constant regions and variable regions not involved in antigen recognition are of human origin.
  • human antibody is meant an antibody containing only human sequences, both for the variable and constant regions of the light chains and for the variable and constant regions of the heavy chains.
  • antibody fragment means any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction comprising at least one disulfide bridge, for example Fab, Fab ', F (ab') 2 , Fab ' -SFI, scFv-Fc or Fc.
  • the enzymatic digestion of the immunoglobulins by papain generates two identical fragments, which are called the Fab fragments (Fragment antigen binding), and an Fc fragment (Crystallizable fragment).
  • Enzymatic digestion of immunoglobulins by pepsin generates an F (ab ') 2 fragment and an Fc fragment split into several peptides.
  • F (ab ') 2 is formed from two Fab' fragments linked by inter-chain disulfide bridges.
  • the Fab parts consist of the variable regions and the CH1 and CL domains.
  • the Fab 'fragment consists of the Fab region and a hinge region.
  • Fab'-SFI refers to an Fab 'fragment in which the cysteine residue of the hinge region carries a free thiol group.
  • the scFv single Chain Fragment variable
  • the structure is stabilized by a short flexible peptide arm, called a linker, which is placed between the two domains.
  • the scFv fragment can be linked to an Fc fragment to give an scFv-Fc.
  • CD56 designates the “Differentiation Cluster 56", which is a membrane glycoprotein member of the superfamily of immunoglobulins (Ig) containing 5 domains of Ig type and two domains of type 3 of fibronectin in its extracellular part. CD56 is also frequently referred to as “Neural-Cell Adhesion Molecule 1 (NCAM 1)”.
  • CD56 + cancer or “CD56 positive cancer” denotes a cancer expressing CD56.
  • CD56 + cancer designates any case of cancer for which cancer cells exhibit CD56 expression. CD56 expression is frequently observed in neuroendocrine carcinomas, pediatric tumors and some blood diseases. It is also reported in certain melanomas and soft tissue sarcomas.
  • the CD56 + cancer is chosen from melanoma, blastema tumors, hemopathies, such as acute myeloid leukemias, myelomas, neoplasms with blast plasmacytoid dendritic cells and neuroendocrine carcinomas, such as small cell carcinoma of the lung and Merkel cell carcinoma.
  • the CD56 + cancer is a carcinoma selected from neuroendocrine carcinomas, such as small cell carcinoma of the lung or Merkel cell carcinoma, preferably Merkel cell carcinoma.
  • identity is calculated by comparing two sequences aligned in a comparison window.
  • the alignment of the sequences makes it possible to determine the number of positions (nucleotides or amino acids) in common for the two sequences in the comparison window.
  • the number of positions in common is therefore divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of identity.
  • the determination of the percent sequence identity can be done manually or by well known computer programs.
  • the identity or the homology corresponds to at least one substitution, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions , of an amino acid residue, preferably at least one substitution of an amino acid residue carried out conservatively.
  • Constantly accomplished amino acid residue substitution involves replacing one amino acid residue with another amino acid residue, having a side chain having similar properties.
  • the families of amino acids possessing side chains with similar properties are well known, for example basic side chains (eg lysine, arginine, histidine), acid side chains (eg aspartic acid, glutamic acid) can be mentioned.
  • polar and uncharged side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g.
  • the antibodies or fragments of homologous antibodies or "variants of antibodies or of fragments of antibodies” that is to say antibodies or fragments of antibodies having the same function
  • a person skilled in the art uses his general knowledge to determine the number of substitutions which can be made and their location in order to be able to preserve the function of the antibody or of the antibody fragment.
  • antibody variants or antibody fragments In order to determine the capacity of one or more antibody variants or antibody fragment to bind specifically to an antigen, several suitable methods, well known to those skilled in the art and described in the prior art, can be used.
  • the antibodies or antibody fragments can therefore be tested by binding methods, such as, for example, the ELISA method, the affinity chromatography method, etc.
  • Variants of antibodies or antibody fragments can be generated for example by the “phage display” method making it possible to generate a phage library.
  • a large number of methods are known in order to generate a “phage display” library and target the antibody variants or antibody fragments having the desired functional characteristics.
  • purified and isolated is meant, with reference to an antibody according to the invention, that the antibody is present in the substantial absence of other biological macromolecules of the same type.
  • purified as used herein preferably means at least 75 wt%, more preferably at least 85 wt%, still more preferably at least 95 wt%, and most preferably at least 98 wt%. antibody, relative to all the macromolecules present.
  • a “pharmaceutically acceptable” means approved by a federal or state regulatory agency or listed in the American or European Pharmacopoeia, or in another generally recognized pharmacopoeia, for use in animals and in humans.
  • a “pharmaceutical composition” means a composition comprising a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may be a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
  • These vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil. , sesame oil, etc. Water is a preferred vehicle when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid vehicles, particularly for injectable solutions.
  • Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, l water, ethanol and the like.
  • tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc or silica); disintegrants (eg potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate).
  • binding agents eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers eg lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants eg, magnesium stearate, talc or silica
  • disintegrants eg potato starch or sodium starch glycolate
  • wetting agents eg, sodium lauryl sulfate
  • Liquid preparations for oral administration may take the form, for example, of solutions, syrups or suspensions, or may be presented as a dry product to be reconstituted with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable vehicles such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (eg methyl or propyl p-hydroxybenzoates or sorbic acid).
  • Pharmaceutical compositions may also contain buffer salts, flavorings, colorants and sweeteners, as appropriate.
  • the composition according to the invention is preferably a pharmaceutical composition.
  • treat encompasses any beneficial or desirable effect on a pathology or condition, and may even include a minimal reduction of one or more measurable markers of the condition or condition. Treatment may, for example, involve either reducing or ameliorating the symptoms of the disease or condition, or delaying the progression of the disease or condition. The term “treatment” does not necessarily mean complete eradication or cure of the condition, or of the associated symptoms.
  • Native mass spectrometry is understood to mean a mass spectrometry analysis carried out under conditions which do not denature the proteins, thus making it possible to retain the non-covalent bonds. From Native mass spectrometry methods are widely described in the literature, for example in reference [4]
  • the invention relates to an antibody-drug conjugate of the following formula (I): in which :
  • A is an anti-CD56 antibody or antibody fragment;
  • the attachment head is represented by one of the following formulas:
  • the linkage arm is a cleavable linkage arm chosen from the following formulas:
  • the spacer is represented by the following formula: m is an integer ranging from 1 to 10, advantageously ranging from 2 to 7, from 3 to 6, advantageously equal to 4 or 5; n is an integer ranging from 1 to 4.
  • the antibody or the anti-CD56 antibody fragment according to the invention can be of mammalian (eg human or mouse), humanized or chimeric origin. It is preferably a monoclonal antibody produced recombinantly by cells genetically modified according to techniques widely described in the prior art.
  • A is an anti-CD56 antibody, it is preferably an IgG, for example lgG1, lgG2, lgG3 or lgG4.
  • A is an antibody or an anti-CD56 antibody fragment which comprises:
  • variable domain of a light chain comprising a CDR1 of amino acid sequence SEQ ID NO: 1, a CDR2 of amino acid sequence SEQ ID NO: 2, and a CDR3 of amino acid sequence SEQ ID NO: 3 ;
  • variable domain of a heavy chain comprising a CDR1 of amino acid sequence SEQ ID NO: 4, a CDR2 of amino acid sequence SEQ ID NO: 5, and a CDR3 of amino acid sequence SEQ ID NO : 6.
  • the light chain variable domain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID NO: 15; and the heavy chain variable domain having at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96%, at least 97%, at least 98% , at least 99% or even 100% homology with the amino acid sequence SEQ ID NO: 16.
  • A can be an antibody or an antibody fragment of amino acid sequence SEQ ID NO: 15 for the variable domain of the light chain and of amino acid sequence SEQ ID NO: 16 for the variable domain of the heavy chain.
  • A is an antibody of amino acid sequence SEQ ID NO: 7 for the light chain and of amino acid sequence SEQ ID NO: 8 for the heavy chain.
  • the heavy chain of amino acid sequence SEQ ID NO: 8 may also have an additional lysine in the C terminal position.
  • A is the antibody described under the reference m906 in application US 2018/0214568 A1 and in reference [1].
  • the m906 antibody is a chimeric anti-CD56 antibody of IgG1 type with amino acid sequence SEQ ID NO: 7 for the light chain and amino acid sequence SEQ ID NO: 8 for the heavy chain.
  • the antibody-drug conjugate of the invention has one of the following formulas
  • the antibody-drug conjugate of the invention has the following formula (la) or has the following formula:
  • the antibody-drug conjugate identified in the examples under the reference "MF-m906-MMAE” corresponds to an antibody-drug conjugate of formula (Ia).
  • “M906” corresponds to the m906 antibody, that is to say an anti-human CD56 antibody of the IgG1 type with amino acid sequence SEQ ID NO: 7 for the light chain and amino acid sequence SEQ ID NO: 8 for the heavy chain.
  • the antibody-drug conjugate according to the invention is purified (or isolated) using known purification techniques, such as purification on a chromatography and / or affinity column.
  • the antibody-drug conjugate When n is equal to 1, the antibody-drug conjugate is commonly called “DAR1". When n is 2, the antibody-drug conjugate is commonly referred to as “DAR2”. When n is 3, the antibody-drug conjugate is commonly referred to as “DAR3”. When n is 4, the antibody-drug conjugate is commonly referred to as "DAR4".
  • the antibody-drug conjugate exhibits one or more effector functions mediated by the attenuated Fc part.
  • the effector function (s) mediated by the Fc part is / are chosen from ADCC (Antibody-dependent cell-mediated cytotoxicity) and CDC (Complement-dependent cytotoxicity).
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • CDC Complement-dependent cytotoxicity
  • Those skilled in the art will have no difficulty in attenuating one or more effector functions mediated by the Fc part with regard to the teaching of the prior art, for example by mutating the Fc part.
  • Many mutations are known to decrease effector functions mediated by the Fc part. It may, for example, be a mutation aimed at deglycosylating the Fc part, in particular at suppressing glycosylation at the level of asparagine 297.
  • the antibody-drug conjugate is deglycosylated at the level of the Fc part, for example the antibody-drug conjugate no longer carries glycosylation at the level of asparagine 297.
  • the invention also relates to a composition comprising one or more antibody-drug conjugate (s) of formula (I), for example of formula (la) or of formula (la ’), as defined above. It may be a pharmaceutical composition comprising one or more antibody-drug conjugate (s) of formula (I) as defined above, for example of formula (la) or of formula (la '), and a pharmaceutically acceptable vehicle.
  • composition according to the invention has the characteristic of being particularly homogeneous, which can result in better stability, better efficiency and / or a reduction in the side effects of the composition, compared to a composition which does not is not homogeneous.
  • the composition according to the invention is characterized by the following characteristics: a) at least 50%, preferably at least 55%, at least 60%, at least 65 %, for example at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least less than 99% of the antibody-drug conjugates of the composition have an n equal to 4; b) the average Drug-to-Antibody Ratio (average DAR) is between 3 and 4.5, preferably between 3.5 and 4, between 3.7 and 4, between 3.8 and 4, for example equal to 3.9 ⁇ 0.1, for example equal to 3.89.
  • average DAR average DAR
  • the average DAR is generally determined by the HIC (Hydrophobia Interaction Chromatography) method or by native mass spectrometry.
  • HIC Hadrophobia Interaction Chromatography
  • native mass spectrometry The HIC method and the native mass spectrometry method are widely described in the literature. Reference may be cited, for example, [3] for the HIC method and reference [4] for the native mass spectrometry method; c) at least 95%, preferably at least 96%, at least 97%, at least 98%, at least 99% of the antibody-drug conjugates are present as a monomer. The percentage of monomer is generally determined by the SEC (Size Exclusion Chromatography) method.
  • the SEC method is widely described in the literature, for example in reference [2]; d) one or more of the ratios of n defined below; or e) a combination of two, three or four characteristics selected from a), b, c) and d).
  • composition according to the invention can be characterized by ratios of n below:
  • composition according to the invention can also be characterized by ratios of n below:
  • the ratios of n can be determined by the HIC (Hydrophobia Interaction Chromatography) method or by native mass spectrometry.
  • composition according to the invention can be characterized by ratios of n determined by the HIC (Hydrophobia Interaction Chromatography) method below:
  • the antibody-drug conjugates of the composition have an n equal to 2,
  • composition according to the invention can also be characterized by ratios of n determined by the HIC (Hydrophobia Interaction Chromatography) method below:
  • n 4
  • composition according to the invention can be characterized by ratios of n determined by native mass spectrometry below:
  • composition according to the invention can also be characterized by ratios of n determined by native mass spectrometry below:
  • n 1
  • n 2
  • the composition according to the invention can also comprise DAROs, that is to say antibodies without cytotoxic conjugate. Preferably less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.2%, less than 0.1% of DARO, for example about 0% of DARO.
  • the percentage of DARO can be determined by the HIC (Hydrophobia Interaction Chromatography) method or by native mass spectrometry.
  • composition according to the invention can also comprise DAR5, that is to say antibody-drug conjugates having 5 cytotoxic conjugates. Preferably less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% of DAR5.
  • DAR5 that is to say antibody-drug conjugates having 5 cytotoxic conjugates.
  • the presence of DAR5 in antibody-drug conjugate compositions is widely described in the literature, however the exact structure of DAR5 is little studied.
  • the composition according to the invention does not comprise a DAR greater than DAR5, for example DAR6, DAR7, etc.
  • the percentages of DAR5 and above can be determined by the HIC (Hydrophobia Interaction Chromatography) method or by native mass spectrometry.
  • the composition according to the invention exhibits the HIC profile of FIG. 1 or the native mass spectrometry profile of FIG. 3.
  • the invention also relates to an antibody-drug conjugate of formula (I) according to the invention or a composition comprising an antibody-drug conjugate of formula (I) according to the invention for use as a drug, for example for use in the treatment of CD56 + cancer, such as melanoma, blastema tumors, blood diseases, such as acute myeloid leukemias, myelomas, plasmacytoid blast dendritic cell neoplasias and neuroendocrine carcinomas.
  • CD56 + cancer is chosen from neuroendocrine carcinomas, such as small cell carcinoma of the lung or Merkel cell carcinoma, preferably Merkel cell carcinoma.
  • the antibody-drug conjugate or the composition according to the invention is preferably formulated for parenteral administration, for example intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • parenterally administered refers to modes of administration other than enteral and topical administration, generally by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intravenous administration.
  • Intravenous administration is preferred in the context of the present invention, for example by intravenous infusion.
  • the dose of antibody-drug conjugate administered to a subject in need thereof will vary depending on several factors, including, without limitation, the route of administration, the type and severity of the condition being treated, the condition. of the patient, the build of the patient, the age of the patient, etc.
  • One skilled in the art can easily determine, based on his knowledge in this field, the required dosage range based on these and other factors.
  • the appropriate dose can also be determined with animal models or with clinical trials.
  • the typical doses of antibody-drug conjugate according to the invention can be from 5 mg / m 2 to 250 mg / m 2 , for example from 30 mg / m 2 to 150 mg / m 2 , from 5 mg / m 2 2 to 75 mg / m 2 , from 75 mg / m 2 to 120 mg / m 2 , by example equal to 100 mg / m 2 , 75 mg / m 2 , 60 mg / m 2 .
  • the administration can be done in one go or, more generally, in several times.
  • the administration schedule may include an initial loading dose followed by maintenance doses, for example weekly, every 2 weeks, every three weeks, every month, or more. The duration of treatment may vary depending on the pathology treated and the subject.
  • the antibody-drug conjugate or the composition according to the invention can be used as monotherapy or in combination with drugs whose therapeutic interest is recognized in the pathology under consideration. It may be, for example, paclitaxel, docetaxel, doxorubicin, cyclophosphamide, lenalidomide, dexamethasone, carboplatin, etoposide, or an antibody used in anti-cancer immunotherapy such as a anti-PD1 or anti-PD-L1.
  • the description also relates to a method of treating CD56 + cancer in a subject comprising the administration to the subject of an effective therapeutic amount of an antibody-drug conjugate of formula (I) according to the invention or of a composition comprising an antibody-drug conjugate of formula (I) according to the invention.
  • the description also relates to a process for the preparation of an antibody-drug of formula (I) as defined above, in which a cytotoxic conjugate of the following formula (II) is reacted: as defined above, with an antibody or an anti-CD56 antibody fragment as defined above.
  • the invention relates to a process for preparing an antibody-drug conjugate according to the invention comprising the following steps:
  • a cytotoxic conjugate by coupling a binding head of formula: with a compound of formula in which: the linkage arm is a cleavable linker arm chosen from the following formulas: the spacer is represented by the following formula: X is Br, Cl, I or F; m is an integer ranging from 1 to 10, advantageously ranging from 2 to 7, from 3 to 6, advantageously equal to 4 or 5; and
  • step (ii) reacting the cytotoxic conjugate obtained in step (i) with an anti-CD56 antibody or an anti-CD56 antibody fragment.
  • the attachment head is described in more detail in the definition of “cytotoxic conjugate”. and in the “antibody-drug conjugate” section above, with the difference that the head attachment used in the method comprises a terminal carboxylic acid function.
  • linker arm is described in more detail in the definition of “cytotoxic conjugate” and in the “antibody-drug conjugate” part above, with the difference that the linker arm used in the method comprises a terminal amine function.
  • cytotoxic drug is described in more detail in the definition of “cytotoxic conjugate” and in the “antibody-drug conjugate” section above.
  • anti-CD56 antibody is described in more detail in the definition of "cytotoxic conjugate” and the “antibody-drug conjugate” section above.
  • step (i) provides a cytotoxic conjugate of formula (IIa) or (IIa ’).
  • the process for preparing a cytotoxic conjugate according to step (i) comprises a step which consists in coupling 6- (2,6-bis (bromomethyl) pyridin- acid. 4-yl) amidohexanoic acid or 6- ((2,6-bis (bromomethyl) pyridin-4-yl) amino) -6-oxohexanoic acid with valine-citrulline-p-aminobenzoyl carbamate of MMAE or a salt of this compound.
  • the process for preparing a cytotoxic conjugate according to the invention makes it possible to obtain 6- (2,6-bis (bromomethyl) pyridin-4-yl) amido- / V-hexanamide-valine -citrulline-p-aminobenzoyl carbamate from MMAE or 6 - ((2,6-bis (bromomethyl) pyridin-4-yl) amino) -6-oxohexanamide-valine-citrulline-p-aminobenzoyl carbamate from MMAE, respectively.
  • the process for preparing an antibody-drug conjugate according to the invention comprises a step which consists in reacting 6- (2,6-bis (bromomethyl) pyridin-4-yl ) amido- / V- hexanamide-valine-citrulline-p-aminobenzoyl carbamate of MMAE or 6- ((2,6-bis (bromomethyl) pyridin-4-yl) amino) -6-oxohexanamide-valine-citrulline-p - MMAE aminobenzoyl carbamate with an anti-CD56 antibody or an anti-CD56 antibody fragment.
  • FIG. 1 represents the HIC (Hydrophobia Interaction Chromatography) profile of an antibody-drug conjugate composition according to the invention.
  • the figure shows that the composition is enriched in DAR4, with more than 50% of DAR4.
  • FIG. 2 represents a SEC (Size Exclusion Chromatography) analysis of an antibody-drug conjugate composition according to the invention. The figure shows that the composition is extremely homogeneous, with more than 99% monomer.
  • FIG. 3 shows a native mass spectrometric analysis on a Vion IMS Qtof mass spectrometer coupled to an Acquity UPLC H-Class system from Waters (Wilmslow, UK) of an antibody-drug conjugate composition according to the invention. This method makes it possible to identify with certainty each species of ARD.
  • the figure shows that the composition is enriched in DAR4, with nearly 75% of DAR4.
  • FIG. 4 shows the expression of CD56 on different cell lines (4 from TLC and one breast cancer control line) by flow cytometry after incubation with the anti-CD56 antibody labeled with the fluorophore FITC (Fluorescein IsoThioCyanate).
  • FIG. 5A], [Fig 5B], [Fig 5C], [Fig 5D], [Fig 5E] represent the performance evaluation of the MF-m906-MMAE ADC, compared to the m906 controls (uncoupled antibody), MF- TTZ-MMAE (ADC Trastuzumab coupled to MMAE) and MMAE (toxin only) on TLC (Figure 5A: WaGa, Figure 5B: PeTa, Figure 5C: MS-1 and Figure 5D: MKL-2) and cancer cell lines breast ( Figure 5E: SK-BR3).
  • the experiments were performed independently twice (6 biological replicates / experiment). The results are expressed as the average (+/- SEM) of the percentage obtained.
  • FIG. 6A-6C represents the evaluation of the specificity of MF-m906-MMAE in the WaGa lines after knock-down of CD56.
  • WaGa cells have been independently transduced with lentiviral vectors containing two distinct doxycycline (Dox) inducible shRNAs (A, B and C) and targeting CD56 sequences. After antibiotic selection (puromycin), cells were exposed to doxycycline for 7 days before evaluation.
  • (ABC): Confirmation of knock-down of CD56 by RT-qPCR in real time (A) ( * : p ⁇ 0.05, the control cells without doxycycline are used as reference), by Western blot (attenuated mass of CD56 140 KDa) (B).
  • FIG. 7 illustrates the performance evaluation of the MF-m906-MMAE conjugate and controls (PBS and MF-TTZ-MMAE a Trastuzumab ADC coupled to MMAE) in TLC xenograft model.
  • Figure 7 shows the relative tumor volume curves (mean +/- SEM) during the study.
  • Each point represents the mean of VTRs for the groups treated with MF-m906-MMAE, MF-TTZ-MMAE and PBS, gray arrows indicate injection times.
  • FIG. 8 illustrates the performance evaluation of the MF-m906-MMAE conjugate and controls (PBS and MF-TTZ-MMAE a Trastuzumab ADC coupled to MMAE) in TLC xenograft model.
  • FIG. 8 represents the weight of the tumors at the end of the study in the experimental group and the control groups ( * : p ⁇ 0.05).
  • the horizontal lines are the means, quartiles and limits.
  • FIG. 9 represents the performance evaluation of MF-m906-MMAE ADC, compared to controls m906 (uncoupled antibody), MF-TTZ-MMAE (ADC Trastuzumab coupled to MMAE) and MMAE (toxin only) on the cell line small cell carcinoma of the lung H69.
  • the experiments were performed independently three times (6 biological replicates / experiment). The results are expressed as the average (+/- SEM) of the percentage obtained. Examples
  • Example 1 Synthesis of a cytotoxic conjugate according to the invention
  • Isonicotinic acid (1) (5.00 g; 40.614 mmol; 1.0 eq) was dissolved in thionyl chloride (15 mL; 206.77 mmol; 5.1 eq) and heated to reflux overnight. After returning to ambient temperature, the excess thionyl chloride was removed by evaporation under reduced pressure, then the residue obtained was dissolved in anhydrous dichloromethane (55 mL). The alcohol Benzyl was added (4.2 ml; 40.614 mmol; 1.0 eq) and the mixture was stirred at reflux for 10 h. After returning to ambient temperature, the reaction medium was neutralized with saturated sodium hydrogencarbonate solution and extracted with dichloromethane (3x100 mL).
  • Benzyl isonicotinate (2) (2.48 g; 11.630 mmol; 1.0 eq) was dissolved in methanol (43 mL), stirred at 50 ° C and concentrated sulfuric acid (320 ⁇ L; 6.016 mmol; 0.52 eq) was added.
  • a solution of ammonium persulfate (26,500 g; 116,000 mmol; 10.0 eq) in water (43 mL) was added in two steps: a first rapid addition of 30 drops, a white suspension forms, then in fast drip for 5 min. The reaction races up to 75 ° C, then the resulting yellow solution was stirred at 50 ° C for an additional 1 h. After returning to room temperature, the methanol was evaporated under reduced pressure.
  • 2,6-bis (hydroxymethyl) isonicotinic acid (4) (50 mg; 0.273 mmol; 1 eq) was dissolved in anhydrous A /, A / -dimethylformamide (3.0 mL), the solution was cooled to 0 ° C, then HATU (156 mg; 0.410 mmol; 1.5 eq) and 2,6-lutidine (147.0 ⁇ L; 1.260 mmol; 4.7 eq) were added. The activating solution was stirred at 0 ° C for 15 min then methyl 6-aminohexanoate (59 mg; 0.322 mmol; 1.2 eq) was added.
  • the walls of the flask were rinsed with 2 mL of anhydrous A /, A / -dimethylformamide and the reaction medium was stirred at room temperature for 15 h.
  • the reaction mixture was diluted with ethyl acetate, washed three times with saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated under reduced pressure.
  • the product was purified by flash chromatography (dichloromethane / methanol, 90:10) to give (5) (76 mg; 91%) as an off-white solid.
  • Antibody used to produce the antibody-drug conjugate according to the invention m906.
  • Bioconjugation buffer 1X saline buffer, for example phosphate, borate, acetate, glycine, tris (hydroxymethyl) aminomethane, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid in a pH range between 6 and 9, with a final NaCl concentration of between 50 and 300 mM and a final concentration of EDTA of between 0.1 and 10 mM.
  • 1X saline buffer for example phosphate, borate, acetate, glycine, tris (hydroxymethyl) aminomethane, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid in a pH range between 6 and 9, with a final NaCl concentration of between 50 and 300 mM and a final concentration of EDTA of between 0.1 and 10 mM.
  • m906 at a concentration of between 1 and 10 mg / mL in the bioconjugation buffer.
  • Reducing agent Solution of a reducing agent chosen from dithiotreitol, b-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride, tris (hydroxypropryl) phosphine at a concentration of between 0.1 and 10 mM in the bioconjugation buffer.
  • a reducing agent chosen from dithiotreitol, b-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride, tris (hydroxypropryl) phosphine at a concentration of between 0.1 and 10 mM in the bioconjugation buffer.
  • Linker solution compound 8 at a concentration of between 0.1 and 10 mM in a mixture of organic solvents chosen from dimethylsulfoxide, A /, A / -dimethylformamide, methanol, tetrahydrofuran, acetonitrile, N, N-dimethylacetamide, dioxane.
  • the reaction mixture was purified on PD-10 (GE Healthcare) with Gibco PBS buffer pH 7.4 the number of times necessary to remove the residual chemical reagents, i.e. purified 3 times.
  • Example 3 analyzes of the antibody-drug conjugate (MF-m906-MMAE)
  • the MF-m906-MMAE ADC was diluted to 1 mg / mL with PBS pH 7.4 before being filtered through 0.22 ⁇ m. 50 ⁇ g of products were injected onto a MAbPac HIC-Butyl column, 5 ⁇ m, 4.6 x 100 mm (ThermoScientific), connected to a Waters Alliance HPLC chain (e2695) equipped with a PDA (e2998) set for detection. at 280 nm.
  • MF-m906-MMAE ADC was eluted at a flow rate of 1 mL / min by gradient from 100% buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate monobasic, 5% isopropanol (v / v), pH 7.0) up to 20% buffer B (50 mM sodium phosphate monobasic, 20% isopropanol (v / v), pH 7.0) in 2 minutes then up to 85% buffer B in buffer A over 30 minutes then this gradient was maintained for 1 min. The temperature was maintained at 25 ° C throughout the separation.
  • the results obtained for MF-m906-MMAE are presented in Figure 1, and in Table 1 below.
  • the MF-m906-MMAE ADC was diluted to 1 mg / mL with PBS pH 7.4 before being filtered through 0.22 ⁇ m. 50 pg of products were injected onto an AdvanceBio SEC 2.7 ⁇ m, 7.8 x 300 mm column (Agilent Technologies), connected to a Waters Alliance HPLC chain (e2695) equipped with a PDA (2998) set for detection at 280 nm.
  • the MF-m906-MMAE ADC eluted at a flow rate of 1 mL / min with isocratic buffer C (1 mM sodium phosphate monobasic, 155 mM sodium chloride, 3 mM sodium phosphate dibasic, 3 mM d (sodium azide, pH 7.0) over 24 minutes. The temperature was maintained at 25 ° C throughout the separation.
  • the mass spectrometric analysis was carried out on a Vion IMS Qtof mass spectrometer coupled to an Acquity UPLC H-Class system from Waters (Wilmslow, UK). Prior to MS analysis, samples (20 ⁇ g) were desalted on a BEH SEC 2.1 x 150 mm 300 ⁇ desalting column by a gradient isocratic (50 mM ammonium acetate, pH 6.5) at 40 pL / min. A bypass valve was programmed to allow solvent to enter the spectrometer for only 6.5-9.5 min. MS data was acquired in positive mode with an ESI source over an m / z range of 500 to 8000 at 1 Hz and analyzed using UNIFI 1.9 software and the MaxEnt algorithm for deconvolution.
  • Example 4 in vitro evaluation of the antibody-drug conjugate MF-m906-MMAE
  • the immunohistochemical labeling with a commercial anti-CD56 antibody (123C3, Ventana, Prediluted) was carried out on a cohort of 90 TLC tumors included in a micro array tissue using a benchMark XT platform following the instructions of the supplier. .
  • CD56 expression was assessed by a person skilled in the art using the following semi-quantitative score: 0: absence of expression, 1: weak and / or heterogeneous positivity; 2: intense and diffuse positivity.
  • the tumor cells were incubated in the presence of an anti-CD56 antibody coupled to FITC (BioLegend, clone: HCD56) or of the control isotype according to the indications provided. by the manufacturer. The cells were then washed twice in PBS + 1% fetal calf serum and then analyzed.
  • FITC BioLegend, clone: HCD56
  • m906 and of trastuzumab were carried out with the m906 antibody used at 650 ng / ml, or the trastuzumab antibody. (Herceptin 150 mg Roche) at 40 ng / ml then revealed using a secondary antibody coupled to peroxidase.
  • WaGa (RRID: CVCL_E998).
  • MS-1 (RRID: CVCL_E995).
  • PeTa (RRID: CVCL_LC73).
  • MKL-2 (RRID: CVCL_D027).
  • the WaGa, MS-1, PeTa and MKL-2 cells are Merkel cell carcinoma cell lines (hereinafter TLC). All of the above lines are CD56-positive ( Figure 4).
  • SK-BR-3 (RRID: CVCL_0033): HER2-positive breast cancer line, chosen as a control because it does not express CD56.
  • the m906 antibody was conjugated with Alexa Fluor 750 using the “SAIVI Rapid Antibody Labeling Kit” (Thermoscientific) according to the instructions given by the manufacturer.
  • the WaGa cells 500,000 cells were incubated for 30 min at room temperature with the m906-Alexa Fluor 750 conjugate in a buffer solution (1X PBS, 2% feta calf serum, 0.1% sodium azide) .
  • the cells were then fixed using BD Cytofix / Cytoperm (BD Biosciences) and permeabilized with Permwash (BD Biosciences, diluted 1/10) according to the manufacturer's instructions.
  • the nuclear and lysosomal compartments were labeled with Hoechst 33342 (BD Pharmigen, 1/10000) and an anti-LAMP1 antibody coupled to phycoerythrin-Cyanine 5 (BD Pharmigen, H4A3), respectively.
  • Analysis of labeled cells was performed using an Amnis ® lmageStream ® X Mark II Imaging Flow Cytometer (Amnis Corp., part of EMD Millipore, Seattle, WA) equipped with 4 lasers (375 nm , 488 nm, 642 nm and 785 nm (SSC)). Images of WaGa were captured with Inspire TM imaging flow cytometry software at 60X magnification and extended depth of field (EDF).
  • EDF extended depth of field
  • the cells of interest were identified using the “Gradient RMS” tool of the Bright Field image (BF: white light). Debris and cell doublets were excluded from analysis based on the aspect ratio to the area plot of the BF image.
  • the area and mean intracellular fluorescence intensity (IMF) of m906 conjugated to Alexa Fluor 750 (channel 12) was evaluated for two different incubation times (5 and 30 min) using the “surface” tools. mask ”and“ cytoplasm mask ”with the IDEAS ® v6.2 software.
  • the internalization score of m906 conjugated to Alexa Fluor 750 (channel 12) was determined using the internalization function to define the ratio of the intensity of intracellular fluorescence to the intensity of whole cells.
  • the co-localization assistant using the 'Bright Detail Similarity' (BDS) function was employed to quantify the level of colocalization between the anti-LAMP1 antibody conjugated to phycoerythrin-Cyanine 5 (channel 5) and m906. conjugated to Alexa Fluor 750 (channel 12).
  • the positive BDS score (n. 1) indicates the lysosomal localization of m906.
  • the anti-CD56 antibody m906 is internalized in the lysosome in the WaGa cells expressing CD56 (Table 5). This is a critical step for drug release from ADC, making m906 a good antibody candidate for ADC development.
  • XTT cytotoxicity test was carried out.
  • the cells were deposited in 7 replicas in a 96-well plate (50,000 cells / well).
  • the MF-m906-MMAE and MF-TTZ-MMAE (ADC control) ADCs were added in incremental concentrations.
  • Culture medium served as a negative control.
  • 25 ⁇ l of XTT reagent was added per well and absorption was measured at 450 nm after 4 hours of incubation at 37 ° C. The absorption at 620 nm was used as a reference.
  • Lentiviral supernatants were produced in HEK293T cells (RRID: CVCL_0063) as previously described [6-7]
  • the harvested supernatant was sterilized by filtration (0.45 ⁇ m) and polybrene was added (1 ⁇ g / ml) before l 'infection.
  • the target cells were washed and then subjected to antibiotic selection (puromycin).
  • antibiotic selection puromycin
  • the cells were exposed to doxycycline for 7 days before analysis.
  • Example 5 in vivo evaluation of the antibody-drug conjugate (MF-m906-MMAE)
  • mice Twenty 7 week old NOD / SCID female mice (Janvier Labs) were maintained under aseptic conditions. All animal related procedures have been approved by the local ethics committee (Apafis-10076- 2017053015488124 v4).
  • the CD56-positive TLC cell line "WaGa" [8] was used for tumor induction.
  • the isoflurane anesthetized mice were injected subcutaneously with 10 7 cells in Matrigel (injection site: back).
  • the size of the tumor determined by measuring the width, length and height with a caliper and the general condition of the animal were monitored every 2 days for the duration of the procedure.
  • the volume of tumor was determined with the following formula width x length x height xw / d. When the tumor volume reached 50 mm 3 , the mice were included in the study and randomly assigned to the experimental or control groups.
  • the animals received an intravenous injection of ADC (either MF-m906-MMAE or MF-TTZ-MMAE, 10 mg / kg) or the injection in equivalent volume of PBS for the control mice.
  • a new injection was given if the tumor volume doubled in the experimental group.
  • Mice were sacrificed 30 days after inclusion or if they reached a critical point (tumor ulceration, 20% weight loss or prostration).
  • An autopsy of the animals was performed and all organs were macroscopically examined by a pathologist. The weight and volume of the tumors were evaluated after dissection. Microscopic examination of tumors, lungs and liver was performed to detect potential metastatic progression.
  • Continuous data are described in averages and limits. Categorical data are described in number and percentage of interpretable cases. Associations were assessed by Fisher's exact test for categorical data or by Mann-Whitney or Kruskall Wallis tests for continuous data. Statistical analyzes were performed using XLStat software (Addinsoft, Paris, France). p ⁇ 0.05 was considered statistically significant.
  • MF-m906-MMAE reduced tumor growth in a mouse model of MCC cell line xenograft.
  • FIG. 7 relative tumor volumes
  • FIG. 8 tumor mass at the end study.
  • similar tumor growth was observed, with an average coefficient of slope of the growth curve of 105 % of initial tumor volume per day (limits 35-166) and 108% of initial tumor volume per day (limits 33-318) for the PBS and MF-TTZ-MMAE group, respectively).

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
EP21714002.9A 2020-02-27 2021-02-26 Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie Pending EP4110405A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR2001974A FR3107648B1 (fr) 2020-02-27 2020-02-27 conjugués anticorps-médicament anti-CD56 et leur utilisation en thérapie
PCT/FR2021/050332 WO2021170961A1 (fr) 2020-02-27 2021-02-26 Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie

Publications (1)

Publication Number Publication Date
EP4110405A1 true EP4110405A1 (fr) 2023-01-04

Family

ID=71894885

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21714002.9A Pending EP4110405A1 (fr) 2020-02-27 2021-02-26 Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie

Country Status (8)

Country Link
US (2) US20230144142A1 (https=)
EP (1) EP4110405A1 (https=)
JP (1) JP2023515845A (https=)
CN (1) CN115515642A (https=)
AU (1) AU2021227413A1 (https=)
CA (1) CA3166699A1 (https=)
FR (1) FR3107648B1 (https=)
WO (1) WO2021170961A1 (https=)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR3096259B1 (fr) * 2019-05-20 2023-12-15 Mc Saf conjugués anticorps-médicament et leur utilisation en thérapie
FR3131835B1 (fr) * 2022-01-17 2025-11-21 Mc Saf Procédé de préparation de conjugués anticorps-médicament
FR3131836B1 (fr) * 2022-01-17 2025-05-23 Mc Saf Conjugués anticorps-médicament pour utilisation thérapeutique
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
AU2024279278A1 (en) 2023-05-31 2025-12-18 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
US20250127728A1 (en) 2023-10-05 2025-04-24 Capstan Therapeutics, Inc. Constrained Ionizable Cationic Lipids and Lipid Nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025149947A1 (en) * 2024-01-12 2025-07-17 Seagen Inc. Antibody-drug conjugates
WO2025149667A1 (en) 2024-01-12 2025-07-17 Pheon Therapeutics Ltd Antibody drug conjugates and uses thereof
US20250302763A1 (en) 2024-02-22 2025-10-02 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104640572B (zh) * 2012-05-15 2018-04-27 索伦托医疗有限公司 药物偶联物，偶联方法，及其用途
WO2013177062A2 (en) * 2012-05-21 2013-11-28 Genentech, Inc. Methods for improving safety of blood-brain barrier transport
FR3008408B1 (fr) * 2013-07-11 2018-03-09 Mc Saf Nouveaux conjugues anticorps-medicament et leur utilisation en therapie
US10548987B2 (en) 2015-07-31 2020-02-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-drug conjugates for targeting CD56-positive tumors
CN107744592B (zh) * 2017-09-15 2020-04-28 四川大学 抗cd56抗体与海兔毒素偶联复合物及其制备方法和用途
CN107715119B (zh) * 2017-09-15 2020-11-10 四川大学 抗cd56抗体与多卡霉素偶联复合物及其制备方法和用途
SG11202006510XA (en) * 2018-01-08 2020-08-28 Regeneron Pharma Steroids and antibody-conjugates thereof

Also Published As

Publication number Publication date
US20230144142A1 (en) 2023-05-11
WO2021170961A1 (fr) 2021-09-02
FR3107648B1 (fr) 2022-03-18
FR3107648A1 (fr) 2021-09-03
AU2021227413A1 (en) 2022-10-20
JP2023515845A (ja) 2023-04-14
US20230405140A1 (en) 2023-12-21
CA3166699A1 (fr) 2021-09-02
CN115515642A (zh) 2022-12-23

Similar Documents

Publication Publication Date Title
EP4110405A1 (fr) Conjugués anticorps-médicament anti-cd56 et leur utilisation en thérapie
JP7158548B2 (ja) Ｍｕｃ１に特異的に結合する抗体及びその用途
CA3082160C (en) Anti-mesothelin antibody and antibody drug conjugate thereof
TW202015739A (zh) 剪接調節抗體-藥物結合物及其使用方法
KR20200121317A (ko) 글리피칸 3 항체 및 그의 접합체
KR20160113623A (ko) 항체-약물 접합체 및 면역독소
JP2017114763A (ja) 抗ｃｄ９８抗体−薬物コンジュゲート
EP3501546A2 (de) Cystein-wirkstoff konjugate und ihre verwendung
JP7846624B2 (ja) ペプチド含有リンカーとの部位特異的抗体－薬物コンジュゲート
RS60389B1 (sr) Vezujući molekuli specifični za asct2 i njihove upotrebe
EP3972652A1 (fr) Conjugues anticorps-medicament et leur utilisation en therapie
US10112999B2 (en) Anti-PRLR antibody-drug conjugates (ADC) and uses thereof
CN119604314A (zh) 抗gd2抗体、免疫偶联物及其治疗用途
JP2025510623A (ja) Ｐｔｋ７を発現する腫瘍を標的化するための抗体コンジュゲート
KR20250137203A (ko) 항체 약물 접합체
WO2025083071A2 (en) Antibody-drug conjugates
JP2025109708A (ja) Ｂリンパ球特異的アマトキシン抗体コンジュゲート
WO2021064332A1 (fr) Conjugues anti-cd30-medicament et leur utilisation en therapie
JP2022500454A (ja) 抗葉酸受容体抗体コンジュゲートによる併用療法
TW202600175A (zh) 用於治療gd2陽性癌症之抗gd2抗體及免疫結合物
HK40123391A (zh) 抗gd2抗体、免疫偶联物及其治疗用途
WO2026082125A1 (zh) 结合folr1的抗体、含有其的药物及其应用
WO2025133322A1 (en) Cnx antigen-binding molecules
JP2025531401A (ja) Ｇｐｃ３抗体薬物複合体及びその使用
WO2026059501A1 (en) Linker moieties

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220810

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)