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
  • A61K47/6801—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
  • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
• • 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
  • 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
• • 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
• • 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
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/524—CH2 domain
• • 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
  • C07K2317/71—Decreased effector function due to an Fc-modification
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• antibody-drug conjugates wherein the antibody specifically binds to folate receptor alpha (FRa), and wherein the drug is preferably chosen among inhibitors of topoisomerase I, for example camptothecine analogues such as exatecan.
• FRa folate receptor alpha
• the drug is preferably chosen among inhibitors of topoisomerase I, for example camptothecine analogues such as exatecan.
• Such antibody-drug conjugates are useful in particular in treating proliferative diseases including cancers, such as ovarian cancer, breast cancer or lung cancer.
• ADCs Antibody-drug conjugates
• Such ADCs comprise at least an antibody and a payload (e.g. a cytotoxic drug), both covalently bonded by a linker.
• ADCs are therefore designed to combine the specificity of antibody target with the efficiency of the payload (e.g. the cytotoxic activity of a chemotherapeutic agent).
• Efficient ADCs should exhibit high specificity and low systemic toxicity.
• antibody used in ADCs needs to bind accurately and efficiency to its antigen, meaning that the suitable target antigen is preferentially or exclusively expressed on targeted cells.
• linker types that have been used to conjugate a cytotoxin or a drug to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
• Linkers are for example chosen among those susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue, for example cathepsins (e.g. cathepsins B, C, D). Efficient linker can ensure an accurate and timely release of the payload.
• a stable linker can release the payload in a targetspecific manner whereas a not-stable linker is more likely to undergo a non-accurate release of the payload (for example due to a non-specific cleavage), leading to a nonspecific systemic toxicity.
• an antibody-drug conjugate comprising an anti-FRa antibody which is efficient, has high specificity, low toxicity (improved therapeutic index) and differentiated mechanism of action of the payload.
• the present disclosure provides topoisomerase I inhibitorbased anti-FRa antibody-drug conjugates with excellent in vivo efficacy in solid tumor cancer models and low toxicity, in particular when compared with reference prior art ADCs targeting FRa-expressing antibody with other payload and drug linkers, such as mirvetuximab soravtansine.
• Ab is an anti-folate receptor alpha (FRa) antibody which binds specifically to SEQ ID NO:12,
• D is a cytotoxic drug moiety bonded to L, p is from 1 to 8, preferably from 6 to 8, and more preferably p is 8.
• Ab is an antibody comprising a human lgG1 isotype constant region.
• Ab is an antibody comprising a mutant or chemically modified constant region of human lgG1 isotype, wherein said mutant or chemically modified constant region confers no or decreased ADCC activity to said antibody when compared to a corresponding antibody with wild type human IgG 1 isotype constant region.
• Ab is an antibody which comprises a human lgG4 isotype constant region, or a mutant or chemically modified lgG4 constant region, wherein said mutant or chemically modified constant region confers no or decreased ADCC activity to said antibody when compared to a corresponding antibody with wild type lgG4 isotype constant region.
• Ab is an anti-FRa antibody comprising either:
• variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:1 , HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3 and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6; or,
• variable heavy chain polypeptide comprising VH of SEQ ID NO:7 and a variable light chain polypeptide comprising VL of SEQ ID NO:8.
• said Ab comprises or essentially consists of a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NQ:10;
• said Ab comprises or essentially consists of a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NQ:10.
• D is an inhibitor of topoisomerase I, preferably selected from the group consisting of camptothecine analogues, and more preferably D is the drug moiety of Exatecan of formula (II) below
• L is a cleavable linker moiety of formula -A-W-, wherein A is an optional stretcher unit linked to Ab, and W is a cleavable moiety linked to D.
• L is a lysosomal protease-sensitive linker, and W comprises a cleavable peptide moiety, for example selected from the group consisting of valinecitrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys).
• L is a protease sensitive cleavable linker and W comprises a sugar cleavable unit, preferably selected from a p-glucuronide or a p-galactoside moiety.
• L is a glutathione-sensitive linker and W comprises a disulfide moiety.
• W is of the following formula (III) wherein each R2 is independently selected from the group consisting of electron-withdrawing groups and C1-C4 alkyl; n is 0, 1 or 2 ;
• T is a sugar cleavable unit or a polypeptide cleavable unit
• Y is O when T is a sugar cleavable unit, or NR3 when T is a polypeptide cleavable unit; and R3 is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole,
• R” and R’ being independently selected from H and Ci-Ce alkyl, and pharmaceutically acceptable salts thereof
• L corresponds to a linker -A-W- of formula (IV) wherein
• Xi is a connector unit
• Z is an optional spacer
• X2 is a connector unit
• K is an optional hydrophobicity masking entity, preferably selected from polysarcosine and polyethylene glycol;
• R1 is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”- C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole;
• R4 is selected from the group consisting of H, C-i-Ce alkyl and C2-C6 alkenyl, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, and -NR”-;
• R 5 is selected from the group consisting of H, C-i-Ce alkyl and C2-C6 alkenyl, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, and -NR”-.
• R’, R” and R’ being independently selected from H and Ci-Ce alkyl.
• Z is independently selected from the group consisting of one or more amino acid(s), one or more N-substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”- , -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene, arylene, cycloalkylene, heterocycl
• R’, R” and R’ being independently selected from H and Ci-Ce alkyl.
• K is a polysarcosine, preferably of the following formula (V) wherein k is an integer between 2 and 50, preferably between 4 and 30, and Re corresponds to OH or NH2.
• T is a sugar cleavable unit which is a glucuronide or a galactoside.
• T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.
• L is covalently bonded to one or more thiol residues of said antibody, preferably, said L corresponding to a linker of formula (VI):
• Ab includes full-length antibodies or antibodies fragments containing antigen binding portions.
• the antibody drug conjugate corresponds to the following formula (VII) wherein Ab is an anti-FRa antibody, such as farletuzumab, or its silent lgG1 variants, typically comprising alanine substitutions in Leucine 234 and Leucine 235 of lgG1 Fc constant region, and p is from 4 to 8.
• the antibody drug conjugate corresponds to the following formula (VII)
• Ab is an anti-FRa antibody, such as farletuzumab, or its silent lgG1 variants, typically a mutant variant of human IgG 1 comprising alanine substitutions in Leucine 234 and Leucine 235 of lgG1 Fc constant region, also commonly called as LALA mutations, and p is 8.
• HCDR2 of SEQ ID NO:2 HCDR3 of SEQ ID NO:3 and
• variable light chain polypeptide comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6;
• L is a cleavable linker of formula -A-W-, wherein A is an optional stretcher unit linked to the Ab, and W is a cleavable moiety linked to D
• D is exatecan; p is from 1 to 8, for example between 4 and 8, preferably between 6 and 8, and for example p is between 7 and 8.
• ADCs for use as a medicament, preferably for use in the treatment of a tumor, for example a solid tumor, more specifically selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.
• Another object of the disclosure relates to the use of above ADCs, in the preparation of a medicament or pharmaceutical composition for the treatment of a tumor, for example a solid tumor, more specifically selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.
• the ADCs may be preferably used in the treatment of a cancer selected from the group consisting of ovarian cancer, triple negative breast cancer, and non-small cell lung cancer.
• the disclosure further relates to a pharmaceutical composition
• a pharmaceutical composition comprising an antibodydrug conjugate as disclosed herein, in combination with one or more pharmaceutical acceptable excipient, diluent or carrier, optionally comprising other active ingredients, for example anti-cancer drugs or immunotherapeutic drugs such as immune checkpoint inhibitors.
• the disclosure also relates to the process for obtaining an ADC of the disclosure, wherein the method comprises:
• Figure 1 represents preclinical rodent efficacy, pharmacokinetic and tolerability data of conjugates, according to example 4.
• Figure 4 represents in vitro ELISA binding experiments of conjugates against recombinant human folate receptor alpha protein, according to example 7.
• Figure 5 represents in vitro surface plasmon resonance (SPR) binding experiments of conjugates against recombinant human folate receptor alpha protein, according to example 8.
• SPR surface plasmon resonance
• Figure 7 represents ex-vivo human plasma stability of conjugates, according to example 10.
• Figure 8 represents in vitro cytotoxicity assay of conjugates on a folate receptor alpha negative breast cancer cell line BT-474, according to example 11.
• Figure 9 represents in vivo efficacy assessment of conjugates in a folate receptor alpha positive SW-620 xenograft cancer model, according to example 12.
• Figure 10 represents in vivo efficacy assessment of conjugates in a second folate receptor alpha positive SW-620 xenograft cancer model, according to example 12.
• Figure 11 represents in vivo efficacy assessment of conjugates in a folate receptor alpha positive OV-90 xenograft cancer model, according to example 12.
• Figure 12 represents in vivo efficacy assessment of conjugates in a second folate receptor alpha positive OV-90 xenograft cancer model, according to example 12.
• Figure 13 represents in vivo efficacy assessment of conjugates in a folate receptor alpha positive KB xenograft cancer model, according to example 12.
• Figure 14 represents in vivo efficacy assessment of conjugates in a folate receptor alpha positive PA-1 xenograft cancer model, according to example 12.
• Figure 15 represents in vivo efficacy assessment of conjugates in a third folate receptor alpha positive OV-90 xenograft cancer model, according to example 12.
• Figure 16 represents in vivo efficacy assessment and tumor re-implantation challenge of conjugates in a folate receptor alpha positive IGROV-1 xenograft cancer model, according to example 12.
• Figure 18 represents in vivo SCID and CD-1 mice tolerability assessment of conjugates at high doses, according to example 13.
• Figure 19 represents in vivo rat pharmacokinetic assessment of total mAb, total ADC and free exatecan sub-components of conjugates, according to example 14.
• Figures 20 and 21 represents in vivo mice pulmonary inflammatory evaluation of conjugates, according to example 15.
• Figure 22 represents in vivo cynomolgus monkey dose-range-finding toxicology study, according to example 16.
• FRa or "folate receptor alpha” refers to the human folate receptor alpha as defined in SEQ ID NO: 12, unless otherwise described. This sequence corresponds to the amino acid sequence of Folate receptor alpha as encoded by FOLR1 gene (Homo sapiens) also available in UniprotKB under entry P15328 (FOLR1_HUMAN).
• antibody as referred to herein includes whole antibodies and any antigen binding fragments (i.e. "antigen-binding portion") or single chains thereof.
• a naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
• the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• antigen-binding portion of an antibody refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. a portion of FRa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a UniBody consisting of a single arm with a modified IgG heavy chain, for example lgG4, at the hinge region, a domain antibody fragment (Ward et al., 1989 Nature 341 :544-546), or a nanobody fragment which consists of a VH domain; and an isolated complementarity determining region (CDR); or any fusion proteins comprising such antigen-binding portion.
• Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domain
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423- 426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883).
• single chain Fv single chain Fv
• Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody.
• These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
• an "isolated antibody”, as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g. an isolated antibody that specifically binds to FRa is substantially free of antibodies that specifically bind to other antigens than FRa).
• An isolated antibody that specifically binds to FRa may, however, have cross-reactivity to other antigens, such as FRa molecules from other species.
• an isolated antibody may be substantially free of other cellular material and/or chemicals.
• an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
• monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of single molecular composition.
• a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
• a method for determining the KD of an antibody is by using surface plasmon resonance, or by using a biosensor system such as a Biacore® (see also for detailed information regarding affinity assesment Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high- throughput protocols for measuring drug/human serum albumin interactions using BIACORE®. Anal Biochem. 2001 ).
• affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, the stronger the affinity.
• an antibody or a protein that "specifically binds to an antigen" is intended to refer to an antibody which detectably bind an epitope presented on an antigen, such as a FRa in the present disclosure. It is typically intended to refer to an antibody, or ADC, that binds to human FRa with a KD of 200 nM or less, 100 nM or less, 50 nM, 40 nM or less, or about 30 nM.
• the KD is comprised between 10' 3 pM and 200 nM, notably between 0.1 pM and 100 nM, notably between 0.1 pM and 50 nM, or between 1 pM and 50 nM notably between 1 pM and 30 nM, between 10 pM and 50 nM, between 0.1 nM and 200nM or between 0.1 nM and 100 nM, or between 1 nM and 50 nM, notably between 1 nM and 30 nM.
• an ADC of the present disclosure is specific for FRa and has a KD as above defined.
• host cell refers to prokaryotic or eukaryotic cells.
• Eukaryotic cells for example mammalian host cells, yeast or filamentous fungi, are preferred, and in particular mammalian cells, because they are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
• ADCC antibody dependent cell cytotoxicity activity refers to cell depleting activity.
• ADCC activity can be measured by ADCC assays commercially available, for example, ADCC Reporter Bioassay as commercialized by Promega under Ref# G7015.
• the term “subject” includes any human or nonhuman animal.
• nonhuman animal includes all vertebrates, e.g. mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, ...
• the term “subjects” also encompasses the term “patient”.
• the term “drug” or D in the formula (I) of the ADC also refers to “a payload”, i.e. a moiety that is conjugated to an antibody (or a fragment).
• the drug D is not to be construed as limited to classical chemical therapeutic agent.
• D can encompass a protein, a peptide or a polypeptide possessing a desired biological activity.
• it refers to a therapeutic moiety, such as a cytotoxin.
• a “cytotoxin” or “cytotoxic agent” includes any agent that is detrimental to (e.g. kills) cells.
• the present disclosure encompasses the compounds or drug or cytotoxin as described herein and, their tautomers, enantiomers, diastereomers, racemates or mixtures thereof, and their hydrates, esters, solvates or pharmaceutically acceptable salts. Any formula given herein is also intended to represent unlabeled as well as isotopically labeled forms of the compounds, like deuterium labeled compounds or 14 C-labeled compounds.
• pharmaceutically acceptable salts refer to salts that retain the biological effectiveness and properties of the compounds of this disclosure and, which typically are not biologically or otherwise undesirable.
• the compounds of the disclosure are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
• Pharmaceutically acceptable acid addition salts can be formed with organic acids and/or inorganic acids.
• Pharmaceutically acceptable base addition salts can be formed with organic bases and/or inorganic bases. Such salts are well-known from thosee skilled in the art.
• the term connector unit refers to a component that connects different parts of the compound together, for example, the connector can connect Ab to a spacer, or a spacer to the amide function -CO-NR1-.
• the connector is a scaffold bearing attachment sites for components of the antibody-drug-conjugate, namely Ab, the spacer, the hydrophobicity masking entity, and/or the amide function -CO-NR1-.
• Non-exhaustive listing of connectors includes: aminoacids, for example lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, homoalanine; amino alcohols; amino aldehydes; polyamines or any combination thereof.
• the connector unit Xi and/or X2 is one or more natural or non-natural aminoacids. In one embodiment, the connector unit Xi and/or X2 is selected from glutamic acid, lysine and glycine.
• the connector units Xi and X2 can be independently selected from the group consisting of one or more amino acid(s), one or more N- substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”- C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene, arylene, cycloalkylene, hetero
• connector units include optionally substituted polyether, aminoacids, benzyl
• aminoacids refers to natural or non-natural aminoacids.
• the CO moiety of the -CONR1- or -CONR1 ’- group can be considered as part of the X2 connector unit when X2 consists of one or more aminoacids.
• Non-exhaustive listing of aminoacids includes lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, and homoalanine.
• a spacer is a divalent arm that covalently binds two components of the antibody-drug- conjugate, such as the 2 connector units.
• Non-exhaustive listing of spacer units includes: alkylene, heteroalkylene (so an alkylene interrupted by at least one heteroatom selected from Si, N, O and S); alkoxy; polyether such as polyalkylene glycol and typically polyethylene glycol; one or more natural or nonnatural aminoacids such as glycine, alanine, proline, valine, N-methylglycine; C3-C8 heterocyclo; C3-C8 carbocyclo; arylene, and any combination thereof.
• a spacer is a divalent linear alkylene group, preferably (CH2)4.
• alkyl refers to a monovalent saturated hydrocarbon chain (having straight or branched chain).
• alkyl refers to Ci-C 2 o alkyl.
• the alkyl is a “lower alkyl”, i.e. an alkyl group having 1 , 2, 3, 4, 5 or 6 carbons (straight or branched chain C-i-Ce alkyl group).
• this includes methyl, ethyl, n-propyl, i- propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.
• Alkylene used alone or as part of alkylene glycol for example, refers to a divalent saturated, straight-chained or branched alkyl group as defined herein.
• Alkenyl and alkynyl refer to at least partially unsaturated, straight-chained or branched hydrocarbon group having 2-20 carbon atoms, preferably 2-12, more preferably 2-6, especially 2-4.
• C3-C8 cycloalkyl or “carbocycle” refer to a saturated or unsaturated cyclic group having 3 to 8 carbon atoms, preferably 3 to 6.
• the cycloalkyl can have a single ring or multiple rings fused together.
• the cycloalkyl can also include spirocyclic rings.
• Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• C3-C8 cycloalkylene or “carbocyclo” refer to a divalent cycloalkyl as defined herein.
• halogen refers to a fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-I) group.
• Ci-Ce haloalkyl refers to a Ci-Ce alkyl as defined herein that is substituted by one or more halogen group as defined herein.
• Suitable C-i-Ce haloalkyl groups include trifluoromethyl and dichloromethyl.
• heteroalkyl refers to a straight or branched hydrocarbon chain consisting of 1 to 12 carbon atoms, preferably 1 to 10, more preferably 1 to 6 carbon atoms, and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized (for example: a sulfoxide or a sulfone) and the nitrogen heteroatom may optionally be quaternized.
• the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
• Heteroalkylene refers to a divalent heteroalkyl as defined above.
• heteroatoms can also occupy either or both of the chain termini.
• Ci-Ce alkoxy refer to a -O-alkyl group, wherein the alkyl group is a Ci-Ce alkyl as defined herein. Suitable Ci-Ce alkoxy groups include methoxy, ethoxy, propoxy.
• Ci-Ce haloalkoxy refer to a Ci-Ce alkoxy group as defined herein, that is substituted by one or more halogen group as defined herein. Suitable haloalkoxy include trifluoromethoxy.
• aryl having 6 to 10 ring atoms refer to a polyunsaturated, aromatic hydrocarbyl group having a single ring or multiple aromatic rings fused together, containing 6 to 10 ring atoms, wherein at least one ring is aromatic.
• the aromatic ring may optionally include one to two additional rings (cycloalkyl, heterocyclyl or heteroaryl as defined herein) fused thereto.
• Suitable aryl groups include phenyl, naphthyl and phenyl ring fused to a heterocyclyl, like benzopyranyl, benzodioxolyl, benzodioxanyl and the like.
• the electron withdrawing group is -NO 2 .
• the electron-withdrawing group is in ortho position with regard to the Y-T substituent of the phenyl ring.
• protecting group refers to a chemical substituent which can be selectively removed by readily available reagents which do not attack the regenerated functional group or other functional groups in the molecule. Suitable protecting groups are known in the art and continue to be developed. Suitable protecting groups may be found, for example in Wutz et al. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-lnterscience, 2007). Protecting group for protection of the amino group as described by Wutz et al. (pages 696-927), are used in certain embodiments.
• amino protecting groups include, but are not limited to, t- butyloxycarbonyl (Boc), 9-fluorenyl methoxycarbonyl (Fmoc), Acetyl (Ac), carboxybenzyl group (Cbz), benzyl group (Bn), allyl, trifluoroacetyl, allyloxy carbonyl (Alloc) group and 2,2,2- trichloroethoxycarbonyl (Troc).
• a hydrophobicity masking entity refers to a group that can reduce the apparent hydrophobicity of the compound.
• the hydrophobicity masking entity can be selected from polysarcosine and polyethylene glycol.
• the number of ethylene glycol or sarcosine moieties may vary in a wide range.
• the number of ethylene glycol or sarcosine moieties in the hydrophobicity masking entity may be comprised between 2 and 500, preferably between 5 and 100, in particular between 5 and 25.
• the hydrophobicity masking entity is a polysarcosine comprising from 6 to 24 sarcosine moieties, preferably comprising from 10 to 12 sarcosine moieties.
• bonds refers to a linkage. This linkage is also represented by the dash in formula (I). Linkage may be a covalent bond, or a non-covalent interaction such as through electrostatic forces. Preferably, bonds are covalent bonds. As used herein, the “wavy lines” on formulas represent the attachment sites between each part (Ab, L, Z, X and D) of the ADC of the disclosure.
• the term “treat” “treating” or “treatment” refers to one or more of (1 ) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease.
• the term “treatment” may refer to the inhibition of the growth of the tumor, or the reduction of the size of the tumor.
• a “therapeutically efficient amount” or “effective amount” of an ADC is an amount sufficient to perform a specifically stated purpose, for example to product a therapeutic effect after administration, such as inhibition or reduction in tumor growth rate or tumor volume, a reduction in a symptom of cancer or some indicia of treatment efficacy.
• a therapeutically effective amount of ADC can reduce the number of cancer cells, reduce the tumor size, inhibit (e.g. slow or stop) tumor metastasis, inhibit (e.g. slow or stop) tumor growth, and/or relieve one or more symptoms.
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.
• % identity number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
• the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, and Wunsch).
• the percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk).
• EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5.
• the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
• the antibody Ab for use in making the ADCs of the disclosure are anti-folate receptor alpha (FRa) antibodies which binds specifically to SEQ ID NO:12.
• Ab is an internalizing antibody fragment of the above-defined recombinant antibodies.
• Internalizing refers to an antibody that is capable of being taken through the cell’s lipid bilayer external membrane to an internal compartment (i.e. “internalized”) upon specific binding to the cell, preferably into a degradative compartment in the cell.
• Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
• a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 ).
• Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells as described herein.
• Ab is a humanized antibody.
• a non-human antibody is humanized to reduce immunogenicity to humans, while having at least the same affinity (or superior affinity) of the parental non-human antibody.
• the antibodies of the present disclosure are humanized antibodies.
• a humanized antibody comprises one or more variable domains in which, CDRs, (or portions thereof) are derived from a non-human antibody, e.g. a murine anti- FRa internalizing antibody, and framework regions (or portions thereof) are derived from human antibody sequences.
• a humanized antibody optionally will also comprise at least a portion of a human constant region.
• Ab is a humanized or human silent antibody, preferably a humanized silent lgG1 antibody.
• silent antibody refers to an antibody that exhibits no or low ADCC activity as measured in an ADCC activity assay.
• the term “no or low ADCC activity” means that the silent antibody exhibits an ADCC activity that is at least below 10%, for example below 50% of the ADCC activity that is observed with the corresponding antibody with wild type corresponding IgG isotype.
• Ab is farletuzumab, or other anti-FRa antibodies as disclosed in W02005080431 or WO2017151979.
• Ab is mirvetuximab or a silent LALA mutant version of mirevutximab.
• Ab has a heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3 sequences and a light chain variable region comprising LCDR1 , LCDR2, and LCDR3 sequences, wherein one or all of these CDR sequences have specified amino acid sequences based on mAb1 (silent LALA mutant version of farletuzumab) antibody described herein, or a functional variant of said antibody with similar CDR sequences differing from the CDR sequences of farletuzumab by 1 , 2 or 3 amino acid conservative modifications, and wherein the antibody or protein retains the desired functional properties of mAb1 antibody, in particular when used as an ADC.
• mAb1 silica mutant version of farletuzumab
• Ab has a heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3 sequences and a light chain variable region comprising LCDR1 , LCDR2, and LCDR3 sequences, wherein one or more of these CDR sequences have specified amino acid sequences based on mirevtuximab (or silent LALA mutant version of mirvetuximab) antibody described herein or a functional variant of said antibody with similar CDR sequences differing from the CDR sequences of farletuzumab by 1 , 2 or 3 amino acid conservative modifications, and wherein the antibody or protein retains the desired functional properties of mAb1 antibody, in particular when used as an ADC.
• mirevtuximab or silent LALA mutant version of mirvetuximab
• Desired functional properties of the anti-FRa antibodies includes without limitation:
• an ADC with such variant anti-FRa antibody provides similar or lower in vitro efficacy in FRa negative cell line (such as BT-474 breast cancer cell lines) as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using an in vitro efficacy assay in FRa negative cell line as used in the Examples; and/or, (vi) an ADC with such variant anti-FRa antibody provides similar or higher in vivo efficacy in tumor-bearing mouse xenograft models as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using one of the xenograft models as used in the Examples.
• conservative sequence modifications is intended to refer to amino acid substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
• Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
• Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• an ADC with such variant anti-FRa antibody provides similar or lower in vitro efficacy in FRa negative cell line (such as BT-474 breast cancer cell lines) as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using an in vitro efficacy assay in FRa negative cell line as used in the Examples; and/or,
• an ADC with such variant anti-FRa antibody provides similar or higher in vivo efficacy in tumor-bearing mouse xenograft models as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using one of the xenograft models as used in the Examples.
• Ab is an anti-FRa antibodies which comprises the 6 CDRs which are 100% identical to corresponding CDRs of mirevtuximab, and the framework amino acid regions which are at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding framework amino acid regions of mirvetuximab, wherein said anti-FRa antibody has the following properties:
• an ADC with such variant anti-FRa antibody provides similar or lower in vitro efficacy in FRa negative cell line (such as BT-474 breast cancer cell lines) as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using an in vitro efficacy assay in FRa negative cell line as used in the Examples; and/or, (vi) an ADC with such variant anti-FRa antibody provides similar or higher in vivo efficacy in tumor-bearing mouse xenograft models as the corresponding control ADC with mAb1 as the anti-FRa antibody, for example using one of the xenograft models as used in the Examples.
• somatic mutations can be "backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.
• site-directed mutagenesis or PCR-mediated mutagenesis.
• Such "backmutated” antibodies are also intended to be encompassed by the invention.
• composition of antibodies of the disclosure may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
• the Fc domain is a silent Fc mutant preventing glycosylation at position 297 of the Fc domain.
• the Fc domain contains an amino acid substitution of asparagine at position 297.
• An example of such amino acid substitution is the replacement of N297 by a glycine or an alanine.
• An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
• the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
• PEG polyethylene glycol
• the pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
• polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci- C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
• the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
• Another modification of the antibodies that is contemplated by the present disclosure is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the disclosure to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule.
• serum protein such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule.
• Antibodies of the disclosure can be obtained using conventional technical known to those of skill in the art. For more information about nucleic acids encoding antibodies of the disclosure as well as generation of transfectomas producing these antibodies, those of skill in the art can also refer to international application number W02005080431 .
• the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
• the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
• the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
• the ADCs of the disclosure contain a cleavable linker moiety L bonded to Ab, said anti- FRa antibody as disclosed in the previous section, on one side, and bonded to the drug D on the other side.
• the linker L is covalently bonded to one or more thiol residues of the antibody Ab, for example from natural or artificial cysteine residues of the antibody sequences.
• cleavable refers to a linker which can be cleaved under specific environmental conditions (such as redox potential or pH) or specific lysosomal enzymes in response to intracellular environments, for example, after internalizing of the ADC in a cell.
• L is a cleavable linker moiety of formula -A-W-, wherein A is an optional stretcher unit linked to Ab, and W is the cleavable moiety linked to D.
• the optional stretcher unit A may be selected from the group consisting of one or more amino acid(s), one or more N-substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3- Cs cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”- C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene, arylene, cycloalkylene,
• R’, R” and R’ being independently selected from H and Ci-Ce alkyl
• W comprises a cleavable peptide moiety which may be selected from the group consisting of valine- citruline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys).
• W comprises a Valine-Alanine peptide moiety.
• W comprises a sugar cleavable unit, preferably selected from 0-glucuronide or 0-galactoside moiety.
• W comprises a disulfide moiety.
• L is a cleavable linker moiety of formula -A-W-, wherein W is of the following formula (III) wherein each R2 is independently selected from the group consisting electron-withdrawing groups and C1-C4 alkyl; n is 0, 1 or 2 ;
• T is a sugar cleavable unit or a polypeptide cleavable unit
• Y is O when T is a sugar cleavable unit, or NR3 when T is a polypeptide cleavable unit;
• R3 is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole,
• R” and R’ being independently selected from H and C1 -C6 alkyl, and pharmaceutically acceptable salts thereof
• T is a sugar cleavable unit which is a glucuronide or a galactoside.
• T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.
• L corresponds to a linker -A-W- of formula (IV) wherein
• Xi is a connector unit
• Z is an optional spacer
• X2 is a connector unit
• K is an optional hydrophobicity masking entity, preferably selected from polysarcosine and polyethylene glycol;
• R1 is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”- C(O)-, -NR”-C(O)-NR'”-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole;
• R4 is selected from the group consisting H, C-i-Ce alkyl and C2-C6 alkenyl, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, and -NR”-;
• R 5 is selected from the group consisting H, C-i-Ce alkyl and C2-C6 alkenyl, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, and -NR”-.
• X1 and X2 are independently selected from the group consisting of one or more amino acid(s), one or more N- substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”- C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene, arylene,
• alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”- C(O)-NR'”-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene being optionally substituted with one or more of the substituents selected from : halogen, oxo, -OH, -NO2, -CN, C-i-Ce
• K is a polyethylene glycol moiety (PEG), preferably comprising between 2 and 50 ethylene-glycol moieties.
• L corresponds to the linker of formula (VI):
• said linker L is covalently bonded to one or more thiol residues of the antibody Ab, for example to the eight thiol residues of the antibody Ab.
• D of the disclosure is a payload which is linked to X on the side of the carbonyl function of X.
• linkage between X and D occurs between carbonyl function of X and amino group of D.
• the payload D is an important component of the ADC design.
• the payload may be a therapeutic agent or a drug.
• drug refers in particular to an agent that is capable of modulating a biological process and/or has biological activity.
• the payload D is a cytotoxic drug which is activated after release from the internalized ADC inside the cytoplasm of tumor cell. It should ideally be able to destroy the tumor cells while not affecting non-tumor cells (when linked to the antibody).
• the payload should also ideally be of high stability in the systemic circulation and lysosomes. It should preferably have an in vitro subnanomolar IC50 value for cancer cell lines and sufficient solubility in the aqueous environment.
• the cytotoxic drug is selected from microtubule-disrupting or anti-mitotic agents such as auristatin (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g. maytansine), DNA-damaging agents such as calicheamicin, duocarmycin, and anthracyclines (e.g. daunorubicin, doxorubicin, dihyrdroxyanthracindione), or inhibitors of topoisomerase I such as camptothecines or their analogues.
• auristatin including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)
• maytansinoids e.g. maytansine
• DNA-damaging agents such as calicheamicin, duocarmycin, and anthracyclines (e.g. daunorubicin, doxorubicin, dihyrdroxy
• D is selected from the group consisting of antimetabolites, (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thioepa chloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin)), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents, e
• the Drug-to-Antibody Ratio has in principle an exact value for a single ADC, it is understood that the value will often be an average value when used to describe a composition containing many ADCs, due to some degree of inhomogeneity, typically associated with the conjugation step.
• the average loading for a sample of an ADC is also referred to herein as the drug to antibody ratio, or "DAR".
• the DAR (p) is between about 1 and about 8 (/.e. 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8), preferably between about 4 and about 8, more preferably between about 6 and about 8, and even more preferably at about 8.
• an antibody When the attachment site is inter-chain cysteine thiol group, an antibody may have only one or few cysteine thiol groups through which a linker may be attached. Indeed, most reactive cysteine thiols generally exist as inter-chain disulfide bridges. Over-attachment of linker-toxin to the antibody may destabilize the antibody by reducing the cysteine residues available to form inter-chain disulfide bridge. Therefore, an optimal drug antibody ratio should increase potency of the ADC (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody moiety and without degrading pharmacokinetics properties.
• -L-D is bonded to the inter-chain reactive thiol residues of the antibody.
• a typical antibody with full length heavy and light chains comprises 8 available inter-chain reactive thiol residues.
• the ADC is a DAR8 ADC wherein the eight -L-D moieties are covalently bonded to the 8 inter-chain reactive thiol residues of the antibody Ab, and, most preferably, wherein -L- corresponds to Formula (VI).
• an ADC of the disclosure corresponds to the following formula (VII) wherein Ab is the anti-FRa as defined in the previous sections and p is from 4 to 8, preferably from 6 to 8.
• an ADC of the disclosure corresponds to the following formula (VII) wherein Ab is the anti-FRa as defined in the previous sections.
• Ab is an anti-folate receptor alpha antibody or an antigen-binding fragment thereof, comprising • a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:
• variable light chain polypeptide comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6;
• L is a cleavable linker of formula -A-W-, wherein A is an optional stretcher unit linked to the Ab, and W is a cleavable moiety linked to D
• D is an inhibitor of topoisomerase I, e.g. exatecan ;
• (iv) p is from 1 to 8, preferably 8.
• the ADC of the disclosure corresponds to formula (I) wherein (i) Ab is an antibody comprising a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10 ;
• L is a cleavable linker of formula -A-W-, wherein A is an optional stretcher unit linked to the Ab, and W is a cleavable moiety linked to D
• D is an inhibitor of topoisomerase I, e.g., exatecan ;
• the ADC of the disclosure is an ADC of formula (I) wherein
• Ab is an antibody comprising a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NQ:10 ;
• (iv) p is from 1 to 8, preferably between 6 and 8, for example about 8.
• the ADC of the disclosure is an ADC of formula (VII) wherein Ab is an antibody comprising a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NQ:10.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the pharmaceutical formulation of the disclosure may further comprise one or more pharmaceutically acceptable excipients selected from stabilizers, surfactants, buffering agents, antimicrobial preservatives, protectants, antioxidants, chelating agents, bulking agents.
• a “solvent” is any pharmaceutically acceptable (i.e., safe and non-toxic for administration to a human or another mammal) and useful ingredient for the preparation of a liquid formulation, such as an aqueous formulation.
• stabilizers are compounds increasing protein stability, especially against unfolding and aggregation.
• the stabilizer is admitted by the authorities as a suitable additive or excipient in pharmaceutical formulations.
• the stabilizer may be a saccharide.
• a "saccharide” herein comprises the general composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars, etc.
• surfactants herein include polysorbate (for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetylbetaine; lauroamidopropyl-, cocamidopropyl-Jinoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- betaine (e.g.
• lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethylglycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc).
• Other examples of pharmaceutically acceptable surfactants include polyoxyethylen-sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycols, polyoxyethylene- stearates, polyoxyethylene alkyl ethers, e.g.
• the concentration of surfactant in the pharmaceutical formulation of the disclosure may be comprised between 0.01 and 0.1 % (w/v),.
• buffering agent refers to an agent which provides that the solution comprising it resists changes in pH by the action of its acid/base conjugate components.
• buffering agents that will control the pH in this range incl ude acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers.
• preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and w-cresol.
• a "protectant”, as generally used herein, is a substance which, when combined with a protein, significantly reduces chemical and/or physical instability of the protein upon lyophilization and/or subsequent refrigerated storage.
• exemplary protectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol); and combinations thereof.
• Additional exemplary of protectants include gelatin, dextrins, modified starch, and carboxymethyl cellulose.
• the antioxidant concentration in the pharmaceutical formulation of the disclosure may be comprised between 5 and 25 mM.
• a “chelating agent” is a pharmaceutically acceptable excipient generally used to maintain the stability of proteins.
• examples of chelating agent include edetate disodium, diethylenetriamine penta-acetic acid, citric acid, hexaphosphate, thioglycolic acid, zinc.
• a "bulking agent,” as generally used herein, is a pharmaceutically acceptable excipient generally used to add mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g. facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure).
• exemplary bulking agents include mannitol, glycine, lactose, modified starch, polyethylene glycol), and sorbitol.
• compositions naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient.
• compositions of the disclosure can be formulated for a topical, oral, parenteral, intraperitoneal, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like, preferably intraperitoneal or intravenous.
• the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• an effective amount of the ADC may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the ADC of the disclosure may be formulated within a therapeutic mixture to comprise about 0.0001 to 1 .0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1 .0 or even 1.0 to about 10 milligrams per dose. Multiple doses can also be administered.
• the pharmaceutical formulation comprising the ADC of the disclosure may be a “Ready- to-Use” injectable formulation or a lyophilized formulation.
• ADC of the present disclosure have therapeutic utilities.
• these molecules can be administered in a subject, e.g. in vivo, to treat, or prevent a variety of disorders.
• the ADC of the present disclosure is a medicament, in particular for use in the treatment of cancer in a subject in need thereof, in particular cancer with tumor cells expressing FRa, more specifically, a cancer with a solid tumor, and more specifically selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.
• cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
• cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer.
• the ADC of the disclosure are particularly useful in the treatment of cancer selected from the group consisting of ovarian cancer, triple negative breast cancer, and non-small cells of
• the disclosure relates to a method of treating cancer, in particular one of the above listed cancers, and more preferably cancer selected from the group consisting of ovarian cancer, triple negative breast cancer, and non-small cell lung cancer, said method comprising administering a therapeutically efficient amount of an ADC of formula (I) as disclosed herein.
• the ADC for use as disclosed above may be used in combination with AZT, IFN-alpha, anti-CD20 mAb, anti-CD25 mAb, anti-PD1 mAb, anti-PDL-1 mAb, anti-CTLA4 mAb, chemotherapy agents.
• Suitable anti-tumor agents may include without limitation, alkylating agents (such as cyclophosphamide, mechloretamine, chlorambucil, melphalan, nitrosureas, temozolomide), anthracyclines (such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (such as paclitaxel, docetaxel), epothilones, inhibitors of Topoisomerase I (such as Irinotecan or Topotecan), inhibitors of Topoisomerase II (such as etoposide, teniposide, or tafluposide), nucleotide analogs and precursor analogs (such as azacitidine, azathioprine, capecitabine, cytarabine, flurouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate,
• the present disclosure provides in a yet further aspect a method comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of an ADC of the disclosure, and at least one second drug substance, said second drug substance being anti-viral, anti-inflammatory agents or cytotoxic, anti-proliferative, chemotherapy or other anti-tumor agents, e.g. as indicated above.
• a process for obtaining ADC of Formula (I) comprises the following steps: culturing a host cell under conditions suitable for expression of a nucleic acid encoding the antibody Ab as defined in the previous sections, isolating the antibody, synthesis of exatecan bonded to the Linker L of formula (VIII): conjugating said antibody to the compound of formula (VIII), thereby obtaining an ADC of formula (I).
• Antibodies can be obtained as explained above. Antibodies contain four accessible interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced, for example by tris(2 -carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT), which results in eight thiol groups that are available for conjugation.
• TCEP tris(2 -carboxyethyl) phosphine
• DTT dithiothreitol
• L is a cleavable linker moiety bonded to said antibody, preferably via a thiol residue,
• D is a cytotoxic drug moiety bonded to L, p is from 1 to 8, preferably from 6 to 8, and more preferably p is 8.
• L is a protease sensitive cleavable linker and W comprises a sugar cleavable unit, preferably selected from a p-glucuronide or a p-galactoside moiety.
• W is of the following formula (III) wherein each R2 is independently selected from the group consisting electron-withdrawing groups and C1-C4 alkyl; n is 0, 1 or 2 ;
• T is a sugar cleavable unit or a polypeptide cleavable unit
• Y is O when T is a sugar cleavable unit, or NR3 when T is a polypeptide cleavable unit;
• R3 is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole,
• R” and R’ being independently selected from H and Ci-Ce alkyl, and pharmaceutically acceptable salts thereof.
• L corresponds to a linker -A-W- of formula (IV) wherein
• Xi is a connector unit
• Z is an optional spacer
• X2 is a connector unit
• K is an optional hydrophobicity masking entity, preferably selected from polysarcosine and polyethylene glycol;
• Ri is selected from the group consisting of H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”-, -NR”- C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole;
• R4 is selected from the group consisting H, C-i-Ce alkyl and C2-C6 alkenyl, said alkyl and alkenyl being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, and -NR”-; and
• Xi and X2 are independently selected from the group consisting of one or more amino acid(s), one or more N-substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”- , -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene,
• Z is independently selected from the group consisting of one or more amino acid(s), one or more N-substituted amino acid, optionally substituted polyether, C1-C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C10 alkenylene, and any combination thereof, said alkylene and alkenylene being optionally interrupted by one or more heteroatom or chemical groups selected from -O-, -S-, -C(O)-, -NR”-, -C(O)NR”- , -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole, and said alkylene,
• R’, R” and R’ being independently selected from H and C1-C6 alkyl.
• K is a polysarcosine, preferably of the following formula (V)
• - Ab is an antibody which comprises a human lgG1 isotype constant region, or a mutant or chemically modified constant region, wherein said mutant or chemically modified constant region confers no or decreased ADCC activity to said antibody when compared to a corresponding antibody with wild type human lgG1 isotype constant region; or,
• - Ab is an antibody which comprises a human lgG4 isotype constant region, or a mutant or chemically modified constant region, wherein said mutant or chemically modified constant region confers no or decreased ADCC activity to said antibody when compared to a corresponding antibody with wild type lgG4 isotype constant region.
• L is a cleavable linker of formula -A-W-, wherein A is an optional stretcher unit linked to the Ab, and W is a cleavable moiety linked to D;
• D is an inhibitor of topoisomerase I, e.g., exatecan ;
• an antibody or antigen-binding fragment comprises a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NQ:10.
• a pharmaceutical composition comprising an antibody-drug conjugate according to any one of embodiments 1-21 , in combination with one or more pharmaceutical acceptable excipient, diluent or carrier, optionally comprising other active ingredients.
• a process for obtaining an antibody-drug conjugate according to any one of claims 1-23, wherein the method comprises: culturing a host cell under conditions suitable for production of an anti-FRa antibody as defined in any one of embodiments 1-21 , isolating said anti-FRa antibody, synthesis of exatecan bonded to the linker L of formula (VIII): conjugating said anti-FRa antibody to the compound of formula (VIII), thereby obtaining an ADC as defined in any one of embodiments 1-21 .
• Sandwich ELISA assays are performed using 96-well high-binding ELISA plates (Corning Inc., New York, NY, USA, Cat#3590). Plates are coated using 100 pL/well of recombinant human FRa protein (Sino Biological cat#11241-H08H) in PBS (pH 7.4) at 2 pg/mL and incubated overnight at 4 °C. After 2 washes with PBS-T (PBS + 0.05% Tween-20), the plates are blocked with 200 pL/well of incubation buffer (PBS-T + 0.1 % BSA) for 1 h at room temperature.
• PBS-T PBS + 0.05% Tween-20
• Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300HD or Bruker AVANCE III HD400 spectrometer, using residual solvent peak for calibration. Mass spectroscopy analysis has been performed by the Centre Commun de Spectrometrie de Masse (CCSM) of the UMR5246 CNRS institute of the University Claude Bernard Lyon 1.
• CCSM Centre Commun de Spectrometrie de Masse
• reaction was filtered over a 0.45pm PTFE filter and purified by chromatography on silica gel (petroleum ether/EtOAc, gradient from 85:15 to 30:70) to afford title compound (380 mg 190%) as a yellow foam.
• Dolomanov et aL, Olex2 A complete structure solution, refinement and analysis program, J. AppL Cryst., 2009, 42, 339-341 ) as the graphical interface.
• the model was refined with ShelXL 2018/3 (Sheldrick, G.M., Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71 , 3-8) using full matrix least squares minimisation on F2. 1.2.2.5.2) Synthesis of stereopure INT2-S and INT2-R compounds
• tert-butyl (2-(4-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)phenyl)-2-hydroxyethyl)carbamate was dissolved in 5 mL of DMF/piperidine 9:1 (v/v) and stirred 15 min at room temperature.
• Chiral separation of racemic tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate was performed using Chiralflash® IC MPLC column 30x100mm, 20pm (Daicel cat#83M73) on a Teledyne Isco CombiFlash® Rf200 system.
• Mobile phase was DCM + 0.2% (v/v) EtOH (isocratic gradient). Flow rate was 12 mL/min.
• Sample solvent was DCM + 0.2% (v/v) EtOH.
• tert-butyl (S)-(2-(4-aminophenyl)-2-hydroxyethyl)carbamate retention time was 21 min
• tert-butyl (R)-(2-(4-aminophenyl)-2-hydroxyethyl)carbamate retention time was 29 min.
• Absolute configuration of the enantiomers previously dissolved in a 1 :1 mixture of heptane/ethanol and allowed to slowly evaporate for 1 week to induce the formation of crystals) was confirmed by x-ray crystallography.
• a block-shaped crystal was mounted on a nylon loop in perfluoroether oil.
• Monoclonal antibodies were produced by transient transfection of CHO K1 cells using art-recognized techniques (outsourced to Evitria AG, Switzerland). cDNAs were cloned into Evitria’s vector system using conventional (non-PCR based) cloning techniques. pDNA was prepared under low-endotoxin conditions based on anion exchange chromatography. DNA concentration was determined by measuring the absorption at a wavelength of 260 nm. Correctness of the sequences was verified with Sanger sequencing (up to two sequencing reactions per plasmid). Suspension-adapted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture at Evitria) were used for antibody production.
• the seed was grown in Evitria’s proprietary animal-component and serum-free medium. Cells were transfected with Evitria’s proprietary transfection reagent. Supernatant was harvested by centrifugation and subsequent filtration (0.2pm filter).
• the antibody was purified using MabSelect SuRe protein A purification resin (Cytiva) and SEC preparative resin. Purity of antibody materials was confirmed by SDS-PAGE and size exclusion chromatography and was over 95%. Endotoxin content was measured using the Charles River Endosafe PTS system.
• a solution of antibody (10 mg/mL in PBS 7.4 + 1 mM EDTA) was treated with a required amount (2.2 molar equivalent for final -DAR4 ADC or 14 molar equivalent for final DAR8 ADC) of tris(2-carboxyethyl)phosphine (TCEP) for 1 -2 hours at 37°C.
• the reduced antibody was buffer-exchanged with potassium phosphate 100 mM pH 7.4 + 1 mM EDTA by three rounds of dilution/centrifugation using Amicon 30K centrifugal filters device (Millipore).
• DAR Drug-Antibody-Ratio assessment by reverse phase liquid chromatography-mass spectrometry (RPLC-MS):
• Denaturing RPLC-QToF analysis was performed using the HPLC method 4 described above in Example 1. Briefly, conjugates were eluted on an Agilent PLRP-S 1000A 2.1x150mm 8pm (80°C) using a mobile phase gradient of water/acetonitrile + 0.1 % formic acid (0.4 mL/min) and detected using a Bruker Impact IITM Q-ToF mass spectrometer scanning the 500-3500 m/z range (ESI+). Data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software.
• Denaturing RPLC-UV analysis was also conducted on an Agilent 1100 HPLC-DAD system, using a slightly modified version of HPLC method 4 described above. Mobile phase modifier 0.1 % formic acid was replaced with 0.1 % TFA, and detection was made using a DAD UV absorbance only (no mass spectrometry detector).
• a solution of antibody mirvetuximab or farletuzumab (10 mg/mL in 100mM KH2PO4 pH 8.0) was treated with a 12mM DMSO solution of sulfo-SPDB-DM4 CAS#1626359-59-8 (MedChem Express), reaching a 7.5 molar equivalent final concentration of sulfo-SPDB- DM4.
• the solution was incubated for 3 hours at room temperature and was filtered using a 0.20pm PES filter.
• the final conjugate was buffer-exchanged/purified with either PBS pH 7.4 buffer or with histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% (w/v) sucrose + 75 mM NaCI) by five rounds of dilution/centrifugation using Amicon 30K centrifugal filters device and were sterile-filtered (0.20pm PES filter).
• ADC was deglycosylated by adding 50U of IgGZERO® (Genovis) enzyme for 50pg of ADC.
• ADC was separated on an Agilent AdvanceBio SEC 200A 1.9pm 2.1x150mm PEEK column (cat# PL1980-3201 PK) maintained at 30°C. The column was equilibrated in 50 mM ammonium acetate + 10% (v/v) HPLC grade isopropanol. The flow rate was maintained at 0.075 mL/min during the run, and the ADC was typically eluted between 3.5 and 4.5 min. The flow and buffer composition were maintained following elution of the mAb or ADC.
• the column eluent was directed into a Bruker Impact IITM Q-ToF mass spectrometer scanning the 300-8000 m/z range (ESP).
• the source capillary voltage was set to 4500 V.
• the source drying gas and nebulizing gas were set at 8.0 L/min and 25 Psi, respectively.
• the source drying temperature was set at 200°C.
• ADC mass spectra were deconvoluted using MaxEnt algorithm included in the Bruker Compass® software, and the DAR was calculated using ion intensity peak height of each ADC sub-species.
• LC-Od light chain
• LC-1d light chain with 1 drug-linker
• HC-Od heavy chain
• HC-1d heavy chain with 1 drug-linker
• HC-2d heavy chain with 2 drug-linkers
• HC-3d heavy chain with 3 drug-linkers.
• Major glycoform is reported for HC.
• Human cancer cell lines used in the present project were purchased from either the American Type Culture Collection (ATCC), The Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) or the European Collection of Authenticated Cell Cultures (ECACC).
• ATCC American Type Culture Collection
• DSMZ The Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH
• ECACC European Collection of Authenticated Cell Cultures
• cells were cultivated following good and established cell culture practices, following instructions from the original ATCC/DSMZ/ECACC supplier for cell culture media & supplement, cryopreservation, and subculturing procedures. Cells were incubated under a 5% CO2 atmosphere at 37°C for no longer than 2 months.
• mice therapeutic index of drug-linker constructs LNK1 (glucuronide-exatecan) and LNK3 (dipeptide Val-Ala-Exatecan) were assessed, to compare mice therapeutic index of the two enzyme-cleavable modalities glucuronide and dipeptide Val-Ala.
• T-GLC-EXA ADC and T-VA- EXA ADCs were formulated, using model human HER2 targeting trastuzumab monoclonal antibody as targeting moiety. These ADCs were compared in terms of in vivo efficacy in a HER2+ gastric cancer model and in terms of tolerability (therapeutic index). As trastuzumab does not cross-react in mice, this assay gives useful information about apparent toxicity of the drug-linker component of the ADC (possible target-mediated toxicities are excluded).
• NCI-N87 gastric cancer cells were implanted subcutaneously in female SCID mice (4 weeks old). ADCs were dosed once intravenously at a sub-curative dose of 1 mg/kg when tumors had grown to approximately 150 mm 3 (6 animals per group, assigned to minimize differences in initial tumor volumes between groups). Tumor volume was measured every 3-5 days by a caliper device and was calculated using the formula (L x W 2 )/2. Mice were sacrificed when the tumor volume exceeded 1000 mm 3 .
• PK profile total Antibody-Drug Conjugate concentration based on mAb component over time
• ADCs were injected at 3 mg/kg in female Sprague-Dawley rats (4-6 weeks old, Charles River) via the tail vein (three animals per group, randomly assigned). Blood was drawn into citrate tubes via retro-orbital bleeding at various time points, processed to plasma and stored at -80°C until analysis.
• ADC concentration based on antibody component was assessed using a human IgG ELISA kit (StemcellTM Technologies) according to manufacturer’s protocol. Standard curves of corresponding monoclonal antibody were used for quantification.
• PK parameters (clearance, half-life and AUC) were calculated by two- compartmental analysis using Microsoft® Excel® software incorporating PK functions (add-in developed by Usansky et aL, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA).
• T-GLC-EXA and T-VA-EXA showed similar in vivo efficacy (Figure 1A) and similar rat PK profile (Figure 1 B) but exhibited significant differences in mice tolerability at high doses (Figure 1 C). It was concluded that, at least for a use with the exatecan payload, the dipeptide Val-Ala cleavable modality provided a better efficacy/tolerability profile. As such, this entity was preferred for other ADC constructs.
• FRa cell surface quantification cells were incubated for 20 min at room temperature with PE anti-FOLR1 antibody (BioLegend cat#908304). Cell viability was assessed using a eBioscienceTM Fixable Viability Dye eFluorTM 780 kit (Thermo Fisher Scientific cat#65-0865- 18), following manufacturer’s instructions. Analysis was performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences) and data were analyzed using FlowJo software (BD Bioscience).
• Sandwich ELISA assays were performed using 96-well high-binding ELISA plates (Corning Inc., New York, NY, USA, Cat#3590). Plates were coated using 100 pL/well of recombinant human FRa protein (Sino Biological cat#11241-H08H); recombinant cynomolgus FRa protein (Sino Biological cat#90950-C08H); recombinant rat FRa protein (Sino Biological cat#81073- R08H) or recombinant mouse FRa protein (Sino Biological cat# 50573-M08H) in PBS (pH 7.4) at 2 pg/mL and incubated overnight at 4 °C.
• SPR Surface plasmon resonance
• recombinant human FRa (Sino Biological cat#11241-H08H) analyte sample has been injected in duplicates at 5 concentrations (0.5, 1 , 2, 4, 8 nM) in series using single cycle kinetics strategy and at constant 70 pL/mL flow rate of running buffer (HBS-EP+, Cytiva cat# BR100669) with contact pulses of 300 sec.
• the dissociation phase was measured by injection of running buffer for 900 sec.
• surfaces/flow cells were regenerated with 3M MgCI? to remove both analyte and ligand, and fresh ligand capture were done using identical conditions.
• the data analysis was performed using the Biacore Evaluation software after subtraction of reference surface and analyte zero-concentration signals. Data were processed and fit to a 1 :1 binding model to determine the binding kinetic rate constants, k a (on-rate) and kd (off-rate), and the KD (equilibrium dissociation constant, also referred to as "affinity"). Mean values and standard deviations over replicates are reported.
• Beads were then washed 3 times with HBS-EP buffer and re-suspended in 1 ,2mL of HBS-EP buffer.
• 100 pL of previous bead solution was added onto 100 pL of HBS-EP in a microcentrifuge tube.
• 10 pL of ADC solution in plasma was added and solution was agitated 2 hours at room temperature.
• the eluted samples containing the deglycosylated ADC were then analyzed by denaturing reversed phase chromatography-mass spectrometry using a Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact IITM Q-ToF mass spectrometer.
• Mobile phase A was water + 0.1 % formic acid and mobile phase B was acetonitrile + 0.1 % formic acid.
• Column was an Agilent PEEK PLRP-S 1000A 2.1x100mm 5pm (80°C). Linear gradient was 20%B to 50%B in 25 min. Flow rate was 0.4 mL/min. UV detection was monitored at 280 nm.
• mice treated with F-LALA-VA-EXA (12mg/kg IV once) were re-implanted with IGROV-1 cells using the same procedure that was used at the beginning of the study (other flank for the animals).
• a control group of 5 new SCID animals were also re-implanted following the exact same procedure.
• FIG 11 is presented a tumor xenograft experiment in the OV-90 cancer model.
• F-VA- EXA, F-LALA-VA-EXA and M-SORAV conjugates were injected IV once at 30 mg/kg. All conjugates were highly active, with 100% remission rate in all groups. However, significant toxicities were observed with the M-SORAV conjugate: 6 out of 6 mice showed unkempt appearances (dull and matted haircoat), 3 out of 6 mice presented signs of prostration and 2 out of 6 mice had diarrhea. No toxicities were observed with F-VA-EXA and F-LALA-VA-EXA conjugates. These data suggest better preclinical therapeutic window for the F-VA-EXA and F-LALA-VA-EXA constructs compared to M-SORAV.
• Total mAb concentration was assessed by ELISA, using a goat polyclonal anti-human IgG (H+L) primary antibody (Jackson Immunoresearch) as capture reagent and a mouse polyclonal anti-human IgG (H+L) HRP conjugate (Jackson Immunoresearch) as secondary detection antibody.
• Total ADC concentration was assessed by ELISA, using a rabbit polyclonal anti-exatecan antibody (ref#6294/00000920, custom-made at Biotem, Apprieu, France) as capture reagent, and a mouse polyclonal anti-human IgG (H+L) HRP conjugate (Jackson Immunoresearch) as secondary detection antibody. Standard curves of ADC were used for quantification.
• Free exatecan concentration was assessed using a LC/MS-MS method, using an Agilent 1100 HPLC system and a Sciex API 4000 MS/MS system.
• Rat plasma samples were protein- precipitated using an organic solution composed of 80:20 (v/v) acetonitrile/methanol + 1 % formic acid + d5-exatecan (20 ng/mL) as internal standard.
• Samples were analyzed using gradient elution mode onto a Phenomenex Kinetex® C8 2.1x30mm 2.6pm 100A column maintained at 45°C (Phenomenex cat# 00D-4497-AN).
• a positive control group was included, treated with clinically approved conjugate Enhertu® (10 mg/kg i.p.), which is known to induce interstitial lung diseases and pneumonitis preclinically and in a small percentage of patients.
• Enhertu® 10 mg/kg i.p.
• broncho-alveolar supernatant was obtained (by broncho-alveolar lavage) and lungs organs were harvested, weighted, and fixed in 0.1 % (w/v) formaldehyde in PBS. After fixation, lungs were rinsed with a 0.02% (w/v) sodium azide in PBS solution and embedded in paraffin for histological staining.
• Inflammatory cytokines levels in broncho-alveolar supernatants obtained from non-bleomycin-conditioned mice were quantified by ELISA.
• IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 were quantified using Bio-Plex ProTM Mouse Cytokine Th2 Panel (Bio-Rad cat# L60000UKVT), following manufacturer’s instructions.
• TGF-U and IL-17 were quantified using Mouse TGF-beta 1 DuoSet and Mouse IL-17 DuoSet ELISA kits respectively (R&D systems cat# DY1679 and DY421 ), following manufacturer’s instructions.
• IHC CD45 quantification results are shown in Figure 20.
• Positive control Enhertu® ADC caused a significant increase in leucocyte inflammatory infiltration when compared to both untreated (***) and bleomycin-conditioned (****) negative control groups.
• F-VA-EXA ADC caused no significant increase in leucocyte inflammatory infiltration when compared to untreated group and caused a significant increase when compared to bleomycin-conditioned (***) control group.
• F-LALA-VA-EXA caused no significant increase in leucocyte inflammatory infiltration when compared to both untreated and bleomycin-conditioned negative control groups. Based on these preclinical results it can be hypothesized that F-VA-EXA and especially F-LALA-VA- EXA (Fc-silent variant) will induce less or no interstitial lung diseases or pneumonitis in a clinical setting.
• Example 16 dose-range-finding toxicology study in cynomolgus monkeys
• a nonhuman primate dose-range-finding toxicology study was conducted using purpose-bred native female cynomolgus monkeys (Macaca fascicularis) of Vietnam origin. The study was performed at Cynbiose SAS (Marcy-l'Etoile, France). The study protocol was approved by the testing facility Institutional Animal Care and Use Committee. F-LALA-VA-EXA ADC was intravenously administered (5mL/kg/h, over a 30 min injection period) at a three-week interval (total of 3 doses) at dose levels of 30, 40, 50 or 60 mg/kg. Two female monkeys/group were used in the study (total of 8 animals).

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