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WO2025230410A1 - Multispecific antibody-drug conjugates - Google Patents

Multispecific antibody-drug conjugates

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Publication number
WO2025230410A1
WO2025230410A1 PCT/NL2025/050198 NL2025050198W WO2025230410A1 WO 2025230410 A1 WO2025230410 A1 WO 2025230410A1 NL 2025050198 W NL2025050198 W NL 2025050198W WO 2025230410 A1 WO2025230410 A1 WO 2025230410A1
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WO
WIPO (PCT)
Prior art keywords
antibody
amino acid
heavy chain
seq
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/NL2025/050198
Other languages
French (fr)
Inventor
Cornelis Adriaan De Kruif
Peter Richard LOWE
Giulio RUSSO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merus BV
Original Assignee
Merus BV
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Filing date
Publication date
Application filed by Merus BV filed Critical Merus BV
Publication of WO2025230410A1 publication Critical patent/WO2025230410A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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
    • A61K47/6835—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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/526—CH3 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/77—Internalization into the cell
    • 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

  • ADCs antibody-drug conjugates
  • ADCs BACKGROUND Antibody-drug conjugates
  • ADCs are complex therapeutic moieties comprising three components: an antibody, a linker, and a cytotoxic, radiotherapeutic, or immunomodulatory agent.
  • ADCs combine the specificity of antibodies and the cytotoxic potency of small molecules, and are increasingly demonstrating benefits for cancer patients.
  • the specificity of antibodies preferentially targets a cytotoxic agent to the tumor microenvironment, thereby reducing cytotoxicity in non-tumor tissues.
  • the linker couples a cytotoxic agent to the antibody and is generally designed such that the cytotoxic agent is not released in the blood but within the tumor microenvironment.
  • the cytotoxic agent induces tumor cell killing by, for example, targeting DNA, microtubules, or topoisomerase 1.
  • the antigen-ADC complex may be internalized into the tumor cell.
  • a non-cleavable linker is used, lysosomal degradation of the antibody takes place and releases the cytotoxic agent in the cell where it can exert its cytotoxic effect.
  • Multispecific antibodies are engineered proteins that can simultaneously bind to two or more different targets (e.g., antigens or to two or more different epitopes of an antigen).
  • targets e.g., antigens or to two or more different epitopes of an antigen.
  • the multispecificity of multispecific antibodies can be used to specifically target cells in the tumor microenvironment as opposed to cells in healthy tissues, and to redirect immune cells to tumor cells.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue.
  • the present disclosure provides an antibody-drug conjugate of the format as described herein comprising a variable domain that binds to EGFR and a variable domain that binds to LGR5.
  • the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody-drug conjugate as described herein. In certain embodiments, the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in therapy. In certain embodiments, the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer. In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof.
  • the present disclosure provides a method for treating cancer, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof.
  • the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody.
  • One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, in particular for the treatment of cancer.
  • This object is met by the provision of a novel antibody-drug conjugate (ADC) format, and ADCs comprising this format.
  • ADC antibody-drug conjugate
  • Bispecific antibodies comprising this format and suitable for use in an antibody-drug conjugate of the present disclosure are for instance described in WO2013/157953 and WO 2013/157954, incorporated herein by reference in their entirety.
  • all CH3 domains and heavy chains comprising such CH3 domains disclosed therein are incorporated herein by reference.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the second CH3 domain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue. Such substitution is made with respect to the wildtype sequence of the CH3 domain.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue.
  • the CH3 domain is a human IgG CH3 domain.
  • the CH3 domain is a human IgG1 CH3 domain.
  • Amino acid residues can be charged or neutral.
  • Neutral amino acid residues are amino acid residues that do not carry electrically charged side chains.
  • Neutral amino acid residues include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), cysteine (Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W). Amino acid residues carrying positively charged side chains, i.e.
  • the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitution T366K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K.
  • the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D. In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E.
  • the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D.
  • the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E.
  • the first CH3 domain or CH3 domain of the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 12 and the second CH3 domain or the CH3 domain of the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 11.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the second CH3 domain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the CH3 domain of the second heavy chain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system.
  • the first CH3 domain or the CH3 domain of the first heavy chain comprises a lysine (K) or an arginine (R) residue at position 364 and the second CH3 domain or the CH3 domain of the second heavy chain comprises an aspartic acid (D) or a glutamine (E) residue at position 368.
  • Multispecific multimer formats suitable for use in an ADC of the present disclosure are for instance described in WO 2019/190327, incorporated herein by reference in its entirety.
  • Other technologies suitable for use in an antibody-drug conjugate of the present disclosure are described in Ha et. al, 2016; Front. Immunol.; Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins; Volume 7, incorporated herein by reference in its entirety.
  • the antibody of the antibody-drug conjugate is a multispecific antibody, in particular a bispecific or trispecific antibody.
  • the multispecific antibody may also be a quadrispecific antibody.
  • a multispecific antibody according to the present disclosure is an antibody that comprises at least two binding domains which have specificity for at least two different targets or epitopes.
  • a bispecific antibody according to the present disclosure is an antibody that comprises at least two binding domains which have specificity for two different targets or epitopes.
  • a trispecific antibody according to the present disclosure is an antibody that comprises at least three binding domains which have specificity for three different targets or epitopes.
  • a quadrispecific antibody according to the present disclosure is an antibody that comprises at least four binding domains which have specificity for four different targets or epitopes.
  • a multispecific antibody according of the present disclosure may be a biparatopic or triparatopic antibody.
  • an “antibody” refers to a proteinaceous molecule and includes for instance all antibody formats available in the art, such as for example a full length IgG, IgA, or IgE antibody, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibody (like VHH and VH), minibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)’2, F(ab)’2-scFv2, and intrabodies.
  • a multispecific antibody of the present disclosure may comprise an Fc region or a part thereof.
  • a multispecific antibody of the present disclosure is an IgG1 antibody.
  • Constant regions of an antibody of the present disclosure may comprise one or more variations that modulate properties of the antibody other than its binding properties to the target antigens.
  • the constant regions may comprise one or more additional variations that favor heterodimerization of the two different heavy chains over homodimerization of each heavy chain, and/or the constant regions may comprise one or more variations that reduce or improve effector function, preferably one or more variations that reduce effector function.
  • An antibody of an antibody-drug conjugate of the present disclosure may comprise a heavy chain comprising a CH3 region as described in WO 2021/235936, incorporated herein by reference in its entirety.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a first heavy chain comprising a CH3 domain comprising amino acid substitutions T366K and L351K and a second heavy chain comprising a CH3 domain comprising amino acid substitution L351D, L368E and an amino acid variation at position S364, K409 and/or K360.
  • the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine, and/or the amino acid at position 409 is isoleucine, leucine or glutamate, and/or the amino acid at position 360 is aspartate.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises amino acid variations in the Fc region that reduce or eliminate effector function. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises at least one amino acid substitution at position 235 and/or 236. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises amino acid substitution L235G and/or G236R, in particular L235G and G236R. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH2 region comprising the amino acid sequence as set forth in SEQ ID NO: 9.
  • An antibody of an antibody-drug conjugate of the present disclosure may comprise a heavy chain comprising a modified constant region, in particular a modified CH1, CH2, or CH3 region, as described in WO 2020/226502, incorporated herein by reference in its entirety.
  • a modified CH1, CH2, and CH3 are modified as compared to the wildtype sequences as set forth in SEQ ID Nos: 7, 8, and 10, respectively.
  • an antibody of an antibody- drug conjugate of the present disclosure may comprise a CH1, CH2, and/or CH3 region comprising one or more variations of an amino acid that is non-surface exposed, wherein the variation is selected from a neutral amino acid to a negatively charged amino acid; a positively charged amino acid to a neutral amino acid; a positively charged amino acid to a negatively charged amino acid; a neutral amino acid to a positively charged amino acid; a negatively charged amino acid to a neutral amino acid; and a negatively charged amino acid to a positively charged amino acid.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from N159, N201, T120, K147, D148, Y149, V154, A172, Q175, S190, and K213.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from D148, Y149, V154, N159, A172, S190, and N201.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from N159 and/or N201.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising variations of amino acids selected from the group A172/S190/N201, T197/K213, D148/Q175, N159/Q213, K147/Q175, Y149/V154/A172/S190, N201/K213, T120/N201, N201/N159, T120/N159, T120/N201/N159 and N201/K213/N159.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH2 region comprising a variation of amino acid V303.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a CH3 region comprising a variation of an amino acid selected from K370, E382 and E388, in particular E388.
  • An antibody of an antibody-drug conjugate of the present disclosure may comprise any suitable light chain, including any suitable common light chain.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a light chain comprising a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 1.
  • the light chain comprises a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 5.
  • a “Fab” typically means a binding domain comprising a heavy chain variable region, a light chain variable region, a CH1 and a CL region.
  • an “Fc region” typically comprises a hinge, CH2, and CH3 region.
  • an antibody of an antibody-drug conjugate of the present disclosure comprises a hinge region having the amino acid sequence as set forth in SEQ ID NO: 6.
  • the present disclosure provides an antibody-drug conjugate comprising a variable domain that binds to EGFR and a variable domain that binds to LGR5.
  • antigen binding can be expressed in terms of specificity and affinity. The specificity determines which antigen or epitope thereof is specifically bound by a variable domain, antibody, or antibody-drug conjugate.
  • the affinity is a measure for the strength of binding to a particular antigen or epitope.
  • variable domain, antibody, or antibody-conjugate is considered to bind an antigen when it has an at least two times higher binding signal than the background signal in the same assay.
  • the variable domain of the antibody drug conjugate that binds to EGFR comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO:13, 17, 21, or 25.
  • each of the HCDRs may comprise at most three, two, or one amino acid variations.
  • only one or two HCDRs may comprise at most three, two, or one non- conservative amino acid variations.
  • HCDR3 does not comprise any amino acid variations.
  • the amino acid variation is a conservative amino acid substitution.
  • a conservative amino acid substitution involves a variation of an amino acid with a homologous amino acid residue, which is a residue that shares similar characteristics or properties.
  • homologous amino acids are known in the art, as are routine methods for making amino acid substitutions in antibody binding domains without significantly impacting binding or function of the antibody, see for instance handbooks like Lehninger (Nelson, David L., and Michael M. Cox.2017. Lehninger Principles of Biochemistry. 7th ed. New York, NY: W.H. Freeman) or Stryer (Berg, J., Tymoczko, J., Stryer, L.
  • an assessment may typically be made of factors such as, but not limited to, (a) the structure of the polypeptide backbone in the area of the substitution, for example, a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and/or (c) the bulk of the side chain(s). If a residue can be substituted with a residue which has common characteristics, such as a similar side chain or similar charge or hydrophobicity, then such a residue is preferred as a substitute.
  • the following groups can be determined: (1) non-polar: Ala (A), Gly (G), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).
  • amino acids may be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) apolar: Leu (L), Val (V), Ile (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gln (Q), Asn (N), Ser (S), Thr (T), Tyr (Y).
  • amino acid residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), Ile (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg R); (5) residues that influence chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F).
  • the substitution of an amino acid residue with another present in the same group would be preferred.
  • conservative amino acid substitution can involve exchanging a member of one of these classes for another member of that same class.
  • the variation results in no, or substantially no, loss in binding specificity of the binding domain to its intended target.
  • Additional types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation.
  • Binding variants encompassed by the present disclosure include somatically hypermutated or affinity matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene segments as the heavy chain variable regions described by sequence herein, the variants having amino acid variations, including non-conservative and/or conservative amino acid substitutions in one, two, or all three HCDRs.
  • variable domain of the antibody drug conjugate that binds to EGFR comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (
  • each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.
  • the variable domain of the antibody drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61.
  • each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.
  • variable domain of the antibody drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and
  • each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.
  • the variable domain of the antibody-drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequences as set forth in SEQ ID NO: 1. In certain embodiments each of the LCDRs may comprise at most three, two, or one amino acid variations.
  • the variable domain of the antibody drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising: light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.
  • each of the HCDRs may comprise at most three, two, or one amino acid variations.
  • only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations.
  • HCDR3 does not comprise any amino acid variations.
  • the amino acid variation is a conservative amino acid substitution.
  • the antibody-drug conjugate comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 18, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 54, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 55, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 56; and wherein the variable domain of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 2, LCDR2 having an amino acid sequence as set forth in SEQ ID NO:
  • variable domain of the antibody-drug conjugate that binds to EGFR comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 13, 17, 21, or 25, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
  • Percent (%) identity as referring to nucleic acid or amino acid sequences herein is defined as the percentage of residues in a candidate sequence that are identical with the residues in a selected sequence, after aligning the sequences for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared.
  • the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
  • the alignment may also be carried out over individual CDR sequences.
  • the sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
  • a comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp.
  • the percent sequence identity between two amino acid sequences or nucleic acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol.48, 443-453).
  • the Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE.
  • the NEEDLE program from the EMBOSS package is used to determine percent identity of amino acid and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A.
  • variable domain of the antibody-drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
  • variable domain of the antibody-drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising the amino acid sequences as set forth in SEQ ID NO: 1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
  • the antibody-drug conjugate comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
  • variable domain of an antibody-drug conjugate of the present disclosure also comprises variable domain variants, which, in addition to the variations in the HCDRs and/or LCDRs referred to above, comprise one or more variations in the framework regions.
  • a variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation.
  • a variable domain variant of an antibody-drug conjugate of the present disclosure comprises no variations in the CDR regions but comprises one or more variations in the framework regions.
  • the antibody-drug conjugate of the present disclosure comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1.
  • the antibody-drug conjugate of the present disclosure comprises: - a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 65; - a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 66; and - two light chains comprising the amino acid sequence set forth in SEQ ID NO: 67.
  • the present disclosure provides an antibody-drug conjugate that competes with an antibody-drug conjugate as described herein for binding to EGFR and/or LGR5.
  • Compet refers to an activity of an antibody-drug conjugate that blocks or displaces an antibody-drug conjugate as described herein from its target antigen(s), in a cross-blocking assay. Therefore, in certain embodiments, an antibody-drug conjugate that competes for binding with an antibody-drug conjugate as described herein, binds to EGFR or LGR5 and blocks or displaces the antibody- drug conjugate as described herein, in a cross-blocking assay.
  • an antibody-drug conjugate that competes for binding with an antibody-drug conjugate as described herein binds to EGFR and LGR5 and blocks or displaces an antibody-drug conjugate as described herein, in a cross-blocking assay.
  • a cross-blocking assay is a competitive ELISA. Methods of performing a competitive ELISA are known to a person of ordinary skill in the art. In brief, in a competitive ELISA, antigen is immobilized on the wells of a microtiter plate and pre-incubated with or without the competing antibody-drug conjugate. This is followed by addition of a biotin-labeled antibody-drug conjugate as described herein.
  • the amount of labeled antibody-drug conjugate bound to the antigen in the wells is measured using avidin-peroxidase conjugate and appropriate substrate.
  • the amount of labeled antibody-drug conjugate that is bound to the antigen has an indirect correlation to the ability of the competing antibody-drug conjugate to compete for binding to the same antigen, i.e., the greater the affinity of the competing antibody-drug conjugate for the same antigen, the less labeled antibody-drug conjugate will be bound to the antigen-coated wells.
  • a candidate competing antibody-drug conjugate is considered to compete for binding to the antigen, if the candidate antibody-drug conjugate can block binding of the antibody-drug conjugate of the present disclosure, to the target antigen, by at least 20%, or by at least 20-50%, or by at least 50%, as compared to the control performed in parallel in the absence of the candidate competing antibody-drug conjugate.
  • Drug-linker combinations the antibody-drug conjugate of the present disclosure comprises a drug which is coupled to the antibody of the antibody-drug conjugate via a linker.
  • linker-drug combination is herein referred to as linker-drug combination.
  • the drug of the linker-drug combination is a cytotoxic or cytostatic drug.
  • Suitable examples of drugs include, but are not limited to, the following: dolastatin-10, auristatin molecules, maytansine, maytansinoids, camptothecin, exatecan, SN-38, IRDye700DX, anthramycin, pyrrolobenzodiazepine (PBD) dimers, calicheamicin, and calicheamicin analogs.
  • Other drugs known to a skilled person may also be used, such as for instance any chemotherapeutic agent, an amanitin, or a microcystin.
  • Auristatin molecules interfere with tubulin polymerization and exert a potent effect by disrupting mitotic spindle formation, resulting in a mitotic block leading to cell death.
  • Auristatin molecules include, but are not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and any derivatives thereof.
  • MMAE is a synthetic antineoplastic agent derived from dolastatin-10, a marine pentapeptide isolated from the Indian Ocean mollusk Dolabella auricularia. Dolastatin-10 is reported to induce apoptosis of lung cancer cells and other tumor cells, and has been developed into commercial drugs for treating lymphomas.
  • MMAE is a potent antimitotic drug and can be conjugated to a multispecific antibody through a protease cleavable peptide linker.
  • vedotin refers to a linker containing a maleimide-caproyl (MC) moiety conjugated to the primary amine of a valine-citrulline (VC) linker linked to a p-aminobenzyloxycarbamoyl (PABC) spacer, and which is linked to a monomethylated amine of MMAE.
  • MC valine-citrulline
  • PABC p-aminobenzyloxycarbamoyl
  • MMAF is also is a synthetic antineoplastic agent derived from dolastatin-10.
  • MMAF can be conjugated to a multispecific antibody through an MC linker. This linker-drug combination is referred to as mafodotin.
  • Maytansine is a highly potent cytotoxin that kills cells by binding to ⁇ -tubulin, preventing microtubules from forming in cells undergoing mitosis during cell division, resulting in cell cycle arrest and eventual cell death.
  • Maytansine can be engineered to introduce a methylene-thiol (-CH2-SH) moiety.
  • -CH2-SH methylene-thiol
  • the combination of maytansine with the methylene-thiol (- CH2-SH) moiety is referred to as mertansine (or DM1), which is a maytansinoid.
  • Maytansine and maytansinoids can be conjugated to a multispecific antibody by using a succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.
  • SMCC succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate
  • This linker-drug combination is referred to as emtansine.
  • Another example of a maytansinoid linker-drug combination is ravtansine (or soravtansine or DM4).
  • Exatecan,Dxd, and DX-8951f are synthetic derivatives of camptothecin, a natural cytotoxin binding to the topoisomerase 1-DNA complex, preventing DNA re-ligation which results in cell cycle arrest and the accumulation of DNA strand breaks and ultimately leads to cell death.
  • Exatecan can be conjugated to a multispecific antibody by a glycine-glycine- phenylalanine-glycine (GGFG) linker.
  • Exatecan can also be conjugated to a multispecific antibody by using a linker comprising an maleimide-caproyl spacer and the GGFG linker, which linker drug combination is referred to as deruxtecan.
  • SN-38 (or 7-ethyl-10-hydroxycamptothecin), is also a synthetic derivative of camptothecin.
  • SN-38 can be conjugated to a multispecific antibody via a CL2A linker.
  • the linker-drug combination is referred to as govitecan.
  • SG3199 (or SCX) is a synthetic dimer analog of the natural pyrrolobenzodiazepine (PBD) cytotoxin, anthramycin.
  • PBD pyrrolobenzodiazepine
  • Anthramycin exerts its cytotoxic mechanism by binding covalently to guanine in the DNA minor groove, inhibiting nucleic acid synthesis and cell division.
  • SG3199 can be conjugated to a multispecific antibody via a maleimide propoyl (MP) moiety conjugated to eight polyethylene glycol (PEG8) monomers linked to a cathepsin B-cleavable valine-alanine (VA) dipeptide with a C-terminal PABC spacer.
  • MP maleimide propoyl
  • PEG8 polyethylene glycol
  • VA valine-alanine
  • This linker-drug combination is referred to as tesirine (or SG3249).
  • Calicheamicin analog which is based on the highly potent cytotoxin calicheamicin, and kills cells by binding to their DNA minor groove and subsequently causing DNA double-strand scission.
  • Calicheamicin analogs can also be conjugated to a multispecific antibody by a hydrazone linker comprising a 4-(4’- acetylphenoxy) butanoic acid moiety, which linker-drug combination is referred to as ozogamicin.
  • the drug of the linker-drug combination is a tubulin inhibitor, a deoxynucleic acid (DNA) damaging agent, a chemotherapeutic agent, a protein degrader, or an immune stimulant.
  • the drug of the linker-drug combination is a tubulin inhibitor that is an auristatin or derivative thereof, a tubulysin or derivative thereof, or a maytansine or derivative thereof.
  • the drug of the linker-drug combination is a DNA damaging agent which is a DNA double-strand breaking agent, a DNA alkylation agent, a DNA intercalator, a DNA cross linker, a topoisomerase I inhibitor, or a topoisomerase II inhibitor.
  • the drug of the linker-drug combination is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), PNU-159682, PE38, IRDye700, proteolysis- targeting chimera (PROTAC), alpha-amanitin
  • the drug of the linker-drug combination is selected from the group consisting of MMAE, MMAF, maytansine, exatecan, SN-38, SCX, and calicheamicin, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is selected from the group consisting of MMAE, MMAF, maytansine, exatecan, SN-38, and SCX, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is MMAE or MMAF, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is MMAE, or a derivative or analog thereof.
  • the drug of an antibody-drug conjugate of the present disclosure can be conjugated to a multispecific antibody by any suitable linker.
  • Linkers known and available in the industry are capable of use in the present disclosure of ADCs.
  • Linkers can be generally divided into two categories: cleavable (such as peptide, hydrazone, or disulfide) or non-cleavable (such as thioether or alkyl or alkoxy).
  • Peptide linkers such as Valine-Citrulline (Val-Cit), that can be hydrolyzed by lysosomal enzymes (such as Cathepsin B) have been used to connect a drug with an antibody.
  • linker can be, in some instances, particularly useful for their relative stability in systemic circulation and the ability to efficiently release the drug in tumor.
  • Suitable linkers include, but are not limited to, those further described herein.
  • the linker may comprise one or more linker components.
  • linker components include 6-maleimido-caproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or“vc”), alanine-phenylalanine (“ala-phe”), p- aminobenzyloxycarbonyl (a“PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”).
  • MC 6-maleimido-caproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • alanine-phenylalanine ala-phe
  • PAB p- aminobenzyloxycarbonyl
  • SPP N-Succinimidyl 4-(2-pyridylthio) pentanoate
  • MCC 4-(N-maleimidomethyl
  • the linker L1 or a fragments thereof comprises MC (6-maleimidocaproyl), MCC (a maleimido-methyl cyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxy-carbonyl), SPP (N- Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-Succinimidyl 4-(N-maleimidomethyl)- cyclohexane-1 carboxylate), SIAB (N-Succinimidyl (4-iodo-acetyl)aminobenzoate.
  • MCC maleimido-methyl cyclohexane-1-carboxylate
  • MP maleimidopropanoyl
  • linkers or fragments thereof include: BS3 ([Bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodimide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([N-e- Maleimidocaproic acid]hydrazide; sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS-ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carbonyl groups of the exposed carbohydrates to primary amines), and SATA (N-succinimidyl- S-acetylthioacetate; SATA (N
  • a chemically reactive group such as a wide variety of active carboxyl groups (e.g., esters), can react with a functional group such as an amine, a thiol or a hydroxyl moiety on the surface of the peptide, where the amine, thiol or hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide.
  • a functional group such as an amine, a thiol or a hydroxyl moiety on the surface of the peptide, where the amine, thiol or hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide.
  • Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide-benzoyl- succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MPA) maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA).
  • NHS N-hydroxysuccinimide
  • MVS gamma-maleimido-butyryloxy succinimide ester
  • MHA maleimido propionic acid
  • MHA maleimido hexanoic acid
  • MUA maleimido undecanoic acid
  • Primary amines are the principal targets for NHS esters. Accessible ⁇ -amino groups present on the N-termini of proteins and the ⁇ -amine of lysine react with NHS esters.
  • an amide bond is formed when the NHS ester conjugation reaction reacts with primary amines releasing N-hydroxysuccinimide.
  • succinimide containing reactive groups are herein referred to as succinimidyl groups.
  • the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide-butrylamide (GMBA or MPA).
  • GMBA or MPA gamma-maleimide-butrylamide
  • maleimide containing groups are referred to herein as maleido groups.
  • the maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is 6.5-7.4.
  • the rate of reaction of maleimido groups with sulfhydryls is 1000-fold faster than with amines.
  • sulfhydryls e.g., thiol groups on proteins such as serum albumin or IgG
  • the linker is attached to the antibody via a lysine residue on the antibody.
  • the linker comprises a MC (6-maleimidocaproyl), a MCC (a maleimidomethyl cyclohexane-1-carboxylate), a MP (maleimidopropanoyl), a val-cit (valine- citrulline), a val-ala (valine-alanine), an ala-phe (alanine-phenylalanine), a PAB (p- aminobenzyloxycarbonyl), a SPP (N-Succinimidyl 4-(2-pyridylthio) pentanoate), 2,5- dioxopyrrolidin-1-yl 4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4- (pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)hex
  • said linker is derived from a cross-linking reagent, wherein the cross-linking reagent comprises N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), 2,5-dioxopyrrolidin-1-yl 3-cyclopropyl-3-(pyridin-2- yldisulfaneyl)propanoate, 2,5-dioxopyrrolidin-1-yl 3-cyclobutyl-3-(pyridin-2- yldisulfaneyl)propanoate, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), 2,5- dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-yldisulfaneyl)butanoate, 2,5-dioxopyrrolidin-1- yl 4-cyclobutyl-4
  • the linker may comprise optionally a substituted (poly)ethylene glycol having from 1 to about 100 ethylene glycol units, from about 1 to about 50 ethylene glycol units, from 1 to about 25 ethylene glycol units, from about 1 to about 10 ethylene glycol units, from 1 to about 8 ethylene glycol units and from 1 to about 6 ethylene glycol units, from 2 to 4 ethylene glycol units, or optionally substituted alkyl groups interdispersed with optionally substituted, O, N, S, P or Si atoms.
  • the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocycle group.
  • the linker may be asymmetric or symmetrical.
  • the linker may be a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, between 1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycol units, between about 2 and 5 ethylene glycol units, between about 2 and 4 ethylene glycol units.
  • a linker or formation of the linker can comprise moieties that can be used in a click conjugation, e.g., in a two-step conjugation in which a first moiety is conjugated to a native or an engineered cysteine or lysine, the first moiety containing a reactive handle, and a second moiety containing the linker-drug component which reacts with the first moiety.
  • An example of a possible reaction between the first moiety’s reactive handle and the second moiety is a metal free click reaction that utilizes strain-promoted azide-alkyne cycloaddition.
  • moieties include, but are not limited to, bicyclononyne (BCN) reacting with an azide or tetrazine, dibenzocyclooctyne (DBCO) reacting with an azide, also denoted as aza-dibenzocyclooctyne (DIBAC), a transcyclooctene (TCO) reacting with a tetrazine (such as methyl tetrazine), or a methyl cycloprene click handle reacting with tetrazine.
  • BCN bicyclononyne
  • DBCO dibenzocyclooctyne
  • DIBAC aza-dibenzocyclooctyne
  • TCO transcyclooctene
  • moieties are as follows, but not limited to: dibenzylcyclooctyne-PegX- carboxylic acid, perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate Chemical Formula: C16H12F5NO4 Molecular Weight: 377.27; 6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanoic acid Chemical Formula: C10H11Br2NO4 Molecular Weight: 369.01; (2- methylcycloprop-2-en-1-yl)methyl carbamate (E)-cyclooct-4-en-1-yl (2-(2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate 3-(5-methylpyridin-2-yl)-6-(pyridin-2-yl)-1,2,
  • the linker is a non-cleavable linker. In certain embodiments, the non-cleavable linker is a covalent linker. In certain embodiments, the non-cleavable linker is attached to the N-terminus, C-terminus or an internal amino acid position of the antibody or an antigen-binding fragment thereof. In certain embodiments, the non-cleavable linker is covalent attached to the drug.
  • the linker is connected with the antibody via a reaction using a wherein LG is halide, triflate, fluorosulfonate, tosylate, mesylate, or besylate;
  • R A is independently hydrogen, C 1 -C 6 alkyl, cycloalkyl-alkylene, C 1 -C 6 haloalkyl, heteroalkyl- alkylene, heteroaryl-alkylene, heterocycloalkyl-alkylene, aryl-alkylene, heteroaryl-alkylene, alkylamino-alkylene, alkylthio-alkylene, alkylcarbonyl, alkoxycarbonyl, or alkylsulfonyl;
  • R B is independently a protecting group, C1-C6 alkyl, cycloalkyl-alkylene, C1-C6 haloalkyl, heteroalkyl-alkylene, heteroaryl-alkylene, heterocycloalkyl-alkylene,
  • the spacer is , wherein m is independently an integer of 0-3, q is independently an integer of 0-12, and r is independently an integer of 1-3.
  • the spacer is , wherein: each Y1 and Y2 is independently a bond, O, S, or NR E ; R E is independently H, deuterium, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, C 1 -C 6 alkyl; C 6 -C 12 aryl, 5-12 membered heteroaryl, C 3 -C 12 cycloalkyl or 3-12 membered heteroalicyclic, or R E together with the nitrogen to which they are bound and another atom of the spacer, be combined to form a 3 to 12 membered heteroalicyclic or 5-12 membered heteroaryl group optionally containing 1 to 3 additional heteroatoms selected from the group consisting of N, O, and S; and m is independently an integer of 0-3, q is independently
  • the linker is a branched linker to connect multiple drug moieties to the antibody.
  • the branched linker comprises , wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e and d is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, f is independently 1, 2, 3, 4, 5, 6, 7 or 8, with the proviso that at least one of e and d is not 0.
  • the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, govitecan, ozogamicin, saratolacan, and soravtansine.
  • the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, and govitecan.
  • the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, and tesirine. In certain embodiments, the antibody-drug conjugate comprises a linker-drug combination selected from vedotin and mafodotin. In certain embodiments, the antibody-drug conjugate comprises linker-drug combination vedotin.
  • Pharmaceutical compositions and methods of use In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier.
  • the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody.
  • the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in therapy, in particular for use in the treatment of cancer.
  • the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody.
  • the present disclosure provides a method for treating a disease, in particular cancer, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof.
  • the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody.
  • the cancer is selected from the group consisting of: colorectal cancer, in particular metastatic colorectal cancer (mCRC), pancreatic cancer, lung cancer, in particular lung squamous cell carcinoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, in particular squamous cell carcinoma of the head and neck (HNSCC), melanoma, testicular cancer, urothelial cancer, renal cancer, stomach cancer, carcinoid cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, in particular esophageal squamous cell carcinoma (ESCC), gastric-esophageal junction cancer, pharyngeal cancer, in particular cancer of the nasopharynx, oropharynx, and hypopharynx, laryngeal cancer, in particular
  • mCRC meta
  • the cancer is an adenocarcinoma.
  • the method or therapy comprises first line treatment or therapy.
  • the method or therapy comprises second line treatment or therapy, in particular following treatment with an immune checkpoint inhibitor.
  • the method or therapy is a monotherapy, only administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, to a subject.
  • the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with an immune checkpoint inhibitor to a subject.
  • the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with a chemotherapy or radiotherapy to a subject.
  • the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with a topoisomerase I inhibitor to a subject.
  • the topoisomerase is irinotecan.
  • the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with fluorouracil, folinic acid, and irinotecan (FOLFIRI) or folinic acid, fluorouracil, and oxaliplatin (FOLFOX).
  • the method or therapy is a monotherapy for the treatment of locally advanced unresectable or metastatic HNSCC, gastric/gastroesophageal junction adenocarcinoma (GEA) with EGFR amplification and/or high EGFR expression, esophageal carcinoma or pancreatic adenocarcinoma.
  • GAA gastric/gastroesophageal junction adenocarcinoma
  • the method or therapy is a second or further line monotherapy for the treatment of HNSCC.
  • the method or therapy is a first line combination therapy with pembrolizumab for the treatment of HNSCC.
  • the method or therapy is a second line combination therapy with chemotherapy for the treatment of metastatic colorectal cancer.
  • the chemotherapy is 5-Fluorouracil, Leucovorin, and Irinotecan (FOLFIRI), or Fluorouracil, Leucovorin, and Oxaliplatin (FOLFOX).
  • the terms “individual”, “subject” and “patient” are used interchangeably and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like, and in particular to a human subject having cancer.
  • the terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on or administering an active agent or combination of active agents to a subject with the objective of curing or improving a disease or symptom thereof or which produces a positive therapeutic response.
  • positive therapeutic response refers to a treatment producing a beneficial effect, e.g.
  • a beneficial effect can take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to initiation of therapy according to the method.
  • a beneficial effect can take the form of slowing, stabilizing, stopping or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, or of a marker of cancer.
  • Effective treatment may, for example, decrease in tumor size, decrease in the presence of circulating tumor cells, reduce or prevent metastases of a tumor, slow or arrest tumor growth and/or prevent or delay tumor recurrence or relapse.
  • therapeutic amount or “effective amount” refers to an amount of an agent or combination of agents that treats a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor development.
  • a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence.
  • an effective amount of the agent or composition is one that, for example, may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • An effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual to be treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can be readily evaluated by the ordinarily skilled physician or other health care worker.
  • An effective amount can be administered to a subject in one or more administrations.
  • An effective amount can also include an amount that balances any toxic or detrimental effects of the agent or combination of agents and the beneficial effects.
  • agent refers to a therapeutically active substance, in the present case an antibody-drug conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.
  • the method for producing an antibody-drug conjugate of the present disclosure is based on hinge and CH1/ CL reduced interchain disulfide bonds conjugation or stochastic lysine conjugation.
  • the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first CH3 domain comprising at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and a second CH3 domain comprising at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody.
  • the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody.
  • an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitution T366K and wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, an antibody is provided wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D.
  • an antibody is provided wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D.
  • an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E.
  • an antibody is provided with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the second CH3 domain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system.
  • an antibody is provided with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the CH3 domain of the second heavy chain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system.
  • an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises a lysine (K) or an arginine (R) residue at position 364 and the second CH3 domain or the CH3 domain of the second heavy chain comprises an aspartic acid (D) or a glutamine (E) residue at position 368.
  • the antibody that is provided is a multispecific antibody, in particular a bispecific or trispecific antibody.
  • the drug of the linker-drug combination is a drug as described herein.
  • the linker-drug combination is a linker-drug combination as described herein.
  • Conjugation sites The linker-drug combination can be conjugated the antibody in any suitable manner. Suitable methods are known to a person skilled in the art and include, but are not limited, to those described herein. Examples of suitable methods involve use of an amino acid present in the antibody, or introduced in the antibody by antibody engineering, that form the conjugation site for coupling a linker-drug combination to the antibody. In certain embodiments, the conjugation site on the antibody is an amino acid.
  • the conjugation site on the antibody is a side chain on an amino acid. In certain embodiments, the conjugation site is a cysteine or lysine residue on the antibody. In certain embodiments, the conjugation site is a reactive group (azide or alkyne) for a click chemistry reaction. In certain embodiments, there are multiple conjugation sites on an antibody for linking multiple of a particular linker-drug combination to the antibody, or the antibody can be engineered to have multiple conjugation sites. In this way, the drug antibody ratio (DAR) can be controlled, e.g. by increasing or decreasing the DAR. In certain embodiments, the conjugation site is an unnatural amino acid.
  • DAR drug antibody ratio
  • the antibody or an antigen binding fragment thereof comprises an unnatural amino acid, and the antibody or antibody fragment and the drug are linked/conjugated via the unnatural amino acid.
  • an unnatural amino acid may be inserted between two naturally occurring amino acids in the antibody or antibody fragment.
  • the unnatural amino acid may replace one or more naturally occurring amino acids in the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated at the N terminus of the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated at the C terminus of the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated distal to the binding region of antibody or antibody fragment.
  • the unnatural amino acid may be incorporated near the binding region of the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated in the binding region of the antibody or antibody fragment.
  • the unnatural amino acid may be p-acetylphenylalanine (pAcF or pAcPhe).
  • the unnatural amino acid may be selenocysteine.
  • the unnatural amino acid may be p- fluorophenylalanine (pFPhe).
  • the unnatural amino acids may be selected from the group consisting of p-azidophenylalanine (pAzF),p-azidomethylphenylalanine(pAzCH2F), p- benzoylphenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF), p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (pCmF), 3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF), o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3- yl)alanine (HQA), selenocysteine, and (2,2'-bipyridin-5-yl)alanine (BipyA).
  • pAzF p-
  • the unnatural amino acids may be 4-(6-methyl-s-tetrazin-3-yl)aminopheynlalanine.
  • the unnatural amino acid may comprise at least one oxime, carbonyl, dicarbonyl, hydroxylamine group or a combination thereof.
  • the one or more unnatural amino acids may comprise at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl/alkyl azide, norbornene, cyclopropene, trans- cyclooctene, or tetrazine functional group or a combination thereof.
  • the unnatural amino acid may be incorporated in a light chain of the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated in a heavy chain of the antibody or antibody fragment.
  • the unnatural amino acid may be incorporated in a heavy chain and a light chain of antibody or antibody fragment.
  • the unnatural amino acid may replace an amino acid in the light chain of the antibody or antibody fragment.
  • the unnatural amino acid may replace an amino acid in a heavy chain of the antibody or antibody fragment.
  • the unnatural amino acid may replace an amino acid in a heavy chain and a light chain of the antibody or antibody fragment.
  • the unnatural amino acid is p-acetylphenylalanine, whose ketone group on the side chain of incorporated unnatural amino acid react with a hydroxylamine functionalized drug to form an oxime bond.
  • the articles “a” and “an” are used herein to refer to one or more of the grammatical object of the article.
  • an element means one or more elements.
  • CDRs and framework regions of antibodies have been described and defined in the art using a number of different systems, including for instance Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991); Kabat et al., J. Biol. Chem.252:6609-6616 (1977)), IMGT (discussed in Giudicelli et al., Nucleic Acids Res.25: 206-2111997), Chothia (Chothia and Lesk J. Mol. Biol.196: 901 -917, 1987; Chothia et al., Nature 342: 877-883, 1989; Al-Lazikani et al., J. Mol. Biol.
  • Honnegher and Plukthun Honnegher and Plukthun, J. Mol. Biol.309: 657- 670, 2001
  • MacCallum MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)
  • Lefranc Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003).
  • the present disclosure encompasses defining the CDRs in accordance with each numbering system available to a skilled person.
  • the present disclosure encompasses defining the CDRs in accordance with the numbering systems of Kabat, IMGT, and Chothia.
  • the HCDRs according to Kabat are provided in the listing of sequences as provided herein.
  • the HCDRs according to IMGT are indicated in bold and underlined in the heavy chain variable region sequences as provided in the listing of sequences as provided herein.
  • the LCDRs according to IMGT are provided in the listing of sequences as provided herein.
  • the LCDRs according to Kabat are indicated in bold and underlined in the light chain variable region sequences as provided in the listing of sequences as provided herein. Amino acids in the constant regions are indicated according to the EU numbering system. Accession numbers are primarily given to provide a further method of identification of a target, the actual sequence of the protein bound may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like.
  • An antigen binding site of an antibody of the disclosure can bind the antigen and a variety of variants thereof, such as those expressed by some antigen positive immune or tumor cells.
  • HGNC stands for the HUGO Gene nomenclature committee.
  • accession number with which information on the gene and protein encoded by the gene can be retrieved from the HGNC database.
  • Entrez Gene provides the accession number or gene ID with which information on the gene or protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database.
  • Ensembl provides the accession number with which information on the gene or protein encoded by the gene can be obtained from the Ensembl database.
  • Ensembl is a joint project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes.
  • the reference is preferably to the human form of the gene or protein.
  • Figure 2 The stability of EGFRxc-MET ADC, EGFRxLGR5 ADC, and trastuzumab ADC (1 mg/ml) in human serum as analyzed at 0, 24, 48 and 96 hours at 37°C by LC-MS, indicated by percentage of conjugated DAR.
  • Figure 3 Freeze-thaw stability of EGFRxc-MET ADC, EGFRxLGR5 ADC, and trastuzumab ADC (2 mg/ml) at -80°C for a minimum of 4 hours as analyzed by HP-SEC, indicated by percentage monomer. Time point 0 (T0) and multiple freeze thaw cycles (FT) are shown.
  • Figure 4 The affinity of EGFRxc-MET ADC and EGFRxLGR5 ADC to soluble c- MET, EGFR, and LGR5 as evaluated in single cycle kinetic analysis using Biacore.
  • the response (response units (RU)) over time (seconds) is indicated in sensorgrams.
  • Figure 5 The binding of soluble EGFRxLGR5 ADC to human Fc ⁇ receptors Fc ⁇ RI (Fig.5A) and Fc ⁇ RIIIA (176 Phe) (Fig, 5B) as evaluated using Biacore.
  • the response (response units (RU)) over time (seconds) is indicated in sensorgrams.
  • Figure 6 The steady state affinity binding of soluble EGFRxLGR5 ADC to FcRn at pH 6.0 (Fig.6A) and pH 7.4 (Fig.6B) as evaluated using Biacore multicycle analysis.
  • the response (response units (RU)) over time is indicated in sensorgrams. Arrowheads in the graphs are indicating the reference timepoint used to derive the binding KD from the steady-state analysis.
  • Figure 7 The binding of EGFRxc-MET ADC (Fig. 7A), EGFRxLGR5 ADC (Fig.7B) and trastuzumab ADC (Fig. 7C), and control antibodies to the C1q component of complement as evaluated by ELISA.
  • Binding at different concentrations is expressed as absorbance read at 450 nm using a SpectraMax M3 plate reader.
  • MFI mean fluorescence intensity
  • bispecific antibody targeting EGFR and c-MET comprises heavy chain variable regions comprising the amino acid sequence as set forth in SEQ ID NO: 17 (EGFR binding domain) and SEQ ID NO: 53 (LGR5 binding domain).
  • Both the EGFR and c-MET binding domains comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1.
  • the heavy chains comprise a CH1 having an amino acid sequence as set forth in SEQ ID NO: 7, a hinge having an amino acid sequence as set forth in SEQ ID NO: 6, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a CH3 region.
  • One of the CH3 regions has an amino acid sequence as set forth in SEQ ID NO: 11 and the other CH3 region has an amino acid sequence as set forth in SEQ ID NO: 12.
  • Both bispecific antibodies are ADCC enhanced. Both bispecific antibodies comprise one heavy chain with a CH3 domain comprising amino acid substitutions T366K and L315K and one heavy chain with a CH3 domain comprising amino acid substitutions L351D and L368E.
  • the aim of this study was to establish conjugation of a therapeutic agent to antibodies comprising the DEKK substitutions, while maintaining sufficient stability, affinity, and exhibiting functional activity.
  • Both bispecific antibodies were coupled with monomethyl auristatin E (MMAE) through a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (VC) linker.
  • MMAE monomethyl auristatin E
  • VC maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl
  • ADCs antibody-drug conjugates
  • trastuzumab-MC-VCP-MMAE a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl linker
  • trastuzumab-MC-VCP-MMAE a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl linker
  • trastuzumab-MC-VCP-MMAE a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl linker
  • EGFRxc-MET bispecific antibody at 11.08 mg/ml in Dulbecco’s PBS, pH 7.5, 5mM EDTA was diluted to 5.06 mg/mL with Dulbecco’s PBS, pH 7.5, 5mM EDTA.
  • a 5 mM solution of TCEP in Dulbecco’s PBS, pH 7.5, 5mM EDTA was added to the diluted antibody solutions. The reduction was allowed to proceed at 40 °C for 1 hour with a final antibody concentration of 5 mg/ml. After 1 hour at 40 °C, the reduction mixture was diluted with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool down to 22 °C.
  • Fractions containing the conjugate were concentrated and buffer-exchanged by ultrafiltration/diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa (EGFRxLGR5) or 50 kDa (EGFRxc-MET) MWCO) equilibrated with Dulbecco’s PBS, pH 7.2.
  • the concentrated samples were sterile filtered through a 0.22 ⁇ m pore size, PVDF membrane filter.
  • Trastuzumab (Roche, lot number N3031H02) at 11.0 mg/ml in Dulbecco’s PBS, pH 7.5, 5mM EDTA was diluted to 5.41 mg/ml with Dulbecco’s PBS, pH 7.5, 5mM EDTA.
  • a 1 mM solution of TCEP in Dulbecco’s PBS, pH 7.5, 5 mM EDTA was added to the diluted antibody solution. The reduction was allowed to proceed at 40 °C for 1 hour with a final antibody concentration of 5 mg/ml.
  • the reduction mixture was diluted with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool down to 22 °C.
  • a portion of the MC- VCP-MMAE linker-payload solution in DMSO was added to the reduced trastuzumab solution resulting in a final concentration of 10% DMSO and a final antibody concentration of 4.0 mg/ml.
  • the conjugation reaction was allowed to proceed at 22 °C for 1 hour.
  • reaction mixture was purified by gel filtration using a Centripure P100 column and further buffer- exchanged by ultrafiltration/diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO) equilibrated with Dulbecco’s PBS, pH 7.2.
  • PES membrane 30 kDa MWCO
  • the concentrated sample was sterile filtered through a 0.22 ⁇ m pore size, PVDF membrane filter.
  • EGFRxLGR5 MC-VCP-MMAE, EGFRxc-MET MC-VCP MMAE, and trastuzumab MC-VCP-MMAE conjugates were characterized by hydrophobic interaction chromatography (HIC), Size-exclusion chromatography (SEC), liquid chromatography–mass spectrometry (LC- MS) and quantified by UV.
  • HIC hydrophobic interaction chromatography
  • SEC Size-exclusion chromatography
  • LC- MS liquid chromatography–mass spectrometry
  • Table 1 shows that the conjugation of MMAE to both bispecific antibodies was successful.
  • EGFRxLGR5 MC-VCP-MMAE and EGFRxc-MET MC-VCP MMAE conjugation resulted in a DAR distribution of between DAR0- DAR8, as determined by HIC and shown in Figure 11.
  • FIG.11A shows the DAR distribution of EGFRxc-MET ADC: 16% of the EGFRxc-MET ADCs in the sample have DAR0, 43.1% have DAR2, 13.8% have DAR4, 22.8% have DAR6, and 4.4% have DAR8.
  • FIG.11B shows the DAR distribution of EGFRxLGR5 ADC: 9.8% of the EGFRxLGR5 ADCs in the sample have DAR0, 22.7% have DAR2, 32.1% have DAR4, 23.9% have DAR6, and 11.7% have DAR8.
  • ADC Average Average % MW [ADC] by Amount Yield DAR DAR monomer (LC-MS) UV (mg) (%) (HIC) (LC-MS) (SEC) (mg/ml) EGFRxc- 3.3 - 93.4 - 3.26 9.1 27 MET ADC EGFRxLGR5 4.1 3.9 97.8 Confirmed 7.12 25 76 ADC (24.6% LMW species) Trastuzumab 4.4 4.3 98.7 Confirmed 3.88 29 80 ADC Table 1. Conjugation of MMAE to bispecific antibodies and trastuzumab. LMW low molecular weight.
  • the antibodies can also be coupled with other drugs, such as for instance MMAF, maytansine, DM1 (mertansine), exatecan, DXd, SN-38, or SG3199 (SCX) or other linker-drug combinations, such as for instance mafodotin, emtansine, deruxtecan, tesirine (SG3249), or govitecan, using methods known in the art, such as for instance those described in the handbook Antibody-Drug Conjugates, Methods and Protocols.
  • drugs such as for instance MMAF, maytansine, DM1 (mertansine), exatecan, DXd, SN-38, or SG3199 (SCX) or other linker-drug combinations, such as for instance mafodotin, emtansine, deruxtecan, tesirine (SG3249), or govitecan, using methods known in the art, such as for instance those described in the handbook Antibody-Drug Conju
  • the Tm (melting temperature) and Tagg (aggregation temperature) of the samples were analyzed running software version 6.0.
  • the T m is defined as the temperature at which half of the protein molecules in solution are unfolded. It is identified as a peak (or peaks) on the first derivative and calculated from a fluorescence curve with the Uncle ⁇ software.
  • the Tagg is defined as the point at which the SLS (Static Light Scattering) signal reaches 10% of the maximum value and is calculated by the Uncle ⁇ software. Formulations that are more stable have higher T m and T agg values. Samples were analyzed in triplicate.
  • the ADCs were spiked in human sera at 1 mg/ml in sterile tubes. All samples were split in 4 aliquots of equal size and transferred to a 37°C incubator. The aliquots were removed from the 37°C incubator at 0, 24, 48 and 96 hours timepoints and transferred at -80°C until they were analyzed.
  • the ADCs were immunocaptured from human sera (0-, 24-, 48- and 96-hours aliquots) using an anti-Human IgG (Fc or CH1 specific) biotin antibody immobilized on streptavidin beads.
  • Capture was performed by mixing 50 ⁇ l of ADC- spiked serum with 50 ⁇ l PBS and 50 ⁇ l beads and incubated for 1 hour at room temperature with shaking (at 700 rpm using Eppendorf thermomixer). After incubation, the supernatant was removed, and the beads were washed three times with 500 ⁇ l PBS. Elution was performed with 100 ⁇ l 2mM HCl, three times (5 min incubation, gentle mix), and the aliquots were pooled. The eluate pH was neutralized with 0.5M Ammonium Bicarbonate (pH 8.0).
  • the ADCs were analyzed by LC-MS using a Waters I Class UPLC in line with a Waters RDa Mass Detector. Chromatographic separation was performed using an Acquity UPLC Protein BEH SEC, 200 ⁇ , 1.7 ⁇ m, 4.6x300mm column (Waters) maintained at RT. Samples were eluted under isocratic conditions in 30% acetonitrile and 0.1% formic and the flow was set at 0.3 ml/min. A volume of 10 ⁇ l was injected per run. The MS spectra were acquired in the 400-7000 m/z range.
  • EXAMPLE 4 Freeze-thaw stability
  • the ADCs were frozen to -80 °C for a minimum of 4 hours and thawed at room temperature before analysis.
  • the ADCs were analyzed after 1, 3, and 5 freeze thaw cycles, and stability was assessed by HP-SEC.
  • HP-SEC analysis was performed using a Thermo Ultimate 3000RS UPLC system (ThermoFisher Scientific, Hemel Hempstead, UK).
  • the column used was a Waters UPLC Protein BEH SEC Column 200 ⁇ , 1.7 ⁇ m, 4.6 x 150 mm (part number 186005225) with a guard column, Waters Acquity UPLC Protein BEH SEC Guard Column, 200 ⁇ , 1.7 ⁇ m, 4.6 mm X 30 mm (part number 186005793).
  • the mobile phase consisted of 200 mM potassium phosphate, 200 mM potassium chloride, 15% (v/v) IPA, pH 6.8.
  • the method was isocratic, with a duration of 10 minutes, ambient column temperature, and a flow rate of 0.35 mL/minute. UV detection was carried out at 280 nm.
  • Single cycle kinetic data was obtained using human c-MET (His tag, Sino Biological, #10692-H08H), EGFR (His tag, Acro Biosystems, #LG5-H52H3), and LGR5 (His tag, Acro Biosystems, #LG5-H52H3) as the analyte injected at a flow rate of 30 ⁇ l/min to minimize any potential mass transfer effects.
  • c-MET His tag, Sino Biological, #10692-H08H
  • EGFR His tag, Acro Biosystems, #LG5-H52H3
  • LGR5 His tag, Acro Biosystems, #LG5-H52H3
  • association phases were monitored for 210 seconds for each of the four injections of increasing concentrations of antigen and a single dissociation phase was measured for 600 seconds following the last injection of antigen.
  • Regeneration of the sensor chip surface was conducted using 3M MgCl2.
  • the signal from the reference channel Fc1 (no IgG captured) was subtracted from that of Fc2, Fc3 and Fc4, to correct for bulk effect and differences in non-specific binding to a reference surface.
  • the signal from each IgG blank run (IgG captured but no antigen) was subtracted to correct for differences in surface stability.
  • the double referenced sensorgrams were fitted with the Langmuir (1:1) binding model where the closeness of fit of the data to the model is evaluated using the Chi square value which describes the deviation between the experimental and fitted (observed and expected) curves.
  • the fitting algorithm seeks to minimize the Chi square value.
  • the data shown in Figure 4 shows that the affinity to each target was unaffected by drug conjugation.
  • EXAMPLE 6 Characterization of binding to human Fc gamma receptors and FcRn
  • human Fc ⁇ RI hCD64, His tag, Sino Biological, Beijing, China, # 10256-H08H
  • human Fc ⁇ RIIIA hCD16A, 176 Phe, His tag, Sino Biological, Beijing, China, # 10389-H08H
  • His-tagged Fc gamma receptors (ligand) diluted in HBS-P + (Cytiva, Uppsala, Sweden) were loaded to a specific RU level onto a CM5 sensor chip pre-coupled to HIS-capture antibody via amine chemistry (His capture kit, Cytiva, Uppsala, Sweden).
  • His capture kit Cytiva, Uppsala, Sweden
  • a five point, three- fold dilution range (0.4111 to 33.33 nM) of antibodies (analyte) without regeneration between each concentration was used for Fc ⁇ RI.
  • the signal from the reference channel Fc1 (blank) was subtracted from that of the Fc loaded with receptor to correct for differences in non-specific binding to the reference surface.
  • the data shown in Figure 6 shows that binding to FcRn was unaffected by drug conjugation at pH 6.0.
  • the EGFRxLGR5 ADCs do not impact the pH sensitive binding of FcRn at pH 6.0 and are expected to allow recycling if internalized into cells, as the absence of binding at pH 7.4 is as normal.
  • EXAMPLE 7 Characterization of binding to C1q
  • Unconjugated parental antibodies and trastuzumab (IgG1 WT antibody) were included as controls.
  • Rituximab human IgG1, 10 mg/ml, Celltrion, #17C2C17DC1
  • IgG4 S241P L248E, 8.4 mg/ml, Abzena, hinge stabilized and Fc silenced
  • Rituximab was used at a starting concentration of 20 ⁇ g/ml and a seven-point, 2.5-fold serial dilution (20 – 0.0819 ⁇ g/mL) was performed. All samples were tested in duplicate. ELISA plates were then incubated at 4 °C overnight. The plates were then blocked using 1x PBS pH 7.4 + 2% (w/v) BSA and incubated for 1 hour at 25°C. After blocking, purified human C1q (Eurobio Scientific, Dorking, Surrey) at 5 ⁇ g/ml was added to the ELISA plates for binding and incubated for 2 hours at 25°C.
  • ELISA plates were washed and the anti-C1q-HRP diluted 1:200 in blocking buffer was added to the ELISA plates for detection and incubated for 1 hour at 25°C. After washing, TMB Microwell Peroxidase substrate (Life Technologies, #002023) was added to the ELISA plates to detect binding. The reaction was stopped with 3NHCl solution (VWR, Lutterworth, Leicestershire, #30018.298) after 5 minutes. Absorbance was read at 450 nm using a SpectraMax M3 plate reader (Molecular Devices, Wokingham, UK). GraphPad Prism 9.0 (GraphPad Software, La Jolla, Ca) was used for data analysis and data were fitted using a four parameters non-linear regression.
  • the ADC and cetuximab (Selleck Chemicals; Product code: A2000; Batch code: A200005) as a reference control antibody were prepared at 10 ⁇ g/ml starting concentration and titrated in a 7-point, 3-fold serial dilution. Cells were re-suspended with either titrated ADC, reference control antibody or flow cytometry buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4 °C.
  • the cells were resuspended in flow cytometry buffer only (unstained) or anti-Human IgG ( ⁇ -chain specific), F(ab′)2 fragment ⁇ R-Phycoerythrin secondary antibody (Sigma-Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After 60 minutes incubation at 4 °C, the cells were washed in flow cytometry buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK). Fluorescence was measured using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific, Loughborough, UK), and data was analyzed using FlowJo v10 (FlowJo, Ashland, OR).
  • Cells were recovered from liquid nitrogen storage and prepared at 1 x 10 6 cells/ml in flow cytometry buffer (PBS (Thermo Fisher, Loughborough, UK) + 1% BSA (Sigma- Aldrich, Poole, UK) + 0.1% Sodium Azide (Sigma-Aldrich, Poole, UK)), and seeded at 100 ⁇ l/well into a low-bind U-bottom 96-well plate (VWR, Lutterworth, UK).
  • the ADC and cetuximab reference antibody were prepared at 10 ⁇ g/ml starting concentration, and subjected to a 10-point, 3-fold serial dilution.
  • Cells were re-suspended with either titrated ADC, reference control antibody buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4°C. After washing with buffer, the cells were resuspended in buffer only (unstained) or anti- Human IgG ( ⁇ -chain specific), F(ab′)2 fragment ⁇ R-Phycoerythrin secondary antibody (Sigma- Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After incubation for 60 minutes at 4°C, the cells were washed in buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK).
  • NCI-1975 (WT) ATCC; cat. no. CRL-5908; lot no.70010182
  • c-MET-KO NCI-1975 c-MET knock-out
  • Cells were re-suspended with either titrated ADC, reference control antibody or flow cytometry buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4 °C. After washing with flow cytometry buffer, the cells were resuspended in flow cytometry buffer only (unstained) or anti-Human IgG ( ⁇ -chain specific), F(ab′)2 fragment ⁇ R-Phycoerythrin secondary antibody (Sigma-Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After 60 minutes incubation at 4 °C, the cells were washed in flow cytometry buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK).
  • EXAMPLE 9 Cytotoxicity The ability of the EGFRxLGR5 ADC to induce killing of A549 target cells was evaluated by a cell viability assay. Cell killing was analyzed by measuring cell viability with CellTiter- Glo ® Luminescent cell viability assay (Promega, #G7573). Cetuximab was used as reference control. A549 WT (ATCC, cat. no. CCL-185) and A549 EGFR KO cells were seeded into 96- well plates at a density of 2 x 10 3 cells per well in complete medium (50 ⁇ l). Plates were then incubated for 24 hours at 37 °C/5% CO2.
  • samples were prepared in complete medium with 8-point 2.5-fold serial dilutions starting at 50 nM and added to the cells in duplicate (25 ⁇ l).
  • Assay plates were analyzed after 120 hours and viability assessment was undertaken utilizing the CellTiter-Glo® luminescence assay. Briefly, assay plates were equilibrated at room temperature for 20 minutes before addition of CellTiter-Glo® reagent (100 ⁇ L per well, Sartorius, #4633). The plates were then shaken on an orbital mixer for 3 minutes at 300 rpm to assist cell lysis and incubated for a further 20 minutes at room temperature to stabilize the signal.
  • Luminescence was recorded using a SpectraMax i3x plate reader (Molecular Devices, Wokingham, UK). Data was then analyzed on GraphPad Prism version 10 (GraphPad Software, La Jolla, CA) using a four-parameter non-linear regression model. Viability of treated cultures was compared to viability of control, untreated cells (100% viable) and expressed as percentage. The percentage viability was plotted against the drug concentration in nM.
  • the results in Figure 9A show that the EGFRxLGR5 ADC induced a dose-dependent reduction in cell viability of WT cells compared to EGFR-KO cells. Cetuximab did not induce cell death of WT or EGFR KO cells.
  • the ability of the EGFRxc-MET ADC to induce killing of A549 target cells was evaluated by a cell viability assay.
  • Cell killing was analyzed by measuring cell viability with CellTiter-Glo ® Luminescent cell viability assay.
  • A549 WT ATCC, cat. no. CCL-185, A549 c- Met KO and A549 EGFR KO cells were seeded into 96-well plates at a density of 2 x 10 3 cells per well in complete medium (50 ⁇ L) and treated as described earlier for EGFRxLGR5 ADC example.
  • ADC and cetuximab control antibody were prepared in complete medium with 8-point 4-fold serial dilutions starting at 66 nM and added to the cells in duplicate.
  • NCI-H1975 WT ATCC; cat. no. CRL-5908; lot no.70010182
  • NCI- H1975 c-Met KO cells were seeded into 96-well plates at a density of 2 x 10 3 cells per well in complete medium (50 ⁇ L). Plates were then incubated for 24 hours at 37 °C/5% CO 2 .
  • the EGFRxc-MET ADC showed selective cytotoxicity at clinically relevant antibody concentrations in this assay. Cetuximab did not induce cell death of WT or c-MET-KO cells.
  • EXAMPLE 10 Internalization The ability of the EGFRxc-MET ADC to induce internalization was evaluated by assessing the level of internalization on A549 WT (ATCC, catalogue nr. CCL-185), A549 c- MET-KO, and A549 EGFR-KO cells. To determine ADC internalization capacity in target cells, cells were monitored live for 24 hours, using Incucyte® live cell imaging. First the cells were seeded onto 96 well plates and incubated overnight, with a media change 1 hour prior to imaging.
  • Antibodies were labelled with FabFluor-pH®, which are Fab fragments directed against a Fc region with a pH sensitive dye attached (Bevan et al., 2018), as per manufacturer’s instructions. Antibodies were labelled with FabFluor-pH® reagent at 3:1 Fabfluor-pH®: antibody molar ratio for 15 minutes at 37°C in complete media. A baseline fluorescence scan was taken 15 minutes before commencing the internalization assay. Once the baseline scan was complete, the labelled antibodies were added to the cells at a final concentration of 10 ⁇ g/ml and 1 ⁇ g/ml. Four hours of scanning took place at approximately 30-minute intervals, followed by a further 20 hours scanning at 1-hour intervals.
  • the internalization of the EGFRxc-MET ADC in A549 WT cells is higher than in EGFR-KO cells.
  • the results in Figure 10B show internalization of EGFRxc-MET ADC in NCI-H1975 WT cells but not in c-MET KO cells.
  • the data show that EGFRxc-MET ADC has comparable A549 WT binding to that of cetuximab, but showed relative higher internalization.
  • the EGFRxc-MET ADC showed relative lower binding than cetuximab on c-MET KO cells, but showed relative higher internalization.
  • exemplary bispecific antibodies comprising the DEKK mutations conjugated to a drug can be produced with clinically relevant DAR, and show preferential binding and activity against dual target expressing cells versus single target expressing cells.
  • the exemplary bispecific ADCs preserve the target binding selectivity, internalization, and cytotoxicity of the naked, unconjugated bispecific antibodies.
  • the exemplary bispecific ADCs preserve the target binding selectivity, internalization, and cytotoxicity of the naked, unconjugated bispecific antibodies.

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Abstract

The present disclosure relates to the field of antibody-drug conjugates (ADCs). In particular it relates to the field of therapeutic antibodies for the treatment of disease, in particular for the treatment of cancer. More particularly it relates to multispecific antibody-drug conjugates.

Description

Title: MULTISPECIFIC ANTIBODY-DRUG CONJUGATES TECHNICAL FIELD The present disclosure relates to the field of antibody-drug conjugates (ADCs). In particular it relates to the field of therapeutic antibodies for the treatment of disease, in particular for the treatment of cancer. More particularly it relates to multispecific antibody-drug conjugates. BACKGROUND Antibody-drug conjugates (ADCs) are complex therapeutic moieties comprising three components: an antibody, a linker, and a cytotoxic, radiotherapeutic, or immunomodulatory agent. ADCs combine the specificity of antibodies and the cytotoxic potency of small molecules, and are increasingly demonstrating benefits for cancer patients. In principle, the specificity of antibodies preferentially targets a cytotoxic agent to the tumor microenvironment, thereby reducing cytotoxicity in non-tumor tissues. The linker couples a cytotoxic agent to the antibody and is generally designed such that the cytotoxic agent is not released in the blood but within the tumor microenvironment. The cytotoxic agent induces tumor cell killing by, for example, targeting DNA, microtubules, or topoisomerase 1. Upon binding to an antigen, the antigen-ADC complex may be internalized into the tumor cell. When a non-cleavable linker is used, lysosomal degradation of the antibody takes place and releases the cytotoxic agent in the cell where it can exert its cytotoxic effect. In case a cleavable linker is used, lysosomal enzymes like cathepsin-L break a specific site in the linker, thereby releasing the drug. Drug release can also be pH dependent. Despite more specific targeting to the tumor microenvironment than chemotherapy, current ADCs are still subject to considerable toxicity, inadequate selectivity, internalization and potency. Multispecific antibodies are engineered proteins that can simultaneously bind to two or more different targets (e.g., antigens or to two or more different epitopes of an antigen). The multispecificity of multispecific antibodies can be used to specifically target cells in the tumor microenvironment as opposed to cells in healthy tissues, and to redirect immune cells to tumor cells. Different technologies for producing multispecific antibodies have been developed over the years, including heterodimerization technology making use of amino acid variations in the CH3. One such technology is described in WO 2013/157953 and WO 2013/157954. This technology is also referred to as the DEKK technology, and is used for producing Biclonics® antibodies, whereas the technology encompasses additional variants and forms of heterodimerization that may also be suitable for CH3 engineering. There remains a need for ADCs that are efficacious in treating cancer with less undesired side-effects, having potential for greater specificity, and enhanced potency than current approaches. SUMMARY One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, in particular for the treatment of cancer. This object is met by the provision of a novel antibody-drug conjugate (ADC) format, and ADCs comprising this format. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue. In certain embodiments, the present disclosure provides an antibody-drug conjugate of the format as described herein comprising a variable domain that binds to EGFR and a variable domain that binds to LGR5. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody-drug conjugate as described herein. In certain embodiments, the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in therapy. In certain embodiments, the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer. In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof. In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof. In certain embodiments, the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody. DETAILED DESCRIPTION One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, in particular for the treatment of cancer. This object is met by the provision of a novel antibody-drug conjugate (ADC) format, and ADCs comprising this format. Bispecific antibodies comprising this format and suitable for use in an antibody-drug conjugate of the present disclosure are for instance described in WO2013/157953 and WO 2013/157954, incorporated herein by reference in their entirety. In particular, all CH3 domains and heavy chains comprising such CH3 domains disclosed therein are incorporated herein by reference. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the second CH3 domain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue. Such substitution is made with respect to the wildtype sequence of the CH3 domain. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue. In certain embodiments, the CH3 domain is a human IgG CH3 domain. In certain embodiments, the CH3 domain is a human IgG1 CH3 domain. Amino acid residues can be charged or neutral. Neutral amino acid residues are amino acid residues that do not carry electrically charged side chains. Neutral amino acid residues include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), cysteine (Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W). Amino acid residues carrying positively charged side chains, i.e. positively charged amino residues, include arginine (Arg, R), histidine (His, H), and lysine (Lys, K). Amino acid residues carrying negatively charged side chains, i.e. negatively charged amino residues, include aspartic acid (Asp, D) and glutamic acid (Glu, E). In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitution T366K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K. In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D. In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, the first CH3 domain or CH3 domain of the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 12 and the second CH3 domain or the CH3 domain of the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 11. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the second CH3 domain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the CH3 domain of the second heavy chain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system. In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises a lysine (K) or an arginine (R) residue at position 364 and the second CH3 domain or the CH3 domain of the second heavy chain comprises an aspartic acid (D) or a glutamine (E) residue at position 368. Multispecific multimer formats suitable for use in an ADC of the present disclosure are for instance described in WO 2019/190327, incorporated herein by reference in its entirety. Other technologies suitable for use in an antibody-drug conjugate of the present disclosure are described in Ha et. al, 2016; Front. Immunol.; Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins; Volume 7, incorporated herein by reference in its entirety. In certain embodiments, the antibody of the antibody-drug conjugate is a multispecific antibody, in particular a bispecific or trispecific antibody. The multispecific antibody may also be a quadrispecific antibody. A multispecific antibody according to the present disclosure is an antibody that comprises at least two binding domains which have specificity for at least two different targets or epitopes. A bispecific antibody according to the present disclosure is an antibody that comprises at least two binding domains which have specificity for two different targets or epitopes. A trispecific antibody according to the present disclosure is an antibody that comprises at least three binding domains which have specificity for three different targets or epitopes. A quadrispecific antibody according to the present disclosure is an antibody that comprises at least four binding domains which have specificity for four different targets or epitopes. A multispecific antibody according of the present disclosure may be a biparatopic or triparatopic antibody. An “antibody” according to the present disclosure refers to a proteinaceous molecule and includes for instance all antibody formats available in the art, such as for example a full length IgG, IgA, or IgE antibody, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibody (like VHH and VH), minibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)’2, F(ab)’2-scFv2, and intrabodies. In certain embodiments, a multispecific antibody of the present disclosure may comprise an Fc region or a part thereof. In certain embodiments, a multispecific antibody of the present disclosure is an IgG1 antibody. Constant regions of an antibody of the present disclosure may comprise one or more variations that modulate properties of the antibody other than its binding properties to the target antigens. For instance, the constant regions may comprise one or more additional variations that favor heterodimerization of the two different heavy chains over homodimerization of each heavy chain, and/or the constant regions may comprise one or more variations that reduce or improve effector function, preferably one or more variations that reduce effector function. An antibody of an antibody-drug conjugate of the present disclosure may comprise a heavy chain comprising a CH3 region as described in WO 2021/235936, incorporated herein by reference in its entirety. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a first heavy chain comprising a CH3 domain comprising amino acid substitutions T366K and L351K and a second heavy chain comprising a CH3 domain comprising amino acid substitution L351D, L368E and an amino acid variation at position S364, K409 and/or K360. In certain embodiments, the amino acid at position 364 is valine, isoleucine, threonine, glutamine or leucine, and/or the amino acid at position 409 is isoleucine, leucine or glutamate, and/or the amino acid at position 360 is aspartate. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises amino acid variations in the Fc region that reduce or eliminate effector function. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises at least one amino acid substitution at position 235 and/or 236. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises amino acid substitution L235G and/or G236R, in particular L235G and G236R. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH2 region comprising the amino acid sequence as set forth in SEQ ID NO: 9. An antibody of an antibody-drug conjugate of the present disclosure may comprise a heavy chain comprising a modified constant region, in particular a modified CH1, CH2, or CH3 region, as described in WO 2020/226502, incorporated herein by reference in its entirety. A modified CH1, CH2, and CH3 are modified as compared to the wildtype sequences as set forth in SEQ ID Nos: 7, 8, and 10, respectively. In certain embodiments, an antibody of an antibody- drug conjugate of the present disclosure may comprise a CH1, CH2, and/or CH3 region comprising one or more variations of an amino acid that is non-surface exposed, wherein the variation is selected from a neutral amino acid to a negatively charged amino acid; a positively charged amino acid to a neutral amino acid; a positively charged amino acid to a negatively charged amino acid; a neutral amino acid to a positively charged amino acid; a negatively charged amino acid to a neutral amino acid; and a negatively charged amino acid to a positively charged amino acid. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from N159, N201, T120, K147, D148, Y149, V154, A172, Q175, S190, and K213. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from D148, Y149, V154, N159, A172, S190, and N201. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising a variation of an amino acid selected from N159 and/or N201. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH1 region comprising variations of amino acids selected from the group A172/S190/N201, T197/K213, D148/Q175, N159/Q213, K147/Q175, Y149/V154/A172/S190, N201/K213, T120/N201, N201/N159, T120/N159, T120/N201/N159 and N201/K213/N159. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH2 region comprising a variation of amino acid V303. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a CH3 region comprising a variation of an amino acid selected from K370, E382 and E388, in particular E388. An antibody of an antibody-drug conjugate of the present disclosure may comprise any suitable light chain, including any suitable common light chain. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a light chain comprising a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 1. In certain embodiments, the light chain comprises a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 5. A “Fab” typically means a binding domain comprising a heavy chain variable region, a light chain variable region, a CH1 and a CL region. An “Fc region” typically comprises a hinge, CH2, and CH3 region. In certain embodiments, an antibody of an antibody-drug conjugate of the present disclosure comprises a hinge region having the amino acid sequence as set forth in SEQ ID NO: 6. In certain embodiments, the present disclosure provides an antibody-drug conjugate comprising a variable domain that binds to EGFR and a variable domain that binds to LGR5. In general, as described herein, antigen binding can be expressed in terms of specificity and affinity. The specificity determines which antigen or epitope thereof is specifically bound by a variable domain, antibody, or antibody-drug conjugate. The affinity is a measure for the strength of binding to a particular antigen or epitope. For the purpose of the present disclosure, a variable domain, antibody, or antibody-conjugate is considered to bind an antigen when it has an at least two times higher binding signal than the background signal in the same assay. In certain embodiments, the variable domain of the antibody drug conjugate that binds to EGFR comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO:13, 17, 21, or 25. In certain embodiments each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non- conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In general, as described herein, typically, a conservative amino acid substitution involves a variation of an amino acid with a homologous amino acid residue, which is a residue that shares similar characteristics or properties. Homologous amino acids are known in the art, as are routine methods for making amino acid substitutions in antibody binding domains without significantly impacting binding or function of the antibody, see for instance handbooks like Lehninger (Nelson, David L., and Michael M. Cox.2017. Lehninger Principles of Biochemistry. 7th ed. New York, NY: W.H. Freeman) or Stryer (Berg, J., Tymoczko, J., Stryer, L. and Stryer, L., 2007. Biochemistry. New York: W.H. Freeman), incorporated herein in its entirety. In determining whether an amino acid can be replaced with a conserved amino acid, an assessment may typically be made of factors such as, but not limited to, (a) the structure of the polypeptide backbone in the area of the substitution, for example, a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and/or (c) the bulk of the side chain(s). If a residue can be substituted with a residue which has common characteristics, such as a similar side chain or similar charge or hydrophobicity, then such a residue is preferred as a substitute. For example, the following groups can be determined: (1) non-polar: Ala (A), Gly (G), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, the amino acids may be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) apolar: Leu (L), Val (V), Ile (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gln (Q), Asn (N), Ser (S), Thr (T), Tyr (Y). Alternatively, amino acid residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), Ile (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg R); (5) residues that influence chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F). The substitution of an amino acid residue with another present in the same group would be preferred. Accordingly, conservative amino acid substitution can involve exchanging a member of one of these classes for another member of that same class. Typically, the variation results in no, or substantially no, loss in binding specificity of the binding domain to its intended target. Additional types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation. Binding variants encompassed by the present disclosure include somatically hypermutated or affinity matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene segments as the heavy chain variable regions described by sequence herein, the variants having amino acid variations, including non-conservative and/or conservative amino acid substitutions in one, two, or all three HCDRs. Routine methods for affinity maturing antibody binding domains are widely known in the art, see for instance Tabasinezhad M, et al. (Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol Lett.2019 Aug;212:106-113). In certain embodiments, the variable domain of the antibody drug conjugate that binds to EGFR comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. In certain embodiments each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In certain embodiments, the variable domain of the antibody drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61. In certain embodiments each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In certain embodiments, the variable domain of the antibody drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; h) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; or i) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64, respectively. In certain embodiments each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In certain embodiments, the variable domain of the antibody-drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequences as set forth in SEQ ID NO: 1. In certain embodiments each of the LCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two LCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, LCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In certain embodiments, the variable domain of the antibody drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising: light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In certain embodiments each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one or two HCDRs may comprise at most three, two, or one non-conservative amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution. In certain embodiments, the antibody-drug conjugate comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 18, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 54, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 55, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 56; and wherein the variable domain of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 2, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 3, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 4. In certain embodiments, the variable domain of the antibody-drug conjugate that binds to EGFR comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 13, 17, 21, or 25, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto. “Percent (%) identity” as referring to nucleic acid or amino acid sequences herein is defined as the percentage of residues in a candidate sequence that are identical with the residues in a selected sequence, after aligning the sequences for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The alignment may also be carried out over individual CDR sequences. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley). The percent sequence identity between two amino acid sequences or nucleic acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol.48, 443-453). The Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this disclosure, the NEEDLE program from the EMBOSS package is used to determine percent identity of amino acid and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For DNA sequences, DNAFULL is used. The parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of this disclosure is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. In certain embodiments, the variable domain of the antibody-drug conjugate that binds to LGR5 comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto. In certain embodiments, the variable domain of the antibody-drug conjugate that binds to EGFR and/or LGR5 comprises a light chain variable region comprising the amino acid sequences as set forth in SEQ ID NO: 1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto. In certain embodiments, the antibody-drug conjugate comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto. In certain embodiments, a variable domain of an antibody-drug conjugate of the present disclosure also comprises variable domain variants, which, in addition to the variations in the HCDRs and/or LCDRs referred to above, comprise one or more variations in the framework regions. A variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation. In certain embodiments, a variable domain variant of an antibody-drug conjugate of the present disclosure comprises no variations in the CDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein, and are expected to retain their binding specificity. In certain embodiments, the antibody-drug conjugate of the present disclosure comprises: - a variable domain that binds to EGFR comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17; - a variable domain that binds to LGR5 comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1. In certain embodiments, the antibody-drug conjugate of the present disclosure comprises: - a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 65; - a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 66; and - two light chains comprising the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the present disclosure provides an antibody-drug conjugate that competes with an antibody-drug conjugate as described herein for binding to EGFR and/or LGR5. For the purpose of the present disclosure, “compete”, “competes”, or “competing” refers to an activity of an antibody-drug conjugate that blocks or displaces an antibody-drug conjugate as described herein from its target antigen(s), in a cross-blocking assay. Therefore, in certain embodiments, an antibody-drug conjugate that competes for binding with an antibody-drug conjugate as described herein, binds to EGFR or LGR5 and blocks or displaces the antibody- drug conjugate as described herein, in a cross-blocking assay. In certain embodiments, an antibody-drug conjugate that competes for binding with an antibody-drug conjugate as described herein, binds to EGFR and LGR5 and blocks or displaces an antibody-drug conjugate as described herein, in a cross-blocking assay. In certain embodiments, a cross-blocking assay is a competitive ELISA. Methods of performing a competitive ELISA are known to a person of ordinary skill in the art. In brief, in a competitive ELISA, antigen is immobilized on the wells of a microtiter plate and pre-incubated with or without the competing antibody-drug conjugate. This is followed by addition of a biotin-labeled antibody-drug conjugate as described herein. The amount of labeled antibody-drug conjugate bound to the antigen in the wells is measured using avidin-peroxidase conjugate and appropriate substrate. The amount of labeled antibody-drug conjugate that is bound to the antigen has an indirect correlation to the ability of the competing antibody-drug conjugate to compete for binding to the same antigen, i.e., the greater the affinity of the competing antibody-drug conjugate for the same antigen, the less labeled antibody-drug conjugate will be bound to the antigen-coated wells. A candidate competing antibody-drug conjugate is considered to compete for binding to the antigen, if the candidate antibody-drug conjugate can block binding of the antibody-drug conjugate of the present disclosure, to the target antigen, by at least 20%, or by at least 20-50%, or by at least 50%, as compared to the control performed in parallel in the absence of the candidate competing antibody-drug conjugate. Drug-linker combinations In certain embodiments, the antibody-drug conjugate of the present disclosure comprises a drug which is coupled to the antibody of the antibody-drug conjugate via a linker. The combination of linker and drug is herein referred to as linker-drug combination. In certain embodiments, the drug of the linker-drug combination is a cytotoxic or cytostatic drug. Suitable examples of drugs include, but are not limited to, the following: dolastatin-10, auristatin molecules, maytansine, maytansinoids, camptothecin, exatecan, SN-38, IRDye700DX, anthramycin, pyrrolobenzodiazepine (PBD) dimers, calicheamicin, and calicheamicin analogs. Other drugs known to a skilled person may also be used, such as for instance any chemotherapeutic agent, an amanitin, or a microcystin. Auristatin molecules interfere with tubulin polymerization and exert a potent effect by disrupting mitotic spindle formation, resulting in a mitotic block leading to cell death. Auristatin molecules include, but are not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and any derivatives thereof. MMAE is a synthetic antineoplastic agent derived from dolastatin-10, a marine pentapeptide isolated from the Indian Ocean mollusk Dolabella auricularia. Dolastatin-10 is reported to induce apoptosis of lung cancer cells and other tumor cells, and has been developed into commercial drugs for treating lymphomas. MMAE is a potent antimitotic drug and can be conjugated to a multispecific antibody through a protease cleavable peptide linker. The name vedotin refers to a linker containing a maleimide-caproyl (MC) moiety conjugated to the primary amine of a valine-citrulline (VC) linker linked to a p-aminobenzyloxycarbamoyl (PABC) spacer, and which is linked to a monomethylated amine of MMAE. MMAF is also is a synthetic antineoplastic agent derived from dolastatin-10. MMAF can be conjugated to a multispecific antibody through an MC linker. This linker-drug combination is referred to as mafodotin. Maytansine is a highly potent cytotoxin that kills cells by binding to β-tubulin, preventing microtubules from forming in cells undergoing mitosis during cell division, resulting in cell cycle arrest and eventual cell death. Maytansine can be engineered to introduce a methylene-thiol (-CH2-SH) moiety. The combination of maytansine with the methylene-thiol (- CH2-SH) moiety is referred to as mertansine (or DM1), which is a maytansinoid. Maytansine and maytansinoids can be conjugated to a multispecific antibody by using a succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker. This linker-drug combination is referred to as emtansine. Another example of a maytansinoid linker-drug combination is ravtansine (or soravtansine or DM4). Exatecan,Dxd, and DX-8951f are synthetic derivatives of camptothecin, a natural cytotoxin binding to the topoisomerase 1-DNA complex, preventing DNA re-ligation which results in cell cycle arrest and the accumulation of DNA strand breaks and ultimately leads to cell death. Exatecan can be conjugated to a multispecific antibody by a glycine-glycine- phenylalanine-glycine (GGFG) linker. Exatecan can also be conjugated to a multispecific antibody by using a linker comprising an maleimide-caproyl spacer and the GGFG linker, which linker drug combination is referred to as deruxtecan. SN-38 (or 7-ethyl-10-hydroxycamptothecin), is also a synthetic derivative of camptothecin. SN-38 can be conjugated to a multispecific antibody via a CL2A linker. When the CL2A linker is attached to SN-38 via a carbonate moiety, the linker-drug combination is referred to as govitecan. SG3199 (or SCX) is a synthetic dimer analog of the natural pyrrolobenzodiazepine (PBD) cytotoxin, anthramycin. Anthramycin exerts its cytotoxic mechanism by binding covalently to guanine in the DNA minor groove, inhibiting nucleic acid synthesis and cell division. SG3199 can be conjugated to a multispecific antibody via a maleimide propoyl (MP) moiety conjugated to eight polyethylene glycol (PEG8) monomers linked to a cathepsin B-cleavable valine-alanine (VA) dipeptide with a C-terminal PABC spacer. This linker-drug combination is referred to as tesirine (or SG3249). Calicheamicin analog, which is based on the highly potent cytotoxin calicheamicin, and kills cells by binding to their DNA minor groove and subsequently causing DNA double-strand scission. Calicheamicin analogs can be conjugated to a multispecific antibody by various linkers containing acylhydrazide (-CO-NH-NH2) and thiol (-SH) moieties, or a linker containing an acid sensitive hydrazone (C=N-N) bond engineered into such linkers. Calicheamicin analogs can also be conjugated to a multispecific antibody by a hydrazone linker comprising a 4-(4’- acetylphenoxy) butanoic acid moiety, which linker-drug combination is referred to as ozogamicin. In certain embodiments, the drug of the linker-drug combination is a tubulin inhibitor, a deoxynucleic acid (DNA) damaging agent, a chemotherapeutic agent, a protein degrader, or an immune stimulant. In certain embodiments, the drug of the linker-drug combination is a tubulin inhibitor that is an auristatin or derivative thereof, a tubulysin or derivative thereof, or a maytansine or derivative thereof. In certain embodiments, the drug of the linker-drug combination is a DNA damaging agent which is a DNA double-strand breaking agent, a DNA alkylation agent, a DNA intercalator, a DNA cross linker, a topoisomerase I inhibitor, or a topoisomerase II inhibitor. In certain embodiments, the drug of the linker-drug combination is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), PNU-159682, PE38, IRDye700, proteolysis- targeting chimera (PROTAC), alpha-amanitin, dimeric amidobenzimidazole (diABZI ), STING agonist-2, STING agonist-3, IMSA172, TLR 7 agonist, or TLR 8 agonist, or a derivative thereof. In certain embodiments the drug of the linker-drug combination is selected from the group consisting of MMAE, MMAF, maytansine, exatecan, SN-38, SCX, and calicheamicin, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is selected from the group consisting of MMAE, MMAF, maytansine, exatecan, SN-38, and SCX, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is MMAE or MMAF, or a derivative or analog thereof. In certain embodiments the drug of the linker-drug combination is MMAE, or a derivative or analog thereof. The drug of an antibody-drug conjugate of the present disclosure can be conjugated to a multispecific antibody by any suitable linker. Linkers known and available in the industry are capable of use in the present disclosure of ADCs. Linkers can be generally divided into two categories: cleavable (such as peptide, hydrazone, or disulfide) or non-cleavable (such as thioether or alkyl or alkoxy). Peptide linkers, such as Valine-Citrulline (Val-Cit), that can be hydrolyzed by lysosomal enzymes (such as Cathepsin B) have been used to connect a drug with an antibody. Such linker can be, in some instances, particularly useful for their relative stability in systemic circulation and the ability to efficiently release the drug in tumor. Suitable linkers include, but are not limited to, those further described herein. In certain embodiments, the linker may comprise one or more linker components. Exemplary linker components include 6-maleimido-caproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or“vc”), alanine-phenylalanine (“ala-phe”), p- aminobenzyloxycarbonyl (a“PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”). Various linker components are known in the art, some of which are described below. In certain embodiments, the linker L1 or a fragments thereof comprises MC (6-maleimidocaproyl), MCC (a maleimido-methyl cyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxy-carbonyl), SPP (N- Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-Succinimidyl 4-(N-maleimidomethyl)- cyclohexane-1 carboxylate), SIAB (N-Succinimidyl (4-iodo-acetyl)aminobenzoate. Further examples of linkers or fragments thereof include: BS3 ([Bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodimide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([N-e- Maleimidocaproic acid]hydrazide; sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS-ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carbonyl groups of the exposed carbohydrates to primary amines), and SATA (N-succinimidyl- S-acetylthioacetate; SATA is reactive towards amines and adds protected sulfhydryls groups). To form covalent bonds, a chemically reactive group, such as a wide variety of active carboxyl groups (e.g., esters), can react with a functional group such as an amine, a thiol or a hydroxyl moiety on the surface of the peptide, where the amine, thiol or hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide. Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide-benzoyl- succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MPA) maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA). Primary amines are the principal targets for NHS esters. Accessible ^-amino groups present on the N-termini of proteins and the ε-amine of lysine react with NHS esters. An amide bond is formed when the NHS ester conjugation reaction reacts with primary amines releasing N-hydroxysuccinimide. These succinimide containing reactive groups are herein referred to as succinimidyl groups. In certain embodiments of the disclosure, the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide-butrylamide (GMBA or MPA). Such maleimide containing groups are referred to herein as maleido groups. The maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls (e.g., thiol groups on proteins such as serum albumin or IgG) is 1000-fold faster than with amines. Thus, a stable thioether linkage between the maleimido group and the sulfhydryl can be formed. In certain embodiments, the linker is attached to the antibody via a lysine residue on the antibody. In certain embodiments, the linker comprises a MC (6-maleimidocaproyl), a MCC (a maleimidomethyl cyclohexane-1-carboxylate), a MP (maleimidopropanoyl), a val-cit (valine- citrulline), a val-ala (valine-alanine), an ala-phe (alanine-phenylalanine), a PAB (p- aminobenzyloxycarbonyl), a SPP (N-Succinimidyl 4-(2-pyridylthio) pentanoate), 2,5- dioxopyrrolidin-1-yl 4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4- (pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 5-ethyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 4- cyclopropyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2- ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopentyl-4-(pyridin-2-ylthio)butanoate, 2,5- dioxopyrrolidin-1-yl 4-cyclohexyl-4-(pyridin-2-ylthio)butanoate, a SMCC (N-Succinimidyl 4- (N-maleimidomethyl)cyclohexane-1 carboxylate), or a SIAB (N-Succinimidyl (4-iodo- acetyl)aminobenzoate). In certain embodiments, said linker is derived from a cross-linking reagent, wherein the cross-linking reagent comprises N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), 2,5-dioxopyrrolidin-1-yl 3-cyclopropyl-3-(pyridin-2- yldisulfaneyl)propanoate, 2,5-dioxopyrrolidin-1-yl 3-cyclobutyl-3-(pyridin-2- yldisulfaneyl)propanoate, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), 2,5- dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-yldisulfaneyl)butanoate, 2,5-dioxopyrrolidin-1- yl 4-cyclobutyl-4-(pyridin-2-yldisulfaneyl)butanoate, N-succinimidyl 4-(2- pyridyldithio)butanoate (SPDB), 2,5-dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2- yldisulfaneyl)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2- yldisulfaneyl)butanoate, N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB), N- succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC), or 2,5- dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13- tetraazaheptadecan-1-oate (CX1-1). In certain embodiments, the linker may comprise optionally a substituted (poly)ethylene glycol having from 1 to about 100 ethylene glycol units, from about 1 to about 50 ethylene glycol units, from 1 to about 25 ethylene glycol units, from about 1 to about 10 ethylene glycol units, from 1 to about 8 ethylene glycol units and from 1 to about 6 ethylene glycol units, from 2 to 4 ethylene glycol units, or optionally substituted alkyl groups interdispersed with optionally substituted, O, N, S, P or Si atoms. In certain instances, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocycle group. The linker may be asymmetric or symmetrical. In some instances, the linker may be a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, between 1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycol units, between about 2 and 5 ethylene glycol units, between about 2 and 4 ethylene glycol units. In certain embodiments, a linker or formation of the linker can comprise moieties that can be used in a click conjugation, e.g., in a two-step conjugation in which a first moiety is conjugated to a native or an engineered cysteine or lysine, the first moiety containing a reactive handle, and a second moiety containing the linker-drug component which reacts with the first moiety. An example of a possible reaction between the first moiety’s reactive handle and the second moiety is a metal free click reaction that utilizes strain-promoted azide-alkyne cycloaddition. Examples of moieties include, but are not limited to, bicyclononyne (BCN) reacting with an azide or tetrazine, dibenzocyclooctyne (DBCO) reacting with an azide, also denoted as aza-dibenzocyclooctyne (DIBAC), a transcyclooctene (TCO) reacting with a tetrazine (such as methyl tetrazine), or a methyl cycloprene click handle reacting with tetrazine. Specific examples of such moieties are as follows, but not limited to: dibenzylcyclooctyne-PegX- carboxylic acid, perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate Chemical Formula: C16H12F5NO4 Molecular Weight: 377.27; 6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanoic acid Chemical Formula: C10H11Br2NO4 Molecular Weight: 369.01; (2- methylcycloprop-2-en-1-yl)methyl carbamate (E)-cyclooct-4-en-1-yl (2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethyl)carbamate 3-(5-methylpyridin-2-yl)-6-(pyridin-2-yl)-1,2,4,5- tetrazine; ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate Chemical Formula: C17H28N2O4 Molecular Weight: 324.42; ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate Chemical Formula: C17H28N2O4 Molecular Weight: 324.42. In certain embodiments, the linker is a non-cleavable linker. In certain embodiments, the non-cleavable linker is a covalent linker. In certain embodiments, the non-cleavable linker is attached to the N-terminus, C-terminus or an internal amino acid position of the antibody or an antigen-binding fragment thereof. In certain embodiments, the non-cleavable linker is covalent attached to the drug. In certain embodiments, the non-cleavable linker comprises C1-C6 alkylene, alkenylene, cycloalkylene with a 3-7 membered ring, alkynylene, arylene, heteroarylene, heterocyclene with a 5-12 membered ring comprising 1-3 atoms of N, O or S, ^O^, ^NH^, ^S^, ^N(C1-6 alkyl)^, ^C(=O)^, ^C(=O)NH^, or combinations thereof, wherein the C1-C6 alkylene, alkenylene, cycloalkylene a 3-7 membered ring, arylene, heteroarylene, and heterocyclene with a 5-12 membered ring comprising 1-3 atoms of N, O or S is unsubstituted or substituted with halide, amino, ^CF3, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkoxy, C1-C3 alkoxy, or C1-C3 alkylthio. In certain embodiments, the linker is connected with the antibody via a reaction using a wherein LG is halide, triflate, fluorosulfonate, tosylate, mesylate, or besylate; RA is independently hydrogen, C1-C6 alkyl, cycloalkyl-alkylene, C1-C6 haloalkyl, heteroalkyl- alkylene, heteroaryl-alkylene, heterocycloalkyl-alkylene, aryl-alkylene, heteroaryl-alkylene, alkylamino-alkylene, alkylthio-alkylene, alkylcarbonyl, alkoxycarbonyl, or alkylsulfonyl; RB is independently a protecting group, C1-C6 alkyl, cycloalkyl-alkylene, C1-C6 haloalkyl, heteroalkyl-alkylene, heteroaryl-alkylene, heterocycloalkyl-alkylene, aryl-alkylene, heteroaryl- alkylene, alkylamino-alkylene, alkylthio-alkylene, alkylcarbonyl, alkoxycarbonyl, or alkylsulfonyl; each of R6, R7 and R8 is independently hydrogen, C1-C6 alkyl, cycloalkyl-alkylene, C1-C6 haloalkyl, heteroalkyl-alkylene, heteroaryl-alkylene, heterocycloalkyl-alkylene, aryl-alkylene, heteroaryl-alkylene, alkylamino-alkylene, alkylthio-alkylene, alkylcarbonyl, alkoxycarbonyl, or alkylsulfonyl; each R9 is independently halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, –C(O)(C1-C4 alkyl), –C(O)O(C1-C4 alkyl), –C(O)NH2, –C(O)NH(C1-C4 alkyl), –C(O)N(C1-C4 alkyl)2, –NH2, –NH(C1-C4 alkyl), –N(C1-C4 alkyl)2, –NH(C2-C4 alkylene)-OH, –NH(C2-C4 alkylene)-O-(C1-C4 alkyl), –OH, –O(C1-C4 alkyl), –O(C1-C4 haloalkyl), –O(C2-C4 alkylene)-NH2, –O(C2-C4 alkylene)-NH-(C1-C4 alkyl), –O(C2-C4 alkylene)-N-(C1-C4 alkyl)2, –O(C1-C4 alkylene)-C(O)OH, –O(C1-C4 alkylene)-C(O)O-(C1-C4 alkyl), –O(C2-C4 alkenyl), –O(C1-C4 alkylene)-(C6-C10 aryl), –O(C1-C4 alkylene)-(5- to 10-membered heteroaryl), –O(C6-C10 aryl), –SH, S(O)2OH, –S(O)2(C1- C4 alkyl), –S(O)2NH2, –S(O)2NH(C1-C4 alkyl), or –S(O)2N(C1-C4 alkyl)2; or two R9, together with atoms to which they are attached, form a C3-C10 cycloalkyl or a 3- to 10-membered heterocycloalkyl ring; and each of v and w is independently an integer of 0-3, In certain embodiments, the reactive group is: In certain embodiments, the linker comprises *–(L1)p–; wherein: * denotes a connection leading to the drug; L1 is independently a bond, –O–, –S–, –NH–, –NRD–, –C(=O)–, –C(=O)O–, –OC(=O)–, – C(=O)NH–, –NHC(=O)–, –C(=O)NRD–, –NRDC(=O)–, –(CH2–O–CH2)n–, C1-C6 alkylene, C1- C6 haloalkylene, –C1-C6 alkoxy–, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10 cycloalkylene, 3- to 10-membered heterocycloalkylene, –C6-C10 aryl–, or 5- to 10-membered heteroarylene, wherein each C1-C6 alkylene, C1-C6 haloalkylene, –C1-C6 alkoxy–, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10 cycloalkylene, 3- to 10-membered heterocycloalkylene, –C6-C10 aryl–, and 5- to 10-membered heteroarylene is optionally substituted with 1, 2, or 3 R9; RD is independently –CR3R4R5; each R3, R4, and R5 is independently hydrogen, halogen, –U, or –G; –U is independently C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 heteroalkyl, C2-C6 alkenyl, or C2-C6 alkynyl; wherein each C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 heteroalkyl, C2-C6 alkenyl, or C2-C6 alkynyl is optionally substituted with 1, 2, or 3 R9 and/or 1 or 2 –G; –G is independently C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10 aryl, or 5- to 10-membered heteroaryl; wherein each C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10 aryl, or 5- to 10-membered heteroaryl is optionally substituted with 1, 2, or 3 R9; each R9 is independently halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, –C(O)(C1-C4 alkyl), –C(O)O(C1-C4 alkyl), –C(O)NH2, –C(O)NH(C1-C4 alkyl), –C(O)N(C1-C4 alkyl)2, –NH2, –NH(C1-C4 alkyl), –N(C1-C4 alkyl)2, –NH(C2-C4 alkylene)-OH, –NH(C2-C4 alkylene)-O-(C1-C4 alkyl), –OH, –O(C1-C4 alkyl), –O(C1-C4 haloalkyl), –O(C2-C4 alkylene)-NH2, –O(C2-C4 alkylene)-NH-(C1-C4 alkyl), –O(C2-C4 alkylene)-N-(C1-C4 alkyl)2, –O(C1-C4 alkylene)-C(O)OH, –O(C1-C4 alkylene)-C(O)O-(C1-C4 alkyl), –O(C2-C4 alkenyl), –O(C1-C4 alkylene)-(C6-C10 aryl), –O(C1-C4 alkylene)-(5- to 10-membered heteroaryl), –O(C6-C10 aryl), –SH, S(O)2OH, –S(O)2(C1- C4 alkyl), –S(O)2NH2, –S(O)2NH(C1-C4 alkyl), or –S(O)2N(C1-C4 alkyl)2; or two R9, together with atoms to which they are attached, form a C3-C10 cycloalkyl or a 3- to 10-membered heterocycloalkyl ring; each of n and p is independently an integer of 1-12. In certain embodiments, the linker further comprises a spacer connected to either end of *–(L1)p–, wherein the spacer comprises an amino acid, dipeptide, tripeptide, tetrapeptide, mono saccharide, disaccharide, polysaccharide, alkylene, alkenylene, cycloalkylene with a 3-7 membered ring, alkynylene, arylene, heteroarylene, heterocyclene with a 5-12 membered ring comprising 1-3 atoms of N, O or S, ^(CH2OCH2)k^, ^O^, ^NH^, ^S^, ^N(C1-6 alkyl)^, ^C(=O)^, ^C(=O)NH^, or combinations thereof, wherein each of the alkylene, alkenylene, cycloalkylene with 3-7 membered ring, arylene, heteroarylene, and heterocyclene with a 5-12 membered ring comprising 1-3 atoms of N, O or S is independently unsubstituted or independently substituted with halide, amino, ^CF3, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkoxy, C1-C3 alkoxy, or C1-C3 alkylthio, wherein k is independently an integer from 1-10. In certain embodiments, the spacer is , wherein m is independently an integer of 0-3, q is independently an integer of 0-12, and r is independently an integer of 1-3. In certain embodiments, the spacer is , wherein: each Y1 and Y2 is independently a bond, O, S, or NRE; RE is independently H, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl; C6-C12 aryl, 5-12 membered heteroaryl, C3-C12 cycloalkyl or 3-12 membered heteroalicyclic, or RE together with the nitrogen to which they are bound and another atom of the spacer, be combined to form a 3 to 12 membered heteroalicyclic or 5-12 membered heteroaryl group optionally containing 1 to 3 additional heteroatoms selected from the group consisting of N, O, and S; and m is independently an integer of 0-3, q is independently an integer of 0-12, and r is independently an integer of 1-3. In certain embodiments, the linker is a branched linker to connect multiple drug moieties to the antibody. In certain embodiments, the branched linker comprises , wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e and d is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, f is independently 1, 2, 3, 4, 5, 6, 7 or 8, with the proviso that at least one of e and d is not 0. In certain embodiments, the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, govitecan, ozogamicin, saratolacan, and soravtansine. In certain embodiments, the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, and govitecan. In certain embodiments, the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, and tesirine. In certain embodiments, the antibody-drug conjugate comprises a linker-drug combination selected from vedotin and mafodotin. In certain embodiments, the antibody-drug conjugate comprises linker-drug combination vedotin. Pharmaceutical compositions and methods of use In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier. In certain embodiments, the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody. In certain embodiments, the present disclosure provides an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, for use in therapy, in particular for use in the treatment of cancer. In certain embodiments, the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody. In certain embodiments, the present disclosure provides a method for treating a disease, in particular cancer, comprising administering an effective amount of an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof. In certain embodiments, the antibody-drug conjugate is a multispecific antibody, such as for instance a multispecific IgG1 antibody. In certain embodiments, the cancer is selected from the group consisting of: colorectal cancer, in particular metastatic colorectal cancer (mCRC), pancreatic cancer, lung cancer, in particular lung squamous cell carcinoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, in particular squamous cell carcinoma of the head and neck (HNSCC), melanoma, testicular cancer, urothelial cancer, renal cancer, stomach cancer, carcinoid cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, in particular esophageal squamous cell carcinoma (ESCC), gastric-esophageal junction cancer, pharyngeal cancer, in particular cancer of the nasopharynx, oropharynx, and hypopharynx, laryngeal cancer, in particular cancer of the paranasal sinuses, nasal cavity, salivary glands or oral cancer, or brain cancer, in particular brain glioma. In certain embodiments, the cancer is an adenocarcinoma. In certain embodiments, the method or therapy comprises first line treatment or therapy. In certain embodiments, the method or therapy comprises second line treatment or therapy, in particular following treatment with an immune checkpoint inhibitor. In certain embodiments, the method or therapy is a monotherapy, only administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, to a subject. In certain embodiments, the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with an immune checkpoint inhibitor to a subject. In certain embodiments, the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with a chemotherapy or radiotherapy to a subject. In certain embodiments, the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with a topoisomerase I inhibitor to a subject. In certain embodiments, the topoisomerase is irinotecan. In certain embodiments, the method or therapy is a combination therapy, administering the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, in combination with fluorouracil, folinic acid, and irinotecan (FOLFIRI) or folinic acid, fluorouracil, and oxaliplatin (FOLFOX). In certain embodiments, the method or therapy is a monotherapy for the treatment of locally advanced unresectable or metastatic HNSCC, gastric/gastroesophageal junction adenocarcinoma (GEA) with EGFR amplification and/or high EGFR expression, esophageal carcinoma or pancreatic adenocarcinoma. In certain embodiments, the method or therapy is a second or further line monotherapy for the treatment of HNSCC. In certain embodiments, the method or therapy is a first line combination therapy with pembrolizumab for the treatment of HNSCC. In certain embodiments, the method or therapy is a second line combination therapy with chemotherapy for the treatment of metastatic colorectal cancer. In certain embodiments, the chemotherapy is 5-Fluorouracil, Leucovorin, and Irinotecan (FOLFIRI), or Fluorouracil, Leucovorin, and Oxaliplatin (FOLFOX). As used herein, the terms “individual”, "subject" and "patient" are used interchangeably and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like, and in particular to a human subject having cancer. The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on or administering an active agent or combination of active agents to a subject with the objective of curing or improving a disease or symptom thereof or which produces a positive therapeutic response. As used herein, "positive therapeutic response" refers to a treatment producing a beneficial effect, e.g. reversing, alleviating, ameliorating, inhibiting, or slowing down a symptom, complication, condition or biochemical indicia associated with a disease, as well as preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease, such as, for example, amelioration of at least one symptom of a disease or disorder, e.g. cancer. A beneficial effect can take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to initiation of therapy according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, stopping or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, or of a marker of cancer. Effective treatment may, for example, decrease in tumor size, decrease in the presence of circulating tumor cells, reduce or prevent metastases of a tumor, slow or arrest tumor growth and/or prevent or delay tumor recurrence or relapse. The term “therapeutic amount" or “effective amount” refers to an amount of an agent or combination of agents that treats a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor development. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence. As used herein, an effective amount of the agent or composition is one that, for example, may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual to be treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can be readily evaluated by the ordinarily skilled physician or other health care worker. An effective amount can be administered to a subject in one or more administrations. An effective amount can also include an amount that balances any toxic or detrimental effects of the agent or combination of agents and the beneficial effects. The term “agent” refers to a therapeutically active substance, in the present case an antibody-drug conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure. Methods for production Great advancements in methods to conjugate cytotoxic agents to antibodies have been made over the recent years, including the use of various different linkers and cytotoxic agents. Any of such methods may be employed for producing an antibody-drug conjugate according to the present disclosure. Suitable examples include, but are not limited to the methods described in for instance Hallam et al. (Hallam et al. Antibody Conjugates with Unnatural Amino Acids. Molecular pharmaceutics 2015; Vol.12; Issue: 6; 1848-1862), Yamada et al. (Yamada et al. AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angewandte Chemie 2019; Vol.: 58; Issue: 17; 5592-5597), Lobba et al. (Lobba et al. Site- Specific Bioconjugation through Enzyme-Catalyzed Tyrosine–Cysteine Bond Formation. ACS Central Science 2020; Vol.: 6; Issue: 9; 1564-1571), Bird et al. (Bird et al. Bridged Cysteine Conjugations. Methods in Molecular Biology 2020; Vol.: 2078; 113-129), van Geel et al. (van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N- Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates. Bioconjugate Chemistry Publisher: American Chemical Society 2015; Vol.: 26; Issue: 11; 2233-2242). In certain embodiments, the method for producing an antibody-drug conjugate of the present disclosure is based on hinge and CH1/ CL reduced interchain disulfide bonds conjugation or stochastic lysine conjugation. In certain embodiments, the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first CH3 domain comprising at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and a second CH3 domain comprising at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody. In certain embodiments, the present disclosure provides a method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitution T366K and wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, an antibody is provided wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D. In certain embodiments, an antibody is provided wherein the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K and the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E. In certain embodiments, an antibody is provided with a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the second CH3 domain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system. In certain embodiments, an antibody is provided with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises a positively charged amino acid residue at position 364 according to the EU numbering system, and the CH3 domain of the second heavy chain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system. In certain embodiments, an antibody is provided wherein the first CH3 domain or the CH3 domain of the first heavy chain comprises a lysine (K) or an arginine (R) residue at position 364 and the second CH3 domain or the CH3 domain of the second heavy chain comprises an aspartic acid (D) or a glutamine (E) residue at position 368. In certain embodiments, the antibody that is provided is a multispecific antibody, in particular a bispecific or trispecific antibody. In certain embodiments, the drug of the linker-drug combination is a drug as described herein. In certain embodiments the linker-drug combination is a linker-drug combination as described herein. Conjugation sites The linker-drug combination can be conjugated the antibody in any suitable manner. Suitable methods are known to a person skilled in the art and include, but are not limited, to those described herein. Examples of suitable methods involve use of an amino acid present in the antibody, or introduced in the antibody by antibody engineering, that form the conjugation site for coupling a linker-drug combination to the antibody. In certain embodiments, the conjugation site on the antibody is an amino acid. In certain embodiments, the conjugation site on the antibody is a side chain on an amino acid. In certain embodiments, the conjugation site is a cysteine or lysine residue on the antibody. In certain embodiments, the conjugation site is a reactive group (azide or alkyne) for a click chemistry reaction. In certain embodiments, there are multiple conjugation sites on an antibody for linking multiple of a particular linker-drug combination to the antibody, or the antibody can be engineered to have multiple conjugation sites. In this way, the drug antibody ratio (DAR) can be controlled, e.g. by increasing or decreasing the DAR. In certain embodiments, the conjugation site is an unnatural amino acid. In certain embodiments, the antibody or an antigen binding fragment thereof comprises an unnatural amino acid, and the antibody or antibody fragment and the drug are linked/conjugated via the unnatural amino acid. In certain embodiments, an unnatural amino acid may be inserted between two naturally occurring amino acids in the antibody or antibody fragment. The unnatural amino acid may replace one or more naturally occurring amino acids in the antibody or antibody fragment. The unnatural amino acid may be incorporated at the N terminus of the antibody or antibody fragment. The unnatural amino acid may be incorporated at the C terminus of the antibody or antibody fragment. The unnatural amino acid may be incorporated distal to the binding region of antibody or antibody fragment. The unnatural amino acid may be incorporated near the binding region of the antibody or antibody fragment. The unnatural amino acid may be incorporated in the binding region of the antibody or antibody fragment. In certain embodiments, the unnatural amino acid may be p-acetylphenylalanine (pAcF or pAcPhe). The unnatural amino acid may be selenocysteine. The unnatural amino acid may be p- fluorophenylalanine (pFPhe). The unnatural amino acids may be selected from the group consisting of p-azidophenylalanine (pAzF),p-azidomethylphenylalanine(pAzCH2F), p- benzoylphenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF), p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (pCmF), 3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF), o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3- yl)alanine (HQA), selenocysteine, and (2,2'-bipyridin-5-yl)alanine (BipyA). ). The unnatural amino acids may be 4-(6-methyl-s-tetrazin-3-yl)aminopheynlalanine. In certain embodiments, the unnatural amino acid may comprise at least one oxime, carbonyl, dicarbonyl, hydroxylamine group or a combination thereof. The one or more unnatural amino acids may comprise at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl/alkyl azide, norbornene, cyclopropene, trans- cyclooctene, or tetrazine functional group or a combination thereof. In certain embodiments, the unnatural amino acid may be incorporated in a light chain of the antibody or antibody fragment. The unnatural amino acid may be incorporated in a heavy chain of the antibody or antibody fragment. The unnatural amino acid may be incorporated in a heavy chain and a light chain of antibody or antibody fragment. The unnatural amino acid may replace an amino acid in the light chain of the antibody or antibody fragment. The unnatural amino acid may replace an amino acid in a heavy chain of the antibody or antibody fragment. The unnatural amino acid may replace an amino acid in a heavy chain and a light chain of the antibody or antibody fragment. In certain embodiments, the unnatural amino acid is p-acetylphenylalanine, whose ketone group on the side chain of incorporated unnatural amino acid react with a hydroxylamine functionalized drug to form an oxime bond. The articles “a” and “an” are used herein to refer to one or more of the grammatical object of the article. By way of example, “an element” means one or more elements. A reference herein to a patent document or other matter is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge at the priority date of any of the claims. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. CDRs and framework regions of antibodies have been described and defined in the art using a number of different systems, including for instance Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991); Kabat et al., J. Biol. Chem.252:6609-6616 (1977)), IMGT (discussed in Giudicelli et al., Nucleic Acids Res.25: 206-2111997), Chothia (Chothia and Lesk J. Mol. Biol.196: 901 -917, 1987; Chothia et al., Nature 342: 877-883, 1989; Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997), and the nomenclatures of Honnegher and Plukthun (Honnegher and Plukthun, J. Mol. Biol.309: 657- 670, 2001), MacCallum (MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)), and Lefranc (Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003)). In general, it is irrelevant which numbering system is used, as an antibody exhibits its properties regardless of the numbering system used. When the amino acid sequence of a variable region is given, a skilled person can readily determine its CDRs based on different numbering systems. Thus, the present disclosure encompasses defining the CDRs in accordance with each numbering system available to a skilled person. In particular, the present disclosure encompasses defining the CDRs in accordance with the numbering systems of Kabat, IMGT, and Chothia. The HCDRs according to Kabat are provided in the listing of sequences as provided herein. The HCDRs according to IMGT are indicated in bold and underlined in the heavy chain variable region sequences as provided in the listing of sequences as provided herein. The LCDRs according to IMGT are provided in the listing of sequences as provided herein. The LCDRs according to Kabat are indicated in bold and underlined in the light chain variable region sequences as provided in the listing of sequences as provided herein. Amino acids in the constant regions are indicated according to the EU numbering system. Accession numbers are primarily given to provide a further method of identification of a target, the actual sequence of the protein bound may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. An antigen binding site of an antibody of the disclosure can bind the antigen and a variety of variants thereof, such as those expressed by some antigen positive immune or tumor cells. HGNC stands for the HUGO Gene nomenclature committee. The number following the abbreviation is the accession number with which information on the gene and protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides the accession number or gene ID with which information on the gene or protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensembl provides the accession number with which information on the gene or protein encoded by the gene can be obtained from the Ensembl database. Ensembl is a joint project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes. When herein reference is made to a gene or a protein, the reference is preferably to the human form of the gene or protein. When herein reference is made to a gene or protein reference is made both to the natural gene or protein and to variant forms of the gene or protein as can be detected in tumors, cancers and the like, preferably as can be detected in human tumors, cancers and the like. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 – Thermal stability of EGFRxLGR5 ADC, EGFRxc-MET ADC, and trastuzumab ADC (1 mg/ml) as assessed by thermal ramp between 25-95°C at a rate of 0.3°C/min. The average Tm (melting temperature in °C) and average Tagg (aggregation temperature in °C) of the samples were analyzed using Uncle^ Biostability Platform. Figure 2 – The stability of EGFRxc-MET ADC, EGFRxLGR5 ADC, and trastuzumab ADC (1 mg/ml) in human serum as analyzed at 0, 24, 48 and 96 hours at 37°C by LC-MS, indicated by percentage of conjugated DAR. Figure 3 – Freeze-thaw stability of EGFRxc-MET ADC, EGFRxLGR5 ADC, and trastuzumab ADC (2 mg/ml) at -80°C for a minimum of 4 hours as analyzed by HP-SEC, indicated by percentage monomer. Time point 0 (T0) and multiple freeze thaw cycles (FT) are shown. Figure 4 – The affinity of EGFRxc-MET ADC and EGFRxLGR5 ADC to soluble c- MET, EGFR, and LGR5 as evaluated in single cycle kinetic analysis using Biacore. The response (response units (RU)) over time (seconds) is indicated in sensorgrams. Figure 5 – The binding of soluble EGFRxLGR5 ADC to human Fc^ receptors FcγRI (Fig.5A) and FcγRIIIA (176 Phe) (Fig, 5B) as evaluated using Biacore. The response (response units (RU)) over time (seconds) is indicated in sensorgrams. Figure 6 – The steady state affinity binding of soluble EGFRxLGR5 ADC to FcRn at pH 6.0 (Fig.6A) and pH 7.4 (Fig.6B) as evaluated using Biacore multicycle analysis. The response (response units (RU)) over time is indicated in sensorgrams. Arrowheads in the graphs are indicating the reference timepoint used to derive the binding KD from the steady-state analysis. Figure 7 – The binding of EGFRxc-MET ADC (Fig. 7A), EGFRxLGR5 ADC (Fig.7B) and trastuzumab ADC (Fig. 7C), and control antibodies to the C1q component of complement as evaluated by ELISA. Binding at different concentrations is expressed as absorbance read at 450 nm using a SpectraMax M3 plate reader. Figure 8 – A) The binding of EGFRxLGR5 ADC and cetuximab to A549 WT and A549 EGFR KO cells as evaluated by flow cytometry, measured at different concentrations and expressed as the mean fluorescence intensity (MFI), measured using Attune NxT Acoustic Focusing Cytometer. B) The binding of EGFRxc-MET ADC and cetuximab to A549 WT, A549 c-MET KO, and A549 EGFR KO cells as evaluated by flow cytometry, measured at different concentrations and expressed as the MFI, measured using Attune NxT Acoustic Focusing Cytometer. C) The binding of EGFRxc-MET ADC and cetuximab to NCI-1975 WT, and NCI- 1975 c-MET KO cells as evaluated by flow cytometry. The MFI was measured at different concentrations using Attune NxT Acoustic Focusing Cytometer. Figure 9 – A) The ability of the EGFRxLGR5 ADC and cetuximab to induce killing of A549 WT or A549 EGFR KO target cells as evaluated by a cell viability assay with CellTiter- Glo®. The percentage viable cells is plotted against the antibody concentration in nM. B) The ability of the EGFRxc-MET ADC and cetuximab to induce killing of A549 WT, A549 c-MET KO, or A549 EGFR KO target cells as evaluated by a cell viability assay using CellTiter-Glo® The percentage viable cells is plotted against the antibody concentration in nM. A single experiment is shown in the upper graph, independent triplicate experiments are shown in the lower graphs. C) The ability of the EGFRxc-MET ADC and cetuximab to induce killing of NCI- H1975 WT or NCI-H1975 c-MET KO target cells as evaluated by a cell viability assay using CellTiter-Glo®. The percentage viable cells is plotted against the antibody concentration in nM. Figure 10 – A) The ability of the EGFRxc-MET ADC and cetuximab at 10 µg/ml (upper graphs) and 1 µg/ml (lower graphs) to induce internalization as evaluated by an internalization assay using A549 WT, A549 c-MET-KO, and A549 EGFR-KO cells. The level of internalization is expressed as the percentage Red area/Phase area at different time points. B) The ability of the EGFRxc-MET ADC to induce internalization as evaluated by an internalization assay using NCI-H1975 WT, and NCI-H1975 c-MET-KO cells. The level of internalization is expressed as the percentage Red area/Phase area at different time points. Figure 11 – A) HIC data showing the DAR distribution of EGFRxc-MET ADC. 16% of the EGFRxc-MET ADCs in the purified sample have DAR0, 43.1% have DAR2, 13.8% have DAR4, 22.8% have DAR6, and 4.4% have DAR8. B) HIC data showing the DAR distribution of EGFRxLGR5 ADC.9.8% of the EGFRxLGR5 ADCs in the sample have DAR0, 22.7% have DAR2, 32.1% have DAR4, 23.9% have DAR6, and 11.7% have DAR8. EXAMPLES EXAMPLE 1 - Production of bispecific Antibody Drug Conjugates Two bispecific antibodies were used in this study: a bispecific antibody targeting EGFR and c-MET and a bispecific antibody targeting EGFR and LGR5. The bispecific antibody targeting EGFR and LGR5 comprises heavy chain variable regions comprising the amino acid sequence as set forth in SEQ ID NO: 17 (EGFR binding domain) and SEQ ID NO: 53 (LGR5 binding domain). Both the EGFR and c-MET binding domains comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1. The heavy chains comprise a CH1 having an amino acid sequence as set forth in SEQ ID NO: 7, a hinge having an amino acid sequence as set forth in SEQ ID NO: 6, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a CH3 region. One of the CH3 regions has an amino acid sequence as set forth in SEQ ID NO: 11 and the other CH3 region has an amino acid sequence as set forth in SEQ ID NO: 12. Both bispecific antibodies are ADCC enhanced. Both bispecific antibodies comprise one heavy chain with a CH3 domain comprising amino acid substitutions T366K and L315K and one heavy chain with a CH3 domain comprising amino acid substitutions L351D and L368E. These amino acid substitutions are also referred to herein as “the DEKK substitutions”. The aim of this study was to establish conjugation of a therapeutic agent to antibodies comprising the DEKK substitutions, while maintaining sufficient stability, affinity, and exhibiting functional activity. Both bispecific antibodies were coupled with monomethyl auristatin E (MMAE) through a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (VC) linker. The resulting antibody-drug conjugates (ADCs) are herein further referred to as EGFRxc-MET-MC-VCP- MMAE or “EGFRxc-MET ADC” and EGFRxLGR5-MC-VCP-MMAE or “EGFRxLGR5 ADC”. Trastuzumab coupled with monomethyl auristatin E (MMAE) through a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (VC) linker, referred to herein as trastuzumab-MC-VCP-MMAE or “trastuzumab ADC”, was generated as a control. EGFRxLGR5 bispecific antibody at 11.3 mg/ml in Dulbecco’s PBS, pH 7.5, 5mM EDTA was diluted to 5.07 mg/ml with Dulbecco’s PBS, pH 7.5, 5mM EDTA. EGFRxc-MET bispecific antibody at 11.08 mg/ml in Dulbecco’s PBS, pH 7.5, 5mM EDTA was diluted to 5.06 mg/mL with Dulbecco’s PBS, pH 7.5, 5mM EDTA. A 5 mM solution of TCEP in Dulbecco’s PBS, pH 7.5, 5mM EDTA was added to the diluted antibody solutions. The reduction was allowed to proceed at 40 °C for 1 hour with a final antibody concentration of 5 mg/ml. After 1 hour at 40 °C, the reduction mixture was diluted with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool down to 22 °C. A 10.0 mg/ml (7.60 mM) solution of MC-VCP-MMAE linker-payload was prepared by dissolving 10.0 mg (7595 nmol) of linker-payload (MW = 1316 g.mol-1) into 1.0 ml of DMSO. A portion of the MC-VCP-MMAE linker-payload solution was added to the reduced antibody solutions resulting in final concentrations of 10% DMSO and final antibody concentrations of 4.0 mg/ml. The conjugation reaction was allowed to proceed at 22 °C for 1 hour for EGFRxLGR5, and at 22°C for 1 hour and at 4 °C for 18 hours for EGFRxc-MET. An additional portion of the MC-VCP-MMAE linker-payload solution was added and the conjugation reaction was allowed to proceed at 22 °C for a further 2 hours for EGFRxLGR5, and for a further 1 hour for EGFRxc-MET. The reaction mixtures were purified by preparative SEC on a HiLoad 26/600 Superdex 200 pg column with DPBS + 10% isopropanol as the elution buffer. Fractions were collected and analyzed by SEC. Fractions containing the conjugate were concentrated and buffer-exchanged by ultrafiltration/diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa (EGFRxLGR5) or 50 kDa (EGFRxc-MET) MWCO) equilibrated with Dulbecco’s PBS, pH 7.2. The concentrated samples were sterile filtered through a 0.22 μm pore size, PVDF membrane filter. Trastuzumab (Roche, lot number N3031H02) at 11.0 mg/ml in Dulbecco’s PBS, pH 7.5, 5mM EDTA was diluted to 5.41 mg/ml with Dulbecco’s PBS, pH 7.5, 5mM EDTA. A 1 mM solution of TCEP in Dulbecco’s PBS, pH 7.5, 5 mM EDTA was added to the diluted antibody solution. The reduction was allowed to proceed at 40 °C for 1 hour with a final antibody concentration of 5 mg/ml. After 1 hour at 40 °C, the reduction mixture was diluted with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool down to 22 °C. A portion of the MC- VCP-MMAE linker-payload solution in DMSO was added to the reduced trastuzumab solution resulting in a final concentration of 10% DMSO and a final antibody concentration of 4.0 mg/ml. The conjugation reaction was allowed to proceed at 22 °C for 1 hour. After 1 hour the reaction mixture was purified by gel filtration using a Centripure P100 column and further buffer- exchanged by ultrafiltration/diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO) equilibrated with Dulbecco’s PBS, pH 7.2. The concentrated sample was sterile filtered through a 0.22 μm pore size, PVDF membrane filter. EGFRxLGR5 MC-VCP-MMAE, EGFRxc-MET MC-VCP MMAE, and trastuzumab MC-VCP-MMAE conjugates were characterized by hydrophobic interaction chromatography (HIC), Size-exclusion chromatography (SEC), liquid chromatography–mass spectrometry (LC- MS) and quantified by UV. The data summarized in Table 1 shows that the conjugation of MMAE to both bispecific antibodies was successful. EGFRxLGR5 MC-VCP-MMAE and EGFRxc-MET MC-VCP MMAE conjugation resulted in a DAR distribution of between DAR0- DAR8, as determined by HIC and shown in Figure 11. Specifically, FIG.11A shows the DAR distribution of EGFRxc-MET ADC: 16% of the EGFRxc-MET ADCs in the sample have DAR0, 43.1% have DAR2, 13.8% have DAR4, 22.8% have DAR6, and 4.4% have DAR8. FIG.11B shows the DAR distribution of EGFRxLGR5 ADC: 9.8% of the EGFRxLGR5 ADCs in the sample have DAR0, 22.7% have DAR2, 32.1% have DAR4, 23.9% have DAR6, and 11.7% have DAR8. ADC Average Average % MW [ADC] by Amount Yield DAR DAR monomer (LC-MS) UV (mg) (%) (HIC) (LC-MS) (SEC) (mg/ml) EGFRxc- 3.3 - 93.4 - 3.26 9.1 27 MET ADC EGFRxLGR5 4.1 3.9 97.8 Confirmed 7.12 25 76 ADC (24.6% LMW species) Trastuzumab 4.4 4.3 98.7 Confirmed 3.88 29 80 ADC Table 1. Conjugation of MMAE to bispecific antibodies and trastuzumab. LMW = low molecular weight. The antibodies can also be coupled with other drugs, such as for instance MMAF, maytansine, DM1 (mertansine), exatecan, DXd, SN-38, or SG3199 (SCX) or other linker-drug combinations, such as for instance mafodotin, emtansine, deruxtecan, tesirine (SG3249), or govitecan, using methods known in the art, such as for instance those described in the handbook Antibody-Drug Conjugates, Methods and Protocols. Methods in Molecular Biology, ISBN 978- 1-4939-9928-6 ISBN 978-1-4939-9929-3 (eBook), https://doi.org/10.1007/978-1-4939-9929-3), in particular Chapter 3 describing conjugation to endogenous cysteines, which method can be used at least for conjugation of MMAF, DXd and tesirine. EXAMPLE 2 - Thermal stability Thermal ramping experiments were performed using Uncle^ Biostability Platform (Unchained Labs, Pleasanton, CA, USA). Each ADC was diluted to 1 mg/ml in PBS. The stability of the formulations was assessed by a thermal ramp between 25-95°C at a rate of 0.3°C/min. The Tm (melting temperature) and Tagg (aggregation temperature) of the samples were analyzed running software version 6.0. The Tm is defined as the temperature at which half of the protein molecules in solution are unfolded. It is identified as a peak (or peaks) on the first derivative and calculated from a fluorescence curve with the Uncle^ software. The Tagg is defined as the point at which the SLS (Static Light Scattering) signal reaches 10% of the maximum value and is calculated by the Uncle^ software. Formulations that are more stable have higher Tm and Tagg values. Samples were analyzed in triplicate. The results as shown in Table 2 and Figure 1 show that the thermal stabilities of the EGFRxc-MET ADC and EGFRxLGR5 ADC are above 55°C, which indicates no concerns in terms of molecule developability. ADC Average Tm1 (^C) Average Tagg (^C) EGFRxc-MET ADC 62.20 55.10 EGFRxLGR5 ADC 56.70 58.30 Trastuzumab ADC 78.40 73.40 Table 2. Thermal stability of ADCs. EXAMPLE 3 - Serum stability The stability of EGFRxc-MET and EGFRxLGR5 ADCs in human serum (IgG depleted) was analyzed over 96 hours at 37°C. The ADCs were spiked in human sera at 1 mg/ml in sterile tubes. All samples were split in 4 aliquots of equal size and transferred to a 37°C incubator. The aliquots were removed from the 37°C incubator at 0, 24, 48 and 96 hours timepoints and transferred at -80°C until they were analyzed. The ADCs were immunocaptured from human sera (0-, 24-, 48- and 96-hours aliquots) using an anti-Human IgG (Fc or CH1 specific) biotin antibody immobilized on streptavidin beads. Capture was performed by mixing 50 μl of ADC- spiked serum with 50 μl PBS and 50 μl beads and incubated for 1 hour at room temperature with shaking (at 700 rpm using Eppendorf thermomixer). After incubation, the supernatant was removed, and the beads were washed three times with 500 μl PBS. Elution was performed with 100 μl 2mM HCl, three times (5 min incubation, gentle mix), and the aliquots were pooled. The eluate pH was neutralized with 0.5M Ammonium Bicarbonate (pH 8.0). After elution from the beads, the ADCs were analyzed by LC-MS using a Waters I Class UPLC in line with a Waters RDa Mass Detector. Chromatographic separation was performed using an Acquity UPLC Protein BEH SEC, 200Å, 1.7 μm, 4.6x300mm column (Waters) maintained at RT. Samples were eluted under isocratic conditions in 30% acetonitrile and 0.1% formic and the flow was set at 0.3 ml/min. A volume of 10 μl was injected per run. The MS spectra were acquired in the 400-7000 m/z range. For DAR evaluation, MS spectra were deconvoluted for the HC and LC peak in the appropriate mass range using the MaxEnt1 algorithm within the UNIFI software. After deconvolution, noise level was evaluated by visual inspection and mass peaks with intensity lower than the observed noise level were excluded from analysis. After noise filtering, signal corresponding to each DAR level were summed and used to calculate the average DAR using the following formula: ^^^^^^= (2^^(Σ^^^^^^^^ℎ^^^^^^ ^^^^^^^^ ^^^^^^^^ ^^^^+ Σ^^^^^^^^ℎ^^^^^^ ^^^^^^^^ ^^^^^^^^ ^^^^))/100. The results as shown in Figure 2 show that more than 70% of the payload remained conjugated to the EGFRxc-MET and EGFRxLGR5 ADCs in human serum for up to 4 days. EXAMPLE 4 - Freeze-thaw stability The stability of EGFRxc-MET and EGFRxLGR5 ADCs (2 mg/ml) to freeze stress was analyzed. The ADCs were frozen to -80 °C for a minimum of 4 hours and thawed at room temperature before analysis. The ADCs were analyzed after 1, 3, and 5 freeze thaw cycles, and stability was assessed by HP-SEC. HP-SEC analysis was performed using a Thermo Ultimate 3000RS UPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The column used was a Waters UPLC Protein BEH SEC Column 200 Å, 1.7 μm, 4.6 x 150 mm (part number 186005225) with a guard column, Waters Acquity UPLC Protein BEH SEC Guard Column, 200 Å, 1.7 μm, 4.6 mm X 30 mm (part number 186005793). The mobile phase consisted of 200 mM potassium phosphate, 200 mM potassium chloride, 15% (v/v) IPA, pH 6.8. The method was isocratic, with a duration of 10 minutes, ambient column temperature, and a flow rate of 0.35 mL/minute. UV detection was carried out at 280 nm. The samples were diluted to 1 mg/ml with PBS, and 10 μl of sample was injected. The percentage of monomer was analyzed by SEC chromatogram. The results as shown in Figure 3 show that the EGFRxc-MET and EGFRxLGR5 ADCs were unaffected by multiple freeze thaw cycles. The percentage of IgG monomer of the EGFRxc-MET ADC was near 100% and comparable to that of trastuzumab-ADC following repeated freeze thaw cycles. These results together with the results obtained in Example 3 indicate that both ADCs are stable in human serum and do not aggregate after multiple cycles of freeze thawing. EXAMPLE 5 - Characterization of target binding The affinity of EGFRxc-MET and EGFRxLGR5 ADCs to soluble recombinant target was evaluated in single cycle kinetic analysis using Biocore (T200) at 25°C running Biocore T200 Evaluation Software (Uppsala, Sweden). HBS-EP+ (Cytiva, Uppsala, Sweden) was used as running buffer as well as for ligand and analyte dilutions. Samples were diluted in running buffer to 1.0 μg/ml. At the start of each cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of a CM5 chip pre-coupled with ~9000 RU anti-Human IgG using standard amine chemistry (Cytiva, Little Chalfont, UK). IgGs were captured at a flow rate of 10 μl/min to give an immobilization level (RL) of ~ 100 RU. The surface was then allowed to stabilize. Single cycle kinetic data was obtained using human c-MET (His tag, Sino Biological, #10692-H08H), EGFR (His tag, Acro Biosystems, #LG5-H52H3), and LGR5 (His tag, Acro Biosystems, #LG5-H52H3) as the analyte injected at a flow rate of 30 μl/min to minimize any potential mass transfer effects. Each sample was tested for binding to all three analytes. A four point, three-fold dilution range of analyte from 3.33 nM to 90 nM (c-MET and LGR5) and 1.11 nM to 30 nM (EGFR) in running buffer was used without regeneration between each concentration. The association phases were monitored for 210 seconds for each of the four injections of increasing concentrations of antigen and a single dissociation phase was measured for 600 seconds following the last injection of antigen. Regeneration of the sensor chip surface was conducted using 3M MgCl2. The signal from the reference channel Fc1 (no IgG captured) was subtracted from that of Fc2, Fc3 and Fc4, to correct for bulk effect and differences in non-specific binding to a reference surface. The signal from each IgG blank run (IgG captured but no antigen) was subtracted to correct for differences in surface stability. The double referenced sensorgrams were fitted with the Langmuir (1:1) binding model where the closeness of fit of the data to the model is evaluated using the Chi square value which describes the deviation between the experimental and fitted (observed and expected) curves. The fitting algorithm seeks to minimize the Chi square value. The data shown in Figure 4 shows that the affinity to each target was unaffected by drug conjugation. EXAMPLE 6 - Characterization of binding to human Fc gamma receptors and FcRn The binding of EGFRxLGR5 to human Fc^RI (hCD64, His tag, Sino Biological, Beijing, China, # 10256-H08H) and human Fc^RIIIA (hCD16A, 176 Phe, His tag, Sino Biological, Beijing, China, # 10389-H08H) was evaluated using Biacore single cycle analysis. At the start of each cycle His-tagged Fc gamma receptors (ligand) diluted in HBS-P+ (Cytiva, Uppsala, Sweden) were loaded to a specific RU level onto a CM5 sensor chip pre-coupled to HIS-capture antibody via amine chemistry (His capture kit, Cytiva, Uppsala, Sweden). A five point, three- fold dilution range (0.4111 to 33.33 nM) of antibodies (analyte) without regeneration between each concentration was used for Fc^RI. Due to afucosylation of the EGFRxLGR5 ADC, a lower concentration range of antibodies (32.9 to 2666.7 nM) was used for FcγRIIIA than that for trastuzumab ADC (98.8 to 8000 nM). In all cases, antibodies were passed over the chip in increasing concentrations at 30 µl/min followed by a single dissociation step. Following dissociation, the chip was regenerated with injection of Glycine pH 1.5. The signal from the reference channel Fc1 (blank) was subtracted from that of the Fc loaded with receptor to correct for differences in nonspecific binding to the reference surface. The data shown in Figure 5 shows that binding to human Fc^RI and human Fc^RIIIA was unaffected by drug conjugation. Similar results were obtained for human Fc^RIIA (hCD32a, 167 arg), Fc^RIIB (hCD32a, 167 his), Fc^RIIB (hCD32B), Fc^RIIIA (hCD16a, 176 Val), and Fc^RIIIB (hCD16b) where binding to the receptors was unaffected by drug conjugation (data not shown). The steady state affinity binding of EGFRxLGR5 ADC to FcRn at pH 6.0 and pH 7.4 was evaluated using Biacore multicycle analysis T200 instrument running Biacore T200 Evaluation Software (Uppsala, Sweden). Human FcRn (FCGRT & β2M Heterodimer, Sino Biological, Beijing, China, # CT009-H08H) was coupled onto a Series S CM5 (carboxymethylated dextran) sensor chip (Cytiva, Little Chalfont, UK) at 10 μg/ml in sodium acetate pH 5.5 using standard amine coupling to ~ 300 RU. Purified antibodies were prepared by a direct dilution of antibody in running buffer. Purified samples were titrated in a five-point three-fold dilution from 12.3 nM to 1000 nM in PBS containing 0.05% Polysorbate 20 (P20) at pH 6.0 or a single 1000 nM point in PBS containing 0.05% Polysorbate 20 (P20) at pH 7.4. Samples were passed over the chip with increasing concentrations at a flow rate of 30 μl/min and at 25°C. The injection time was 30 s per concentration and the dissociation time was 60 s. Following a single dissociation, the chip was regenerated with 0.1 M Tris pH 8.0. The signal from the reference channel Fc1 (blank) was subtracted from that of the Fc loaded with receptor to correct for differences in non-specific binding to the reference surface. The data shown in Figure 6 shows that binding to FcRn was unaffected by drug conjugation at pH 6.0. The EGFRxLGR5 ADCs do not impact the pH sensitive binding of FcRn at pH 6.0 and are expected to allow recycling if internalized into cells, as the absence of binding at pH 7.4 is as normal. EXAMPLE 7 - Characterization of binding to C1q The binding of EGFRxc-MET and EGFRxLGR5 ADCs to the C1q component of complement was evaluated by ELISA, which measures the binding of purified C1q to the bispecific ADCs directly coated on a plate. Unconjugated parental antibodies and trastuzumab (IgG1 WT antibody) were included as controls. Rituximab (human IgG1, 10 mg/ml, Celltrion, #17C2C17DC1) was included as positive control, and IgG4 (S241P L248E, 8.4 mg/ml, Abzena, hinge stabilized and Fc silenced) as a negative control. Titrations of bispecific ADCs and controls were directly coated on an ELISA plate, and binding of purified human C1q (1.12 mg/ml, Pathway Diagnostics, #A400) was detected using an antihuman C1q-HRP conjugate (1 mg/ml, Abcam, #ab46191). Nunc Maxisorp™ ELISA plates (ThermoFisher Scientific, Loughborough, UK) were coated with serial dilutions of the antibodies, rituximab positive control or the IgG4 negative control in duplicate. The antibodies and the negative control were coated at a starting concentration of 100 μg/mL, with a seven point, four-fold serial dilution (100 – 0.0244 μg/ml) performed. Rituximab was used at a starting concentration of 20 μg/ml and a seven-point, 2.5-fold serial dilution (20 – 0.0819 μg/mL) was performed. All samples were tested in duplicate. ELISA plates were then incubated at 4 °C overnight. The plates were then blocked using 1x PBS pH 7.4 + 2% (w/v) BSA and incubated for 1 hour at 25°C. After blocking, purified human C1q (Eurobio Scientific, Dorking, Surrey) at 5 μg/ml was added to the ELISA plates for binding and incubated for 2 hours at 25°C. ELISA plates were washed and the anti-C1q-HRP diluted 1:200 in blocking buffer was added to the ELISA plates for detection and incubated for 1 hour at 25°C. After washing, TMB Microwell Peroxidase substrate (Life Technologies, #002023) was added to the ELISA plates to detect binding. The reaction was stopped with 3NHCl solution (VWR, Lutterworth, Leicestershire, #30018.298) after 5 minutes. Absorbance was read at 450 nm using a SpectraMax M3 plate reader (Molecular Devices, Wokingham, UK). GraphPad Prism 9.0 (GraphPad Software, La Jolla, Ca) was used for data analysis and data were fitted using a four parameters non-linear regression. The results shown in Figure 7 show that binding to C1q was unaffected by drug conjugation. The trastuzumab-ADC did show reduced binding to C1q. EXAMPLE 8 - Characterization of binding to dual- and single target expressing cell lines The binding of the EGFRxLGR5 ADC to A549 wildtype (WT) (ATCC, cat.no. CCL- 185) and A549 EGFR knock-out (EGFR-KO) cells was evaluated by flow cytometry. Cells were harvested and prepared at 0.6 x 106 cells/ml in flow cytometry buffer and seeded at 100 μl/well into a low-bind U-bottom 96-well plate (VWR, Lutterworth, UK). The ADC and cetuximab (Selleck Chemicals; Product code: A2000; Batch code: A200005) as a reference control antibody were prepared at 10 µg/ml starting concentration and titrated in a 7-point, 3-fold serial dilution. Cells were re-suspended with either titrated ADC, reference control antibody or flow cytometry buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4 °C. After washing with flow cytometry buffer, the cells were resuspended in flow cytometry buffer only (unstained) or anti-Human IgG (γ-chain specific), F(ab′)2 fragment−R-Phycoerythrin secondary antibody (Sigma-Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After 60 minutes incubation at 4 °C, the cells were washed in flow cytometry buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK). Fluorescence was measured using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific, Loughborough, UK), and data was analyzed using FlowJo v10 (FlowJo, Ashland, OR). Data was fitted using a 4PL in GraphPad Prism 10 (GraphPad Software, La Jolla, CA). The results shown in Figure 8A show that the EGFRxLGR5 ADC binds EGFR positive cells but does not bind to EGFR negative cells. Drug conjugation did not introduce any undesired reactivities in the tested experimental set-up. The binding of the EGFRxc-MET ADC to A549 wildtype (WT) (ATCC, cat.no. CCL- 185), A549 c-MET knock-out (c-MET-KO), and A549 EGFR knock-out (EGFR-KO) cells was evaluated by FACS. Cells were recovered from liquid nitrogen storage and prepared at 1 x 106 cells/ml in flow cytometry buffer (PBS (Thermo Fisher, Loughborough, UK) + 1% BSA (Sigma- Aldrich, Poole, UK) + 0.1% Sodium Azide (Sigma-Aldrich, Poole, UK)), and seeded at 100 μl/well into a low-bind U-bottom 96-well plate (VWR, Lutterworth, UK). The ADC and cetuximab reference antibody were prepared at 10 µg/ml starting concentration, and subjected to a 10-point, 3-fold serial dilution. Cells were re-suspended with either titrated ADC, reference control antibody buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4°C. After washing with buffer, the cells were resuspended in buffer only (unstained) or anti- Human IgG (γ-chain specific), F(ab′)2 fragment−R-Phycoerythrin secondary antibody (Sigma- Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After incubation for 60 minutes at 4°C, the cells were washed in buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK). Fluorescence was measured using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific, Loughborough, UK), and data was analyzed using FlowJo v10 (FlowJo, Ashland, OR). Data was fitted using a 4-parameter logistic (4PL) regression in GraphPad Prism 10 (GraphPad Software, La Jolla, CA). The results shown in Figure 8B show that the EGFRxc-MET ADC have a relative higher binding signal on double positive (EGFR+c-MET+) than on single positive (EGFR+c-MET-, or EGFR-c-MET+) cells. Cetuximab showed reduced binding only to EGFR KO cells. The binding of the EGFRxc-MET ADC to NCI-1975 (WT) (ATCC; cat. no. CRL-5908; lot no.70010182), and NCI-1975 c-MET knock-out (c-MET-KO) cells was evaluated by FACS. Cells were harvested and prepared at 1 x 106 cells/ml in flow cytometry buffer (and seeded at 100 μl/well into a low-bind U-bottom 96-well plate (VWR, Lutterworth, UK). The ADC and cetuximab reference control antibodies were prepared at 20 µg/mL starting concentration and titrated in a 7-point, 6-fold serial dilution. Cells were re-suspended with either titrated ADC, reference control antibody or flow cytometry buffer in duplicate (buffer; control wells) before incubation for 60 minutes at 4 °C. After washing with flow cytometry buffer, the cells were resuspended in flow cytometry buffer only (unstained) or anti-Human IgG (γ-chain specific), F(ab′)2 fragment−R-Phycoerythrin secondary antibody (Sigma-Aldrich, Poole, UK) diluted 1 in 100 in binding buffer. After 60 minutes incubation at 4 °C, the cells were washed in flow cytometry buffer and fixed with 10% BD CellFix (BD Biosciences, Oxford, UK). Fluorescence was measured using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific, Loughborough, UK), and data was analysed using FlowJo v10 (FlowJo, Ashland, OR). Data was fitted using a 4PL in GraphPad Prism 10 (GraphPad Software, La Jolla, CA). The results shown in Figure 8C show that the EGFRxc-MET ADC have a relative higher binding signal on double positive (EGFR+c-MET+) than on single positive (EGFR+c-MET-) cells. Cetuximab binding showed similar binding to both WT and c-MET KO cells. The EGFRxc-MET ADC shows selective binding to dual target expressing cells at clinically relevant antibody concentrations. EXAMPLE 9 – Cytotoxicity The ability of the EGFRxLGR5 ADC to induce killing of A549 target cells was evaluated by a cell viability assay. Cell killing was analyzed by measuring cell viability with CellTiter- Glo® Luminescent cell viability assay (Promega, #G7573). Cetuximab was used as reference control. A549 WT (ATCC, cat. no. CCL-185) and A549 EGFR KO cells were seeded into 96- well plates at a density of 2 x 103 cells per well in complete medium (50 µl). Plates were then incubated for 24 hours at 37 °C/5% CO2. After 24 hours, samples were prepared in complete medium with 8-point 2.5-fold serial dilutions starting at 50 nM and added to the cells in duplicate (25 µl). Assay plates were analyzed after 120 hours and viability assessment was undertaken utilizing the CellTiter-Glo® luminescence assay. Briefly, assay plates were equilibrated at room temperature for 20 minutes before addition of CellTiter-Glo® reagent (100 µL per well, Sartorius, #4633). The plates were then shaken on an orbital mixer for 3 minutes at 300 rpm to assist cell lysis and incubated for a further 20 minutes at room temperature to stabilize the signal. Luminescence was recorded using a SpectraMax i3x plate reader (Molecular Devices, Wokingham, UK). Data was then analyzed on GraphPad Prism version 10 (GraphPad Software, La Jolla, CA) using a four-parameter non-linear regression model. Viability of treated cultures was compared to viability of control, untreated cells (100% viable) and expressed as percentage. The percentage viability was plotted against the drug concentration in nM. The results in Figure 9A show that the EGFRxLGR5 ADC induced a dose-dependent reduction in cell viability of WT cells compared to EGFR-KO cells. Cetuximab did not induce cell death of WT or EGFR KO cells. The ability of the EGFRxc-MET ADC to induce killing of A549 target cells was evaluated by a cell viability assay. Cell killing was analyzed by measuring cell viability with CellTiter-Glo® Luminescent cell viability assay. A549 WT (ATCC, cat. no. CCL-185, A549 c- Met KO and A549 EGFR KO cells were seeded into 96-well plates at a density of 2 x 103 cells per well in complete medium (50 µL) and treated as described earlier for EGFRxLGR5 ADC example. ADC and cetuximab control antibody were prepared in complete medium with 8-point 4-fold serial dilutions starting at 66 nM and added to the cells in duplicate. Assay plates were analyzed after 120 hours and viability assessment was undertaken utilizing the CellTiter-Glo® luminescence assay as described earlier for the EGFRxLGR5 ADC. The results in Figure 9B show that the EGFRxc-MET ADC induced concentration- dependent cell death of WT and c-MET KO A549 cells, but not of EGFR-KO cells. At concentrations where bivalent binding versus monovalent is predicted to occur, cell death levels trended to higher levels on A549 WT vs A549 c-MET-KO cells. Cetuximab did not induce cell death of WT, EGFR KO, or c-MET-KO cells. The ability of the EGFRxc-MET ADC to induce killing of NCI-H1975 target cells was evaluated by a cell viability assay measured by cell viability with CellTiter-Glo® Luminescent cell viability assay. NCI-H1975 WT (ATCC; cat. no. CRL-5908; lot no.70010182) and NCI- H1975 c-Met KO cells were seeded into 96-well plates at a density of 2 x 103 cells per well in complete medium (50 µL). Plates were then incubated for 24 hours at 37 °C/5% CO2. After 24 hours, samples were prepared in complete medium at final assay concentrations starting at 4 nM with a 5-fold 4-point serial dilution and added to the cells in duplicate (25 µL). The cells were then incubated for 120 hours and further treated and analyzed as described before for the EGFRxLGR5 ADC. The results in Figure 9C show that EGFRxc-MET ADC induced concentration-dependent cell death of WT and c-MET KO NCI-H1975 cells. EGFRxc-MET induce more cell death in WT cells compared to c-MET KO cells. These results indicate that selective cytotoxicity, i.e. cytotoxicity of dual target expressing cells versus single target expressing cells, diminish at increasing antibody concentrations. The EGFRxc-MET ADC showed selective cytotoxicity at clinically relevant antibody concentrations in this assay. Cetuximab did not induce cell death of WT or c-MET-KO cells. EXAMPLE 10 - Internalization The ability of the EGFRxc-MET ADC to induce internalization was evaluated by assessing the level of internalization on A549 WT (ATCC, catalogue nr. CCL-185), A549 c- MET-KO, and A549 EGFR-KO cells. To determine ADC internalization capacity in target cells, cells were monitored live for 24 hours, using Incucyte® live cell imaging. First the cells were seeded onto 96 well plates and incubated overnight, with a media change 1 hour prior to imaging. Antibodies were labelled with FabFluor-pH®, which are Fab fragments directed against a Fc region with a pH sensitive dye attached (Bevan et al., 2018), as per manufacturer’s instructions. Antibodies were labelled with FabFluor-pH® reagent at 3:1 Fabfluor-pH®: antibody molar ratio for 15 minutes at 37°C in complete media. A baseline fluorescence scan was taken 15 minutes before commencing the internalization assay. Once the baseline scan was complete, the labelled antibodies were added to the cells at a final concentration of 10 µg/ml and 1 µg/ml. Four hours of scanning took place at approximately 30-minute intervals, followed by a further 20 hours scanning at 1-hour intervals. To analyze, an image threshold was established from the F0 scans. Any red intensity above this threshold recorded was noted as “red area.” This value was normalized to the area of cells within the image (“Phase area”) to give a “Red area/Phase area (%)” value. The ability of EGFRxc-MET ADC to induce internalization was further evaluated by assessing the level of internalization on NCI-H1975 WT, and NCI-H1975 c-MET-KO cells using Incucyte as described above. The results shown in Figure 10A show that EGFRxc-MET ADC induced internalization in A549 cells, at 10 μg/ml and 1 μg/ml, is comparable to the internalization in c-MET-KO cells. The internalization of the EGFRxc-MET ADC in A549 WT cells is higher than in EGFR-KO cells. The results in Figure 10B show internalization of EGFRxc-MET ADC in NCI-H1975 WT cells but not in c-MET KO cells. Overall, the data show that EGFRxc-MET ADC has comparable A549 WT binding to that of cetuximab, but showed relative higher internalization. The EGFRxc-MET ADC showed relative lower binding than cetuximab on c-MET KO cells, but showed relative higher internalization. The above experiments show that exemplary bispecific antibodies comprising the DEKK mutations conjugated to a drug can be produced with clinically relevant DAR, and show preferential binding and activity against dual target expressing cells versus single target expressing cells. In particular, the exemplary bispecific ADCs preserve the target binding selectivity, internalization, and cytotoxicity of the naked, unconjugated bispecific antibodies. In particular, the exemplary bispecific ADCs preserve the target binding selectivity, internalization, and cytotoxicity of the naked, unconjugated bispecific antibodies. In silico pharmacological modelling (data not shown) indicates that in tumor peak and trough concentrations of an EGFRxc-MET ADC (conjugated to MMAE) correspond to those shown experimentally to result in selective binding, internalization and cytotoxicity.
SEQUENCES SEQ ID NO: 1 – Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 2 – Light chain CDR1 according to IMGT QSISSY SEQ ID NO: 3 – Light chain CDR2 according to IMGT AAS SEQ ID NO: 4 – Light chain CDR3 according to IMGT QQSYSTPPT SEQ ID NO: 5 – CL region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 6 – hinge region EPKSCDKTHTCPPCP SEQ ID NO: 7 - CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 8 – CH2 region APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 9 – CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 10 – CH3 region GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 – CH3-DE region GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 – CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNG NTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDY WGQGTLVTVSS SEQ ID NO: 14 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 15 – Heavy chain CDR2 according to Kabat WISAYNGNTNYAQKLQG SEQ ID NO: 16 – Heavy chain CDR3 according to Kabat DRHWHWWLDAFDY SEQ ID NO: 17 – Heavy chain variable region QVQLVQSGSELKKPGASVKISCKASGYDFTNYAMNWVRQAPGHGLEWMGWINANTG DPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDSAVYYCTRERFLEWLHFDYWGQGT LVTVSS SEQ ID NO: 18 – Heavy chain CDR1 according to Kabat NYAMN SEQ ID NO: 19 – Heavy chain CDR2 according to Kabat WINANTGDPTYAQGFTG SEQ ID NO: 20 – Heavy chain CDR3 according to Kabat ERFLEWLHFDY SEQ ID NO: 21 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEY GKTFFAQNFQGRVTMTEDTSADTAYMELSSLRSEDTAVYYCATEGYYETTTYYYNLF DSWGQGTLVTVSS SEQ ID NO: 22 – Heavy chain CDR1 according to Kabat ELSMH SEQ ID NO: 23 – Heavy chain CDR2 according to Kabat GFDPEYGKTFFAQNFQG SEQ ID NO: 24 – Heavy chain CDR3 according to Kabat EGYYETTTYYYNLFDS SEQ ID NO: 25 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKTSGYTFTDYAMTWVRQAPGQGLEWMGWITTNTG DPTYAPGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVYHWIRGFEFWGQGTL VTVSS SEQ ID NO: 26 – Heavy chain CDR1 according to Kabat DYAMT SEQ ID NO: 27 – Heavy chain CDR2 according to Kabat WITTNTGDPTYAPGFTG SEQ ID NO: 28 – Heavy chain CDR3 according to Kabat VYHWIRGFEF SEQ ID NO: 29 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSGGSFSSSSSYWGWIRQPPGKGLEWIGSFYYSGNT YYNPSLKSRVTISEDTSKNQFSLKLSSVTAADTAVYYCARQTYSSSWDGVLYYFDYWG QGTLVTVSS SEQ ID NO: 30 – Heavy chain CDR1 according to Kabat SSSSYWG SEQ ID NO: 31 – Heavy chain CDR2 according to Kabat SFYYSGNTYYNPSLKS SEQ ID NO: 32 – Heavy chain CDR3 according to Kabat QTYSSSWDGVLYYFDY SEQ ID NO: 33 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSNGSISTYYWSWIRQPPGKGLEWIGYVYYTGRTKY NPSLKSRVTISVDTSKNQFSLNLSSVTAADTAVYYCARGGIVVVPAARDYYYYMDVW GKGTTVTVSS SEQ ID NO: 34 – Heavy chain CDR1 according to Kabat TYYWS SEQ ID NO: 35 – Heavy chain CDR2 according to Kabat YVYYTGRTKYNPSLKS SEQ ID NO: 36 – Heavy chain CDR3 according to Kabat GGIVVVPAARDYYYYMDV SEQ ID NO: 37 – Heavy chain variable region EVQLVQSGAEVKKPGESLKIACKGSGFSFTSHWIGWVRQKPGRGLEWMGVIYPGDSD TRYSPSFQGQVTVSADKSINTAYLQWNSLKASDTAIYYCARPNSGSPRYFEFWGRGTL VTVSS SEQ ID NO: 38 – Heavy chain CDR1 according to Kabat SHWIG SEQ ID NO: 39 – Heavy chain CDR2 according to Kabat VIYPGDSDTRYSPSFQG SEQ ID NO: 40 – Heavy chain CDR3 according to Kabat PNSGSPRYFEF SEQ ID NO: 41 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSFYYSGNT YYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQEYYYGSGSPSYYFDYWG QGTLVTVSS SEQ ID NO: 42 – Heavy chain CDR1 according to Kabat SSSYYWG SEQ ID NO: 43 – Heavy chain CDR2 according to Kabat SFYYSGNTYYNPSLKS SEQ ID NO: 44 – Heavy chain CDR3 according to Kabat QEYYYGSGSPSYYFDY SEQ ID NO: 45 – Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKGSGDSFISHWIAWVRQMPGKGLEWMGIVYPGDSDT RYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHEWELLGPFDYWGQGTL VTVSS SEQ ID NO: 46 – Heavy chain CDR1 according to Kabat SHWIA SEQ ID NO: 47 – Heavy chain CDR2 according to Kabat IVYPGDSDTRYSPSFQG SEQ ID NO: 48 – Heavy chain CDR3 according to Kabat HEWELLGPFDY SEQ ID NO: 49 – Heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGTSTNDAISWVRQTPGQGLEWMGSIIPILDTT DHAQKFQGRVTITADKSTNTAYMELNSLRSDDTAVYYCAREHIAARQDYFDYWGQGT LVTVSS SEQ ID NO: 50 – Heavy chain CDR1 according to Kabat NDAIS SEQ ID NO: 51 – Heavy chain CDR2 according to Kabat SIIPILDTTDHAQKFQG SEQ ID NO: 52 – Heavy chain CDR3 according to Kabat EHIAARQDYFDY SEQ ID NO: 53 – Heavy chain variable region EVQLVQSGSKLKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMGWINTDTG DPTYAQGFTGRFVFSLDTSVSTAFLQINSLKAEDTAVYYCARGDCDSTSCYRYSYGYE DYWGQGTLVTVSS SEQ ID NO: 54 – Heavy chain CDR1 according to Kabat SYTMN SEQ ID NO: 55 – Heavy chain CDR2 according to Kabat WINTDTGDPTYAQGFTG SEQ ID NO: 56 – Heavy chain CDR3 according to Kabat GDCDSTSCYRYSYGYEDY SEQ ID NO: 57 – Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKVSGGTFRSYAISWVRQAPGQGLEWMGGIIPIFDTR NYAQILQGRVTITADLSTSTAYMELNSLRSEDTAIYYCARGSDEGDWFDPWGQGTLVT VSS SEQ ID NO: 58 – Heavy chain CDR1 according to Kabat SYAIS SEQ ID NO: 59 – Heavy chain CDR2 according to Kabat GIIPIFDTRNYAQILQG SEQ ID NO: 60 – Heavy chain CDR3 according to Kabat GSDEGDWFDP SEQ ID NO: 61 – Heavy chain variable region EVQLVQSGTEVRKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGSIIPILGTT DHAQKFQDRVTITADKSSNTTYMELSSLRSDDTAVYYCAREYIAARLDYFDSWGQGTL VTVSS SEQ ID NO: 62 – Heavy chain CDR1 according to Kabat NYAIS SEQ ID NO: 63 – Heavy chain CDR2 according to Kabat SIIPILGTTDHAQKFQD SEQ ID NO: 64 – Heavy chain CDR3 according to Kabat EYIAARLDYFDS SEQ ID NO: 65 – Heavy chain EVQLVQSGSKLKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMGWINTDTG DPTYAQGFTGRFVFSLDTSVSTAFLQINSLKAEDTAVYYCARGDCDSTSCYRYSYGYED YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 66 – Heavy chain QVQLVQSGSELKKPGASVKISCKASGYDFTNYAMNWVRQAPGHGLEWMGWINANTG DPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDSAVYYCTRERFLEWLHFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 67 – Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

CLAIMS 1. An antibody-drug conjugate comprising an antibody with a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue.
2. The antibody-drug conjugate according to claim 1, wherein the CH3 domain of the first heavy chain comprises amino acid substitution T366K and the CH3 domain of the second heavy chain comprises amino acid substitution L351D.
3. The antibody-drug conjugate according to claim 2, wherein the CH3 domain of the first heavy chain further comprises amino acid substitution L351K.
4. The antibody-drug conjugate according to claim 2 or 3, wherein the CH3 domain of the second heavy chain further comprises amino acid substitution Y349E, Y349D, or L368E.
5. The antibody-drug conjugate according to claim 4, wherein the CH3 domain of the second heavy chain further comprises amino acid substitution L368E.
6. The antibody-drug conjugate according to any one of claims 1-5, wherein the antibody of the antibody-drug conjugate is a multispecific antibody, in particular a bispecific or trispecific antibody.
7. The antibody-drug conjugate according to any one of claims 1-6, wherein the antibody of the antibody-drug conjugate comprises a variable domain that binds to EGFR and a variable domain that binds to LGR5.
8. The antibody-drug conjugate according to claim 7, wherein the variable domain that binds to EGFR comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO: 13, 17, 21, or 25.
9. The antibody-drug conjugate according to claim 7 or 8, wherein the variable domain that binds to LGR5 comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61.
10. The antibody-drug conjugate according to any one of claims 7-9, wherein the variable domain that binds to EGFR and/or LGR5 comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequences as set forth in SEQ ID NO: 1.
11. The antibody-drug conjugate according to any one of claims 7-10, wherein - the variable domain that binds to EGFR comprises a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 18, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 20; - the variable domain that binds to LGR5 comprises a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 54, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 55, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 56; and wherein the variable domain of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 2, LCDR2 having an amino acid sequence as set forth in SEQ ID NO:3, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 4.
12. The antibody-drug conjugate according to any one of claims 7-11, wherein the variable domain that binds to EGFR comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 13, 17, 21, or 25, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
13. The antibody-drug conjugate according to any one of claims 7-12, wherein the variable domain that binds to LGR5 comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 29, 33, 37, 41, 45, 49, 53, 57, or 61, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
14. The antibody-drug conjugate according to any one of claims 1-13, wherein the variable domain that binds to EGFR and/or LGR5 comprises a light chain variable region comprising the amino acid sequences as set forth in SEQ ID NO: 1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
15. The antibody-drug conjugate according to any one of claims 7-14, wherein - the variable domain that binds to EGFR comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; - the variable domain that binds to LGR5 comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.
16. The antibody-drug conjugate according to any one of claims 7-15, wherein - the variable domain that binds to EGFR comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 17; - the variable domain that binds to LGR5 comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 53; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to LGR5 comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1.
17. The antibody-drug conjugate according to any one of claims 1-16, wherein the antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, govitecan, ozogamicin, saratolacan, and soravtansine.
18. An antibody-drug conjugate that competes with an antibody-drug conjugate according to any one of claims 1-17 for binding to EGFR and/or LGR5.
19. A pharmaceutical composition comprising an effective amount of an antibody-drug conjugate according to any one of claims 1-18, and a pharmaceutically acceptable carrier.
20. An antibody-drug conjugate according to any one of claims 1-18, or a pharmaceutical composition according to claim 19, for use in therapy.
21. The antibody-drug conjugate antibody-drug conjugate according to any one of claims 1-18, or the pharmaceutical composition according to claim 19, for use in the treatment of cancer.
22. A method for treating a disease, comprising administering an effective amount of an antibody-drug conjugate according to any one of claims 1-18, or the pharmaceutical composition according to claim 19, to a subject in need thereof.
23. A method for treating cancer, comprising administering an effective amount of an antibody-drug conjugate according to any one of claims 1-18, or the pharmaceutical composition according to claim 19, to a subject in need thereof.
24. A method for producing an antibody-drug conjugate, the method comprising: - providing an antibody comprising a first and a second heavy chain, each heavy chain comprising a CH3 domain, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue; and - coupling a linker-drug combination to the antibody.
25. The method according to claim 24, wherein the CH3 domain of the first heavy chain comprises amino acid substitution T366K and wherein the CH3 domain of the second heavy chain comprises amino acid substitution L351D.
26. The method according to claim 25, wherein the CH3 domain of the first heavy chain further comprises amino acid substitution L351K.
27. The method according to claim 25 or 26, wherein the CH3 domain of the second heavy chain further comprises amino acid substitution Y349E, Y349D, or L368E.
28. The method according to claim 27, wherein the CH3 domain of the second heavy chain further comprises amino acid substitution L368E.
29. The method according to any one of claims 24-28, wherein the antibody of the antibody-drug conjugate is a multispecific antibody, in particular a bispecific or trispecific antibody.
30. The method according to any one of claims 24-29, wherein the drug of the linker- drug combination is a cytotoxic drug.
31. The method according to claim 30, wherein the cytotoxic drug is selected from the group consisting of MMAE, MMAF, maytansine, exatecan, SN-38, SCX, and calicheamicin, or a derivative or analog thereof.
32. The method according to any one of claims 24-31, wherein the linker-drug combination is selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, govitecan, ozogamicin, saratolacan, and soravtansine.
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